SLOVENSKÁ POĽNOHOSPODÁRSKA UNIVERZITA V NITRE FAKULTA BIOTECHNOLÓGIE A POTRAVINÁRSTVA KATEDRA FYZIOLÓGIE ŽIVOČÍCHOV Z FYZIOLÓGIE ŽIVOČÍCHOV

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1 SLOVENSKÁ POĽNOHOSPODÁRSKA UNIVERZITA V NITRE FAKULTA BIOTECHNOLÓGIE A POTRAVINÁRSTVA KATEDRA FYZIOLÓGIE ŽIVOČÍCHOV VII. CELOSLOVENSKÝ SEMINÁR Z FYZIOLÓGIE ŽIVOČÍCHOV mája 2007, Topolčianky, Nitra - Slovenská republika

2 SLOVENSKÁ POĽNOHOSPODÁRSKA UNIVERZITA V NITRE FAKULTA BIOTECHNOLÓGIE A POTRAVINÁRSTVA KATEDRA FYZIOLÓGIE ŽIVOČÍCHOV ZBORNÍK z vedeckého seminára s medzinárodnou účasťou VII. CELOSLOVENSKÝ SEMINÁR Z FYZIOLÓGIE ŽIVOČÍCHOV mája 2007 Topolčianky, Nitra, Slovenská republika

3 Redakčná rada: Ing. Adriana Kolesárová, PhD doc. MVDr. Peter Massányi,PhD. doc. Ing. Norbert Lukáč, PhD. Schválil rektor Slovenskej poľnohospodárskej univerzity v Nitre dňa ako zborník za vedeckého seminára s medzinárodnou účasťou ISBN

4 Vedecký výbor: prof. Ing. Jozef Bulla, DrSc. Slovenská poľnohospodárska univerzita v Nitre SR prof. MVDr.László Bárdos, Ph.D. Szent István University, Budapest, Hungary doc. MVDr. Viera Cigánková, PhD. Univerzita veterinárneho lekárstva, Košice, SR prof. MVDr., Ing. Pavel Jelínek, DrSc. Mendělevova univerzita poľnohospodárstva a lesníctva v Brne, Česká republika prof. Ing. Jaroslav Kováčik, Ph.D. Slovenská poľnohospodárska univerzita v Nitre SR prof. MVDr. Imrich Maraček, DrSc. Univerzita veterinárneho lekárstva, Košice, SR prof. MVDr. Juraj Pivko, DrSc. Slovenská poľnohospodárska univerzita v Nitre SR Dr. Robert Stawarz, Ph.D. Pedagogical University, Krakow, Poland doc. Ing. Miroslav Valent, Ph.D. West Virginia University, Morgantown, USA prof. dr. hab. Wladyslaw Zamachowski, Ph.D. Pedagogical University, Krakow, Poland prof. RNDr. Michal Zeman, DrSc. Univerzita Komenského, Bratislava, SR Organizačný výbor: prof. Ing. Jaroslav Kováčik, PhD. doc. MVDr. Peter Massányi, PhD. Ing. Adriana Kolesárová, PhD. doc. Ing. Norbert Lukáč, PhD. Ing. Marcela Capcarová, PhD. Ing. Anna Kalafová. Ing. Jiřina Kročková Ing. Monika Schneidgenová Ing. Peter Čupka Alena Balážová Viera Bösel Recenzenti: prof. Ing. Jaroslav Kováčik, PhD. prof. MVDr. Juraj Pivko, DrSc. doc. MVDr. Peter Massányi, PhD. Dr. Robert Stawarz, PhD. Dr. Grzegorz Formicki, PhD. RNDr. Janka Drábeková, PhD. Ing. Miroslav Juráček, PhD.

5 VPLYV HYPODYNAMIE NA OBLIČKY JAPONSKÝCH PREPELÍC (COTURNIX COTURNIX JAPONICA) EFFECT OF HYPODYNAMY ON THE KIDNEYS OF JAPANESE QUAILS (COTURNIX COTURNIX JAPONICA) Almášiová, V., Cigánková, V., Holovská, K., Zibrín, M. Katedra anatómie, histológie a fyziológie, Univerzita veterinárskeho lekárstva Košice Abstract The aim of present study was to observe the influence of simulated weightlessness on the structure and ultrastructure of kidneys of Japanese quails reared under the conditions of experimental long-term hypodynamy. Hypodynamy is a method to simulate weightlessness in the earth laboratory conditions. Two days after hatching the quails were suspended in special shirts below the ceiling of a cage so their feet did not touch the floor. They could consume food and water ad libitum. Experimental animals were sacrificed after 7, 14, 21, 28, 35, 42, 49, 56, and 63 d of hypodynamy. Animals of the same age, hatched at the same time, and fed the same diet were used as controls. The samples of kidney were fixed and routinely processed for light (LM) and transmission electron microscopy (TEM). It has been found that at short-term hypodynamy (7 21 days) there is not a more pronounced damage of the kidney structure. Due to long-term hypodynamy (28 63 days) there are some morphological changes in the proximal and distal tubular cells as well as in collecting ducts. However, the structure of renal corpuscles was not damaged. Key words: microgravity, kidney, morphology, Japanese quails Úvod Pokroky v kozmickom výskume a pravdepodobnosť budúcich dlhotrvajúcich pobytov v kozme vyvolávajú záujem o ich účinky na človeka a zvieratá. Kozmické experimenty sú náročné a drahé, preto sa častejšie využívajú modelové pokusy. Jednou z metód imitujúcich stav beztiaže na Zemi v laboratórnych podmienkach je hypodynamia [15]. U vtákov sa používajú špeciálne košieľky zo sieťoviny, pomocou ktorých sú zvieratá zavesené na strope klietky tak, aby sa nemohli dotýkať podlahy, avšak aby mohli prijímať krmivo a vodu ad libitum [7]. Veľmi vhodným aviárnym biologickým objektom na študovanie vplyvu hypodynamie je Japonská prepelica. Je to vták s nízkou hmotnosťou tela, vysokou produkčnou a reprodukčnou schopnosťou, krátkym individuálnym vývojom, dobrou toleranciou na stiesnené podmienky a adaptabilitou na mikrogravitáciu [8, 17]. Zároveň má slúžiť ako nevyhnutný heterotrofný článok v uzavretom umelom ekosystéme kozmických lodí 5

