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No part of this publication may be reproduced, stored in a retrieval system or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission of the publisher. 106 «2 2015 ACTA AGRICULTURAE SLOVENICA Biotehniška fakulteta Univerze v Ljubljani Biotechnical Faculty University of Ljubljana Acta agriculturae Slovenica • ISSN 1581-9175 • 106 - 2 • Ljubljana, december 2015 Acta agriculturae Slovenica Volume / Letnik 106 • Number / Številka 2 • 2015 VSEBINA / CONTENTS MIKROBIOLOGIJA / MICROBIOLOGY David VOGRINC, Maša VODOVNIK, Romana MARINŠEK-LOGAR 67 Microbial biosensors for environmental monitoring Mikrobni biosenzorji za monitoring okolja EKONOMIKA / ECONOMICS Jaka ŽGAJNAR 77 Ekonomska analiza organiziranosti jahalnega centra; primer uporabe linearnega programiranja Economic analysis of the organisation of a riding centre; application of the linear programming approach ŽIVINOREJA / ANIMAL SCIENCE Jernej OGOREVC, Tjaša LAPANJA, Klavdija POKLUKAR, Natalija TOMINŠEK, Peter DOVČ 87 Establishment of primary keratinocyte culture from horse tissue biopsates Vzpostavitev primarne celične kulture konjskih keratinocitov iz biopsij Martina PLANINC, Milena KOVAČ, Špela MALOVRH 93 Analysis of backfat thickness in on-farm tested gilts in Slovenia using reaction norms Reakcijske norme za debelino hrbtne slanine pri testiranih mladicah v Sloveniji Dušan TERČIČ, Mirjana ŽOLGER, Mojca PESTOTNIK 97 Effect of different litter materials on foot pad dermatitis, hock burn and feather coverage in broiler chickens Vpliv različnih vrst nastila na pojav vnetja kože na blazinicah stopal, vnetja kože skočnih sklepov ter na operjenost pri pitovnih piščancih Mojca SIMČIČ, Miran ŠTEPEC, Betka LOGAR, Klemen POTOČNIK 103 Analiza lastnosti zunanjosti pri plemenskih bikih cikastega goveda Analysis of type traits of cika sires Tomaž BARTOL 113 Subject index by AGROVOC descriptors Predmetno kazalo po deskriptorjih AGROVOC Nataša SIARD 115 Subject index by AGRIS category codes Vsebinsko kazalo po predmetnih kategorijah AGRIS 117 Abecedno kazalo avtorjev Author's index 119 Navo dila avtorj em 121 Notes for authors COBISS: 1.02 Agris category code: T01 MICROBIAL BIOSENSORS FOR ENVIRONMENTAL MONITORING David VOGRINC Maša VODOVNIK 2, Romana MARINŠEK-LOGAR 3 Received September 10, 2015; accepted September 30, 2015. Delo je prispelo 10. septembra 2015, sprejeto 30. septembra 2015. Microbial biosensors for environmental monitoring Microbial biosensors are analytical devices capable of sensing substances in the environment due to the specific biological reaction of the microorganism or its parts. Construction of a microbial biosensor requires knowledge of microbial response to the specific analyte. Linking this response with the quantitative data, using a transducer, is the crucial step in the construction of a biosensor. Regarding the transducer type, biosensors are divided into electrochemical, optical biosensors and microbial fuel cells. The use of the proper configuration depends on the selection of the biosensing element. With the use of transgenic E. coli strains, bioluminescence or fluorescence based biosensors were developed. Microbial fuel cells enable the use of the heterogeneous microbial populations, isolated from wastewater. Different microorganisms are used for different pollutants - pesticides, heavy metals, phenolic compounds, organic waste, etc. Biosensing enables measurement of their concentration and their toxic or genotoxic effects on the microbes. Increasing environmental awareness has contributed to the increase of interest for biomonitoring. Although technologies, such as bioinformatics and genetic engineering, allow us to design complex and efficient microbial biosensors for environmental pollutants, the transfer of the laboratory work to the field still remains a problem to solve. Key words: microbiology / environmental protection / microbial biosensors / environmental pollutants / microbial fuel cells / bioluminescence / genetics / bioinformatics / genetic engineering Mikrobni biosenzorji za monitoring okolja Mikrobni biosenzorji so analitske naprave, ki nam omogočajo zaznavanje snovi v okolju zaradi specifične biološke reakcije, ki poteka v mikroorganizmu ali njegovem delu. Če želimo takšno napravo uporabiti za monitoring onesnažil v okolju, moramo dobro poznati odziv mikroorganizma na specifičen analit in ga preko pretvornika signala spremeniti v kvantitativno obliko. Poznamo različne konfiguracije mikrobnih biosenzorjev glede na pretvornik signala - elektrokemijske in optične biosenzorje ter mikrobne gorivne celice. Vsaka ima svoje prednosti in slabosti, katero uporabimo, je v veliki meri odvisno od izbora biosenzorskega organizma. Transgene celice E. coli nam omogočajo bioluminescenčno ali fluorescenčno zaznavo, v mikrobne gorivne celice pa lahko vključimo mešane mikrobne združbe. Z izborom organizma se prilagajamo tudi onesnažilu. Med najpogostejšimi onesnažili so pesticidi, težke kovine, fenoli, organski odpadki. Z biosenzorji ne spremljamo le njihovih koncentracij v okolju, pač pa beležimo tudi toksične in genotoksične vplive analitov na mikroorganizme. S povečevanjem skrbi za okolje narašča tudi pomen mikrobnih biosen-zorjev. Razvoj tehnologij, kot sta bioinformatika in genetski inženiring, nam omogoča temeljitejše in uspešnejše načrtovanje uporabe mikrobnih biosenzorjev v okoljskih aplikacijah. Izziv za prihodnje pa ostaja prenos mikrobne biosenzorske tehnologije na teren. Ključne besede: mikrobiologija / varstvo okolja / mikrobni biosenzorji / okoljska onesnažila / mikrobne gorivne celice / bioluminescenca / genetika / bioinformatika / genetski inženiring 1 Univ. of Ljubljana, Biotechnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia 2 Same address as 1 3 Same address as 1, e-mail: romana.marinsek@bf.uni-lj.si 1 INTRODUCTION A biosensor is a self-contained integrated device, capable of providing specific quantitative or semi-quantitative analytical information using a biosensing element connected with a transducer (IUPAC 1996, Thevenot et al., 2001). Biosensor construction, a three-step process, involves combining two elements with different characteristics. First, a biological sensing element is chosen, then a transducer is selected, and finally the biological component (detection element) is fixed to the transducer (Xu and Ying, 2011). Enzymes, antibodies, cell receptors, microorganisms, animal and plant cells or tissue cultures can be used as biorecognition components of a biosensor. Microorganisms have a huge potential for detection of a wide spectrum of chemical substances and their mixtures, they are adjustable to different reaction conditions and compared to enzymes or antibodies do not require expensive preparation processes (Shin 2010, Xu and Ying, 2011). They can be genetically modified, too. This characteristic enables the use of microbial biosensors in the fields of environmental monitoring, food safety and medicine. 2 CHOICE AND APPLICATION OF A PROPER ORGANISM Choice of a proper microorganism for the detection of pollutants and their effects in the environment and its incorporation with the competent transducer is a key step in the development of an environmental biosensor. Bacteria and yeast are the most commonly used (Xu and Ying, 2011). The chosen microorganism must be robust and capable of specific pollutant detection in small concentrations, to ensure price efficient detection. Recently whole-cell biosensors (Chan et al., 2013; Niazi et al., 2008; Mulchandani and Rajesh, 2011; Anu Prathap et al., 2012) and microbial fuel cells (Di Lorenzo et al., 2014; Shen et al., 2013; Liu et al., 2014; Ayyaru and Dharmaligman, 2013) draw special attention on the field of environmental monitoring. Genetic engineering became important, too. We can manipulate organisms to improve mechanisms of analyte detection or express them in new organisms (Mulhandani and Rajesh, 2011). DNA segments coding for detection mechanisms can be transferred into model organisms with optimized growing conditions, such as Escherichia coli and Saccharomyces cerevisiae. The organism and the detection configuration should be combined properly to achieve the best possible detection of the signal. 3 BIOSENSOR CONFIGURATION There are three main types of microbial biosensors classified, based on different signal transducers: electrochemical, optical and microbial fuel cells (Xu and Ying, 2011). Electrochemical transducers use the change of the electric current, potential and conductivity, caused by mi-crobial-analyte contact. They can be further divided into amperometric, potentiometric and conductometric biosensors. Amperometric microbial biosensors operate at a fixed potential with respect to a reference electrode, and then the corresponding current is obtained due to the oxidation or reduction of electroactive species at the surface of the electrode (Xu and Ying, 2011). This configuration has been described by Yong et al. (2011), Anu Prathap et al. (2012) and Wang et al. (2013); on the other hand, the potentiometric transducer was constructed by Mulchan-dani and Rajesh (2011). Transducers of this kind use ion-selective electrodes to transmit the biological signal into an electric signal. They are less sensitive, produce higher relative error and a worse linear relationship between the exporting signal and the concentration of the detected analyte (Xu and Ying, 2011). As it is obvious from their name, conductometric biosensors measure changes in conductivity of the media, caused by the target analyte. Although the conductance measurements are extremely sensitive, the detection of solution conductance is considered to be nonspecific (Xu and Ying, 2011). Optical biosensors can be defined as sensor devices that make use of optical principles, such as biolumines-cence, fluorescence and colorimetry for transduction of a biochemical interaction into a suitable output signal (Xu and Ying, 2011). The use of genetic engineering enables an expression of fluorescence and bioluminescence in the target organism. Scientists report of luciferase (Niazi et al., 2008; Shin, 2010; Chan et al., 2013) and green fluorescent protein (GFP) applications (Wei et al., 2013; Kim et al., 2015). Microbial fuel cells (MFC) are bioelectro-chemical devices that produce electrical energy through the action of specific microbes (known as anodophiles), capable of transferring the electrons generated from the oxidation of organic compounds (the fuel) to an anode electrode (Di Lorenzo et al., 2014). In a typical two-chamber MFC (Fig. 1), the electrons are absorbed by the anode and are transported to the cathode through an external circuit. After crossing a proton exchange membrane, the protons enter the cathodic chamber where they combine with oxygen to form water (Du et al., 2007). Electric current, produced by fuel oxidation, can serve as a transducer of a microbial response to the analyte. Due to their simple design and low cost, single-chamber MFC, where the cathode is exposed to air, are extensively used in envi- Figure 1: A scheme of a two-chamber microbial fuel cell Slika 1: Shema dvoprekatne mikrobne gorivne celice ronmental applications (Du et al., 2007). They are useful for the detection of biochemical oxygen demand (BOD) (Liu et al., 2013, Ayyaru and Dharmaligman, 2014), heavy metals and their toxicity (Shen et al., 2013; Liu et al., 2014; Di Lorenzo et al., 2014). MFC enable the use of heterogeneous microbial populations, isolated from wastewater plants and other working MFC. This characteristic makes them very successful for the development of sensitive, specific and cost efficient biosensors. 4 ENVIRONMENTAL APPLICATIONS Organisations, like WHO and FAO, have realised the negative effect of pollutants on human health (Bereza-Malcolm et al., 2015). Together with their concern grows the need for monitoring of dangerous substances in the environment. Pollutant residues can eventually accumulate in our food and drinking water. Food quality control systems are established to prevent that kind of cases. A biosensor, used instead of conventional chemical monitoring methods, must be easy to use, cost-efficient, stable when stored and capable of the detection of small amounts of the analyte. It must have a low detection limit and a short response time (Kumar et al., 2006). Transferring a working microbial biosensor under controlled laboratory conditions onto the field requires a lot of work and optimization. What follows is an overview of current trends in the field of environmental microbial monitoring. 4.1 PESTICIDES Pesticides are chemical or biological substances meant for pests control. Considering the target organism, we can divide them into insecticides, herbicides, fungicides, bacteriocides, nematocides and others. The use of the first three listed above represent 95 % of world consumption (Aktar et al., 2009). Insecticides are the most acute toxic group of pesticides. Their extensive use has a major environmental impact, resulting in water and ground accumulation. The negative effect of the pesticides rose awareness decades ago and led to the development of detection methods like gas and liquid chromatography for the monitoring of organophosphate pesticides. These methods have brought high selectivity and sensitivity, but are inappropriate for field detection, require expensive equipment and a skilled technician (Mulchandani and Rajesh, 2011). Furthermore, chemical analytical methods only provide the information of pesticide identity and quantity, but no information about their toxicity. Some biosensors are capable to detect pesticide toxicity and therefore they are suitable for their detection. Biosensors represent their alternative. Kumar et al. (2006) report of an optical biosensor with bacteria Flavobacterium cells, adsorbed on the glass fiber. The cells express organophosphate hydrolase on their surface, an enzyme, capable of the hydrolysis of the organophosphate pesticides to the optically measurable colour products. Synthetic samples of the methyl parathion have been tested with the biosensor and a gas chromatography analysis. Biosensoric analysis was based on the ratio between the hydrolysed methyl parathion and the amount of the colourful product - p-nitrophe-nol. The methods have comparable results, but the biosensoric analysis is more cost-efficient. The developed biosensor showed a lower detection limit compared to other similar devices. The same detection principle of the p-nitrophenol as a hydrolysing product of the methyl parathion, was applied by Kumar and D'Souza (2010). They immobilized bacteria Sphingomonas JK1 on the bottom side of the microplate and linked them with an optical plate reader, to form an optical biosensor. This system enables multiple sample detection on one plate. Biosensor can detect 4-80 цЫ concentrations of methyl parathion and can be reused up to 75 times. Kumar and D'Souza (2011) also report of a recombinant E. coli, peri-plasmically expressing enzymes for methyl parathion hydrolysis, as a biorecognition element. Microbial cells were immobilized on a screen printed electrode, using glutaraldehyde. The researchers observed the changes in the electric current, caused by different concentrations of the methyl parathion. Biosensor showed good selectivity - it did not react to the addition of glucose, sucrose and endosulfan; the response to the phenol and p-ami-nophenyl sulfate was insignificant and had good stability - it preserved 80 % of the enzymatic activity after being used in 32 reactions. Expression of the organophosphate hydrolase on the surface of the cells is efficient, its sensitivity can be improved by the application of the genetic engineering methods. Recombinant biofilm forming bacteria Moraxella, containing the ICN protein from the Pseudosomonas syringae INA5, are capable of the detecting 1 цЫ methyl parathion and 0.2 цЫ paraoxon (Mulchandani and Rajesh, 2011). Overexpression of the linA2 gene, encoding the Y-hexachlorocyclohexane dehydrochlorinase (LinA2) in E. coli BL21, has been used by Anu Prathap et al. (2012) for the development of a sensitive, selective and fast electrochemical biosensor. LinA2 protein catalyses the dehydrochlorination of lindane into trichlo-robenzene, forming HCl as a by-product and causing an increase in the conductivity of the cell microenvironment that can be detected with the pulse amperometry. Authors report about the detection limit of 2 ppt for lin-dane. E. coli is among the most frequently used organisms in the field of microbial biosensors. A bioluminescent strain PGRFM, including luxCDABE operon and promotor region of the pgi gene, important for the metabolic answer to the oxidative stress, was applied by Niazi et al. (2008) for the construction of an optical biosensor. The sensor showed excellent response to the methyl viologen, pesticide that causes the induction of reactive oxygen species, with the detection limit of 0.6 ppm, when exposed to starving conditions. Another detection principle for the toxicity of ametryn, fenamiphos and endosulfan was reported by Yong et al. (2011). Amperometrically working ferricyanid was used as a redox probe to measure the overall toxicity of the chemicals on the E. coli respiration. Endosulfan was the most toxic, with the IC50 = 5.7 mg/L. 4.2 HEAVY METALS Heavy metals are extensively used in several industry branches such as mining, metallurgical, electronics, electroplating and metalfinishing (Wang et al., 2013). The main threats to human health from heavy metals are associated with exposure to lead, cadmium, mercury and arsenic (Järup, 2003). Standard detection techniques - spectrometry, ionic chromatography, potentiometric electrodes - are expensive, sometimes time consuming and require high skilled technicians. Development of simple methods, suitable for field application, is the priority in the field of heavy metal analysis. Biosensor detection is among them. Yüce et al. (2010) reported on the inclusion of the cyanobacterium Phormidium as the biosensing element of an amperometrical biosensor. Heat treated dead cy-anobacterial biomass was mixed with carbon dust and added to a steal rod to form an electrode, capable of the detection of Pb(II) in water solution. The Ag/AgCl reference electrode and platinum wire as counter electrode were also the part of the apparatus that measures the changes in the electric field, induced by heavy metal water solution. Results showed good stability and repeatability, a hypothetical limit of detection was set for 5 X 10-8 M. Microbial fuel cells (MFC) became important as well. The presence of a pollutant in wastewater can inhibit the metabolic activity of the electrochemically active bacteria, leading to the reduced electron transfer and weak current production. Single-chamber air-cathode MFC, enriched with real domestic wastewater have been applied by Shen et al. (2013) for the detection of Cu (II). They were interested in the response of a biofilm, formed by microorganisms in wastewater, at different flow rates. Higher feed rate causes higher shear rate in the surrounding of the MFC, leading to the overproduction of the extracellular polymeric substances and reduced biosensor sensitivity. The effect of metals on microbes can be also measured with an oxidative stress biosensor. Ooi et al. (2015) constructed a biosensor, using E. coli DH5a™ transformed with pRSET-roGFP2 plasmid that enables fluorescent de- tection of arsenic induced oxidative stress. The biosensor is fast, efficient and enables detection down to 0.2 ^g/l of arsenic. The same microorganism was used by Arias-Barierro et al. (2010) for the detection of Cd2+, Cu2+, Pb2+, Zn2+ and arsenite. The described biosensor is even more sensitive and enables the detection down to 1*10-7 mg/l arsenite, 0.001 ppm copper and zinc ions, 0.01 ppm cadmium ion and 5 ppm lead ions. A continuous flow of the analyte to the biosensor is the most recent improvement in the field of biomonitoring. Kim et al. (2015) incorporated E. coli DH5 in a microfluidic device, capable of feeding nutrients and various concentrations of heavy metals ions under continuous-feed mode, for the detection of Pb2+ and Cd2+. The detection mechanism is based on the negative control of the GFP reporter gene, mediated by CadC-type transcriptional repressors, which bind to Pb2+ or Cd2+ divalent ions and derepress the GFP reporter promoters. They observed 3-4 fold increase in the sensitivity of the biosensor and good specificity dynamics to detect Pb2+ in Cd2+, comparing to conventional batch-type detection modes. An alternative approach in the construction of a biosensor for the detection of heavy metal pollution on the field enables synthetic biology. The environmental pressure - oscillation in the temperature, pH, access to the nutrients and toxicants in the environment affect a diverse set of regulatory elements, controlling the downstream signal cascade (Bereza-Malcolm et al., 2015). Microbial biosensor can be constructed de novo, using regulatory elements for the production of new genetic circuits. The authors estimate that this biosensor application can solve the problem of weak specificity and the toxic nature of heavy metals to the microbial chassis in real world applications. 