Biotehniška fakulteta Univerze v Ljubljani Biotechnical Faculty University of Ljubljana AACTA AGRICULTURAE SLOVENICA94•22009 Acta agriculturae Slovenica • ISSN 1581-9175 • 94 – 2 • Ljubljana, december 2009 Acta agriculturae Slovenica Volume / Letnik 94 · Number / Številka 2 · 2009 VSEBINA / CONTENTS Jože OSTERC 85 Prof. dr. Jože FERČEJ (1917–2009) In Memoriam Prof. Dr. Jože FERČEJ (1917–2009) PREHRANA / NUTRITION Gregor TORKAR, Maja KOROŠEC, Stojan KOSTANJEVEC, Alenka POLAK, Verena KOCH 87 Odpor do živil med slovenskimi osnovnošolci Food dislikes among Slovenian schoolchildren Tamara FRANKIČ, Mojca VOLJČ, Janez SALOBIR, Vida REZAR 95 Use of herbs and spices and their extracts in animal nutrition Uporaba zelišč in začimb ter njihovih ekstraktov v prehrani živali Tanja PAJK ŽONTAR, Janez SALOBIR, Vida REZAR 103 Time dependent formation of markers of oxidative stress induced by a high fat diet supplemented or unsupplemented with vitamin E in pigs Časovna odvisnost tvorbe kazalcev oksidacijskega stresa pri prašičih, povzročenega s prehrano, obogateno z maščobami ter z ali brez dodatka vitamina E Tatjana PIRMAN, Philippe PATUREAU MIRAND, Andrej OREŠNIK, Janez SALOBIR 111 Effects of dietary pectin on protein digestion and metabolism in growing rats Vpliv pektina na prebavo beljakovin in metabolizem pri rastočih podganah GENETIKA / GENETICS Tomaž VOLČIČ, Drago KOMPAN, Peter DOVČ, Simon HORVAT 121 Uporaba DNA označevalcev za preverjanje porekla pri ovcah in določanja očetovstva v čredah z več plemenskimi ovni Molecular genetics markers used for parentage verification and paternity determination in multiple-sire sheep pedigrees Jernej OGOREVC, Sonja PRPAR, Peter DOVČ 133 In vitro mammary gland model: establishment and characterization of a caprine mammary epithelial cell line In vitro model mlečne žleze: vzpostavitev in določitev značilnosti epitelne celične linije iz kozje mlečne žleze MIKROBIOLOGIJA / MICROBIOLOGY Blaž STRES, Boštjan MUROVEC 139 New primer combinations with comparable melting temperatures detecting highest numbers of nosZ sequences from sequence databases Nove kombinacije začetnih oligonukleotidov s primerljivimi temperaturami taljenja zaznavajo najvišje število sekvenc nosZ v podatkovnih bazah Tomaž ACCETTO 143 Isolation and use of Prevotella ruminicola TC18 plasmid pTC18 in Escherichia coli-P. ruminicola shuttle vector construction Osamitev plazmida pTC18 seva Prevotella ruminicola TC18 in njegova uporaba v razvoju prenosljivih vektorjev Escherichia coli-P. ruminicola Gregor GORENC, Tomaž ACCETTO, Gorazd AVGUŠTIN 147 The search for conjugative transposon in rumen bacterium Prevotella bryantii B14 Iskanje konjugativnega transpozona v vampni bakteriji Prevotella bryantii B14 ŽIVALSKA PROIZVODNJA / ANIMAL PRODUCTION Abdulmojeed YAKUBU, Jafaru Ari GWASKA, Adebowale Emmanuel SALAKO 153 Strain and placement density effects on welfare, haematological and serum biochemical indices of broilers in north central Nigeria Vpliv genotipa in gostote naselitve na počutje, serumske in biokemijske parametre pri brojlerjih v severni in osrednji Nigeriji Matej VIDRIH, Anton VIDRIH, Milan POGAČNIK, Drago KOMPAN 159 Minerals management in silvopastoral system of karst pasture Drevesno pašna raba in rudnine na kraškem pašniku EKOLOGIJA / ECOLOGY Rajko BERNIK, Aleš ZVER 167 Introduction pilot biogas reactors and application to define biogas potential of basic substrat, swine slurry Predstavitev pilotnega reaktorja in aplikativna določitev bioplinskega potenciala osnovenga substrata, prašičje gnojevke Tomaž BARTOL 173 Subject index by Agrovoc descriptors Predmetno kazalo po deskriptorjih Agrovoc Nataša SIARD 175 Subject index by Agris category codes Vsebinsko kazalo po predmetnih kategorijah Agris 177 Abecedno kazalo avtorjev Author’s index 179 Navodila avtorjem 181 Notes for authors Acta argiculturae Slovenica, 94/2, 85 –86, Ljubljana 2009 PROF. DR. JOŽE FERČEJ (1917–2009) Štirinajstega oktobra smo se na ljubljanskih Žalah poslovili od staroste slovenskih govedorejcev, profesorja dr. Jožeta Ferčeja. Profesor Ferčej je bil zadnji iz skupine strokovnjakov, ki so v Sloveniji po drugi svetovni vojni izvedli pasemsko rajonizacijo in razširili umetno osem- enjevanje krav. Rodil se je 28. februarja 1917 na Blejski Dobravi. Gi- mnazijo je končal v Kranju, študij kmetijstva pa 1941, dva dni pred napadom na Jugoslavijo, na Kmetijsko – goz- darski fakulteti v Beogradu. Tam je leta 1941 diplomiral in 1963 tudi doktoriral s področja selekcije rjave pasme v Sloveniji. Po vojni je bil referent za živinorejo v Celju in Mariboru ter na Ministrstvu za kmetijstvo. Bil je tudi tajnik Zveze živinorejskih zadrug za sivo-rjavo govedo in tajnik zveze živinorejskih selekcijskih zadrug. Od leta 1953 do leta 1974 je bil znanstveni sodelavec, znanstveni svetnik in predstojnik Zavoda za živinorejo na Kmetij- skem inštitutu Slovenije. V letih 1955 in 1956 je bil pol leta na izpopolnjevanju v Kopenhagnu na Danskem. Od leta 1974 in do upokojitve v letu 1987 je bil redni profe- sor za govedorejo na Oddelku za živinorejo, Biotehniške fakultete v Ljubljani. V letih 1971 in 1972 je bil profesor Ferčej predsednik Jugoslovanske skupnosti znanstveno- raziskovalnih organizacij za področje živinorejskih ved. Večkrat je bil tudi delegat Jugoslavije na sestankih in kon- ferencah Evropske zootehnične federacije. Intenzivno je deloval v Evropskem združenju rejcev rjavega goveda, v dveh obdobjih je bil tudi predsednik tega združenja in je organiziral konferenci združenja v Sloveniji. Za realizacijo razvojnih programov na področju govedoreje se je znal povezati z govedorejskimi strokov- njaki po Sloveniji, poskrbel pa je tudi za razvoj mladih sodelavcev, Janeza Pogačarja in Slavka Čepina, ki sta ka- sneje doktorirala in postala univerzitetna profesorja. S skrbjo za mlajše sodelavce je nadaljeval tudi na Bioteh- niški fakulteti. V Sloveniji bi težko našli govedorejca, ki se s profesorjem ni kdaj srečal. Dobre, prijateljske stike je znal navezovati tudi z naprednimi kmeti. Aktiven je ostal tudi po upokojitvi leta 1987. Izjemno plodno delo profesorja ni ostalo neopaže- Acta agriculturae Slovenica, 94/2 – 200986 J. OSTERC no v široki javnosti. Prejel je številna domača in medna- rodna odlikovanja, priznanja in nagrade, med njimi Red dela z zlatim vencem (1968), Red zaslug za narod z zlato zvezdo (1987), Priznanje švicarske zveze rejcev rjavega goveda (1969), Jesenkovo priznanje Biotehniške fakul- tete (1977), Nagrado Sklada Borisa Kidriča za tehnično izboljšavo – s sodelavci (1981), Kidričevo nagrado za ži- vljenjsko delo v govedoreji (1987). Profesor je bil ploden pisec. Seznam bibliografskih navedb od leta 1947 presega 450 enot. Slovenski gove- dorejci, kmetijski strokovnjaki in prijatelji smo prof. dr Jožetu Ferčeju hvaležni za vse kar je naredil in napisal za slovenskega kmeta, kmetijstvo in zlasti za govedorejo. S svojim delom si je postavil trajen spomenik. In Memoriam Prof. Dr. Jože FERČEJ (1917–2009) Professor Jože Frečej, the doyen of Slovenian cat- tle breeders was buried on Ljubljana cemetery Žale on October 14. Professor Ferčej belonged to the group of experts who after the Second World War introduced ar- tificial insemination and regionalisation of cattle breeds to Slovenia. He was born on February 28 1917 at Blejska Do- brava, finished high school in Kranj and university study at the Faculty for Agriculture and Forestry in Belgrade in 1941, two days before German air force attacked Yugosla- via. From the same Faculty he received his PhD in 1963. After the Second World War, he was employed as an expert for cattle breeding in Celje, Maribor and at the Slovenian Ministry for agriculture. He was also secretary of the Society for brown cattle breeding and secretary of the Society for cattle selection. Between 1955 and 1974 he was researcher and head of the unit for animal breed- ing at the Agricultural institute of Slovenia. In the years 1955 and 1956, he spent six months as a guest researcher in Copenhagen, Denmark. From 1974 to his retirement in 1987 he vas full professor for cattle breeding at the Department of Animal Science at Biotechnical Faculty, University of Ljubljana. In the years 1971 and 1972 was professor Ferčej president of the Yugoslav zootecnical research community. He represented Yugoslavia several times at the meetings of the European brown cattle As- sociation, was two times its president and organized two annual meetings of the Association in Slovenia. He developed vivid contacts to researchers and experts in the field of cattle breeding in Slovenia as well as abroad. He was mentor of several graduate students, among tem also Janez Pogačar and Slavko Čepin, who both later became university professors. Professor Ferčej remained active also after his retirement in 1987. For his professional and research work he received numerous awards, among them “Red dela z zlatim vencem” (1968), “Red zaslug za narod z zlato zvezdo” (1987), Award of the Swiss Brown Cattle Breeder Association (1969), Jesenko Award of the Biotechnical Faculty (1977), Award of the Boris Kidrič Foundation for technical innovation – with co-workers (1981) and Kidrič award for his lifework in the field of cattle breeding (1987). Professor Ferčej was a fruitful writer. His entire bib- liography contains more than 450 units and Slovenian cattle breeders, experts, farmers and friends appreciate his contribution to the development of Slovenian agricul- ture. With his work he will remain with us. Prof. dr. Jože Osterc Acta argiculturae Slovenica, 94/2, 87–93, Ljubljana 2009 COBISS: 1.01 Agris category code: S40 ODPOR DO ŽIVIL MED SLOVENSKIMI OSNOVNOŠOLCI Gregor TORKAR 1, Maja KOROŠEC 2, Stojan KOSTANJEVEC 3, Alenka POLAK 4, Verena KOCH 4 Delo je prispelo 16. septembra 2008, sprejeto 15. septembra 2009. Received September 16, 2008; accepted September 15, 2009. 1 Tacenska cesta 135 a, SI-1133 Ljubljana, Slovenija, dr., e-pošta: gregor.torkar@guest.arnes.si 2 Ljubljanska 80 a, SI-3000 Celje, Slovenija 3 Univ. v Ljubljani, Pedagoška fak., Kardeljeva ploščad 16, SI-1000 Ljubljana, Slovenija, mag. 4 Isti naslov kot 3, doc. dr. Odpor do živil med slovenskimi osnovnošolci Članek predstavlja analizo vzrokov za odpor do živil med slovenskimi osnovnošolci, starimi od 10 to 15 let, glede na spol, razred in kraj bivanja. V anketni raziskavi je sodelova- lo 628 osnovnošolcev iz 16 naključno izbranih šol v Sloveniji. Ugotovili smo, da osnovnošolci največ odklanjajo živila zaradi senzoričnih lastnosti, kot sta okus in vonj. Med dejavniki oko- lja imajo na odklanjanje živil največji vpliv prehranske navade v družini. Anketirana dekleta so bolj izbirčna kot fantje. Manj odklonilen odnos imajo le do zelenjave. Starejši učenci bolj od- klanjajo živila živalskega izvora, predvsem notranjih organov (npr. možgani, jetrca, vampi), kot njihovi mlajši kolegi. Razlike med osnovnošolci iz zahodne in vzhodne Slovenije se kažejo v odporu do mesa mehkužcev in dvoživk. Osnovnošolci v zaho- dni Sloveniji imajo manjši odpor do tovrstne hrane. Ključne besede: prehrana ljudi / osnovnošolci / odpor do hrane / prehranske navade / Slovenija Food dislikes among Slovenian schoolchildren The article presents analysis of reasons for food dislikes among Slovenian primary schoolchildren aged 10 to 15, by gender, age and permanent residence. Altogether 628 primary school children from 16 randomly chosen schools in Slovenia filled the questionnaire. We found out that the most influen- tial origins of food dislikes among schoolchildren were sensory characteristics, especially taste and smell. Among environmen- tal factors were the most influential eating habits in families. Girls were more particular about their food then boys, except when it comes to vegetables. The study revealed that older schoolchildren disliked more organ meat than their younger colleagues. Differences among schoolchildren from western and eastern Slovenia were significant in dislikes toward meat from molluscs and amphibians. Schoolchildren from western Slovenia were less rejectable toward this kind of food. Key words: human nutrition / primary school children / food dislikes / eating habits / Slovenia 1 UVOD Pravilna prehrana in redna telesna dejavnost sta ključna dejavnika za optimalno rast in razvoj mlado- stnika (Reinhardt in Brevard, 2002; Story in Neumark- Sztainer, 1999). Naklonjenost oziroma odpor do hrane pomembno vplivata na prehranske navade, še posebej v otroštvu (Drewnowski in Hann, 1999; Wardle in Cooke, 2008). Prehranske navade in vedenje so odraz številnih dejavnikov, ki jih lahko v grobem delimo na genetske in okoljske (Wardle in Cooke, 2008). Med genetskimi dejavniki, ki so predvsem odvisni od naše dedne zasnove, gre izpostaviti tri. Na prvem mestu je brez dvoma okus. Raziskovalci ugotavljajo, da imajo otroci in odrasli raje sladke in slane okuse (Beau- champ in Moran, 1982; Destor in sod., 1977) kot pa kisle ali grenke (Destor in sod., 1975; Steiner, 1979). Naklo- njenost do nekaterih okusov najverjetneje kaže človeko- vo evolucijsko prilagajanje na s sladkorjem in energijo bogata živila ter odklanjanje kisle in grenke hrane, ki bi lahko bila okužena z nevarnimi bakterijami ali toksini (Wardle in Cooke, 2008). Kot vse kaže je dedno pogoje- na tudi želja po uživanju gostih, visoko energijskih živil (Birch, 1992; Gibson in Wardle, 2003). Genetski dejavnik Acta agriculturae Slovenica, 94/2 – 200988 G. TORKAR in sod. naj bi bil, vsaj deloma, tudi izbirčnost in neofobija (strah oziroma odklanjanje nove, nepoznane hrane). Neofobija naj bi negativno vplivala na sam vnos zelenjave, sadja in mesnin ter skupen vnos energije z živili (Cooke, 2004; Cooke in sod., 2006), nima pa vpliva na količino zaužitih mlečnih izdelkov in škrobnih živil (Cooke in sod., 2003). Rozin in Fallon (1980), Rozin (1991) ter Letarte in sod. (1997) ugotavljajo, da so meso in mesni izdelki najpo- gosteje omenjena skupina živil, ki je ljudje ne marajo. Najbolj pogosto odklanjajo meso organov, medtem ko sta perutnina in govedina najbolj priljubljeni vrsti mesa (Letarte in sod., 1997). Odpor do mesa in mesnih izdel- kov je pogostejši pri ženskah (Kubberod in sod., 2002; Letarte in sod., 1997). Otroci v procesu interakcije z okoljem razvijajo svo- jo naklonjenost do živil; izpostavljeni so novim živilom, teksturam, okusom in vonjem (Birch, 1999). Med sen- zoričnimi lastnostmi ponujenih živil je za sprejemanje pomemben videz in barva živila oziroma jedi (Cardelo, 1996). Učijo se doma, v šoli in pri prijateljih (Skinner in sod., 1998). Zelo pomemben okoljski dejavnik je družin- sko okolje, še posebej prehranske navade mater (Scaglio- ni et al., 2008). Starši oblikujejo otrokove prehranske na- vade in vedenja s svojim lastnim zgledom in izbiro živil, ki jim jih ponujajo (Cutting et al., 1999), kot tudi z ve- denjem in načinom hranjenja otroka (Birch et al., 2001; Johnson in Birch, 1994). Nezanemarljivi dejavniki okolja so tudi vpliv medijev (Byrd-Bredbener in Grasso, 2000), prehransko izobraževanje v šoli in šolska prehrana (Neu- mark-Sztainer et al., 1999, Skinner et al., 2002). V raziskavi smo obravnavali nenaklonjenost do ži- vil pri slovenskih osnovnošolcih, starih med 10 in 15 let. Zanimalo nas je, kateri so najpomembnejši dejavniki, ki vplivajo na nenaklonjenost ter kakšne so razlike v ne- naklonjenosti do živil glede na spol in razred, ki ga obi- skujejo. Želeli smo tudi ugotoviti, ali so opazne razlike v odporu do hrane med učenci iz vzhodnega in zahodnega dela Slovenije. 2 MATERRIAL IN METODE 2.1 VZOREC Anketni vprašalnik je izpolnilo 628 učencev iz 16-ih naključno izbranih osnovnih šol iz različnih krajev Slo- venije (Murska Sobota, Maribor, Novo mesto, Ljubljana, Kranj, Nova Gorica, Celje, Veržej, Slovenska Bistrica, Dolenjske Toplice, Dol pri Ljubljani, Bled, Ajdovščina, Štore, Marezige, Mirna). Podatki o spolu in starosti an- ketiranih učencev so v preglednicah 1 in 2. 2.2 OPIS INSTRUMENTA Anketni vprašalnik smo sestavili na podlagi rezul- tatov preizkusnega vprašalnika. V preizkusnem vprašal- niku so bila navedena živila, za katere smo na podlagi poklicnih izkušenj predvidevali, da jih učenci odklanjajo. V anketni vprašalnik smo vključili 20 živil, za katere so učenci v preizkusnem vprašalniku najpogosteje označili, da jih ne marajo. Za oceno odpora do živil smo uporabili Likertovo petstopenjsko lestvico (Likert, 1932). V nada- ljevanju so nas zanimali vzroki, zakaj ne jedo naštetih živil. Izbirali so lahko med 15-imi navedenimi vzroki, ki smo jih prav tako oblikovali na osnovi rezultatov preiz- kusnega vprašalnika. Anketirani so imeli na voljo tudi odgovor drugo, kjer so lahko dodali vzrok, ki ni bil na- veden. Od anketiranih smo pridobili podatek o starosti, spolu, razredu in kraju šolanja. Anketirani so podali tudi svoje podatke o višini in teži. 2.3 POSTOPEK IZVAJANJA ANKETE Anketni vprašalnik smo preizkusili na skupnem vzorcu 80-tih učencev. Na osnovi rezultatov smo obliko- vali končni vprašalnik in ga poslali po pošti na 16 na- ključno izbranih slovenskih osnovnih šol. V navodilih za izvajalce anket na osnovnih šolah je bilo navedeno, da naj anketo izvedejo v enem šestem razredu in v enem devetem razredu. Do razlik v številu anketirancev med kraji šolanja je prišlo zaradi različnega števila učencev na posamezni šoli in posledično različnega števila učencev v razredih na šolah. Izpolnjene anketne vprašalnike smo po pošti prejeli z vseh 16-ih osnovnih šol. Spol Frekvenca Delež Fantje 298 47,5 Dekleta 330 52,5 Preglednica 1: Spol Table 1: Gender Starost Frekvenca Delež Kumulativni delež 10 32 5,1 5,1 11 277 44,1 49,2 12 18 2,9 52,1 13 109 17,4 69,4 14 185 29,5 98,9 15 7 1,1 100,0 Preglednica 2: Starost Table 2: Age Acta agriculturae Slovenica, 94/2 – 2009 89 ODPOR DO ŽIVIL MED SLOVENSKIMI OSNOVNOŠOLCI 2.4 OBDELAVA PODATKOV Podatki vprašalnika so bili obdelani na nivoju de- skriptivne in inferenčne statistike. Pri tem smo uporabili frekvenčno distribucijo spremenljivk, osnovno deskrip- tivno statistiko spremenljivk (mere srednje vrednosti, mere razpršenosti), Levene preskus homogenosti varianc (F-preskus), t-preizkus za neodvisne vzorce, faktorsko analizo (z varimax rotacijo). Podatki in rezultati so pred- stavljeni v preglednicah in z grafi. 3 REZULTATI IN DISKUSIJA 3.1 NENAKLONJENOST / ODKLONILEN ODNOS DO ŽIVIL IN VZROKI Za 20 vrst živil, ki so jih osnovnošolci v preizku- snem vprašalniku najpogosteje označili, da jih ne marajo, so anketirani podali svojo oceno na petstopenjski lestvici (ocena 1 pomeni maksimalno nenaklonjenost; ocena 5 pomeni maksimalno naklonjenost do živila). Iz pregle- dnice 3 je razvidno, da anketirani osnovnošolci ocenju- jejo z največjim odporom polže, možgane, žabje krake in vampe. Anketirani so si najbolj enotni glede odpora do polžev (SD = 0,821) in možganov (SD = 0,858), ki, glede na njihove ocene, veljata za najbolj nepriljubljeni živili. Med navedenimi živili anketirani najmanj odklo- nilno ocenjujejo rozine, bučke, ocvirke, krvavice, školjke, vampe in kozje mleko. Z ozirom na povprečne vrednosti za posamezne spremenljivke (živila) in interval zaupanja lahko ugotovimo, da anketirani, razen v primeru bučk in rozin, večinoma odklanjajo navedena živila. S faktorsko analizo (z varimax rotacijo) smo izraču- nali manjše število linearnih kombinacij merjenih spre- menljivk (pregl. 4) z lastno vrednostjo (ang. eigenvalue) večjo od 1,5. Tri živila (olive, rozine, kozje mleko), kate- rih vrednosti so bile manjše od 0,35, so bila izločena iz nadaljnje obravnave (Anastasi, 1996). Izračunani faktorji so “odpor do zelenjave” (α = 0,79) s šestimi spremenljiv- kami, “odpor do mesa, predvsem notranjih organov” (α = 0,68) s šestimi spremenljivkami in “odpor do mesa meh- kužcev ali dvoživk” (α = 0,68) s štirimi spremenljivkami. Ena spremenljivka (račje meso) je bila izločena iz nadalj- nje obravnave, ker so bile njene vrednosti večje od 0,35 v več kot enem od treh izračunanih faktorjev (Palaigeor- Povpr. SD Standardna napaka Brokoli 2,289 1,421 0,056 Cvetača 2,571 1,510 0,061 Ohrovt 2,065 1,325 0,052 Olive 2,412 1,578 0,062 Por 2,420 1,463 0,058 Bučke 2,998 1,588 0,063 Jajčevci 2,421 1,535 0,061 Rozine 3,496 1,511 0,063 Krvavice 2,761 1,535 0,063 Ocvirki 2,847 1,589 0,063 Kunčje meso 2,377 1,576 0,062 Račje meso 2,590 1,605 0,064 Polž 1,221 0,820 0,033 Kraki 1,399 1,055 0,042 Vampi 1,757 1,350 0,053 Možgani 1,226 0,858 0,034 Jetra 2,382 1,601 0,063 Školjke 2,761 1,771 0,071 Hobotnica 2,507 1,681 0,067 Kozje mleko 2,746 1,531 0,061 Preglednica 3: Odpor do hrane pri slovenskih osnovnošolcih (N = 628) Table 3: Food dislike among Slovenian schoolchildren (N=628) Odpor do zelenjave Odpor do mesa, predvsem notra- njih organov Odpor do mesa mehkužcev in dvoživk Bbrokoli 0,819 0,037 0,013 Cvetača 0,783 0,045 −0,031 Ohrovt 0,735 0,052 −0,032 Por 0,603 0,100 0,128 Bučke 0,659 0,051 0,141 Jajčevci 0,538 0,056 0,262 Krvavice 0,011 0,719 −0,113 Ocvirki 0,031 0,651 0,013 Kunčje meso 0,122 0,552 0,274 Račje meso 0,088 0,488 0,388 Vampi 0,075 0,570 0,211 Možgani −0,116 0,379 0,158 Jetrca 0,085 0,616 0,132 Polži 0,012 0,108 0,718 Žabji kraki −0,040 0,152 0,737 Školjke 0,284 0,091 0,654 Hobotnica 0,220 0,146 0,653 Lastna vrednost 4,314 2,413 1,524 Preglednica 4: Faktorska analiza (z varimax rotacijo) Table 4: Factorial analysis (using varimax rotation) Acta agriculturae Slovenica, 94/2 – 200990 G. TORKAR in sod. vali, ki jo čutijo anketirani osnovnošolci. Jedi, kot so npr. vampi, možgani, žabji kraki, polži in krvavice, anketira- ni odklanjajo tudi zaradi gnusa. Odklanjanje živil zaradi nepoznavanja le teh, kar v strokovni literaturi imenujejo neofobija (Cooke, 2004; Cooke in sod., 2006), lahko opa- zimo v odporu anketiranih osnovnošolcev do živil, kot so npr. možgani, žabji kraki in polži. Iz slike 1 je razvidno, da sta med slovenskimi osnov- nošolci okus in vonj daleč najpomembnejša vzroka za odklonilen odnos do zelenjave, kar je najverjetneje po- sledica dovzetnosti/naklonjenosti otrok in odraslih za sladke in slane okuse ter odpor do kislih in grenkih oku- sov. Pri odporu do mesa, predvsem notranjih organov, so vzroki bolj enakomerno razporejeni (slika 2). Poleg vonja in okusa pomembno vplivata na odpor tudi videz in bar- va. Med drugimi vzroki (17 %) anketirani omenjajo ve- getarijanstvo, ime jedi, nepoznavanje jedi, verske razloge, ljubezen do živali itd. Tudi v odporu do mesa mehkuž- cev in dvoživk so vzroki raznoliki (slika 3). Poleg okusa, vonja, videza in barve živila anketirani odklanjajo živila tudi zato, ker jih ne pripravljajo doma, se jim žival (jed) gnusi oziroma so sluzaste. Med drugimi vzroki (18  %) pogosteje omenjajo nepoznavanje jedi, njeno sestavo, ime živila in ljubezen do živali. 3.2 RAZLIKE GLEDE NA SPOL IN RAZRED OSNOVNE ŠOLE S pomočjo t-testa smo ugotavljali, kakšne so razli- ke v odklanjanju živil glede na spol. Iz preglednice 5 je razvidno, da so razlike med spoloma statistično značilne. Fantje imajo bolj odklonilen odnos do navedenih vrst zelenjave (p = 0,020). Dekleta pa imajo bolj odklonilen Slika 1: Glavni vzroki za odpor do zelenjave. Figure 1: Main reasons for resufing vegetables. Slika 2: Glavni vzroki za odpor do mesa, predvsem notranjih organov. Figure 2: Main reasons for refusing meat, mainly inner organs. Slika 3: Glavni vzroki za odpor do mesa mehkužcev in dvoživk. Figure 3: Main reasons for refusing meat from molluscs and amphibians. giou in sod. 2005). Skupni Crombach alfa koeficient za instrument (brez štirih spremenljivk) je 0,87. V nadaljevanju smo anketirane osnovnošolce spra- ševali po vzrokih za odklonilen odnos do živil. Najpo- membnejši vzroki za odklanjanje navedenih živil so njihove senzorične lastnosti. Iz primerjave posameznih dejavnikov (slika 1, slika 2, slika 3) je razvidno, da sta glavna vzroka za odpor do živil okus in vonj, s čimer se naše ugotovitve ujemajo z rezultati tujih raziskav (Bea- uchamp in Moran, 1982, Destor in sod., 1977). Najpo- membnejši okoljski dejavnik, ki vpliva na odklonilen odnos osnovnošolcev do možganov, žabjih krakov, pol- žev ter še nekaterih drugih živil živalskega izvora, so prehranske navade v družini. Nezanemarljiv vzrok za odklanjanje kunčjega in račjega mesa je ljubezen do ži- Acta agriculturae Slovenica, 94/2 – 2009 91 ODPOR DO ŽIVIL MED SLOVENSKIMI OSNOVNOŠOLCI odnos do živil živalskega izvora – odpor do mesa, pred- vsem notranjih organov (p = 0,000), ter odpor do mesa mehkužcev in dvoživk (p = 0,005). Po rezultatih sodeč so dekleta veliko bolj izbirčna od fantov in odklanjajo večje število živil obravnavanih v raziskavi. Rezultati potrjujejo ugotovitve raziskav Kubberod in sod. (2002) in Letarte in sod. (1997), da ženske pogosteje odklanjajo meso in mesne izdelke kot moški. Manj odklonilen odnos imajo le do zelenjave, kar je verjetno posledica sodobnih mo- dnih trendov, ki dekletom zapovedujejo suhe postave. Uživanje nizko kalorične hrane, kot je zelenjava, pomaga ohranjati želena telesna razmerja. Ugotavljali smo tudi razlike v odporu do hrane med šestošolci (Mo = 11 let) in devetošolci (Mo = 14 let). Ugotovili smo, da imajo devetošolci večji odpor do mesa, predvsem notranjih organov, (t = 2,126; df = 624; p = 0,034) kot pa šestošolci (pregl. 6). Druge razlike niso statistično značilne. 3.3 RAZLIKE MED VZHODNO IN ZAHODNO SLOVENJO Pri ugotavljanju razlik v odporu do hrane med osnovnošolci iz vzhodnega in zahodnega dela Sloveni- je smo v analizo izmed 16 šol vključili samo tri najbolj vzhodne (Murska Sobota, Maribor, Veržej) in tri najbolj zahodne (Marezige, Ajdovščina, Nova Gorica) šole. S pomočjo t-testa smo primerjali regionalne razlike za tri izračunane faktorje odpora do hrane. Spol N Povpr. SD t df p-vrednost Odpor do zelenjave M 298 2,360 0,9812 −2,329 626 0,020 Ž 330 2,552 1,0888 Odpor do mesa, predvsem notranjih organov M 298 2,540 0,9421 8,763 566 0,000 Ž 330 1,940 0,7491 Odpor do mesa mehkužcev in dvoživk M 298 2,091 1,0405 2,825 605 0,005 Ž 330 1,865 0,9581 Preglednica 5: Odklanjanje živil glede na spol Table 5: Food dislike regarding sex Razred N Povpr. SD t df p-vrednost Odpor do zelenjave 6. 317 2,459 1,039 0,058 624 0,954 9. 309 2,454 1,040 Odpor do mesa, predvsem notranjih organov 6. 317 2,297 0,921 2,126 624 0,034 9. 309 2,145 0,865 Odpor do mesa mehkužcev in dvoživk 6. 317 1,950 0,976 −0,420 624 0,675 9. 309 1,983 1,019 Preglednica 6: Odpor do hrane glede na razred osnovne šole Table 6: Food dislike regarding age (school grade) Slovenija N Povpr. SD t df p-vrednost Odpor do zelenjave Vzh. 143 2,374 0,975 −1,188 250 0,236 Zah. 109 2,527 1,066 Odpor do mesa, predvsem notranjih organov Vzh. 143 2,301 0,775 0,248 250 0,804 Zah. 109 2,275 0,848 Odpor do mesa mehkužcev in dvoživk Vzh. 143 1,902 0,951 −2,292 250 0,023 Zah. 109 2,172 0,891 Preglednica 7: Razlike med osnovnošolci iz vzhodne (vzh) in zahodne (zah) Slovenije v odporu do hrane Table 7: Differences in food dislike between schoolchildren from eastern and western part of Slovenia Vzhodna (Vzh) Slovenija: Murska Sobota, Maribor, Veržej; Zahodna (Zah) Slovenija: Marezige, Ajdovščina, Nova Gorica Acta agriculturae Slovenica, 94/2 – 200992 G. TORKAR in sod. Iz preglednice 7 je razvidno, da so razlike med vzho- dno in zahodno Slovenijo statistično značilne pri odpo- ru do mesa mehkužcev in dvoživk (t = −2,292; df = 250; p  =  0,023). Osnovnošolci iz zahoda Slovenije imajo manjši odklonilen odnos do tovrstne hrane kot osnov- nošolci iz vzhoda Slovenije. Razlike so bile pričakovane, saj so školjke, polži in hobotnice mediteranska hrana. Ta je veliko bolj dostopna v zahodni Sloveniji, ki se razteza proti Jadranskemu morju. Tudi sama kulinarična razno- vrstnost slovenskega prostora vpliva na razlike v odnosu do mediteranske hrane v prid učencev iz zahodnega dela Slovenije. 4 SKLEPI Na osnovi rezultatov raziskave lahko naše ugoto- vitve strnemo v naslednje sklepe: –– osnovnošolci odklanjajo živila največkrat zaradi senzoričnih lastnosti, kot sta okus in vonj; –– med dejavniki okolja imajo največji vpliv na sprejemanje ali odklanjanje živil prehranske na- vade v družini; –– anketirana dekleta so bolj izbirčna kot fantje; manj odklonilen odnos imajo le do zelenjave; –– devetošolci bolj odklanjajo živila živalskega iz- vora, predvsem notranjih organov (npr. možgani, jetrca, vampi), kot šestošolci; –– razlike med osnovnošolci iz zahodne in vzhodne Slovenije se kažejo v odporu do mesa mehkužcev in dvoživk. Osnovnošolci v zahodni Sloveniji imajo manjši odpor do tovrstne hrane, kar je lahko odraz poznavanja tradicionalne prehrane okolja in boljše dostopnosti mediteranske hrane na zahodu Slovenije. Ker preko 95  % učencev v Sloveniji uživa dopol- dansko malico, ki jo pripravljajo v šolski kuhinji, okoli 60 % učencev pa tudi kosila (Zavod Republike Slovenije za šolstvo, 2009), rezultati raziskave lahko služijo za us- merjanje načrtovanja šolskih jedilnikov, predvsem pri ponudbi živil. 5 VIRI Anastasi A. 1996. Psychological Testing. 7th edn. New York: Macmillan. Beauchamp G.K., Moran M., 1982. Dietary experience and sweet taste preference in human infants. Appetite, 3, 139– 152. Birch L.L. 1999. Development of food preferences. Annual Re- view of Nutrition, 19, 41–62. Birch L.L. 1992. Children’s preferences for high-fat foods. Nu- trition Reviews, 50, 249–255. Birch L.L., Fisher J.O., Markey C.N., Grimm-Thomas K., Saw- yer R., Johnson S.L. 2001. 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The relationship between child food neophobia and everyday food consumption. Ap- petite, 41, 205–206. Cutting T.M., Fisher J.O., Grimm-Thomas K., Birch L.L. 1999. Like mother, like daughter: familial patterns of overweight are mediated by mothers dietary disinhibition. American Journal of Clinical Nutrition, 69, 608–613. Destor J.A., Maller O., Turner R.E. 1977. Preference for sweet in humans: infants, children, and adults. V: Taste and De- velopment: the Genesis of Sweet preference (J.M. Weiffen- bach, ur.). Washington, US Government Printing Office, 161–172. Destor J.A., Maller O., Andrews K. 1975. Ingestive responses of human newborns to salty, sour, and bitter stimuli. Journal of comparative and physiological psychology, 89, 966–970. Drewnowski A., Hann C. 1999. Food preferences and reported frequencies of food consumption as predictors of current diet in young women. American Journal of Clinical Nutri- tion, 70, 28–36. Gibson E.L., Wardle J. 2003. Energy density predicts preferenc- es for fruit and vegetables in 4-year-old children. Appetite, 41, 97–98. Johnson S.L., Birch L.L. 1994. Parents’ and children’s adiposity and eating style. Pediatrics, 94, 635–661. Likert R. 1932. A Technique for the Measurement of Attitudes. Archives of Psychology 140: 1–55. Neumark-Sztainer D., Story M., Perry C.L., Casey M. 1999. Factors influencing food choices of adolescents: findings from focus group discussions with adolescents. Journal of American Dietetic Association, 102, 929–937. Palaigeorgiou G.E., Siozos P.D., Konstantakis N.I., Tsoukalas I.A. 2005. A computer attitude scale for computer science freshmen and its educational implications. Journal of Com- puter Assisted Learning 21: 330–342. Reinhardt W.C., Brevard P.B. 2002. Integrating the Food Guide Pyramid and Physical Activity Pyramid for positive dietary and physical activity behaviors in adolescents. Journal of American Dietic Association, 102, S96–S99. Acta agriculturae Slovenica, 94/2 – 2009 93 ODPOR DO ŽIVIL MED SLOVENSKIMI OSNOVNOŠOLCI Scaglioni S., Salvioni M., Galimberti C. 2008. Influence of pa- rental attitudes in the development of children eating be- haviour. British Journal of Nutrition, 99, 22–25. Skinner J.D., Carruth B.R., Bounds W., Ziegler P.J. 2002. Chil- dren’s food preferences: a longitudinal analysis. Journal of American Dietetic Association, 102, 1638–1647. Skinner J.D., Carruth B.R., Moran J., Houk K., Schmidhammer J., Reed A., Coletta F., Cotter R., Ott D. 1998. Toddlers’ food preferences: concordance with family members’ preferenc- es. Journal of Nutrition Education, 30, 17–22. Steiner J.E. 1979. Facial expressions of neonate infant indicating the hedonics of food related stimuli. V: Taste and Develop- ment: the Genesis of Sweet preference (J.M. Weiffenbach, ur.). Washington, US Goverment Printing Office, 173–189. Story M., Neumark-Sztainer D. 1999. Promoting healthy eating and physical activity in adolescents. Adolescence Medicine, 10, 109–123. Wardle J., Cooke L. 2008. Genetic and environmental determi- nants of children’s food preferences. British Journal of Nu- trition, 99, 15–21. Zavod Republike Slovenije za šolstvo 2009. http://www.zrss.si/ doc/GOS_PREHRANA%20V%20OŠ.doc (vstop 11.2.2009) Acta argiculturae Slovenica, 94/2, 95–102, Ljubljana 2009 COBISS: 1.02 Agris category code: L02 USE OF HERBS AND SPICES AND THEIR EXTRACTS IN ANIMAL NUTRITION Tamara FRANKIČ 1, Mojca VOLJČ 2, Janez SALOBIR 3, Vida REZAR 4 Delo je prispelo 16. septembra 2008, sprejeto 15. septembra 2009. Received September 16, 2008; accepted September 15, 2009. 1 Univ. of Ljubljana, Biotechnical Fac., Zootechnical Dep., Groblje 3, SI-1230 Domžale, Slovenia, Ph.D., e-mail: tamara.frankic@bfro.uni-lj.si 2 Same address, e-mail: mojca.voljc@bfro.uni-lj.si 3 Same address, Assoc. Prof., Ph.D., e-mail: janez.salobir@bfro.uni-lj.si 4 Same address, Ph.D., e-mail: vida.rezar@bfro.uni-lj.si Use of herbs and spices and their extracts in animal nutrition The ban on nutritive antibiotic use in Europe and the in- creased awareness of the consumers triggered a need for natu- ral and safe feed additives to achieve better production results of farm animals. Plant extracts are used in animal nutrition as appetite and digestion stimulants, stimulants of physiological functions, for prevention and treatment of certain pathologi- cal conditions, as colorants and antioxidants. This article is a review of present literature data on the usage of plant extracts in poultry, pig and ruminant nutrition. Key words: animal husbandry / pigs / ruminants / poul- try / animal nutrition / herbs / spices / plant extracts Uporaba zelišč in začimb ter njihovih ekstraktov v prehrani ži- vali Prepoved uporabe nutritivnih antibiotikov v prehrani ži- vali v Evropi in naraščajoča zavest potrošnikov je sprožila po- trebo po uporabi naravnih in zdravih prehranskih dodatkov za doseganje boljših proizvodnih rezultatov. Rastlinske izvlečke v prehrani živali uporabljamo kot stimulatorje apetita in preba- ve, za preprečevanje in zdravljenje nekaterih bolezenskih stanj, za stimuliranje fizioloških funkcij, kot barvila in kot antioksi- dante. Predstavljen članek je pregled dosedanjih znanstvenih dognanj o uporabi rastlinskih ekstraktov v prehrani perutnine, prašičev in prežvekovalcev. Ključne besede: živinoreja / prašiči / prežvekovalci / pe- rutnina / prehrana živali / zelišča / začimbe / rastlinski izvlečki 1 INTRODUCTION Only quality feed together with proper hygiene, po- table water and management can ensure the production of nutritious animal products with desired organoleptic properties (Saxena, 2008). Keeping farm animals healthy is necessary to obtain healthy animal products. For the last decade the use of additives of natural origin in animal and human nutrition has been encouraged. Numerous researches focused on the clarification of the biochemi- cal structures and physiological functions of various feed additives like probiotics, prebiotics, organic acids and plant extracts. Herbs, spices and their extracts were already used thousands of years ago in Mesopotamia, Egypt, India, China and old Greece, where they were appreciated for their specific aroma and various medicinal properties (Greathead, 2003). When discussing the use of herbs and spices as feed additives, we can hardly rely only on old believes about health impact of certain herbs and spices or their active components. We need a scientific proof of their beneficial effect on health and performance of the animals to justify their use. The technological progress enables us to more easily determine the structure and function of yet unidentified active molecules of plant ori- gin. To gain advantageous effects of herbs and spices, they can be added to feed as dried plants or parts of plants and as extracts. The composition of extracts from the same plant depends on the method of extraction and Acta agriculturae Slovenica, 94/2 – 200996 T. FRANKIČ et al. the properties of the extraction solvent used. Depending on the chemical characteristics of extraction solvents we can extract only certain molecules. There is also a dif- ference between purified and unpurified extracts. Unpu- rified extracts contain a number of different molecules extracted with certain solvent, which can affect the ac- tion of each other, while purified extracts contain only one active component. The purified active molecules extracted from plants can be sometimes substituted by synthetic naturally identical molecules. Plants mainly contain one or some predominant active molecules (sec- ondary metabolites), which are responsible for certain biological effects. The amount of these molecules varies depending on the variety of plant, growing conditions, harvest time etc. When we need the effect of a specific active component, it is more efficient to use a purified molecule alone than a dried plant or unpurified extract. But we have to be aware, that the potency of an unpuri- fied extract often exceeds the potency of a purified one because of synergistic effect among the molecules in it. When talking about plant extracts, we must mention also essential oils. These are extracts of vaporous oils of strong taste and smell, which are still usually extracted by dis- tillation with steam. Essential oils are very potent mol- ecules and must be used in small quantities. Adversaly they can affect the function of intestinal microflora, can cause allergies, suppress feed intake and can be stored in tissues. With the proper usage, most of essential oils are recognized as GRAS (generally recognized as safe). Today the market offers different extracts of certain aro- matic plants, combinations of extracts of different plants, purified active components or combinations of purified active components and synthesized active molecules (naturally identical) (Indresh, 2007). The effect of active components from herbs and spices depends largely on the dosage used. No effect Table 1: Often used plants, its active components and functions (Loo and Richard, 1992; Charalambous, 1994; Kamel, 2000) Preglednica 1: Pogosto uporabljene rastline, njihove aktivne komponente in funkcije (Loo in Richard, 1992; Charalambous, 1994; Kamel, 2000) Plant Used parts Mayor active component Function Aromatic spices Nutmeg Seed Sabinene Digestion stimulant, antidiarrhoeic Cinnamon Bark Cimetaldehyde Appetite and digestion stimulant, antiseptic Cloves Cloves Eugenol Appetite and digestion stimulant, antiseptic Cardamom Seed Cineol Appetite and digestion stimulant Coriander Leaves, Seed Linalol Digestion stimulant Cumin Seed Cuminaldehyde Digestive, carminative, galactagogue Anise Fruit Anethol Digestion stimulant, galactagogue Celery Fruit, Leaves Phtalides Appetite and digestion stimulant Parsley Leaves Apiol Appetite and digestion stimulant, antiseptic Fenugreek Seed Trigonelline Appetite stimulant Pungent spices Capsicum Fruit Capsaicin Digestion stimulant Pepperr Fruit Piperine Digestion stimulant Horsradish Root Allyl izotiocianat Appetite stimulant Mustard Seed Allyl izotiocianat Digestion stimulant Ginger Rizom Zingerone Gastric stimulant Garlic Bulb Allicin Digestion stimulant, antiseptic Herbs Rosemary Leaves Cineol Digestion stimulant, antiseptic, antioxidant Thyme Whole plant Thymol Digestion stimulant, antiseptic, antioxidant Sage Leaves Cineol Digestion stimulant, antiseptic, carminatif Laurel Leaves Cineol Appetite and digestion stimulant, antiseptic Mint Leaves Menthol Appetite and digestion stimulant, antiseptic Acta agriculturae Slovenica, 94/2 – 2009 97 USE OF HERBS AND SPICES AND THEIR EXTRACTS IN ANIMAL NUTRITION whatever can be observed at small doses; on the other hand, large amounts can be even toxic. The search for nutritive antibiotic alternatives in EU and increased awareness and concern of the consumers, further encouraged the precise researches on the possi- bilities of plant extract use in animal nutrition. The main scope in animal husbandry – to ensure good perform- ance of farm animals and get quality animal products, can be achieved only with the effort to keep the animals healthy. In this aspect, herbs and spices are not just ap- petite and digestion stimulants, but can, with impact on other physiological functions, help to ensure good health and welfare of the animals, what can positively affect their performance. 