6 na zabezpečenie produkcie živočíšnych bielkovín pre kozmonautov a vytvoriť psychologicky príjemnejšie prostredie pri dlhodobých pobytoch v kozme [1]. Materiál a metodika Experimenty boli robené na Ústave biochémie a genetiky živočíchov SAV v Ivanke pri Dunaji. Kurčatá japonských prepelíc oboch pohlaví neselektovanej línie na hypodynamiu dva dni po vyliahnutí boli premiestnené do špeciálnych klietok a zavesené do košieľok zo sieťoviny tak, aby sa nemohli dotýkať podlahy, avšak aby mohli prijímať krmivo a vodu ad libitum. Prepelice toho istého veku a rovnako kŕmené slúžili ako kontrolné. Hneď po usmrtení zvierat sme odobrali excízy z obličiek pokusných a kontrolných prepelíc na 7., 14., 21., , 42., 49., 56. a 63. deň pokusu. Tkanivo sme spracovali na svetelnú (SM) a transmisnú elektrónovú mikroskopiu (TEM). Vzorky na SM sme spracovali rutinnou metódou fixáciou vo formaldehyde a zaliatím do parafínu. Excízy na TEM sme fixovali v 3% glutaraldehyde v kakodylátovom pufri, postfixovali 1% OsO 4, odvodnili v acetóne a zaliali do Durcupanu. Ultratenké rezy kontrastované uranylaceátom a citrátom olovnatým sme pozorovali a fotografovali TEM Tesla BS 500. Histologické preparáty ofarbené H-E a polotenké rezy ofarbené toluidínovou modrou sme pozorovali svetelným mikroskopom Jenamed. Výsledky Pri krátkodobej (7 až 21 dňovej) hypodynamii nedochádza k výraznejšiemu poškodeniu štruktúry obličiek. Ich mikroskopický a submikroskopický obraz je v podstate rovnaký ako u kontrolných prepelíc. SM sme v kôrovej časti lalôčikov pozorovali početné obličkové telieska nepravidelného tvaru a v dreni lalôčikov väčšie, menej početné okrúhle obličkové telieska. TEM sme v kortikálnych aj medulárnych obličkových telieskach pozorovali glomeruly na povrchu ktorých boli podocyty. Tieto bunky mali veľké oválne jadro a obsahovali pomerne málo cytoplazmatických organel. Ich výbežky trabekuly a menšie pedikuly medzi ktorými sa nachádzali štrbinky neboli porušené. Bazálna membrána bola tenká a súvislá. Fenestrované krvné kapiláry, ktoré sú súčasťou filtračno-štrbinovej membrány nejavili nijaké známky poškodenia ich štruktúry. Kanáliky proximálneho segmentu, v ktorých prebieha tubulárna rezorpcia predstavujú najdlhšie úseky močového kanálika. Tvorili ich bunky cylindrického tvaru, ktoré v SM mali výrazne acidofilnú cytoplazmu a na apikálnom povrchu mali súvislý kefkovitý lem. Kanáliky 6

7 mali nepravidelný štrbinovitý lúmen a bočné hranice buniek boli málo zreteľné. Obsahovali typické tyčinkovité mitochondrie, ktoré bunkám dávali jemne prúžkovaný vzhľad. V TEM mali stredne elektróndenznú cytoplazmu a okrúhle jadro s difúzne rozptýleným chromatínom a výrazným jadierkom sa nachádzalo v bazálnej časti bunky. V cytoplazme obsahovali početné veľké mitochondrie pretiahleho tvaru s mitochondriovými kristami, ktoré boli orientované kolmo na bazálnu membránu. Bazálna membrána vytvárala početné invaginácie, ktoré zväčšujú jej povrch a sú dôležité pri zahusťovaní moču. Golgiho komplex tvorený cisternami a váčikmi bol uložený supranukleárne. V cytoplazme sa nachádzali aj cisterny granulovaného endoplazmatického retikula a lyzozómy. Na apikálnom povrchu buniek bolo veľké množstvo vysokých tenkých, paralelne usporiadaných mikroklkov. Početné pinocytárne váčiky, ktoré boli prítomné v cytoplazme svedčia o veľkej endocytóznej aktivite týchto buniek. Kanáliky distálneho segmentu tvorili bunky kubického tvaru. Lúmen distálnych kanálikov bol oproti lúmenu proximálnych kanálikov väčší. V SM mali bunky distálneho segmentu stredne acidofilnú cytoplazmu a na ich apikálnom povrchu sa nachádzali iba ojedinelé mikroklky. Mitochondrie usporiadané do stĺpcov podmieňujú pásikovaný vzhľad bazálnej časti týchto buniek. Hranice medzi bunkami boli zretelnejšie ako v proximálnom kanáliku. V TEM mali bunky stredne elektróndenznú cytoplazmu, jadro okrúhleho tvaru s difúzne rozptýleným chromatínom a výrazným jadierkom sa nachádzalo uložené v apikálnej časti bunky. Typické boli aj dlhé tyčinkovité mitochondrie a bunková membrána, ktorá v bazálnej časti buniek vytvárala početné hlboké invaginácie. O nižšej aktivite endocytózy týchto buniek svedčí aj prítomnosť ojedinelých pinocytárnych vezikúl a lyzozómov. Henleova slučka spája proximálny a distálny stočený kanálik a jej dĺžka sa líši podľa typu nefrónu. Kôrové nefróny majú iba krátky kanálik vystlaný jednou vrstvou plochých buniek, kým dreňové nefróny majú dlhú Henleovu slučku pokračujúcu smerom do drene lalôčika a vracajúcu sa opäť do kôry lalôčika. Preto sa nazýva častejšie medulárnou slučkou a je zložená z tenkého a hrubého segmentu. Intermediárne nefróny majú medulárnu slučku krátku. Bunky tenkého segmentu boli dlaždicovitého tvaru a mali okrúhle alebo oválne centrálne uložené jadro. Na apikálnej časti buniek sa nachádzali ojedinelé krátke nepravidelné mikroklky. V cytoplazme boli roztrúsené malé mitochondrie, granulované endoplazmatické retikulum a voľné ribozómy. Bazálna membrána netvorila interdigitáciíe a prstovité výbežky. Bunky hrubého segmentu mali podobnú štruktúru, len boli tvorené vyššími bunkami. 7