4.3 TOXICITY AND GENOTOXICITY The overall effect of the pollutants on the environment cannot be determined without an estimation of their toxicity. For the measurements of toxicity of water and ground samples, we use commercially developed tests - Mictorox® and ToxAlert® with Vibrio fischeri, Cellsense® with Escherichia coli (Rodriguez-Mozaz et al., 2004). They use fluorescent and amperometric detection. These systems no longer fulfil the need for monitoring of the toxicants in the environment, so the development of new methods is of great interest. The secondary plant metabolites can show antimicrobial activity. Chan et al. (2013) developed two biosensors for the evaluation of aldehyde and phenolic terpenes and isothiocyanate on the microbes. The first biosensor combined the characteristics of commercial biosensors -they used E. coli HB101 with the luxCDABE gene from V. fischeri, the other biosensor consisted of Acinetobacter baylyi ADP1_recA_lux, transformed with the luxCDABE gene from Photorhabdus luminescens. These transgenic bacteria produce light in the presence of toxicants, damaging the DNA; the intensity of the light is directly correlated with the recA expression level. RecA is an essential DNA repair gene. Isothiocyanate and cinnamaldehyde are the most toxic substances for E. coli - they mechanically damage plasmalemma, weaken the cell metabolism and the production of the energy, but they do not activate recA A. baylyi - it is less plausible that they damage the microbial genome. Many studies examine the toxicity of heavy metals. An amperometrical microbial biosensor ToxTell applies different microbial species as a biosensing element, giving the optimal results of the toxicity of the real samples (Wang et al., 2013). The test organisms, Psychrobacter bacteria, isolated from the wastewater plant were immobilized on a polycarbonated screen printed electrode membrane. They investigated the toxicity of Cu2+, Cd2+, Zn2+, Cr6+, Hg2+ and Pb2+ to determine the EC50 value. The highest EC50 value was observed for Pb - 110 mg/l, the lowest for Hg - 0.8 mg/l. The toxicity of the metals increases with the decrease of particle size, as shown by Ivask et al. (2014). They investigated the toxicity of the silver nanoparticles, according to their size, to bacteria E. coli and Pseudomonas fluorescens, yeast S. cerevisiae and microalgae Pseudokirchneriella subcapitata. The latter showed the highest sensitivity. Liu et al. (2014) reported on the use of MFC as a real-time wastewater toxic shock biosensor. They monitored the response of the microbes, isolated from a wastewater plant, to the shocks of Cr6+, Fe3+, NO3- and sodium acetate. The growth of a biofilm on the anodic electrode was observed after five days. The biofilm enables the support and protection for electro-genic bacteria and improves the biosensor specificity - it can differentiate the chromium, iron, nitrate and sodium acetate shock. Single-chamber air-cathode MFC was used by Di Lorenzo et al. (2014) for the detection of cadmium. At optimal pH and temperature, the addition of cadmium in feeding water caused immediate change in the outgoing current. The biosensor enabled the detection of cadmium in the range of 1 to 50 ^g/l. In an extensive study of the application of yeast in a hypersensitive biosensor, capable of automatic detection of a broad spectrum of genotoxic pollutants, Wei et al. (2013) used transformed and mutated S. cerevisiae BY4741 cells to establish their response on genotoxic chemicals (methyl sulfonyl methane (MMS), 4-nitroqui-noline-oxide (4-NQO), phleomycin, hydrogen peroxide, tert butyl hydroperoxide methil viologen, chlorambucil Table 1: Overview of the biosensors, according to the target analyte, used microorganism, transducer type and detection specificity Preglednica 1: Pregled biosenzorjev po tarčnih analitih, uporabljenem mikroorganizmu, vrsti prevodnika in specifičnosti detekcije Analyte Microorganism Transducer tyPe Detection limit (LOD), EC50 or IC50 Reference Methyl parathion Flavobacterium Optical LOD = 0.3 ||M Kumar et al. (2006) Methyl parathion Sphingomonas JK1 Optical Detection range: 4-80 |M Kumar and D'Souza (2010) Methil parathion Recombinant E. coli Electrochemical LOD = 0.5 |M Kumar and D'Souza (2011) Methyl parathion, paraoxon Recombinant Moraxella Electrochemical Methil parathion: LOD = 1 |M, paraoxon: LOD = 0.2 ||M Mulchandani and Rajesh (2011) Lindane Recombinant E. coli BL21 Electrochemical LOD = 2 ppt Anu Prathap et al. (2012) Methyl viologen Recombinant E. coli Optical LOD = 0.6 ppm Niazi et al. (2008) DCP, ametryn, endosulfan, fenamiphos E. coli Electrochemical IC50 = 5.7-22 mg/L Yong et al. (2011) Lead Phormidium Electrochemical LOD = 2.5*10-8 M Yüce et al. (2010) Copper Heterogeneous microbial populations MFC LOD = 5 ppm Shen et al. (2013) Arsenite, selenite Recombinant E. coli DH5a™ Optical Arsenite: LOD = 0.2 |g/l, selenite: LOD = 5.8 ng/l Ooi et al. (2015) Cadmium, copper, lead, zinc, arsenite Recombinant E. coli DH5a™ Optical Pb: LOD = 5 ppm, Cd: LOD = 0.01 ppm, Cu, Zn: LOD = 0.001 ppm, arsenite: LOD = 1*10-7 mg/l Arias-Barierro et al. (2010) Zinc, cadmium Recombinant E. coli DH5 Optical / Kim et al. (2015) Isothiocyanate, cinnamaldehyde Recombinant E. coli HB101 Optical / Chan et al. (2013) Isothiocyanate, cinnamaldehyde Recombinant Acinetobac-ter baylyi ADP1 Optical / Chan et al. (2013) Copper, cadmium, zinc, chromium, mercury, lead Psychrobacter Electrochemical EC50: 0.8-110.1 mg/l Wang et al. (2013) Silver nanoparticles E. coli, P. fluorescens, S. cerevisiae, P. subcapitata Optical EC50: 0.01-8.17 mg/l Ivask et al. (2014) Chromium, iron, nitrate, sodium acetate Heterogeneous microbial populations MFC / Liu et al. (2014) Cadmium Electroactive mixed bacteria MFC Detection range: 1-50 |g/l Di Lorenzo et al. (2014) MMS, 4-NQO, phleomy-cin, hydrogen peroxide, tert butyl hydroperoxide methyl viologen, chlo-rambucil and cisplatin S. cerevisiae BY4741 Optical 4-NQO: LOD = 0.12 ng/ml MMS: LOD = 0.36 |g/ml Wei et al. (2013 Zinc, copper, 3,5-DCP, benzene, toluene, bro-nopol Recombinant E. coli HB101 Optical EC50: 0.09-21.0 mg/l Horsburgh et al. (2002) Catechol Lactobacillus Electrochemical Detection range: 0.5-5.0 mM Sagiroglu et al. (2011) Continued overleaf / nadaljevanje na naslednji strani 72 Acta agriculturae Slovenica, 106/2 - 2015 Salicylate Recombinant E. coli Optical LOD = 0.1 ||M Shin (2010) DH5a BOD P. putida SG10 Electrochemical Detection range: 0.5-10 mg/l Chee (2013) BOD Electroactive mixed bacteria MFC Detection range: 100-750 ppm Ayyaru and Dhar-maligman (2014) BOD Heterogeneous microbial populations MFC Detection range: 3-164 ppm Di Lorenzo et al. (2014) BOD Heterogeneous microbial Electrochemical Detection range: Vaiopoulou et al. populations 20-450 mg/l (2005) and cisplatin). Transcripts for RNR3 and HUG1 genes that work as a sensor due to their overexpression induced with DNA damage were linked with the yEGFP reporter gene, enabling the fluorescent detection. They report of improved detection at mutants with five or seven genes deleted. The highest sensitivity was observed with quintuple and septuple mutants. The septuple mutant of the HUG1 sensor gene showed the greatest sensitivity (relative sensitivity: 0.12 ng/ml for 4-NQO and 0.36 |g/ml for MMS). Real-time monitoring is a key in the cases of large spills of toxicants to ensure an immediate response and reduce the negative effects on the environment (Di Lorenzo et al., 2014). On-line microbial biosensor can be used for automatized detection of toxicity. The E. coli HB101 cell suspension transformed with the pUCD607 plasmid with a lux CDABE insert was applied by Hors-burgh et al. (2002) for the detection of the toxicity of environmental samples from a metal plating plant, a paper mill and a distillery. They constructed a pump system, enabling continuous flow of the cells, mixed with the samples to a light detection unit. This biosensor is sensitive on a broad spectrum of chemicals (zinc, copper, 3,5-DCP, benzene, toluene, bronopol), the EC50 values measured with a biosensor for zinc and bronopol were significantly more reliable than EC50 measured by batch mode in a cuvette. The biosensor of this kind enables quick and cheap making of environmental samples fingerprint, without the use of chemicals. 4.4 PHENOLIC COMPOUNDS Phenolic compounds that appear in the environment originate from the paper and pulp industry and from the production of drugs, dyes, and antioxidants (Rodriguez-Mozaz et al., 2004). Lyophilised cells of the bacteria Lactobacillus, that were immobilised on a teflonmembrane oxygen electrode, work as a practical biosensor, suitable for the detection of catechol in wastewater and dairy products (Sagiroglu et al., 2011). The sensor measures the difference in the concentration of dissolved oxygen depending on the concentration of catechol and shows good sensitivity, substrate specificity, repeatability and cost-efficiency. Aromatic compounds raise special awareness due to their toxicity and environmental resistance. The microbial activation mechanism, triggering the NahR regulatory protein synthesis in the presence of salicylate, was used by Shin (2010) for the construction of a biosensor. The E. coli DH5a was transformed with a pNRSAL plasmid containing the nahR gene and lucif-erase reporter gene, for the bioluminescent detection of salicylate. The response of the mutants, introduced by side directed mutagenesis at the residues 169 and 248 of the nahR gene was compared to the response of the wild type organism. The substitution of the amino acids leads into drastic changes in the microbial response to salicy-late, including the 50-fold increase of sensitivity. 4.5 BIOCHEMICAL OXYGEN DEMAND Biochemical oxygen demand (BOD or BOD5) can be measured by a dedicated BOD test that applies aerobic microorganisms that consume the organic compounds in water systems for biochemical decomposition (Chee, 2013). BOD represents the oxygen used for neutralisation of organic compounds in 5 days, at 20 °C. Its conventional determination is time consuming and requests an expert to achieve repeatable results (Ayyaru and Dharmaligman, 2013). The use of biosensor enables us to avoid long-lasting incubation. They are mainly appropriate for the detection of BOD in samples with high concentration of easy-degradable organic compounds. Chee (2013) used five microorganisms (P. putida SG10, P. fluorescens IAM12022, P. putida IAM1236, B. subtilis IAM12118, T.cutaneum IFO10466) that were immobilized on a porous cellulose-nitrate membrane of an oxygen electrode for the detection of BOD in river samples. All of the organisms were exposed to artificial wastewater and standard solutions of glucose and glutaminic acid. The most sensitive one (P putida SG10 with the detection limit of 0.5 mg/l) was applied for the characterization of river samples. This biosensoric method is comparable with the determination of BOD5 by the standard method. MFC are also suitable for the detection of BOD. Ayyaru and Dharmaligman (2014) report of a single chamber MFC, enriched with electrochemically active bacteria, isolated from the University of Anna water treatment plant as a suitable biosensor for the characterization of the unstable BOD. They monitored the electric current, produced by MFC at continuous feeding of the properly diluted samples of artificial wastewater. The anodic electrode senses the BOD as a current, produced by electrogenic bacteria, when in contact with organic compounds. A similar principle was used by Di Lorenzo et al. (2014) for the evaluation of a single-chamber air-cathode MFC with multilayer 3D printing. MFC was enriched with heterogeneous microbial populations from another working MFC. They monitored the amperomet-rical response of the sensor on increasing concentration of acetate in water. The biosensor enabled a fast linear detection 3-164 ppm of chemical oxygen demand (due to the acetate, used in the study, it is similar to BOD5) The CO2 concentration in gas phase, a by-product of microbial respiration activity during the catalysis of organic compounds, can be measured for the determination of current BOD values in wastewater samples. This principle was used by Vaiopoulou et al. (2005) for the development of a microbial biosensor, consisting of a conical fluidized bed reactor and cylindrical oxygen saturation chamber. The cell biomass from the activated sludge was used as an immobilized biosensing component. The biosensor was firstly calibrated in a laboratory with artificial wastewater with the addition of glucose and acetic acid and later used for the detection of BOD in a wastewater treatment plant Xianthi. The biosensor is adjustable for a broad range of wastewater.It enables the use of microbial populations from existing wastewater treatment plant and shows high activity of the immobilized cells, due to the continuous oxygen feeding. 5 SUMMARY Microorganisms are appropriate biosensing elements for the construction of environmental pollutants biosensors. They are used for the detection of heavy metals, pesticides, phenolic compounds, BOD and toxicity or genotoxicity. MFC and whole cell biosensors are the most frequently used biosensor types. 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Received November 30, 2015; accepted December 15, 2015. Ekonomska analiza organiziranosti jahalnega centra; primer uporabe linearnega programiranja V prispevku analiziramo ekonomiko konjeniškega centra, ki poleg storitev šole jahanja vključuje tudi rejo in oskrbo konj. Temeljimo na predpostavkah hipotetičnega kmetijskega gospodarstva, ki deluje v osrednji Sloveniji. Priprave optimalnega načrta gospodarjenja se v prispevku lotevamo s pomočjo metod matematičnega programiranja. Z njihovo uporabo lahko v danih razmerah z različnih zornih kotov ovrednotimo razvojno perspektivnost in pokažemo na možnosti povečanja dodane vrednosti. Na podlagi maksimiranja pokritja poizkušamo odgovarjati na različna vprašanja in izzive, ki se pojavijo pri vodenju in načrtovanju takšnega konjeniškega centra. Dobljeni rezultati kažejo, da so dana cenovno-stroškovna razmerja neugodna za lastno vzrejo konj, in sicer tako za namen obnove lastne črede šolskih konj kot tudi za prodajo. Slednje odraža trenutno neugodne razmere na področju konjereje. Na takšnih gospodarstvih je oskrba konj pomembna aktivnost in preko oportunitetnega vidika določa optimalni nabor dejavnosti. Tako z vidika diverzifikacije dohodka kot dodatnega vira prihodka so zanimive tudi aktivnosti male šole jahanja za otroke. Šola jahanja, ki ima zaposlenega enega inštruktorja jahanja, lahko ob predpostavki pokritega jahališča stroške polne zaposlitve pokrije s 4,6 šolskih konjev. Ključne besede: konji / konjeništvo / jahalni centri / ekonomska analiza / linearno programiranje 1 UVOD V zadnjih desetletjih se je uporaba konj drastično spremenila. Konj je izgubil svoj prvotni pomen kot delovna in transportna žival (Vejnovič, 2008). V današnjem času se konje uporablja predvsem kot rekreacijske živali za jahanje v prostem času, za uporabo v zdravstvene Economic analysis of the organisation of a riding centre; application of the linear programming approach In this contribution, the economics of an equestrian centre, which in addition to a variety of riding school activities also includes breeding and livery, are analysed. We consider the conditions for a hypothetical holding operating in central Slovenia. Methods of mathematical programming are applied in order to attempt to optimise the holding's activities. Their use may in the given situation facilitate the evaluation of development prospects from different perspectives and indicate the opportunities of increasing value-added. On the basis of maximizing the gross margin, we try to address the various questions and challenges that arise in managing and planning for such an equestrian centre. The obtained results indicate that breeding is unfavourable in the given price-cost ratio, both for the renewal of the working horses herd and for sale. This reflects the current adverse situation in the field of horse breeding. Livery is an important activity on such holdings and through opportunity perspective provides an optimal set of activities. Activities of the riding school for children are interesting in terms of income diversification, as well as an additional source of revenue. A riding school with one riding instructor and an indoor arena needs 4.6 horses in order to cover the costs of full-time employment. Key words: horses / horseback riding / economic analysis / riding centers / linear programming in terapevtske namene ter v vrhunskem športu (Hess in sod., 2014). Avtorji tudi izpostavljajo, da je v zadnjem obdobju ključen dejavnik povečanja staleža konj predvsem povečano povpraševanje po storitvah konjeništva za zabavo in prosti čas. Ob tem Zasada (2013) ugotavlja, da je vse večja težnja urbanega prebivalstva po preživljanju prostega časa v naravi pripeljala do razvoja kmetijskih 1 Univ. v Ljubljani, Biotehniška fak., Odd. za zootehniko, Groblje 3, SI-1230 Domžale, Slovenija gospodarstev, ki v obliki jahalnih centrov ponujajo različne konjeniške storitve. Ljudje iz mest zaradi pomanjkanja ustrezne infrastrukture, časa ter znanja o skrbi za živali, konje prepustijo v oskrbo kmetijskim gospodarstvom, ki izpolnjujejo te kriterije in s tem zagotavljajo ustrezne življenjske pogoje za živali (Birke in sod., 2010). Ta dejavnost tako na mnogih gospodarstvih že predstavlja osrednjo oziroma dopolnilno dejavnost. Ob tem Estwood in sod. (2008) navajajo različne oblike oskrbe konj, ki jih lahko srečamo v praksi. Dodaten vir prihodkov pa na takšnih gospodarstvih pogosto predstavlja ponudba jahalnih tečajev, treniranja konj in podobnih aktivnosti, vezanih na konjeniško dejavnost. Slednje je nedvomno tudi posledica dejstva, da se bistveno več ljudi vključuje v ta šport, kot pa jih dejansko ima lastnega konja (Hess in sod., 2014). Takšna kmetijska gospodarstva s šolo jahanja tako pokrijejo razliko v povpraševanju in omogočajo ježo in aktivnosti s konji tudi tistim, ki nimajo lastnega konja. Konjeništvo v Evropi postaja pomembna gospodarska panoga. Nix (2015) ugotavlja, da gre v primeru Anglije celo za drugo najpomembnejšo zaposlitveno dejavnost na podeželju. Poleg najrazličnejših storitev jahanja in vožnje vpreg, ta vključuje tudi druge aktivnosti in sicer od izdelave in trženja opreme za jahače, voznike in konje, veterinarstva, kovaštva pa vse do pridelovanja krme oziroma nakupa krme (Estwood in sod., 2008). El-gaker in sod. (2012) ocenjujejo, da se tovrstni prihodki iz konjeništva na Švedskem uvrščajo na peto mesto med najpomembnejšimi prihodki iz kmetijstva in po njihovi oceni znašajo 5 milijard €. Podobno vrednost dosegajo tudi v Nemčiji, kjer naj bi s tremi do štirimi konji ustvarili polno delovno mesto. Nadalje pa Liljenstolpe (2009) ugotavlja, da je v Veliki Britaniji za polno delovno mesto potrebnih pet do sedem konjev. V praksi srečamo različne oblike