2 POSSIBLE USE OF HERBS AND SPICES 2.1 HERBS AND SPICES AS APPETITE AND DI- GESTION STIMULANTS When considering supplementing the feed with herbs and spices or their extracts to stimulate the appe- tite, we have to know the taste preferences of different an- imal species. Janz et al. (2007) found that pigs preferred the feed supplemented with garlic or rosemary over the feed supplemented with oregano or ginger. Furthermore, Jugl-Chizzola et al. (2006) noticed that weaned pigs con- sumed significantly less feed if it was supplemented with thyme or oregano. If pigs in this experiment had the pos- sibility to choose among feed with or without above men- tioned spices, they had chosen the unsupplemented feed. The spices known for their appetite stimulant effect are cinnamon, cloves, cardamom, laurel and mint (Loo and Richard, 1992). Due to the wide variety of active components, differ- ent herbs and spices affect digestion processes differently. Most of them stimulate the secretion of saliva. Curcuma, cayenne pepper, ginger, anis, mint, onions, fenugreek, and cumin enhance the synthesis of bile acids in the liver and their excretion in bile, what beneficially effects the digestion and absorption of lipids. Most of the prelisted spices stimulate the function of pancreatic enzymes (li- pases, amylases an proteases), some also increase the ac- tivity of digestive enzymes of gastric mucosa (Srinivasan, 2005). Besides the effect on bile synthesis and enzyme ac- tivity, extracts from herbs and spices accelerate the diges- tion and shorten the time of feed/food passage through the digestive tract (Platel and Srinivasan, 2001; Suresh and Srinivasan, 2007). 2.2 ANTIMICROBIAL ACTION OF HERBS AND SPICES Feed supplements with growth promoting activity increase stability of feed and beneficially influence the gastrointestinal ecosystem mostly through growth inhi- bition of pathogenic microorganism’s growth. Due to im- proved health status of digestive system, animals are less exposed to the toxins of microbiological origin. Conse- quently herbs and spices help to increase the resistance of the animals exposed to different stress situations and in- crease the absorption of essential nutrients, thus improv- ing the growth of the animals (Windisch et al., 2008). Numerous secondary metabolites formed by plants serve as defence agents against physiological and environ- mental stressors, predators and pathogenic microorgan- isms. Several in vitro studies showed strong antimicro- bial activity of certain plant extracts against Gram− and Gram+ bacteria. Pasqa et al. (2006) found a change in long chain fatty acid profile in the membranes of E. coli grown in the presence of limonene or cinnamaldehyde. Similar observations were made with Salomonella en- terice grown in the presence of carvacrol or eugenol and with Bronchotrix thermosphacta grown in the presence of either limonene, cinnamaldehyde, carvacrol or eugenol. In the case of Pseudomonas fluorescens in Staphylococcus aureus none of the tested phytochemicals changed the fatty acid profile. The changes in fatty acid composition can affect surviving ability of microorganisms. The studies measuring hydrophobicity of E. coli (test for measuring the ability of microbial attachment) showed a large increase of hydrophobicity of E. coli grown in the presence of St. John’s wort or Chinese cin- namon and a moderate increase when medium was sup- plemented with thyme or Ceylon cinnamon. The differ- ences in hydrophobicity were in good correlation with MIC50 values (minimal inhibitory concentration). This confirms the fact that herbs and spices act as antimicro- bial agents by changing the characteristics of cell mem- branes, and causing ion leakage, thus making microbes less virulent (Windisch et al., 2008). The exact antimicro- bial action of herbs and spices in in vivo situations is hard to evaluate, because of the very complex and balanced microbial populations in gastrointestinal tract and the interaction of active components from herbs and spices with other nutrients. Castillo et al. (2006) reported that the mixture of cinnamaldehyde, capsicum oleoresin and carvacrol enhances the growth of lactobacilli, and so in- creases the ratio of lactobacilli to enterobacteria. So herbs and spices do not posses only the antimicrobial activity, but also modulate the composition of microbial popula- tion by prebiotic activity. Acta agriculturae Slovenica, 94/2 – 200998 T. FRANKIČ et al. 2.3 ANTI-INFLAMMATORY ACTION Extracts of curcuma, red pepper, black pepper, cu- min, cloves, nutmeg, cinnamon, mint and ginger showed anti-inflammatory effect in the studies on rats (Srini- vasan, 2005; Manjunatha in Srinivasan, 2006). The ma- jor active molecules with anti-inflammatory action are terpenoids and flavonoids. These molecules suppress the metabolism of inflammatory prostaglandins. The most known herbs and spices with anti-inflammatory poten- tial in our area are chamomile, marigold, liquorice and anis (Craig, 2001). 2.4 ANTIOXIDATIVE ACTION Many active components of herbs and spices can prevent lipid peroxidation through quenching free radi- cals or through activation of antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase. Main molecules responsible for the antioxidative properties of herbs and spices are phenolic substances (flavonoids, hydrolysable tannins, proanthocianidins, phenolic acids, phenolic terpenes) and some vitamins (E, C and A). Often used herbs rich in phenolics are: rosemary, thyme, oregano, sage, green tea, chamomile, ginko, dandelion and marigold (Halliwell et al., 1995; Craig, 2001; Ćetković et al., 2004; Škerget et al., 2005; BakIrel et al., 2008; Fasseas et al., 2008). Herbs and spices can protect the feed against oxida- tive deterioration during storage. This is a widely used practice in pet food and human food industry. The herb commonly used for feed/food preservation is rosemary (Rosmarinus officinalis). It can be used alone or in combi- nation with tocopherols or synthetic antioxidants (Jacob- sen et al., 2008). 2.5 IMMUNOSTIMULANT FUNCTION The immune system generally benefits from the herbs and spices rich in flavonoids, vitamin C and caro- tenoids. The plants containing molecules which possess immunostimulatory properties are echinacea, liquorice, garlic and cat’s claw. These plants can improve the activ- ity of lymphocytes, macrophages and NK cells, they in- crease phagocytosis or stimulate the interpheron synthe- sis (Craig, 1999). 3 THE USE OF HERBS AND SPICES IN NUTRITION OF DIFFERENT ANIMAL SPECIES 3.1 POULTRY How to replace antibiotic growth promoters is also a question for the poultry industry. Some studies on plant extracts are showing promising results. Çabuk et al. (2006) measured production parameters of broilers which were supplemented by a mixture of oregano, lau- rel, sage, anis and citrus essential oils. The mixture of es- sential oils significantly improved feed conversion, what can be attributed to more effective availability of nutri- ents due to the changes in intestinal ecosystem. Lippens et al. (2005) tested the efficacy of a mixture of cinnamon, oregano, thyme, cayenne pepper and citrus extracts and a mixture of plant extracts and organic acids in comparison to nutritive antibiotic avilamicin in broil- er chickens. Chickens supplemented with plant extracts reached significantly higher body weight than the ones in the control or avilamicin group. Higher body weight was a consequence of increased feed consumption. Feed conversion in group fed plant extracts was 0.4% better than in the group with avilamicin and 2.9% better than in the control group. The authors noticed no synergistic effect between plant extracts and organic acids. Resistance of coccidia to currently used coccidi- ostatics to treat coccidiosis represents a serious problem in poultry industry. The use of plant extracts to treat coc- cidiosis is not a new approach. When searching for the best natural extract to treat coccidiosis, we have to take into account that the extract needs to be at least partially soluble in lipids to penetrate the cellular membrane, be- cause coccidia are located inside the cells. Two Chinese plants, Dichroa febrifuga and Sophora flavescens are rich in alkaloids which are effective in treating coccidiosis (Youn in Noh, 2001). As infections with Emeria tenela include also lipid peroxidation in the intestine, herbs and spices with strong antioxidant potency may represent a good supportive treatment. In one of the latest studies Naidoo et al. (2008) studied the capacity of four African plants which would be appropriate to treat coccidiosis: leaves of Combretum woodii, leaves and stem of Artemi- sia afra, a whole plant and seeds of Vitis vinifera. Extracts of all chosen plants improved the feed conversion to the same extent as coccidiostatic toltrazuril. The best effect was seen with Tulbaghia violacea, which also partially lowered the shedding of oocysts. The use of herbs and spices as antioxidants is not important only for the health of the animals, but also for the oxidative stability of their products. The effect of oregano essential oil on oxidative stability of chicken and Acta agriculturae Slovenica, 94/2 – 2009 99 USE OF HERBS AND SPICES AND THEIR EXTRACTS IN ANIMAL NUTRITION turkey meat was well studied in the past. Supplementa- tion of turkeys with 200 mg/kg of oregano essential oil significantly decreased lipid peroxidation of cooked and fresh meat during refrigerated storage (Botsoglou et al., 2003b). Essential oil of oregano also efficiently preserved the quality of chicken meat during frozen storage (Bot- soglou et al., 2003a). Extracts from herbs and spices in combination with vitamins C and E even more effectively prevent lipid peroxidation in tissues, what was shown in the studies on chickens and turkeys (Papageorgiou et al., 2003; Young et al., 2003). At this time the use of plant extracts instead synthetic or semi-synthetic antioxidants represents higher economical costs, however, this could be avoided with systematic intensified growing of needed plants and new technological processes of extraction. The colorants for increasing yolk colour in laying hens or skin colour in broilers in intensive production can be of natural (carotenoids) or synthetic origin. Of- ten used forage plants rich in carotenoids are maize and alfalfa. Besides these there are several other plants used for isolation of natural pigments like tagetes and red pep- per. The main yellow pigments in tagetes are zeaxantin and lutein, while red pepper contains two important red pigments – capsantin and capsorubin. The extract from tagetes colours the yolk three times less effectively in comparison with the synthetic apo-ester of carotenic acid. Pigments from natural origin also degrade during the feed storage up to 30% (Sirri et al., 2007). Neverthe- less, pigments obtained from tagetes or calendula species and red pepper are very suitable as yolk colorants in or- ganic farming. 3.2 PIGS In the pig production, most problems can be expect- ed in the time of weaning. Weaning can be accompanied by infections, especially with enterotoxic Escherichia coli. The use of herbs and spices in piglet nutrition can reduce the incidence of infections. Results from Roselli et al. (2007) showed that alicin from garlic protects intestinal cells from increased permeability of membrane in pigs infected with E. coli. Garlic also contains active substanc- es which suppress the action of fungi and viruses (Zigger, 2001) and improve the feed intake and daily weight gain of piglets (Janz et al., 2007). Cinnamaldehyde, an active component of cinnamon, possesses antibacterial proper- ties. Zigger (2001) observed larger feed intake and live weight gain of weaned pigs fed feed supplemented with garlic and cinnamon extracts. The mortality due to intes- tinal disorders dropped from 3.9 to 1.2%. Namkung et al. (2004) found that a mixture of cinnamon, thyme and oregano extracts inhibited the growth of coliform bacte- ria. A brown algae Ascophyllum nodosum could be a good feed supplement with growth promoting activity of pigs infected with E. coli (Turner et al., 2002). Combination of carvacrol, cinnamaldehyde and capsicum oleoresin beneficially effected gastrointestinal ecosystem and gastric emptying of weaned pigs (Man- zanilla et al., 2004). The same mixture was tested for its antioxidative properties in our laboratory. The mixture effectively protected pig’s blood lymphocytes against oxi- dative DNA damage at the concentration of 271.2 mg/ kg of feed. Its effect was comparable to that of 90.4 mg/ kg of vitamin E. The concentration of spice mixture sup- plemented to pigs in this study was not sufficient to fully prevent lipid peroxidation induced by high intake of lightly oxidizable PUFA. Frankič et al. (in press) studied antioxidant capacity of propylene glycol extracts of Calendula officinalis (Cal- endula off. 1 – extract from petals, 3 ml/day; Calendula off. 2 – extract from whole flowers tops, 3 ml/day) and vitamin E (38.4 mg/day) in the case of oxidative stress induced by high PUFA intake in pigs. The extracts ef- fectively prevented oxidative DNA damage in peripheral lymphocytes (measured as % DNA in the tail of the com- et and OTM (Olive tail moment), but did not prevent lipid peroxidation, measured by 8-OHdG (8-hidroxy- deoxyguanosine) (Table 2). Although most studies concerning the effect of herbs and spices in pig production have been conducted on piglets, Allan et al. (2005) carried out an experiment on swine. Swine were fed 1000 ppm of dried oregano leaves and flowers enriched with 500 g/kg of oregano es- sential oil. Observed beneficial effects of oregano supple- mentation were: lower mortality rate, less culling during Table 2: Lymphocyte DNA damage and urinary 8-OHdG excre- tion of pigs fed a high PUFA diet with or without Calendula off. extracts Preglednica 2: Poškodbe DNA limfocitov in količina s sečem izločenega 8-OHdG, pri prašičih, krmljenih z visoko vsebnostjo PUFA v krmi z oziroma brez dodatka ekstrakta Calendula off. Group % DNA in the tail of the comet OTM 8-OHdG (µg/24 h) Control 7.8a 1.74a 149.9ab Oil 12.0b 4.68b 269.4b Calendula off. 1 6.8a 1.46a 138.9a Calendula off. 2 8.2a 2.05a 150.6ab Vitamin E 6.6a 1.54a 216.5b SEM 0.65 0.372 31.44 P-value < 0.01 < 0.01 0.02 abc LS-means – without the same superscript differ significantly, P < 0.05; OTM – Olive tail moment; 8-OHdG – 8-hidroxy-deoxyguanosine. Acta agriculturae Slovenica, 94/2 – 2009100 T. FRANKIČ et al. lactation period, shorter service interval, more live born and less stillborn piglets. 3.3 RUMINANTS Herbs and spices have been introduced also to ru- minant nutrition. Microbial ecosystem in the rumen is composed from complex anaerobic microbial population of bacteria, fungi, protozoa, methanogeneous arhea and bacterifagi. Numerous metabolites produced in rumen during microbial fermentation affect the basic digestive and metabolic functions and productivity of the host. Researchers have been searching for new possibilities to modulate the microbial fermentation in the rumen. The main goal of manipulating the rumen fermentation is to increase the effectiveness of digestion and metabolism of nutrients, to increase the productivity of the animals and to suppress the undesirable processes as methanogenesis. In intensive farming systems the feed additives, includ- ing antibiotics, were used to increase the production of milk, meat and wool. The ban on antibiotic use in Europe increases the production costs what triggered the need to search for antibiotic alternatives also in ruminant nutri- tion. There are numerous studies showing beneficial ef- fects of herbs and spices on feed intake, immune func- tions and health, rumen fermentation and productivity of calves, dairy cows, heifers and also beef cattle (Krasze- wski et al., 2002; Greathead, 2003; Wawrzynczak et al. 2000; Cardozo et al. 2006). There are some data of the positive effect of plant supplements in nutrition of sheep and goats (Butter et al., 1999). Extracts of yucca plant contain saponnins and glico-components which are re- sponsible for the increase of rumen fermentation and in some cases for reduction of ammonium synthesis (Ryan, P. and Quinn, T.: http://www.irishscientist.ie/P175.htm). Kudke et al. (1999) supplemented calves with powder of Azadirachta indica tree. Supplemented calves had higher weight gain than unsupplemented ones. The unsupple- mented group had much higher incidence of parasite infections. Gladine et al. (2007) tested the antioxidant effect of marigold, grape, rosemary and citrus extracts in sheep. Lipid peroxidation was induced by continuous infusion of linseed oil into the duodenum. The extracts were ap- plied directly into rumen through the rumen cannula. The results showed that all tested plant extracts kept their antioxidant capacity in vivo in sheep. The most bioeffi- cient in limiting lipid peroxidation was marigold extract. Cardozo et al. (2006) studied the effect of alfalfa ex- tract, anise, capsicum, and a mixture of cinnamaldehyde end eugenol on ruminal fermentation in beef heifers. The results indicated that tested concentrations of cinnamal- dehyde and eugenol mixture, anise oil and capsicum oil may be used as modifiers of rumen fermentation in beef production systems. Same authors tested six natural plant extracts (garlic, cinnamon, anise, yucca, oregano and capsicum extract) and three secondary plant metabolites (cinnamaldehyde, eugenol, anethole) at five doses and two different pH (7.0 and 5.5) to determine their effect on in vitro microbial fermentation using ruminal fluid of heifers (Cardozo et al., 2005). Results demonstrated that the effect of herbs and spices on ruminal fermentation in beef cattle may differ depending on ruminal pH. At pH 5.5, garlic, capsicum, yucca and cinnamaldehyde altered ruminal fermentation in favour of propionate, which is more energetically efficient. Results obtained in the research of Benchaar et al. (2007) showed limited effects of 750 mg/day of essen- tial oil mixture (thymol, eugenol, vanillin, guaiacol and limonene) on nutrient utilization, ruminal fermentation, and milk performance of cows fed diets containing alfal- fa or corn silage as a sole forage source. Polish research- ers showed that 2% of mixture of Urtica dioica, Pradix teraxci, Agrimonia eupatoria, Fructus carvi and Matrica Chamonilla improves the quality of milk (Kraszewski et al., 2002). Tannins, the secondary plant metabolites found in stem, wood, leaves, fruits and seeds of many plant species can positively affect the protein digestion in ruminants. Tannins bind to proteins and form complexes which pass trough the rumen undegraded. These proteins which pass the microbial degradation in the rumen are then success- fully utilized by the animal and provide the proteins nec- essary especially in the special physiological states (like early lactation) and in the cases when feed is not of the best quality (Waghorn et al., 1990). Tannins also prevent bloat of the rumen (Butter et al., 1999) and possess anti- helmitic properties (Barry and McNabb, 1999). Extracts from herbs and spices help to prevent and alleviate different kinds of health problems. They are ef- fective in treatment of endometritis (inflammation of the endometrium) in cows. Esparza-Borges and Ortiz-Már- quez (1996) evaluated the effect of extrats of garlic (Al- lium sativum, L), eucalypt (Eucalyptus globulus, Labill.) and Gnaphalium conoideum on acute endometritis of Holstein cows. The most effective of all extracts was the garlic extract, however, also eucalypt worked beneficially. 4 CONCLUSIONS The main scope of animal production is to ensure the high productivity, healthy animals and quality ani- mal products, which are stable and appropriate for fur- Acta agriculturae Slovenica, 94/2 – 2009 101 USE OF HERBS AND SPICES AND THEIR EXTRACTS IN ANIMAL NUTRITION ther processing. In this aspect, herbs and spices are not just appetite and digestion stimulants, but can, with im- pact on other physiological functions, help to sustain good health and welfare of the animals and improve their performance. 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Poultry Science, 82: 1343–1351 Acta argiculturae Slovenica, 94/2, 103–110, Ljubljana 2009 COBISS: 1.01 Agris category code: L51 TIME DEPENDENT FORMATION OF MARKERS OF OXIDATIVE STRESS INDUCED BY A HIGH FAT DIET SUPPLEMENTED OR UNSUPPLEMENTED WITH VITAMIN E IN PIGS Tanja PAJK ŽONTAR 1, Janez SALOBIR 2, Vida REZAR 3 Delo je prispelo 16. septembra 2008, sprejeto 15. septembra 2009. Received September 16, 2008; accepted September 15, 2009. 1 Univ. of Ljubljana, Biotechnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia, Ph.D., e-mail: tanja.pajk@amis.net 2 Same address, Prof., Ph.D., e-mail: janez.salobir@bfro.uni-lj.si 3 Same address, Ph.D., e-mail: vida.rezar@bfro.uni-lj.si Time dependent formation of markers of oxidative stress induced by a high fat diet supplemented or unsupplemented with vitamin E in pigs The time dependent formation of oxidative damage induced by polyunsaturated fat in the diet was investigated in an experi- ment with pigs as a model for humans. The role of vitamin E in the prevention of oxidative stress was also studied. Twenty-four growing pigs were penned individually and after an adaptation period divided into three groups. All groups received isocaloric daily rations composed of a basal diet isocalorically supplement- ed with: starch, linseed oil or linseed oil and vitamin E. Oxidative stress was evaluated by measuring the degree of lymphocyte and granulocyte nuclear DNA damage, concentration of malondial- dehyde (MDA) in blood plasma, 24-hour urine MDA excretion rate and concentration of vitamin E isomers in the blood at the beginning, after 24 hours, after 6 days and at the end of the 22 day experimental period. The results confirmed that a high pro- portion of polyunsaturated fat in the diet increased lymphocyte and granulocyte DNA damage only after 6 days. The lymphocytes appear to be more sensitive to this type of oxidative stress than granulocytes. The MDA concentration in the blood and urinary MDA excretion after 24 hours of oxidative stress seem to be more accurate indicators than the rate of lymphocyte and especially granulocyte DNA damage. Vitamin E supplementation effectively protects the blood cells against increased DNA damage during the whole course of the experiment, but failed to reduce MDA formation significantly 24 hours and 6 days after the beginning of oxidative load. The study further suggests that supplementa- tion of vitamin E is able to completely prevent DNA damage of both types of investigated blood cells at any time, but is only able to reduce the formation of lipid peroxidation products after pro- longed treatment. Key words: pigs / animal nutrition / oxidative stress / DNA damage / polyunsaturated fatty acids / PUFAs / vitamin E / comet assay / malondialdehyde Časovna odvisnost tvorbe kazalcev oksidacijskega stresa pri pra- šičih, povzročenega s prehrano, obogateno z maščobami ter z ali brez dodatka vitamina E V raziskavi smo spremljali časovno odvisnost oksidacij- skega stresa, povzročenega z dodatkom večkrat nenasičenih maščobnih kislin (VNMK) ter vlogo vitamina E pri njegovem zmanjšanju. Poskus smo izvedli na prašičih kot modelu za člove- ka. V individualne bilančne kletke smo uhlevili 24 mladih rasto- čih prašičev ter jih po obdobju prilagajanja razdelili v tri skupine. Vse tri skupine so dobivale enake osnovne izokalorične dnevne obroke z dodatki škroba, lanenega olja ali lanenega olja in vita- mina E. Oksidacijski stres smo ovrednostili kot stopnjo poškodb jedrne DNK limfocitov in gralulocitov, koncentracijo malondial- dehida (MDA) v krvni plazmi, 24-urno izločanje MDA s sečem in koncentracijo izomer vitamina E v krvni plazmi. Vrednosti navedenih parametrov so bile določene na začetku poskusa, po 24 urah, po 6 dneh in na koncu 22 dnevnega poskusa. Rezultati so pokazali, da je visoka vsebnost večkrat nenasičenih maščobnih kislin (VNMK) v prehrani povečala poškodbe jedrne DNK lim- focitov in granulocitov že po 6 dneh. Limfociti so se, v primerjavi z granulociti, izkazali kot bolj občutljivi. Koncentracija MDA v krvni plazmi in v 24-tih urah izločena količina MDA s sečem se je v našem primeru izkazala kot boljši pokazatelj oksidacijskega stresa v primerjavi s stopnjo poškodb DNK limfocitov, še poseb- no granulocitov. Dodatek vitamina E je učinkovito zaščitil krvne celice pred povečanimi poškodbami DNK v celotnem poskusnem obdobju, medtem ko smo po 24 urah in tudi po 6 dneh ugoto- vili statistično značilno večjo koncentracijo MDA v primerjavi z začetnimi vrednostmi. Na osnovi dobljenih rezultatov lahko sklepamo, da dodatek vitamina E oba tipa preiskovanih krvnih celic v celoti zaščiti pred poškodbami DNK, nastajanje produktov lipidne peroksidacije pa se lahko zmanjša le po dolgotrajnejšem dodajanju. Ključne besede: prašiči / prehrana živali / oksidacijski stres / poškodbe DNK / večkrat nenasičene maščobne kisline / vitamin E / kometni test / malondialdehid Acta agriculturae Slovenica, 94/2 – 2009104 T. PAJK ŽONTAR et al. 1 INTRODUCTION Diet and nutritional-related lifestyle factors have a great influence on the formation of free radicals in humans and animals, and they are also important for protection against the harmful effects of radicals. Previ- ous studies have shown that oxidative stress induced by too high an intake of dietary polyunsaturated fatty ac- ids (PUFAs) enhances damage to DNA, while increased intake of antioxidants may have a protective function (RDA, 1989). However, to our knowledge no studies were performed to investigate the time dependent course of oxidative stress induced by a high dietary intake of polyunsaturated fat. At the same time, the protective ef- fect of antioxidants in this process has also not been elu- cidated. Differences in the time dependent formation of various parameters of oxidative stress could be of interest not only from the theoretical point of view but also from practical considerations. Questions such as how fast does oxidative stress occur after increased oxidative load caused by high PUFA intake, how efficiently are antioxi- dants able to play a protective role, what is the response of different indicators of oxidative stress. The aim of the present study was to investigate time dependent changes of some markers of oxidative stress induced by a high in- take of dietary polyunsaturated fat in pig, as a model for humans. At the same time, the potency of vitamin E in preventing these changes was studied. The hypothesis of the study was that time depend- ent appearance of different markers of oxidative stress (malondialdehyde concentration in blood plasma and 24-hour urine MDA excretion rate, degree of leukocyte and granulocyte nuclear DNA damage, concentration of vitamin E isomers in the blood) is not the same, and that supplementation of feed with vitamin E provides protec- tion against some of the damaging effects of PUFAs. 2 MATHERIAL AND METHODS 2.1 EXPERIMENTAL ANIMALS, DIETS, BLOOD AND URINE SAMPLES Twenty-four young growing castrated male cross- breed pigs (live weight 11.9 kg ± 1.0) were included in the experiment. The animals were penned individually in balance cages that allowed separate collection of urine. The experiment was divided into adaptation and experi- Group LowFat HighFat HighFat+Vit-E Wheat starch, g/day 227.78 111.58 111.58 Linseed oil, g/day 0.0 53.12 53.12 Maize, g/day 59.80 59.80 59.80 Soybean meal, g/day 117.60 117.57 117.57 Skimmed milk powder, g/day 91.11 91.11 91.11 Mineral – vitamin supplement 1, g/day 2.53 2.53 2.53 Vitamin E, mg/day 0.0 0.0 57.64 Daily feed intake, g/day 498.82 435.71 435.71 Nutritive value: Metabolisable energy 2, KJ/day 7423.50 7423.50 7423.50 Proportion of energy from fat 3, % 5 30 30 Proportion of energy from PUFA3, % 2.9 20.9 20.9 Protein, g/day 86.5 88.7 87.9 Fat, g/day 8.7 56.0 57.1 Total dietary fibre, g/day 44.0 45.2 45.9 Table 1: Composition and content of energy and nutrients in daily rations of experimental groups of pigs (estimated for a 12 kg pig) Preglednica 1: Sestava ter energijska in hranilna vrednost dnevnega obroka posamezne poskusne skupine (preračunano na 12 kg prašiča) 1 Calculated to meet nutritional requirements according to NRC (1998). Mineral-vitamin supplement provided daily: 2.0 g Ca, 3.4 g P, 0.15 g Na, 5500 IU vitamin A, 7.6 IU vitamin E. 2 The energy value of feedstuffs and diets was estimated according to GEH (1988). 3 Estimated. Acta agriculturae Slovenica, 94/2 – 2009 105 TIME DEPENDENT FORMATION OF MARKERS OF … SUPPLEMENTED OR UNSUPPLEMENTED WITH VITAMIN E IN PIGS mental periods that lasted for 14 and 22 days, respec- tively. The animals were fed 2.5 times the maintenance requirement (Prosky et al., 1992). At the beginning of the experimental period, the animals were randomly assigned to three groups. All the groups received isoca- loric daily rations composed of an equal amount of the basal diet which was supplemented according to the dif- ferent dietary treatments: LowFat with starch, HighFat with linseed oil, HighFat+VitE with linseed oil and vita- min E (Table 1). The part of energy requirements which were met by fat in the LowFat group and both linseed oil supplemented groups was 5 and 30%, respectively. The amount of vitamin E in the HighFat+Vit-E diet should cover the increased needs for vitamin E because of the higher PUFA intake was calculated according to Muggli (1994). The composition and analysis of daily rations in dif- ferent groups is presented in Table 1. The feed was fed in the form of a feed mixture. All ingredients of the mixture, except linseed oil, were mixed together weekly. The lin- seed oil was added and mixed to the diet of individual animals before every feeding. During the adaptation period the animals adapted to the rearing system and all of them received the same diet (LowFat). The animals were fed twice a day. Water was provided at libitum. At the beginning and at the end of the experiment, the pigs were weighted. At the beginning, after 24 hours, after 6 days and at the end of the 22 days experimental period blood sam- ples were taken from the jugular vein and 48-h urine was collected (except 24 hours after the beginning of the ex- periment when the collection time was 24 hours). The content of protein, fat and fibre was determined by standard procedures published by Neumann and Bassler (1997). The fatty acid composition of diets was analyzed by a gas chromatographic method after trans- esterification of lipids as described previously (Fidler et al., 2000). 2.2 LYMPHOCYTE AND GRANULOCYTE DNA DAMAGE – SINGLE-CELL GEL ELECTRO- PHORESIS – COMET ASSAY Blood samples for single cell gel electrophoresis (Comet assay) were collected in 4.5 ml evacuated tubes containing EDTAK3 anticoagulant. Blood samples were stored on ice for a maximum of 1 hour before the sepa- ration procedure. Lymphocytes and granulocytes were separated from the blood samples on a discontinuous Percoll gradient according to a modified procedure de- scribed by Hjorth et al. (1988) and Kjeldsen et al. (1999). A partially modified procedure of Singh et al. (1988) was implemented for the comet assay. Olympus CH 50 epiflu- orescent microscope at 200 × magnification was used for the examination of leukocyte nuclei in the microgels (100 W Hg lamp, excitation filter of 480–550 nm and barrier filter of 590 nm). The images were captured by Hama- matsu Orca 1 CCD camera, analyzed and the nuclear DNA damage estimated by a dedicated computer pro- gram Comet 4 (Single Cell Gel Electrophoresis, Kinetic Imaging Ltd., 2000). For each treatment, two slides were prepared and 50 cells (total 100 cells) were examined. 2.3 PLASMA AND URINE MALONDIALDEHYDE (MDA) CONCENTRATION The blood samples for MDA concentration analy- sis and urine were collected and prepared as described previously (Pajk et al., 2006). The methodology of Wong et al. (1987) modified by Chirico (1994) and Fukunaga et al. (1995) was used to measure the concentrations of malondialdehyde (MDA) in blood plasma and urine by HPLC using a Waters Symmetry C18 chromatography column (5 mm, 4.6 × 150 mm) and a Waters Symmetry C18 guard column (5 mm, 3.9 × 20 mm). A Waters Alli- ance 2690 apparatus equipped with a Waters Dual l Ab- sorbance Detector 2487 was applied. The results of the analysis were evaluated by the Millenium32 Chromatog- raphy Manager program. 2.4 VITAMIN E CONCENTRATION IN PLASMA Blood samples for vitamin E concentration were collected in 10 ml evacuated tubes containing EDTAK3 anticoagulant. Plasma was prepared by centrifugation (400 × g for 10 min.) at 4  °C and transferred to micro centrifuge tubes. The samples were stored at −70 °C. Ac- cording to Abidi (2000) and Aust et al. (2001) vitamin E (as α– and β+γ- tocopherols) was extracted from plasma by hexane, after precipitation of proteins with ethanol containing 0.3% (w/v) tert.-butyl-p-cresol (BTH) to pre- vent oxidation. Samples were analyzed by HPLC (Waters Alliance 2690), using a Waters Symmetry C18 chromatog- raphy column (5 mm, 4.6 × 150 mm) and an ODS C18 guard column (4 mm L × 3.0 mm ID). A Waters Dual l Absorbance Detector 2487 and Waters Scanning Fluores- cence Detector 474 were used. 2.5 STATISTICAL ANALYSIS The data were analyzed by the General Linear Mod- el (GLM) procedures from SAS® software (SAS, 2000). Acta agriculturae Slovenica, 94/2 – 2009106 T. PAJK ŽONTAR et al. Comparisons between the different treatments were made by contrasts provided by the GLM procedure. The data were expressed as least square means ± standard er- ror. A least significant difference of 0.05 was used to sepa- rate the treatment means. 3 RESULTS During the experiment, the animals had no health or other problems, consumed feed without residues and normal body weight gain was observed in all groups (337 ± 61 g per day). While at the beginning of the experimental pe- riod no statistical differences among groups in any of the measured parameters could be observed, already 24 hours after the nutritional intervention some very impor- tant differences among dietary treatments were found. 3.1 PLASMA AND URINE MALONDIALDEHYDE CONCENTRATION The concentration of MDA in plasma and the uri- nary MDA excretion rate in the LowFat group remained at almost the same level during the whole experimen- tal period (Table 2). In contrast, both MDA parameters increased in both linseed oil supplemented groups sig- nificantly already 24 hours after dietary intervention. Six days afterwards and at the end of the 22 day experimental period, the MDA concentration in plasma and the MDA excretion rate in urine in HighFat and HighFat+Vit-E groups were also significantly higher than in the LowFat group. While 24 hours and six days after the beginning of the experiment increased MDA excretion rates in urine were at the same level in the HighFat and HighFat+Vit-E groups, on the 22nd day the value in the HighFat group Group LowFat HighFat HighFat+Vit-E MDA in plasma, nmol/ml: At the beginning 0.25 ± 0.09 0.26 ± 0.09 0.28 ± 0.07 After 24 hours 0.22a ± 0.10 0.64b ± 0.28 0.47c ± 0.18 After 6 days 0.24a ± 0.14 0.67b ± 0.35 0.69b ± 0.25 After 22 days 0.24a ± 0.12 0.66b ± 0.31 0.48c ± 0.12 MDA urine excretion, nmol/24 hour: At the beginning 2 225 ± 1 261 2 193 ± 1 089 2 315 ± 877 After 24 hours 2 115a ± 652 8 589b ± 1 679 7 363b ± 1 519 After 6 days 2 519a ± 1 126 10 067b ± 5 342 8 659b ± 5 279 After 22 days 3 402a ± 1 421 20 588b ± 11 362 10 704ab ± 5 100 Table 2: Effect of high polyunsaturated fat and vitamin E intake on plasma malondialdehyde concentration and malondialdehyde excretion in urine during the experiment Preglednica 2: Vpliv zauživanja večkrat nenasičenih maščobnih kislin in vitamina E na koncentracijo malondialdehida v krvni plazmi in količino dnevno izločenega malondialdehida v obdobju poskusa a, b Means with different superscripts in the same line differ significantly; P≤0.05 Group LowFat HighFat HighFat+Vit-E Percentage of DNA in head of comets in lymphocytes At the beginning 95.1 ± 0.83 94.9 ± 1.30 94.9 ± 0.66 After 24 hours 93.6a ± 1.06 91.9b ± 1.71 93.9a ± 1.14 After 6 days 93.3a ± 0.94 88.3b ± 0.68 92.9a ± 0.77 After 22 days 91.6a ± 1.85 82.7b ± 2.72 91.3a ± 0.93 Table 3: The percentage of DNA in head of comets in lymphocytes during the experiment Preglednica 3: Odstotek DNK v glavi kometov v limfocitih v obdobju poskusa a, b Means with different superscripts in the same line differ significantly; P≤0.05 Acta agriculturae Slovenica, 94/2 – 2009 107 TIME DEPENDENT FORMATION OF MARKERS OF … SUPPLEMENTED OR UNSUPPLEMENTED WITH VITAMIN E IN PIGS was significantly higher. At this time MDA excretion with urine in the vitamin E supplemented HighFat+Vit- E group did not significantly differ from either the Low- Fat or the HighFat group. Also the value for plasma MDA concentration was in between that of the other two groups. In this case the difference from the LowFat and to HighFat groups was significant. 