8 Distálny kanálik ústi do zberacích kanálikov ktoré prebiehajú medzi jednotlivými lalôčikami, vytvárajú medulárny konus a ústia do močovodu. V SM boli bunky zberacích kanálikov svetloružovo sfarbené, výrazne ohraničené a kupolovite vyčnievali do lúmenu. V TEM sme pozorovali, že stenu zberacích kanálikov tvorili dva typy buniek, svetlé a tmavé. Svetlé viacpočetné bunky kubického tvaru mali pomerne veľké centrálne uložené jadro a v cytoplazme obsahovali málo cytoplazmatických organel. Pomerne malé mitochondrie boli orientované rôznym smerom a bazálna membrána nevytvárala záhyby. Na apikálnom povrchu sa nachádzali len ojedinelé malé mikroklky. Tmavé ojedinelé bunky obsahovali viac mitochondrií a vezikúl a na povrchu mali vyššie mikroklky. V obličkách prepelíc pri dlhodobej (28 až 63 dňovej) hypodynamii sme SM nepozorovali výraznejšie morfologické zmeny. Avšak TEM sme zistili nasledovné zmeny: V proximálnych kanálikoch sa nachádzali dva typy buniek. Svetlé bunky s elektrónlucentnou cytoplazmou a tmavé bunky s elektróndenznou cytoplazmou. Svetlé bunky mali okrúhle jadro s difúzne rozptýleným chromatínom a v cytoplazme obsahovali početné mitochondrie oválneho tvaru s mitochondriovými kristami. V niektorých bunkách sa nachádzali mierne dilatované mitochondrie a početné svetlé vakuoly s obsahom vločkovitého materiálu. Na apikálnom povrchu boli mikroklky bez výraznejších známok poškodenia. Tmavé bunky mali elektróndenznú cytoplazmu a obsahovali dlhé tyčinkovité mitochondrie s tenkými kristami a menšie vakuoly. Mali výrazne tmavé scvrknuté jadro. Mikroklky na apikálnom povrchu týchto buniek boli tmavé so zastretou štruktúrou. Niektoré mikroklky vytvárali závitnicové štruktúry. V obličkách pokusných zvierat na 63. deň hypodynamie niektoré proximálne kanáliky obsahovali len tmavé bunky. U pokusných prepelíc sa v cytoplazme apikálnej časti buniek distálneho kanálika nachádzal vločkovitý materiál. Na 42. až 63. deň hypodynamie sme v cytoplazme zberacích kanálikov pozorovali veľké svetlé vakuoly, ostatné organely nejavili známky poškodenia. Diskusia Renálne funkcie, ako metabolizmus vody a solí sú v podmienkach mikrogravitácie extrémne zaťažené, pretože telové tekutiny lokalizované v nižších oblastiach tela sa presúvajú smerom ku hlave a do hrudnej oblasti blood shift [14, 15]. Je zvláštne, že počas skorých fáz simulovaných experimentov napríklad u bed rest a water immersion bola zaznamenaná zvýšená diuréza [2, 15, 18], ale v priebehu reálnych letov do kozmu bola diuréza naopak znížená [6, 12, 13, 14]. Vysvetlením môže byť skutočnosť, že na organizmus pri kozmickom lete významne vplýva stres a ten aktiváciou sympatika zapríčiní zvýšenie hladín 8

9 glukokortikoidov a katecholamínov, ktoré sekundárne cestou konstrikcie obličkových ciev znížia diurézu [6]. Kostra a svalstvo, ktoré zabezpečujú podpornú funkciu organizmu, reagujú na mikrogravitáciu atrofiou svalstva končatín a osteoporózou [21, 22]. U ľudí, ktorí absolvovali pobyt v kozme, sa v dôsledku zvýšenej rezorbcie kostného tkaniva zistilo aj riziko tvorby močových kameňov. K ich tvorbe významne prispieva saturácia moča soľami Ca (hyperkalciúria), pokles obsahu citrátov v moči, redukcia objemu a zmena ph moča [19, 20]. Pri dlhodobých kozmických letoch ako aj pri dlhotrvajúcich modelových pokusoch nastávajú zmeny vodno- elektrolytovej rovnováhy, exkrécia Na a diuréza klesá, naopak exkrécia Ca a produkcia ADH a hormónov zahrnutých v RAAS (renín-angiotenzín-aldosterón-systém) stúpa [12]. Viacerí autori zistili morfologické zmeny v dôsledku mikrogravitácie v kozme a hypodynamie na Zemi aj v obličkách potkanov. Počas kozmického letu sa ich obličky, ale aj pľúca, srdce a nadobličky značne zväčšili [10, 11]. Už po 1 hodine po zavesení potkanov za chvost došlo medzi proximálnymi kanálikmi k rozšíreniu intersticiálnych priestorov [16]. Štruktúru a ultraštruktúru obličiek dospelých japonských prepelíc, ktoré boli 7 dní v kozme študovali Cigánková a kol. a Kočišová a kol. [3, 9]. Prepelice rovnakého veku odchované v laboratóriu slúžili ako kontrola. Autori zistili, že krátkodobá mikrogravitácia nemá nepriaznivý dôsledok na štruktúru, ani funkciu obličiek [3, 9]. Cigánková a kol. [4, 5] zistili morfologické zmeny aj v nadobličkách japonských prepelíc, ktoré sa vyliahli na orbitálnej stanici MIR. Zmeny mali podobný charakter aký je známy pri strese. Svedčí to o tom, že už aj krátkodobý pobyt v kozme má nepriaznivý vplyv na organizmus kurčiat japonských prepelíc. Zistené zmeny sú autormi považované za reverzibilné [6, 15]. Mikrogravitácia v kozme a hypodynamia na Zemi zapríčinila zvýšenie koncentácie katecholamínov a kortikosteroidov v krvnej plazme japonských prepelíc, naopak koncentrácia estradiolu v krvnej plazme prudko klesla [6, 8]. Záver Z našich pozorovaní môžeme usúdiť, že krátkodobá (7 až 21 dňová) hypodynamia nespôsobuje v obličkách japonských prepelíc výraznejšie morfologické zmeny. Vplyvom dlhodobej (28 až 63 dňovej) hypodynamie vznikajú morfologické zmeny v bunkách proximálnych a distálnych kanálikov ako aj v bunkách zberacích kanálikov. Štruktúra obličkových teliesok nebola poškodená. Zmeny pokladáme za reverzibilné a pripisujeme ich 9