gospodarstev, ki se tako ali drugače ukvarjajo s konjeništvom in konjerejo. Ta gospodarstva se po strukturi in velikosti pomembno razlikujejo (Elgaker in sod., 2012). Veliko je klasičnih oziroma ljubiteljskih kmetij, ki konje uporabljajo za delo oziroma prosti čas, manj pa je kmetijskih gospodarstev, specializiranih v panogi reje konj. Slednje se pojavljajo v obliki kobilarn ali jahalnih centrov, na katerih je prisotno večje število konj (Žgajnar in Zupan, 2014). Sama struktura kmetijskega gospodarstva, gledano z vidika aktivnosti, pa navadno definira tudi usmeritev le-te bodisi v oskrbo konj, rekreacijsko jahanje, šole jahanja oziroma profesionalno treniranje konjeniških disciplin. Jahalni centri storitve jahanja tržijo pod skupnim imenom »šola jahanja«, ki vključujejo različne programe, od individualnih ur jahanja, jahanja v skupini, terenskega jahanja, jahanja ponijev za najmlajše ipd., ki so namenjeni različnim starostnim skupinam z različnim predznanjem. V zadnjih letih vse več jahalnih centrov ponuja tudi jahalne tabore in tako imenovane konjičkove delavnice, preko katerih otroci poleg jahanja spoznavajo tudi vsakdanje delo in skrb za konje (Žagar Hribar in Žgajnar, 2015). Nabor aktivnosti kot tudi storitev je tako zelo pester. Od šole jahanja, oskrbe konj do reje in vzre-je konj. Tako kot pri ostalih gospodarskih panogah pa je za uspešno in učinkovito upravljanje potrebno natančno poznavanje posamezne aktivnosti tudi s stroškovno-pri-hodkovnega vidika. Slednje je ključno pri izbiri kombinacij aktivnosti, ki bodo ob danih pogojih omogočila dosego najvišje ravni dohodka. Za izpolnjevanje tega cilja je poleg potrebnega znanja s področja tehnologije, reje konj, konjeništva in upravljanja takšnega kmetijskega gospodarstva, potrebno tudi natančno poznavanje in obvladovanje stroškov. Iz tega naslova se bomo v danem prispevku osredotočili na podrobno analizo priprave načrta gospodarjenja ter izbiro ustreznega nabora storitvenih aktivnosti. Pri uspešnosti delovanja takšnega gospodarstva nedvomno pomembno vlogo igra povpraševanje po njihovih storitvah. Ob tem pa velja izpostaviti, da za trg s konjeniškimi dejavnostmi veljajo določene posebnosti. Tako Hess in sod. (2014) poudarjajo, da gre pri tovrstnih aktivnostih, vezanih na konjeništvo, v večini primerov za lokalni trg in ne za nacionalni trg, kar načeloma velja za kmetijske trge. Tako je po ekonomski teoriji trg z jahalnimi storitvami pomembno odvisen od cen posameznih storitev. Te se razlikujejo med državami kot tudi med posameznimi regijami znotraj držav. Cene so odvisne od več dejavnikov, med katerimi poleg ponudbe in povpraševanja na nekem območju, ki ga določata povprečen prihodek gospodinjstev in gostota prebivalstva, pomembno vlogo igrajo zlasti kvalitativni dejavniki, kot so izkušenost in ugled učitelja jahanja, razpoložljiva infrastruktura, ne nazadnje pa tudi lokacija centra (Hess in sod., 2014). Pomembno vlogo pri definiranju cen storitev igra tudi tradicija določenega jahalnega centra (Hess in sod. 2014). Glede na njihove analize jahalnih šol na Švedskem so ugotovili, da višje cene dosegajo centri z daljšo tradicijo. Tako so cene storitev pri novoustanovljenih oziroma mlajših centrih, ki so svoje storitve pred kratkim ponudile na trg, značilno nižje. Povprečne cene mesečne oskrbe konj se v EU gibljejo od 150 € do 750 €, cena povprečne ure jahanja pa med 10 € in 45 €. Slovenija po opravljeni mednarodni analizi s svojimi cenami zavzema srednje vrednosti med evropskimi državami (Liljenstolpe, 2009). Ob tem pa Hess in sod. (2014) ugotavljajo, da je v bližini urbanih naselij povprečni dohodek gospodinjstva višji in posledično imajo tudi šole jahanja nekoliko višje cene svojih storitev. K zagotavljanju čim boljšega ekonomskega položaja in blaginje kmetijskih gospodarstev oziroma jahalnih centrov so poleg znanj tehnologije, reje konj in ekono- mike potrebne tudi dodatne informacije o dostopnosti razpoložljivih virov in stroškovno-cenovnih razmerij. Slednje lahko pridobimo tudi s pomočjo podrobnih analiz storitveno-proizvodnega načrta. Ugotavljamo, da je v Sloveniji pomanjkanje tovrstnih analiz, neupoštevanje ekonomskih načel pri analizi poslovanja na področju ko-njeništva pa še otežuje optimalno načrtovanje aktivnosti, s katerimi bi najbolje izkoristili dane možnosti posameznega jahalnega centra oziroma pokazali na razvojne perspektivnosti. Eden izmed pristopov reševanja in analiziranja tovrstnih problemov je modeliranje z elektronskimi preglednicami. Modele, ki jih zapišemo v obliki elektronskih preglednic, namreč lahko enostavno rešujemo z metodami matematičnega programiranja (Powell in Baker, 2009). Izmed slednjih je za tovrstne probleme najpogosteje uporabljeno predvsem linearno programiranje (v nadaljevanju LP), kjer optimiranje poteka na podlagi minimiranja oziroma maksimiranja namenske funkcije (Žgajnar in sod., 2011). Iz rešitev LP dobimo informacije o optimalni razporeditvi proizvodnih virov, o ključnih omejujočih virih, proizvodnih dejavnikih v presežku in smiselnih vrednostih za pridobitev dodatnih enot omejenih virov (Boehlje in Eidman, 1984). LP omogoča enostavno analizo morebitnih sprememb vhodnih parametrov, kot so cene inputov oziroma opravljenih storitev. Enostaven je tudi izračun oportunitetnih stroškov ob različnih tehnoloških učinkovitostih ob izbiri alternativne aktivnosti (Winston, 2004). Dodatno pa z analizo občutljivost lahko pridobimo tudi informacije o stabilnosti dobljenih rezultatov (Hazell in Norton, 1986). Na področju analize konjeniške dejavnosti v Sloveniji je bil podoben pristop uporabljen pri diplomskem delu, ki ga je opravil Stariha (2015). Namen danega prispevka je predstaviti razvito modelno orodje za analize na področju konjeniške dejavnosti in sicer zlasti z vidika organiziranja centra, ki se poleg različnih oblik oskrbe in vzreje konj ukvarja tudi s šolo jahanja. Na primeru hipotetičnega gospodarstva želimo pokazati na nekatere zakonitosti, ki se kažejo v danih ekonomskih razmerah in ki jih ob določeni predpostavkah lahko posplošimo tudi na druge podobne primere. Prispevek nadaljujemo s kratkim opisom pristopa modeliranja ter razvitega modelnega orodja za ekonomsko analizo optimalne organiziranosti aktivnosti jahalnega centra. V prvem delu na kratko opišemo strukturo modela ter ključne aktivnosti, ki lahko vstopajo v optimalno rešitev. Sledi opis ključnih skupin omejitev. Preko opisa hipotetičnega jahalnega centra podajamo osnovne predpostavke modeliranja. Tako poizkušamo na danem hipotetičnem primeru predstaviti nekatere zakonitosti, ki veljajo pri izbiri aktivnosti na takšnih gospodarstvih. Po predstavitvi rezultatov prispevek zaključujemo z diskusijo ključnih ugotovitev naše analize. 2 MATERIAL IN METODE Problem optimalne organizacije jahalnega centra obravnavamo kot primer eno-kriterijskega problema, ki ga z vidika ekonomske teorije lahko razporedimo v skupino normativnih pristopov. Za potrebe dane analize temeljimo na pristopu omejene optimizacije in sicer determinističnega linearnega programiranja. Z optimizacij-skim potencialom LP ob danih omejitvah iščemo maksimum namenske funkcije, ki v našem primeru predstavlja najvišje pokritje (POK), ki bi ga na danem gospodarstvu ob predvidenih pogojih lahko dosegli. Modelno orodje je razvito v Excelovem programskem okolju. To omogoča njegovo enostavno povezovanje, dopolnjevanje in prilagajanje posameznemu analiziranemu primeru. Povezave in večina potrebnih operacij je avtomatiziranih s pomočjo makrov, zapisanih v obliki kode VBA (Visual Basic for Applications) v urejevalniku za Visual Basic. Zapis osnovnih matrik za reševanje matematičnega modela kot tudi njegovo formiranje in množenje je tako sorazmerno enostavno. Razvito modelno orodje je z vidika uporabnika odprt sistem, saj omogoča dopolnjevanje in prilagajanje posameznim analiziranim primerom. V prvi vrsti seveda s spreminjanjem proizvodnih parametrov obstoječim aktivnostim in prilagajanjem omejitev, kot tudi z dodajanjem novih aktivnosti in omejitev. Z modelnim orodjem tako lahko analiziramo različne tipe kmetijskih gospodarstev, usmerjenih v konjeništvo in konjerejo. 2.1 MATEMATIČNI ZAPIS MODELA Namen modela je poiskati optimalen proizvodno--storitveni načrt, s katerim dosežemo najboljši finančni rezultat na ravni celotnega gospodarstva. S tem pa tudi analizirati in iskati ključne zakonitosti, ki jih je potrebno upoštevati pri načrtovanju takšnega centra. Model omogoča iskanje optimalne rešitve z maksimiranjem pokritja (POK). Dobljena rešitev tako odslikava odločanje gospodarja, ki je ravnodušen do tveganja in kot edino vodilo pri načrtovanju proizvodnje upošteva ekonomski vidik, seveda ob vseh danih tehnoloških zakonitostih in omejitvah razpoložljivih virov. Matematično lahko LP zapišemo, kot je prikazano v enačbi (1): n max POK = У POK x L-! i i =1 tako, da je n Za..x. < b za vse i = 1 dom i i i =1 x. > 0 j pri čemer najvišje POK izračunamo kot vsoto pokritji posameznih storitvenih aktivnosti (POK), pomnoženih z obsegom j-te aktivnosti (x.), ki vstopa v optimalni pro-izvodno-storitveni načrt. a. zajema tehnološko-rejske zakonitosti in pri naboru danih omejitev (bi) omogoča iskanje maksimuma namenske funkcije (maxPOK). 2.2 OPIS AKTIVNOSTI IN NJIHOVIH ZNAČILNOSTI Za potrebe analize smo se omejili na del najpogostejših oblik storitvenih aktivnosti na področju konjeni-štva in delom tudi konjereje. Model v dani verziji tako omogoča analizo zlasti rekreativnih tipov jahalnih centrov, usmerjenih v šolo jahanja, oskrbo konj ter deloma tudi lastno vzrejo konj. Ker je tokratni poudarek prispevka tudi na uporabljeni metodologiji, nabora aktivnosti nismo širili na druge tipe, ki se nanašajo predvsem na profesionalne športno-jahalne centre in specializirana gospodarstva za rejo in vzrejo konj. Pri razvoju LP je ključno dobro poznavanje tehnoloških zakonitosti aktivnosti. Za vsako od aktivnosti, ki lahko vstopajo v optimalno rešitev, smo tako pripravili podrobno kalkulacijo v obliki ekonomsko-tehnološke karte. Iz slednje se izračunavajo potrebni tehnološki koeficienti (aij), kot tudi vzročni koeficienti (POKj) za potrebe optimizacije. Osnovne tehnološke zakonitosti, opredeljene s pomočjo tehnoloških kart, na katerih temeljijo posamezne aktivnosti, smo zasnovali po vzoru klasičnih kalkulacij pokritja. Pri analizi gospodarstev, usmerjenih v konjeništvo in konjerejo, in pri pripravi proizvodno--storitvenih načrtov je z danim modelom možno izbirati med različnimi aktivnostmi. V grobem jih lahko razdelimo na tri glavne skupine: [1] aktivnosti oskrbe konj, [2] aktivnosti reje in vzreje konj, [3] aktivnosti šole jahanja (klasična šola jahanja ter aktivnosti male šole jahanja). Prva skupina vključuje aktivnosti, ki se nanašajo na oskrbo konj. Pri tem imamo dve osnovni skupini in sicer oskrbo zasebnih konj ter oskrbo lastnih konj za potre- be šole jahanja. Aktivnosti smo opredelili do te mere, da omogočajo izbiro različnih tehnologij in sistemov reje (npr. klasični angleški boksi, prosta reja). Pri vseh rejah je predvideno, da se za nastil uporablja žagovina. Klasična oskrba konj vključuje dvakrat dnevno čiščenje boksov ter krmljenje voluminozne in močne krme. Dodatno vključuje tudi enkrat dnevno vodenje konj v izpust (215 dni v letu) ter sprehajalno napravo (200 dni v letu). Pri predvideni prosti reji predpostavljamo, da so konji uhle-vljeni v posebnih skupinskih boksih z dostopom do pašnikov in so več časa zunaj. Posledično se stroški takšne oskrbe znižajo. Porabi se namreč manj nastilja, manj je dnevnega dela s čiščenjem ter v vegetacijski dobi pretežen del krmnega obroka predstavlja paša. Konj v takšni oskrbi tudi ne vodimo v sprehajalno napravo in seveda je bistveno manj premikov živali med pašniki. Tako znaša predvidena prodajna cena za klasično oskrbo 285 €, za prosto rejo 180 € oziroma za kobile z žrebetom v prosti reji 200 € mesečno. Pri oskrbi lastnih konj za potrebe izvajanja aktivnosti šole jahanja smo dodatno vključili tudi stroške kovača, veterinarskih storitev in zavarovanja. Pri teh aktivnostih smo vključili tudi stroške dela za korekcijsko prejahova-nje (skupno 30 ur letno) ustrezno usposobljene osebe, hkrati pa smo predpostavili, da se te konje vodi v sprehajalno napravo polovico manj kot zasebno uhlevljene konje. Pri reji lastnih konj za potrebe izvajanja aktivnosti šole jahanja je ključna predpostavka, da lahko kupimo že izšolanega konja ali pa le-ta izhaja iz lastne vzreje. V obeh primerih predpostavljamo, da je konj v uporabi 10 let in sicer med petim in petnajstim letom starosti, nato ga nadomestimo z novim. Tako stroške obnove in prihodke izločene živali v prvem primeru zajamemo na ravni aktivnosti, v drugem primeru pa preko dodatnih aktivnosti reje in vzreje konj. Slednja skupina vključuje tako rejo plemenske kobile oziroma žrebice za razplod, kot tudi rejo letnika, dvoletnika, triletnika in štiriletnika. Dodatno pa vstopajo tudi aktivnosti prodaje doma vzrejenih živali in sicer v kateri koli fazi od žrebeta do štiriletnika. Pri definiranju aktivnosti »vzreje lastnega delovnega konja« smo predpostavili, da z ujahovanjem pričnemo v četrtem letu in ga v enem letu tudi zaključimo. Osnovno ujahan konj tako lahko (i) vstopa v šolo jahanja ali pa (ii) ga prodamo po predvideni ceni. Poleg klasičnih šolskih konj smo med aktivnosti vključili tudi aktivnost reje ponija za otroke. Pri tem veljajo podobne zakonitosti, le da ponijev ne vzrejamo sami, pač pa jih lahko le dokupimo. V vseh primerih delovnih konj smo predpostavili, da konj letno lahko opravi 900 delovnih ur. Ob tem smo upoštevali, da lahko zaradi manjše poškodbe ali bolezni pride do izpada uporabe konja za določeno krajše obdobje. Pomembno skupino storitvenih aktivnosti pa v našem modelu predstavljajo aktivnosti klasične šole jahanja in male šole jahanja za otroke. Pri snovanju teh aktivnosti smo izhajali iz izkušenj najpogosteje prisotnih oblik v tovrstnih šolah jahanja1. Načrtovanje in izvedba posameznih aktivnosti namreč definira tip jahalnega centra. Ta je lahko organiziran kot rekreativni ali kot profesionalni športni center, možne pa so seveda vse kombinacije med tema skrajnostma. V modelno orodje smo tako v prvem delu vključili: individualno uro jahanja (40 €), skupinsko uro jahanja (21 €) ter terensko jahanje v skupini (18 €/ osebo). Pri zadnjih dveh aktivnostih smo predpostavili povprečno tri jahače na posamezni storitvi. Drugi del pa zajema skupino aktivnosti v okviru male šole jahanja za otroke. Na tovrstnih centrih je namreč zanimiva tudi ponudba male šole jahanja oziroma jahanje ponijev, jahalni tabori med počitnicami, konjič-kove delavnice (Žagar Hribar in Žgajnar, 2014; 2015) ter v zadnjem času tudi vse pogosteje organizacija roj-stnodnevnih zabav za otroke. Dane aktivnosti smo tako vključili v nabor možnosti, ki lahko vstopajo v optimalno rešitev. Od tega z izjemo male šole jahanja na poniju, ki traja pol ure (15 €/storitev), konjičkove delavnice obsegajo dve uri (18 € na posamezno storitev, oziroma 110 € na paket 10 obiskov), praznovanje rojstnodnevne zabave tri ure (150 €) in tedenski tabor 8 ur dnevno (150 €). V vseh primerih šola jahanja poteka z ustrezno usposobljeno osebo - inštruktorjem jahanja. Pri kalkuliranju stroškov dela smo ovrednotili strošek dela inštruktorja na bruto 8,5 € na uro. Prisotnost inštruktorja je obvezna tudi pri jahalnih aktivnostih, ki so vključene v jahalne tabore, konjičkove delavnice in rojstnodnevne zabave. Zaradi večjega števila otrok pri slednjih aktivnostih, pa pri izvajanju storitev sodeluje tudi dodatna oseba. Predviden strošek znaša 6,5 € na uro bruto. 2.3 NABOR OMEJITEV Kmetijsko gospodarstvo, ki se ukvarja s konjeni-štvom, je tako kot vsaka gospodarska družba omejen sistem v smislu količine dostopnih proizvodno-storitvenih sredstev, kot so površine, delo in kapital v različnih oblikah (npr. hlevi, odprto jahališče, pokrito jahališče, sprehajalna naprava, različna druga oprema in stroji, čreda šolskih konj). Poleg danih notranjih omejitev pa se takšno gospodarstvo sooča tudi z zunanjimi omejitvami. V našem primeru so primer slednjih predvsem tržne omejitve, ki definirajo obseg povpraševanja po posameznih storitvah in aktivnostih jahalnega centra. Namen omeji- 1 Pri tem smo izhajali predvsem iz izkušenj na Pedagoško raziskovalnem centru za konjerejo Krumperk. tev, ki jih vključujemo v tovrstne modele, je kar najbolje povzeti dano situacijo obravnavanega subjekta. V primeru našega modelnega orodja bi osnovni nabor omejitev (bi) lahko združili v naslednje skupine: - Infrastrukturne omejitve (število boksov po kategorijah konj). - Tržne omejitve - po posameznih kategorijah storitev in aktivnosti (šola jahanja in oskrba zasebnih konj). - Omejitve ustrezno kvalificirane delovne sile (hl-evar in inštruktor jahanja) na ravni leta in najem dodatne delovne sile. - Bilance za zagotavljanje ustrezne delovne obremenitve šolskih konj. - Bilanca živali po kategorijah v primeru razčlenjenih živinorejskih aktivnosti (reja in vz-reja) ter minimalen stalež posameznih kategorij živali. 2.4 OPIS ANALIZIRANEGA PRIMERA Uporabo modelnega orodja predstavljamo na hipotetičnem kmetijskem gospodarstvu. Predpostavljamo, da se le-to nahaja v osrednji Sloveniji, v bližini srednje velikega urbanega naselja. Na danem gospodarstvu imajo vso potrebo infrastrukturo za izvajanje šole jahanja kot tudi oskrbe konj. Sama infrastruktura za izvajanje aktivnosti šole jahanja pri naši analizi ni obravnavana kot omejitev. Predvideno je, da pri polno zasedenem hlevu kapacitete zadostujejo za vse potrebe šole jahanja, kot tudi za uporabo zasebno uhlevljenih konj. Na strani oskrbe konj smo predvideli, da ima gospodarstvo možnost izpustov za vse konje in jih različno, glede na sezono, hlevar vodi v izpust. Med potrebno infrastrukturo imajo poleg izpustov in pašnikov tudi sprehajalno napravo za konje. Pri tem predpostavljamo, da imajo kapacitete boksov za 25 individualno uhlevljenih konj, od tega je pet primernih tudi za kobile z žrebeti. Ob tem potencialne stranke lahko izbirajo med različnimi tipi oskrbe, tudi prosto rejo v obsegu do 5 živali. Skupno tako lahko uhlevijo do 30 živali. Center ima na razpolago za izvajanje šole jahanja ustrezna jahališča kot tudi pokrito jahališče. Slednje omogoča, da lahko svoje aktivnosti izvajajo tudi v primeru slabega vremena, kar bi v nasprotnem primeru vnašalo precej negotovosti. Pri analizi smo predvideli, da vso voluminozno krmo (seno, pašo, zeleno krmo) pridelajo na gospodarstvu. Ker ta krma z izjemo sena navadno ni predmet trgovanja, smo stroške njihove pridelave ovrednotili s pomočjo modelnih kalkulacijah Kmetijskega inštituta Slovenije. Vso ostalo močno krmo in mineralno vitaminske dodatke pa smo ovrednotili po tržnih cenah. Pri tem se seveda poraja vprašanje kako se spremeni morebitna organiziranost centra in aktivnosti pri načrtovanju gospodarjenja, če se spremenijo cenovno-stroškovna razmerja, tudi kot posledica, da več oziroma manj krme pridelajo sami oziroma dokupijo. Slednji vidik ni predmet obravnave v tem prispevku, je pa lahko izhodišče za nadaljnjo analizo. Pri dani analizi smo predpostavili, da je število lastnih konj kot tudi njihova namembnost v modelu neznanka. Osnovna predpostavka je, da se na centru za potrebe šole jahanja, kot tudi reje in vzreje, redi lipicanske konje. Pri tem predpostavljamo, da je ustrezno izšolan konj za potrebe šole jahanja na trgu dostopen po ceni 12.000 €. Prav takšna je tudi predvidena prodajna cena lastno vzrejenega štiriletnega konja, ki opravi šolanje. Zaradi poenostavitev smo upoštevali, da je strošek plemenske kobile 10.000 €, nižji pa je strošek žrebice (6.000 €). Ob tem predpostavljamo, da lahko rejec proda žrebe po ceni 1.500 €, letnika po ceni 2.500 €, dvoletnika po ceni 3.000 €, triletnika preden začne z ujahovanjem pa po ceni 4.500 € 1. Na danem gospodarstvu imajo stalno razpoložljivo delovno silo in sicer enega hlevarja (v obsegu 1.900 ur), ki skrbi za oskrbo konj, premike konj (izpust, sprehajalna naprava), čiščenje hlevov, po potrebi pa lahko sodeluje tudi kot pomočnik pri nekaterih aktivnostih male šole jahanja. Pri tem smo pavšalno ocenili tudi obseg dela, ki vključuje pripravo krme na lastnih površinah, razvoz gnoja ter ostalih potrebnih opravil. V obsegu 1.900 ur letno pa je na razpolago tudi inštruktorja jahanja, ki izvaja aktivnosti klasične šole jahanja in male šole jahanja ter po potrebi ujahuje in prejahuje lastne konje. V primeru dodatnih potreb po delovni sili lahko le-to gospodarstvo tudi najame. Pri tem smo predpostavili, da gre za ustrezno usposobljeno delovno silo, pri čemer dodaten strošek posamezne ure v izhodiščnem scenariju znaša 5,5 € za hlevarska opravila in 7,8 € za pomoč pri šoli jahanja. 