3.2 NUCLEAR DNA DAMAGE OF LYMPHOCYTES AND GRANULOCYTES The results of DNA damage of lymphocytes and granulocytes are presented in Table 3 and 4 as a percent- age of DNA in the head of the comet. The experiment confirmed that a high proportion of polyunsaturated fat in the diet (group HighFat) in- creased lymphocyte and granulocyte DNA damage. An absolutely small, but significant decrease of degree of lymphocytes DNA damage was observed even after 24 hours. In both types of cells the effect was more strongly expressed after six days. While the decrease in the per- centage of DNA in the head of lymphocyte and granu- locyte on the 6th day was similar, on the 22nd day of the experimental period the percentage of lymphocyte DNA in the head was lower. In both types of cells degree of lymphocytes DNA damage in the HighFat+Vit-E group remained on the same level during the experiment as in the LowFat group. 3.3 VITAMIN E CONCENTRATION IN PLASMA Plasma concentrations of α- and β+γ-tocopherol throughout the experiment are reported in Table 5. Dur- ing the experimental period there was a significant effect of the type of diet consumed. The effect was observed even after 24 hours. While the plasma α–tocopherol con- centration in the LowFat and HighFat groups was un- changed during the whole experimental period, the con- Group LowFat HighFat HighFat+Vit-E Percentage of DNA in head of comets in granulocytes At the beginning 92.9 ± 1.2 92.6 ± 1.9 93.0 ± 2.2 After 24 hours 92.0 ±0.45 91.0 ± 2.15 91.7 ± 0.65 After 6 days 92.3a ±1.0 88.6b ± 0.4 92.0a ± 1.1 After 22 days 92.3a ± 1.8 87.6b ± 2.0 91.7a ± 1.2 Table 4: The percentage of DNA in head of comets in granulocytes during the experiment Preglednica 4: Odstotek DNK v glavi kometov v granulocitih v obdobju poskusa a, b Means with different superscripts in the same line differ significantly; P≤0.05 Group LowFat HighFat HighFat+Vit-E α–tocopherol (ppm) At the beginning 2.46 ± 1.01 2.45 ± 1.03 2.17 ± 0.41 After 24 hours 2.21a ± 1.12 2.02a ±0.79 3.23b ± 1.14 After 6 days 2.08a ± 0.80 2.02a ± 0.93 5.33b ± 1.93 After 22 days 1.83a ± 0.87 1.86a ± 0.99 4.53b ± 1.37 β+γ-tocopherol (ppm) At the beginning 0.034 ± 0.011 0.041 ± 0.019 0.051 ± 0.029 After 24 hours 0.032a ± 0.013 0.249b ± 0.0114 0.252b ± 0.112 After 6 days 0.034a ± 0.013 0.180b ± 0.108 0.123c ± 0.097 After 22 days 0.029a ± 0.015 0.237b ± 0.198 0.135c ± 0.068 Table 5: Concentration of α- and β+γ-tocopherol in plasma during the experiment Preglednica 5: Koncentracija α- in β+γ-tokoferola v plazmi v obdobju poskusa a, b, c Means with different superscripts in the same line differ significantly; P≤0.05 Acta agriculturae Slovenica, 94/2 – 2009108 T. PAJK ŽONTAR et al. centration significantly increased in the HighFat+Vit-E group even 24 hours after the beginning of the experi- ment and remained so afterwards. The concentration of β+γ-tocopherol in plasma in the LowFat group remained at the same level during the whole experimental period. β+γ-tocopherol con- centration significantly increased in the HighFat and HighFat+Vit-E groups even 24 hours after dietary inter- vention and remained so also on the 6th and 22nd days of the experiment. At this time the concentration in the HighFat+Vit-E group was significantly lower than in the HighFat group. 4 DISCUSSION The time dependent formation of oxidative stress induced by a high dietary intake of polyunsaturated fat is currently not well known. At the same time, the protec- tive effect of vitamin E in this process has not yet been elucidated. In the present study oxidative stress was induced by the selection of linseed oil which contains 73 wt. % of PUFA (Rezar et al., 2003). The energy supply from PUFA was approximately 19% (Table 1). It is known that a high intake of PUFA increases the nutritive requirements for antioxidative vitamins (Muggli, 1994). The oxidative stress in both groups fed linseed oil was additionally in- creased by the fact that the supply of supplemented anti- oxidative vitamins was not increased. As expected, feeding linseed oil in the HighFat group increased the oxidative stress by increasing not only the formation of products of lipid peroxidation but also the rate of blood cell DNA damage. The increased presence of MDA in plasma and urine reflects the products of li- pid oxidation originating from diet and formed in the tissues (Guichardant et al., 1994). Our previous studies (Rezar et al., 2003) showed plasma MDA concentration and especially MDA excreted in the urine to be sensitive biochemical markers of the extent of lipid peroxidation. A study by Marnett (2002) found that lipid peroxi- dation is one of the major sources of endogenous DNA damage in humans that may contribute to cancer and other chronic diseases linked to lifestyle and dietary fac- tors. The results of the present experiment show that the high intake of PUFA in the HighFat group significantly increased not only the concentration of MDA in blood plasma and the urinary MDA excretion rate but also the degree of both lymphocyte and granulocyte DNA dam- age. The results obtained clearly demonstrate the harmful effects of polyunsaturated fat in the diet on the oxidative status of pigs, which in view of their metabolism and di- gestion may serve as a good model for humans (Darcy- Vrillon et al., 1993). While the effect of oxidative stress induced by a high fat intake on lipid peroxide formation has already been shown (Yang et al., 1997; Rezar et al., 2003), the present study is, to our knowledge, the first to demonstrate in vivo time dependent effects and the different effects on MDA formation and lymphocyte and granulocyte DNA integrity. As assumed, the degree of lymphocyte and granulo- cyte DNA damage was not the same. According to their different physiological roles and life span, these two cell types can exhibit rather different sensitivities to chemi- cal, physical or biological insults in DNA damage (Gio- vannelli et al., 2003) and probably different DNA repair capacities (Šrám et al., 1998). A difference between the DNA damage of lymphocyte and granulocyte was ob- served even after 24 hours and was even greater on the 22nd day of the experiment (Table 2, 3). In the present study, lymphocytes, which have a longer life-span, were found to respond faster and to accumulate DNA damage with prolonged PUFA exposure. The rate of DNA dam- age in lymphocytes increased with prolonged oxidative stress. Tice (1995) found that short-lived granulocytes may provide information only on current exposure whereas lymphocytes might also give information on past exposure. The results thus provide some evidence that lymphocytes are more sensitive to oxidative stress caused by PUFA than granulocytes, as a result of their DNA repair system and/or longer life span. While the level of lymphocyte and granulocyte DNA damage did not change 24 hours after the beginning of the experiment, the plasma MDA concentration and urine MDA excretion rate already at that time showed a significantly higher rate of lipid oxidation (Table 4). This indicates that at least in the early stage of such a type of increased oxidative stress MDA measurements are more sensitive parameters of increased oxidative stress. Dur- ing the experiment the MDA concentration in the blood and the rate of MDA excretion with urine increased. It is obvious that the increase in the latter was much more pronounced and correlates better with the rate of DNA damage of lymphocytes than granulocytes. Determination of α- and β+γ-tocopherols in plasma may contribute information on the antioxidant status of an individual and may be useful for evaluation of nutri- tional status and risk of degenerative diseases (Aust et al., 2001). It is known from other investigations (Mileva et al., 2002) that as a consequence of increased oxidative load occurs a decrease in the concentration of antioxida- tive substances in the blood. On that account it was ex- pected that the concentration of a-tocopherol in plasma would decrease in the HighFat group. But that was not Acta agriculturae Slovenica, 94/2 – 2009 109 TIME DEPENDENT FORMATION OF MARKERS OF … SUPPLEMENTED OR UNSUPPLEMENTED WITH VITAMIN E IN PIGS the case. The reason for this might be a low a-tocopherol concentration of the basal diet and might indicate that in the experiment the applied NRC (1998) recommen- dations are too low. Since linseed oil is a poor source of a-tocopherol (8.59 mg/100 g) and a good source of β+γ- tocopherol (106, 93 mg/100 g), an increase in plasma β+γ-tocopherol concentration in the HighFat group was expected and actually observed. As expected, the consumption of vitamin E in the form of α-tocopherol in the HighFat+Vit-E group also significantly increased the concentration of α-tocopherol in plasma during the whole course of the experiment (Ta- ble 5). The increase in plasma α-tocopherol was also pos- itively associated with the observed parameters of oxida- tive stress (Tables 2, 3, 4). The degree of lymphocyte and granulocyte DNA damage in the HighFat+Vit-E group was significantly lower than in the HighFat group. More- over, the degree of lymphocyte and granulocyte DNA damage in the HighFat+Vit-E group was at the same level as in the LowFat group and was not influenced by the high unsaturated fat intake during the whole experimen- tal period. The positive effect of vitamin E on oxidative stress was also demonstrated as a reduction in MDA con- centration in plasma and urine (HighFat+Vit-E group) (Table 4). But in contrast to the protective effect in blood cells, a significant protective effect was not observed ear- lier than at the 22nd day of the experiment. At this point the vitamin E supplementation was able to reduce MDA formation by approximately 50%. Some other investiga- tors also found that supplementing the diet with vitamin E reduces the plasma or urinary MDA level as well as liver MDA concentration (Cadenas et al., 1996; Naidoo et al., 1998; Kirimlioglu et al., 2006). Since the amount of MDA excreted in the urine correlates positively with its synthesis in the body (Siu and Draper, 1982), and the measurement of urinary-excreted MDA is a more precise indicator of the plasma MDA concentration (Guichard- ant et al., 1994; Kosugui et al., 1994), the reduced oxida- tive load in the vitamin E supplemented group could be regarded as even more important. 5 CONCLUSIONS The results confirmed that a high proportion of polyunsaturated fat (PUFA) in the diet increased the measured parameters of oxidative stress. The lym- phocytes proved to be a more sensitive indicator of this type of oxidative stress but the difference was observed only after longer exposure to this type of oxidative stress (22 days). 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Effect of di- etary fiber on antioxidation in rats. Journal of Hygiene Re- search, 26: 318–320 Wong S.H.Y., Knight J.A., Hopfer S.M., Zaharia O., Leach C.N., Sunderman F.W.J. 1987. Lipoperoxides in plasma as mea- sured by liquid-chromatographic separation of malondi- aldehyde – thiobarbituric acid adduct. Clinical Chemistry, 33: 214–220 Acta argiculturae Slovenica, 94/2, 111–119, Ljubljana 2009 COBISS: 1.01 Agris category code: L51 EFFECTS OF DIETARY PECTIN ON PROTEIN DIGESTION AND METABOLISM IN GROWING RATS Tatjana PIRMAN 1, Philippe PATUREAU MIRAND 2, Andrej OREŠNIK 3, Janez SALOBIR 3 Delo je prispelo 16. septembra 2008, sprejeto 15. septembra 2009. Received September 16, 2008; accepted September 15, 2009. 1 Univ. of Ljubljana, Biotehnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia, e-mail: tatjana.pirman@bfro.uni-lj.si 2 INRA, Unité Nutrition Humaine, INRA Clermont-Ferrand – Theix, F-63122 Theix, France 3 Same address as 1, Prof., Ph.D. Effects of dietary pectin on protein digestion and metabolism in growing rats In an attempt to clarify the relationships between the digestive and metabolic effects of additional feeding of citrus pectin, the intestinal contents, tissues weights, nitrogen excre- tion and retention were studied in laboratory rats. Twenty-four growing male Wistar rats (98.8 g ± 5.3 g of body weight) were fed ad libitum for 20 days with balanced diets containing casein as the source of protein. In the experimental diet 96 g of wheat starch was replaced by 80 g of citrus pectin and 16 g of vegetable oil. Apparent digestibility and apparent protein biological value were calculated, the weights of digestive tissues and digestive organ content weights were also determined. All tissues of dif- ferent parts of the digestive tract were heavier in the pectin fed group, and small intestine and caecum were statistically sig- nificant different as compared to control group. The contents of the small intestine and caecum were significant heavier in the pectin group. Pectin significantly lowered dry matter intake and growth rate and significantly increased faecal excretion of nitrogen and significantly decreased urinary nitrogen excre- tion. The consequence of alteration in the nitrogen excretion route was significantly lower apparent protein digestibility and apparent net protein utilisation, but apparent protein biological value was unchanged. Key words: proteins / digestibility / metabolism / pectin / small intestine / large intestine / laboratory rats Vpliv pektina na prebavo beljakovin in metabolizem pri rasto- čih podganah Da bi poskusili razložiti povezavo in vpliv krmljenja pek- tina iz limonine lupine na prebavo in presnovo, smo izmerili maso vsebine prebavil, maso tkiv prebavil ter izločen in absor- biran dušik pri laboratorijskih podganah. Štiriindvajset rastočih laboratorijskih podgan moškega spola seva Wistar (s povpreč- no telesno maso 98,8 g ± 5,3 g) smo 20 dni krmili ad libitum z uravnoteženo krmo, ki je vsebovala kazein kot vir beljakovin. V poskusni krmi smo 96 g pšeničnega škroba zamenjali z 80 g pektina iz limonine lupine in 16 g mešanice rastlinskih olj. Izračunali smo navidezno prebavljivost beljakovin in navide- zno biološko vrednost beljakovin ter določili maso posameznih tkiv in vsebine prebavil. V poskusni skupini, ki je imela v krmi pektin, smo izmerili večjo maso tkiv posameznih delov prebavil v primerjavi s kontrolno skupino, s statistično značilnimi raz- likami pri tankem in slepem črevesju. Masa vsebine tankega in debelega črevesja je bila statistično značilno večja pri skupini s pektinom. V skupini s pektinom v krmi so živali zaužile zna- čilno manj suhe snovi krme in imele manjše priraste, značilno več izločenega dušika v blatu, pa tudi značilno manj izločenega dušika preko seča. Posledica razlik v izločanju dušika preko bla- ta ali seča je značilno manjša navidezna prebavljivost in navi- dezna neto izkoristljivost beljakovin, medtem, ko je navidezna biološka vrednost beljakovin ostala nespremenjena. Ključne beseda: beljakovine / prebavljivost / presnova / pektin / tanko črevo / debelo črevo / laboratorijske podgane Acta agriculturae Slovenica, 94/2 – 2009112 T. PIRMAN et al. 1 INTRODUCTION Pectin is a component of many feedstuffs. Especial- ly citrus and sugar beet pulp are rich in pectin and to a limited degree it is present in grains and legumes. Pectin improves the stability and tight connection of plant cells, their osmolality and water content, and together with hemicellulose, cellulose and lignin it reinforces the cell matrix. The name pectin is derived from the Greek word “pectos”, which means gelatinizing or swelling and is re- lated to the main physical and chemical character of this molecule in establishing gels. Most authors point out the resistance of pectin to the digestive enzymes of mammals and include it among the non-starch polymers (East- wood, 1992). A logical approach is the classification of pectin as part of dietary fibre (Trowell et al., 1976; East- wood, 1978; Schneemann, 1986; McDougall et al., 1996), but only a small part of pectin belongs to the cell wall. The rest is part of the cell cytoplasm and most fractions of pectin are soluble. After isolation, pectin can be used as a special dietetic feed composition. A large number of investigations have been carried out to study the effect of dietary fibre on the digestibility of nutrients. Most authors report that the fibre content of diet can impair the apparent digestibility of nutrients. In particular, the effect of dietary fibre differs with the source and nature of the fibre and relates to its chemi- cal composition as well as to its physical and chemical properties. The effect of the viscous nature of dietary fi- bre, like pectin, on digestibility is contradictory. Already Murray et al. (1977) reported a decrease in the apparent digestibility of nitrogen in pigs after feeding gel-forming polysaccharides (methyl-cellulose or pectin), whereas the replacement of starch by cellulose caused no de- crease. Other authors observed no effect of gel-forming polysaccharides on nitrogen digestion in rats (Larsen et al., 1994) and in pigs (Li et al., 1994). Soluble polysaccha- rides have been found generally to be without significant effect on the apparent digestibility of nutrients in pigs (Huisman et al., 1985). For other authors, not only the amount of nitrogen excreted in faeces, but also the ni- trogen excreted in urine was found to be affected by the type and fermentability of carbohydrate. Pastuszewska et al. (2000a) found that faecal nitrogen excretion in rats was increased by all carbohydrates (potato starch, pectin or cellulose) when substituted for cereal starch, but only pectin decreased urinary nitrogen excretion. Carbohy- drates significantly altered the routes of nitrogen excre- tion in protein-free diets too (Pastuszewska et al., 2000b). The present study was designed to examine the ef- fect of pectin on protein utilisation and on the develop- ment of the gastrointestinal tract to obtain a better pic- ture of the nutritive functions of pectin in growing rats. 2 MATERIAL AND METHODS 2.1 DIETS Two diets were prepared, a control diet and a pectin diet in which a fraction of the wheat starch was replaced by pectin from citrus peel (Fluka ref. No. 76280, degree of esterification 63–66%, MW 30 000–100 000). The diets contained different amounts of an oil mixture to adjust the diets’ energy concentrations. The protein source in the diets was casein purchased from Union des Caséiner- ies de Charente Maritime (La Rochelle, France) and the diets were calculated to contain 110 g of crude protein. Diets (Table 1) were designed to meet the nutritional re- quirements of growing rats (NRC, 1995). Weende analy- sis, mineral content and dietary fibre analysis (Lee et al., 1992) of the diets were performed. 2.2 ANIMALS AND EXPERIMENTAL PROCE- DURE All procedures were performed according to cur- rent legislation on animal experimentation in Slovenia. Permission for the experiment was granted by the Veteri- nary Administration of the Republic of Slovenia (VURS) under the number 323-02-215/2004/2. Twenty four male Wistar rats (98.8 g ± 5.3 body weight) reared in the Lek laboratory animal unit (Ljubljana, Slovenia) were housed in cages placed in a room kept at about 21 °C and 60% humidity (checked and recorded each day), with light automatically regulated on a 12-hour light/dark cycle starting at 7.00 a.m. After a 4 days adaptation period, in which the rats received a control diet, rats were separated into two equal groups (n = 12), with average body weight 120.1 ± 5.5 g and 120.1 ± 5.2 g in the control and pectin groups, respectively. Animals were individually housed in metabolic cages which permitted the collection of urine and faeces separately during the experiment, and had free access to drinking water. They received ad libi- tum control or pectin semi-synthetic diet during an 18 or 20-day period. On the 6th day of the experimental period the 5-day balance study began (Orešnik and Cvirn, 1984; Orešnik et al., 1982; Stekar et al., 1984). Each day animals received a new weighed daily meal and the residue from the day before was weighed. Body weights were recorded on the first day of the balance experiment, on the third day and on the last day. Urine was collected in a bottle after filtra- Acta agriculturae Slovenica, 94/2 – 2009 113 EFFECTS OF DIETARY PECTIN ON PROTEIN DIGESTION AND METABOLISM IN GROWING RATS tion. This bottle contained 10 ml of 6 M HCl for each cage to stop all reactions in the urine and to prevent losses of nitrogen. The faeces samples were collected in a different vessel. On the last day (5th) of the balance study, the urine was transferred to a prepared plastic bottle, weighed and stored at −20  °C until analyses were performed. Fae- ces samples were also stored in prepared plastic bottle, weighed and frozen. Before taking an aliquot of the sample for analysis, faeces were homogenised in a ceramic holder. Urine was homogenised by shaking to prevent stratification. In di- ets, faeces and urine nitrogen was determined by the Kjel- dahl method. Dietary crude protein (CP) was evaluated as N × 6.25. Dry matter and crude ash were determined in diets and faeces. Based on the amount of N intake, N in faeces and N in urine, the following indices of N uti- lisation were calculated: nitrogen apparent digestibility = (N intake − N in faeces) / N intake; apparent protein bio- logical value = (N intake − N in faeces − N in urine) / (N intake − N in faeces) and apparent net protein utilisation (NPU) = (N intake − N in faeces − N in urine) / N intake; protein efficiency ratio (PER) = growth rate / CP intake. Digestibility of organic matter (OM) = OM intake − OM in faeces / OM intake and dry matter efficiency: growth rate / dry matter intake × 100 were also calculated. 2.3 SAMPLING OF TISSUES Half of the animals in each group were sacrificed on the 18th day of the experiment and the other half on the 20th day in the morning between 9 and 11 o’clock. Rats were anaesthetized with an abdominal injection of Pen- tothal (0.1 ml/100 g of body weight, Sigma, Saint Louis, Mo., USA). The digestive tract was quickly removed. Each part of the digestive tract (stomach, small intestine, colon and caecum) was weighed with its content. The di- gestive content of the small intestine was collected. The small intestine wall was rinsed with a cold 2% TCA so- lution, wiped and weighed. Stomach, colon and caecum treatment was similar to treatment of the small intestine. 2.4 DATA ANALYSIS Data were analysed by the General Linear Models (GLM) procedure (SAS/STAT, 2000), taking into consid- eration the diet as the main effect, and in the case of the relative weight of intestinal tissues and gastrointestinal content also the day of slaughter. Data are expressed as least square means (LSM) ± standard deviation (SD). Sig- nificance was considered established at P < 0.05. Table 1: Diets (g/kg) Preglednica 1: Krma (g/kg) Control Kontrola Pectin Pektin Casein / Kazein 120 120 Wheat starch / Pšenični škrob 616 520 Premix / Premiks 1 40 40 Sugar (sucrose) / Sladkor (saharoza) 50 50 Mixture of vegetable oils Mešanica rastlinskih olj 2 54 70 Agar-agar / Agar-agar 40 40 Pectin / Pektin 0 80 Mineral mixture Mešanica rudninskih snovi 3 70 70 Vitamin mixture Mešanica vitaminov4 10 10 Sum / Vsota 1000 1000 1 Mixture of wheat starch and L-cystine 55 mg/g (2.2 g of cystine added in each diet) / Mešanica pšeničnega škroba in L-cistina 55 mg/g (2,2 g cistina dodanega vsaki krmi) 2 Rape oil, groundnut oil and sunflower oil (50:45:5) / Repično olje, arašidovo olje in sončnično olje (50:45:5) 3 Minerals UAR 205 b (UAR, Villemoisson-sur-Orge, France) / Rudnin- ske snovi UAR 205 b 4 Vitamins UAR 2.00 / Vitamini UAR 2,00 Control / Kontrola (12) Pectin / Pektin (12) P-value / p-vrednost * Stomach / Želodec 0.63 ± 0.14 0.66 ± 0.06 0.5410 Small intestine / Tanko črevo 2.94 ± 0.29 3.63 ± 0.84 0.0151 Caecum / Slepo črevo 0.41 ± 0.07 0.65 ± 0.10 < 0.0001 Colon / Kolon 0.63 ± 0.07 0.70 ± 0.11 0.0656 Whole intestine / Celotno črevesje 3.97 ± 0.33 4.97 ± 0.88 0.0016 Digestive tract / Prebavni trakt 4.60 ± 0.42 5.63 ± 0.88 0.0016 Table 2: Relative weight of digestive tissues (g per 100 g of the body weight) (average ± SD) Preglednica 2: Relativna masa tkiv prebavnega trakta (g / 100 g telesne mase) (povprečje ± SD) * Diet and day of slaughtering as two main effects / Krma in dan žrtvovanja kot dva glavna vpliva Acta agriculturae Slovenica, 94/2 – 2009114 T. PIRMAN et al. 3 RESULTS In Table 2 the weights of different organs of the di- gestive tract are expressed as relative weights (g/100 g of body weight) to minimise the body size effect. The av- erage relative weights of the small intestine and caecum were significantly (P < 0.05) higher in the pectin group as compared to the control. The relative weight of the stomach was similar in both groups. The average colon relative weight was higher in the pectin group than in the control, but the difference was not significant. The average relative weight of the whole intestine or whole digestive tract was also significantly (P < 0.05) higher in the pectin group. Similar results were found for the weights of the di- gestive contents. There was significantly (P < 0.05) more digestive contents in the small intestine, caecum and whole intestine in the pectin fed rats compared to the control group (Table 3). After the 5-day pre-experimental period, the aver- age body weight of animals in the pectin group was sig- nificantly (P < 0.05) lower as compared to the control group. Since the dry matter intake in pectin group was lower all the time of experimental period, the difference become significant already at the beginning of the bal- ance experiment (15.9 g ± 11.04 g ) and increased (30.7 g ± 13.98 g) until the end of the balance experiment (Table 4). The result was significantly lower growth rate in pec- tin group as compared to control group. The problem of different body weight could be minimise, if animals of the control group have some lower body weight at the beginning of experimental period, but in this case the age of animals will be different and the difference in the growth rate even bigger. The differences in the dry matter efficiency and PER value were also significantly lower in the pectin group than in the control group. Since the dry matter intake was lower in the pectin group, the nitrogen intake was also significantly (P < 0.05) lower than in the control group (Table 5). The amount of faeces excreted per day, the amount of N excreted in faeces per day and the amount of urine excreted per day were significantly (P < 0.05) higher in the pectin group as compared to the control group. On the other hand, the nitrogen excreted in urine was significantly lower in Control Kontrola (12) Pectin Pektin (12) P-value p-vrednost * Content of stomach / Vsebina želodca 4.40 ± 5.71 3.06 ± 3.08 0.1634 Content of small intestine / Vsebina tankega črevesja 1.42 ± 0.78 3.34 ± 2.57 0.0069 Content of caecum / Vsebina slepega črevesja 2.36 ± 0.72 3.91 ± 0.72 < 0.0001 Content of colon / Vsebina kolona 1.44 ± 0.58 1.60 ± 0.50 0.4928 Content of whole intestine / Vsebina celotnega črevesja 5.23 ± 1.40 8.85 ± 3.07 0.0002 Content of digestive tract / Vsebina prebavnega trakta 9.70 ± 6.21 11.91 ± 5.69 0.1028 Table 3: Weights (g) of the gastrointestinal content (average ± SD) Preglednica 3: Mase (g) vsebine prebavil (povprečje ± SD) * Diet and day of slaughtering as two main effects / Krma in dan žrtvovanja kot dva glavna vpliva Control Kontrola (6) Pectin Pektin (6) P-value p-vrednost Initial body weight (g) / Telesna masa ob začetku poskusa (g) 149.3 ± 7.11 133.4 ± 6.27 0.0021 Final body weight (g) / Telesna masa ob koncu poskusa (g) 179.2 ± 8.10 148.5 ± 7.22 < 0.0001 Dry matter intake (g DM/day) / Zaužita suha snov (gSS/dan) 17.0 ± 0.70 13.9 ± 0.46 < 0.0001 Growth rate (g/day) / Prirast (g/dan) 6.0 ± 0.51 3.0 ± 0.40 < 0.0001 DMI/average body weight (g/g) / ZSS/povprečno telesno maso (g/g) 0.103 ± 0.004 0.099 ± 0.005 0.0734 Dry matter efficiency (%) / Izkoristek suhe snovi krme (%) 35.22 ± 2.34 21.75 ± 2.32 < 0.0001 PER (g growth/g CP) / PER (g prirasta/g SB) 3.0 ± 0.20 1.6 ± 0.20 < 0.0001 Table 4: Body weight, dry matter intake and growth rate (average ± SD) in 5 days balance measurements Preglednica 4: Telesna masa, zaužita suha snov in prirast (povprečje ± SD) v 5 dneh bilančnih meritev DMI – dry matter intake / ZSS – zaužita suha snov; PER – protein efficiency ratio / učinkovitost beljakovin za prirast; CP – crude protein / SB – surove beljakovine Acta agriculturae Slovenica, 94/2 – 2009 115 EFFECTS OF DIETARY PECTIN ON PROTEIN DIGESTION AND METABOLISM IN GROWING RATS the pectin group. Consequently, the nitrogen balance was significantly decreased in the pectin group (on average only 71.5% of the value in the control group). Results ex- pressed per 1 g of average body weight show significant increase excretion of N in faeces (for 84%) and decrease consumed N (for 10%) and N balance (for 16%) in pec- tin group, but no significant differences in excretion of N through urine. The apparent protein digestibility, digestibility of dry matter and organic matter decreased significantly (P < 0.0001) as a result of pectin addition to the diet (Ta- ble 5). On the contrary, the apparent biological value of protein was not affected by pectin, the average values were not different in the two groups (P = 0.4519), but apparent net protein utilisation was also significantly de- creased in the pectin group as compared to the control. 4 DISCUSSION It is well recognized that dietary non-starch polysac- charides, especially soluble ones, such as pectin, can de- crease the apparent digestibility of whole protein or of amino acids in pigs (de Lange et al., 1989; Mosenthin et al., 1994; Zhu et al., 2005; Libao-Mercado et al., 2006). Such effects are likely to be related to endogenous ni- trogen losses, which can be seen in increased secretion and impaired reabsorption in the lower part of the gas- trointestinal tract in pigs (Grela et al., 1998) and in rats (Larsen et al., 1993), and in stimulation of the rate of mi- crobial fermentation in the gut of monogastric animals (Eggum, 1995; Schulze et al., 1995; McCullough et al., 1998). The present study demonstrates that the course of Control Kontrola (6) Pectin Pektin (6) P-value p-vrednost N intake (mg/day) Zaužiti N (mg/dan) 323 ± 13 259 ± 9 < 0.0001 N intake/average body weight (mg/g) Zaužiti N/povprečno telesno maso (mg/g) 2.0 ± 0.1 1.8 ± 0.1 0.0188 Excreted faeces (g fresh mass/day) Izločeno blato (g svežega blata/dan) 2.4 ± 0.2 3.1 ± 0.6 0.0016 N in faeces (mg/day) N v blatu (mg/dan) 28.0 ± 3.2 44.1 ± 5.9 0.0002 N in faeces/average body weight (mg/g) N v blatu/povprečno telesno maso (mg/g) 0.171 ± 0,023 0.314 ± 0.050 < 0.0001 Excreted urine (g/day) Izločen seč (g/dan) 19.5 ± 2.9 24.4 ± 2.7 0.0120 N in urine (mg/day) N v seču (mg/dan) 86.8 ± 9.3 66.2 ± 7.1 0.0015 N in urine/average body weight (mg/g) N v seču/povprečno telesno maso (mg/g) 0.529 ±0.053 0.471 ± 0.057 0.0980 N balance (mg/day) Bilanca N (mg/dan) 208 ± 10 149 ± 13 < 0.0001 N balance/average body weight (mg/g) Bilanca N/povprečno telesno maso (mg/g) 1.27 ±0.05 1.06 ± 0.08 0.0003 Apparent digestibility of protein (%) Navidezna prebavljivost beljakovin (%) 91 ± 1 83 ± 2 < 0.0001 Digestibility of organic matter (%) Prebavljivost organske snovi (%) 95 ± 1 92 ± 1 < 0.0001 Apparent protein biological value (%) Navidezna biološka vrednost beljakovin (%) 71 ± 2 69 ± 4 0.4519 Apparent net protein utilisation (%) Navidezna neto izkoristljivost beljakovin (%) 64 ± 2 57 ± 4 0.0047 Table 5: Balance experiment, digestibility, protein biological value and apparent net protein utilisation (average ± SD) Preglednica 5: Bilančni poskus, prebavljivost, biološka vrednost beljakovin in navidezna neto izkoristljivost beljakovin (povprečje ± SD) N – nitrogen / dušik Acta agriculturae Slovenica, 94/2 – 2009116 T. PIRMAN et al. digestion and also the enlargement of the intestinal tis- sues are influenced by pectin. It has been found that the addition of pectin to the diet significantly increases endogenous nitrogen flow in rats (Pastuszewska et al., 2000b) and in pigs (de Lange et al., 1989; Libao-Mercado et al., 2006) which includes digestive gland secretions, desquamated cells from ac- tive replacement of the gastrointestinal mucosal lining, and secretion of plasma components (urea and a small amount of plasma protein) (Shah et al., 1982). Indeed pectin has been shown to stimulate enterocyte turno- ver (Fukunaga et al., 2003; Chun et al., 1989), and may increase mucin secretions because of stimulation by the increase in caecal short chain fatty acid production (Bar- celo et al., 2000) in laboratory rats. The effect of pectin on protein flow (of dietary and endogenous origin) could also be the result of an interfer- ence with luminal protein digestion either of dietary or of endogenous origin, as shown for other dietary fibers that enhance endogenous nitrogen secretion (Schulze et al., 1995). Endogenous amino acids may not be available for absorption because of the physical and chemical adsorp- tive properties of pectin (Souffrant, 2001). El Kossori et al. (2000) suggested that protein hydrolysis could be pre- vented by interactions between protein or enzyme and fiber without modification of the viscosity, and would depend on the kind of fiber. Animals producing more mucus could have a slower absorption rate, because it has been postulated that mucus contributes to the apparent thickness of the unstirred layer and affords protection to the mucosal surface (Nimmerfall and Rosenthaler, 1980). However, increased luminal mucin did not disturb glu- cose or ovalbumin absorption (Morita et al., 2006). Undigested endogenous or dietary nitrogenous compounds can be transformed by microorganisms in the large intestine. In the intestinal tract of pigs, mi- crobes degrade up to 90% of pectin by fermentation (Drochner et al., 2004). Such microbes use pectin as an energy source and also use most of the luminal nitrogen, which is consequently excreted in faeces. However, this phenomena cannot be responsible for the enlargement of excreted nitrogen in the faeces unless the increased microbial fermentation, evidenced by the increase in the production of short chain fatty acids (SCFA) in the large intestine (Pirman et al., 2007), induces a stimulation of endogenous nitrogen secretions. Beside that, SCFAs have some other important roles in the intestinal lumen. Re- search on rats showed that an increased concentration of SCFA in the intestinal lumen, because of the ingestion of non-starch polysaccharides or non-digestible oligosac- charides decreased the pH value in the large intestine, leading to the conversion of NH3 to NH4 +. This form of ammonia cannot diffuse through the intestinal wall (Younes et al., 1995), leading to the change of the nitro- gen metabolism, since the more nitrogen is excreted by faeces, the less nitrogen is transferred to urea in the liver and consequently less nitrogen is excreted by urine (Mo- senthin et al., 1992). On the other hand, where microbial fermentation is very intensive (because of the presence of soluble dietary fiber, like pectin), the utilization of urea by microflora in the intestinal lumen is increased and again nitrogen is excreted through faeces (microbial mass) and less nitrogen is excreted through urine. In the present study, the enlarged faecal nitrogen loss seems to be of endogenous origin. Indeed, faecal nitrogen loss was increased by 57% by pectin feeding, which is very similar to the increase described for endog- enous losses (53%) in rats fed a pectin nitrogen–free diet when compared with a control nitrogen-free diet (Pas- tuszewska et al., 2000b). This conclusion suggests that the increase in nitrogen faecal loss induced by pectin feeding is unlikely to result from an increase in faecal loss of di- etary nitrogen. Consequently most dietary amino acids in our experiment would have been absorbed as with the control diet. All the carbohydrates (potato starch, pectin, cel- lulose and tannic acid) used in the study on laboratory rats of Pastuszewska et al. (2000a) tended to decrease the blood urea nitrogen concentration as compared to the control (wheat starch as carbohydrate), and nearly 20% nitrogen excreted by urine in a control group (319 mg vs. 254 mg in urine of pectin group) was obviously metabo- lized in the large intestine (72 mg vs. 110 mg in faeces of pectin group). If such a proportion was applied in the present study, on average 16.54 mg of endogenous urea nitrogen (blood urea nitrogen excreted by faeces) would be redirected from urinary excretion to fecal excretion. This value is close to the difference between nitrogen fae- cal excretion in the pectin (28.00 mg/d) and control rats (44.05 mg/d). Nitrogen originating from endogenous urea may be a major component of the increase in nitro- gen faecal losses by pectin feeding. Another consequence of this exchange is that the assessments of protein nu- tritional value need to be corrected to take into account this exchange between urine and faecal nitrogen excre- tion. In this case, the corrected nitrogen digestibility be- comes 91.31% and 89.37% for the control and the pectin diets, respectively, while the corrected biological value becomes 70.58% and 64.21% in the control and pectin diets, respectively. Consequently the main difference in protein and diet nutritional value between the control and the pectin diets seems to be not at absorption level but rather in differences in urea excretion pathways and possibly in amino acid metabolism. This could be a consequence of an increase in the requirements for a specific nutrient (energy, amino ac- Acta agriculturae Slovenica, 94/2 – 2009 117 EFFECTS OF DIETARY PECTIN ON PROTEIN DIGESTION AND METABOLISM IN GROWING RATS ids like threonine) due to the high protein turnover in intestinal tissues observed in pectin fed rats, which may impair amino acid utilization in other organs (Pirman et al., 2007). Indeed the weights of intestinal tissues in rats were increased as a result of addition of pectin to the diet, as it was found in a previous study (Pirman et al., 2007). When different fibers were compared, the great- est enlargement of caecal digests and tissue was observed after addition of pectin to the diet (Pastuszewska et al., 2000a). This intestinal hypertrophy corresponded to hy- perplasia in the small intestine (Brown et al., 1979), to an increase in villous cell exfoliation and crypt cell prolifera- tion (Jacobs 1983), and to alteration of gut morphology by increasing the number of goblet cells and mucus pro- duction (Cassidy et al., 1981) in laboratory rats. In our previous study on addition of pectin to the diet of the laboratory rats (Pirman et al., 2007) the villous height in the small intestine and crypt depth in small intestine, caecum and colon were significantly increased as com- pared to the control diet. In a previous study, the strong stimulation of intestinal protein turnover corresponded to a slight decrease in muscle protein turnover (Pirman et al., 2008). In the study of Zhu et al. (2005) on pigs the utilization of threonine in whole body protein deposition was linearly decreased with the dietary pectin level, but not of lysine. This was connected to the high amount of threonine in mucoproteins, so the influence of pectin on digestive physiology operates through amino acid and nitrogen utilization at the whole body level. 