10 skôr regulačnému pôsobeniu obličiek v rámci adaptácie na hypodynamiu ako poruchám funkcie nefrónov. Pokusy dokázali, že japonské prepelice žijúce v nepriaznivých podmienkach hypodynamie rástli, vyvíjali sa a dosiahli produkčný vek. Práca bola finančne podporená Grantovou agentúrou VEGA 1/3495/06 Literatúra 1. Boďa K.: Kozmická biológia. Gravitačná fyziológia. Vydavateľstvo SAV, Košice, 1984, s Baisch F.J.: Head down tilt combined with breathing assistance by the Iron lung. A new simulation model for cardiovascular deconditioning, skin and kidney function in weightlessness. J. Gravit. Physiol. 2002, 9,1, Cigánková V., Kočišová J., Boďa K., Tomajková E., Dadasheva O. A.: Morphological changes in tissue of the testes and kidneys in Japanese quail exposed to microgravity. Acta Vet., Brno, 1993, 62, Cigánková V., Lenhardt Ľ., Zibrín M., Kočišová J., Tomajková E., Komorová T., Sabo V., Boďa K., Dadasheva O.A., Guryeva T.S.: Histological changes in the adrenal glands of newly hatched Japanese quail chicks during a short stay on the orbital station Mir. Folia Veter., 2001, 45, 1, Suppl., Cigánková V., Zibrín M., Kočišová J., Tomajková E., Sabo V., Boďa K., Dadasheva, O.A., Guryeva, T.S.: Morphological changes in the adrenals of Japanese quail chickens hatched out in microgravity. Aviakosm. Ekolog. Med., 2002, 36, 5, Christensen N.J., Drummer C., Norsk P.: Renal and sympathorenal responses in space. Am. J. Kidney Dis. 2001, 38, 3, Juráni M.., Výboh P., Lamošová D., Barošková Ž., Somogyiová E., Boďa K., Gažo M.: The effect of a 90- day hypodynamy on the neurohumoral system, egg laying and metabolism of proteins in Japanese quail. Physiologist 1983, 26, 6, Juráni M., Boďa K., Výboh P., Zeman M., Lamošová D., Somogyová E., Košťál Ľ., Baumgartner J.: An endocrine response to short-term hypodynamy in Japanese quail selected for resistance to hypodynamy. Physiologist, 1991, 34, Kočišová J., Cigánková V., Marcaník J., Boďa K., Tomajková E., Dadasheva O.A., Sabo V.: Ultrastructural and histochemical changes in tissues of Japanese quails exposed to 7- days microgravity. In: Current Trends in Cosmic Biology and Medicine, (Eds.) Boďa, Baranov, Slovak Academy of Science, Ivanka pri Dunaji, 1991, pp Miyake M., Yamasaki M., Hazama A., Nielsen S., Shimizu T.: Effect of microgravity on organ development of the neonatal rat. Biol. Sci. Space 2004, 18, 3, Miyake M., Yamasaki M., Hazama A., Nielsen S., Shimizu T.: Effect of microgravity on organ development of the neonatal rat. Biol. Sci. Space 2004, 18, 3, Morukov B.V., Noskov V.B., Larina I.M., Natochin I.V.: The water-salt balance and renal function in space flights and in model experiments. Ross. Fiziol. Zh. Sechenova 2003, 89, 3, Norsk P., Christensen N.J., Bie P., Gabrielsen A., Heer M., Drummer C.: Unexpected renal responses in space 2000, Lancet. 356, Norsk P.: Cardiovascular and fluid volume control in humans in space. 2005, Curr. Pharm. Biotechnol., 6, 4, Planel H.: Space and Life. An Introduction to Space Biology and Medicine. Gravity and Weightlessness CRC Press, Boca Raton-London-New York-Washington, D.C., 2004, p Pettis C.R., Drake M., Witten M.L., Truitt J., Braun E., Lindberg K., McNeil G., Hall J.N.: Early renal changes in 45 degrees HDT rats. Acta Astronaut. 2002, 50, 6, Škrobánek P., Hrbatá M., Baranovská M., Juráni M.: Growth of Japanese quail chicks in simulated weightlessness. Acta Vet. (Brno), 2004, 73, Wang T.J., Wade C. E : Renal function of rats in response to 37 days of HDT. J. Gravit. Physiol. 2001, 8, 2, Whitson P.A., Pietrzyk R.A., Morukov B.V., Sams C.F.: The risk of renal stone formation during and after long duration space flight. Nephron 2001, 89, 3, Zerwekh J.E.: Nutrition and renal stone disease in space. Nutrition 2002, 18, 10, Zibrín M., Cigánková V., Kočišová J., Tomajková E., Komorová T., Lenhardt Ľ., Sabo V., Boďa K., Dadasheva O.A., Guryeva T.A.: The structure of some tissues and organs of Japanese quail hatched on the orbital space station Mir. Folia Vet., 2001, 45, Suppl., Zibrín M., Boďa K., Cigánková V., Kočišová J., Tomajková E., Komorová T., Sabo V., Pivko J.: Long term experimental hypodynamy affects the structure of spongy bone and osteoclasts in Japanese quail. Acta Vet. (Brno), 2003, 72,