2.5 SCENARIJSKA ANALIZA S pomočjo razvitega modelnega orodja smo za namen danega prispevka analizirali tri izhodiščne primere. V prvem primeru (A) predpostavljamo, da imamo opravka z gospodarstvom, ki v nabor svojih aktivnosti lahko vključiti oskrbo zasebnih konj, ter rejo in vzrejo lastnih konj za prodajo. Prisoten je učitelj, ki je vešč uja-hovanja konj in je tako ključen 'produkt', poleg storitev oskrbe, predvsem ujahan konj. Seveda pa lahko prodajo tudi še neujahane mlade živali, v kateri koli fazi od žre- 1 Predpostavljene cene ne odražajo podrobne analize razmer na trgu, pač pa so določene hipotetično za potrebe dane analize. beta do ujahanega štiriletnika. V drugem primeru (B) smo analizirali primer, ko ima kmetijsko gospodarstvo možnost dodatno izvajati tudi aktivnosti klasične šole jahanja za odrasle, bodisi na lastno vzrejenem konju ali pa na kupljenem šolskem konju. V tretjem primeru (C) pa smo nabor možnih aktivnosti razširili tudi na malo šolo jahanja za otroke. Dodatno smo analizirali, kako se spremeni optimalni proizvodni načrt pri različnih postavkah najetega dela hlevarja in inštruktorja jahanja. Pri analizi smo predpostavili tri možnosti in sicer: (1) kmetijsko gospodarstvo nima možnosti najema dodatne delovne sile, (2) kmetijsko gospodarstvo lahko najame dodatno delovno silo, pri čemer strošek dodatne hlevar-ske ure znaša 5,5 € ter inštruktorske ure 7,8 €. (3) V zadnjem primeru pa smo predpostavili, da se zniža zgolj strošek hlevarske ure za 0,3 €/h. 3 REZULTATI IN RAZPRAVA V nadaljevanju prikazujemo rezultate analize hipotetičnega gospodarstva (pregl. 1). Ker pri prvem (A) in drugem (B) proizvodnem načrtu razpoložljiva delovna sila ni bila izkoriščena, posledično ni razlik med rezultati ob predpostavkah (1) in (2). Zato za ta dva primera prikazujemo zgolj rezultate ob predpostavki (2). Kot izhaja iz preglednice 1, na višino realiziranih prihodkov pomembno vpliva nabor aktivnosti, ki vstopajo v optimalno rešitev. V prvem primeru (A2) tako gospodarstvo dosega skupne prihodke iz naslova oskrbe zasebnih konj in proste reje v višini 44.750 €. Ob danih stroških bi tako gospodarstvo doseglo POK v višini dobrih 15 tisoč €. Zanimiv je rezultat, ki kaže, da bi z možnostjo najema dodatne delovne sile (hlevarja) stalež povečali do zapolnitve razpoložljivih kapacitet (skupno 30 konj), kar bi prihodke zvišalo za dobrih 46 %, vendar zaradi hkratnega povišanja spremenljivih stroškov, bi se doseženo POK zvišalo zgolj za 1 %. Zanimiv rezultat, ki ga nakazuje prvi scenarij (A2 in A3), kjer predpostavljamo, da se na takšnem centru lahko izvaja le oskrba zasebnih konj (klasična in prosta reja) ter reja in vzreja konj, slednja ob dani stroškovno-cenovnih razmerjih ne vstopa v optimalno rešitev. Ta v takšnem primeru vključuje le aktivnosti oskrbe zasebnih konj, pri čemer je potrebno poudariti, da se v vseh primerih (tudi B in C) v rešitev vključuje zgolj klasična prosta reja, ne pa tudi prosta reja kobile z žrebetom. Za slednjo znaša zmanjšan strošek v vseh treh primerih -430 €, za kolikor bi se poslabšalo skupno doseženo pokritje, če bi aktivnost vstopala v optimalno rešitev. Preglednica 1: Optimalen proizvodni načrt organiziranosti jahalnega centra pri različnih izhodiščih in različnih stroških najete delovne sile Table 1: Optimal production plans to organise riding centre with different assumptions and different costs of hired labour Doseženi rezultati pri različnih izhodiščih A2 A3 B2 B3 C1 C2 C3 Ekonomski kazalniki na ravni KMG (€) Skupni prihodki 44.750 96.300 106.652 156.987 119.518 131.679 181.436 Skupni VC 29.735 81.119 52.556 102.728 58.592 69.031 118.627 Skupni strošek krme 12.692 27.904 12.302 27.156 12.175 11.881 26.564 Strošek nastilja 3.450,7 7.972,6 3.557,3 7.972,6 3.564,7 3.607,9 7.972,6 Strošek dela hlevarja 12.350 25.747 12.350 25.431 12.350 12.350 25.281 Strošek dela inštruktorja 0 0 13.912 13.912 16.150 20.349 20.349 Pokritje 15.014 15.181 54.096 54.258 60.926 62.649 62.809 Seznam aktivnosti Vzreja in reja (št.)* Oskrba konj (št.) Klasična oskrba 9,9 25,0 5,7 20,4 5,9 4,8 19,3 Prosta reja - oskrba 5,0 5,0 5,0 5,0 5,0 5,0 5,0 Delavni konj (št.) Šolski konj (nakup) 0,0 0,0 4,6 4,6 4,0 4,6 4,6 Šolski konj (vzreja) 0,0 0,0 0,0 0,0 0,0 0,0 0,0 Poni 0,0 0,0 0,0 0,0 0,4 1,0 1,0 Aktivnosti šole jahanja (h) Individualna ura jahanja 500,0 500,0 500,0 500,0 500,0 Jahanje v skupini 2.100,0 2.100,0 2.100,0 2.100,0 2.100,0 Terensko jahanje - skupina 600,0 600,0 160,4 600,0 600,0 Mala šola jahanja 0,4 300,0 300,0 KD - posamično vplačilo 150,0 150,0 150,0 KD - paketna storitev 60,0 60,0 60,0 Jahalni tabor 4,0 4,0 4,0 Rojstno dneva zabava 20,0 20,0 20,0 Delovna sila (h) Hlevar 1.900,0 1.900,0 1.900,0 1.900,0 1.900,0 1.900,0 1.900,0 Inštruktor 0,0 0,0 1.636,7 1.636,7 1.900,0 1.900,0 1.900,0 Najem delovne sile - hlevar 0,0 2.576,3 0,0 2.515,5 0,0 0,0 2.486,6 Najem delovne sile - inštruktor 0,0 0,0 0,0 0,0 0,0 538,3 538,3 Delo hlevarja - razčlenjeno (h) Čiščenje boksov 664,7 1.581,7 686,3 1.581,7 687,8 696,6 1.581,7 Nastiljanje boksov 211,6 486,7 218,1 486,7 218,5 221,1 486,7 Čiščenje hleva 151,2 334,6 155,5 334,6 155,8 157,6 334,6 Krmljenje skupaj 302,4 669,2 311,0 669,2 311,6 315,1 669,2 Premiki živali - izpust 305,4 737,5 315,6 737,5 316,3 320,4 737,5 Sprehajalna naprava 264,7 666,7 213,5 605,9 209,9 189,1 577,1 Legenda: KD - konjičkove delavnice; VC - spremenljivi stroški;* Ker nobena od aktivnosti reje in vzreje ne vstopa v rešitev, smo jih pri prikazu izpustili; A - možne samo aktivnosti reje in vzreje, B - vključuje dodatno aktivnosti šole jahanja, C - pogoji B ter možnost aktivnosti male šole jahanja; (1) ni možnosti najema dodatne delovne sile; (2) možnost najema dodatnega hlevarja (5,5 €/h) ter dodatne inštruktorske ure (7,8 €/h); (3) isti pogoji, kot v primeru 2, le da strošek dodatne ure hlevarja znaša 5,2 €/h. Iz rezultatov (pregl. 1) zelo jasno izhaja, da v danih cenovno-stroškovnih razmerjih in predvideni tehnologiji, da so konji ves čas vzreje individualno uhlevljeni v boksih, reja ekonomsko ni upravičena. Tako v nobenem od predvidenih scenarijev reja in vzreja ne vstopata v optimalno rešitev, zato rezultatov po posameznih kategorijah posebej tudi ne prikazujemo. Senčne cene kažejo na problem visokih oportunitenih stroškov. Z dodatnimi analizami smo ugotovili, da se aktualnost sicer nekoliko izboljša, če uspemo pomembno znižati stroške reje (zlasti delo hlevarja, deloma pa tudi krmnega obroka). Denimo v primeru predpostavk C, bi to pomenilo skoraj 50 % znižanje stroškov štiriletne vzreje. Oziroma bi v takšnem primeru moral vzrejeni konj dosegati sorazmerno visoko prodajno ceno, da bi se gospodarstvu splačalo vzrejati živali bodisi za prodajo oziroma lastne šolske konje. Modelni rezultati kažejo, da v kolikor bi bil v danih razmerah ustrezno izšolan šolski konj dosegljiv po ceni 12.000 €, se lastna vzreja in ujahovanje ne splačata (pregl. 1). Ta vstopi v rešitev šele, če se predvidena nabavna cena šolskega konja zviša. Zanimivo je, da je zmanjšan strošek, izračunan pri postoptimalni analizi, bistveno višji pri uporabi takšnega konja za šolo jahanja (7.761 €), kot pa če bi ujahanega prodali (465 €). Seveda bi bil rezultat drugačen, v kolikor bi predpostavili, da je strošek dela inštruktorja stalni strošek. Vendar, ker naš model temelji na konceptu POK, takšna analiza ni možna. Slednje lepo odslikava trenutne razmere na področju konjereje, ki jih je gospodarska kriza nedvomno še poslabšala. V kolikor predpostavimo, da ima gospodarstvo tudi ustrezno povpraševanje po storitvah šole jahanja za odrasle (scenarij B), se doseženo POK na gospodarstvu izboljša za dobrih 72 %, prihodki pa v obeh primerih (2 in 3) presežejo 100 tisoč €. Tudi v tem primeru se pokaže ekonomsko upravičena potreba po dodatnem delu le v primeru nižjega stroška dela (5,2 €/h), se pa v obeh primerih B2 in B3 stalež privatno uhlevljenih konj zniža na račun lastno uhlevljenih šolskih konj (4,6 konjev). Število slednjih determinira predvideno povpraševanje po storitvah šole jahanja. To je v našem primeru zunanja (eksogena) spremenljivka in jo preprosto določimo. Ker je povpraševanje nižje od razpoložljivih letno ocenjenih ur inštruktorja jahanja, je pričakovano, da omejitve niso omejujoče in lahko opravimo vse storitve. S pomočjo po-stoptimalne analize smo tako ugotovili, da znaša senčna cena vsake dodatne enote povpraševanja po storitvah ob pogojih B (isto za 2 in 3) za individualno uro jahanja 25,87 €, 10,66 € za skupinsko uro jahanja ter 6,25 € za posamezno uro skupinske ježe. Kar logično nakazuje tudi na to, da bi se s povečanim povpraševanjem dosežen rezultat izboljšal, oziroma koliko lahko odštejemo za dodatno enoto povpraševanja po danih storitvah. Iz tega vidika je zanimivo, da se pri pogojih (C) te vrednosti zni- žajo, kar je seveda posledica oportunitenega vidika aktivnosti male šole jahanja. Nadalje je zanimivo, da se tudi v primeru pogojev (B) stalež zasebno uhlevljenih konj poveča, če je dodatno delo moč dobiti po ceni, manjši ali enaki 5,2 €/h (B2). Vendar z dodatnim izračunom lahko ugotovimo, da se doseženo POK na vloženo povprečno uro zniža z dobrih 15 € na 8,96 €. Vse bolj pogosto pa na kmetijskih gospodarstvih, usmerjenih v konjeništvo, lahko srečamo poleg že analiziranih aktivnosti tudi najrazličnejše aktivnosti male šole jahanja (C), kar lahko pomembno izboljša dosežen rezultat. Kot izhaja iz preglednice 1, v vseh treh primerih predvideni prihodki presežejo 100 tisoč €. Za ta primer prikazujemo tudi scenarij, kjer predpostavljamo, da ni možno najeti dodatne delovne sile. Če primerjamo C1 in B2, lahko vidimo, da je skupni stalež (nekoliko poenostavljeno) enak, se pa na račun manjšega števila šolskih konj vključi tudi poni. Slednje lepo kaže na pomen oportuni-tetnega vidika. Tako se pomembno zniža obseg izvajanja terenske ježe na zgolj 26,7 %, vendar se do zapolnitve kapacitet inštruktor jahanja vključi v storitve male šole jahanja. In sicer tiste, katerih cenovno-stroškovno razmerje je ugodnejše. Torej skoraj nič male šole jahanja (30 min ježe na poniju), v celoti pa ostale predvidene (skupinske) storitve. Problem, ki se sicer pojavi v praksi, je predvsem ta, da je pri skupinskih aktivnostih večji riziko prepozne odpovedi oziroma stranka ne pride, kar pa pomembno poslabša (predpostavljeno) ekonomiko. Seveda pa bi bil v takšnem primeru pričakovano ta trend obraten ali vsaj manj izrazit. Nadalje je razvidno, da v kolikor imamo možnost najema dodatne delovne sile, se v optimalni načrt vključijo vse aktivnosti in v primeru nižjega stroška hlevarja tudi v tem primeru pride do popolne zapolnitve razpoložljivih kapacitet za oskrbo konj. Senčna cena je tudi v tem primeru enaka kot v primeru B. Tudi pri scenariju (C) bi gospodarstvo kljub sicer precej različnemu obsegu dela doseglo zelo podobno višino skupnega POK. Posledično je pričakovano, da se doseženo POK na vloženo uro poslabša. Ob pogojih C1 bi znašalo 16,03 € na vloženo uro, pri možnosti dodatnega najema delovne sile (C2) pa 14,44 €. V kolikor bi strošek dela znašal manj ali enako 5,2 € na dodatno uro hlevarja, se stalež živali poveča do maksimuma, kar pa zniža doseženo pokritje na opravljeno povprečno uro na 9,2 €. 4 ZAKLJUČKI V prispevku smo predstavili razvito modelno orodje za analizo optimalne organiziranosti jahalnega centra na hipotetičnem primeru. Uporabljen pristop matematičnega programiranja, ki temelji na LP, se je izkazal kot učinkovit, saj omogoča študiranje in analiziranje zakoni- tosti na kmetijskih gospodarstvih, umerjenih v konjerejo in konjeništvo. Poslovanje hipotetičnega konjeniškega centra smo ovrednotili v različnih okoliščinah s pomočjo treh osnovnih scenarijev (A, B in C). Za vsak scenarij smo izdelali osnovno analizo poslovanja, iz katere izhaja, katere aktivnosti lahko vstopajo v optimalno rešitev in kakšen je njihov predviden obseg. Proizvodni načrt posameznega scenarija smo nato nadgradili s postoptimal-no analizo, iz katere dobimo informacije o občutljivosti dobljene rešitve na morebitne spremembe, hkrati pa tudi, koliko lahko odštejemo za dodatno enoto posameznega omejenega resursa oziroma za koliko bi se morala spremeniti višina stroškov ali prodajne cene, da bi določena aktivnost vstopala v rešitev. Rezultati kažejo na nekatere zakonitosti, ki jih lahko opazimo tudi v praksi. Tako želimo izpostaviti predvsem trenutno izrazito neugodne pogoje za rejo in vzrejo konj na takšnih centrih. Pri tem je potrebno iskati možnosti čim cenejše tehnologije, kar uhlevljanje v boksu zagotovo ni. Z izrazitim znižanjem stroškov vzreje in ob ustrezno visokih prodajnih cenah je to lahko tudi ekonomsko upravičeno. Izkaže se, da imajo stroški dela lahko pomemben vpliv na obseg optimalne oskrbe konj. Seveda se obseg dela med gospodarstvi lahko precej razlikuje, ključno pa je predvsem, kako dolge poti mora hlevar pri posameznem opravilu opraviti. Z aktivnostmi šole jahanja lahko značilno izboljšamo ekonomiko na konkretnem gospodarstvu. POK se lahko izboljša tudi za 72 %. Pri tem pa je zelo pomembno, da stalež šolskih konj prilagodimo kapacitetam inštruktorja jahanja. Izkaže se, da so za polno zaposlitev dovolj 4,6 konj. Da bo z modelom možno analizirati tudi ostala gospodarstva, usmerjena v konjeništvo, bo v nadaljnji fazi razvoja modelnega orodja potrebno razširiti nabor aktivnosti. Pri nadaljnjem razvoju modelnega orodja bi bilo zanimivo vključiti tudi vpliv sezone in razčleniti povpraševanje kot tudi razpoložljive kapacitete preko tedna in vikenda. Namreč med temi obdobji prihaja do pomembnih razlik v obsegu povpraševanja na eni strani, po drugi strani pa tudi razpoložljivi kapaciteti infrastrukture. Slednje je po naših pričakovanjih izraziteje na tovrstnih centrih, kot smo ga obravnavali v naši analizi, v primerjavi s profesionalnimi športnimi centri. Poleg predstavljenega pa dodaten izziv predstavlja tudi vzreja konj ter iskanje dovoljene višine stroškov. Po eni strani je potrebno najti ekonomsko vzdržno rešitev, po drugi strani pa gre za izrazit problem vezanega kapitala, katerega bi lahko obrav- navali tudi z dinamičnimi metodami vrednotenja naložb, kar vzreja in ujahovanje konj zagotovo je. 5 VIRI Birke L., Hockenhull J., Creighton E. 2010. The horse's tale: narratives of caring for/about horses. Society and Animals, 18: 331-347. doi:10.1163/156853010X524307 Boehlje M.D., Eidman V.R.J. 1984. Farm Management. New York, Wiley and Sons: 806 str. Elgàker H., Pinzke S., Nilsson C., Lindholm G. 2012. Horse riding posing challenges to the Swedish Right of Public Access. Land Use Policy, 29: 274-293. doi:10.1016/j.landuse-pol.2011.06.005 Estwood S., Jensen A-L.R., Jordon A. 2008. Business management for the Equine industry. 2nd edition. Estwood. Blackwell Publishing: 158 str. Hess S., Surry Y., Kron R., Liljenstolpe C., Lindberg G., Anders-son H. 2014. A hedonic analysis of the price for horse riding lesson in Sweden. 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Horsekeep-ing and the peri-urban developement in the Berlin Metropolitan Region. Journal of Land Use Science, 8, 2: 199-214. doi:10.1080/1747423X.2011.628706 Žagar Hribar S., Zgajnar J. 2014. Konjičkove delavnice za otroke, 1. Del. Revija o konjih, 22, 12: 46-47 Žagar Hribar S., Zgajnar J. 2015. Konjičkove delavnice za otroke, 2. Del. Revija o konjih, 23, 1: 45-47 Zgajnar J., Erjavec E., Kavčič S., Zadnik S.L. 2011. Uporaba metod operacijskih raziskav pri načrtovanju kmetijske proizvodnje. Uporabna informatika, XIX, 3: 125-135 Zgajnar J., Zupan M. 2014. Ekonomska upravičenost uporabe gum pri uhlevitvi konj. Revija o konjih, 22, 12: 39-41 COBISS: 1.01 Agris category code: L50 ESTABLISHMENT OF PRIMARY KERATINOCYTE CULTURE FROM HORSE TISSUE BIOPSATES Jernej OGOREVC 1 2, Tjaša LAPANJA 2, 3, Klavdija POKLUKAR 2, 4, Natalija TOMINŠEK 2, 5, Peter DOVČ 6 Received October 23, 2015; accepted November 20, 2015. Delo je prispelo 23. oktobra 2015, sprejeto 20. novembra 2015. Establishment of primary keratinocyte culture from horse tissue biopsates Primary cell lines established from skin tissue can be used in immunological, proteomic and genomic studies as in vitro skin models. The goal of our study was to establish a primary keratinocyte cell culture from tissue biopsates of two horses. The primary keratinocyte cell culture was obtained by mechanical and enzymatic dissociation and with explant culture method. The result was a heterogeneous primary culture comprised of keratinocytes and fibroblasts. To distinguish epithelial and mesenchymal cells immunofluorescent characterisation was performed, using antibodies against cytokeratin 14 and vimentin. We successfully at attained a primary cell line of keratinocytes, which could potentially be used to study equine skin diseases, as an animal model for human diseases, and for cosmetic and therapeutic product testing. Key words: molecular biology / primary cell culture / skin / horses / keratinocytes 1 INTRODUCTION Because of the economic value and the number of animals (in 2013 there were almost 60 million registered horses) (Faostat, 2015), horses (Equus caballus) represent a group of economically important animals. A significant share of veterinary research has focused on researching and treating diseases in horses. In some cases, a horse also became an animal model for human diseases, for example in melanoma studies (Seltenhammer et al., 2004) and muscle, joints, tendons, ligaments, and bone injuries Vzpostavitev primarne celične kulture konjskih keratinocitov iz biopsij Primarna celična linija iz kožnega tkiva lahko služi za imunološke, proteomske in genomske raziskave kot in vitro model kože. Cilj naše študije je bila vzpostavitev primarne celične linije keratinocitov iz biopsije kožnega tkiva dveh konjev. Primarno celično kulturo smo pridobili z mehansko in encimsko razgradnjo tkiva in z metodo izraščanja celic. Rezultat je bila heterogena kultura keratinocitov in fibroblastov. Za imunološko karakterizacijo smo uporabili protitelesa proti citokeratinu 14 in vimentinu ter poskusili obogatiti delež ke-ratinocitov v kulturi. Uspešno smo pridobili primarno celično linijo keratinocitov, ki je potencialno uporabna za preučevanje kožnih bolezni konjev, kot živalski model za preučevanje človeških bolezni in za testiranje kozmetičnih izdelkov ter zdravil. Ključne besede: molekularna biologija / primarna celična kultura / koža / konji / keratinocite (Nagy et al., 2014). In research that is focusing on analysing various diseases, their mechanisms of action, and subsequent physiological changes and potential treatments, the use of in vitro models is crucial. It enables the application of immunological, genomic and proteomic studies on a tissue culture of an organ, excluding the systemic and environmental effects and providing more comparable results as in in vivo studies. Additionally, in vitro experiments are more reasonably priced and less ethically controversial. The skin represents the largest organ in mam- 1 Univ. of Ljubljana, Biotechnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia, e-mail: jernej.ogorevc@bf.uni-lj.si 2 The authors contributed equally to the work 3 Same address as 1, Study program in biotechnology, e-mail: tjasalapanja@gmail.com 4 Same address as 3, e-mail: klavdija.poklukar@gmail.com 5 Same address as 3, e-mail: natalija.tominsek@gmail.com 6 Same address as 1, peter.dovc@bf.uni-lj.si malian body that is divided in three major layers: dermis, epidermis and subcutis. The major cell type in the outer layer of skin are keratinocytes, which represent a protective layer. The most prevalent cell type in dermis are fibroblasts, which produce different growth factors, needed for proliferation of keratinocytes. The types of cells found in subcutis are macrophages, fibroblasts and adipose cells (Orazizadeh et. al., 2015). Research on horse skin cell lines resulted in a range of applications, including establishment of primary keratinocyte cultures for researching extracellular matrix proteins, associated with skin disseases (Visser and Pollit, 2010) and development of an equine skin-equivalent model that could be used in regenerative medicine, pathogenesis research, and biological and pharmacological studies (Cerrato et al., 2014). However, very few protocols for preparation and sustainment of horse primary cell lines have been developed (e.g. Wunn et al., 1999; Aasen and Bellmonte, 2010). The purpose of this study was to establish a primary horse skin epithelial cell culture and to characterise cell types present in the derived culture. The study is interesting for researchers studying degenerative skin and hair diseases in horses and possibly in other species. For example, in human medicine optimisation of protocols for sustaining keratinocyte cell lines became important as a part of production process of skin substitute that is used for treatment after burns and other larger tissue damages. Various diseases in horse, such as hereditary equine regional dermal asthenia (HERDA), alopecia areata (AA) and incontinentia pigmenti (IP), known in different breeds, are associated with skin lesions, hair or mane loss and other related health problems. Detecting skin and hair diseases is usually a big economic burden for breeders. At the same time, these diseases are mostly incurable, largely due to the lack of information about the illness. Skin models employing primary cells could be used for pathogenesis research and understanding of such diseases and could be useful as a starting point in drug screening/testing studies (Guo and Jahoda, 2009). 