5 CONCLUSIONS Pectin is an important factor affecting the propor- tions of faecal and urinary nitrogen excretion and ulti- mately both apparent protein digestibility and corrected apparent protein biological value. This effect is related to the fermentability of pectin, especially in the large intestine. Furthermore, the study confirmed the effects of pectin on digestive physiology, namely increased urea excretion from blood to intestine and reduced urea ex- cretion by urine. Both consequences (in digestive tract and in kidney function) of the presence of pectin in the diet are of benefit for health status in animals and men. 6 POVZETEK Pektin spada v skupino topne vlaknine in ga naj- demo v mnogih in različnih vrstah hrane in krme. V lit- eraturi najdemo različne in nasprotujoče učinke na preb- avljivost hranljivih snovi pri dodajanju topne vlaknine v obrok. V večini primerov povečajo izločanje dušika preko blata, a le redko zmanjšajo izločanje dušika preko seča. Z našo raziskavo smo želeli ugotoviti vpliv pektina na izkoristljivost beljakovin in na razvoj posameznih delov prebavil, s tem pa bi dobili boljšo predstavo o pre- hranski vlogi pektina. Pripravili smo dve krmi, v katerih je bil kazein kot vir beljakovin. V poskusni krmi smo del pšeničnega škroba zamenjali s pektinom iz limonine lu- pine in mešanico rastlinskih olj, da sta bili krmi izoen- ergijski. 24 rastočih laboratorijskih podgan moškega spola smo po 4 dnevih predposkusa razdelili v dve ho- mogeni skupini (telesna masa 120,1 g ± 5,5 g in 120,1 g ± 5,2 g za kontrolno in pektinsko skupino) in jih 20 dni ad libitum krmili z eno od pripravljenih krmnih mešanic in merili zauživanje krme in priraste. V času poskusa smo 5 dni zaporedoma ločeno zbirali blato in seč (bilančni poskus). Na koncu poskusa smo živali žrtvovali, odvzeli tkiva prebavil in jih stehtali polne in prazne. Izračunali smo navidezno prebavljivost beljakovin in organske snovi, navidezno biološko vrednost beljakovin in navi- dezno neto izkoristljivost beljakovin. Pektin vpliva na povečanje mase tkiv prebavil in vsebine prebavil, kar v največji meri velja za slepo črevo. Pektin je tudi pomem- bno vplival na porazdelitev izločenega dušika preko blata oz. preko seča, kar ima vpliv na navidezno prebavljivost beljakovin in korigirano biološko vrednost beljakovin. To je povezano s fermentabilnostjo pektina. Naši rezul- tati potrjujejo vpliv pektina na fiziologijo prebave, saj povečuje izločanje sečnine (dušika) preko krvnega ob- toka nazaj v prebavila in zmanjšuje izločanje le-te preko seča. Oboje ima ugoden vpliv na zdravstveno stanje živali in ljudi. 7 ACKNOWLEDGMENTS The authors are grateful to Christian Lafarge for diet preparation. Assistance in the laboratory provided by Marko Kodra is gratefully acknowledged. 8 REFERENCES Barcelo A., Claustre J., Moro F., Chayvialle J.A., Cuber J. C., Plaisancié P. 2000. Mucin secretion is modulated by lu- minal factors in the isolated vascularly perfused rat colon. Gut, 46: 218–284 Brown R.C., Kelleher J., Losowsky M.S. 1979. The effect of pec- tin on the structure and function of the rat small intestine. 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Journal of Animal Science, 83, 5: 1044–1053 Acta argiculturae Slovenica, 94/2, 121–131, Ljubljana 2009 COBISS: 1.01 Agris category code: L10 UPORABA DNA OZNAČEVALCEV ZA PREVERJANJE POREKLA PRI OVCAH IN DOLOČANJA OČETOVSTVA V ČREDAH Z VEČ PLEMENSKIMI OVNI Tomaž VOLČIČ 1 2, Drago KOMPAN 3, Peter DOVČ 4, Simon HORVAT 5 Delo je prispelo 16. septembra 2008, sprejeto 15. septembra 2009. Received September 16, 2008; accepted September 15, 2009. 1 Univ. v Ljubljani, Biotehniška fak., Odd. za zootehniko, Groblje 3, SI-1230 Domžale, Slovenija 2 Sedanji naslov: e-pošta: tomaz.volcic@go.kgzs.si 3 Isti naslov kor 1, doc. dr. e-pošta: drago.kompan@bfro.uni-lj.si 4 Isti naslov kor 1, prof., dr. e-pošta: peter.dovč@bfro.uni-lj.si 5 Isti naslov kor 1, izr. prof., dr. e-pošta: simon.horvat@bfro.uni-lj.si Uporaba DNA označevalcev za preverjanje porekla pri ovcah in določanja očetovstva v čredah z več plemenskimi ovni Rodovniški podatki kontroliranih tropov so bistvenega pomena za rejce ovc in izvajanje selekcijskih programov. V tej študiji smo preverili, če je možno z osmimi molekularno ge- netskimi označevalci ter uporabo računalniškega programa ATLAS preveriti skladnost podatkov v več-generacijskih ro- dovnikih jezersko-solčavske in oplemenjene jezersko-solčavske pasme. Ugotovili smo 90,9  % točnost beleženja rodovniških podatkov, medtem ko smo neskladja ugotovili pri štirih druži- nah od 44-ih. V dveh primerih je bil potomcu nepravilno pri- pisan oče ali mati, pri ostalih dveh primerih pa sta bila jagnjetu napačno pripisana oba starša. V drugem delu raziskave smo poskušali z istim nizom mikrosatelitnih označevalcev določiti očetovstva štirim jagnjetom istrske pramenke, kjer v času pa- ritev v trop pripuščajo dvajset ovnov in tako jagnje nima po- znanega očeta. Trem jagnjetom smo lahko nedvoumno določili očeta z izločitvijo ostalih devetnajstih. V enem primeru smo ugotovili, da niz osmih označevalcev ni dovolj informativen za določitev očeta potomcu, zato smo analizo razširili in z vklju- čitvijo dodatnih štirih mikrosatelitnih označevalcev uspešno določili očetovstvo tudi v tem primeru. Z izbranim nizom mo- lekularnih označevalcev in obdelave podatkov je možno učin- kovito preverjati obstoječe rodovniške podatke in napovedati očetovstvo jagnjetom v tropih, kjer pripuščajo večje število ov- nov. Rezultati take analize so lahko v pomoč rejcem in selekcij- skim službam za izboljšanje točnosti rodovniških podatkov in učinkovitejše načrtovanje reje in selekcije pri ovcah. Ključne besede: ovce / poreklo / očetovstvo / molekularna genetika / genetski označevalci Molecular genetics markers used for parentage verification and paternity determination in multiple-sire sheep pedigrees Pedigree data from recorded flocks are of importance for the proper flock management as well as for the selection pro- grammes and accurate performance prediction. In this study we examined if the use of eight molecular markers and com- puter analysis package ATLAS can be applied to verify known pedigrees of Jezersko-Solčava and Improved Jezersko-Solčava sheep breed from Slovenia. 90.9  % of pedigree data were in concordance with molecular genetic tests whereas in four pedi- grees discordant parentage tests were obtained. In two cases, a different father or mother was assigned, whereas in the other two pedigrees both parents were discordant with molecular test results. In the second part of this study we aimed to deter- mine the paternity for four lambs of Istrian Pramenka breed, in which a random mating scheme with 20 rams was used and hence the lambs had no father assigned. Using the same set of eight microsatellite markers, we were able to unequivocally de- termine paternity for 3 out of 4 lambs. In one case the analysis was not informative enough but with inclusion of 4 more mic- rosatellite markers its sire could be determined. With the cho- sen set of microsatellite markers and data analysis programme ATLAS it is therefore possible to efficiently perform pedigree data validation as well as paternity prediction for lambs from flocks, where a large number of rams are used in a random mating system. Applying such molecular tests could help sheep breeders in flock management and improve efficiency of selec- tion programmes. Key words: sheep / parentage / paternity / molecular ge- netics / genetic markers Acta agriculturae Slovenica, 94/2 – 2009122 T. VOLČIČ in sod. 1 UVOD Preverjanje porekla, še zlasti očetovstva, z uporabo molekularnih označevalcev je relativno uveljavljena me- toda pri nekaterih vrstah domačih živalih, kot so konji, govedo, psi in eksotične živali. Pri teh vrstah živali se je ugotavljanje porekla z uporabo molekularnih označeval- cev razvilo predvsem zaradi cenovne vrednosti plemen- skih živali ter zaradi omejitve pri trgovanju in prometu z le tistimi linijami živali, ki imajo predložen rodovniški certifikat. Velik pomen ima tudi pri laboratorijskih ozi- roma poskusnih živalih predvsem zaradi večje zaneslji- vosti in predstavljivosti rezultatov poskusa. Pri ovcah je imelo v preteklosti preverjanje porekla manjši pomen. Molekularno genetske študije so bile bolj v rabi za oceno genetske sorodnosti oziroma diverzitete populacij ovc. V sedanjem času pa, ko je vse več populacij v selekcijskih programih ter v programih obveznega genotipiziranja za prionski gen (PrP), bi bilo zaželeno imeti metode za do- datno preverjanje porekla. Na primer, v velikih čredah ovc, kjer v čredi plodi več ovnov hkrati, je celo nemogoče določiti očeta, poleg tega pa se tudi v tropih s kontroli- ranimi pripusti zgodi, da je zapisan napačen oče. Če je jagnje, ki ima dvomljiv rodovnik, nato izbrano za test, je zaželeno, da z dodatno molekularno genetsko meto- do ugotovimo poreklo vsaj po očetu. To je pomembno tako z vidika ocenjevanja plemenske vrednosti očeta kot nadzora inbridinga, torej zaradi izogibanja nadaljnjega parjenja v sorodstvu in zanesljivejše genotipizacije za PrP lokus. Preverjanje porekla, gensko kartiranje, forenzične preiskave in genska diagnostika so le nekatere izmed mo- žnosti za uporabo tipiziranja DNA (Dovč, 1994). Ker je identiteta očeta velikokrat bolj vprašljiva kot identiteta matere, je večina postopkov bolj usmerjena v preverjanje očetovstva. V ta namen uporabljamo molekularno ge- netske metode na osnovi genetskih označevalcev (Dovč, 1994). Genetski označevalec je lahko katerikoli odsek v genomu, kjer obstajajo razlike v zaporedju nukleotidov v DNA. Na ravni populacije se ta variabilnost kaže v razli- kah med osebki znotraj populacije. Vendar razlike nima- jo bistvenega vpliva na fenotip živali, kljub temu pa nam razkrivajo genetsko sorodnost med osebki. Pomembne značilnosti, s katerimi ocenjujemo informativne genetske označevalce, so velika stopnja variabilnosti, enakomerna in številčna razporeditev po genomu ter enostavnost in cenenost identifikacije ter uporabe. Mikrosatelitni ozna- čevalci izpolnjujejo vse te zahteve, in sicer so visoko va- riabilni, številni, razporejeni so naključno po genomu ter relativno tehnično nezahtevni (Kavar in Dovč, 1999). Z uporabo sodobne tehnologije za analizo mikrosatelitov lahko postopke standardiziramo in avtomatiziramo ter tako hitro in enostavno pridemo do rezultatov. Pri od- čitavanju genotipa (števila baznih parov) posameznega označevalca upoštevamo, da potomec podeduje dva ale- la, enega po očetovi, drugega pa po materini strani. Če posameznemu osebku združeno obravnavamo genotipe na več lokusih, dobimo sestavljen genotip, ki predstavlja molekularni opis osebka na izbranih lokusih. S primer- janjem sestavljenih genotipov osebkov, ki so domnevno sorodstveno povezani, lahko sorodstvene zveze potrdimo ali ovržemo. Z uporabo večjega števila mikrosatelitov (8 in več) se zelo zmanjša verjetnost, da bi imela dva osebka po naključju enak sestavljen genotip. S kombinacijo več visoko polimorfnih lokusov lahko dobimo specifičen ge- notip za vsako žival, razen za enojajčne dvojčke ali visoko inbridirane linije (Kavar, 2001). Informativnost oz. poli- morfnost označevalca v populaciji lahko predstavimo z vrednostjo PIC (Polymorphism Information Content), ki je odvisna od števila alelov na lokusu in porazdelitve ale- lov v populaciji. Pri ovcah je doslej znanih že skoraj tisoč mikro- satelitov (http://rubens.its.unimelb.edu.au/~jillm/jill. htm) in so jih že uporabili v študijah preverjanja pore- kla (Achmann in sod., 1998). V čredah drobnice, kjer je pri parjenju prisoten en plemenski oven, običajno ne prihaja do rodovniških neskladij vsaj glede določanja očetovstva.. Drugače je v rejah, kjer se v času pripustov velikih čred uporablja več plemenskih ovnov hkrati. V teh primerih lahko predstavlja rodovništvo in kontrola porekla problem. Take črede lahko z leti dosežejo visoko stopnjo inbridiranosti, čigar posledice se lahko kažejo v slabši plodnosti, slabši proizvodnosti, pogostejšem poja- vljanju raznih infektivnih in dednih bolezni, povezanih z večjo pogostostjo izražanja recesivnih mutacij (Laughlin in sod., 2003). Laughlin in sod. (2003) so v ta namen z uporabo mikrosatelitnih označevalcev ugotavljali očeto- vstvo na potomcih iz čred, kjer je bilo pri pripustih pri- sotnih več samcev hkrati. Ugotovitve raziskave kažejo, da so lahko ob primerjavi genotipov na treh mikrosatelitnih označevalcih lahko določili očeta 73 jagnjetom od 79 ja- gnjet (92  %), če so poznali tudi genotip matere. Kadar so bili genotipi označevalcev ocenjeni brez informacije genotipa matere, so lahko določili očeta 58 jagnjetom od 79 jagnjet (73 %). V tem primeru torej očetovstva niso mogli določiti pri 27 % potomcev. Najnovejše raziskave pri genetskem kontroliranju porekla drobnice so usmerjene v pomnoževanje večjega števila mikrosatelitnih lokusov hkrati v eni PCR reakciji. Do sedaj so bile pri ovcah izvedene kontrole porekla z mikrosateliti v laboratoriju Gruppi Sanguigni (LGS, Cre- mona, Italija), kjer so razvili dvodelni sistem, pri katerem so v eno PCR reakcijo v prvem setu združili 7, v drugem pa 8 mikrosatelitov (Glowatzki-Mulis in sod., 2007). Štu- dija je uspešno dodelila posamezne živali pravi pasmi. Ocenjena verjetnost, da sta genotipa dveh naključno iz- Acta agriculturae Slovenica, 94/2 – 2009 123 UPORABA DNA OZNAČEVALCEV ZA PREVERJANJE POREKLA PRI OVCAH ... V ČREDAH Z VEČ PLEMENSKIMI OVNI branih posameznikov iz populacije identična, je nihala med 5,23 × 10(−7) in 2,24 × 10(−18). Leta 2004 je v Sloveniji stekel postopek kontrole porekla z molekularnimi metodami za trop istrskih pra- menk iz Centra za sonaravno rekultiviranje Vremščica. Analizo izvajajo na Veterinarski fakulteti v Ljubljani, kjer uporabljajo standardni set mikrosatelitnih lokusov (ena- ke označevalce, kot smo jih uporabljali v naši raziskavi), ki omogoča izločitev napačnih prednikov z več kot 99 % verjetnostjo (Cotman, 2007). V genetskem laboratoriju Oddelka za zootehniko so bili prvi poskusi genotipizacije z mikrosateliti usmerjeni na določanje genetske variabil- nosti med avtohtonimi populacijami ovc ter identifikaci- jo osebkov in preverjanje porekla (Kavar in Dovč, 1999; Kavar in sod. 2002). Z namenom, da bi ocenili genetsko sorodnost med tremi slovenskimi avtohtonimi pasmami ovc, so Kavar in sod. (2002) z uporabo mikrosatelitov ge- notipizirali ovce istrskih pramenk, bovških ovc in jezer- sko solčavskih ovc (Kavar in sod., 2002). Rezultati kažejo, da razlike med pasmami niso zelo velike in da sta si bov- ška in jezersko solčavska ovca genetsko bližji kot istrska pramenka. Rezultati analize AMOVA so pokazali, da ve- čina variance izhaja iz razlik znotraj pasem, le približno 6 % se je da razložiti z razlikami med pasmami (Kavar in sod., 2002). Zato predpostavljajo skupen nastanek vseh treh pasem oz. še posebej bovške in jezersko-solčavske ovce. Našo raziskavo smo izvedli z namenom, da bi z uporabo molekularnih genetskih označevalcev preverili poreklo pri jezersko-solčavski in oplemenjeni jezersko- solčavski pasmi ter primerjali rezultate z že znanimi rodovniškimi podatki. Ocenili smo tudi informativno vrednost osmih mikrosatelitnih označevalcev za istrsko pramenko in s programom ATLAS določili očetovstvo v tropu istrskih pramenk, kjer je v času parjenja priso- tnih več plemenskih ovnov. Z analizo frekvenc alelov in stopnje heterozigotnosti pri istrski pramenki, jezersko- solčavski in oplemenjeni jezersko solčavski pasmi smo ocenili tudi informativnost uporabljenega niza mikrosa- telitnih označevalcev. 2 MATERIAL IN METODE 2.1 VZORČENJE V študijo je bilo vključenih 24 družin (oče, mati in njuni potomci) oplemenjene jezersko-solčavske pasme, 19 družin jezersko-solčavske pasme in 4 družine istrske pramenke. Skupno smo genotipizirali 124 živali, vzreje- nih pri osmih rejcih (pregl. 1). Vsi potomci so bili vključeni v kontrolo porekla in proizvodnje za drobnico. Potomcem (odbrana moška ja- gnjeta za testiranje), starim od 90 do 120 dni, je veterinar odvzel kri na karantenski postaji Horjul in testni postaji v Logatcu iz zunanje vratne vene (v. jugularis externa). Staršem, to je plemenskim ovcam in ovnom pri rejcih in Centru za sonaravno rekultiviranje Vremščica, smo s posebnim ščipalcem odvzeli majhen košček rovaša (pri- bližno 4 mm2). 2.2 IZOLACIJA DNA, IZBIRA MIKROSATELITOV IN VERIŽNA REAKCIJA S POLIMERAZO PCR Odmrznjeni košček ušesnega tkiva smo po odstra- nitvi dlak lizirali z dodatkom 300 µl pufra za lizo s 5 µl proteinaze K (Kavar s sod. 2002). Vzorec smo inkubirali na 55 °C za najmanj 4 ure pri občasnem mešanju ter izoli- rali DNA s standardno fenolno ekstrakcijo ter alkoholno precipitacijo. Pri vzorcih krvi smo v reagenčne posodice odpipetirali 200 µl krvi in dodali 800 µl TE pufra. Razto- pino smo centrifugirali 30 sekund na 12000g in superna- tant odlili. Sediment smo ponovno resuspendirali v 800 µl TE pufra in prejšnji korak trikrat ponovili. Očiščeni celični sediment smo resuspendirali z 200 µl liznega pu- fra in 4 µl proteinaze K. Ostali del protokola je enak kot pri ekstrakciji DNA iz ušesnega tkiva. Mikrosatelitne lokuse smo določili glede na pred- hodne študije (Kavar in sod. (2002) in izbrali osem po- limorfnih mikrosatelitnih označevalcev (pregl. 2). PCR reakcijsko mešanico smo pripravili v ločenem prostoru, kjer ni nevarnosti kontaminacije s produkti predhodnih PCR reakcij in ostalih DNA molekul. Reakcijski pogoji so prikazani v preglednici 3. Reakcijo PCR smo izvajali v mikrotiterskih ploščah v mikroprocesorsko vodenem cikličnem termostatu PTC – 100 (MJ Research, Watertown MA, USA) in uporabili program, ki se je začel z denaturacijo na 95  °C 5 min, sledilo je 13 ciklov denaturacije pri 95 °C 15 s, prileganja Rejec Pasma Št. potomcev Št. mater Št. očetov Rejec 1 JSR 13 12 3 Rejec 2 JSR 9 8 1 Rejec 3 JSR 2 2 1 Rejec 4 JS 4 4 1 Rejec 5 JS 3 3 1 Rejec 6 JS 10 9 2 Rejec 7 JS 3 3 1 Rejec 8 IP 4 4 20 Preglednica 1: Živali v študiji po pasmah in rejcih Table 1: Animals used in the study listed by breed and breeder JS – jezersko-solčavska; JSR – oplemenjena JS; IP – istrska pramenka Acta agriculturae Slovenica, 94/2 – 2009124 T. VOLČIČ in sod. začetnih oligonukleotidov pri 60 °C 30 s, in podaljševa- nja pri 72 °C 1 min ter 21 ciklov denaturacije pri 95 °C 15 s, prileganja pri 52 °C 30 s, in podaljševanja pri 72 °C 1 min, ter zaključnim podaljševanjem pri 72 °C, 10 min. Genotipizacijo smo izvedli z avtomatskim sekvenatorjem ABI Prism 310 (Perkin – Elmer Applied Biosystems, Fo- ster City, ZDA). Ker aparat ABI PRISMTM 310 omogoča istočasno zaznavanje štirih različnih fluorescentnih bar- vil v eni kapilari, smo pri analizi združevali po štiri PCR produkte v dve mešanici, ki sta vsebovali različno ozna- čene začetne oligonukleotide (pregl. 4). Mešanico, ki smo jo nanašali na sekvenator, smo pripravili z združitvijo 3µl PCR produkta štirih mikrosatelitnih označevalcev. Pred nanosom v sekvenator smo odpipetirali 5 µl združene PCR mešanice v reagenčno posodico, dodali 12 µl for- mamida in 0.4 µl standarda Rox 350. Po kratkem centri- fugiranju smo vzorce za 3 min. denaturirali na 95 °C in jih takoj po tem postavili na led. Tako pripravljene vzorce smo razvrstili v sekvenator za genotipizacijo. 2.3 ANALIZA GENOTIPOV S PROGRAMOM AT- LAS IN STATISTIČNE OBDEALVE Zbiranje podatkov in določanje genotipov smo izve- dli z uporabo programske opreme GENESCAN Analysis Software, verzija 3.7. Zbrane podatke genotipov (alel smo označili kot velikost produkta PCR) v Excelu smo vnesli v program ATLAS (Perez in sod., 2004), s katerim smo preverili ujemanje genotipov med starši in potom- ci. Pri določanju očetovstva v tropu istrske pramenke, kjer smo imeli na razpolago 20 potencialnih plemen- skih ovnov, smo z orodjem predlaganje prednikov (ang.: Propose parents) potomcu določili ovna znotraj skupine Ime označevalca Dolžine PCR produktov Zaporedje začetnih oligonukleotidov (Primers)Min. Max. MAF214 181 250 5’- GGG TGA TCT TAG GGA GGT TTT GGA GG -3’ 181 250 5’- AAT GCA GGA GAT CTG AGG CAG GGA GG -3’ MAF65 124 142 5’- AAA GGC CAG AGT ATG CAA TTA GGA G -3’ 124 142 5’- CCA CTC CTC CTG AGA ATA TAA CAT G -3’ McM42 79 101 5’- CAT CTT TCA AAA GAA CTC CGA AAG TG -3’ 79 101 5’- CTT GGA ATC CTT CCT AAC TTT GGG -3’ McM527 168 178 5’- GTC CAT TGC CTC AAA TCA ATT C -3’ 168 178 5’- AAA CCA CTT GAC TAC TCC CCA A -3’ TGLA53 Govedo 116 136 5’- CAG CAG ACA GCT GCA AGA GTT AGC -3’ 116 136 5’- CTT TCA GAA ATA GTT TGC ATT CAT GCA -3’ OarAE119 144 181 5’- CTC AGC AAA TGG TTC CTG GGC ACC -3’ 144 181 5’- TTT TAT AGT GAG GTG ACC ACT TGA TG -3’ OarCP49 77 103 5’- CAG ACA CGG CTT AGC AAC TAA ACG C -3’ 77 103 5’- GTG GGG ATG AAT ATT CCT TCA TAA GG -3’ OarFCB11 119 142 5’- GGC CTG AAC TCA CAA GTT GAT ATA TCT ATC AC-3’ 119 142 5’- GCA AGC AGG TTC TTT ACC ACT AGC ACC -3’ Preglednica 2: Seznam uporabljenih mikrosatelitnih označevalcev, predviden razpon dolžin PCR produktov, ter zaporedja začetnih oligonukleotidov Table 2: List of microsatellite markers, range of PCR product lenghts and nucleotide sequences of both primers Koncentracije PCR reagentov Količina reagenta / vzorec, µL Deionizirana H2O (Sigma) 1,42 1 × Taq pufer (Feramentas) 1,00 25 mM MgCl2 (Feramentas) 1,00 2 mM dNTP 1,00 0,25 uM 5’ začetni oligonukleotid 0,25 0,25 uM 3’ začetni oligonukleotid 0,25 0,4 enote polimeraze Ampli Taq (Fermentas) 0,08 PCR mešanica skupaj 5,00 DNA (5 ng/µl) 5,00 Celotna količina reakcijske mešanice 10,00 Preglednica 3: PCR reakcijska mešanica Table 3: Reaction mixture for the PCR reaction Acta agriculturae Slovenica, 94/2 – 2009 125 UPORABA DNA OZNAČEVALCEV ZA PREVERJANJE POREKLA PRI OVCAH ... V ČREDAH Z VEČ PLEMENSKIMI OVNI potencialnih staršev. Na koncu, ko so bili očetje določe- ni ustreznim družinam, smo z orodjem izris rodovnika (ang.: draw pedigree) grafično predstavili izris genotipov osebka in njegovih najbližjih sorodnikov (staršev, bratov, sester ipd.) Da bi določili stopnjo polimorfizma označevalcev pri pasmah, smo izračunali frekvence alelov in delež he- terozigotnosti z uporabo programa GENETIX (Belkhir in sod., 1998). Preučili smo odnose med osebki znotraj po- pulacij oz. informativnost (polimorfnost) označevalcev za posamezne pasme. Pomembna značilnost genetskega označevalca je njegova heterozigotnost, torej verjetnost, da je nek osebek heterozigoten za ta označevalec. Mera za informativnost označevalca nam pove pričakovan de- lež heterozigotnih osebkov. Večja, kot je heterozigotnost, bolj je označevalec informativen. Visoko polimorfni označevalec naj bi imel odstotek heterozigotnosti večji od 70 % (Ott, 1992). Pričakovani delež heterozigotnosti smo izračunali iz števila posameznih alelov v populaciji glede na Hardy-Weinbergovo ravnotežje po naslednjem obrazcu: k H = 1 − ∑ × pi 2 i = 1 k = število alel pi = pogostost i-te alele 3 REZULTATI 3.1 ANALIZA POREKLA ZA JEZERSKO- SOLČAVSKO IN OPLEMENJENO JEZERSKO- SOLČAVSKO PASMO Z namenom, da bi preverili rodovniške podatke, smo s pomočjo programa ATLAS najprej testirali tiste živali, za katere smo imeli znano poreklo. Preverili smo rodovniške podatke, ki jih zapisujejo rejci sami ter po- datke selekcijske službe. Oboje smo primerjali z rezultati, ki smo jih dobili pri testiranju genotipov. V 40-tih od 44- tih družin smo potrdili pravilno zapisovanje rodovniških podatkov. Na sliki 1(levo) je prikazana jezersko-solča- vska družina rejca 6, desno pa oplemenjena jezersko- solčavska družina rejca 1. Vsaka žival je predstavljena z Mešanica 1 / Mix1 Mešanica 2 / Mix2 FAM MCM 42 (79–101) bp OarCP 49 (77–103) bp FAM TGLA 53 (116–136) bp OarAE 119 (144–181) bp TAMRA MAF 65 (124–142) bp OarFCB11 (119–142) bp TAMRA McM 527 (168–178) bp MAF 214 (181–250) bp JOE Preglednica 4: Shema združevanja mikrosatelitnih označevalcev za analizo na sekvenatorju Table 4: Pooling of microsatellite PCR products for genotyping Slika 1: Slikovni prikaz (program ATLAS) dveh rodovnikov, kjer je genotipska analiza potrdila pravilno zapisovanje rodovniških podatkov. Figure 1: Scheme drawn by programme ATLAS for two pedigrees with concordant results between listed pedigree and genotype test. Acta agriculturae Slovenica, 94/2 – 2009126 T. VOLČIČ in sod. identifikacijsko številko, npr. OČE rejca 6 ima ID 293542. Pri potomcu so za vsakega od osmih lokusov (alel prika- zan kot število CA ponovitev) prikazani aleli, ki jih poto- mec podeduje po strani očeta (zgornja vrsta) in po strani matere (spodnja vrsta). V 4 primerih od 44 –tih (9,1  %) smo odkrili raz- hajanja med rezultati genetske analize rodovnikov in za- pisanih rodovniških podatkov. V dveh primerih sta bila potomcu nepravilno pripisana oče ali mati, pri ostalih dveh primerih pa sta bila jagnjetu napačno pripisana oba starša (slika 2). Pri dveh družinah smo opazili neskladja na šestih mikrosatelitnih lokusih, pri ostalih dveh druži- nah pa na petih oziroma štirih lokusih. 3.2 DOLOČANJE OČETOVSTVA ZA ISTRSKO PRAMENKO Analiza je temeljila na določanju očetovstva v čredi ovc istrske pramenke iz Centra za sonaravno rekultivira- nje Vremščica, kjer v času paritev v čredo ovc pripušča- jo dvajset plemenskih ovnov hkrati. Z namenom, da bi jagnjetom določili očete, smo genotipizirali dvajset ple- menskih ovnov na osmih mikrosatelitnih lokusih ter re- zultate primerjali z genotipi potomcev in njim pripisanih mater z uporabo programa ATLAS. Z osmimi mikrosa- teliti smo trem jagnjetom (št. 11344, 11345, 11342) lahko nedvoumno določili očeta izmed dvajset potencialnih očetov (slika 3). Izbrani set osmih označevalcev ni bil dovolj infor- mativen za določitev očeta potomcu 11346 (pregl. 5), saj je analiza s programom ATLAS od dvajsetih ovnov do- ločila dva verjetna očeta. Da bi lahko jagnjetu 11346 kot očeta določili enega izmed dveh kandidatnih ovnov, smo v analizo dodali še štiri označevalce OarCP20, Oar116, Oar109 ter Oar50 (pregl. 6). Zaradi podobnosti v genotipu na osmih lokusih med kandidatnima očetoma 1 in 2 (pregl. 5; 11211 in 151982) lahko domnevamo, da obstajajo sorodstvene povezave med njima, kar je za majhno populacijo istrske pramenke pričakovano (velika verjetnost je, da sta brata ali polbrata). Uporaba dodatnih štirih mikrosatelitnih lo- kusov je potomcu 11346 kot očeta nedvoumno določila kandidatnega ovna št. 151982 (pregl. 6). Mikrosatelitni lokus Oar109 je izločil ovna 11211 in se tako izkazal za odločilnega. Lokusa OarCP20 in Oar116 sta za to pa- smo brez informativne vrednosti zaradi homozigotnosti na vseh alelih. Z določitvijo očetovstva potomcu 11346 smo tako lahko določili očetovstvo vsem preiskovanim jagnjetom istrskih pramenk. 3.3 ANALIZA FREKVENC ALELOV IN INFORMA- TIVNOSTI ZA POSAMEZNE MIKROSATELI- TNE LOKUSE PO PASMAH V analizo frekvenc alelov smo vključili pasme je- zersko-solčavska, oplemenjena JS in istrska pramenka Slika 2: Primer neskladja genotipov staršev z genotipom potomca pri rejcu 1 – jagnje je bilo pripisano napačni družini (zgoraj). V spodaj primeru je bila jagnjetu pripisana napačna mati. Osenčeni lokusi pri določenem osebku predstavljajo neskladja genotipov med predvidenimi starši in potomcem. Figure 2: A case of two pedigrees where genotype analysis demonstrated discordant results with enlisted pedigress. Progeny of the left pedigree was assigned to a wrong family (shadowed boxes), whereas progeny of the right pedigree was not assigned to correct dam. Acta agriculturae Slovenica, 94/2 – 2009 127 UPORABA DNA OZNAČEVALCEV ZA PREVERJANJE POREKLA PRI OVCAH ... V ČREDAH Z VEČ PLEMENSKIMI OVNI ter osem mikrosatelitnih lokusov. Število alelov, ki smo jih za posamezen mikrosatelitni lokus našli pri določeni pasmi ovc, je prikazano na sliki 4. Na vsakem od pre- učevanih lokusov smo pri vseh treh pasmah našli veči- no alelov. Poleg tega smo identificirali od dva do pet za pasmo specifičnih alelov (tako imenovani privatni aleli). Za istrsko pramenko je bilo za vseh osem lokusov ugo- tovljenih skupno trinajst privatnih alelov, osem za jezer- sko-solčavsko in šest za oplemenjeno jezersko-solčavsko ovco. Povprečno število alelov na mikrosatelitni lokus je za OPLEMENJENA JEZERSKO-SOLČAVSKA znašalo 7,5, prav toliko za JS, za IP pa 7,63 alelov na lokus. Z namenom določitve stopnje polimorfnosti in s tem informativne moči posameznih lokusov znotraj pre- učevanih pasem smo izračunali delež heterozigotnosti. Glede na povprečno heterozigotnost smo označevalce (slika 4) razdelili v tri skupine, visoko polimorfne (hete- rozigotnost nad 70 %), srednje polimorfne (heterozigo- Slika 3: Uspešno določanje očetovstva (od 20 možnih) z osmimi mikrosatelitnimi označevalci trem potomcem istrske pramenke. Figure 3: Successfull assignment of the sire out of 20 potential fathers to three progeny. ID   OarCP49 OarAE119 MAF65 MAF214 TGLA53 McM42 McM527 OarFCB11 11346 SIN 2 19 50 50 36 40 67 70 27 27 7 8 57 58 26 30 143132 MATI 2 2 49 50 27 36 67 70 27 38 7 14 57 58 26 26 11211 Kandidatni oče 1 3 19 49 50 40 42 62 70 27 36 7 8 55 57 30 39 151982 Kandidatni oče 2 17 19 50 50 34 40 68 70 27 27 8 8 55 57 30 30 Preglednica 5: Rezultati genotipizacije družine z osmimi lokusi, ki prikazuje dva možna očeta Table 5: Raw genotype data for eight markers that could not distinguish amongst two potential fathers Acta agriculturae Slovenica, 94/2 – 2009128 T. VOLČIČ in sod. tnost med 50–70 %) in nižje polimorfne (heterozigotnost od 0–50  %). Za vse tri pasme ovc smo opazili visoko polimorfnost označevalcev na lokusu MAF65, TGLA53 in OarFCB11 (slika 5). Pri posameznih pasmah je pri je- zersko-solčavski izstopal lokus OarFCB11 (H= 0,86), pri oplemenjeni jezersko-solčavski lokus TGLA53 (0,84) in pri istrski pramenki lokus OarCP49 (0,86). V skupino srednje polimorfnih sta se pri jezersko-solčavski uvrstila lokusa OarCP49 (0,59) in McM42 (0,68), pri oplemenje- ni jezersko-solčavski lokus OarAE119 (0,66), medtem ko smo pri istrski pramenki zabeležili tri lokuse OarAE119 (0,68), McM42 (0,63) in McM527 (0,51). V najmanj in- formativni skupini mikrosatelitnih označevalcev (he- terozigotnost od 0–50 %) imamo en sam lokus, in sicer pri pri oplemenjeni jezersko-solčavski lokus MAF214 z deležem heterozigotnosti 0,32 – pri tej pasmi smo na tem lokusu zasledili le 3 alele, kar je najmanjše število alelov na vseh lokusih. 4 RAZPRAVA IN SKLEPI 4.1 ANALIZA RODOVNIKOV Pri preverjanju rodovniških podatkov smo z analizo genotipov ugotovili, da prihaja pri vodenju rodovniških podatkov drobnice do napak. V štirih družinah od 44- ID   OarCP20 Oar116 Oar109 Oar50 11346 SIN 72 72 38 38 73 75 76 76 143132 MATI 72 72 38 38 73 73 74 76 11211 Kan. 1 72 72 38 38 73 78 76 77 151982 Kan. 2 72 72 38 38 74 75 76 76 Preglednica 6: Genotipi na dodatnih lokusih za potomca 11346 Table 6: Genotyping for four additional markers for progeny number 11346 Slika 5: Povprečna heterozigotnost na lokusih pri jezersko-solčavski (JS), oplemenjeni JS (JSR) in istrski pramenki (IP). Figure 5: Average heterozygosity for analysed markers in Jezersko-solčava (JS), Improved Jezersko-solčava (JSR) and Bela Krajina pramenka (IP) breeds. Slika 4: Število alelov za posamezene mikrosatelitne lokuse pri jezersko-solčavski (JS), oplemenjeni jezersko-solčavski (JSR) in istrski pramenki (IP). Figure 4: Number of alleles for genetic markers in Jezersko-Solčava (JS), Improved Jezersko-Solčava (JSR) and Bela krajina pramenka (IP) breeds. Acta agriculturae Slovenica, 94/2 – 2009 129 UPORABA DNA OZNAČEVALCEV ZA PREVERJANJE POREKLA PRI OVCAH ... V ČREDAH Z VEČ PLEMENSKIMI OVNI ih (9,1 %) smo zasledili neskladja v rodovnikih pri štirih od skupno sedmih rejcev, vključenih v analizo. V dveh primerih (rejec 1 in 3) sta bila potomcu napačno pripisa- na oba starša. Pri primeru rejca 1 smo kasneje dodatno primerjali genotip jagnjeta (294651) z genotipi preostalih živali, ki smo jih pri tem rejcu imeli na razpolago. Rezul- tat primerjave je pokazal, da jagnje v resnici pripada ma- teri (31065) in očetu (11083) in ne staršem, ki so mu bili ob rojstvu pripisani (slika 6). Poleg tega smo ugotovili, da ima jagnje št. 294651 brata (294649) z enakim geno- tipom, zato lahko predvidevamo, da sta enojajčna dvojč- ka. Podobno smo poskusili tudi za rejca 3, vendar zaradi majhnega števila živali (5), vključenih v analizo, pravih staršev nismo mogli določiti. Razlogov, zaradi katerih prihaja pri vodenju ro- dovnika do napak, je lahko več. Po podatkih selekcijske službe za leto 2006 je v kontroliranih tropih v Sloveniji vključenih 106 tropov JS pasme, pri kateri je registriranih 2.969 ovc, ki so imele 3.784 jagnjitev in 116 tropov ople- menjene jezersko-solčavske pasme s 3.690 registriranimi ovcami s 4.762 jagnjitvami. Največ jagnjitev je bilo zabe- leženih v spomladanskem obdobju z vrhoma v mesecu marcu in maju, z okrog 2500 rojenimi jagnjeti na mesec (Kompan s sod., 2006; Zajc in Komprej, 2007). V velikih tropih to lahko predstavlja problem, saj na isti dan lahko jagnji več ovc hkrati. Posledice tega so lahko zamenjave med materjo in novorojencem, kjer jagnje vzredi ovca, ki v resnici ni njegova mati. Do podobnih ugotovitev so prišli tudi Laughlin in sod. (2003). Lahko sklepamo, da se taki primeri največkrat dogajajo pri jagnjitvah na pašni- ku ali v neprimerno urejenih hlevih, kjer je nadzorovanje poteka jagnjitev omejeno. Za naša dva primera bi lahko predpostavili, da je šlo za samo napako pri zapisovanju rojstnih podatkov, ali pa je na isti dan jagnjilo več ovc, med katerimi je mati (31065) jagnjila dvojčka, eden od njiju (294651) pa se je pomešal z drugimi novorojenimi jagnjeti in ga je posvojila druga ovca. Rejec v tem prime- ru ni mogel predvideti, da jagnje pripada drugi materi in ga je avtomatsko pripisal materi, ki ni bila prava. Podob- no bi lahko trdili tudi za rejca 2 (slika 2), ki je jagnjetu (294664) pripisal napačno mater (52848). Četrti primer neskladja genotipov smo ugotovili pri rejcu 5, ki ima v tropu stalno prisotnega ovna in ga zaradi preprečevanja parjenja v sorodstvu vsakih nekaj paritvenih obdobij menjuje. Problem se pojavi ravno v času menjave enega plemenskega ovna z drugim, ko sta v tropu prisotna oba ovna. Možnosti, da je prišlo do napake pri določanju geno- tipov ali pri našem vzorčenju tkiv, kar je prispevalo k ne- skladju rezultatov, tudi ne moremo povsem izključiti. Ker smo določali genotipe na več lokusih, je možnost vpliva posamezne napake pri določanju genotipov majhna, saj neskladje navadno ugotovimo na več lokusih hkrati. Bolj verjetna bi bila napaka zaradi napačnega vzorčenja. Le ta je zaradi načina vzorčenja tudi malo verjetna, saj sta bila pri pregledu ušesne številke prisotna najmanj dva člove- ka – identifikaciji številke je takoj sledil odvzem vzorca. Obstaja sicer še možnost napačnega odčitavanja ušesne Slika 6: Določanje pravih staršev (oven 11083 in ovca 31065) za potomca 294651, ki je bil v rodovniku pripisan napačni družini (ovnu 11158 in ovci 31068). Figure 6: Identification of correct parents (sire 11083 and dam 31065) for the progeny 294651 that was assigned to a wrong family (sire 11158 and dam 31068). Acta agriculturae Slovenica, 94/2 – 2009130 T. VOLČIČ in sod. številke in napačni pripis laboratorijske številke posame- znemu vzorcu. Čeprav možnosti napak pri vzorčenju in obdelavi vzorcev v laboratoriju ne moremo povsem iz- ključiti, menimo, da je večina neskladij (9,1 %) posledica napak pri vpisovanju in vodenju rodovnikov. Zaključimo lahko z ugotovitvijo, da je natančnost rodovniških podatkov s strani rejcev in ob pomoči se- lekcijskih služb 90,9  %. Iz zgornje razprave sledi, da se nekatere napake s posegi v tehnologijo reje da odpraviti, nekatere pa bodo rejcem ostale prikrite. Zato se tu ponu- jajo alternativne metode ugotavljanja porekla, kot je opi- sana v tej študiji, ki bi omogočala, da bi odstotek točnosti rodovnikov približali 100 % in s tem dosegli vodenje ro- dovnikov brez napak. 4.2 DOLOČANJE OČETOVSTVA V tropih, kjer se pri pripustih uporablja več ple- menskih ovnov hkrati, so podatki o poreklu živali zaradi nezmožnosti nadzorovanja pripustov osiromašeni s po- datkom očeta. Zato v teh tropih predstavlja velik pomen možnost določanja očetovstva in nadzora rodovniških podatkov s pomočjo tipizacije DNA. Poleg tega pa je za rejce, ki so vključeni v kontrolo porekla in proizvodnje, pogoj, da so vse živali predstavljene z znanim poreklom. Tako smo v tej študiji preverjali, če je možno z izbra- nih setom osmih mikrosatelitov in uporabo programa ATLAS jagnjetom istrske pramenke določiti enega od možnih dvajsetih očetov. Z izbranimi osmimi mikrosa- teliti smo lahko trem jagnjetom istrske pramenke nedvo- umno določili očeta izmed potencialnih dvajsetih ovnov. V primeru enega jagnjeta smo lahko izločili 18 potenci- alnih očetov, dva očeta sta bila enako verjetna. Zato smo analizo razširili in vanjo vključili dodatne štiri mikrosa- telitne označevalce ter s tem uspešno določili očetovstvo tudi temu potomcu. 