11 Viera Almášiová, MVDr., Univerzita veterinárskeho lekárstva, Ústav histológie a embryológie, Komenského 73, Košice, THE EFFECT OF INCLUDING FABA BEAN IN THE DIET AND HEN S AGE ON EGGS ALBUMIN QUALITY AL-Sardary Sardar Y. T. - Abas. Kamaran Abduljalil Animal Department, Agricultural College, Salahaddin University- Iraq Abstract The aim of this experiment was to study the influence of field beans (FB) and also birds age on some albumin parameters of commercial layers. Seven different experimental diets (Raw Faba Beans RFB 10%, 20% and 30% compared with Roasted Faba Beans RoFB 10%, 20% and 30%) and both groups compared with commercial layers diet labeled control (C). The experiment was performed in a seven group laying test with 3 replicates, with a total number of 630 birds aged 43 weeks. The control group and six trial groups were enclosed in 3 floor caged batteries. To test the effect of the birds age on the albumin quality, 28 days during the experiment period (P) were observed. For the feeding we used a feed mixture of wheat, barley, soybean meal, RFB or RoFB, vegetable fat with an addition of minerals, vitamins and enzymes. The influence of the diet and ages on Haugh Unit (HU), Albumin Index (AI) and Albumin Percentage (AP) were studied. HU increased significantly with including RFB and RoFB in the diet compared with C. And the values were increased linear with increasing RoFB levels. AI also increased generally, and the values were significant at the level of 10% and 20% for both RFB and RoFB groups compared with C. But was insignificant at the level of 30% for both RFB and RoFB groups. HU decreased with the increasing of the bird s age but AI and AP increased significantly by advanced ages of the bird. Key words: vicia faba, Layers, Haugh Unit, Albumin Index, Albumin Percentage. Introduction The use of legumes as sources of protein for the animal feed industry is expected to increase further in the near future. Rising incomes in the Asian region are increasing the demand for meat products, and hence the requirement for animal feeds. There have been changes in public perception and some unfortunate developments, such as the consequences of mad cow disease (i.e. bovine spongiform encephalopathy or BSE) in UK. This has resulted in many poultry feed industries either choosing to, or being banned from, using 11

12 animal by-products as a source of protein (Farrell, 1997). Single source of plant protein is not comfortable to provide the exact amino acids required for all animals. It is, therefore, preferable to include a range of protein sources in diet formulations, each complementing the other (Hanburya, 2000). The feeding value of the egg and marketing interest depending on the inner egg quality, which are the physical characteristics like Haugh Unit, Albumin index and Yolk index (Card and Nesheim,1971). Eggs inner composition affected by the food, birds age, strain, seasons and storage (Panda,1998). Prochaska et al (1996) observed a significant increase in albumin weight by increasing lysine content of layers diet. Silversides and Scott (2001) pointed out that the diet dos not affected the HU values. But Mateos and Puchal (1982) found that including faba bean in the diet increased significantly (p 0.05) the HU value. In addition the HU value increased with increasing of faba bean content in layers diet. Mateos and Puchal (1982) and Robblee et al (1977) pointed out significant results by consuming diets containing 10%, 20% and 30% of RFB, and HU increased progressively. Also Campbll et al (1980) found an increase in HU in groups consumed diet with 25.5% RFB compared with control. And Mateos and Puchal (1982) observed a significant increase in HU in groups consumed diets containing 55% RFB. But in other experiment Mateos and Puchal (1982) observed that diets containing 4%, 8% and12% RFB has not affected HU value. In general HU value will decrease by increasing birds age ( Austic and Nesheim,1990: Solomn,1997: Lapao et al, 1999: Tharrington, 1999). While AI value increase with advanced ages of the bird. Silversides (1994) demonstrated that total albumin content in the egg increase with the advanced ages of the bird But not significant (Lee and Garlich, 1992: Hussein et al,1993). And the AP decreased significantly (Anderson et al, 1978: Fletcher et al, 1981and 1983: Haq et al,1996: Silversides and Scott, 2001). While Rossi and Pompei (1995) and Al-haweizy (2002) observed a significant increase in AP with increasing birds age. Materials and methods The experiment was carried out within the Erbil poultry project, and the laboratory analysis in the nutrition laboratory of the Agriculture College of the University of Salahaddin. The study comprised the determination of the effects of using 10%, 20% and 30% of RFB and the same levels of RoFB compared with the control group, and the effect of the birds age, 12