2 MATERIALS AND METHODS 2.1 ESTABLISHMENT AND PROPAGATION OF THE CELL CULTURE Skin biopsates containing epidermis and dermis were aseptically removed from neck area below the mane of two Appaloosa breed horses (Equus caballus). From each horse two samples of approximately 2 cm2 were taken and transferred into phosphate buffered saline (PBS) with added antibiotics (100 mg/mL streptomycin and 100 mg/mL penicillin). Tissue was stored at 4 °C during transport and processed immediately. Enzymatic and tissue explant methods were used for establishment of primary cell culture. For enzymatic digestion, the tissue biopsates were incubated in dispase II (2.4 U/mL) at 4 °C for 18 hours. After incubation of tissue epidermis was separated from dermis. The epidermis was cut into small pieces and incubated in 0.25 % trypsin with 0.05 % EDTA for 30 minutes at 37 °C (Vissar and Pollitt, 2010). During the incubation, the samples were shook every several minutes. Trypsin was inactivated with addition of medium containing serum. The liquid phase was separated from the remaining tissue pieces and centrifuged at 1000 rpm for 10 minutes to collect the dissociated cells in suspension for seeding. Fresh trypsin can be added to the processed tissue and the process repeated for several times, until the tissue is fully dissociated. The collected cells were seeded in untreated plastic culture flasks and Geltrex (Gibco)-covered culture flasks. Thin-layer Geltrex method (described in the manufacturer's protocol) was used to cover plastic surface with a mixture of DMEM and Geltrex in 1:100 ratio. DMEM/F12 (D6421, Sigma) supplemented with 10 % FBS, 10 ng/mL EGF, 5 |g/mL insulin, 0.4 |g/mL hydrocortisone, 0.1 |g/10 mL cholera toxin, 2 mmol/L glutamine, and 5 |g transferrin was used to grow the cells (growth medium). The cells were grown at 37 °C and 5 % CO2. Differential trypsinisation was used to obtain enriched keratinocyte culture. The primary culture, containing keratinocytes and fibroblasts, was incubated in a solution of 0.125 % trypsin-EDTA for several minutes, with continuous microscopic observation. When most of the fibroblasts detached the reaction was stopped. The solution containing fibroblasts was removed, while keratinocytes remain attached. Because of the difference in the adhesion potential of the two predominant cell types, we were able to obtain two separate, almost pure keratinocyte and fibroblast cell lines. Solution of 0.25 % trypsin-EDTA was used to detach the cells for subculturing. The cells were split in a 1:5 ratio and cultivated further in the growth medium. A mixture of basic medium, 20 % FBS and 10 % DMSO was used for freezing the cells. The cells were frozen in a freezing container that provides a cooling rate of 1 °C per minute, kept at -80 °C overnight, and transferred in liquid nitrogen for long term storage. Alternatively, for explant culture method tissue was mechanically minced and small parts of the tissue seeded directly in culture dishes, containing the growth medium. All other procedures were the same as described previously. 2.2 IMMUNOFLUORESCENT STAINING The cells, grown for seven days, were washed in PBS and fixed in 4 % formalin for 5 minutes. Cells were per-meabilised with 1 % Triton-X in PBS and incubated for 20 minutes. The fixed cells were washed with PBS and blocked with 10 % goat serum for 30 minutes at room temperature. Monoclonal mouse (Santa Cruz Biotech sc-53253) and polyclonal rabbit (Covance PRB-155P) antibodies against cytokeratin 14 and monoclonal mouse antibody against vimentin (Santa Cruz Biotech sc-73262) were used to distinguish between keratinocytes and mes-enchymal (fibroblast) cells. The cells were incubated overnight at 4 °C in a solution of primary antibodies (1:100). After three 5-minute washes in PBS, they were incubated for 60 minutes in a dilution (1:200) of labelled secondary goat-anti-rabbit antibody (Invitrogen, Alex-afluor 488, A11008) to visualize attachment of polyclonal cytokeratin 14 and in goat-anti-mouse (Alexafluor 594, A11037) antibody to visualize attachment of primary mouse monoclonal antibodies (cytokeratin 14 and vi-mentin). After incubation in secondary antibodies, the cells were washed again for several times in PBS and counterstained with DAPI (1:1000) for 3 minutes to visualize nuclear DNA. After washing in PBS, the cells were observed under fluorescent microscope (Nikon Eclipse TE, 2000). 3 RESULTS AND DISCUSSION Tissue explant and enzymatic method were used for establishment of the primary keratinocyte cell culture. The enzymatic dissociation of the tissue resulted in a heterogeneous mixture of cells, predominantly consisting of keratinocytes and fibroblasts. Adherent cells that explanted from attached pieces of the tissue consisted mainly of keratinocytes, however the yield of cells was much lower, compared to enzymatic method. In tissue explant method, cell outgrowths growing from mechanically processed tissue were observed after several days; e.g. five days as showed in Figure 1. In enzymatic method, the dissociated cells attached, but the proliferation rate was low in the first few days. After several days in culture the cells started to proliferate and formed islands of densely packed keratinocytes and more sparsely growing fibroblasts (Fig. 2a). The cells attached to both - Geltrexmatrix and plastic surface, however, proliferation was faster on Geltrex. Witte and Kao (2005) showed that extracellular matrix is extremely important in cell adhesion and proliferation. It can also affect the development of epithelial cells, their morphogenetic activity and even survival. Evidence suggests that the absence of appropri- ate matrix might be the reason for progressive necrosis of epithelial cells on plastic surface (Freeman et al., 1978). After 7 days of cultivation, keratinocytes formed large clusters (Fig. 2b). Orazizadeh et al., 2015 compared enzymatic and explant methods for human foreskin derived keratinocytes in the absence of feeder layer. They noticed that after enzymatic treatment no attached cells were found after 7 to 10 days. With explant method they found separated keratinocytes after 24 hours that latter formed large clusters of cells. Our findings differ from that, namely, the yield of cells was higher when enzymatic method was used. After 10 days in culture we observed large colonies of keratinocytes, surrounded by fibroblasts. Witte and Kao (2005) reported that growth and adhesion of keratinocytes can be increased in the presence of mesenchymal cells (fibroblasts). Interleukin I beta (IL-1ß) is released by keratinocytes and stimulates production of diverse fibroblast growth factors (FGF) in mesenchymal cells, including keratinocyte growth factor (KGF) that enhances migration and proliferation of keratinocytes (Witte and Kao, 2005). Fibroblasts are normally unwanted in primary cell cultures. Fibroblast contamination in the culture was decreased with mechanical removal of dermis in the skin biopsate (using scalpel) and with incubation in dispase II that enabled physical separation of epidermal and dermal tissue. For additional separation of keratinocytes from fibroblasts the differential trypsinisation was used. In case of tissue explant method, we obtained a relatively pure keratinocyte culture. Guo and Jahonda (2009) reported that fibroblasts migrate out of adult skin explant later than keratinocytes. With well-timed removal of tissue pieces from the culture, we were able to avoid fibro-blast contamination. Figure 1: Explant culture of keratinocytes with visible piece of the attached skin tissue (magnification x 40) Slika 1: Kultura keratinocitov, pridobljena z metodo izraščanja celic, z vidnim delom kožnega tkiva (40 x povečava) /йьжг ■. ■ кз ияии'^тк: —i _ JI Щш цицицимц , ' ' а: У' ■ t , ~ - ■ i'/vi;- - Л " Jgtó К ' У К , *.** -i» , ABC Figure 2: Keratinocytes and fibroblasts in the culture established with enzymatic method during day 2 (A, magnification x 40), day 7 (B, magnification x 40), and day 10 (C, magnification x 40) Slika 2: Kultura keratinocitov in fibroblastov, pridobljena z encimsko metodo drugi dan (A, 40 x povečava), 7 dan (B, 40 x povečava ) in 10 dan (40 x povečava) and mesenchymal (fibroblast) cells (Fig. 3). Epithelial cell morphology and expression of cytokeratin 14 proves that cells in the primary culture belong to keratinocyte lineages. Vimentin was expressed only in fibroblasts, which can also be recognised by their elongated spindle shaped morphology (Vissar and Pollit, 2010). We found that keratinocytes from equine skin were able to proliferate in the primary culture for at least four passages, without any visible change in the morphology and proliferation rate, estimated by the time needed to overgrow the growth surface between the passages. Cells adhere well to Geltrex, which we used instead of collagen type I (Vissar and Pollit, 2010), but can attach and proliferate also on plastic surface. The established cell culture can be frozen and recovers with approximately 80 % viability rate. The cells are available for further research, for example, as a research model of horse skin diseases, product testing and toxicity screens in pharmaceutical and cosmetic industry, or as an animal model for studying certain human skin diseases. л ' > i t äSm ■ L irfm V* A B Figure 2: Mixed culture of keratinocytes and fibroblasts after immunostaining under bright field (A) and fluorescent (B) illumination (magnification x 200). Keratinocytes stained with antibodies against cytokeratin 14 (green) and fibroblasts stained with antibodies against vimentin (red). Nuclei were counterstained with DAPI (4', 6-diamidine-2'-phenylindole dihydrochloride). Slika 2: Mešana kultura keratinocitov in fibroblastov pod svetlobnim (A) in fluorescentnim mikroskopom (B) (magnification x 200). Keratinociti so obarvani s protitelesom proti citokeratinu 14 (zelena), fibroblasti pa s protitelesom proti vimentinu (rdeča). Jedra so obarvana z DAPI (4', 6-diamidin-2'fenilindol dihidroklorid). Cells were characterised based on morphological characteristics and by immunofluorescent staining. Antibodies against cytokeratin 14, distinct for keratinocytes, and antibody against vimentin, distinct for fibro-blasts, were used for basic characterisation. Cytokeratins are cytoskeletal proteins distinctive for epithelial cells. Cytokeratin 14 forms hemidesmosomes together with cytokeratin 5, the structures on the inner basal surface of keratinocytes that attach cells to the extracellular matrix, and consequently maintain integrity of the basal layer (Nobusawa et. al., 2014). Positive staining against vimentin is often used for identification of fibroblasts. It is a type III cytoskeletal intermediate filament (IF) that is expressed in mesenchymal cells. Furthermore, vimentin is used in identifying cells that undergo an epithelial-to-mesenchymal transition (EMT). During EMT epithelial cells drastically change their shape and motile behaviour on the point of differentiating into mesenchymal cells (Mendez et al., 2010). Immunofluorescent co-staining showed presence of both markers in the culture - epithelial (keratinocytes) 4 REFERENCES Aasen T., Izpisua Belmonte J.C. Isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells. 2010. Nature Protocols, 5: 371-382. doi:10.1038/nprot.2009.241 Cerrato S., Ramio-Lluch L., Brazis P., Rabanal R.M., Fondevila D., Puigdemont A. 2014. Establishment and characterisation an of equine skin-equivalent model. Veterinary Dermatology, 25: 475-e77. doi:10.1111/vde.12134 FAOSTAT. 2013. Production/Live Animals. http://faostat3.fao. org/browse/Q/QA/E (16. Oct. 2015) Freeman A.E., Igel H.J., Herrman B.J., Kleinfeld K.L. 1976. Growth and characterization of human skin ephitelial cell cultures. In vitro, 12: 352-362. doi:10.1007/BF02796313 Guo A., Jahoda C.A.B. 2009. An improved method for homan keratinocyte culture from skin explants: Cell expansion is linked to markers of activated progenitor cells. Experimental Dermatology, 18: 720-726. doi:10.1111/j.1600-0625.2009.00900.x Mendez M.G., Kojima, S.I., Goldman, R.D. 2010. Vimentin induces changes in cell shape, motility, and adhesion during the epithelial to mesenchymal transition. The FASEB Journal, 24, 6: 1838-1851. doi:10.1096/fj.09-151639 Nagy K., Sung H.K., Zhang P., Laflamme S., Vincent P., Agha-Mohammadi S., Woltjen K., Monetti C., Michael I.P., Smith L.C., Nagy A. 2011. Induced pluripotent stem cell lines de- rived from equine fibroblasts. Stem Cell Reviews, 7: 693702. doi:10.1007/s12015-011-9239-5 Nobusawa A., Sano T., Negishi A., Yokoo S., Tetsunari O. 2014. Immunohistochemical staining patterns of cytokeratins 13, and 17 in oral epithelial dysplasia including orthokeratotic dysplasia. Pathology International, 64: 20-27. doi:10.1111/ pin.12125 Orazizadeh M., Hashemitabar M., Bahramzadeh S., Dehbashi F.N., Saremy S. 2015. Comparison of the enzymatic and ex-plant methods for the culture of keratinocytes isolated from human foreskin. Biomedical Reports, 3, 3: 304-308 Seltenhammer M.H., Heere-Ress E., Brandt S., Druml T., Jansen B., Pehamberger H., Niebauer G.W. 2004. Comparative histopathology of grey-horse-melanoma and human malignant melanoma. Pigment Cell Research, 17: 674-81. doi:10.1111/j.1600-0749.2004.00192.x Visser M.B., Pollitt C.C. 2010. Characterization of extracellular matrix macromolecules in primary cultures of equine keratinocytes. BMC Veterinary Research, 6, 16: 1-8. doi:10.1186/1746-6148-6-16 Witte R.P., Kao W.J. 2004. Keratinocyte-fibroblast paracrine interaction: the effects of substrate and culture condition. Biomaterials, 26: 3673-3682. doi:10.1016/j.biomateri-als.2004.09.054 Wunn D., Wardrop K.J., Meyers K., Kramer J., Ragle C. 1999. Culture and characterisation of equine terminal arch en-dotelial cells and hoof keratinocytes. American Journal of Veterinary Research, 60, 1: 128-132 COBISS: 1.01 Agris category code: L01, L10 ANALYSIS OF BACKFAT THICKNESS IN ON-FARM TESTED GILTS IN SLOVENIA USING REACTION NORMS Martina PLANINC Milena KOVAČ 2, Špela MALOVRH 3 Received June 09, 2015; accepted November 20, 2015. Delo je prispelo 09. junija 2015, sprejeto 20. novembra 2015. Analysis of backfat thickness in on-farm tested gilts in Slovenia using reaction norms Animals have the ability to respond to differences in environment, which can be called phenotypic plasticity or environment sensitivity. Robust animal, that show little variability across environments, are less sensitive. Therefore, the aim of this study was to investigate genotype by environment interaction for backfat thickness using reaction norm. Random regression model have become common for analyzing data on individuals over time. Reaction norms were predicted for 239 sires. Results show how sires differ across environments for backfat thickness. Key words: pigs / gilts / genetics / environment / maternal genotype / backfat thickness / reaction norms / Slovenia 1 INTRODUCTION The ability of living organisms to respond to changes in their environments is called phenotypic plasticity or environmental sensitivity (de Jong and Bijma, 2002). Genotypes with highly variable production across different environments are characterized as 'plastic'. However, genotypes with little variability across environments are called as 'robust'. Population under high selection pressure become more sensitive and as such, robustness has rapidly become a term with high interest in animal production (Knap, 2005; ten Napel et al., 2006). Robustness is not a trait which is easily measured. Thus, there are many definitions for it. Among others, it is defined as the ability of even production potential through a wide range of environments. Robust animas should be less sensitive Reakcijske norme za debelino hrbtne slanine pri testiranih mladicah v Sloveniji Živali imajo možnost, da se na razlike v okolju odzivajo različno. To imenujemo fenotipska plastičnost ali okoljska občutljivost. Robustne živali, ki kažejo majhno variabilnost med okolji, so manj občutljive. Namen prispevka je z reakcijskimi normami preučiti interakcijo med genotipom in okoljem za debelino hrbtne slanine. Za analizo podatkov smo uporabili model z naključno regresijo, reakcijske norme smo napovedali 239 očetom. Rezultati kažejo, kako se preučevana lastnost za posameznega očeta skozi okolja spreminja. Ključne besede: prašiči / mladice / genetika / okolje / ma-ternalni genotip / debelina hrbtne slanine / reakcijske norme / Slovenija to stress and are expected to recover more quickly than less robust animals. This indicates that robust animals function well under a wide range of environments. Phenotypic plasticity is related to genotype by environment interaction. In pig breeding, genotype by environment interaction could reduce genetic improvement if breeding values for any trait used as breeding goal are predicted on records obtained in specific test environments. However, productive animals are raised in different environments. A key problem is to decide under which conditions animals should be tested and how genotype by environment interaction can be included in selection procedure (de Jong and Bijma, 2002). The evaluation of genotypes in only one environment cannot be used to predict the performance of pigs reared in different environments whenever genotype by environment 1 Univ. of Ljubljana, Biotechnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia 2 Same address as 1, e-mail: milena.kovac@bf.uni-lj.si 3 Same address as 1, e-mail: spela.malovrh@bf.uni-lj.si interaction exists. Genetic evaluation requires a possibility of sire ranking in each environment. Kolmodin et al. (2002) applied random regression models as reaction norm models to express performance of a genotype as a function of the environment. If different genotypes have different reaction norms, then there is genotype by environment interaction (de Jong and Bi-jma, 2002). However, these models require definition of environment constraints on a continuous scale. Random regression models have become common for the analysis of longitudinal data or repeated records on individuals over time (Schaeffer, 2004). Some studies on genotype by environment interaction using reaction norm models have been reported for litter size in pigs (Knap and Su, 2008), for growth traits in pigs (Hermesch et al., 2008; Li and Hermesch, 2012), and for milk production in dairy cattle (Kolmodin et al., 2002; Shariati et al., 2007, Logar at al., 2007). The objective of this study was to apply reaction norm model to evaluate genotype by environment interaction for backfat thickness in gilts in Slovenia. var 2 MATERIAL AND METHODS Individual records and pedigree information were collected for 19053 on-farm tested animals. Data comprising Slovenian Landrace - line 11, Slovenian Large White 22 and Hybrids 22x11 (21) and 11x22 (12) gilts Table 1: Descriptive statistics for body weight, daily gain, and backfat thickness (N = 18805) Preglednica 1: Opisna statistika za telesno maso, dnevni prirast in debelino hrbtne slanine (N = 18805) Variable Mean SD Min. Max. Body weight (kg) 108.8 13.8 80 200 Daily gain (g/day) 554 46 375 708 Backfat (mm) 10.5 2.2 4 24 were routinely recorded from 2000 to 2013. Animals were raised on 46 family farms under production conditions. Records were included if body weight was at least 80 kg, and age at the end of the test was at most 300 days. The environmental variable was defined as herd-year average of daily gain. After editing, 18805 records were obtained on gilts weighing on average 108.8 kg at the end of test (Table 1). The average backfat thickness was 10.5 mm (±2.2). The observations were spread over 1864 herd-year seasons. The pedigree file contained animals with records and up to five generations of ancestors. There were 239 different sires included. The variable use to characterize the environment of animal was defined as deviation of herd-year daily gain on test and backfat thickness. Linear regression was applied to describe random sire effect over environments. The following mixed model was used: yijklmno - И + Gi + Sj + Rk + b(xijklmn - X ) + hk + s0im + s1im + Wijklmn + lijklmn + eijklmno (1) where yijkimno is a trait, ^ is overall mean for trait. The fixed part of model included genotype Gi (i - 1, 2, 3, 4), season Sj j - 1, 2, ... 