4.3 INFORMATIVNOST Najvišji nivo heterozigotnosti in s tem informativno- sti lokusa smo pri vseh treh pasmah dosegli z označeval- cem TGLA53, medtem ko se je za najmanj informativne- ga izkazal lokus MAF214. Lokusi OarCP49, OarAE119, MAF65 in OarFCB11 se pri teh treh pasmah uvrščajo v razred visoko polimorfnih mikrosatelitov, visok delež heterozigotnosti imata tudi McM42 in McM527, ki za najvišjim razredom ne zaostajata veliko. Rezultati dele- žev heterozigotnosti in števila alelov kažejo, da je najver- jetneje stopnja inbridinga populacije istrske pramenke iz CSR Vremščice podobna kot pri jezersko-solčavski in oplemenjeni jezersko-solčavski pasmi. To ugotovitev utemeljujemo z rezultatom, da smo pri tej pasmi dobili v povprečju 7,6 alelov na lokus, kar je sploh največ od vseh pasem, vključenih v raziskavo, heterozigotnost alelov pa je znašala 71 %. Poudariti pa je potrebno, da je bilo v našo analizo zajetih za populacijsko-genetske študije še vedno relativno majhno število vzorcev in bi tako za bolj kon- kretne sklepe o inbridiranosti populacije istrske pramen- ke morali analizirati večje število živali. Vendarle smo v enem od štirih družin odkrili višjo stopnjo sorodstva, kar lahko kaže, da znotraj pasme istrske pramenke lah- ko obstajajo visoko inbridirane pod-populacije oziroma družine – v tem primeru je visoka stopnja sorodstva v tej družini istrske pramenke privedla k zmanjšanju stopnje polimorfnosti mikrosatelitnih lokusov in s tem njihove informativnosti. Za nadaljnje analize predlagamo, da se našemu uporabljenemu setu doda še označevalec Oar109 ali zamenja označevalec MAF512, ki je s petimi aleli in 51  % heterozigotnostjo na zelo nizkem informativnem nivoju. Mikrosateliti, uporabljeni v tej študiji in analiza s programom ATLAS sta torej primerna pri preverjanju pravilnosti rodovniških podatkov in pri določanju očeto- vstva. V zadnjem času se v Sloveniji na področju selekcije v ovčereji vse bolj uveljavlja uporaba molekularno-ge- netskih metod. Molekularni podatki so dober pokazatelj variabilnosti v populacijah, kar nam omogoča zasnovo programov za ohranjanje genetskih raznovrstnosti v po- pulacijah živali. Eden takih pristopov je prav gotovo tudi kontrola porekla z uporabo molekularnih markerjev, ki se je v Sloveniji že dobro uveljavila pri gospodarsko po- membnih domačih živalih (konji, govedo, prašiči itd.). Tudi pri drobnici se srečujemo s čedalje strožjim selek- cijskim nadzorom, zato so analize te vrste preizkušene, v nekaterih primerih pa tudi uporabljene. Ker pa je vse več populacij ovc, vključenih v programe obveznega ge- notipiziranja za prionski gen, bi bilo zaželeno dodatno preverjanje porekla. To je pomembno tako z vidika oce- njevanja plemenske vrednosti jagnjeta kot z vidika kon- trole inbridinga, torej izogibanja nadaljnjega parjenja v sorodstvu, ter z vidika zanesljivejše genotipizacije za lo- kus PRNP. Ta študija je pokazala, da kljub skrbi za natančno vodenje rodovniških knjig pri teh še vedno prihaja do napak. Da bi se napakam izognili, se selekcijskim služ- bam ponuja možnost nadzorovanja porekla z uporabo mikrosatelitnih označevalcev. V ta namen velja omeni- ti, da lahko postopek, s katerim smo izvedli analizo ro- dovništva, še nekoliko posodobimo s pomnoževanjem večjega števila mikrosatelitnih lokusov hkrati v eni PCR reakciji. To nam omogoči hitro, cenejšo ter enostavno izvedbo genotipizacije. Zaključimo lahko z dejstvom, da uporaba visoko polimorfnih DNA označevalcev omogo- ča učinkovito in objektivno preverjanje rodovniških po- Acta agriculturae Slovenica, 94/2 – 2009 131 UPORABA DNA OZNAČEVALCEV ZA PREVERJANJE POREKLA PRI OVCAH ... V ČREDAH Z VEČ PLEMENSKIMI OVNI datkov, predvsem pomemben pa je podatek, da postajajo ta orodja vse pomembnejša pri projektih ohranjanja ge- netske raznovrstnosti. 5 ZAHVALA Zahvaljujemo se rejcem in Centru za sonaravno rekultiviranje Vremščica za omogočanje raziskave z do- voljenjem za vzorčenje tropov. Domnu Drašlerju smo hvaležni za pomoč pri zbiranju vzorcev, Dušanu Birtiču za koristne nasvete ter dr. Andreju Razpetu za strokovno pomoč pri genotipiziranju. 6 VIRI Achmann R., Dworak E., Muller M., Brem G. 1998. Parentage control in Austrian domestic mountain sheep (Ovis aries) using DNA microsatellite analysis. Animal Genetics, 29, 1: 12–13 Belkhir K., Borsa P., Goudet J., Chikhi L., Bonhomme F. 1998. GENETIX. Logical sous Windows TM pour la genetiqe des populations. CNRS UPR 9060 University Montpellier, Laboratoire Genome et Populations Cotman M. 2007. »Določanje porekla z molekularnimi meto- dami pri Istrski pramenki«. Ljubljana, Univerza v Ljubljani, Veterinarska fakulteta (osebni vir, marec 2007) Dovč P. 1994. Tipiziranje DNA kot metoda za preverjanje porekla živali. Zbornik Biotehniške fakultete Univerze v Ljubljani, Kmetijstvo, Zootehnika, Suplement 64: 45–56 Dovč P. 1996. Molekularna genetika v ovčereji. V: Možnosti razvoja reje drobnice v Sloveniji, Postojna, 27-29 nov. 1996. Slovenj Gradec, Kmetijska založba: 93–98 Glowatzki-Mulis M.L., Muntwyle J. Gaillard C. 2007. Cost – efective parentage verification with 17- plex PCR for goats and 19-plex PCR for sheep. Animal genetics, 38, 1: 86–88 Kavar T., Dovč P. 1999. Uporaba mikrosatelitov za opis genetske raznolikosti. Sodobno kmetijstvo, 32, 6: 290–292 Kavar T. 2001. Ocena genetske raznolikosti v populaciji konj lipicanske pasme. Doktorska disertacija. Ljubljana, Univer- za v Ljubljani, Biotehniška fakulteta: 12 str. Kavar T., Kompan D., Dovč P. 2002. Genske razlike med istrsko pramenko, bovško ovco in jezersko solčavsko ovco. Zborn- ik Biotehniške fakultete Univerze v Ljubljani, Kmetijstvo, Zootehnika, Suplement 80: 192–201 Kompan D., Lotrič Žan M., Birtič D., Cividini A. 2006. Register pasem z zootehniško oceno, vrsta: ovce. http://www.bfro. uni-lj.si/Kat_center/genska_banka/Register2005/Ovce.pdf (28. mar. 2007) Laughlin A.M., Waldron D.F., Craddock B.F., Engdahl G.R., Dusek R.K., Huston J.E., Lupton C.J., Uckert D.N., Shay T.L., Cockett N.E. 2003. Use of DNA markers to Determine Paternity in a Multiple-Sire Mating Flock. American Sheep Industry. http://www.sheepusa.org/index.phtml?page=site/ news_details&nav_id=fe6dfef3f426db0c74270baae708b64 8 (15. apr. 2007) Ott J. 1992. Strategies for characterizing highly polymorphic markers in human gene mapping. Human genetics, 51: 283–290 Perez-Encisio E., Perez-Encisio M., Garcia-Bernal P. 2004. At- las. Perez Enciso, Miguel. http//:www.icrea.es/pag.asp?id=Miguel.Perez (7. dec. 2006) Zajc P., Komprej A. 2007. Plodnost ovc in koz v kontroliranih tropih v Sloveniji za obdobje 2006. Drobnica, 12, 2: 7–9 Acta argiculturae Slovenica, 94/2, 133–138, Ljubljana 2009 COBISS: 1.01 Agris category code: L10 IN VITRO MAMMARY GLAND MODEL: ESTABLISHMENT AND CHARACTERIZATION OF A CAPRINE MAMMARY EPITHELIAL CELL LINE Jernej OGOREVC 1, Sonja PRPAR 2, Peter DOVČ 3 Delo je prispelo 16. septembra 2008, sprejeto 15. septembra 2009. Received September 16, 2008; accepted September 15, 2009. 1 Univ. of Ljubljana, Biotechnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia, email: jernej.ogorevc@bfro.uni-lj.si 2 same address, email: sonja.prpar@bfro.uni-lj.si 3 same address, Prof., Ph.D., email: peter.dovc@bfro.uni-lj.si In vitro mammary gland model: establishment and character- ization of a caprine mammary epithelial cell line Demanding transcriptomic studies in combination with challenging experiments in livestock animal species could be replaced by good in vitro models mimicking the function of ruminant mammary gland. The objective of our study was to establish epithelial cell line obtained from primary cell culture of lactating goat mammary gland. Mammary tissue from lac- tating Saanen goat (Capra hircus) was digested in collagenase and hyaluronidase solution and plated on plastic flasks. When growing on plastic, typical cobblestone morphology of epithe- lial cells and larger irregularly shaped cells, corresponding to myoepithelial cells were observed. When growth medium was supplemented with lactogenic hormones (insulin, hydrocorti- sone, and prolactin) and cells were cultured on plastic for ex- tended period of time at high density, dome-like structures ap- peared as a result of cell to cell contact induced differentiation. Immunofluorescence staining using antibodies against smooth muscle α-actin, vimentin and various cytokeratins were used to distinguish between different cell types. Cell types of epithelial and myoepithelial cells were confirmed. Complete differentia- tion of cells was achieved when growing them on a commercial basal membrane matrix preparation which contains laminin, collagen IV, and various growth factors. Cells grown on basal membrane matrix in growth medium supplemented with lacto- genic hormones differentiated morphologically and function- ally. Spherical structures that resembled the alveoli of lactating mammary gland were observed. Reverse transcription PCR (RT-PCR) was performed on the total RNA extracted from the cultured cells in order to detect the potentially present milk protein mRNAs. Key words: goats / mammary gland / molecular genetics / cell culture / lactogenesis / caseins / expression / immunofluo- rescence In vitro model mlečne žleze: vzpostavitev in določitev značilno- sti epitelne celične linije iz kozje mlečne žleze Zahtevne transkriptomske študije, ki vključujejo posku- se na živih živalih, je mogoče nadomestiti z ustreznimi in vi- tro modeli. Cilj naše raziskave je bil vzpostaviti celično linijo epitelnih celic, pridobljenih iz primarne celične kulture kozje mlečne žleze v laktaciji. Žlezno tkivo koze (Capra hircus) sanske pasme smo razgradili v raztopini kolagenaze in hialuronidaze in nacepili v plastične posodice. Pri rasti na plastični podlagi so se pojavile značilne epitelne strukture v obliki tlakovcev in ve- čje celice nepravilnih oblik, ki so po zunanjosti ustrezale mioe- pitelnim celicam. Ob dodatku laktogenih hormonov (inzulin, hidrokortizon, prolaktin) v medij in po daljšem obdobju rasti ter ob visoki gostoti celic na plastični podlagi so se oblikovale kupolaste strukture, ki so posledica diferenciacije zaradi med- celičnih interakcij. Za karakterizacijo različnih celičnih tipov smo uporabili imunofluorescenčno barvanje za α-aktin gladkih mišic, vimentin in različne citokeratine. Z barvanjem smo po- trdili prisotnost epitelnih in mioepitelnih celic. Popolno dife- renciacijo celic smo dosegli z gojenjem na komercialno pripra- vljenem matriksu, ki posnema bazalno membrano in vsebuje laminin, kolagen IV in različne rastne faktorje. Celice so se na podlagi iz ekstracelularnega matriksa in ob dodatku laktogenih hormonov morfološko in funkcionalno diferencirale. Nastale so sferične strukture, podobne alveolam mlečne žleze v laktaci- ji. Z reverzno verižno reakcijo s polimerazo (RT-PCR) smo na izolirani RNA preverili prisotnost mRNA za mlečne proteine. Ključne besede: koze / mlečna žleza / molekularna gene- tika / celična kultura / laktogeneza / kazeini / ekspresija / imu- nofluorescenca Acta agriculturae Slovenica, 94/2 – 2009134 J. OGOREVC et al. 1 INTRODUCTION Because of the commercial value of milk there is a great interest in understanding mechanisms involved in milk protein expression and udder resistance to patho- gens which cause infectious agalactia or secretion of abnormal milk. Demanding transcriptomic studies in- vestigating mechanisms influencing mammary gland metabolism usually involve in vivo experiments. Ad- ditionally, treatments in vivo can have systemic effects which make controlling the environment of epithelial cells in a predictable way very difficult (Rose & McCo- noche, 2006). For this reasons adequate in vitro model mimicking the function of the mammary gland would be of great importance for the study of physiological, bio- chemical and immunologic functions of the mammary gland. Furthermore, there are almost no techniques that would allow the maintenance of organs ex vivo long enough to permit necessary molecular biological investi- gations. As such, an enormous potential exists in the use of three-dimensional (3-D) cell culture models as surro- gates for tissues. In the recent years, mammary cell cul- ture models were mainly used to study cell differentiation during lactation, innate immune response to infections and response to hormonal induction of lactogenesis in mammary epithelial cells (MECs). Several ruminant immortalized cell lines such as MAC-T (Huyhn et al., 1991) and BME-UV (Zavizion et al., 1996) have been established by stable integration of the simian virus large T-antigen (SV40LTA). However, because of their low responsiveness to lactogenic hor- mones, transformed mammary cell lines were mainly used to study insulin growth factor 1 (IGF-1) metabolism (German & Barash, 2002). It is still not clear how modi- fications in immortalized cell lines alter physiological pathways of transformed cells, therefore the use of pri- mary cell lines is much more representative of the in vivo system maintaining organ-specific functions and signal transduction pathways (Pantschenko et al., 2000). Growth of primary mammary cell cultures from lactating mammary gland on plastic usually results in loss of tissue specific functions. Cells in this state do not synthesize any of the milk components nor do they have the cellular response of in vivo cells (Blum et al., 1989). On the other hand the growth of MECs on pre-formed extracellular matrices results in morphological differen- tiation as well as in synthesis of milk components (Rose et al., 2002). Kabotyanski et al. (2009) studied transcrip- tion of β-casein (CSN2) and suggested that the expres- sion of CSN2 is induced synergistically by lactogenic hor- mones together with local growth factors, cell-cell and cell-substratum interactions. The objective of our study was to establish goat MEC (GMEC) line, from primary cell culture, that is re- sponsive to lactogenic hormonal induction and capable of expressing milk protein genes. The established GMEC line will be used for further studies of mammary gland differentiation, induction of lactation and infection re- sponse. 2 MATERIALS AND METHODS 2.1 ESTABLISHMENT OF CELL CULTURE Mammary tissue was aseptically removed from the mammary gland of lactating Saanen goat (Capra hircus) immediately after slaughter. The gland was wiped with 70% ethanol and chopped up in chunks which were washed in HBSS (Hank’s Buffered Salt Solution) me- dium containing penicillin (200 µg/ml), streptomycin (200 µg/ml), gentamicin (200 µg/ml), ampicilin (200 µg/ ml) and amphotericin B (10 µg/ml). Tissue was further sliced in smaller pieces and digested in 100 ml of colla- genase (Biochrom AG) and hyaluronidase (Sigma) solu- tion (400 U/ml of each) prepared in HBSS with HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) containing all of the above listed antibiotics in the same concentrations. The digestion was carried out at 37  °C with gentle shaking. The digesta were collected at 60, 120 and 180 minutes after the initiation of digestion and were filtered through a steel mesh. The filtrates were put in a 50 ml tube and washed several times with HBSS followed by centrifuging at 1200 rpm for 5 minutes. The cell suspen- sion was filtered through 40 µm mesh, centrifuged and plated on plastic or further resuspended in 90% FBS and 10% DMSO for freezing in liquid nitrogen. Aliquots of cell suspensions (fractions 1–3) were plated on plastic flasks in growth medium RPMI 1640 (Sigma) supplemented with lactogenic hormones insulin (1 µg/ml), hydrocortisone (1 µg/ml), and prolactin (1 µg/ ml). Cells were incubated in a CO2 incubator at 37 °C, 5% CO2 and saturated humidity. The medium was changed every 2 to 3 days. For observing dome formation cells were maintained in culture for at least 20 days. When performing passaging the cells were treated with 0.05% trypsin-EDTA (Sigma) and incubated at 37 °C until the cells detached from the plastic dish. The cells were than resuspended in growth medium. Characteristics of the cell line were observed under light microscope. 2.2 IMMUNOFLUORESCENT STAINING Cells were seeded in 6-well plates on cover glasses and cultured till they nearly reached confluence. They Acta agriculturae Slovenica, 94/2 – 2009 135 IN VITRO MAMMARY GLAND MODEL: ESTABLISHMENT ... CAPRINE MAMMARY EPITHELIAL CELL LINE were washed with cold phosphate buffered saline (PBS) and fixed in a mixture of cooled acetone and methanol (dilution 1:1) at −20 °C. Monoclonal antibodies against smooth muscle α-actin (sc-58669), vimentin (sc-73262) and cytokeratins (K) 14, 18, and 19 (sc-53253; sc-51582; sc-6278; all Santa Cruz Biotechnology) were used to dis- tinguish between different cell types. The cover glasses were covered with solution of primary antibodies (dilu- tion 1:200) in PBS with BSA (3%) and incubated over- night at room temperature. For unspecific binding con- trol cover glass was incubated with PBS – BSA (3%). As a secondary antibody goat anti–mouse–FITC (F4143, Sigma) was used in dilution 1:500 in PBS – BSA (3%). Incubation was carried out in dark for 1 hour, and cells were observed under fluorescent microscope (Nikon Eclipse TE, 2000). 2.3 THIN LAYER METHOD AND 3D CULTURE METHOD GMECs were grown on Geltrex Reduced Growth Factor Basement Membrane Matrix (Invitrogen) which is a soluble form of basement membrane purified from Engelbreth-Holm-Swarm tumor that gels at 37 °C form- ing a reconstituted basement membrane. The major components of Geltrex (GT) include laminin, collagen IV, entactin and various growth factors. In both methods GT was thawed at 2 to 8  °C overnight on ice in refrig- erator. In thin layer method, used for culturing primary cell line, GT was diluted in cold serum-free RPMI 1640 medium in a concentration of 0.1 mg/ml and sufficient amount was used to cover the entire growth surface. Coated object was placed at 37 °C for 60 minutes or un- til dry. In 3D culture method 50 µl of GT was used per well of 24-well plate and left at 37 °C to promote gelling of matrix. GMECs were suspended in RPMI 1640 media containing 2% of GT and approximately 105 cells were plated per well. The cells were grown at 37 °C in humidi- fied atmosphere of 5% CO2 in air and observed through microscope. 2.4 LACTATION INDUCTION (RT-PCR) Total RNA was extracted from confluent second passage GMECs, grown on thin layer GT in lactogenic growth medium (as described previously), using TRI Reagent (Ambion) in accordance to manufacturer’s in- structions. In order to detect potentially present milk Figure 1: Goat mammary cell line (second passage) growing as a monolayer on plastic. A. Colony morphology observed after 18 days in culture. Islands of epithelial cells surrounded with myoepithelial cells (magnification × 40). B. Island of densely packed epithelial cells (magnification × 200). C and D. Dome-like structures in 30 day old post-confluent cell line made by light microscopy using differ- ent light polarisation (magnification × 40). Slika 1: Kozja celična linija (druga pasaža) v obliki monosloja na plastični podlagi. A. Morfologija kolonij v kulturi po 18. dneh. Otoki epitelnih celic obkroženi z mioepitelnim celicami (40 x povečava). B. Otoček epitelnih celic (200 x povečava). C in D. Kupolaste strukture v 30 dni stari postkonfluenti kulturi – svetlobna mikroskopija z uporabo različnih polarizacijskih filtrov (40 x povečava). Acta agriculturae Slovenica, 94/2 – 2009136 J. OGOREVC et al. protein mRNAs reverse transcription-polymerase chain reaction (RT-PCR) was performed using OneStep RT- PCR kit (Qiagen). The PCR primers for amplification of β-casein (CSN2) and housekeeping gene β-actin (ACTB) were as follows: CSN2a-F: 5’-ACAGCCTC- CCACAAAACATC-3’, CSN2a-R: 5’-AGGAAGGT- GCAGCTTTTCAA-3’ with product length 206 bp; ACTBa-F: 5’-CCAACCGTGAGAAGATGACC-3’, ACT- Ba-R: 5’-CGCTCCGTGAGAATCTTCAT-3’ with prod- uct length 247 bp. RT-PCR products for β-casein and β-actin (ACTB) were isolated from agarose gel using gel extraction kit (Jetquick) and confirmed by sequencing. 3 RESULTS Digestion of mammary tissue in collagenase and hy- aluronidase solution resulted in isolation of heterogenous culture which contained mixed population of epithelial and myoepithelial (smooth muscle α-actin positive) cells. When grown on plastic, typical cobblestone morphology of epithelial cells and larger irregularly shaped cells cor- responding to myoepithelial cells were observed (Figures 1 A and B). Dome-like structures appeared as a result of cell to cell contact induced differentiation, when cells were grown for extended period of time at high density (Figures 1 C and D). The presence of cells representing epithelial and myoepithelial cell type was confirmed by immunostain- ing. Myoepithelial cells stained positively for smooth muscle α-actin whereas proposed luminal epithelial cells did not (Fig. 2). Cells of caprine cell line stained variously for cytokeratins (K14, K18, and K19) and negatively for mesenchymal intermediate filament protein vimentin. Complete differentiation of cells was achieved when growing them on basal membrane (GT) matrix. GMECs grown on thin layer of GT matrix in growth medium supplemented with lactogenic hormones were able to express β-casein. Expression of β-casein was confirmed Figure 2: Cell line labelled with antibodies for smooth muscle α-actin. Arrows indicate island of epithelial cells surrounded with myo- epithelial cells. A. light microscopy (magnification ×100). B. fluorescent microscopy (magnification ×100). Slika 2: Celična linija označena s protitelesi proti α-aktinu gladkih mišičnih vlaken. Puščici prikazujeta otok epitelnih celic obkrožen z mioepitelnimi celicami. A. svetlobna mikroskopija (100 x povečava). B. fluorescentna mikroskopija (100 x povečava). Figure 3: Agarose gel electrophoresis of RT-PCR products for β-casein (lanes 2,3) and β-actin (lane 4) as control marker. Slika 3: Agarozna gelska elektroforeza RT-PCR produktov za β-casein (stolpec 2,3) in β-aktin (stolpec 4), ki smo ga uporabili za kontrolo. Acta agriculturae Slovenica, 94/2 – 2009 137 IN VITRO MAMMARY GLAND MODEL: ESTABLISHMENT ... CAPRINE MAMMARY EPITHELIAL CELL LINE with reverse transcription PCR (Fig. 3). Products for β-casein (CSN2) and housekeeping gene β-actin (ACTB), which we used as control, were confirmed by sequenc- ing. This indicates that under lactogenic conditions milk proteins are being produced by GMECs. When grown on GT matrix using 3-D culture meth- od and supplemented with medium containing lactogen- ic hormones three-dimensional spheroids resembling acini of lactating mammary gland were formed (Fig. 4). 4 DISCUSSION We describe the establishment of GMEC line fo- cusing on growth morphology, expression of cytoskel- etal proteins and evidence of differentiation. When the dissociated mammary gland cells were grown in vitro some of the cells formed island monolayer aggregates while others existed in free single-cell form. The GMECs were of different types. Epithelial type of cells depended closely on one another, were connected to each other and formed islands of similar densely packed cuboidal cells. Myoepithelial cell growth was observed in individual, random cell pattern at lover density compared to their epithelial counterparts. Spontaneous domes are formed in post-confluent cell line which in a way is reminiscent of 3D organiza- tion of cells. It has been previously shown that formation of dome-like structures was connected with fluid under the epithelial cells that grew on plastic (Pickett et al., 1975). Functional and structural changes that take place Figure 4: 3D culture of GMECs in GT matrix supplemented with growth medium containing lactogenic hormones (magnification ×100). Slika 4: 3D organizacija epitelne celične linije v GT matriksu, gojena v gojišču z dodanimi laktogenimi hormoni (100 x povečava). in dome-forming cells correspond to cellular changes oc- curring in vivo when tubules and alveoli are developed in the mammary gland at pregnancy (Zucchi & Dulbecco, 2002). Specific smooth muscle α-actin monoclonal anti- body reactivity was shown in myoepithelial cells. Actin is observed as sheets of filaments in the myoepithelial cell cytoplasm, whereas epithelial cells did not react with this antibody. Since vimentin is a marker of nonepithelial cells (i.e. cells of mesenchymal origin) non-staining of GMECs indicate that there are no fibroblasts in the cell line. Cytoskeletal protein expression is very much de- pendent on culture conditions and substrate of growth, thus we were not able to determine specific staining for cytokeratins. The cells isolated from the goat mammary gland undergo three-dimensional organization in Geltrex. We observed formation of mammospheres or acinus-like structures, morphologically similar to those described as deriving from MECs (German & Barash, 2002, Rose et al., 2002). Under this condition casein secretion by GMECs was dependent on the presence of lactogenic hormones. We were able to prove expression of β-casein, which is the major milk protein in goat milk, however we were not able to prove expression of other milk proteins. When growing in culture, GMECs closely mimic the in vivo state of mammary gland, thus providing a suitable cell system model to study complex biological processes and pathways. Compared with monolayer cells in 2D culture, 3D cell culture provides physiologically much more relevant model for studying mammary cell Acta agriculturae Slovenica, 94/2 – 2009138 J. OGOREVC et al. function. Our GMEC line will be exploited in transcrip- tomic studies focused on host response during infection, replacing challenging in vivo experiments. 5 REFERENCES Blum J.L., Wicha M.S. 1988. Role of the cytoskeleton in laminin induced mammary gene expression. J. Cell. Physiol., 135: 13–22 German T., Barash I. 2002. Characterization of an epithelial cell line from bovine mammary gland. In vitro Cell. Dev. Biol. Anim., 38: 282–292 Huynh H.T., Robitaille G., Turner J. 1991. Establishment of bo- vine mammary epithelial cells (MAC-T): an in vitro model for bovine lactation. Experimental Cell Research, 197: 191–199 Kabotyanski E.B., Rijnkels M., Freeman-Zadrowski C., Buser A.C., Edwards D.P., Rosen J.M. 2009. Lactogenic hormonal induction of long distance interactions between beta-casein gene regulatory elements. J. Biol. Chem., 284: 22815–24 Pantsckenko A.G., Woodcock-Mitchell J., Bushmich S.L., Yang T.J. 2000. Establishment and characterisation of a caprine mammary epithelial cell line (CMEC). In Vitro Cell. Dev. Biol. – Animal, 36: 26–37 Pickett P.B., Pitelka D.R., HamamotoT., Misfeldt D.S. 1975. Oc- cluding junctions and cell behavior in primary cell culture of normal and neoplastic mammary gland cells. The Jour- nal of Cell Biology, 66: 316–332 Rose M.T., Aso H., Yonekura S., Komatsu T., Hagino A., Ozut- sumi K., Obara Y. 2002. In vitro differentiation of a cloned bovine mammary epithelial cell. J. Dairy Res., 69: 345–355 Rose M.T., McConoche H. 2006. The long road to a representa- tive in vitro model of bovine lactation. JIFS, 3: 67–72 Zavizion B., van Duffelen M., Schaeffer W., Politis I. 1996. Es- tablishment and characterization of a bovine mammary ep- ithelial cell line with unique properties. In Vitro Cell. Dev. Biol. Anim., 32: 138–148 Zucchi I., Dulbecco R. 2002. Proteomic dissection of dome for- mation in a mammary cell line. Journal of Mammary gland Biology and Neoplasia, 7, 4: 373–384 Acta argiculturae Slovenica, 94/2, 139–142, Ljubljana 2009 COBISS: 1.01 Agris category code: / NEW PRIMER COMBINATIONS WITH COMPARABLE MELTING TEMPERATURES DETECTING HIGHEST NUMBERS OF nosZ SEQUENCES FROM SEQUENCE DATABASES Blaž STRES 1, Boštjan MUROVEC 2 Delo je prispelo 16. septembra 2008, sprejeto 15. septembra 2009. Received September 16, 2008; accepted September 15, 2009. 1 Univ. of Ljubljana, Biotechnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia, Ph.D., M.Sc., e-mail: blaz.stres@bfro.uni-lj.si 2 Univ. of Ljubljana, Fac. of Electrical Engineering, Tržaška 25, SI-1000 Ljubljana, Slovenia, Assist.Prof., Ph.D., M.Sc. New primer combinations with comparable melting tempera- tures detecting highest numbers of nosZ sequences from se- quence databases We explored existing primer sequences targeting nitrous oxide reductase (nosZ) gene in order to explore their capability to recognize variant nosZ sequences. Published nosZ sequences longer than 380 AA residues were obtained from Functional- Gene Database /Repository (http://flyingcloud.cme.msu.edu/ fungene/) and used for explorations with PrimerChart pro- gram. The numbers of sequences recovered using all possible forward and reverse primer combinations were determined and the stringency of primer site recognition was further varied by allowing 1, 2, or 3 primer mismatches to DNA binding site. We identified novel primer combinations resulting in satisfactory amplicon length (> 500 bp) and increased sequence recognition capabilities at comparable forward and reverse primer melting temperatures. Overall, this study indicates that current state of the art molecular methods can be and should frequently be further refined by the use of targeted bioinformatic approaches. Key words: microbiology / molecular biology / denitrifi- cation / nitrous oxide reductase / melting temperature / detec- tion Nove kombinacije začetnih oligonukleotidov s primerljivimi temperaturami taljenja zaznavajo najvišje število sekvenc nosZ v podatkovnih bazah V tej študiji sva raziskala obstoječe sekvence začetnih oli- gonukleotidov, s katerimi se pomnožujejo fragmenti gena za reduktazo N2O (nosZ), da bi proučila njihovo zmožnost pre- poznavanja variant sekvenc nosZ. Objavljene sekvence gena nosZ daljše od 380 aminokislninskih ostankov sva pridobila od FunctionalGene Database /Repository (http://flyingcloud. cme.msu.edu/fungene/) in jih analizirala s programom Primer- Chart. Raziskala sva število, ki ga prepoznajo posamične mo- žne kombinacije začetnih oligonukleotidov. V nadaljevanju sva spreminjala natančnost prileganja začetnih oligonukleotidov na tarčno DNK tako, da sva dovolila 1, 2, or 3 napačna parjenja med začetnim oligonukleotidom in DNK. Tako sva identifici- rala nove kombinacije začetnih oligonukleotidov, ki ustvarijo ustrezno dolge fragmente (> 500 bp), s povišano sposobnostjo prepoznavanja sekvenc pri primerljivi temperaturi taljenja za- četnih oligonukleotidov. Prav tako so se nakazale nove možno- sti za izboljšanje začetnih oligonukleotidov z vnosom novih degeneriranih mest. Ta študija nakazuje, da je novejše moleku- larne metode možno in tudi potrebno pogosto nadgrajevati s ciljanimi bioinformatskimi pristopi. Ključne besede: mikrobiologija / molekularna biologija / denitrifikacija / dušikov oksid / reduktaza / temperatura talje- nja / zaznavanje be readily studied by cultivation dependent approaches, culture-independent methods have been widely em- ployed to explore microbial diversity and understand community dynamics. These methods are believed to provide a snapshot of the relative abundances of under- lying microbial populations. However, as the number of 1 INTRODUCTION Knowledge of abundances and kinds of organisms in an ecosystem is widely recognized as an important step towards understanding the ecology of the system (Prosser et al., 2007). As most prokaryotic species cannot Acta agriculturae Slovenica, 94/2 – 2009140 B. STRES and B. MUROVEC sequences deposited to public databases increases and basic molecular approaches to studying microbial com- munities continue to differ, it is ever increasingly hard to grasp and contemplate the outcomes of numerous stud- ies. A closer inspection of published literature (http:// www.ncbi.nlm.nih.gov/PubMed or http://www.sciencdi- rect.com) reveals that a number of studies deployed un- balanced tools, the unbalance spanning from sampling, over DNA extraction to molecular tool development, their use and interpretation. NosZ is the crucial enzyme in denitrification that is responsible for conversion of potent greenhouse gas N2O to molecular dinitrogen. In an accompanying paper (Stres and Murovec, 2007) we explored the differences in predicted melting temperatures of available forward and reverse primers used in amplification of nosZ sequences. In this work we explored the capability of these prim- ers to recognize nosZ sequences using 0, 1, 2, 3 primer mismatch thresholds in order to identify novel primer combinations resulting in increased sequence recogni- tion capabilities at comparable melting temperatures. To achieve this in controlled manner, the aligned pub- lished sequences of nitrous oxide reductase (nosZ) gene of sufficient length were obtained from FunctionalGene Database /Repository (http://flyingcloud.cme.msu.edu/ fungene/) and used as a model community. 2 MATERIALS AND METHODS 2.1 DATA SELECTION Literature on the molecular methods used for am- plification of target denitrification genes from environ- mental samples was explored as described in accom- panying paper (Stres and Murovec, 2007). The primer sequences were extracted and organized into two dic- tionaries, containing forward and reverse primer se- quences. Each primer sequence was characterized with a primer published name, first base binding location and its DNA binding sequence. A selection of HMM aligned nosZ database cur- rently containing 2025 sequences was downloaded from FunGene Repository / Pipeline (http://flyingcloud.cme. msu.edu/fungene/). The sequences were selected accord- ing to primary criterion length (L > 380 AA) and HMM score (s > 20) thus resulting in a dataset containing1985 sequences. 2.2 DATA ANALYSIS Newly developed software PrimerChart (Murovec and Stres, unpublished) was used for analysis. The num- bers of sequences recovered using all possible forward and reverse primer combinations were explored. The stringency of primer site recognition was varied by al- lowing 1, 2, or 3 primer mismatches to DNA binding site. The combinations of forward and reverse primer pairs were ranked according to the number of sequences de- tected. 3 RESULTS AND DISCUSSION In the present study we used previously described primer sets to sample a model microbial community comprised of the longest available and aligned nosZ sequences. Figure 1 shows schematic distribution of primer binding sites to the gene of nosZ according to Pseudomonas aeruginosa 2192 complete genome full ni- trous oxide reductase sequence under accession number NZ_AAKW01000028. As it can be seen, the primer binding sites are mainly designed and distributed in the center region of nosZ gene spanning between ~1000 bp and ~2000 bp covering roughly 1000 kb region between two conserved CuA and CuZ sites (Hoeren et al., 1993). In this respect, this region contains the highest sequence coverage. Figure 2 shows the distribution of hits as detected by different forward and reverse primer combinations used in this study. Each primer combination was as- signed a number key after they were sorted according to the number of recognized sequences. As it can be seen, a small number of primer sets could be identified as po- tential candidates for primers with highest recognition capabilities of sequences from model nosZ community. Figure 1: The schematic representaion of primer-binding sites of primers used in this study. For more details on primers please see Stres and Murovec (2007). Slika 1: Shematičen prikaz mest naleganja začetnih oligonukleotidov, uporabljenih v tej študiji. Za podrobnosti glede začetnih oli- gonukleotudov glej Stres and Murovec (2007). Acta agriculturae Slovenica, 94/2 – 2009 141 NEW PRIMER COMBINATIONS WITH COMPARABLE ... NUMBERS OF nosZ SEQUENCES FROM SEQUENCE DATABASES As primers were covering comparable gene region we explored the effect of additional primer mismatches to DNA primer binding site. Increasing the number of recognition sites from 0 to 3 resulted in roughly 50% more detected sequence (dat not shown). This indicates that the degeneracy of already degenerated primers should and could be further increased to incorporate un- accounted variations in primer binding sites. However, this was not the scope of our current research. In theory, the melting temperature of forward and reverse primers used in a pair should be kept as compara- ble as possible (Ausubel et al., 1999). Therefore the in-sil- ico melting temperatures (Stres and Murovec, 2007) were taken as a measure of comparability of primers melting temperatures and a measure of their compatibility in or- der to be used as a primer pair in amplification. In this re- spect the following combination of forward and reverse primers can be suggested for further use in molecular studies (Table 1). However, differences in average melting temperatures of novel primer combinations should be Figure 2: The schematic representaion of primer-binding sites of primers used in this study. For more details on primers please see Stres and Murovec (2007). Slika 2: Shematičen prikaz mest naleganja začetnih oligonukleotidov, uporabljenih v tej študiji. Za podrobnosti glede začetnih oli- gonukleotudov glej Stres and Murovec (2007). 0 100 200 300 400 500 600 700 0 50 100 150 200 250 No. of primer combinations No . o f d et ec te d se qu en ce s – Table 1: The combination of forward and reverse primers suggested for further use in molecular studies exploring nosZ diversity in complex samples. Designations ?, +, ++, +++ indicate > 10 °C, < 6 °C,< 4 °C and < 2 °C difference in average melting temperatures of paired oligonucleotides, respectively. Preglednica 1: Predlagane kombinacije začetnih oligonukleotidov za uporabo v molekularnih študijah raznolikosti nosZ in complex samples. Oznake ?, +, ++, +++ kažejo > 10 °C, < 6 °C,< 4 °C in < 2 °C razlike v povprečnih temperaturah taljenja začetnih oligonu- kleotidov v paru. nameF average FTm sd nameR average RTm sd DNA Matches ∆ Tm ∆ Tm suitability Fragment lenght 24nosZf436 48.52 2.59 1nos1319R 61.94 2.34 580 −13.42 ? 883 24nosZf436 48.52 2.59 4nos1527R 59.33 1.97 474 −10.81 ? 1 091 35PsNosZ175F 60.07 0.00 1nos1319R 61.94 2.34 454 −1.87 +++ 1 144 2Nos1527F 64.65 1.87 17nosZ1773b 61.07 2.84 442 3.58 ++ 246 27nosZ-F-1181 66.01 1.83 19nosZ1R1421 65.42 2.31 441 0.58 +++ 240 27nosZ-F-1181 66.01 1.83 17nosZ1773b 61.07 2.84 439 4.94 + 592 25nosZ-F1211 66.18 1.61 19nosZ1R1421 65.42 2.31 436 0.76 +++ 210 25nosZ-F1211 66.18 1.61 17nosZ1773b 61.07 2.84 432 5.11 + 562 2Nos1527F 64.65 1.87 1773R 61.18 2.19 426 3.47 ++ 246 27nosZ-F-1181 66.01 1.83 1773R 61.18 2.19 423 4.83 + 592 25nosZ-F1211 66.18 1.61 1773R 61.18 2.19 418 5.00 + 562 Acta agriculturae Slovenica, 94/2 – 2009142 B. STRES and B. MUROVEC noted before adopting these combinations for research. Two of the most promising primer combinations resulted in more than 10  °C difference in melting temperatures rendering them least suitable. Both have numerous de- generated sites as can be seen from accompanying stand- ard deviations of their average melting temperatures. The most suitable primer pair satisfying (i) the need for suf- ficient amplicon length, (ii) comparable average melting temperatures, and (iii) sequence recognition capabilities appears to be NosZ175F and nosZ1319R. However, the following problems still remain: (i) relatively low number of sequences deposited to pub- lic databases, (ii) low number of sequences of sufficient quality (containing only characters A, T, G, C), (iii) un- equal melting temperatures of most suitable suggested primer pairs and (iv) relatively low resolution resulting from short sequences amplified by some of the suggested primers. Future studies, especially metagenomic studies and direct reconstructions of genomes from environ- ment, are going to provide valuable data on the uncov- ered nosZ gene variants in environment. 