13 with commercial Hy-Line W98 layers, on Haugh Unit (HU), Albumin index (AI) and Albumin Percentage (AP) were studied. 630 hens aged 43 weeks were used. The birds were housed in automatic controlled housings in 3 floor caged batteries ( cm) for each cage. And they were distributed randomly into seven groups, each group comprised 90 birds distributed in 18 cages (3 replications 6 cages 5 birds) where feed with different feed mixtures as illustrated in (table 1). The experiment period was 140 days. Diets were fed to birds ad libitum, and at the end of the week remaining diets were collected and weighed to determine the daily feed consumption for each replication. The daily light period was 17 hours with light density 10 lux. Heating temperature was (126 c 130oc) for the toasting of the fb seeds. Chemical analyses of the seeds were carried out according to a.o.a.c Moisture was 7.08%, crude protein was 23.5%, crude fat was 1.0%, crude fiber was 7.72%, and ash was 3.7%. Statistical analyses were carried out by a factorial experiment conducted in c.r.d, with the model equation yijk = µ + ti + pj + eijk. Analyzed results and compared means by the duncan multiple range test (duncan, 1955), on a probability level of 5%. Results and discussion Significant lower value of HU observed in the control group compared to the other groups (table 2). This is could be attributed to the contribution of FB content in the other groups diet. Due to the effect of contribution of FB proteins, which are rich in lysine amino acid ( Prochska et al,1996), and contribute in the structure of the Ovomucin protein, which is responsible on the frim of the albumin (Tossant and Latshow,1999). In the other hand HU value decreased with increasing RFB level in the diet and the opposed with RoFB. This is could be attribute to the effect of the heat treatment, which may be reduce ANFs effects, thus lead to the best utilization of lysine amino acid by the intestine mucosal, and due to the protein degradation. Insignificant depression observed with the increasing of birds ages during the first four periods. And values were significantly higher than P5 (table 3). This is could be attributed to the depression of feed efficiency during the last periods of birds age (Davidson, 1973: Klieste,1976: Scott et al,1982).these results confirm the results obtained by (Canningham et al, 1960: Izat,1985: Silversides and Scott,2001). AI values were higher in all treatments compared to the control (table 1). But T3 and T6 were insignificantly higher than the control. And also the values in T1, T2 and T3 are higher 13

14 insignificantly than T4, T5 and T6. this is attributed to the improving in the utilization of the protein included in the diet due to the low level of ANFs in these diets, but with increasing the content of the FB the AI value will decrease. A relative increase of AI was observed with the increase of bird s age. These results conformable to (Canningham et al,1960: Izat, 1985: Lapao et al, 1999: AL-Haweizy, 2002). AP values in the treatments containing RFB or RoFB were higher than control, and tend to be higher in which treatments contained RoFB in the diet. This is attributed to the improving of utilization of protein content of the diet. AP increased significantly with advanced ages of the bird. This is attributed to the increase of eggs weight (Ahn et al,1997), in addition decreasing of egg yolk (Rose, 1997). Values in P3, P4 and P5 were significantly higher than P1 and P2. This is attributed to the increase of androgen and progesterone hormones with the increase of birds age, which affecting the secretion of the albumin in to the oviduct. Conclusions There is possibility to including the Faba Bean seed as raw or toasted at 10 % level in layers diet to obtain higher quality of table eggs for consumption, and also obtain a safely products by feeding hens, diets without animal protein sources. The age of the hen doesn t affect the eggs albumin quality in generally and tend to be better by advanced ages of the bird particularly during our experiment period. Table 2 The effect of the treatments on Haugh Unit, Albumin Index and Albumin Percentage. Treatments Albumin characters Haugh Unit Albumin Index percentage Control 78.87±0.82 c 41.9±1.7 cd 63.28±0.29 cd T ±0.61 a 48.2±1.5 a 63.17±0.23 cd T ±0.71 a 49.28±1.5 a 64.38±0.22 a T ±091 ab 45.43±1.8 bcd 63.32±0.23 cd T ±0.90 b 46.6±1.7 ab 63.95±0.22 ab T ±0.91 a 45.4±1.8 ac 63.58±0.29 bc T ±0.87 a 45.1±1.9 ad 63.51±0.25 bd 14

15 Table 3 The effect of bird s age on Haugh Unit, Albumin Index and Albumin Percentage. Periods in weeks Albumin Characteristics Haugh unit Albumin index percentage P1 (43-46) 83.57±0.72 a 38.28±1.1 d 61.96±0.21 c P2 (47-50) 82.15±0.73 ac 51.2±1.4 a 62.96±0.27 b P3 (51-54) 82.93±0.71 ab 46.0±1.5 bc 64.11±0.41 a P4 (55-58) 81.53±0.66 ad 46.7±1.2 ac 64.52±0.12 a P5 (59-62) 80.67±0.70 bcd 47.7±1.5 ab 64.35±0.13 a Numbers with small letters means, differences are significant ( P 0.05). Table 1 Compositions %, and calculated chemical analyses of the diets. Treatments Feed staffs % T1 T2 T3 T4 T5 T6 Cont. Broad bean % SBM 44%* Wheat Barley Vegetable Fat Methionine Lysine Limestone Di Cali Pho** Salt [Cx-Layer]*** Avienzyme**** Choline Chloride Minerals***** Calculated Chemical analyses of diets MJ, ME/Kg Diet Crud Protein %

16 Crud Fiber% Calcium % Phosphorus% Lysine Methionine+Cystin% Sodium ME / CP *Soy bean meal ** Di Calcium Phosphate ***Vitamin Complex, K3, E, Biotin, Pantothenic acid, Niacin, Folic acid, B12, B6, B2, B1, D3, A **** Xylanase, β-glucanase ***** Mineral Complex, Cu, Fe, I, Mn, Si VPLYV EXPERIMENTÁLNEHO PODANIA NIKLU NA KVALITU BIELKA A HMOTNOSŤ KONZUMNÝCH VAJEC EFFECT OF EXPERIMENTAL ADMINISTRATION OF NICKEL ON EGG ALBUMEN QUALITY AND EGG WEIGHT OF MARKET EGGS Arpášová, H., Massányi, P., Capcarová, M., Kolesárová, A., Kalafová, A. Slovenská poľnohospodárska univerzita Abstrakt The experiment was implemented on the efficient Isabrown laying hens producing brown shell eggs. We used 53 weeks old hens. The hens were fed by the standard feed mixture HYD 10. Animals accepted fodder ad libitum. Hens (n=20) were divided into four groups (K, E1, E2, E3). Experimental hens (5 in each group) received nickel (NiCl 2 ) per os in drinking water for 28 days. In control group K hens taked drinking water without additions, in experimental groups was supplement in the drinking water nickel (NiCl 2 ) in different concentrations. In experimental group E1 in concentration 0.02g.l -1, in experimental group E2 in concentration 0.2g.l -1 and in experimental group E3 in concentration 2g.l -1. In all groups laying hens accepted drinking water ad libitum. The experiment lasted 28 days. The following efficiency indexes were monitored, namely egg weight, albumen weight, index of albumen, percentage and the Haugh units of the albumen. The greatest differences during experiment were achieved between control group C and experimental group E3 with the highest concentration 2 g.l -1 NiCl 2. Different in values in the other groups with low concentrations NiCl 2 weren t so bold in comparison with control group. The egg weight was in order groups: 16