160) and herd Rk (k - 1, 2, ... 46). Animal weight at the end of test (xijklmr) was described by linear regression with b as linear regression coefficient. Random part of model included common herd-year environment (hkl), common litter environment (ljHm„), intercept (level) of reaction norm for sire (s0im), random regression coefficient (slope) for sire (s1im) with environmental variable (Wjklmn) expressed as deviation of herd-year average of daily gain. Variable (eijklmno) is random residual. Sire effect s0 and s1 were assumed to be normally distributed with mean zero and covariance structure: ) A = S„ ® A = ®A (2) where s0 and s1 are vectors of unknown parameters for intercept and slope in random regression for sire effect. Genetic variance was partitioned into three components: variance for level (ffo), for slope (ff^), and covariance between the two (ffrfn). Matrix A is the additive relationship matrix for sire and the matrix S0 is equal to one quarter of the genetic variance matrix for level and slope. The environmental variable herd-year averages were grouped into six classes allowing for heterogeneous residual variances across environments. The observations were assigned to classes by their environment value. The lowest values formed first class and the highest values formed the sixth class. The classes had equal range of environmental variables. The residuals were assumed to be independently distributed with mean zero and variance a]e within environmental class t. In the reaction norm models, breeding values are described by linear regression with level and slope as parameters. Predicted breeding values within environment wt could be expressed as predicted offspring performance POPim/w; Kolmodin et al., 2002) calculated as: POP im ' = s, 0im + siimWt (3) SAS software (SAS Inst. Inc., 2008) was used for data editing and finalization of the results. Dispersion parameters were estimated using residual maximum likelihood methodology as applied in VCE-5 (Groeneveld et al., 2010). 3 RESULTS AND DISCUSSION Estimates of genetic (co)variances and correlations together with residual variance estimates in the reaction norm model are shown in table 2. Estimates of residual variances decrease progressively with increasing environmental variable for backfat thickness suggesting heterogeneous residual variances. In better environments, testing conditions of gilts are Table 2: Variance components and genetic correlations between level and slope for backfat thickness Preglednica 2: Komponente variance in genetska korelacija med stopnjo in naklonom za debelino hrbtne slanine (Co)variance component Estimate ± SEE Genetic correlation Sire level 0.30 ± 0.04 0.14 ± 0.13 slope 23.84 ± 7.26 Residual variances in class 1 2.14 ± 0.04 2 2.63 ± 0.13 3 1.63 ± 0.02 4 1.53 ± 0.02 5 1.37 ± 0.03 6 1.20 ± 0.07 -0.5 -1.0 -1.5 -2.0 Environment Figure 1: Reaction norms for sample of 43 sires for backfat thickness (mm) in different herd environments (x-axe shows deviation from average environment in SD units of herd-year daily gain average) Slika 1: Reakcijske norme za debelino hrbtne slanine (mm) v različnih okoljih pri 43 očetih (na x-osi je prikazan odklon od povprečnega okolja, ki je definirano kot povprečni dnevni prirast v čredi znotraj leta) more standardized and the gilts express more uniformity as in barren environments. Estimated sire variance for slope was 23.84 (mm/(g/ day))2. Genetic correlation between level and slope was estimated to 0.14, which is an indication of potential reranking. As long as the genetic variance of the reaction norm slope is greater than zero and genetic correlation among environments is smaller than one, there is genotype by environment interaction (de Jong and Bijma, 2002). Genetic correlation between the reaction norm of level and slope far from 1 will cause re-ranking of animals among environments (Su et al., 2006). Reaction norm was used also to describe genetic variability of pig carcass weight as a function of heat stress of crossbreed pigs in North Carolina (Zumbach et al., 2008). They estimated negative correlation between the intercept and slope. This could be due to increased sensitivity of animals to heat stress. Sire effects for 43 sires are illustrated by linear regression on Figure 1 showing re-ranking of sires across environments. For backfat thickness, the best animals have the most negative breeding values predicted, because selection is for thinner backfat. Sires A and B are very sensitive, in other words not robust to the environmental changes. Sire A is more superior with less backfat thickness in rich environment (Fig. 1, right) and is ranked on the 27th place only in the most barren environment (Fig. 1, left), while sire B fits better to barren than rich environment. Both are very specific and not well suited for the whole specter of environmental conditions. Nevertheless, sire D performed well and has excellent as well as steady genetic merit across all environments. Sire C has a robust genotype as well. Its breeding values seems to be more or less constant over environments considered, but the level of breeding value is worse that the level for sire D. In general, reranking observed was greater than expected in such small population. Re-ranking of boars in organic and conventional pig production was studied in Swedish Lan-drace (Wallenbeck et al., 2009) for growth rate and backfat thickness. Wallenbeck et al. (2009) found re-ranking of boars between the two production systems. In their study, the best boar in the conventional environment (boar X) was ranked on place 11th in the organic environment. At the same time, the lowest ranking boar in the conventional environment was almost as good as boar boar X in the organic environment. But, the positive correlations mean that boars that are ranked highly on conventional breeding value are in many cases ranked highly on organic breeding value. This indicates that interactions GxE are week. Reaction norms were used to investigate sire by environment interactions for growth rate and backfat thickness in Australian pigs (Li and Hermesch, 2012). Li and Hermesch (2012) reported, that Large White was the most sensitive breed for growth rate and backfat thickness, while Duroc was the most robust breed across their production systems. 4 CONCLUSIONS Robustness of gilts in Slovenia was studied using reaction norm model. Analyses were performed using random regression model for backfat thickness. Breeding value as well as ranking of some sires (example sire A and B) changed over environments. There was a group of sires which breeding values and rank did not change much over environments. Overall results proved existence of genotype by environment interaction. This information is useful to setup strategic performance recording procedures for genetic improvement of productivity and robustness. 5 REFERENCES de Jong G., Bijma P. 2002. Selection and phenotypic plasticity in evolutionary biology and animal breeding. Livestock Production Science, 78: 195-214. doi:10.1016/S0301-6226(02)00096-9 Groeneveld E., Kovač M., Mielenz N. 2010. 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Journal of Animal Science, 86: 2076-2081. doi:10.2527/jas.2007-0282 COBISS: 1.01 Agris category code: L01, L73 EFFECT OF DIFFERENT LITTER MATERIALS ON FOOT PAD DERMATITIS, HOCK BURN AND FEATHER COVERAGE IN BROILER CHICKENS 1 Dušan TERČIČ 2, Mirjana ŽOLGER 3, Mojca PESTOTNIK 4 Received November 30, 2015; accepted December 15, 2015. Delo je prispelo 30. novembra 2015, sprejeto 15. decembra 2015. Effect of different litter materials on foot pad dermatitis, hock burn and feather coverage in broiler chickens An experiment was conducted to evaluate the feather coverage on breast and thighs in meat type chickens, reared on three different types of litter materials, including wood shavings, shredded paper and chopped wheat straw. The experiment lasted for 72 days. A total of 447 one-day-old meat type chickens were randomly allotted to the three litter materials, giving 149 birds per litter. On days 24, 47 and 72 of the experimental period, feathering on the breast and thighs was scored according to a 1-4 scale. In addition, the severity of foot pad dermatitis (FPD) and hock burn (HB) was evaluated at the same days by attributing scores from 1-3. Litter characteristics (moisture content, water absorbing capacity, bulk density, pH) were determined at the end of the experiment. Severity of FPD varied significantly (P < 0.016) among the litter materials with chopped straw showing the highest severity scores and wood shavings showing the lowest. Litter material had no effect (P > 0.016) on the severity of HB and on thigh feathering. Birds reared on chopped wheat straw presented the lowest breast feather coverage. Straw litter had the highest moisture content and pH value. In conclusion, wood shavings proved to be best litter material for meat type chickens rearing among the three tested litter materials. Key words: poultry / broiler chickens / litter / foot pad dermatitis / hock burn / feather coverage Vpliv različnih vrst nastila na pojav vnetja kože na blazinicah stopal, vnetja kože skočnih sklepov ter na operjenost pri pitov-nih piščancih Poskus je bil zasnovan z namenom ocenitve operjenos-ti področja prsi in beder pitovnih piščancev, vzrejenih na treh vrstah nastila, vključujoč lesne oblance, razrezan papir in rezano pšenično slamo. Poskus je trajal 72 dni. Štiristo sedemin-štirideset en dan starih pitovnih piščancev je bilo naključno razdeljenih v 3 skupine, 149 živali na posamezno vrsto nastila. V poskusnem obdobju smo trikrat (24., 47. in 72. dan) ocenili operjenost področja prsi in beder z uporabo lestvice z ocenami od 1 do 4. Istočasno smo ocenili intenzivnost vnetja kože na blazinicah stopal in intenzivnost vnetja kože na skočnih sklepih z uporabo lestvice z ocenami od 1 do 3. Na koncu poskusa smo vzorcem nastila določili gostoto, vsebnost vode, pH vrednost ter sposobnost zadrževanja vode. Intenzivnost vnetja kože na blazinicah stopal je bila značilno različna (p < 0,016) med posameznimi vrstami nastila in sicer so najvišje (najslabše) ocene dosegli piščanci, uhlevljeni na rezani pšenični slami, in najnižje (najboljše) piščanci, uhlevljeni na lesnih oblancih. Vrsta nasti-la ni značilno vplivala (p < 0,016) niti na operjenost področja beder niti na pojavljanje vnetij kože na skočnih sklepih. Pri piščancih, uhlevljenih na pšenični slami, je bila opažena najslabša operjenost področja prsi. Vzorec nastila s pšenično slamo je imel najvišjo vsebnost vode ter najvišjo pH vrednost. Izmed treh proučevanih materialov so se kot najboljši material za na-stiljanje v rejah pitovnih piščancev izkazali lesni oblanci. Ključne besede: perutnina / pitovni piščanci / nastil / blazinice stopal / skočni sklepi / vnetje kože / operjenost 1 This article is part of a Master thesis entitled »The use of different types of litter material for rearing broiler breeders«, issued by Mirjana Žolger, supervisor Assist. Prof. Dušan Terčič, Ph. D. / Prispevek je del magistrskega dela Mirjane Žolger z naslovom »Uporaba različnih vrst nastila pri vzreji piščancev kokoši težkega tipa«, mentor doc. dr. Dušan Terčič 2 Univ. of Ljubljana, Biotechnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia, e-mail: dusan.tercic@bf.uni-lj.si 3 Šmartno pri Slovenj Gradcu 154, SI-2383 Šmartno, Slovenia, e-mail: mikicatayson@gmail.com 4 Same address as 2, e-mail: mojca.pestotnik@bf.uni-lj.si 1 INTRODUCTION Foot pad dermatitis (FPD), hock burn (HB) and breast blisters (BB) are dermatological problems with similar pathologies and are collectively known as a contact dermatitis (Greene et al., 1985). Contact dermatitis is an ulcerative condition of the skin affecting the plantar surface of the feet (FPD), the hock (HB) and the breast (BB) (Haslam et al., 2007). It is seen in growing broiler chickens and turkeys, and in broiler parents. Some lesions are superficial, whereas others progress to deep ulcers and cause discomfort and pain (Cengiz et al., 2011). Besides the negative effect on welfare, different forms of contact dermatitis affect farmer income, and in the future it is likely to have increasing importance in terms of legislation. Several major factors are associated with the occurrence of contact dermatitis including type, depth and condition of litter, stocking density, feed composition, light and climate (Meluzzi and Sirri, 2009). Of these, litter may be the most important because meat type chickens spend most of their time on the litter and their foot pads, hock and breast are in constant contact with the material on the floor. Therefore, if the type, quantity and quality of litter material are not optimal there is a considerable risk that birds will develop contact dermatitis and breast blisters (Meluzzi and Sirri, 2009). Various types of litter materials are used in different countries. In Slovenia, wood shavings and sawdust are the most common materials used as litter in commercial broiler production. However, these preferred litter materials are becoming limited in supply and expensive. Therefore, appropriate substitutions need to be considered. Various forms of recycled paper and chopped straw have proven to be good litter materials in research and commercial situations. With those idea in view, the present study was designed to evaluate the effects of different litter substrates on feather coverage and on the severity of FPD and HB in meat type chickens. 2 MATERIAL AND METHODS The experiment was carried out at a poultry research station (Biotechnical Faculty, University of Ljubljana, Slovenia) with the approval of the Commission for Animal Experiments of the Institution. Four hundred forty seven newly hatched parent stock chicks of Slovenian traditional meat type breed »Slovenian Late Feathering Hen« were randomly assigned, as mixed sex, to the three litter materials (wood shavings, chopped wheat straw, shredded paper), giving 149 birds per litter (pen). Each group was kept in a pen measuring 30 m2 resulting in a flock density of 5 birds m-2. Experimental pens were located side-by-side within the same environmentally controlled poultry house. Feeder and drinker spaces were identical in each pen. Day old chicks were individually identified by toe clipping. The depth of litter in all pens was approximately 8-10 cm. The brooding temperatures were kept at 32 to 33 °C from day 1 to 7; thereafter, the temperature was reduced by 3 °C/week until it reached 21 to 23 °C, and was maintained at that temperature thereafter. During the first month, all birds consumed feed for ad libitum intake. From the age of 30 days all birds were fed on restricted rations according to breeder recommendations. The feed was supplied in a single daily feed and was generally consumed in less than 20 minutes. The chickens were given a standard grower feed (11.9 MJ ME/kg, 20.0 % CP) containing an anticoccidial additive. Access to water was unlimited. Fresh litter materials were added to pens whenever damp litter resulting from excessive drinking was observed. The photoperiod was 23 h light (L) and 1 h dark (D) during the first week. Thereafter, light was decreased by 3 to 4 h/week to 8 L:16 D at week 8. The trial lasted 72 days. The severity (i.e., extent of lesions) of FPD and HB and the degree of feathering on the breast and thighs were determined at 24, 47 and 72 days. At 24 and 47 days approximately 50-60 birds per pen were randomly chosen, while at 72 days of age all birds were evaluated. The foot pad and hock lesions were assessed using a 3-point scoring system, in which 1 = no lesions; no or very small superficial lesions, slight discoloration on a limited area, mild thickening of the skin; 2 = mild lesion; discoloration of the foot pad, superficial lesions, dark papillae; and 3 = severe lesion; ulcers or scabs, signs of haemorrhages or swollen foot pads. The foot pad and hock scores were evaluated by using the mean of both feet. Breast and thighs feathering were scored according to the following 1-4 score scale: 1 = skin is not visible, given area completely feathered; 2 = less than 25 % of feathers missing from a given area; 3 = 25-50 % of feathers missing from a given area; and 4 = more than 50 % of feathers missing from a given area. At the end of experiment, litter samples were collected from five randomly chosen locations within each pen and thoroughly mixed to obtain material representative of the entire pen. Each litter material was analyzed for moisture content, pH value and moisture retention capacity. Litter samples were dried at 100 °C for 18 hours to determine moisture content which is expressed on a fresh matter basis. The pH of each litter type was measured after litter samples of nearly 10 g were suspended for 30 min in 100 mL of distilled water. pH was recorded until constant values were obtained. In order to determine water absorbing Table 1: Scores of FPD and HB severity and feathering scores on the breast and thighs of meat type chickens reared on different litter materials over 72 days Preglednica 1 : Ocene poškodb kože na blazinicah stopal oziroma na področju skočnih sklepov in ocene operjenosti področja prsi oziroma bederpri pitovnih piščancih, rejenih 72 dni na različnih vrstah nastila FPD scores HB scores Feathering scores on the breast Feathering scores on the thighs Experimental group Number of chickens Mean value P value Mean value P value Mean value P value Mean value P value Wood shavings 243 1.00a 0.0001 1.00a 1.000 1.62a 0.0001 1.46a 0.449 Shredded paper 254 1.15b 1.00a 1.81a 1.50a Chopped wheat straw 245 1.48c 1.00a 2.71 1.57a ,bc Means followed by the same letters in the same column are not significantly different (P > 0.016) 3 RESULTS AND DISCUSSION capacity, dried samples of pure litter materials were weighed and placed in pans. Moisture holding capacity was determined by filling the pan with water and letting it stand for 90 minutes. Excess water was then drained for 3 minutes and the sample was then weighed again. The percentage of water absorbed was then calculated on dry matter basis. The results were analysed using the statistical SAS program (SAS Institute, 2008). Ordinal variables (feather scores, foot pad and hock