4 CONCLUSIONS The analysis of primer combinations revealed that existing primers sequence could be further modified to accommodate novel degenerated sites and thus be able to detect a broader sequence diversity. Further, some previously untested primer combinations were explored resulting in higher number of recognized sequences, suf- ficient length of amplicon (> 500 bp) and comparable melting temperatures, thus indicating their potential for future use in molecular studies. Future work is going to be directed towards detailed analysis of primer binding sites in order to generate combinations of primers target- ting widest array of availabe sequences. Overall, this study indicates that current state of the art molecular methods can be and should frequently be further refined by the use of targeted bioinformatic ap- proaches. 5 REFERENCES Ausubel F.M., Brent R., Kingston R.E., Moore D.D. Seidman J.G., Smith J.A., Struhl K. 1999. Current protocols in mo- lecular biology. New York, John Wiley and Sons, N. Y. Hoeren F.U., Berks B.C., Ferguson S.J., McCarthy J.E.G. 1993. Sequence and expression of the gene coding the respiratory nitrous-oxide reductase from Paracoccus denitrificans: new and conserved structural and regulatory motifs. Eur. J. Bio- chem., 218: 49–57 Prosser J.I., Bohanan B.J.M., Curtis T.P., Ellis R.J., Firestone M.K., Freckleton R.P., Green J.L., Green L.E., Killham K., Lennon J.J., Osborn A.M., Solan M., van der Gast C.J., Young J.P.W. 2007. The role of ecological theory in micro- bial ecology. Nature Rev. Microbiol., 5: 384–392 Stres B., Murovec B. Melting temperatures of degenerated oli- gonucleotides targetting nitrous oxide reductase (nosZ) genes. Acta Agric. Slov. (submitted) Acta argiculturae Slovenica, 94/2, 143–146, Ljubljana 2009 COBISS: 1.01 Agris category code: / ISOLATION AND USE OF Prevotella ruminicola TC18 PLASMID pTC18 IN Escherichia coli-P. ruminicola SHUTTLE VECTOR CONSTRUCTION Tomaž ACCETTO 1 Delo je prispelo 16. septembra 2008, sprejeto 15. septembra 2009. Received September 16, 2008; accepted September 15, 2009. 1 Univ. of Ljubljana, Biotechnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia, Ph.D., M.Sc., e-mail: tomaz.accetto@bfro.uni-lj.si cies to be genetically manipulated are needed and restric- tion barriers must be characterized and circumvented in order to develop a successful gene transfer system. To construct shuttle vectors for P. ruminicola, native P. ruminicola plasmids are therefore needed. Plasmids, however are surprisingly scarce in this bacterial genus (Peterka et al., 2003). One of the few reported P. rumini- cola plasmids was found in P. ruminicola strain TC18 but was not characterized nor exploited as a shuttle vector (Avguštin, 1992). Recently, the type II restriction-modi- fication system of P. ruminicola 23 was described as well as a procedure using HaeIII methylase to protect DNA against it was developed (Accetto et al., 2005). Isolation and use of Prevotella ruminicola TC18 plasmid pTC18 in Escherichia coli-P. ruminicola shuttle vector con- struction A cryptic plasmid of approximately 3 kilobases named pTC18 was discovered in a ruminal Prevotella ruminicola TC18 strain and cloned into Escherichia coli. Based on pTC18, several shuttle vectors, containing Prevotella/Bacteroides tetQ selection marker and E. coli vector pUC19 inserted at two different posi- tions in pTC18 were constructed. The shuttle vectors, protected with HaeIII methylase against the P. ruminicola 23 restriction were electroporated into P. ruminicola. Despite numerous at- tempts a tetracycline resistant recombinant strain 23 was not obtained. The possible causes for electroporation failure are discussed. Key words: microbiology / anaerobic bacteria / Prevotella ruminicola / shuttle vector / rumen Osamitev plazmida pTC18 seva Prevotella ruminicola TC18 in njegova uporaba v razvoju prenosljivih vektorjev Escherichia coli-P. ruminicola V vampnem sevu Prevotella ruminicola TC18 smo odkrili 3 kilobazne pare dolgo plazmidno DNA, jo poimenovali pTC18 in klonirali v Escherichia coli. Na njeni osnovi smo razvili več različic prenosljivega plazmida, ki je poleg pTC18 vseboval še selekcijski marker tetQ iz sevov rodu Bacteroides in plazmidni vektor E. coli pUC19. Prenosljive vektorje smo s HaeIII meti- lazo zaščitili proti restrikciji v P. ruminicola 23 in jih nato po- skusili vnesti v P. ruminicola 23 z elektrotransformacijo. Kljub mnogim poskusom nismo uspeli pridobiti proti tetraciklinu odpornih sevov P. ruminicola 23. Ključne besede: mikrobiologija / anaerobne bakterije / Prevotella ruminicola / prenosljivi vektor / vamp 1 INTRODUCTION Prevotella ruminicola is thought to be the most nu- merous among the strictly anaerobic gram negative ru- men bacteria from the genus Prevotella which apparently play important roles in the rumen ecosystem (Tajima et al., 2001; Miyazaki et al., 2003). The genome of the P. ru- minicola type strain 23 is currently being sequenced at former TIGR, now J. Craig Venter Institute (http://www. jcvi.org/rumenomics/). However, even the most basic genetic tools such as gene introduction system, which would enable verification of ideas that may originate from the genome data analysis, are undeveloped for this bacterial species. It was shown previously (Purdy et al., 2002) in Clostridium difficile that plasmids, native to spe- Acta agriculturae Slovenica, 94/2 – 2009144 T. ACCETTO 2 MATERIAL AND METHODS 2.1 STRAIN, PLASMID, MEDIUM AND GROWTH P. ruminicola TC18 (van Gylswyk, 1990) was grown anaerobically in M2 medium (Hobson, 1969) according to the Bryant’s modification of the Hungate technique (Bryant, 1972). Source of tetQ alele was E. coli-Bacter- oides shuttle plasmid pRH3 (Daniel et al., 1995). The plasmid DNA was extracted using standard alkaline lysis. Cleavage with restriction endonucleases, ligation and transformation of Escherichia coli were all done using standard molecular biology techniques (Sam- brook, 2001). The DNA was protected against the P. ru- minicola 23 restriction using HaeIII methylase (NEB, USA) according to manufacturers instructions in reac- tions which contained S-adenosyl methionine as the methyl donor. The protected plasmid DNA was electro- porated into P. ruminicola TC18 as described previously (Accetto et al., 2005). Briefly: growth of P. ruminicola TC18 culture was stopped during exponential growth at OD600 = 0.5 by chilling on ice. The cells were then washed three times in anaerobic ice-cold 10% glycerol, electro- porated at 12.5 kV/cm, resuspended in fresh M2 medium and left at 37  °C for an hour. Subsequently, the 0.1 ml portions of cells were transferred on tetracycline contain- ing M2 agar plates in an anaerobic chamber. 3 RESULTS AND DISCUSSION Plasmid DNA was isolated from P. ruminicola TC18 (Fig. 1A). Restriction enzymes HindIII, BamHI, KpnI in XhoI all convert plasmid DNA into a linear, approximate- ly 3100 base pairs long DNA. The plasmid was named pTC18 and its restriction map is presented in Fig. 1B. HindIII cleaved pTC18 was ligated into multi- ple cloning site of pUC19 and transformed into E. coli TOP10 (Invitrogen, USA). The resulting construct was cleaved using SstI and ligated to tetQ allele. The latter was obtained by cleavage of pRH3 with SstI and subsequent isolation of 2.6 kilobase pair fragment from the agarose gel. The ligation products were transformed into E. coli TOP10 and restriction analysis of plasmid DNA was per- formed on several recombinant strains to obtain strains harbouring both possible tetQ orientations (Fig. 2) Since it is possible that HindIII site lies within the pTC18 replication region and thus cloning into this site would most likely inactivate replication in Prevotella hosts, we have also constructed shuttle vectors using the pTC18 KpnI site. The procedures were essentially the same as above yielding constructs presented in figure 3. All four shuttle vector constructs were subsequently protected against the P. ruminicola 23 restriction enzyme Pru2I using HaeIII methylase and electroporated into P. ruminicola 23 cells. Despite numerous attempts we were unable to obtain a tetracycline resistant P. ruminicola 23 strain harbouring the shuttle vector. The electroporation parameters i.e. DNA concentration, electrocompetent cells density and electroporation time constant were es- sentially the same as in the previously described suc- cessful electroporation of plasmid pRH3 into P. bryantii TC1-1 strain (Accetto et al., 2005). Several explanations for the failure of electroporation are possible: (i) both, HindIII and KpnI site are placed within the region essen- tial for pTC18 replication (ii) P. ruminicola 23 harbours A– 1– 2– B – Figure 1: A: Plasmid DNA isolated from P. ruminicola TC18, agarose DNA electrophoresis. 1: marker generuler 1kb dna ladder (Fer- mentas); 2: plasmid DNA isolated from P. ruminicola TC18. B: Restriction map of pTC18. Slika 1: A: Plazmidna DNA iz P. ruminicola TC18, agarozna DNA elektroforeza. 1: velikostni standard generuler 1kb dna lestvica (Fermentas); 2: plazmidna DNA, osamljena iz P. ruminicola TC18. B: Restrikcijska mapa pTC18. Acta agriculturae Slovenica, 94/2 – 2009 145 ISOLATION AND USE OF Prevotella ruminicola TC18 PLASMID … SHUTTLE VECTOR CONSTRUCTION another, non type II restriction system (iii) P. ruminicola 23 contains a cryptic plasmid that cannot be isolated by ordinary means or its relicts, but in both cases they be- long to the same incompatibility group as pTC18 does and (iv) tetQ gene is lethal to or does not function in P. ruminicola 23. 4 CONCLUSIONS The novel Prevotella plasmid pTC18 based shut- tle vectors were unable to transform P. ruminicola 23. Several strategies to overcome this may be envisaged: transformation of other P. ruminicola strains preceded by protection of transforming DNA using cell free ex- tract of strains to be transformed (Accetto et al., 2005); the tetQ antibiotic resistance gene can be exchanged with cfXA2 cephalosporinase resistance gene, known to reside in several oral Prevotella isolates (Giraud-Morin et al., 2003) and finally, the other two unique restriction sites BamHI and XhoI can be exploited as cloning sites for antibiotic resistance gene and E. coli replicon in order to evade the pTC18 replication region supposedly inacti- vated by cloning into HindIII and KpnI sites. – Figure 2: Restriction maps of shuttle vectors based on pTC18 cleaved with HindIII and with different orientations of tetQ gene. Slika 2: Restrikcijska mapa prenosljivih vektorjev osnovanih na pTC18 cepljenim s HindIII z različnima usmeritvama tetQ. – Figure 3: Restriction map of shuttle vectors based on pTC18 cleaved with KpnI and with different orientation of tetQ gene. Slika 3: Shema prenosljivih vektorjev osnovanih na pTC18 cepljenim s KpnI z različnima usmeritvama tetQ. Acta agriculturae Slovenica, 94/2 – 2009146 T. ACCETTO 5 REFERENCES Accetto T., Peterka M., Avguštin G. 2005. Type II restriction modification systems of Prevotella bryantii TC1-1 and Prevotella ruminicola 23 strains and their effect on the ef- ficiency of DNA introduction via electroporation. FEMS Microbiology Letters, 247: 177–183 Avguštin G. 1992. Analysis of the role of bacterium Prevotella (Bacteroides) ruminicola in rumen ecosystem using molec- ular genetic techniques. Doctoral dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Fac.: 184 p. Bryant M.P. 1972. Commentary on the Hungate technique for culture of anaerobic bacteria. American. Journal of Clinical Nutrition, 25: 1324–1328 Daniel A.S., Martin J., Vanat I., Whitehead T.R., Flint H.J. 1995. Expression of cloned cellulase/xylanase gene from Prevotel- la ruminicola in Bacteroides vulgatus, Bacteroides uniformis and Prevotella ruminicola. Journal of Applied Bacteriology, 79: 417–424 Giraud-Morin C., Madinier I., Fosse T. 2003. Sequence analysis of cfxA2-like beta-lactamases in Prevotella species. Journal of Antimicrobial Chemotherapy, 51: 1293–1296 van Gylswyk N.O. 1990. Enumeration and presumptive identi- fication of some functional groups of bacteria in the rumen of dairy cows fed grass silage-based diets. FEMS Microbiol- ogy ecology, 73: 243–254 Hobson P.N. 1969. Rumen bacteria. In: Methods in Microbiol- ogy. Vol 3B. Norris J.R., Ribbons D.W. (eds.). London and New York, Academic press: 133–149 Miyazaki K., Miyamoto H., Mercer D.K., Hirase T., Martin J.C., Kojima Y., Flint H.J. 2003. Involvement of the multidomain regulatory protein XynR in positive control of xylanase gene expression in the ruminal anaerobe Prevotella bryantii B14. Journal of Bacteriology, 185: 2219–2226 Peterka M., Tepšič K., Accetto T., Kostanjšek R., Ramšak A., Lipoglavšek L., Avguštin G. 2003. Molecular microbiology of gut bacteria: genetic diversity and community structure analysis. Acta Microbiologica et Immunologica Hungarica, 50: 395–406 Purdy D., O´Keeffe T.A.T., Elmore M., Herbert M., Mcleod A., Bokori-Brown M., Ostrowski A., Minton N.P. 2002. Conju- gative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the re- striction barrier. Molecular Microbiology, 46: 429–452 Sambrook J., Russel D.W. 2001. Molecular Cloning: A Labora- tory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press Tajima K., Aminov R.I., Nagamine T., Matsui H., Nakamura M., Benno Y. 2001. Diet-dependent shifts in the bacterial ppopulation of the rumen revealed with real-time PCR. Applied and Environmental Microbiology, 67: 2766–2774 Acta argiculturae Slovenica, 94/2, 147–152, Ljubljana 2009 COBISS: 1.01 Agris category code: / THE SEARCH FOR CONJUGATIVE TRANSPOSON IN RUMEN BACTERIUM Prevotella bryantii B14 Gregor GORENC 1, Tomaž ACCETTO 1, Gorazd AVGUŠTIN 2 Delo je prispelo 16. septembra 2008, sprejeto 15. septembra 2009. Received September 16, 2008; accepted September 15, 2009. 1 Univ. of Ljubljana, Biotechnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia, Ph.D. 2 Same address, Prof., Ph.D., e-mail: gorazd.avgustin@bfro.uni-lj.si The search for conjugative transposon in rumen bacterium Pre- votella bryantii B14 Only few plasmids and bacteriophages have been de- scribed to date in ruminal prevotella strains, therefore it ap- pears plausible that the genetic exchange in these organisms must exploit other routes. Large conjugative transposons make possible the gene exchange process in bacteria from the genus Bacteroides, the phylogenetic relatives of ruminal prevotellas. The access to fully or partially finished genome sequences of Bacteroides and Prevotella representatives made possible the search for conserved regions within putative conjugative trans- posons. Multiple sequence alignment of known and putative conjugative transposon gene sequences of Bacteroides thetaio- taomicron, Prevotella intermedia, Bacteroides fragilis and Tan- erella sp. was used to locate partially conserved regions within most preserved conjugative transposition genes, traG, and to construct appropriate degenerated oligonucleotide primers. These were used to amplify genome fragments from ruminal prevotella strains. Sequence analysis of the subcloned PCR products revealed the presence of a hypothetical gene in the genome of Prevotella bryantii B14, similar to the ORF BF2880 from B. fragilis YCH46, which is a part of a large conjugative transposon. Inverse PCRs were designed and performed to confirm the initial findings. A partial map of P. bryantii B14 pu- tative conjugative transposon region was constructed, indicat- ing an intergeneric horizontal gene transfer. Key words: microbiology / molecular genetics / conjuga- tive transposon / Prevotella bryantii / Bacteroides fragilis Iskanje konjugativnega transpozona v vampni bakteriji Prevo- tella bryantii B14 Ker sevi rodu Prevotella iz vampa le izjemoma posedujejo plazmide in je opisanih le nekaj bakteriofagov, je zelo verjetno, da izmenjava genov pri teh organizmih vključuje druge poti. Veliki konjugativni transpozoni omogočajo prenos genov pri rodu Bacteroides, filogenetskih sorodnikih vampnih prevotel. Dostop do delno ali v celoti sekvenciranih genomov predstav- nikov Bacteroides in Prevotella je omogočil iskanje ohranjenih regij znotraj domnevnih konjugativnih transpozonov. S porav- navo več sekvenc znanih ali domnevnih genov konjugativnih transpozonov iz vrst Bacteroides thetaiotaomicron, Prevotella intermedia, Bacteroides fragilis in rodu Tanerella smo določili delno ohranjene regije v najbolj ohranjenem genu konjugativne transpozicije, traG, in jih uporabili za izdelavo primernih zače- tnih oligonukleotidov za pomnoževanje dela gena pri vampnih sevih iz rodu Prevotella. Analiza sekvenc subkloniranih po- množkov je pri P. bryantii B14 razkrila prisotnost hipotetičnega gena, podobnega odprtemu čitalnemu okvirju BF2880 seva B. fragilis YCH46, ki je del velikega konjugativnega transpozona. Z inverzno verižno reakcijo s polimerazo smo potrdili prvotne ugotovitve. Izdelali smo delno mapo regije domnevnega konju- gativnega transpozona pri P. bryantii B14, ki nakazuje, da je pri- šlo do medrodovnega horizontalnega prenosa genov. Ključne besede: mikrobiologija / molekularna genetika / konjugativni transpozon / Prevotella bryantii / Bacteroides fra- gilis Acta agriculturae Slovenica, 94/2 – 2009148 G. GORENC et al. 1 INTRODUCTION Prevotella bryantii is a Gram-negative, strictly anaerobic member of the evolutionary distinct phylum Bacteroidetes, also known as the CFB group (Paster et al., 1994; Shah and Collins, 1990). It is an important envi- ronmental commensal bacterium inhabiting the rumen (Avguštin, 1992; Hobson, 1997; Ramšak et al., 2000). In this complex ecosystem some important functions have been assigned to ruminal prevotellas, e.g. degradation of proteins and certain plant cell polysaccharides (Daniel et al., 1995; Hobson, 1997; Teather et al., 1997). Due to its rather distinct capability to survive at lower pH values than other rumen bacteria, this organism was proposed as a suitable model organism for genetic manipulation studies in ruminal ecosystem (Russel and Dombrowski, 1980). The substantial evolutionary distance to well stud- ied bacteria like E.coli is probably the main reason for poor understanding of prevotella genetics (Avguštin et al., 1994, Accetto and Avguštin, 2007). The lack of suit- able genetic tools hindered the progress and brought it almost to standstill in the mid 1990’s. The recent progress in the genetic research of their relatives i.e. the prevotel- las and the bacteroides from the human colon revived the interest in the genetics of ruminal prevotellas in last cou- ple of years (Shoemaker et al., 1992; Nikolich et al., 1994; Vercoe and White, 1997; Flint and Scott, 2000; Accetto et al., 2005, Accetto and Avguštin, 2007). If an organism is to be a suitable genetic model, its horizontal gene transfer capacity should be known as well potential barriers for it. In addition to strong non- specific deoxyribonuclease activity of Prevotella bryan- tii B14 (Flint and Thomson, 1990), only few plasmids and bacteriophages have been described from ruminal prevotella strains (Flint and Stewart, 1987; Ogata et al., 1996; Accetto and Avguštin, 1997; Ambrožič et al., 2001), therefore it appears plausible that the genetic exchange in these organisms must exploit other routes. Conjugative transposons are distinct DNA segments that are normally integrated into bacterial chromosome and transfer by conjugation from donor to recipient bac- terium. These genetic elements are integrated in the host genome except during transfer, therefore no method for their identification exists, analogous to plasmid isolation (Salyers et al., 1995). All studies of conjugative transpo- sition in ruminal Prevotella strains were linked to con- jugative transposition elements in Bacteroides spp. In vitro experiments showed that bidirectional transfer of native conjugative transposition element Tcr Emr 12256 from B. fragilis clinical isolate can occur between closely related human commensal species Bacteroides uniformis and B. thetaiotaomicron, and ruminal P. bryantii strains (Shoemaker et al., 1992). Additionally, mating experi- ments confirmed in vitro mobilization of Tcr Emr 7853 conjugative transposition element in P. bryantii B14 strain (Nikolich et al., 1994). Nevertheless, to date the presence and identity of a conjugative transposon in any ruminal Prevotella species remains unproven. 2 MATERIAL AND METHODS 2.1 BACTERIAL STRAINS AND DNA ISOLATION Two strictly anaerobic ruminal strains from genus Prevotella were used in this study: P. bryantii B14 (Rus- sel, 1983) and P. bryantii TC 1-1 (Van Gylswyk, 1990). Strains were grown under strict anaerobic conditions in M2 medium (Hobson, 1969) by modification of the Hungate technique for cultivation of anaerobic bacteria, as described by Bryant (1972). Eight ml of M2 medium was inoculated and incubated under anaerobic condi- tions for 14–24 hrs at 37 °C, until optical density at 654 nm reached 0.9–1.4. Total genomic DNA was isolated by modification of the CTAB/NaCl isolation protocol, as described in “Current Protocols in Molecular Biology” (Ausubel et al., 1987). 2.2 MULTIPLE SEQUENCE ALIGNMENT AND DEGENERATE PRIMER CONSTRUCTION Multiple sequence alignment program tool Clustal X (Thompson et al., 1997) was used to align known and putative conjugative transposon transfer gene sequences of B. thetaiotaomicron, P. intermedia, B.  fragilis and Ta- nerella sp. Largest consensus regions within traG gene were located and, considering their appropriate length and reciprocal location, used to construct a pair of de- generate oligonucleotide primers CTf1310 and CTr2270 (see results). 2.3 DEGENERATE PCR, DNA CLONING, SE- QUENCING AND SEQUENCE ANALYSIS Degenerate PCR was used to amplify the putative homologs of the traG gene in P. bryantii B14 and P. bry- antii TC 1-1 strains. 25 ml of reaction mixture contained 2.5 ml of the 10x Taq Buffer (Fermentas), 2 mM MgCl2, 0.2  mM deoxynucleoside triphosphate mixture (dATP, dGTP, dTTP, dCTP), 1.4 mM of each degenerate primer, 0.75 U Taq DNA polymerase (Fermentas) and 20 ng of genomic DNA, extracted from cultured ruminal prevo- tella strains. A series of PCR reactions was performed to determine the final amplification conditions: an ini- Acta agriculturae Slovenica, 94/2 – 2009 149 THE SEARCH FOR CONJUGATIVE TRANSPOSON IN RUMEN BACTERIUM Prevotella bryantii B14 tial denaturation step of 94  °C for 3 min, followed by 40 cycles of denaturation at 94 °C for 45 s, annealing at 53 °C for 1 min and extension at 72 °C for 1 min. PCR was prolonged by final extension at 72 °C for 7 min. PCR products were separated by electrophoresis on 1% agar- ose gels, stained with ethidium bromide and visualized under UV light using GelDoc 1000 trans-illuminator (BioRad). Degenerate PCR products containing specif- ic DNA bands were excised from the gel, purified with QIAQUICK Gel Extraction Kit (Qiagen) and subcloned with TOPO TA Cloning Kit (Invitrogen). DNA sequenc- es of the pBAD/Thio-TOPO vector inserts were deter- mined by Microsynth GmbH (Switzerland). Sequence data were analyzed using BLAST analysis and compared to sequences from the NCBI and TIGR CMR databanks. 2.4 INVERSE PCR AND PRIMER WALKING Retrieved sequences were used as a template for construction of specific primer pair, oriented outwards of the retrieved sequence, and used for inverse PCR (Och- man et al., 1988). 5 mg of genomic P. bryantii B14 DNA was partially digested using restriction endonucleases EcoRI (Gibco), HindIII (Promega), NotI or PstI (Fermen- tas), followed by purification, agarose electrophoresis size selection and quantification of digested DNA. A se- ries of self-ligation reactions was set, using 0.1, 0.25, 0.5, 0.75, 1.0; 5 and 10 ng/μl DNA with 5 U of T4 DNA ligase (Fermentas) per reaction. Self-ligation reaction was car- ried out at 22 °C for 1 h, again followed by purification, agarose electrophoresis size selection and DNA quanti- fication. A series of inverse PCRs with an annealing tempera- ture span of 50–60 °C was set. 50 ml of reaction mixtures contained 5.0 ml of the 10x Long PCR Buffer+Mg (Fer- mentas), 0.5  mM deoxynucleoside triphosphate mix- ture (dATP, dGTP, dTTP, dCTP), 0.5 mM of each inverse primer, 2.5 U Long PCR Enzyme Mix (Fermentas) and 1.0 ml of purified self-ligation reactions containing 0,1 to 10 ng/μl DNA per reaction. Amplification conditions consisted of an initial denaturation step of 94  °C for 2 min, followed by 10 cycles of denaturation at 94 °C for 15 s, annealing for 30 s and extension at 68 °C for 10 min. 27 cycles followed, consisted of denaturation at 94 °C for 15 s, annealing for 30 s and extension at 68 °C for 10 min, with extra 5 s added each cycle. PCR was prolonged by final extension at 68 °C for 10 min. PCR products were examined by electrophoresis, purified and sequenced (Microsynth GmbH, Switzerland). The acquired se- quences were analyzed and used to construct primers for next part of the sequence in primer walking procedure. Sequences were linked and compared to known genome sequences of related species. 3 RESULTS AND DISCUSSION Multiple sequence alignment of known and putative conjugative transposition traG genes from sequenced members of the Bacteroidetes phylum showed that no conserved oligonucleotides larger than 5 nucleotides ex- ist within the aligned 2.5 kbp region (complete alignment not shown). Thus two largest partially conserved regions were identified, spanning approx. 960 bp long region (Fig. 1). The mismatch positions were used as 2–4-base degeneracies for the primer construction. 19 bp forward primer CTf1310 (5’-CSA-AYM-GHA-ACA-ART-TYR- T-3’) with 192-fold degeneracy and 17 bp reverse primer CTr2270 (5’-TCC-TTN-TCS-GTC-AGN-CC-3’) with 32-fold degeneracy were constructed and subsequently used in degenerate PCR. Figure 1: Multiple sequence alignment of known and putative conjugative transposition transfer genes from sequenced members of the Bacteroidetes phylum. Partial display of the aligned 2.5 kbp region with the two largest partially conserved regions, used for construc- tion of degenerated oligonucleotide primers CTf1310 and CTr2270, is shown. Slika 1: Poravnava sekvenc znanih in domnevnih genov prenosa konjugativne transpozicije članov debla Bacteroidetes, pri katerih je že znana celotna sekvenca genoma. Prikazan je delni prikaz poravnave 2,5 kbp regije z dvema najobsežnejšima delno ohranjenima regijama, ki smo ju uporabili za izdelavo degeneriranih začetnih oligonukleotidov CTf1310 in CTr2270. Acta agriculturae Slovenica, 94/2 – 2009150 G. GORENC et al. In degenerate PCR the competitive inhibition due to high primer degeneracy may occur. Primers anneal to the correct template but are not extended by the polymer- ase due to unstable 3’-ends, which results in high ineffi- ciency of the first few PCR cycles. This can be overcome by increasing PCR cycles, which in return may increase nonspecific background and decrease the yield of specific PCR product. Only P. bryantii B14 reaction showed the presence of expected fragment of approximately 1 kbp (Fig. 2), which was isolated and subcloned. The analysis of the sequenced region revealed the presence of a complete open reading frame sharing 24% identity with traI gene from P. intermedia strain 17 coju- gative transposon and 58% identity with the ORF BF2880 from the putative conjugative transposon of the B. fragilis strain YCH46, at the amino acid level. The 24% identity with the traI gene from P. intermedia 17 is on the border as far as assigning of the function or recognition of ho- mology is concerned. However, the rather high degree of similarity with the ORF BF2880 from B. fragilis provides strong evidence for intergeneric horizontal gene transfer, especially if we bare in mind that the average DNA:DNA homology of total chromosomal DNA from Bacteroides species and ruminal Prevotella species is less than 5% (Johnson in Harich, 1986). Determination of the complete sequence of the cloned P. bryantii B14 genome fragment made possible the construction of specific primer set, which was sub- sequently used for inverse PCR in order to extend the known sequence in both directions. Figure 3 shows a series of inverse PCR amplifications from self-ligation reactions under most suitable conditions. All inverse PCR reactions showed the presence of 2.5 kbp products. The products of 0.1 ng/μl DNA reac- tion without additional multiple or smeared bands were used for subsequent sequencing, primer construction and gene walking procedure. The retrieved sequence data was analyzed and used to construct a partial map of P.  bryantii B14 genomic region homologous to the putative conjugative transposon region of the B. fragilis YCH46 . Its structure and organization is shown in Fig- ure 4. Five ORFs with 162, 231, 108, 102 and 164 amino- acid residues were identified, with the first two being completely sequenced. BLAST analysis at amino-acid Figure 2: Agarose gel electropho- resis of degenerate PCR multipli- cation of putative traG homologs in P. bryantii B14 and P. bryantii TC1-1. M – DNA size marker O’GeneRuler 1 kb DNA Ladder, 2 ml; 1 – Prevotella bryantii B14; 2 – Prevotella bryantii TC 1-1. Slika 2: Agarozna gelska elektroforeza pomnoževanja domnevnega homologa gena traG z degenerirano verižno reakcijo s polimerazo pri P. bryantii B14 in P. bryantii TC1-1. M – veliko- stni standard O'GeneRuler 1 kb DNA Ladder, 2 ml; 1 – Prevotella bryantii B14; 2 – Prevotella bryan- tii TC 1-1. Figure 3: Agarose gel electrophoresis of inverse PCR amplifica- tions from self-ligation reactions of P. bryantii B14 genomic DNA flanking the targeted region. Reactions were performed at 54 °C annealing temperature, using 0,1 to 10 ng/μl DNA per reaction. M – DNA size marker O’GeneRuler 1 kb DNA Ladder, 2 µl; 10 µl reaction samples: 1 – 0,1 ng/μl DNA; 2 – 0,25 ng/μl DNA; 3 – 0,5 ng/μl DNA; 4 – 0,75 ng/μl DNA; 5 – 1,0 ng/μl DNA; 6 – 5 ng/μl DNA; 7 – 10 ng/μl DNA. Slika 3: Agarozna gelska elektroforeza produktov inverzne verižne reakcije genomske DNK P. bryantii B14. Reakcije so bile izvedene pri 54 °C z 0,1 do 10 ng/μl DNA na reakcijo. M – ve- likostni standard O’GeneRuler 1 kb DNA Ladder, 2 µl; vzorci po 10 µl na jamico: 1 – 0,1 ng/μl DNA; 2 – 0,25 ng/μl DNA; 3 – 0,5 ng/μl DNA; 4 – 0,75 ng/μl DNA; 5 – 1,0 ng/μl DNA; 6 – 5 ng/μl DNA; 7 – 10 ng/μl DNA. Acta agriculturae Slovenica, 94/2 – 2009 151 THE SEARCH FOR CONJUGATIVE TRANSPOSON IN RUMEN BACTERIUM Prevotella bryantii B14 level (TIGR; http://tigrblast.tigr.org/cmr-blast/) showed the highest similarity with BF2879 – BF2884 ORFs (with 69%, 61%, 45%, 71% and 44% identities, respectively) in B. fragilis YCH46, which are placed within the larg, 120 kbp putative conjugative transposon CTn3Bf (Kuwahara et al., 2004). Comparison of the mapped P. bryantii B14 region to the available genomic sequences of the relat- ed Bacteroidetes (TIGR CMR; http://cmr.tigr.org/tigr- scripts/CMR/CmrHomePage.cgi) shows a similar gene organization. These findings confirm that these genetic elements were likely transferred to or from P.  bryantii B14. Nevertheless, additional work is needed to reveal the nature of conjugative transposition in ruminal Prevotella species, to determine the presence of an active conjuga- tive transposition element and its complete sequence. We are currently optimizing the genomic DNA primer walk- ing procedure and preparing a cosmid genomic library in order to reveal the sequence of this interesting region in P. bryantii B14. 4 CONCLUSIONS Multiple sequence alignment of known and putative conjugative transposition traG genes from sequenced members of the Bacteroidetes phylum showed that no conserved oligonucleotides larger than 5 nucleotides ex- ist within the aligned region, therefore appropriate de- generate oligonucleotide primers had to be constructed to amplify homologous gene fragments from ruminal prevotella strains. Sequence analysis of degenerate PCR products revealed the presence of an ORF homologous with ORF BF2880 from the putative conjugative trans- poson of B.  fragilis YCH46, showing 58% identity at the amino acid level. Inverse PCRs making possible the amplification of the flanking regions in P.  bryantii B14 were set and their products sequenced. A partial map of P. bryantii B14 genomic DNA homologous to the putative conjugative transposon region of B.  fragilis YCH46 was constructed, showing the same gene organization and high gene similarity. The observed indices suggest that the conjugative transposition elements of P. bryantii B14 were introduced through an intergeneric horizontal gene transfer. 5 REFERENCES Accetto T., Peterka M., Avguštin. 1999. Deoxyribonuclease ac- tivities of rumen bacteria from the genus Prevotella. Zborn- ik Biotehniške fakultete Univerze v Ljubljani, 74: 83–88 Accetto T., Avguštin G. 2007. Studies on Prevotella nuclease us- ing a system for the controlled expression of cloned genes in P. bryantii TC1-1. Microbiology-SGM, 153: 2281–2288 Accetto T., Peterka M., Avguštin G. 2005. Type II restriction modification systems of Prevotella bryantii TC1-1 and Prevotella ruminicola 23 strains and their effect on the ef- ficiency of DNA introduction via electroporation. 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Hob- son P.N., Stewart C.S. (eds.). New York, Chapman and Hall: 425–466. Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin F., Hig- gins D.G. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25: 4876–4882 Vercoe P.E., White B.A. 1997. Genetics of ruminal anaerobic bacteria. In: Gastrointestinal microbiology. Volume II. Mackie R.I., White B.A., Isaacson R.E. (eds.). New York, Chapman & Hall microbiology series: 321–372 Acta argiculturae Slovenica, 94/2, 153–158, Ljubljana 2009 COBISS: 1.01 Agris category code: L01, L50 STRAIN AND PLACEMENT DENSITY EFFECTS ON WELFARE, HAEMATOLOGICAL AND SERUM BIOCHEMICAL INDICES OF BROILERS IN NORTH CENTRAL NIGERIA Abdulmojeed YAKUBU 1 2, Jafaru Ari GWASKA 1, Adebowale Emmanuel SALAKO 3 Delo je prispelo 16. septembra 2008, sprejeto 15. septembra 2009. Received September 16, 2008; accepted September 15, 2009. 1 Nasarawa State University, Faculty of Agriculture, Department of Animal Science, Keffi, Shabu-Lafia Campus, P.M.B. 135, Lafia, Nigeria 2 Corresponding author’s e-mail: abdul_mojeedy@yahoo.com 3 University of Ibadan, Department of Animal Science, Animal Breeding and Genetics Laboratory, Ibadan, Nigeria Strain and placement density effects on welfare, haematologi- cal and serum biochemical indices of broilers in north central Nigeria This study aimed at evaluating the influence of strain and stocking density on welfare, haematological and serum biochemical indices of broilers in a 28-day trial. Two hundred and seven 4-week old birds each of Anak Titan and Arbor Acre genetic provenience were randomly allocated to three housing densities of 8.3, 11.1 and 14.3 birds/m2.These corresponded to 17, 22 and 30 birds per pen ( 2.01 × 1.00 m ) in a 2x3 factorial experiment. Each treatment group was replicated three times. The welfare parameters estimated were gait score, feather score, foot and hock burns, pecking, pushes, chases, fights and mor- tality. Blood samples were tested for packed cell volume (PCV), red blood cells (RBC), white blood cells (WBC), haemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC). Similarly, sera were utilized for the determination of total protein, albumin, globulin, glucose, cho- lesterol, and creatinine contents. There were no genotype-asso- ciated differences (P > 0.05) in the welfare indicators examined. However, placement density significantly (P < 0.05) influenced incidence of pushes, chases and fights, with higher values in most cases recorded for birds housed at the highest density. The strains and population densities were similar (P > 0.05) in hae- matological profile. Strain and stocking density also exerted no influence (P > 0.05) on serum biochemical components. Strain × stocking density interaction effects were not observed in all the parameters. Consequently, the two strains could be reared at a density of 14.3 birds/m2 since density did not lead to a great degree of stress. Key words: poultry / broilers / genotype / stocking den- sity / blood parameters / animal welfare Vpliv genotipa in gostote naselitve na počutje, serumske in bio- kemijske parametre pri brojlerjih v severni in osrednji Nigeriji V raziskavi smo skušali oceniti vpliv genotipa in gosto- te naselitve na počutje živali, hematološke in biokemijske pa- rametre seruma brojlerjev v 28 dnevnem poskusu. Dvesto sedem štiri tedne starih brojlerjev provinienc Anak Titan in Arbor Acre smo naključno porazdelili v tri oddelke z gostoto naselitve 8,3, 11,1 in 14,3 ptic/m2, kar je ustrezalo 17, 22 in 30 pticam na oddelek (2,01 × 1,00 m) v faktorskem poskusu 2x3. Za vsako obravnavo smo imeli po tri ponovitve. Počutje živali smo ocenjevali z ocenami za držo telesa, perje, žulje na nogah, kljuvanje, odrivanje, preganjanje, spopade in smrtnost. V vzor- cih krvi smo določili hematokrit (PCV), število rdečih krvničk (RBC), belih krvničk (WBC), hemoglobin (Hb), srednji volu- men eritrocitov (MCV), srednjo maso hemoglobina na eritrocit (MCH) in srednjo koncentracijo hemoglobina v hematokritu (MCHC). Krvni serum smo uporabili za določitev vsebnosti skupnih beljakovin, albumina, globulina, glukoze, holesterola in kreatinina. V poskusu nismo opazili z genotipom povzroče- nih razlik v indikatorjih počutja (P > 0,05). Gostota naselitve je značilno (P < 0,05) vplivala na pogostnost odrivanja, lovljenja in bojev, ki smo jih najpogosteje opazovali pri najgosteje nase- ljenih živalih. Med različnimi genotipi in naselitvenimi različi- cami nismo opazili razlik v hematološkem profilu (P > 0,05). Genotip in gostota naselitve ravno tako nista vplivala na bioke- mijske parametre v krvnem serumu (P > 0,05). Pri nobenem od proučevanih parametrov nismo opazili interakcij med genoti- pom in gostoto naselitve. Zaključujemo, da tudi najvišja gostota naselitve,14,3 živali /m2 ni povzročala značilnega stresa. Ključne besede: perutnina / pitovni piščanci / genotip / gostota naselitve / krvni parametri / dobro počutje živali Acta agriculturae Slovenica, 94/2 – 2009154 A. YAKUBU et al. 1 INTRODUCTION Animal welfare has generated concerns from the do- mestic and global market sectors. According to English Federation for Humane Treatment of Animals (Jensens and Toates, 1997), animal welfare is accomplished if there are lack of hunger and thirst; lack of discomfort; lack of pain, injury or sickness; freedom for normal be- havior; and lack of fear, anxiety and depression. Welfare of birds is to a large extent regulated by various intrin- sic and extrinsic factors, among which stocking density plays a pivotal role. In the broiler industry, the major wel- fare concern is the effect of high stocking densities on the welfare of birds, especially during the final weeks of the growing period when body weight per unit area is high (Ravindran et al.,2006). There are conflicting reports on the effects of high placement density on the welfare, performance and im- mune status of birds. Dozier et al. (2005) reported that body weight gain and feed consumption were adversely affected by increasing the housing density from 30 to 45kg of BW/m2 of floor space. High rearing densities in broilers are associated with an increased incidence of leg problems (Sorensen et al., 2000). However, Ravindran et al. (2006) reported that weight gain, feed intake, livability and carcass characteristics of broilers grown at densities of 16, 20 and 24 birds/m2 were similar over the whole 35-day trial. Thaxton et al. (2006) reported that stock- ing density did not cause adaptive changes indicative of stress in birds. The European Union is currently adopting stand- ards for broilers aimed at a chief welfare concern, namely overcrowding by limiting maximum stocking density (Dawkins et al., 2004). Focusing research on adequate space requirements may lead to management changes that could help diminish stress and subsequently lead to improved growth and survivability. In North Central Nigeria, there appeared to be virtually no documented evidence on the appropriate placement density for broil- ers. The current practice involves the indiscriminate al- location of birds to floor space based on the imagination (subjective evaluation) of the farmers. This tends to un- dermine animal welfare and hence, profitability of the enterprise. Therefore, the present investigation set out to de- termine the effects of genotype and stocking density on welfare indicators, haematological and serum biochemi- cal parameters of broilers. The result so obtained could contribute to the knowledge on optimal floor space of broilers in the semi-humid tropics characterized by high environmental temperature and relative humidity. 