17 62.27; 59.11; 64.90; 50.97g. Statistically significant differences were achieved among the following combinations of the groups: very high significant difference (P 0.001) between control group and experimental group E3, between experimental groups E3 and E2 respectively E3 and E1; high significant difference (P 0.01) between experimental groups E1 and E2. Different between control group and experimental group E3, between experimental groups E3 and E2 respectively E3 and E1 were statistically very high significant (P 0.001) for albumen weight. Statistically high significant difference (P 0.01) was observed between experimental groups E2 and E1 and significant difference (P 0.05) was observed between control group and experimental group E2. Different between control group and experimental group E2 respectively experimental groups E3 and E2 were statistically significant (P 0.05) for percentage albumen from egg weight. By evaluation of Index albumen and Haugh units were found no significant differences between control and experimental groups (P>0.05). Úvod Stopové prvky ako kadmium, olovo, chróm, nikel, ortuť, arzén majú na živý organizmus toxický efekt a sú často považované za kontaminanty (He et al. 2005). Nikel je toxický prvok, zlúčeniny dvojmocného niklu sú karcinogénne ako pre ľudí tak aj pre zvieratá. (Prokeš et al. 2005). Nikel sa zúčastňuje pri tvorbe krvi a ovplyvňuje hladinu inzulínu v krvi. Najviac ho obsahuje pečeň a perie, je považovaný za slabý karcinogén. Je známe, že môže určitým spôsobom ovplyvniť DNA (Matkar et al. 2006). Výsledky práce Martinez, Diaz (1996) ukázali, že doplnok vysokých dávok Ni a Mn signifikantne zvyšujú obsah hemoglobínu v krvi a tiež ph. Jeho toxická dávka je pomerne vysoká a prejavuje sa nechutenstvom, znížením úžitkovosti a vyššou mortalitou. Toxicita niklu vyvoláva veľký záujem kvôli rozšírenému výskytu Ni v prostredí. Možným vysvetlením ako Ni spôsobuje poškodenie a smrť buniek, je peroxidácia lipidov a tvorba reaktívnych foriem kyslíka. V experimentoch sa už po 1 hodine od aplikácie Ni ľudským lymfocytom zistila zvýšená tvorba hydroxylových radikálov, čo hrá úlohu pri oxidatívnom poškodení ľudských lymfocytov akútnym účinkom niklu (Chang-Yu Chen et al. 2003). Potrava s nízkym obsahom niklu, menej ako 0,1 mg.kg -1 sušiny, vedie k symptómom deficiencie. Na nikel sa môže vytvoriť alergia, vonkajšie vystavovanie sa zliatinám niklu môže spôsobiť zápaly kože dermatitídy. Experiment so štyrmi druhmi zvierat ukázal, že vystavovanie sa niklu vedie k poruchám v metabolizme horčíka a predovšetkým zinku (Anke et al. 1995). Výsledky experimentu autorov Kpomblekou et al. (2002), ktorí skúmali vzorky podstielky brojlerových kurčiat ukázali širokú variabilitu koncentrácie minerálií, pri nikle bol rozsah 0,2-25,1 mg.kg -1. Wilson et al. (2001) skúmali vo svojich pokusoch efekt niklu v dávkach 0; 25; 50; 75; 100 a 17

18 150 mg.kg -1 na pevnosť kostí a úžitkové parametre kohútov broilerového typu ustajnených na hlbokej podstielke a v klietkach. Štandardná kŕmna zmes v tomto experimente bola obohatená o 25 mg niklu na kg krmiva. Nikel nemal významný efekt na živú hmotnosť brojlerových kurčiat v klietkach ani na podstielke, neovplyvnil ani pevnosť holennej kosti u kurčiat, podobne merateľná hustota kostí nebola niklom ovplyvnená. Fakayode et al. (2003) analyzovali obsah ťažkých kovov vo vajciach sliepok. Autori našli silné pozitívne korelácie medzi hladinami kovov v krmive a vo vajciach, priemerné koncentrácie niklu vo vajciach boli 0,03 mg.kg -1. Meluzzi et al. (1996) sledovali transfér ťažkých kovov (Cr, Ni, Pb) do vajca, ťažké kovy boli podávané sliepkam perorálne, krmivom obohateným troma rozdielnymi dávkami, nižšou, zhodnou a vyššou ako je prípustná hranica pre každý kov. Pre nikel boli dávky 100, 300 a 500 ppm ako NiSO 4. Koncentrácie niklu boli v širokom rozpätí, od 71 do 964 ppb pre bielok, ppb pre žĺtok a ppb pre škrupinu. Hladina niklu vo vajciach sa zvyšovala predlžovaním pokusu z 30 na 75 dní. Podiel olova transferovaného do jedlých častí vajca bol v porovnaní s niklom vyšší. Znáška a kvalita vajec boli ťažkými kovmi signifikantne negatívne ovplyvnené. Materiál a metodika Do experimentu bolo zaradených 20 nosníc znáškového hybrida Isabrown vo veku 53 týždňov. Pokusné nosnice boli ustajnené v konvenčnej trojetážovej klietkovej batérii, kŕmené boli ad libitum, kompletnou kŕmnou zmesou pre chov nosníc na produkciu konzumných vajec HYD 10. Sliepky boli rozdelené do 4 skupín, do všetkých skupín boli vybraté sliepky s približne rovnakou živou hmotnosťou. V kontrolnej skupine zvieratá prijímali pitnú vodu bez akýchkoľvek prídavkov, v pokusných skupinách bol do pitnej vody pridaný nikel vo forme NiCl 2 v rôznych koncentráciách. V pokusnej skupine P1 v koncentrácii 0,02 g.l -1, v pokusnej skupine P2 v koncentrácii 0,2 g.l -1 a v pokusnej skupine P3 v koncentrácii 2 g.l -1. Vo všetkých skupinách prijímali sliepky pitnú vodu ad libitum. Sledované ukazovatele : hmotnosť vajec, hmotnosť bielka, percentuálny podiel bielka, index bielka, Haughove jednotky bielka. V závere pokusu bola uskutočnená kompletná analýza vajec, analyzovali sme po 15 vajec z každej skupiny. V skupine P1 s najvyššou koncentráciou niklu bol počet vajec nižší, nakoľko vplyvom vysokej dávky sa u väčšiny sliepok v skupine znáška zastavila, prípadne znášali neštandardné vajcia. Rozdiely medzi jednotlivými ukazovateľmi v rámci pozorovaných skupín sme testovali jednofaktorovou analýzou variancie jednostupňového triedenia. Výsledky jednofaktorovej analýzy variancie boli doplnené Scheffeho testom. Pokus trval 28 dní. 18