scores) were analyzed using Shapiro-Wilk test to verify the normality of residuals, and Levene test for the homogeneity of variances. Comparisons of the results were done using the Kruskal-Wallis test. When the effects were significant they were tested with the Wilcoxon nonparametric rank test within procedure NPAR1WAY. To counteract the problem of multiple comparisons, all statements of significance for ordinal variables were based upon P < 0.016 (Bonferroni correction). The severity scores of FPD and HB and feathering scores on the breast and on the thighs are presented in Table 1. Wood shavings were ranked as the litter material with the lowest FPD severity. Chopped wheat straw had the worst FPD score (Table 1). It was observed that feathering scores on the breast were significantly (P < 0.016) higher in birds on chopped wheat straw in comparison with birds od shredded paper and wood shavings. The presence of breast blisters was not observed in any experimental group. Feathering scores on the thighs were not affected by the litter materials (P > 0.016). Initial (at 24 days) and final (at 72 days) FPD scores were similar among birds placed on the shredded paper and chopped straw (Table 2). At 47 days of age, FPD scores were higher (P < 0.016) among birds Table 2: Effect of litter type on the severity of foot pad dermatitis and hock burn at different ages Preglednica 2: Vpliv vrste nastila na obseg poškodb kože na blazinicah stopal in kože na področju skočnih sklepov pri različnih starostih FPD scores HB scores Age of chickens Experimental group Number of animals Mean value P value Number of animals Mean value P value 24 days Wood shavings 55 1.00 0.0001 55 1.00a 1.000 Shredded paper 57 1.19a 57 1.00a Chopped straw 52 1.32a 52 1.00a 47 days Wood shavings 58 1.00a 0.0001 58 1.00a 1.000 Shredded paper 58 1.36b 58 1.00a Chopped straw 58 2.62c 58 1.00a 72 days Wood shavings 130 1.00 0.032 130 1.00a 1.000 Shredded paper 139 1.05a 139 1.00a Chopped straw 135 1.05a 135 1.00a a,b,c Means followed by the same letters in the same column and within the same age are not significantly different (P > 0.016) Table 3: Effect oflitter type on thefeather coverage ofthe breast and thighs at different ages Preglednica 3: Vpliv vrste nastila na oper.enostpodroč.a prsi oziroma bederpri različnih starostih Feathering scores on the breast Feathering scores on the thighs Age of Experimental Number of Mean Number of Mean chickens group animals value P value animals value P value 24 days Wood shavings 55 2.85a 0.0001 55 3.07a 0.0001 Shredded paper 57 3.57b 57 3.22a Chopped straw 52 3.86c 52 3.71 47 days Wood shavings 58 1.18a 0.0001 58 1.00a 1.000 Shredded paper 58 1.25a 58 1.00a Chopped straw 58 2.58 58 1.00a 72 days Wood shavings 130 1.30a 0.0001 130 1.00a 0.369 Shredded paper 139 1.31a 139 1.00a Chopped straw 135 2.31 135 1.00a a,b,c Means followed by the same letters in the same column and within the same age are not significantly different (P > 0.016) raised on chopped straw than those raised on shredded paper. Throughout the whole trial period FPD scores were significantly (P < 0.016) lower in those animals that were placed on wood shavings. Greater values of breast and thighs feather scores were present at 24 days of age. Beyond this age, the values lowered in all experimental groups. This may be attributed to the fact that chicks moult the natal plumage formed in embryonic development into juvenile feathers between 21-35 days of age. Breast feather cover as indicated by feather score was significantly (P < 0.016) better in chicks placed on wood shavings compared with birds placed on chooped straw at every age (Table 3). At the ages of 47 days and 72 days all of the evaluated birds achieved a thighs feather score of 1, or full thighs feather cover. Today reduced feathering is considered beneficial when broilers are reared in hot climates, as it increases heat dissipation; however, it impairs carcass quality (Garcia et al., 2012). Characteristics of litters are summarized in Table 4. Chopped straw on one hand contained the highest percentage of moisture, on the other hand chicks grown on the chopped straw showed the most severe foot pad lesions. This is not surprising because it is well known that excessive contact with wet and am-moniacal litter is generally considered to be the primary cause of foot and hock burn (Tucker and Walker, 1992). The water holding capacity of litter is a fundamental factor in preserving the foot in a good state (Meluzzi and Sirri, 2009). Foot pad burn scores were lowest in the pen littered with wood shavings. Wood shavings as a litter material with the highest waterholding capacity and consisted of small particles resulted in lower moisture content, minimizing the incidence of lesions in foot pads and breast. Oliveira et al. (2004) found the similar results. Meluzzi et al. (2007) raised birds on chopped straw or wood shavings both in winter and in summer seasons and observed that birds kept on wood shavings exhibited a reduction of 35 % in foot pad dermatitis than those kept on straw (Meluzzi and Sirri, 2009). Tucker and Walker (1992) found lower hock burn scores in birds reared on wood shavings rather than straw, but this effect was not seen in the study of Su et al. (2000). Table 4: Chemical and physical characteristics of three litter materials Preglednica 4: Kemi.ske in fizikalne lastnosti treh vrst nastila Characteristics of litter materials Litter material / Experimental group Moisture content (%) Water absorbing capacity (%) Bulk density (kg/m3) pH of litter material at 72 days Shredded paper 9.41 ± 0.17 37.32 ± 4.10 350.2 ± 18.24 8.51 ± 0.64 Chopped straw 21.93 ± 3.67 56.84 ± 2.41 368.5 ± 42.67 9.02 ± 0.82 Wood shavings 7.62 ± 0.14 86.36 ± 7.23 317.8 ± 11.53 8.71 ± 0.34 a,b,c Means followed by the same letters in the same column and within the same age are not significantly different (P > 0.016) 100 Acta agriculturae Slovenica, 106/2 - 2015 4 CONCLUSIONS From all collected data we can conclude that: - Of the three litter materials tested, wood shavings showed the lowest FPD severity and the highest feather coverage ob breast, whereas chopped wheat straw showed the highest FPD severity and the lowest feather coverage of breast. - Type of litter material had no significant effect on HB and feathering score on the thighs. - Wood shavings as litter substrate have been found to be better than chopped wheat straw and shredded paper with regard to moisture content and water retention capacity. 5 REFERENCES Cengiz Ö., Hess J.B., Bilgili S.F. 2011. 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SAS/STAT User's Guide, Version 9.02. SAS Institute Inc., Cary, NC, USA Su G., Sorensen P., Kestin S.C. 2000. A note on the effects of perches and litter substrate on leg weakness in broiler chickens. Poult. Sci., 79: 1259-1263. doi:10.1093/ps/79.9.1259 Oliveira M.C., Ferreira H.A., Cancherini L.C. 2004. Efeito de condicionadores quimicos sobre a qualidade da cama de frango. Arq. Bras. Med. Vet. Zoo., 56: 536-541. doi:10.1590/ S0102-09352004000400016 Tucker S.A., Walker A.W. 1992. Hock burn in broilers. In: Recent Advances in Animal Nutrition. Garnsworthy P.C., Ha-resign W., Cole J.D.A. (eds.). Oxford, Butterworth Heinemann: 33-49. doi:10.1016/B978-0-7506-0714-8.50006-0 COBISS: 1.01 Agris category code: L10 ANALIZA LASTNOSTI ZUNANJOSTI PRI PLEMENSKIH BIKIH CIKASTEGA GOVEDA 1 Mojca SIMČIČ 2, Miran ŠTEPEC 3, Betka LOGAR 4, Klemen POTOČNIK 3 Delo je prispelo 15. oktobra 2015, sprejeto 15. decembra 2015. Received October 15, 2015; accepted December 15, 2015. Analiza lastnosti zunanjosti pri plemenskih bikih cikastega goveda V raziskavi smo analizirali lastnosti zunanjosti pri 330 plemenskih bikih cikastega goveda. Sistem ocenjevanja zunanjosti pri cikastem govedu vključuje merjene, posamezne ocenjevane in sestavljene ocenjevane lastnosti. Posamezne ocenjevane lastnosti so razdeljene v dva sklopa, in sicer avtohtonost in telesne oblike. Tri sestavljene ocenjevane lastnosti so: avtohtonost, omišičenost in telesne oblike. Sistematski del statističnega modela smo analizirali s proceduro GLM v programskem paketu SAS in je vključeval vpliv leta ocenjevanja in starost živali ob ocenjevanju kot linearno regresijo. V povprečju so bili 14,6 mesecev stari plemenski biki v vihru visoki 117,1 cm. Na podlagi višine vihra in posameznih ocenjevanih lastnosti iz sklopa avtohtonost so bili potencialni plemenski biki neposredno ob koncu ocenjevanja razvrščeni v cikasti, delni cikasti in pincga-vski tip. Tak način razvrščanja ne upošteva vplivov okolja na izraženost lastnosti zunanjosti. Na lastnosti zunanjosti in posledično na razvrstitev plemenskih bikov v tip pomembneje vplivata vsaj leto ocenjevanja in starost ob ocenjevanju. Razvrstitev živali v ustrezen tip bi bilo zaradi tega potrebno opraviti po izvrednotenju ocen za lastnosti zunanjosti ob upoštevanju vplivov okolja. Ključne besede: govedo / pasme / cikasto govedo / plemenski biki / tipi / lastnosti zunanjosti / ocenjevanje Analysis of type traits of cika sires The aim of the study was to analyse type traits in 330 Cika sires. Scoring system of Cika cattle includes measured, individual scored and composite scored traits. Individual scored traits are divided into two groups, autochthonousness and form. Three composite traits are autochthonousness, muscularity and form. Fixed part of the model was analysed by GLM procedure in SAS software package, which included the effect of the year and animal age on the scoring day as linear regression. On average, 14.6 months old sires were 117.1 cm high at withers. Immediately, at the end of the scoring procedure all sires were classified into Cika, Semi-Cika and Pinzgauer type, based on the height at withers and individual scored traits from the autochthonous group. This method of classification does not take into account the environmental effects. We found significant effects of the year of scoring and animal age on type traits and therefore on the classification into the type. Sires classification should be carried out after the type traits data evaluation and after the exclusion of environmental effects. Key words: cattle / breeds / Cika / sires / type traits / scoring 1 Prispevek je del doktorske disertacije Mojce Simčič z naslovom »Fenotipske in genetske značilnosti cikastega goveda«, mentor prof. dr. Dragomir Kompan in somen-tor doc. dr. Gregor Gorjanc / This article is part of a doctoral dissertation entitled »Phenotypic and genetic characterisation of Cika cattle«, issued by Mojca Simčič, supervisor Assoc. Prof. Dragomir Kompan, Ph. D. and co-supervisor Assist. Prof. Gregor Gorjanc, Ph. D. 2 Univ. v Ljubljani, Biotehniška fak., Odd. za zootehniko, Groblje 3, SI-1230 Domžale, Slovenija, e-naslov: mojca.simcic@bf.uni-lj.si 3 Isti naslov kot 2 4 Kmetijski inštitut Slovenije, Hacquetova ulica 17, SI-1000 Ljubljana, Slovenija 1 UVOD Cikasto govedo je slovenska avtohtona kombinirana pasma goveda s poudarkom na prireji mleka. Rejski cilj je ohranjanje pasme v prvotnem tipu in preprečevanje parjenja v sorodstvu. Cikasto govedo je razširjeno skoraj po vsej Sloveniji, vendar je številčno najbolj zastopano na območju, od koder izvira (Bohinj, okolica Kamnika in okolica Kobarida). Barvni vzorec je tipičen in značilen za to pasmo ter se zelo razlikuje od drugih pasem goveda v Sloveniji. Nekatere živali cikastega goveda so po barvnem vzorcu bolj podobne pincgavskemu govedu, druge pa tux-zillertaler govedu iz Avstrije (Sambraus, 1999). Osnovna barva plašča je rumenordeča do temno rdeče rjava, največkrat kostanjevo rjava, ki mora prevladovati nad belo barvo. Glava mora biti osnovne barve. Po hrbtu imajo živali širši ali ožji bel pas, ki se nadaljuje pod repom do vimena ali mod in po trebuhu naprej do prsi. Rep je bele barve. Ožji ali širši beli pasovi so prisotni tudi čez zgornji del prednjih nog (v predelu komolčnega sklepa) in zadnjih nog (v predelu kolenskega sklepa). Barva vseh sluznic je rožnata. Barva rogov ob korenu je belo rumena, na gornji tretjini rjavkasta, konice pa so svetlejše. Parklji so temne barve (Navodila za presojo ..., 1935; Žan Lotrič in sod., 2010). Zunanji znak pasemske pripadnosti je bela ožja ali širša podolžna lisa po hrbtu in trebuhu (Navodila za presojo ..., 1935). Selekcija cikastega goveda temelji na lastnostih zunanjosti, ki so bile pri tej pasmi prvič ocenjene v letih 2002 in 2003, ko so ugotovili veliko fenotipsko raznolikost med živalmi znotraj populacije. Poleg tega je prvo ocenjevanje cikastega goveda potekalo po sistemu ocenjevanja, ki je bil takrat v uporabi za tri pasme goveda (rjavo, lisasto, črno-belo) za prirejo mleka v Sloveniji (Pogačar in sod., 1995; Pogačar in Potočnik, 1997). Na podlagi ugotovljene večje raznolikosti med živalmi so v letu 2006 vse plemenske živali ocenili še enkrat. Ocenjevanje je takrat potekalo na podlagi posebej prirejenega obrazca za cikasto govedo. Posebnost sistema ocenjevanja cikastega goveda je, da so v ocenjevanje lastnosti zunanjosti vključeni tudi vsi potencialni plemenski biki, ki jih ocenimo v starosti od 12 do 20 mesecev. Za cikasto govedo je bil leta 2005 sprejet in potrjen rejski program (Žan in sod., 2005). Leta 2010 je bil rejski program nekoliko dopolnjen in ponovno sprejet (Žan Lotrič in sod., 2010). V okviru tega ocenjevanje zunanjosti vključuje merjene in ocenjevane lastnosti. Pri bikih je merjenih lastnosti sedem. Na podlagi obsega prsi se določi (odčita z merilnega traku) tudi telesna masa. Posebnost ocenjevanja v primerjavi z drugimi pasmami je dvanajst tako imenovanih posameznih lastnosti za avtohtonost, ki opisujejo pasemske značilnosti. Poleg tega ocenimo tudi šest posameznih lastnosti za telesne oblike in tri sestavljene lastnosti. Na podlagi posameznih ocen za avtohtonost, ocene za izraženost skoč-nega sklepa in višine vihra so potencialni plemenski biki na koncu ocenjevanja razvrščeni v tri tipe (cikasti, delni cikasti, pincgavski). Na podlagi ocene za tip je žival odbrana za pleme. Čas ocenjevanja je prilagojen tehnologiji reje, zato živali ocenjujemo izven pašne sezone. Velikost populacije cikastega goveda je v primerjavi z ostalimi pasmami v Sloveniji majhna, zato vse plemenske živali ocenjuje samo en ocenjevalec. Analiza lastnosti zunanjosti je namenjena spremljanju in ugotavljanju primernosti porazdelitve ocen posameznih lastnostih in je hkrati podlaga za oceno genetskih parametrov, ki so potrebni za napovedovanje plemenskih vrednosti. Namen te raziskave je bil analizirati lastnosti zunanjosti za merjene in ocenjevane lastnosti ter poiskati vplive okolja, ki imajo neposreden vpliv na te lastnosti. 2 MATERIAL IN METODE Podatke smo pridobili iz Centralne podatkovne zbirke (CPZ) Govedo (Logar in sod., 2005), ki jo vodijo na Kmetijskem inštitutu Slovenije (KIS), ki je druga priznana organizacija v živinoreji in pooblaščena tudi za arhiviranje podatkov. Pridobljeni podatki, zabeleženi v okviru ocenjevanja lastnosti zunanjosti, so vključevali 375 plemenskih bikov cikastega goveda, ocenjenih v letih od 2002 do 2014. V nadaljnjo analizo smo vključili samo živali, ki so bile ocenjene po novem načinu ocenjevanja. Živali, ki so bile ocenjene pred letom 2006, smo iz obdelave izključili, tako pri analizi nismo uporabili sistematskega vpliva načina ocenjevanja. V skladu z omejitvami v rej-skem programu glede starosti ob ocenjevanju bi v analizo vključili zelo majhno število plemenskih bikov, saj v praksi ocenjujejo mlajše bike, kot je priporočeno. Da bi dosegli boljšo ocenljivost posameznih vplivov, smo v analizo vključili vse plemenske bike, ki so bili na dan ocenjevanja stari od 10 do 20 mesecev. Navedenim zahtevam je ustrezalo 330 plemenskih bikov cikastega goveda, zato smo jih vključili v končno analizo. Plemenski biki so bili v povprečju stari 446,2 ± 81,3 dni. Najmlajši je bil star 307 dni, najstarejši pa 850 dni. Vsi so bili ocenjeni v letih od 2006 do 2014 (slika 1). Sistem ocenjevanja zunanjosti vključuje sklop merjenih lastnosti (7), sklop posameznih ocenjevanih lastnosti za avtohtonost (12), sklop posameznih ocenjevanih lastnosti za telesne oblike (6) in sklop sestavljenih lastnosti (3). Istočasno na podlagi izjave rejca ocenjujejo še temperament bika in zabeležijo napake posameznih lastnosti zunanjosti. Ocenjevane napake v sklopu za avtohtonost so: temen gobec, neustrezna barva plašča, beli znaki na 60 л 1/1 50 С e fc 40 Ш Q. Л > 30 С -D 20 tu (J o 10 o. 0 et > 0,05) avtohtonost je bil med 3 in 13 %, pri posameznih lastnostih za telesne oblike pa med 2 in 9 %. Delež pojasnjene variabilnosti za sestavljeno lastnost avtohtonost je bil 6 %. ICAR (International agreement ..., 2012) priporoča analizo lastnosti zunanjosti z modelom, kjer naj bi bili vključeni vplivi starosti in sezone. Ocenjevalci naj ne bi korigirali ocen glede na starost, sezono, očeta in tehnologijo reje v času postopka ocenjevanja, ampak morajo oceniti le biološko izraženost lastnosti. Zabeležiti bi morali še način uhlevitve (prosta reja, vezana reja, reja z izpustom) in tip tal (beton, cement, les, pesek, guma, slama, pašnik), ker bi lahko vplivali na nekatere lastnosti zunanjosti. V zadnjem obdobju so tudi pri analizah variance lastnosti zunanjosti pri prvesnicah italijanskih avtohtonih pasem valdostana (Mazza in sod., 2013), rendena (Mazza in sod., 2014) in piemontese (Mantovani in sod., 2010) vključili vpliv starosti ob ocenjevanju. Poleg tega so vključili še interakcijo čreda-leto-ocenjevalec. Z navedenim modelom so pojasnili tudi večji delež variabilnosti (R2 = 0,19-0,36). V model za analizo lastnosti zunanjosti španske pasme asturiana de los valles so vključili vplive črede, interakcijo ocenjevalec-leto-sezona in starost (Gu-tiérrez in Goyache, 2002). V našem primeru je ocenjevalec samo eden, po čredi pa je premalo meritev, da bi jo lahko obravnavali kot sistematski vpliv. Nekateri avtorji (npr. Dal Zotto in sod., 2007, Klop-čič in Hamoen, 2010) priporočajo tudi vključitev telesne kondicije v model kot vpliv pri analizi lastnosti zunanjosti. Telesna kondicija predstavlja pokritost s podkožno maščobo oziroma lojem, ki ga otipamo na predelu korena repa in ledvenih vretenc in lahko vpliva na ocene zunanjosti, še posebno, ko so živali v preskromni ali v predobri kondiciji. Ocenjevanje telesne kondicije bi zato kazalo vpeljati tudi v ocenjevanje zunanjosti plemenskih bikov cikaste pasme. 4 SKLEPI Razvrščanje plemenskih bikov cikastega goveda v tip poteka ob zaključku ocenjevanja, kar onemogoča, da bi upoštevali morebitne vplive okolja. Pri razvrščanju se upošteva oceno za sestavljeno lastnost avtohtonost. Ugotovili smo, da na lastnosti zunanjosti in posledično na razvrstitev plemenskih bikov v tip značilno vplivata vsaj leto ocenjevanja in starost ob ocenjevanju. Razvrstitev živali v ustrezen tip bi bilo zaradi tega potrebno opraviti računsko, po izvrednotenju ocen za lastnosti zunanjosti, pri katerem se v veliki meri izključi vplive okolja. S tem bi dobili bolj pravilne razvrstitve živali v tip. Analiza lastnosti zunanjosti pri plemenskih bikih cikastega goveda je bila narejena kot prvi korak pred oceno fenotipskih in genetskih parametrov, ki so osnova za napoved plemenskih vrednosti. Plemenske vrednosti bi lahko bolje služile kot osnova za selekcijo, saj predstavljajo aditivni genetski vpliv in hkrati omogočajo razvrščanje živali. Tak parameter je s selekcijskega vidika učinkovitejši od razvrstitve v tri tipe na osnovi fenotipskih vrednosti, ki poleg tega, da ne upošteva vplivov okolja, vključuje tudi tiste dele genetske vrednosti, ki se ne dedujejo. Predlagamo, da bi ob ocenjevanju zunanjosti v celoti upoštevali pravila ICAR in zabeležili tehnologijo reje ter ocenili tudi telesno kondicijo živali, kar bi bilo mogoče vključiti kot vpliv v postopku genetskega vrednotenja. Pri ocenjevanju zunanjosti nekaterih pasem goveda v Sloveniji že nekaj let spremljajo telesno kondicijo. 