2 MATERIALS AND METHODS 2.1 STUDY LOCATION The research was conducted in the Poultry Unit of the Teaching and Research Farm, Faculty of Agriculture, Nasarawa State University, Keffi, Shabu-Lafia Campus, located in the guinea savanna agro-ecological zone of northern Nigeria.The mean monthly environmental temperature during the study which lasted four weeks was 32.750C, while the monthly relative humidity, rain- fall and evaporation were 79.00%, 207.45mm and 2.5ml respectively. 2.2 EXPERIMENTAL DESIGN Four hundred and fourteen broiler chickens con- sisting of equal number of Anak Titan and Abor Acre strains were utilized for the investigation. Birds were raised on conventional starter ration (22.00% crude pro- tein and 2800kcal/kgME) from day old to 4-week of age. Birds were randomly allocated to three stocking density treatments vis: 8.3, 11.1 and 14.3 birds /m2. These corre- sponded to 17, 22 and 30 birds per pen in a 2 × 3 factorial arrangement. Each treatment group was replicated three times. The dimension of each pen made of wooden plank and wire netting was 2.01m2 (2.01m × 1.00m), and was constructed in such a way as to permit straight-through ventilation. From week five to week eight, the birds were fed commercial broiler finisher ration (2900kcal/kg ME and 20.00% crude protein). Feed and fresh clean water were supplied ad libitum. The feeders and waterers were allotted proportionately depending on the number of birds in each pen. Vaccination schedule and other man- agement practices were strictly adhered to. 2.3 DATA COLLECTION Birds were assessed on a weekly basis for gait score, feather score and foot and hock burn as described by Ravindran et al. (2006). Number of pecking, pushes, fights and chases were recorded per pen per replicate during feeding and at 3-day interval, following the pro- cedure adopted by Olukosi et al. (2001). Gait score was assessed for six randomly selected birds per pen. Birds were watched by two observers walking in the run within the poultry house, and their walking ability was scored on a three-point scale (0, normal gait, bird walks freely and has regular and even strides and is well balanced; 1, bird walks with irregular and uneven strides and appears unbalanced; 2, bird is reluctant to move and is unable to Acta agriculturae Slovenica, 94/2 – 2009 155 STRAIN AND PLACEMENT DENSITY EFFECTS ON WELFARE … INDICES OF BROILERS IN NORTH CENTRAL NIGERIA walk many strides before sitting down). A score was as- cribed only when there was consensus between the two observers. Feather score or the degree of feather coverage over the breast was recorded for six birds per pen. Each bird was stroked over the keel with the palm of the hand in an anterior or posterior direction, and the amount of flesh showing was scored on a three -point scale (1, no visible skin, complete feather cover; 2, relatively small amount of skin showing; 3, relatively large amount of skin show- ing). Foot and hock burn was recorded for all the birds in a pen using a three -point scale (1, no burns; 2, mild burns; 3, severe burns). 2.4 HAEMATOLOGICAL AND BIOCHEMICAL ANALYSES At the end of the experimental period, blood sam- ples were collected from four randomly selected birds per treatment combination. Five-ml of blood was collected through the jugular veins in immobilized animals. Half of the sample was expelled gradually into vacutainer glass tubes containing ethylene diamine tetra acetic acid (EDTA) for the determination of haematological compo- nents following standard procedures described by Davice and Lewis (1991). The rest of the sample was collected in a second set of vacutainer glass tubes without EDTA for serum biochemical parameters. The haematological indices investigated were packed cell volume (PCV), red blood cells (RBC), white blood cells (WBC), haemo- globin (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscu- lar haemoglobin concentration (MCHC).Similarly, sera were used for the determination of total protein, albu- min, globulin, glucose, cholesterol and creatinine con- tents. 2.5 STATISTICAL ANALYSIS Data collected were subjected to multivariate analy- sis of variance using SPSS (2001) statistical package. The separation of means was effected using least significant difference (LSD) method and tested at probability level of 5%. 3 RESULTS The influence of genotype and stocking density on the welfare parameters of broilers is shown in Table 1. Genotype did not significantly (P > 0.05) affect the wel- fare parameters investigated. Stocking density had no effect (P  >  0.05) on gait score, foot and hock burns, feather score and pecking. However, housing density significantly (P < 0.05) influ- enced number of pushes, chases and fights, with higher mean values recorded for birds stocked at 14.3 birds/m2. Effect of genotype and population density on the haematological parameters of broilers is shown in Table 2. Strain and density effects on PCV, RBC, WBC, Hb, MCV, MCH and MCHC were not observed (P > 0.05). Effect of strain and placement density on serum bio- chemical parameters of broilers is presented in Table 3. Genotype exerted no significant influence (P > 0.05) on the parameters estimated. There was also no placement density influence (P  >  0.05) on total protein, albumin, globulin, glucose, cholesterol and creatinine contents. Strain × stocking density interaction effects were not ob- served in all the parameters investigated. Parameters Genotype Stocking density (bird/m2) Anak Titan Abor Acre Prob. S.E.M 8.3 11.1 14.3 Prob. S.E.M Gait score 1.19 1.17 0.85 0.14 1.21 1.04 1.29 0.36 0.13 Food and hock burns 1.47 1.39 0.59 0.15 1.33 1.33 1.63 0.28 0.13 Feather score 1.86 1.84 0.90 0.13 1.88 1.71 1.97 0.13 0.11 Pecking 1.77 1.52 0.32 0.24 1.72 1.53 1.71 0.56 0.30 Pushes 2.36 2.34 0.88 0.15 1.99 2.31 2.25 0.01 0.13 Chases 1.78 1.83 0.72 0.14 1.48 1.83 2.11 0.01 0.12 Fights 1.94 1.83 0.37 0.12 1.53 1.79 2.34 0.05 0.10 Mortality 0.01 0.01 0.85 0.006 0.01 0.01 0.008 0.65 0.007 Table 1: Effect of genotype and stocking density on welfare indices of broilers Preglednica 1: Vpliv genotipa in gostote naselitve na počutje brojlerjev S.E.M: Standard error of means Acta agriculturae Slovenica, 94/2 – 2009156 A. YAKUBU et al. 4 DISCUSSION Genotype-associated significant differences did not manifest in the welfare parameters investigated. This is an indication of similarity in the ranking of the two strains under consideration, although better numerical mean values were recorded for Abor Acres. The present result is in consonance with that of Albentosa et al. (2003), where strain of birds did not influence fearful- ness and exploratory behaviour. Similarly, Anderson et al. (2004) reported that appetitive behaviours and feather pecking were not affected by strain, and that the patterns and number of aggressive acts did not increase to com- promise the welfare status of the birds. In contrast to the present findings, Kjaer and Sorensen (2002) and Aerni et al. (2005) reported effect of genotype on feather pecking, mortality and cannibalism respectively. Gait score repre- sents subjective method of evaluation of the walking abil- ity of birds. Genetic differentials in gait score, foot and hock burns, feather pecking and cannibalism in Ross 208 and Labresse cross had been documented (Nielsen et al., 2003). The performance of the birds in gait score, feather score, foot and hock burns and pecking was independent of placement density. The present findings are consistent with that of Ravindran et al. (2006) where placement den- sity did not exert any influence on leg and feather scores. In contrast to the current observations, Sorensen et al. (2000) reported poorer walking ability in birds reared at higher densities and attributed this to constrained mo- bility and reduced opportunity for activity, especially as birds approach the end of the grow out phase. Dozier et al. (2005) reported that foot pad lesson score increased progressively as placement density in- creased from 30 to 45kg of BW/m2. The submission of Muniz et al. (2008) confirms this, as percentage foot-pad dermatitis in broilers increased linearly with increasing stocking density. The negative effect of high housing den- sity on hock burns had also been reported (Thomas et al., 2004), reflecting poorer litter quality and the increased time that the birds spend sitting, in contact with the litter. The differential effects of stocking rate on gait score and foot and hock burn of the present investigation and others might be partly attributed to the length of rearing, environment and management practices. Under the con- Parameters Genotype Stocking density (bird/m2) Anak Titan Abor Acre Prob. S.E.M 8.3 11.1 14.3 Prob. S.E.M PCV (%) 31.25 31.17 0.92 0.85 31.50 31.13 31.00 0.64 1.03 RBC (x 106/μL) 2.46 2.47 0.84 0.05 2.48 2.47 2.45 0.76 0.07 WCB (x 103/μL) 24.61 24.48 0.64 2.76 24.64 24.61 24.37 0.45 3.38 Hb (g/dl) 9.42 9.89 0.23 0.38 9.73 9.64 9.59 0.85 0.47 MCV (fl) 128.87 130.06 0.45 1.52 129.95 130.50 127.93 0.18 1.86 MCH (pg) 38.86 39.58 0.25 0.62 39.38 39.40 38.87 0.50 0.76 MCHC (g/dl) 30.30 30.60 0.36 0.33 30.63 30.13 30.61 0.23 0.41 Table 2: Effect of genotype and stocking density on haematological indices of broilers Preglednica 2: Vpliv genotipa in gostote naselitve na hematološke parametre pri brojlerjih S.E.M: Standard error of means Parameters Genotype Stocking density (bird/m2) Anak Titan Abor Acre Prob. S.E.M 8.3 11.1 14.3 Prob. S.E.M Total protein (g/dl) 5.76 5.91 0.54 0.26 5.98 5.89 5.63 0.29 0.32 Albumin (g/dl) 2.48 2.77 0.18 0.33 2.54 2.98 2.35 0.13 0.40 Globulin (gdl) 3.28 3.14 0.67 0.30 3.44 2.92 3.28 0.18 0.37 Glucose (mmol/L) 12.72 13.43 0.32 0.70 13.86 12.45 12.91 0.12 0.87 Cholesterol (mmol/L) 4.91 5.60 0 19 0.51 5.45 5.08 5.24 0.55 0.63 Creatinine (μmol/L) 71.83 71.33 0.90 4.02 70.25 75.00 69.50 0.28 4.92 Table 3: Effect of genotype and stocking density on serum biochemical indices of broilers Preglednica 3: Vpliv genotipa in gostote naselitve na biokemijske parametre pri brojlerjih S.E.M: Standard error of means Acta agriculturae Slovenica, 94/2 – 2009 157 STRAIN AND PLACEMENT DENSITY EFFECTS ON WELFARE … INDICES OF BROILERS IN NORTH CENTRAL NIGERIA ditions of the current study, more of mild feather pecking and less of aggressive pecking which was mainly directed at the head was observed. The other agonistic behaviours such as pushes, chases and fights were not similar in the three stocking density treatments, as they were more as- sociated with the highest housing density. The competi- tion for feed could partly be responsible for the observed responses. Similar findings have been reported in broil- ers where increasing the feeder space reduced agonistic acts during the feeding period from 7.8 (at 2.4cm feeder space to 4.5 (at 3.6cm/bird) (Olukosi et al. 2001). Accord- ing to Spinu et al. (2003), stereotyped pecking increased with an increase in density. Mortality is one of the most obvious measures of bird welfare. However, population density was not a significant explanatory factor in it in the current investigation. This concurs with the report of Thomas et al. (2004). In contrast, Imaeda (2000) found that mortality was markedly increased at higher animal densities. Measurement of haematological indices provides valuable information on the immune status of animals. The literature provides varying evidence concerning the effect of stocking density on birds’ physiological re- sponse and stress. The present findings are comparable to the report of Talebi et al. (2005) which revealed that the haematological values of four main broiler strains (Ross, Cobb, Abor Acres and Arian) showed slight but non-significant differences, indicating that the broiler strains are nearly similar to each other in haematologi- cal indices. Conversely, Manzoor et al., (2003) reported genotype-associated differences in the haematological parameters of broiler lines. Placement density did not ex- ert any influence on the haematological characteristics, as very similar values were recorded for the three stock- ing rates investigated. The two strains under consideration appeared to be similar in their serum biochemical values. However, Abor Acres seemed to have better numerical mean values for total protein, albumin, glucose and creatinine. Serum biochemical parameters were also not density depend- ent. This is an indication that the body homeostasis, and hence health of the birds were not adversely disturbed as a result of housing the birds up to 14.3 birds/m2. Using linear trend analysis, Thaxton et al. (2006) reported that stocking density did not cause physiological adaptive changes indicative of stress. Skomorucha and Muchacka (2007) submitted that the level of biochemical indicators was affected by animal density, although this manifested greatly in birds placed under housing density of 17 birds/ m2, which is quite higher than the 14.3 birds/m2 reported in the present study. 5 CONCLUSIONS The study has shown that genotype had no signifi- cant effect on gait score, foot and hock burns feather score, pecking, pushes, chases, fights and mortality of broilers. Conversely, stocking density significantly influ- enced incidence of pushes, chases and fights with higher mean values recorded for birds reared at 14.3 birds/m2. There was no genotype and stocking density effects on the haemotological parameters. Serum biochemical in- dices were also not significantly affected by placement density. Genotype × stocking interaction effects were not observed in all the parameters investigated. It is con- cluded that the two strains could be reared at a stocking density of 14.3 birds/m2, since density did not negatively affect the physiological adaptive responses of birds. This will eventually guarantee high yield per unit area, which could assist livestock farmers and the entire populace in poverty alleviation under the climatic and production conditions of this study. 6 REFERENCES Aerni V., Brinkhof M.W.G., Wechsler B., Oester H., Frohlich E. 2005. Productivity and mortality of laying hens in aviaries: a systematic approach.World’s Poult. Sci. J., 61: 130–142 Albentosa M.J., Kjaer J.B., Nicol C.J. 2003. Strain and age differ- ences in behaviour, fear response and pecking tendency in laying hens. Br. Poult. Sci., 44: 333–344 Anderson K.E., Davis G.S., Jenkins P.K., Carrol A.S. 2004. Ef- fects of bird age, density and molt on behavioural profiles of two commercial layer strains in cages. Poult. Sci., 83: 15–23 Davice J.U., Lewis S.M. 1991. Practical haematology. 8th Edi- tion. London, Longman Ltd.: 22–68 Dawkins C., Donnelly A., Jones T.A. 2004. Chicken welfare is influenced more by housing conditions than by stocking density. Nature, 27: 342–344 Dozier III W.A., Thaxton J.P., Branton S.L., Morgan G.W., Miles D.M., Roush W.B., Lott B.D., Vizzier-Thaxton Y. 2005. Stocking density effects on growth performance and pro- cessing yields of heavy broilers. Poult. Sci., 84: 1332–1338 Imaeda N. 2000. Influence of the stocking density and rearing season on incidence of sudden death syndrome in broiler chickens. Poult. Sci., 79: 201–204 Jensens P., Toates F.M. 1997. Stress as a state of motivational system. Appl. Anim. Behav. Sci., 53: 145–146 Kjaer J.B., Sorensen P. 2002. Feather pecking and cannibalism in free-range laying hens as affected by genotype, dietary level of methionine + cystine, light intensity during rearing and age at first access to the free range. Appl. Anim. Behav. Sci., 76: 21–39 Manzoor A., Cheema M.A., Qureshi A., Havenstein G.B. 2003. A comparison of the immune profile of commercial broiler strains when raised on marginal and high protein diets. Int. J. Poult. Sci., 2: 300–312 Acta agriculturae Slovenica, 94/2 – 2009158 A. YAKUBU et al. Nielsen B.L., Thomsen M.G., Sorensen P., Young J.F. 2003. Feed and strain effects on the use of outdoor areas by broilers. Br. Poult. Sci., 44: 161–169 NIMET. 2008. Nigerian Meteorological Agency, Lafia, Nasar- awa State Olukosi O.A., Daniyan O.C., Matanmi O. 2001. Effects of feeder space allowance on agonistic behaviour and growth perfor- mance of broilers. Livestock Research for Rural Develop- ment, 13. http://www.cipav.org.co/lrrd/lrrd13/1/oluk131.htm Ravindran V., Thomas D.V., Thomas D.G., Morel P.C.H. 2006. Performance and welfare of broilers as affected by stocking density and zinc bacitracin supplementation. Anim. Sci. J., 77: 110–116 Skomorucha I., Muchacka R. 2007. Effect of stocking density and management on the physiological response of broiler chickens. Annals Anim. Sci., 7: 321–328 Sorensen P., Su G., Kestin S.C. 2000. Effects of age and stocking density on leg weakness in broiler chickens. Poult. Sci., 79: 864–870 Spinu M., Benveneste S., Degen A.A. 2003. Effect of density and season on stress and behaviour in broiler breeder hens. Br. Poult. Sci., 44: 170–174 SPSS. 2001. Statistical Package for the Social Sciences. New York, SPSS Inc. Talebi A., Asri-Rezaei S., Rozeh-Chai, Sahraei R. 2005. Com- paratative studies on haematological values of broiler strains (Ross, Cobb, Arbor-acres and Arian). Int. J. Poult. Sci., 4: 573–579 Thaxton J.P., Dozier III W.A ., Branton S.L., Morgan G.W., Miles D.M., Roush W.B., Lott B.D., Vizzier-Thaxton Y. 2006, Stocking density and physiological adaptive responses of broilers. Poult. Sci., 85: 819–824 Thomas D.G., Ravindran V., Thomas D.V., Camden B.J., Cot- tam Y.H., Morel P.C.H., Cook C.J. 2004. Influence of stock- ing density on the performance, carcass characteristics and selected welfare indicators of broiler chickens. N. Z. Vet. J., 52: 76–81 Acta argiculturae Slovenica, 94/2, 159–166, Ljubljana 2009 COBISS: 1.01 Agris category code: L02, P30 MINERALS MANAGEMENT IN SILVOPASTORAL SYSTEM OF KARST PASTURE Matej VIDRIH 1, Anton VIDRIH 2, Milan POGAČNIK 3, Drago KOMPAN 4 Delo je prispelo 16. septembra 2008, sprejeto 15. septembra 2009. Received September 16, 2008; accepted September 15, 2009. 1 Univ. of Ljubljana, Biotechnical Fac., Agronomy Dept., Jamnikarjeva 101, SI-1111 Ljubljana, Slovenia, Prof., Ph.D. 2 Same address as 1, Ass., Ph.D. 3 Univ. of Ljubljana, Veterinary Fac., Gerbičeva 60, SI-1000 Ljubljana, Slovenia, Prof., Ph.D. 4 Univ. of Ljubljana, Biotechnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia, Ass.Prof., Ph.D. Minerals management in silvopastoral system of karst pasture A survey of mountain pasture topsoil was undertaken first to set up field experiment in karst region on effects of applied P on minerals concentration in herbage. Content of SOM, C, N, CEC of soil and its base saturation are presented in the article. Great variability in depth, pH value and K level was found in soil. Low base saturation and high deficit of P was more com- mon for all soil samples. Six different plant species presenting a great portion of herbage available for grazing and browsing at different occasions during grazing season were sampled and analysed for macro- and some microminerals. Concentration of P was very low in perennial grasses (1.1 g P kg−1 of DM). In leaves of common hazel (Corylus avellana L.) and common beech (Fagus silvatica L.) the concentration of P was identical as in white clover (Trifolium repens L.) and the level was high enough to cover animal needs when intake of herbage was suf- ficient. Leaves of woody plants were high in Mn concentration, but still below the levels that reduced growth rate in lambs. Ap- plication of P fertilizer had only small effect on increase of P in herbage, but large one on decrease on concentration of Ca in herbage. There was not clear effect of added P on concentration of Zn, Mn, Fe and Cu. Higher yield of DM induced with added fertilizer had not have any dilution effect on concentration of those minor elements in herbage. Key words: animal husbandry / animal nutrition / pastur- ing / karst pastures / soil / herbage / minerals / superphosphates Drevesno pašna raba in rudnine na kraškem pašniku Na območju planinskega pašnika je bil napravljen pregled rodovitnosti zemlje in nato izveden poljski poskus o vplivu gnojenja s P na vsebnost rudnin v zelinju kraške vegetacije. V prispevku so predstavljeni podatki o vsebnosti organske sno- vi v tleh, C in N, kapacitete sorpcije ter zasičenost z bazami sorptivnega dela tal. Ugotovljena je bila velika variabilnost v debelini vrhnje plasti zemlje, pH vrednosti in oskrbljenosti tal s K. Značilna je tudi nizka zasičenost z bazami sorptivnega dela tal in veliko pomanjkanje rastlinam dostopnega P v zemlji. Za določanje vsebnosti rudnin v zelinju razpoložljivem za pašo in smukanje, je bilo vzorčeno šest različnih vrst rastlin, ki pred- stavljajo znaten delež krme v različnih delih pašne sezone. V travah, ki predstavljajo ob koncu pomladi znaten delež raz- položljive krme je bila ugotovljena zelo nizka vsebnost P (1,1 g kg−1 SS). V listju navadne leske (Corylus avellana L.) in na- vadne bukve (Fagus sylvatica L.) je bila vsebnost P enaka kot v zelinju plazeče detelje (Trifolium repens L.) in je bila dovolj visoka za pokritje potreb pašnih živali po P, če je v obroku do- volj zaužitega zelinja. V listih lesnatih rastlin je bila ugotovljena visoka vsebnost Mn, toda še vedno pod vrednostjo, ki vpliva na zmanjšanje dnevnih prirastov pri jagnjetih. Gnojenje s fosfati ni imelo značilnega vpliva na povečanje vsebnosti P v zelinju, toda močno je vplivalo na zmanjšanje vsebnosti Ca v zelinju rastlin ruše. Uporabljena fosfatna gnojila niso značilno vplivala na spremembo vsebnosti Zn, Mn, Fe in Cu v zelinju razpoložlji- vem za pašo. Višji pridelek zelinja dosežen z uporabo fosfatnih gnojil ni učinkoval razredčitveno na vsebnost mikroelementov v zelinju kraškega pašnika. Ključne besede: živinoreja / prehrana živali / paša / kraški pašniki / tla / zelinje / minerali / superfosfati Acta agriculturae Slovenica, 94/2 – 2009160 M. VIDRIH et al. 1 INTRODUCTION The grazing animals will play very important role in the process of farming restoration on hill and karst grass- land of Slovenia. The land under consideration was main- ly used as cut meadows (hand cut) in the past. The plant minerals such as phosphorus (P), calcium (Ca), magne- sium (Mg), sodium (Na) and sulphur (S) were drained out from the grassland soils because the hay produced there was fed to animals in confinement to get large vol- ume of yard manure, which was used then on arable land to keep their fertility at the level high enough for good crop production (Gruden, 1910). Seminatural grasslands have longer history than is often realized (Walter, 1973), and karst grassland was a subject to depletion of plant available minerals most of the time (Vidrih A, 2005). An additional reason for depletion of plant minerals from karst hay grassland is strong wind bora (Mihevc, 1997). Most of nutrients in plants are organic and recycled to grassland soils through the decomposition of herbage. That is why this material must come in good contact with the soil, and mineralized by soil microorganisms. This way dead leaves and tillers can not be blown away by wind. Trampling of grazing animals is most efficient method for to incorporate organic matter into top layer of soil. But this trampling was absent from hay grassland, and wind erosion took its share of minerals from karst grassland. Occasional burning (accidentally or purpose- ly) of vegetation over abandoned karst grassland was an additional drawn out of minerals from karst soil. As a consequence of all this a large area of karst grassland has been abandoned and converted to woody plant types of ecosystem. It has become increasingly evident that re- gion is loosing its typical landscape, and that important habitat of very large proportion of wild fauna and flora is decreasing in size very much (Grove and Rackham, 2001; Kaligarič et al., 2006). Very variable soil profile, stoniness as well as topog- raphy are serious limitations for levels of production and potential for improvement of karst pasture vegetation. Whilst it is recognised that these limitations exist, it is also clear that they are of a kind which cannot readily be changed by improvement measures used under more favourable conditions for farming. Thus greater attention must be given to the nature of chemical limitation of soil fertility and the methods for their correction (Vidrih M et al., 2007). Sima et al. (2004) is suggesting that this soil factors are probably more important in limiting produc- tion of grazing animals on potentially improvable soils than are climatic factors. Concentration of minerals in both soil and plant, influences the mineral status of graz- ing animals, and considering all these facts, the soil and herbage available for grazing on karst pasture were ana- lysed for concentration of minerals, and the effect of P fertilization on mineral status of herbage is reported. 2 MATERIAL AND METHODS A study was conducted on a herbaceous community of the Festuco-Brometea Br.-Bl. (Kaligarič, 1997) in the Dinaric karst site of Slovenia (lat. 45.41 °N, long. 14.12 °E, alt. 820 m) with 1500 mm rainfall. The experimental site was part of the mountain pasture Vremščica for milking sheep. Field experiment was set in the farmlet that has North exposition, with moderate slope and was subdi- vided into 6 paddocks and lasted from 2002 to 2005. The herbage was utilized by grazing only in a 6 week rotation during 100 to 120 day-long grazing season. Soil samples were collected in spring before onset of the grazing sea- son from sites appropriate to locate the planned field ex- periment. To determine the concentration of minerals in herbage available for grazing and browsing, samples were collected from formerly not fertilized area. The grass spe- cies: sheep’s fescue (Festuca ovina L.), chalk false brome (Brachypodium pinnatum L.) and erect brome (Bromus erectus L.) were collected in spring, because of their high abundance for grazing during that period. Young branch- es and leaves of common hazel (Corylus avellana L.) and common beech (Fagus sylvatica L.) were sampled during summer grass dormant period, as leaves of shrubs repre- sented supplemental feed at that time. Legume white clo- ver (Trifolium repens L.) was sampled at beginning of sec- ond rotation from grazed but not fertilized area. As very valuable feed white clover is grazed soon after the sheep enter the paddock. All together six different plant species were collected separately (three grass species, two shrub species and a legume species). Within a paddock where lambs (sheep replacement) were grazed under regular ro- tation, an experiment of latin square design consisting of four treatments with four replicates was set. Four levels of P (0, 30, 90, 270 kg P2O5 ha− 1) were under investigation in the first year. Next year superphosphate was applied again only at treatment 30 and 90 kg P2O5 ha− 1. In spring of the third year the superphosphate was applied equally as in a first year; all three fertilized treatments received superphosphate at the same rate as the first year. Total amount of applied P during four years of investigation was 0, 90, 270 and 540 kg P2O5 ha− 1 respectively for treat- ment 1, 2, 3 and 4. Major soil physical and chemical properties were analysed in soil samples (Egner et al., 1960; Janitzky, 1986; SIST ISO 13878, 1998; Soil Survey Staff, 2009). The concentration of minerals in herbage samples was determined by standard procedures for plant tissue (Ca, Mg, Na, Mn, Zn, Fe and Cu were determined by atomic Acta agriculturae Slovenica, 94/2 – 2009 161 MINERALS MANAGEMENT IN SILVOPASTORAL SYSTEM OF KARST PASTURE absorption spectrometry [Varian Model AA240, Palo Alto, CA], P by spectrophotometric determination [Fias, Perkin Elmer, USA], K by flame photometry [FP6410, Unicom Optics, PRC]). To determine concentration of minerals in herbage from fertilized experiment, samples were obtained immediately before grazing (pre-grazing). To represent the herbage grazed by the animals, samples were obtained by hand plucking at the end of the spring. Sampling units (n = 25) were randomly selected out of the population from each plot, and combined in one sample for each treatment. Herbage samples were oven dried for approximately 12h at 100 °C and ground in a hammer mill to pass a 1-mm sieve. These samples were chemically analysed and the results are assumed to relate closely to the composition of the herbage eaten. The data obtained from our study were subjected to analysis of variance for random block and latin square design with GenStat Release v7.1 (Genstat 7 Committee, 2003) and Duncan’s post hoc test at P < 0.05 probability level to de- termine significant differences between the treatments. 3 RESULTS AND DISCUSSION 3.1 MINERALS IN SOIL Most frequent type of soil found over pasture Vremščica is typically brown rendzina. This is less pro- ductive soil, where nutrient concentration and water holding capacity are low. Reasons for this are slow proc- ess of soil formation and very long history of utilisation of vegetation that existed there in the past (Lovrenčak, 1993). The top layer of soil – A1 horizon, where most roots of herbal vegetation can be found is in average 12.6 cm tick, has low pH, and is deficient in P available to plants (2.7 mg P2O5 100 g− 1 of soil). Level of K in soil is low to moderate (Table 1). Content of soil organic matter (SOM) in top layer is high (10%). This and substantial content of clay in soil (> 20%), is the reason for the high cation exchange capacity (CEC) of the soils of karst pas- ture. Clay in rendzina is not only a source of nutrients, its also determine the degree to which organically bound P can accumulate in the soil (Table 2). Because of very high CEC the base saturation of this soil is low (32.3%). Ca and Mg together present most of this base saturation, which should be round 80% to assure balanced soil for good growth of white clover and efficient fixation on N by symbiotic bacteria. C to N ratio is too high (13.2 :1) for to accomplish efficient transformation of dead organic matter into humus. The very high saturation of soil with hydrogen ion (67.8% H+ ) is in accordance with soil acidity (4.5 pH). The rank of saturation of exchange capacity with hydrogen ion should be 10 to 15% H+ as stated by Kinsey and Walters (2006). 3.2 MINERALS IN HERBAGE From results obtained in current research it is evi- dent that great differences exist in concentration of min- erals between three kinds of available herbage for animals grazing or browsing within vegetation of karst pasture. Native perennial grasses which are adapted to grow on P deficient soil with a pronounced dry season spell have very low concentration of minerals in total (49.4 g ash kg−1 of DM on average). In leaves of shrubs the concen- Table 1: Chemical properties of soil (n=30) on mountain pasture Vremščica (SD – standard deviation, SE – standard error of average) Preglednica 1: Fizikalne in kemijske lastnosti tal (n = 30) planinskega pašnika Vremščica (SD – standardna deviacija, SE – stan- dardna napaka povprečja) Soil depth cm pH in KCl Clay % P2O5 K2O SOM % C % C:N ratiomg 100 g-1 of soil Average 12.6 4.5 21.1 2.7 14.4 9.9 5.7 13.2 SE 0.8 0.1 1.3 0.4 1.6 0.7 0.4 0.7 Table 2: Cation exchange capacity (CEC), base saturation and mineral composition in the soil (n = 30) on mountain pasture Vremščica (SD – standard deviation, SE – standard error of average) Preglednica 2: Kapaciteta sorpcije, zasičenost z bazami in koncentracija mineralov v tleh (n = 30) planinskega pašnika Vremščica (SD – standardna deviacija, SE – standardna napaka povprečja) Base CEC Base saturation Ca Mg K Na H eq mmol H 100 g-1 soil % Average 9.7 30.0 32.2 24.6 6.3 1.2 0.2 67.8 SE 1.2 1.3 3.4 2.9 0.8 0.1 0.07 3.4 Acta agriculturae Slovenica, 94/2 – 2009162 M. VIDRIH et al. tration of minerals is similar (53.2 g ash kg−1 of DM) as in grasses. In the herbage of white clover was found 104.8 g ash kg−1 of DM, which is twice as much ash as have the formers (Table 3). The average concentration of P in grasses under in- vestigation was 1.1 g P kg−1 of DM. This is half of what is requirement for sheep and cattle in their diet. Only herbage containing at least 2.5 g P kg−1 of DM will en- sure that the P requirement for all classes of sheep will be met if their DM intakes are adequate (Whitehead, 2000). For rapidly growing animals the herbage on offer should contain at least 3.0 g P kg−1 of DM (Ozanne and Howes, 1971). Leaves of common hazel and common beech were much better source of P for animals grazing karst pasture vegetation than perennial grasses. On average the con- centration of P in leaves of shrubs was 2.6 g P kg−1 of DM. This is very important to know, because during dry sum- mer period when grasses are seed set and such a herb- age has very low concentration of P, animals must have possibility to browse leaves of woody plants. Herbage of white clover had enough P (2.6 g kg−1 of DM) for the need of grazing animals. The problem is that white clover is very sparsely found and less abundant over karst pasture. During the period of improvement of karst pasture (con- trol grazing, fertilizer or lime application) legumes are distributed as patches over area and their proportion in total in the sward is still very low. Only for a short period of time after entering the new paddock, the intake of leg- umes may be sufficient high by grazing animals. Concentration of Ca was lowest in herbage of sheep’s fescue (2.7 g Ca kg−1 of DM) and highest in white clo- ver (15.9 g Ca kg−1 of DM). Leaves of common hazel had more Ca than leaves of common beech. Values for Ca in herbage of white clover were adequate for maintenance, growth and pregnancy of grazing animals. But when diet as a whole is deficient in P, as this is the case when peren- nial grasses present bulk of diet, than the concentration of Ca must be higher, because its absorption in animals is decreased due to P deficiency in herbage. A K level in herbage under investigation was high and exceeded the requirements of grazing animals (Whitehead, 2000). High concentration of K in herbage of white clover (29.6 g K kg−1 of DM was indication that samples of this herbage were collected from the patches where animal dung was left formerly. Animal diet low in Na level, but high in K level could further reduce the Na intake of the grazing animals (Aspinall et al., 2004). This was the case with grasses and leaves of shrubs in present research. Only herbage of white clover had adequate con- centration of Na (0.89 g Na kg−1 of DM) to maintain full Na status of lactating ewes with lambs (Gillespie et al., 2006). Since the micronutrients in soils are derived almost entirely from the parent material, the soils on limestone are normally low in all micronutrients except Mn. Con- centration of Zn, Mn, Fe and Cu elements in herbage were within the lower part of values reported elsewhere (Spears, 1994; Grace, 1983). There was less Zn in grasses and more in leaves of woody plants. Symptoms of Zn deficiency in sheep and cattle may occur when animals graze herbage with less than about 20 mg Zn kg−1 of DM. Table 3: Concentration of minerals (g kg−1 or mg kg−1 of DM) in six different plant species of sward available for grazing and browsing from vegetation most abundant on karst pasture. Means followed by the same letter in a row are not significantly different based on Duncan’s test (P < 0.05) Preglednica 3: Koncentracija mineralov (g kg−1 ali mg kg−1 SS) v šestih različnih vrstah rastlin ruše, ki so bile na razpolago za pašo in smukanje iz najbolj obilne vegetacije na kraškem pašniku. Povprečja z enako črko v vrstici se ne razlikujejo statistično značilno (p < 0,05) Herbage content Sheep’s fescue Chalk false brome Upright brome Common hazel Common beech White clover Ash (g kg−1) 42.8 a 55.8 c 49.7 b 60.0 c 46.4 b 104.8 d Phosphorus (g kg−1) 0.98 a 0.97 a 1.25 b 2.65 c 2.63 c 2.64 d Calcium (g kg−1) 2.74 a 5.33 b 4.11 b 13.2 d 8.1 c 15.9 d Magnesium (g kg−1) 1.30 a 1.19 a 1.48 a 3.58 c 2.82 b 3.74 c Potassium (g kg−1) 9.1 a 11.3 b 14.1 b 10.9 a 10.9 a 29.6c Sodium (g kg−1) 0.04 a 0.16 b 0.24 b 0.17 b 0.15 b 0.89 c Zink (mg kg−1) 15.8 a 22.6 b 21.2 b 32.9 c 26.7 b 27.9 b Manganese (mg kg−1) 42.2 a 49.9 a 94.1 b 310.9 d 199.6 c 107.5 c Iron (mg kg−1) 84.1 b 98.6 c 91.4 c 75.0 a 89.8 b 112.9 d Copper (mg kg−1) 3.7 a 8.0 c 7.1 b 11.9 d 14.7 d 7.4 b Acta agriculturae Slovenica, 94/2 – 2009 163 MINERALS MANAGEMENT IN SILVOPASTORAL SYSTEM OF KARST PASTURE Herbage of sheep’s fescue had less than this and the val- ues for Zn of other herbage was little above this. Very high was the concentration of Mn (311 mg Mn kg−1 of DM) in leaves of common hazel and similar high was for common beech leaves. The average value for three grasses under survey was 62.1 mg Mn kg−1 of DM, and for herbage of white clover was 107.5 mg Mn kg−1 of DM. Limestone as a parent material where rendzina soil is formed has high content of Mn. With increasing acid- ity of top soil, leaching of Mn occurs and this element accumulates on clay particles in lower soil horizons. Be- cause the roots of woody plants search for minerals deep- er in the soil, the higher absorption of Mn is achieved by the roots of woody plants. Mn content was high in white clover too, because this valuable plant can be very easy introduced on places where woody plants were growing. When shrubs are thinned and open space with enough light is formed, white clover has an opportunity for fast establishment. Decomposing shrub leaves are additional source of Mn for white clover grown on shrub cleared areas. Several studies, in which Mn cycling was re- searched, have indicated that 20 to 25 mg Mn kg−1 of DM is adequate for growth and reproduction (Grace, 1983). Concentration of Mn in leaves of shrubs exceeded 10 to 14 times the values required in animal diet for optimum skeletal development and to prevent reproductive prob- lems. Variability of Mn concentration in different grass species grown at the same location can be very high as reported by Orešnik et al. (1999). But all these concentra- tions of Mn in herbage are still well below the levels that reduced growth rate observed in lambs grazing pastures which contained 400 mg Mn kg−1 of DM (Grace, 1983). Sheep may be somewhat more susceptible than cattle to excessive Mn intake. As reported by Grace (1983) the animal growth rates were only significantly reduced where concentra- tions of Mn were above 1200 mg kg−1 of DM for the full 2 week grazing period. The soil pH strongly influences the Mn level in plants with the Mn uptake being greatest in acid soil. Liming tends to decrease herbage concentra- tion of Mn through its effect on soil pH. Change from pH 5.2 to 6.2 reduced the concentration of Mn in the herb- age of mixed sward from an average of 290 to 130 mg Mn kg−1 of DM. To lessen the amount of lime required to lower herbage Mn concentration, the improved grazing management which limits the dead material content ap- pear to be effective as was found by Smith et al. (2006). Variability in concentrations of iron (Fe) between kinds of herbage under investigation is very small. In leaves of common hazel is the lowest concentration of Fe (75.0 mg Fe kg−1 of DM) and in white clover herbage is the highest (112.9 mg Fe kg−1 of DM). These concentrations in herb- age are adequate to cover animal requirements upon Fe, and are not to high to interfere with the Cu metabolism in animals. Leaves of common beech have highest concentra- tion of Cu (14.7 mg Cu kg−1 of DM), lesser is in common hazel and much lower in white clover (7.4 mg Cu kg−1 of DM). Provided that the availability of the Cu is not greatly influenced by the presence of high S supply and the DM intakes are adequate, then herbage containing 5 to 6 mg Cu kg−1 of DM for sheep and 7 to 10 mg Cu kg−1 of DM for cattle requirements of Cu should meet (Spears, 1994). 