19 Výsledky a diskusia Zmeny hmotnosti vajec sú uvedené v tabuľke 1 a obr. 1. Hmotnosť vajec v jednotlivých skupinách sa v priebehu pokusu najvýraznejšie menila v tretej pokusnej skupine P3, v ktorej bol zaznamenaný najvýraznejší pokles hmotností. V kontrolnej skupine K bola hmotnosť 62,27 g, v tretej pokusnej skupine P3 s koncentráciou niklu 2 g.l -1 bol výrazný pokles v porovnaní s kontrolnou skupinou aj s ďalšími pokusnými skupinami, hmotnosť analyzovaných vajec dosiahla len 50,97 g, v pokusnej skupine P2 bola zaznamenaná hmotnosť 64,90 g a v pokusnej skupine P1 bola hmotnosť vajec 59,11 g. Tieto výsledky sa zhodujú s výsledkami autorského kolektívu Meluzzi et al. (1996), ktorí podobne zaznamenali štatisticky významne negatívne ovplyvnenie znášky, hmotnosti vajec aj ich kvality ťažkými kovmi. Pri štatistickom vyhodnotení boli zaevidované veľmi vysoko významné rozdiely (P 0,001) medzi skupinami K : P3; P3 : P2; P3 : P1 a vysoko významné rozdiely (P 0,01) medzi skupinami P1 : P2. Najnižšia hmotnosť bielka (tab. 2, obr. 2) bola v tretej pokusnej skupine P3, čo súvisí s najvýraznejším poklesom hmotnosti celých vyprodukovaných vajec v tejto skupine spôsobeným najvyššou koncentráciou niklu v tejto skupine (2,0 g.l -1 ). Rozdiely hmotností bielkov vajec v ostatných skupinách s nižšími koncentráciami NiCl 2 neboli tak výrazné v porovnaní s kontrolnou skupinou. Pri štatistickom vyhodnotení boli zaevidované veľmi vysoko významné rozdiely (P 0,001) medzi skupinami K : P3; P3 : P2; P3 : P1, vysoko významné rozdiely (P 0,01) medzi skupinami P2 : P1 a významné rozdiely (P 0,05) medzi skupinami P2 : K. V percentuálnom podiele bielka (tab. 2, obr. 3) boli výsledky v poradí skupín: 61,81; 63,15; 64,67; 61,63 %. Ako vyplýva z obrázku a z tabuľkových hodnôt, v percentuálnom podiele bielka z hmotnosti celého vajca boli hodnoty v pokusnej skupine P3 s najvyššou koncentráciou niklu takmer zhodné s kontrolnou skupinou, vyšší percentuálny podiel bielka z hmotnosti celého vajca bol v skupinách P2 (64,67 %) a P1 (63,15%) čomu zodpovedá aj vyhodnotenie štatistickej významnosti rozdielov, ktoré boli významné ((P 0,05) len medzi skupinami P2 : K a P2 : P3. V indexe bielka (tab. 2, obr. 4) boli hodnoty veľmi vyrovnané (86,94; 85,50; 86,78; 84,67), medzi všetkými kombináciami skupín bol rozdiel štatisticky nevýznamný (P>0,05). Podobne v Haughových jednotkách (83,61; 82,02; 81,23; 80,19 HJ) bola najvyššia hodnota v kontrolnej skupine, v ostatných skupinách sa hodnota znižovala so zvyšujúcou sa 19

20 koncentráciou NiCl 2 v skupine. Rozdiel medzi skupinami bo štatisticky nevýznamný (P>0,05) (tab.2, obr. 5). Tabuľka 1 - Zmeny hmotnosti vajec (g) počas experimentu UKAZOVATEĽ SKUPINA K P1(0,02 g.l -1 ) P2 (0,2 g.l -1 ) P3 (2 g.l -1 ) x 62,26 59,11 64,90 50,97* SD 4,85 2,45 3,94 3,03 Hmotnosť vajca CV 7,79 4,14 6,07 5,95 (g) min 55,5 54,0 56,8 47,5 max 70,8 64,1 73,1 56,5 Hmotnosť vajca: *P 0,01 (P1 P2); P 0,001 (P3 K, P1, P2) Obrázok 1 Dynamika zmien hmotnosti vajec 80 hmotnosť vaj ec (g ) K P1 P2 P3 Obrázok 2 Dynamika zmien hmotnosti bielka 50 no sť bielka (g) hmot K P1 P2 P3 20

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