5 VIRI Alberti P., Panea B., Sa-udo C., Olleta J.L., Ripoll G., Ertbjerg P., Christensen M., Gigli S., Failla S., Concetti S., Hocquette J.F., Jailler R., Rudel S., Renand G., Nute G.R., Richardson R.I., Williams J.L. 2008. Live weight, body size and carcass characteristics of young bulls of fifteen European breeds. Livestock Science, 114: 19-30, doi:10.1016/j.livs-ci.2007.04.010 Communod R., Guida S., Vigo D., Beretti V., Munari E., Co-lombani C., Superchi P., Sabbioni A. 2013. Body measures and milk production, milk fat globules granulometry and milk fatty acid content in Cabanina cattle breed. Italian Journal of Animal Science, 12, e18: 107-115, doi:10.4081/ ijas.2013.e18 Dal Zotto R., De Marchi M., Dalvit C., Cassandro M., Gallo L., Carnier P., Bittante G. 2007. Heritabilities and genetic correlations of body condition score and calving interval with yield, somatic cell score, and linear type traits in Brown Swiss cattle. Journal of Dairy Science, 90: 5737-5743, doi:10.3168/jds.2007-0280 Felius M. 1995. Cattle breeds - an encyclopedia. Doetinchem, Misset: 799 str. Gutiérrez J.P., Alvarez I., Fernandez I., Royo L.J., Diez J., Goyache F. 2002. Genetics relationships between calving date, calving interval, age at first calving and type traits in beef cattle. Livestock Production Science, 78: 215-222, doi:10.1016/S0301-6226(02)00100-8 International agreement of recording practices. ICAR Recording Guidelines. 2014. Berlin, ICAR - International Committee for Animal Recording: 619 str. http://www.icar. org/Documents/Rules%20and%20regulations/Guidelines/ Guidelines_2014.pdf (5. feb. 2015) Klopčič M., Hamoen A. 2010. Linearno ocenjevanje krav črno-bele pasme. Domžale, Biotehniška fakulteta, Oddelek za zootehniko: 28 str. Logar B., Podgoršek P., Jeretina J., Ivanović B., Perpar T. 2005. Online - available milk - recording data for efficient support of farm management. V: Knowledge transfer in cattle husbandry. Publication - European Association for Animal Production. Wageningen, Wageningen Academic Publishers: 227-230 Mantovani R., Cassandro M., Contiero B., Albera A., Bittante G. 2010. Genetic evaluation of type traits in hypertrophic Piemontese cows. Journal of Animal Science, 88: 35043512, doi:10.2527/jas.2009-2667 Mazza S., Guzzo N., Sartori C., Berry D.E., Mantovani R. 2014. Genetic parameters for linear type traits in the Rendena dual-purpose breed. Journal of Animal Breeding and Genetics, 131, 1: 27-35, doi:10.1111/jbg.12049 Mazza S., Sartori C., Berry D., Mantovani R. 2013. Factors affecting linear type traits of Valdostana cattle. Agriculturae Conspectus Scientificus, 78, 3: 207-2011 Navodila za presojo barvnih znakov cikastega goveda. 1935. Službeni list kraljevske banske uprave dravske banovine, 6, 28: 212-213 Pogačar J., Potočnik K. 1997. Linearno opisovanje in ocenjevanje zunanjosti krav. Sodobno kmetijstvo, 30, 11: 470-473 Pogačar J., Kunstelj P., Zupančič A., Čeh J. 1995. Linearno opi- sovanje in ocenjevanje krav. Domžale, Govedorejska služba Slovenije, Biotehniška fakulteta, Oddelek za zootehniko: 21 str. Polc F. 1958. Značilnejše telesne mere volov in bikov v Bohinju in primerjava teh mer s kravami. Diplomska naloga, Ljubljana, Fakulteta za agronomijo, gozdarstvo in veterino, Katedra za živinorejo: 39 str. Sambraus H.H. 1999. Gefährdete Nutztierrassen. Ihre Zuchtgeschichte, Nutzung und Bewahrung. Stuttgart, Ulmer: 384 str. SAS Institute Inc. 2001. The SAS System for Windows, Release 8.02. Cary, NC, SAS Institute Inc. Simčič M., Gorjanc G., Čepon M., Dovč P., Kompan D. 2013a. Genetske značilnosti populacije cikastega goveda v Sloveniji. Kmetovalec, 5: 16-18 Simčič M., Lenstra J.A., Baumung R., Dovč P., Čepon M., Kompan D. 2013b. On the origin of Slovenian Cika cattle. Journal of Animal Breeding and Genetics, 130: 487-495, doi:10.1111/jbg.12034 Simčič M., Smetko A., Sölkner J., Seichter D., Gorjanc G., Kompan D., Medugorac I. 2015. Recovery of native genetic background in admixed populations using haplotypes, phe-notypes, and pedigree information - using Cika cattle as a case breed. PloS ONE, 10, 4: e0123253, 20 str., doi:10.1371/ journal.pone.0123253 Žan M., Čepon M., Kompan D. 2005. Rejski program za cikasto govedo: dopolnjen v skladu s pripombami MKGP z dne 05. 01. 2005. Mengeš, Govedorejska zadruga: 92 str. Žan Lotrič M., Čepon M., Kompan D, Klinkon Z., Rokavec R., Jeretina J., Logar B., Mrkun J., Burja A. 2010. Rejski program za cikasto govedo: dopolnjen v skladu s pripombami MKGP z dne 27. 09. 2010. Medvode, Združenje rejcev avtohtonega cikastega goveda v Sloveniji: 83 str. SUBJECT INDEX BY AGROVOC DESCRIPTORS PREDMETNO KAZALO PO DESKRIPTORJIH AGROVOC Tomaž BARTOL 1 animal breeding 77-85 gilts 93-96 animal housing 97-101 horses 77-85, 87-91 animal models 87-91 human pathology 87-91 backfat thickness 93-96 income 77-85 bioinformatics 67-75 land races 103-112 biosensors 67-75 linear programming 77-85 body condition 103-112 litter for animals 97-101 body conformation 103-112 luminescence 67-75 body measurements 103-112 materials 97-101 body measurements 93-96 meat animals 97-101 cattle 103-112 microorganisms 67-75 cell culture 87-91 models 87-91, 93-96, 103-112 chickens 97-101 molecular biology 87-91 cost benefit analysis 77-85 mothers 93-96 economic analysis 77-85 optimization methods 77-85 environmental protection 67-75 pollutants 67-75 fathers 93-96, 103-112 recreation 77-85 broiler chickens 97-101 saddle horses 77-85 feathers 97-101 skin diseases 87-91 foot diseases 97-101 skin 87-91 genetic engineering 67-75 sows 93-96 genetics 67-75, 93-96 swine 93-96 genotype environment interaction 93-96 testing 87-91 genotypes 93-96 tissue culture 87-91 1 Univ. of Ljubljana, Biotechnical Fac, Dept. of Agronomy, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia, e-mail: tomaz.bartol@bf.uni-lj.si SUBJECT INDEX BY AGRIS CATEGORY CODES VSEBINSKO KAZALO PO PREDMETNIH KATEGORIJAH AGRIS Nataša SIARD 1 Animal husbandry - L01 77-85, 93-96, 97-101 Animal genetics and breeding - L10 93-96, 103-112 Animal physiology and biochemistry - L50 87-91 Animal diseases - L73 97-101 Pollution - T01 67-75 Mathematical and statistical methods - U10 77-85 1 Univ. of Ljubljana, Biotechnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia, e-mail: natasa.siard@bf.uni-lj.si ABECEDNO KAZALO AVTORJEV AUTHOR'S INDEX Št. No. Avtor Author Stran primarnega prispevka Page of the primary source 1. BARTOL Tomaž 113 2. DOVČ Peter 87-91 3. KOVAČ Milena 93-96 4. LAPANJA Tjaša 87-91 5. LOGAR Betka 103-112 6. MALOVRH Špela 93-96 7. MARINŠEK-LOGAR Romana 67-75 8. OGOREVC Jernej 87-91 9. PESTOTNIK Mojca 97-101 10. PLANINC Martina 93-96 11. POKLUKAR Klavdija 87-91 12. POTOČNIK Klemen 103-112 13. SIARD Nataša 115 14. SIMČIČ Mojca 103-112 15. ŠTEPEC Miran 103-112 16. TERČIČ Dušan 97-101 17. TOMINŠEK Natalija 87-91 18. VODOVNIK Maša 67-75 19. VOGRINC David 67-75 20. ŽGAJNAR Jaka 77-85 21. ŽOLGER Mirjana 97-101 NAVODILA AVTORJEM PRISPEVKI Sprejemamo izvirne znanstvene članke, predhodne objave in raziskovalne notice s področja zootehni-ke (genetika, mikrobiologija, imunologija, prehrana, fiziologija, ekologija, etologija, mlekarstvo, ekonomika, bioinformatika, živalska proizvodnja in predelava živalskih proizvodov, tehnologija in dokumentalistika) v slovenskem in angleškem jeziku, pregledne znanstvene članke pa samo po poprejšnjem dogovoru. Objavljamo tudi prispevke, podane na simpozijih, ki niso bili v celoti objavljeni v zborniku simpozija. Če je prispevek del diplomskega, magistrskega ali doktorskega dela, navedemo to in tudi mentorja v sprotni opombi na dnu prve strani. Navedbe morajo biti v slovenskem in angleškem jeziku. Pri prispevkih v slovenskem jeziku morajo biti preglednice, grafikoni, slike in priloge dvojezični, povsod je slovenščina na prvem mestu. Naslovi grafikonov in slik so pod njimi. Preglednice, slike in grafikoni so v besedilu. Grafikoni morajo biti črno-beli. Latinske izraze pišemo ležeče. V slovenščini uporabljamo decimalno vejico, v angleščini decimalno piko. Prispevki naj bodo strnjeni, kratki, največ 12 strani, napisani z urejevalnikom besedil in oddani v doc ali rtf formatu (Windows). Izgled strani naj bo čim bolj enostaven; v besedilo ne vstavljajte glave in noge. Pisava v besedilu in preglednicah je Times New Roman, velikost črk 12, v obsežnih preglednicah je lahko 10, pisava v grafikonih in slikah je Ariel, velikost črk najmanj 8, pisava za primerjave nukleotidnih in aminokislinskih zaporedij je Courier; zunanji rob 2,0 cm, notranji 2,5 cm. PRVA STRAN Na prvi strani prispevka na desni strani označimo vrsto prispevka, sledi naslov prispevka, pod njim avtorji. Ime avtorjev navedemo v polni obliki (ime in priimek). Vsakemu avtorju dodamo sprotno opombo, ki je vidna na dnu strani, in vsebuje polni naslov ustanove ter znanstveni in akademski naslov; vse v jeziku prispevka. Navedemo sedež ustanove, kjer avtor dela. Če je raziskava opravljena drugje, avtor navede tudi sedež te inštitucije. Na željo avtorjev bomo navedli naslov elektronske pošte. Pod imeni avtorjev je datum prispetja in datum sprejetja prispevka, ki ostaneta odprta. Sledi razumljiv in poveden izvleček v enem odstavku (skupaj s presledki do 1400 znakov). Vsebuje namen in metode dela, rezultate, razpravo in sklepe. Sledijo ključne besede. Izvlečku v jeziku objave sledi naslov in izvleček s ključnimi besedami v drugem jeziku. VIRI V besedilu navajamo v oklepaju avtorja in leto objave: (priimek, leto). Če sta avtorja dva, pišemo: (priimek in priimek, leto), če je avtorjev več, pišemo: (priimek in sod., leto). Sekundarni vir označimo z »navedeno v« ali »cv.«. Seznam virov je na koncu prispevka, neoštevilčen in v abecednem redu. Vire istega avtorja, objavljene v istem letu, razvrstimo kronološko z a, b, c. Primer: 1997a. Nekaj primerov navajanja virov: Vodovnik M., Marinšek-Logar R. 2008. Način delovanja in učinki probiotikov v prehrani živali. Acta agriculturae Slo-venica, 92, 1: 5-17 Fraser A.F., Broom D.M. 1990. Farm animal behaviour and welfare. London, Bailliere Tindall: 437 str. Hvelplund T. 1989. Protein evaluation of treated straws. V: Evaluation of straws in ruminant feeding. Chenost M., Reiniger, A. (ur.). London, Elsevier Applied Science: 66-74 Žgajnar J., Kermauner A., Kavčič S. 2007. Model za ocenjevanje prehranskih potreb prežvekovalcev in optimiranje krmnih obrokov. V: Slovensko kmetijstvo in podeželje v Evropi, ki se širi in spreminja. 4. konferenca DAES, Ljubljana, 8.-9. sep. 2007. Kavčič S. (ur.). Domžale, Društvo agrarnih ekonomistov Slovenije: 279-288 ISO 5534 / IDF 4. Cheese and processed cheese - Determination of the total solids content - Reference method. 2004: 1-7 Frajman P., Dovč P. 2004. Milk production in the post-genomic era. Acta agriculturae Slovenica, 84, 2: 109-119. http://aas.bf.uni-lj.si/zootehnika/84-2004/PDF/84-2004-2-109-119.pdf (15. mar. 2009) Prispevke recenziramo in lektoriramo. Praviloma pošljemo mnenje prvemu avtorju, po želji lahko tudi drugače. Če urednik ali recenzenti predlagajo spremembe oz. izboljšave, vrne avtor popravljeno besedilo v 10 dneh v natisnjenem in elektronskem izvodu. Ko prvi avtor vnese še lektorjeve pripombe, odda popravljeno besedilo v natisnjenem in elektronskem izvodu. Pri oddaji končne verzije avtor priloži jasno označene izvirnike slik (ločene grafične datoteke ali fotografije). Datoteke slik poimenuje enako kot v tekstu (npr. Slika1. jpg, Slika2.eps, Slika3.bmp ...). Originalne fotografije na avtorjevo željo vrnemo. Vektorske slike sprejemamo samo v eps (Encapsulated Postscript) formatu, s tekstom, ki je spremenjen v krivulje. Rasterske slike morajo biti v enem od običajnih formatov (npr. tiff, jpg, bmp). Ločljivost naj bo vsaj 300 dpi. Prispevke sprejemamo vse leto. ODDAJA Avtorji prispevke oddajo v natisnjenem in elektronskem izvodu. Priložijo tudi izjavo s podpisi vseh avtorjev, da avtorske pravice v celoti odstopajo reviji. NOTES FOR AUTHORS PAPERS We publish original scientific papers, preliminary communications and research statements on the subject of animal science (genetics, microbiology, immunology, nutrition, physiology, ecology, ethology, dairy science, economics, bioinformatics, animal production and food processing, technology and information science) in Slovenian and English languages while scientific reviews are published only upon invitation. Reports presented on conferences that were not published entirely in the conference reports can be published. If the paper is part of BSc, MSc or PhD thesis, this should be indicated together with the name of the mentor at the bottom of the front page and will appear as foot note. All notes should be written in Slovenian and English language. Papers in Slovenian language should have tables, graphs, figures and appendices in both languages, Slovenian language being the first. Titles of graphs and figures are below them. Figures and graphs are part of the text. Clearly marked original figures should be added (photographs or separate graphic files); they can be returned upon request. Latin expressions are written in italics. Decimal comma is used in Slovenian and decimal point in English. The papers should be condensed, short and should not exceed 12 pages, edited with word processor and submitted as doc or rtf file (Windows). Text formatting should be as simple as possible, without headers and footers. Font Times New Roman, size 12 should be used for text and tables (in large tables size 10 is allowed), Ariel should be used for graphs and figures (letter size at least 8) and Courier for nucleic- and amino acid sequence alignments. Right margin is 2.0 cm, left margin 2.5 cm. FIRST PAGE The type of the paper should be indicated on the first page on the right side following by the title of the paper and authors. Full names of the authors are used (first name and surname). Each name of the author should have been added an index, which is put immediately after the author's name and displayed in the footnote. It contains address of the institution and academic degree of the author, in the language of the paper. The address of the institution in which the author works is indicated. If the research was realised elsewhere, the author should name the headquarters of the institution. E-mail is optional. Under the address of the authors some space for dates of arrival and acceptance for publishing should be left. A comprehensive and explicit abstract in one paragraph (up to 1400 characters, including spaces) follows indicating the objective and methods of work, results, discussion and conclusions. Key words follow the abstract. The abstract in the language of the paper is followed by the title, abstract and key words in the alternative language. REFERENCES References should be indicated in the text by giving author's name, with the year of publication in parentheses, e.g. (surname, year). If there are two authors, the following form is used: (surname and surname, year). If there are more than two authors, we use (surname et al., year). Secondary sources should be quoted in the form "cited in". The references should be listed at the end of the paper in the alphabetical order and not numbered. If several papers by the same author and from the year are cited, a, b, c, etc. should be put after the year of the publication: e.g. 1997a. Some examples: Simončič M., Horvat S., Stevenson P.L., Bünger L., Holmes M.C., Kenyon C.J., Speakman J.R., Morton N.M. 2008. Divergent physical activity and novel alternative responses to high fat feeding in polygenic fat and lean mice. Behavior Genetics, 38, 3: 292-300 Fraser A.F., Broom D.M. 1990. Farm animal behaviour and welfare. London, Bailliere Tindall: 437 p. Hvelplund T. 1989. Protein evaluation of treated straws. In: Evaluation of straws in ruminant feeding. Chenost M., Reiniger, A. (eds.). London, Elsevier Applied Science: 66-74 Žgajnar J., Kermauner A., Kavčič S. 2007. Model za ocenjevanje prehranskih potreb prežvekovalcev in optimiranje krmnih obrokov. In: Slovensko kmetijstvo in podeželje v Evropi, ki se širi in spreminja. 4. konferenca DAES, Ljubljana, 8.-9. sep. 2007. Kavčič S. (ed.). Domžale, Društvo agrarnih ekonomistov Slovenije: 279-288 ISO 5534 / IDF 4. Cheese and processed cheese - Determination of the total solids content - Reference method. 2004: 1-7 Frajman P., Dovč P. 2004. Milk production in the post-genomic era. Acta agriculturae Slovenica, 84, 2: 109-119. http://aas.bf.uni-lj.si/zootehnika/84-2004/PDF/84-2004-2-109-119.pdf (15. mar. 2009) DELIVERY Papers should be delivered as a printed and electronic copy. A statement signed by all authors transfers copy rights on the published article to the Journal. Papers are reviewed and edited. First author receives a review if not defined otherwise. If reviewers suggest some corrections, the author should forward them within 10 days in printed and electronic form. After the first author considers the referee's notes, the corrected paper should be sent in printed and electronic form to the Editor. Submission of the final version must contain properly labelled original figures (separate files or photographs). The figure files should be labelled as they appear in the text (Figure1.jpg, Figure2.eps, Figure3.bmp ...). Original photographs can be returned to the author upon request. Vector graphics have to be in eps (Encapsulated Postscript) format with the text transformed in curves. Raster figures and photos should be in one of common formats (e.g. tiff, jpg, bmp) with at least 300 dpi resolutions. Papers are accepted all the year. Biotehniška fakulteta Univerze v Ljubljani Biotechnical Faculty University of Ljubljana ISSN 1581-9175 9 Spletna izdaja - Online: ISSN 1854-1941 Acta agriculturae Slovenica ISSN 1581-9175 • letnik / Volume 106 • številka / Number 2 • 2015 VSEBINA / CONTENTS David Vogrinc, Maša Vodovnik, Romana Marinšek-Logar, Microbial biosensors for environmental monitoring / Mikrobni biosenzorji za monitoring okolja...................................................................67 Jaka Zgajnar, Ekonomska analiza organiziranosti jahalnega centra; primer uporabe linearnega programiranja / Economic analysis of the organisation of a riding centre; application of the linear programming approach ...........................................................................................................77 Jernej Ogorevc, Tjaša Lapanja, Klavdija Poklukar, Natalija Tominšek, Peter Dovč, Establishment of primary keratinocyte culture from horse tissue biopsates / Vzpostavitev primarne celične kulture konjskih keratinocitov iz biopsij ............................................................................................87 Martina Planinc, Milena Kovač, Špela Malovrh, Analysis of backfat thickness in on-farm tested gilts in Slovenia using reaction norms / Reakcijske norme za debelino hrbtne slanine pri testiranih mladicah v Sloveniji ...........................................................................................................93 Dušan Terčič, Mirjana Žolger, Mojca Pestotnik, Effect of different litter materials on foot pad dermatitis, hock burn and feather coverage in broiler chickens / Vpliv različnih vrst nastila na pojav vnetja kože na blazinicah stopal, vnetja kože skočnih sklepov ter na operjenost pri pitovnih piščancih .............................................................................................................................97 Mojca Simčič, Miran Štepec, Betka Logar, Klemen Potočnik, Analiza lastnosti zunanjosti pri plemenskih bikih cikastega goveda / Analysis of type traits of cika sires ........................................... 103 Tomaž Bartol, Subject index by Agrovoc descriptors / Predmetno kazalo po deskriptorjih Agrovoc...........................................................................................................................................113 Nataša Siard, Subject index by Agris category codes / Vsebinsko kazalo po predmetnih kategorijah Agris ............................................................................................................................. 115 Abecedno kazalo avtorjev / Author's index.......................................................................................... 117 Navodila avtorjem ..............................................................................................................................119 Notes for authors................................................................................................................................ 121 9771581917001