3.3 P FERTILIZATION AND CONCENTRATION OF MINERALS IN HERBAGE The regular application of different rate of super- phosphate did not have any pronounced effect on ash content. It is normal with soils in the pH range from 4.5 to 5.5 which are deficient in P, that the application of P produces positive yield response. This is the reason that content of ash in herbage is not much increased because of dilution effect. But the application of superphosphate increased the concentration of P in herbage. On control treatment there was 3.4 g P kg−1 of DM of herbage. An increase of 23% for concentration of P in herbage was achieved in average on treatments where superphosphate was applied at the rates of 90 to 540 kg ha−1 P2O5 during three year time. Contrary to the statement for P was found for the concentration of Ca in herbage. At the control treatment, there was 9.0 g Ca kg−1 of DM. Application of P fertilizer decreased the Ca concentration in herbage. With highest rate (540 kg ha−1 P2O5) of fertilizer use, the content of Ca in herbage was 6.1 g P kg−1 of DM. Changes in concentra- tion of Mg in herbage through applied superphosphate are smaller than for Ca, but are in same direction; higher yield of DM, less Mg in herbage on weight basis. Concentration of K is within the range found in grasses collected from grazed sward only (12.2 g K kg−1 of DM). Increased concentration of K in herbage from plots with higher rate of superphosphate fertilization can only be explained through effect of deposited sheep urine; more of it was left on plots with higher amount of herbage available for grazing and longer stay of animals on those plots. Concentration of Na in the herbage, was in average 1.2 g Na kg−1 of DM. At low rate of applied P fertilizer the dilution effect through higher yield is more distinct. At heavier fertilization and higher yield the animals inter- fere with their excreta on concentration of Na in herbage. Sheep were all the time supplemented with common salt Acta agriculturae Slovenica, 94/2 – 2009164 M. VIDRIH et al. and this might have an effect on concentration of Na in herbage. There is not clear effect of added P on concentra- tion of Zn, Mn, Fe and Cu (Table 4). Higher yield of DM induced with added fertilizer didn’t have any dilution ef- fect on concentration of those minor elements in herb- age. Concentration of Fe is higher than found in different plant species at the onset of experiment. The reason for this might be the sampling of herbage closer to ground and possible contamination of sward with trampling of grazing animals. The soils of karst grassland as such are very un- suitable for to improve their fertility and to increase the abundance of legumes in the sward on the short time. Slow decomposition of soil organic matter (SOM) is the limiting factor to the cycling nutrients in the soil-plant- animal system. As reported by Clark and Woodmansee (1992) the microorganisms have a substantial require- ment for P. Net mineralization of SOM conducted by them, occurs only when their need on P has been met. The critical ratio of C to P for mineralization to occur must be less than 100:1 (Tate, 1985). In the soil of karst grassland dead SOM has the C to P ratio well above this. On addition the high content of SOM of karst grassland acts as big sink for P added with fertilization. As dead plant material of karst vegetation, largely present in the rooting zone, is insufficient in P, initially immobilisation of this element occurs when added to the soil, even in a large amount of P fertilizer. Many different plant species can be found within the indigenous karst sward (Kaligarič, 1997; Vidrih M, 2003). Unfortunately, most of the grasses are of low feeding quality (Brachypodium spp., Festuca ovina L., Koeleria pyramidata L., Bromus erectus L., Carex spp.). Further obstacle for higher stocking rate or better pro- duction is low portion of legumes in existing sward. It almost never exceeds 6% (Vidrih and Kotnik, 1995; Batič et al., 1999). Among different species of herbs 41 of them are medicinal plants, which can have distinct effect on animal health due to their higher concentration of min- erals or any of secondary substances (Kotnik and Vidrih A, 1995). As reported by Jones and Thomas (1987) there are several abiotic and biotic mechanisms that redistribute clay and nutrients in the landscape, similar to karst pas- tures, resulting in nutrient rich and nutrient poor patches which are differentially exploited by herbivores. Much of the spatial pattern of plant communities in karst pasture is similarly linked to variations in soil nutrient supply, which is in addition affected by deposition of excreta by grazing animals. When a nutrient is extremely deficient, as a P in karst soil, the addition of a small amount of nutrient will increase the yield but actually cause a small decrease in concentration of minerals. Due to high Ca level, con- sumed leaves of woody plants may result in a diet with an excessively Ca to P ratio compared to grasses. Intake of leaves of woody plants would be advantageous and would balance a selected diet on pasture dominated by grasses, is a statement made by Garmo (1999). A large amount of forage for animals in former times comprised leaves of shrubs and trees. Thus herbivores are adapted to uti- lize the foliage of woody plants in amount not harmful to them. Patchy pattern of grazing even on ideal ryegrass - Table 4: Effect of superphosphate application on concentration of minerals (g kg−1 or mg kg−1 of DM) in herbage available for grazing (pre-grazing) on the karst pasture. Means followed by the same letter in a row are not significantly different based on Duncan’s test (P < 0.05) Preglednica 4: Vpliv gnojenja z superfosfatom na koncentracijo mineralov (g kg−1 ali mg kg−1 SS) v razpoložljivem zelinju za pašo na kraškem pašniku. Povprečja z enako črko v vrstici se ne razlikujejo statistično značilno (p < 0,05) Herbage content Fertilizer treatments – total applied kg P2O5 ha −1 in three years not fertilized 90 kg ha−1 270 kg ha−1 540 kg ha−1 Ash (g kg−1) 87.2 a 84.2 a 85.4 a 84.9 a Phosphorus (g kg−1) 3.40 a 3.38 a 4.93 b 4.18 b Calcium (g kg−1) 8.97 b 6.84 a 6.03 a 6.08 a Potassium (g kg−1) 12.22 b 9.85 a 10.53 a 12.53 b Magnesium (g kg−1) 1.96 b 1.93 b 1.80 a 1.85 a Sodium (g kg−1) 1.21 a 1.06 a 1.13 a 1.24 a Zink (mg kg−1) 22.60 a 22.82 a 21.52 a 22.85 a Manganese (mg kg−1) 60.2 b 60.4 b 50.1 a 62.6 b Iron (mg kg−1) 371.8 b 503.7 c 274.4 a 379.1 b Copper (mg kg−1) 13.06 b 10.18 a 10.93 a 10.90 a Acta agriculturae Slovenica, 94/2 – 2009 165 MINERALS MANAGEMENT IN SILVOPASTORAL SYSTEM OF KARST PASTURE white clover pasture is clear evidence that animals needs more than just an abundance of most palatable herbage for to satisfy their physiological requirements. Mineral concentration in both soil and plant influ- ences the mineral status of grazing animals and many oth- er factors, such as selective grazing, interaction between minerals and production level. The important concept is that it is the concentration of the P in soil solution and not the total potentially available P that determines the rate at which the plant will grow (Mouat, 1984). A large amount of added P on small scale is achieved through the dung patch and at the site where the animals are camping or have drinking facilities. Since most nutrients ingested by grazing animals are returned to the soil in excreta, it is very important to achieve even distribution of it and to prevent nutrients transfer or accumulation on camp sites (Rigueiro-Rodríguez et al., 2007). Nutrients in urine and dung patches within the main grazing area cause a mosaic of nutrient levels and may lead to nutrient loss- es, even from unfertilized soils. Leaching losses may be smaller when woody plants are present on pasture. Trees and shrubs can further sequester nutrients from deeper soil layers as reported by Lehmann et al. (1997). Increased nutrient availability often causes or ac- celerates grass encroachment and a decrease in species diversity. However, grazing has been found to decrease grass encroachment and increase species diversity (Batič et al., 1999), a finding attributed to improved light access to plant species of low growing habit. Such conditions fa- voured a number of plant species which had otherwise low competitive ability under conditions of higher nutri- ent availability and denser vegetation like the white clo- ver has. 4 CONCLUSIONS The mineral composition of plants collected from karst pasture vegetation vary widely, so any changes in botanical composition of the vegetation can lead to ma- jor changes in mineral intake of grazing animals. In karst environment seldom all necessary growth factors are ad- equate to meet the needs of all plants within a sward. The large amount of P needs to be applied to achieve better plants growth on karst pastures. This is often uneconom- ic in term of cost of input and value of current return. But the beneficial effect of P fertilization will still be felt in later years and if this residual effect is used as a base for continued application, more and more efficient systems may be developed. Even with higher rates of applied P initially there will be no much increase in the concentra- tion of minerals in herbage available for grazing. To im- prove the efficiency of the system in shorter time, there is a need to exploit more efficiently all different groups of plants presented in vegetation of karst pasture and to enhance the speed of mineralisation of large amount of SOM. Since most nutrients ingested by grazing animals are returned to the soil in excreta it is very important to know the content of the minerals in different plants the animals have on offer. And at the same time managing animal grazing the way to exploit most efficiently the available minerals in dung and urine and to increase the activity of soil microorganisms to achieve more efficient decomposition of dead organic matter. It is likely that the creation and maintenance of such patches is necessary for the survival of grazers in nutrient poor environment as the karst pasture is. The importance of nutrient return through dung and urine of grazing and browsing animals will have great value when feeding animals during the winter on grazing land. 5 ACKNOWLEDGEMENT This work was done within Animal health, environ- ment and food safety No P4-0092, a program funded by the Slovenian Research Agency. 6 REFERENCES Aspinall R., Mandaluniz N., Hight L.J., Lucas R.J. 2004. 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Acta argiculturae Slovenica, 94/2, 167–172, Ljubljana 2009 COBISS: 1.01 Agris category code: P05 INTRODUCTION PILOT BIOGAS REACTORS AND APPLICATION TO DEFINE BIOGAS POTENTIAL OF BASIC SUBSTRAT, SWINE SLURRY Rajko BERNIK 1, Aleš ZVER 2 Delo je prispelo 16. septembra 2008, sprejeto 15. septembra 2009. Received September 16, 2008; accepted September 15, 2009. 1 Univ. of Ljubljana, Biotechnical Fac., Dept. of Agonomy, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia, Prof., Ph.D., M.Sc., e-mail: rajko.bernik@bf.uni-lj.si 2 Same address, Assist., e-mail: ales.zver@bf.uni-lj.si Introduction pilot biogas reactors and application to define biogas potential of basic substrat, swine slurry Cooperation between the ‘Panvita Group’ and the Bio- technical Faculty, University of Ljubljana, resulted in the con- struction of a pilot biogas reactor – a miniaturised version of the economical biogas reactor. The aim of construction was to support scientific research in the field of biogas generation, while, at the same time, optimising the processes conducted in economical reactors and testing the new substrates in the field of biogas generation. A 2500-litre reactor, containing a 500-li- tre gasholder, was built to this purpose. In the first operating period, biogas-generation potential of the raw and partially pu- rified swine slurries was tested. The three repetitions allowed us to generate an average of 529 litres CH4 per 1 kilogram of organic dry matter. Key words: biogas reactor / pig slurry / biogas / methane / electric energy Predstavitev pilotnega reaktorja in aplikativna določitev bio- plinskega potenciala osnovenga substrata, prašičje gnojevke Biotehniška fakulteta in Skupina Panvita smo v sodelo- vanju postavili preizkusni bioplinski reaktor, ki je pomanjšana različica gospodarskega bioplinskega reaktorja. Namen gradnje preizkusnega reaktorja je bil znanstveno raziskovalnem delo na področju pridobivanja bioplina in hkrati optimiziranje proce- sov v gospodarskih reaktorjih ter preizkušanja novih substra- tov v proizvodnji bioplina. Zgradili smo 2500 litrski reaktor, od celotnega volumna reaktorja je 500 litrov plinohrama. Kot cilj v prvem obratovalnem obdobju smo ovrednotili bioplinski potencial surove prašičje gnojevke in delno prečiščene prašičje gnojevke. Pravilno ovrednoten osnovni substrat je izhodišče za vse nadaljnje preizkuse, saj je to medij v katerem se bodo vrednotili vsi substrati ali mešanice substratov. S tremi pono- vitvami smo dosegli v povprečju 524 litrov CH4 na kilogram organske suhe snovi (OSS). Ključne besede: bioplinski reaktor / prašičja gnojevka / bioplin / metan / električna energija 1 INTRODUCTION The reduction of CO2 values in the atmosphere is a world goal (UNFCCC, 1997). The EU member states have clear instructions to substitute the current fossil en- ergy with renewable sources up to the value of 12% of the used gross energy by 2010. This means three times increasing the energy production from existent renew- able sources in the EU. In Slovenia there are already some existent biogas stations and some are being built. Biogas plants im- mensely help reducing greenhouse gas emissions and concurrently contribute a certain amount of renewable energy. The more biogas plants we have the bigger the effects of the test reactors will be, with which we can optimise the performance of biogas plants and test new substrates (Zver, 2005). Above all, the test reactors are in- tended for fast evaluation of a new organic mixture. This new organic mixture would be used in the actual reactor after the analysis of all the significant parameters for an economical and ecological performance of the reactor. The essential function of the test reactors is to enhance the economy of the real biogas plants. Biogas is the mixture of gasses, which originate Acta agriculturae Slovenica, 94/2 – 2009168 R. BERNIK and A. ZVER from anaerobic fermentation. Anaerobic fermentation is a biological process which is based on methanogenesis, where bacteria decompose organic material. The prod- ucts of the process are mainly methane and carbon diox- ide. The process can be simplified with the reaction: 2C + 2H2O à CH4 + CO2 (Đulbić, 1986). There are many factors to ensure optimal biogas production. They fall into three categories: –– Physical : imported heat in the reactor, mixing of the organic mixture in the reactor, density, struc- ture of the organic mixture in the reactor, specific yield (1/kg DM) –– Chemical : C:N ratio, pH, content of macro and micro elements, toxins, heavy metals –– Biological: type of microorganisms, their quality and quantity It is reasonable to search for and to optimise the mixture of different substrates in a test reactor. Thus we can ensure optimal exhaustion of substrates for biogas production and our findings can be applied in the eco- nomical devices. KG Rakičan EKOTEH d.o.o. is a member of the Panvita Group. They already operate a 1.3 MW biogas plant and they are also currently building a new smaller biogas plant. The following substrates are imported in the existent biogas plant: 13t of raw slurry, 8.5 t of by-products of animal ori- gin (BPOAO2 and BPOAO3), 35.6t of maize silage, and 60t of slurry. The article focuses on the biogas yield produced from swine slurry. Therefore, the results of preceding re- search will be presented as well. Different authors quote various biogas yields. We could practically say that the span of results is too big and unrealistic. Furthermore, the reason for the span is not given and this is the key factor for future assessment and planning of the device. 2 METHODS 2.1 INTRODUCTION OF THE TEST REACTOR A pilot reactor was built in the immediate vicinity of the existent biogas plant (Fig. 1). Its purpose is opti- mising the functioning of economical biogas plants and testing new substrates. The volume of the pilot reactor is 2500 litres, from which the working volume is 2000 litres and the remaining 500 litres is intended for the gas- holder. It is a miniature version of the economical biogas reactor. In it we can test substrates processed in the same manner as in the economical biogas reactor. 2.2 COMPONENTS OF THE REACTOR –– The stirring device is placed horizontally and driven by a 500W electro engine. –– The heating appliance is placed inside on the walls of the reactor; it is heated by water from an outer heating source. –– The additional – auxiliary stirring device is ad- justable in the spill-over edge, which prevents clogging of the exit; it is driven by a 350W engine. –– The swine slurry pump is driven by an electrical engine and is used to pump the purified slurry into the reactor. It pumps 2 litres of slurry per minute, taking into account the losses in the pipeline. We can set the intervals of the pumping of the slurry and the amount of it. –– The opening for adding additional substrates is a funnel-shaped opening into which, at certain in- tervals, substrates are added. Due to stirring the opening is being gradually emptied and thus it ensures a continuous filling. –– The biogas flow-metre is a mechanical counter. It notes the biogas flow on the basis of the turns of the spades. In our case the spades are being driven by the biogas. –– The analyser of the biogas quality is portable. It Author Extent of biogas production (l/kg organic substance) Average biogas production (l/kg ODM) Swine slurry Beck, 1997 340–550 450 Swine slurry Gaćeša, 1985 550 Swine slurry Gobec, 2005 200–350 275 Swine slurry Karpenstein-Machan, 2005 250–350 300 Table 1: Efficacy of biogas production from swine slurry Preglednica 1: Izpleni bioplina iz prašičje gnojevke po različnih avtorjih l/kg – litres of biogas per kilogram of organic substance; ODM organic dry matter Acta agriculturae Slovenica, 94/2 – 2009 169 INTRODUCTION PILOT BIOGAS REACTORS AND APPLICATION TO DEFINE BIOGAS ... SUBSTRAT, SWINE SLURRY is used to analyse the biogas quality in certain in- tervals or when needed. We can measure % CH4, % CO2, % O2, and ppm H2S. –– The control box with a computer controls all the devices in the reactor except for the analyser of the biogas quality. The software enables the set- ting of the intervals for the stirring devices and the pump. It also provides a simultaneous regis- ter of the data during the intervals. –– Other equipment includes the laboratory with all the necessary equipment and other equipment for storage and handling of substrates. –– Sampling pints. The reactor has six exits with ball-bearing valves, which enable sampling for the analysis. Two of these are 80mm in diameter. –– The window is a glass door. We can observe the occurrence in the reactor from the top. 2.3 WORKING WITH THE REACTOR The working volume of the reactor (2000 litres) is so big that the processes in it cannot be completely control- led and directed. Processes which are equal to those in the economical reactor are: complete stirring (usually a crust occurs on the substrates with a major proportion of dryness and fibres), emissions of biogas, comparable fluctuation of pH, structures of different microorgan- isms, etc. The test reactor gives us more tangible results for a certain substrate in comparison to the results given in certain literatures, which usually contain average results gained in smaller reactor laboratories under optimal conditions. The results are then used directly in the eco- nomical reactor. Thus the fluctuation of the economical reactor is optimised. Concurrently, we can analyse the functioning of the test device. 2.4 INITIATION OF THE TEST REACTOR The pilot reactor was filled with the mixture of bac- teria from the anaerobic part of a purification device or an old biogas plant. The temperature was set to 37.5 °C with ±1.5 °C deviation. The stirring was adjusted to the economical devices. The swine slurry was gradually add- ed. After having established that the test reactor func- Figure 1: Test reactor (on the left: view from the front, on the right: view from the back). Slika 1: Preizkusni reaktor (levo: prednja stran, desno: zadnja stran). Acta agriculturae Slovenica, 94/2 – 2009170 R. BERNIK and A. ZVER tions perfectly, we started with the systematic research of the test reactor. 2.5 CHOOSING THE BASIC SUBSTRATE During the first weeks of the operation we deter- mined daily fluctuation of biogas production. We started researching the reasons for the unbalanced biogas pro- duction. The cause for this was the quality of the raw swine slurry and the changing dryness from 0.5% to 7%. The dryness of the swine slurry depended on the day of the week. During weekends the stables are not washed. During weekdays the stables are being washed, adding the water used for washing to the common reservoir of the slurry. From there the slurry is taken for the use in the experimental reactor. Another occurrence of unbal- anced production was detected when the pipe for add- ing raw swine slurry was clogged by a ball of swine hair. The flow was lower than we expected. However, these are technical problems which can be solved by designing the device correctly. According to the above described technical problems we decided to use partially purified swine slurry with dryness between 1% and 1.5%. Thus we ensured that the input material is homogeneous and avoided technical stirring problems. The swine slurry under consideration has a very specific composition. On average it contains 41% of ash in dry matter. 2.6 EVALUATION OF SWINE SLURRY The purified swine slurry will in future be the basic medium in the reactor. Thus we needed to assess its en- Figure 2: Sketch of the test reactor (front view and ground plan). Slika 2: Skica preizkusnega reaktorja (naris in tloris). Acta agriculturae Slovenica, 94/2 – 2009 171 INTRODUCTION PILOT BIOGAS REACTORS AND APPLICATION TO DEFINE BIOGAS ... SUBSTRAT, SWINE SLURRY ergy potential at a certain residence time. We decided on a 30-day residence time. The purified swine slurry had 41% of ash in dry matter. After months of evaluation we gained the sought data for this swine slurry, biogas yield per kilogram of dry matter, and biogas yield per kilogram of organic dry matter. However, we did not seek for non- degradable organic dry matter. 3 RESULTS AND DISCUSSION During the first three months of production, we were evaluating biogas potential of the raw swine slurry and partially purified swine slurry. Figure 3 and Table 2 show biogas productions in time periods 1, 2, 3 and 4. In the time periods 1 and 2 we used raw swine slurry. How- ever, due to its physical characteristics it was not suitable to include it in further experiments. The main problem was the unbalanced amount of dry matter. In the time periods 3 and 4 we evaluated partially purified swine slurry. This is shown in Figure 3. Curve 1 shows that raw swine slurry is not suitable as the basic substrate because of dryness volatility. Curve 2 shows operation with raw slurry. However, in this case the slurry was left in a con- tainer for the thick part to sediment. We only used the cleaner upper part of the slurry, but the volatility of the dryness was still too high. Thus it is not suitable to be used as the basic substrate. Table 2 shows data about the biogas yield from slur- ry tested during certain time periods. The standard de- viation with raw slurry is immense. Thus it is not suitable to be used as the basic substrate in further experiments. 3.1 BIOGAS POTENTIAL OF PARTIALLY PURI- FIED SWINE SLURRY Due to technical problems and homogeneity of the basic substrate we decided to use partially purified swine slurry. In Table 3 we see the average biogas yield for a Substrate Biogas production in 10 days Min. Max. Average Median Standard deviation Produced biogas (l) Slurry 1 70 510 240 200 141 2405 Slurry 2 280 420 379 398 53 3790 Partially purified slurry 3 250 320 284 298 28 2840 Partially purified slurry 4 225 320 267 265 33 2665 Table 2: Basic statistics of biogas produced from partially purified swine slurry Preglednica 2: Osnovna statistika bioplina iz surove in delno prečiščene prašičje gnojevke Figure 3: Biogas production per day during the evaluation of biogas potential of raw swine slurry (1 and 2) and partially purified swine slurry (3 and 4). Slika 3: Produkcija bioplina po dnevih pri vrednotenju bioplinskega potenciala surove gnojevke (1 in 2) in delno prečiščene prašičje gnojevke (3 in 4). Acta agriculturae Slovenica, 94/2 – 2009172 R. BERNIK and A. ZVER 20-day period. We used a total of 1040 litres of slurry and produced 5505 litres of biogas or 3854 litres of methane. We reached the average biogas yield of 275 litres per day, or 756 litres of biogas per kilogram of organic dry matter, or 423 litres per dry matter. The average biogas composi- tion is: CH4 70%, CO2 30%, O2 0% in 1500 ppm H2S. 4 CONCLUSION We evaluated the biogas potential of specific swine slurry. Partially purified swine slurry has specific charac- teristics in comparison with other slurries mentioned by other authors. It was reasonable to use partially purified slurry. It gave us valuable information concerning its bi- ogas potential. In future, partially purified swine slurry will be used as the basic medium for testing and determining the bi- ogas potential of different substrates. One of the exam- ples is the mixture of partially purified slurry and silage. From the gross produced biogas, gained from this mix- ture, we mathematically subtract the produce of partially purified swine slurry. Thus we gain the net biogas yield from certain substrate. Partially purified slurry will also hold the function of a means of transportation into the biogas reactor for different substrates. Thus, with the help of the fluid partially purified slurry, for which the energy potential is already known, we can import into the reac- tor organic material with bigger content of dry matter. This enables us to define the common or partial energy potential, or we can anticipate the energy utility of a cer- tain organic material. 5 REFERENCES Beck J. 1997. Anaerobic treatment. In: Manure managment. Treatment strategies for sustainable agriculture. Burton C.H. (ed.). Bia, Silsoe Research Institute: 79–88 Đulbić M. 1986. Biogas: dobijanje korišćenje i gradnja uređaja. Beograd, Tehnička knjiga: 171 p. Gobec I. 2005. (So)substrati pri proizvodnji bioplina. Društvo za energetsko ekonomiko in ekologijo. http://www.ljudmila.org/sef/stara/bioplin05/predstavitve_ in_fotografije/predstavitve/postojna/so)substrati%20 pri%20proizvodnji%20bioplina%20postojna.pdf (7. nov. 2007) Gaćeša S., Vrbaški L., Baras J., Knežić L., Kašnja M., Zidanski F. 1985. Biogas-proizvodnja i primena. Novi Sad, Tehnološki fakultet: 233 p. Karpenstein-Machan M. 2005. Energiepflanzenbau für Biogas- anlgenbetreiber. Frankfurt am Main. DLG-Verlags-GmbH: 191 p. Medved S., Novak P. 2000. Biomasa. In: Varstvo okolja in ob- novljivi viri energije. Ljubljana, Fakulteta za strojništvo: 231 p. Polprasert C. 1996. Organic waste recycling. Technology and management. 2nd edition. West Sussex, John Wiley & Sons: 412 p. Rempel H. 2003. Ressourcen und Verfügbarkeit von Ener- gierohstoffen 2003. – XXVIII. 426 S., E. Schweizerbart’sche Verlagsbuchhandlung (Nägele und Obermiller), ISBN 3-510-95900-0, Stuttgart. UNFCCC. 1997. The Kyoto Protocol to the United nations Framework Convention on Climate Change. FCCC/CP 1997/7Add.1,4. Zver A. 2005. Obnovljivi viri energije, rastlina kot energija in rastlina kot hrana. Diplomsko delo. Ljubljana, Univ. v Lju- bljani, Biotehniška fakulteta, Odd. za zootehniko: 67 p. Substrate Partially purified swine slurry % DM 1.0–1.5 % CA* in DM 44 C : N 36 Biogas / litre of slurry (l) 5.29 Biogas / kg DM (l) 423 Biogas / kg ODM (l) 756 CH4 / litre of slurry (l) 3.71 CH4 / kg DM (l) 296 CH4 / kg ODM (l) 529 Table 3: Chemical characteristics of partially purified swine slurry and biogas yield Preglednica 3: Kemične lastnosti delno prečiščene prašičje gnojevke in izplen bioplina *CA = crude ashes, BG; We gained comparable results with previous researchers on the test biogas reactor. Acta argiculturae Slovenica, 94/2, 173–174, Ljubljana 2009 SUBJECT INDEX BY AGROVOC DESCRIPTORS PREDMETNO KAZALO PO DESKRIPTORJIH AGROVOC Tomaž BARTOL 1 1 Univ. of Ljubljana, Biotechnical Fac, Dept. of Agronomy, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia, Assoc.Prof., Ph.D., e-mail: tomaz.bartol@bf.uni-lj.si Acta agriculturae Slovenica, 94/2 – 2009174 T. BARTOL Acta argiculturae Slovenica, 94/2, 175, Ljubljana 2009 SUBJECT INDEX BY AGRIS CATEGORY CODES VSEBINSKO KAZALO PO PREDMETNIH KATEGORIJAH AGRIS Nataša SIARD 1 1 Univ. of Ljubljana, Biotechnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia, Ph.D., M.Sc., e-mail: natasa.siard@bfro.uni-lj.si Animal husbandry – L01: 153–158 Animal feeding – L02: 95–102, 159–166 Animal genetics and breeding – L10: 121–131, 133–138 Animal physiology and biochemistry – L50: 153–158 Animal physiology – nutrition – L51: 103–110, 111–119 Energy resources and management – P05: 167–172 Soil science and management – P30: 159–166 Nutrition programmes – S40: 87–93 Acta argiculturae Slovenica, 94/2, 177–178, Ljubljana 2009 ABECEDNO KAZALO AVTORJEV AUTHOR’S INDEX Št. No. Avtor Author Stran primarnega prispevka Page of the primary source 1. ACCETTO Tomaž 143–146, 147–152 2. AVGUŠTIN Gorazd 147–152 3. BARTOL Tomaž 173–174 4. BERNIK Rajko 167–172 5. DOVČ Peter 121–131, 133–138 6. FRANKIČ Tamara 95–102 7. GORENC Gregor 147–152 8. GWASKA Jafaru Ari 153–158 9. HORVAT Simon 121–131 10. KOCH Verena 87–93 11. KOMPAN Drago 121–131, 159–166 12. KOROŠEC Maja 87–93 13. KOSTANJEVEC Stojan 87–93 14. MUROVEC Boštjan 139–142 15. OGOREVC Jernej 133–138 16. OREŠNIK Andrej 111–119 17. OSTERC Jože 85–86 18. PAJK ŽONTAR Tanja 103–110 19. PATUREAU MIRAND Philippe 111–119 20. PIRMAN Tatjana 111–119 21. POGAČNIK Milan 159–166 22. POLAK Alenka 87–93 23. PRPAR Sonja 133–138 24. REZAR Vida 95–102, 103–110 25. SALAKO Adebowale Emmanuel 153–158 Acta agriculturae Slovenica, 94/2 – 2009178 Št. No. Avtor Author Stran primarnega prispevka Page of the primary source 26. SALOBIR Janez 95–102, 103–110, 111–119 27. SIARD Nataša 175 28. STRES Blaž 139–142 29. TORKAR Gregor 87–93 30. VIDRIH Matej 159–166 31. VIDRIH Anton 159–166 32. VOLČIČ Tomaž 121–131 33. VOLJČ Mojca 95–102 34. YAKUBU Abdulmojeed 153–158 35. ZVER Aleš 167–172 Acta argiculturae Slovenica, 94/2, 179–180, Ljubljana 2009 NAVODILA AVTORJEM PRISPEVKI Sprejemamo izvirne znanstvene članke, predhodne objave in raziskovalne notice s področja zootehnike (ge- netika, mikrobiologija, imunologija, prehrana, fiziologi- ja, ekologija, etologija, mlekarstvo, ekonomika, živalska proizvodnja in predelava živalskih proizvodov, tehno- logija in dokumentalistika) v slovenskem in angleškem jeziku, pregledne znanstvene članke pa samo po poprej- šnjem dogovoru. Objavljamo tudi prispevke, podane na simpozijih, ki niso bili v celoti objavljeni v zborniku sim- pozija. 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 slo- venskem in angleškem jeziku. Pri prispevkih v slovenskem jeziku morajo biti pre- glednice, 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 eno- staven; 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 gra- fikonih 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 znan- stveni in akademski naslov; vse v jeziku prispevka. Na- vedemo 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 z do 250 besedami. 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 ob- jave: (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. Ne- kaj 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 Acta agriculturae Slovenica, 94/2 – 2009180 Fraser A.F., Broom D.M. 1990. Farm animal behaviour and wel- fare. London, Bailliere Tindall: 437 str. Hvelplund T. 1989. Protein evaluation of treated straws. V: Evaluation of straws in ruminant feeding. Chenost M., Rei- niger, 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 eko- nomistov Slovenije: 279–288 ISO 5534 / IDF 4. Cheese and processed cheese – Determina- tion 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) ODDAJA Avtorji prispevke oddajo v natisnjenem in elektron- skem izvodu. Priložijo tudi izjavo s podpisi vseh avtorjev, da avtorske pravice v celoti odstopajo reviji. Prispevke recenziramo in lektoriramo. Praviloma pošljemo mnenje prvemu avtorju, po želji lahko tudi drugače. Če urednik ali recenzenti predlagajo spremem- be oz. izboljšave, vrne avtor popravljeno besedilo v 10 dneh v natisnjenem in elektronskem izvodu. Ko prvi av- tor vnese še lektorjeve pripombe, odda popravljeno bese- dilo v natisnjenem in elektronskem izvodu. Pri oddaji končne verzije avtor priloži jasno označe- ne izvirnike slik (ločene grafične datoteke ali fotografije). Datoteke slik poimenuje enako kot v tekstu (npr. Slika1. jpg, Slika2.eps, Slika3.bmp). Originalne fotografije na av- torjevo 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. Acta argiculturae Slovenica, 94/2, 181–182, Ljubljana 2009 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, 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. 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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. Di- vergent 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 wel- fare. London, Bailliere Tindall: 437 p. Hvelplund T. 1989. Protein evaluation of treated straws. In: Evaluation of straws in ruminant feeding. Chenost M., Rei- niger, 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 eko- nomistov Slovenije: 279–288 ISO 5534 / IDF 4. Cheese and processed cheese – Determina- tion 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 elec- tronic copy. A statement signed by all authors transfers copy rights on the published article to the Journal. Papers are reviewed and edited. First author re- ceives a review if not defined otherwise. If reviewers sug- gest 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. 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