Annals of Warsaw University of Life Sciences – SGGW Science No 52 Warsaw 2013 Contents

BRZOZOWSKI M., STRZEMECKI P. GŁOGOWSKI R., DZIERŻANOWSKA- Estimation the effectiveness of probiot- -GÓRYŃ D., RAK K. The effect of di- ics as a factor infl uencing the results of etary fat source on feed digestibility in fattening rabbits 7 chinchillas (Chinchilla lanigera) 23 DAMAZIAK K., RIEDEL J., MICHAL- GRODZIK M. Changes in glioblastoma CZUK M., KUREK A. Comparison of multiforme ultrastructure after diamond the laying and weight of laying hens nanoparticles treatment. Experimental in two types of cages 13 model in ovo 29 JARMUŁ-PIETRASZCZYK J., GÓR- ŁOJEK J., ŁOJEK A., SOBORSKA J. SKA K., KAMIONEK M., ZAWIT- Effect of classic massage therapy on the KOWSKI J. The occurrence of ento- heart rate of horses working in hippo- mopathogenic fungi in the Chojnowski therapy. Case study 105 Landscape Park in 37 ŁUKASIEWICZ M., MROCZEK- KAMASZEWSKI M., OSTASZEW- -SOSNOWSKA N., WNUK A., KAMA- SKA T. The effect of feeding on ami- SZEWSKI M., ADAMEK D., TARASE- nopeptidase and non-specifi c esterase WICZ L., ŽUFFA P., NIEMIEC J. Histo- activity in the digestive system of pike- logical profi le of breast and leg muscles -perch (Sander lucioperca L.) 49 of Silkies and of slow-growing KNIŻEWSKA W., REKIEL A. Changes Hubbard JA 957 113 in the size of population of the European MADRAS-MAJEWSKA B., OCHNIO L., wild boar Sus scrofa L. in the selected OCHNIO M., ŚCIEGOSZ J. Comparison voivodeships in Poland during the years of components and number of Nosema sp. 2000–2011 59 spores of wintering Carniolan and Italian KOŚLA T., POROWSKA A. The wel- bees debris 121 fare of horses assessed by the investiga- tions of chosen parameters of the stable MAZURKIEWICZ A., TUMIALIS D., microclimate 67 PEZOWICZ E. New sites of the rare Mi- crolepidoptera on Warsaw basin KOŚLA T., POROWSKA A. The welfare 129 of horses assessed by the zoohygienic in- ventory method 77 MAZURKIEWICZ A., TUMIALIS D., PEZOWICZ E., URBAŃSKI J., GA- KUCZYŃSKA B.A., PUPPEL K.S., LEWSKI P., GÓRAL K. The effect of METERA E., SAKOWSKI T., KAPUS- density on the breeding optimization of TA A., BUDZIŃSKI A., GRODZKI H. the tropical house cricket Gryllodes sig- Effect of cow’s breed and feeding season illatus (Walker) (Orthoptera: Gryllidae) on the content of bioactive whey protein of milk produced according to principles 135 of the biodynamic farming 85 MROCZEK-SOSNOWSKA N., BA- KUŹNICKA E., GRONDKOWSKA A. TORSKA M., ŁUKASIEWICZ M., The quality of camel wool held in the WNUK A., SAWOSZ E., JAWOR- Tunisian Sahara Desert 91 SKI S., NIEMIEC J. Effect of nanopar- ticles of copper and copper sulfate ad- ŁOBODZIŃSKA A., GRUSZCZYŃ- ministered in ovo on hematological and SKA J. Canine von Willebrand’s disease biochemical blood markers of – knowledge and awareness among dog chickens 141 breeders and owners in Poland 95 NIEMIEC T., SZMIDT M., KRUK-ROSZ- SZMIDT M., NIEMIEC T., SAWOSZ- KOWSKA A., SAWOSZ-CHWALI- -CHWALIBÓG E., MITURA K. The in- BÓG E., MITURA K. Carbon synthetized fl uence of nanodiamond particles on rat by RF PACVD method enhances the ac- health status 195 tivity of antioxidants 151 WASILEWSKA-NASCIMENTO B. NIŻNIKOWSKI R., CZUB G., GŁO- Occurrence of entomopathogenic fungi WACZ K., ŚWIĄTEK M., ŚLĘ- in soil of Santiago and Fogo islands (Re- ZAK M. Polymorphism of insulin like public of Cape Verde) 203 growth factor IGF-1 in position 211 in WNUK A., MROCZEK-SOSNOW- national sheep breeds with carped wool SKA N., ADAMEK D., KAMASZEW- compared to Polish Merino and Euro- SKI M., ŁUKASIEWICZ M., NIEMIEC pean Mufl on (Ovis aries musimon) 157 J. Effect of rearing system and gender on PEZOWICZ E., TUMIALIS D., MA- histological profi le of breast and ZURKIEWICZ A. Sensitivity of imago leg muscles in hybrid (Cobb×Zk) 211 and larvae of the lesser mealworm Alphi- WNUK A., MROCZEK-SOSNOW- tobius diaperinus (Panzer 1797) in a saw- SKA N., ŁUKASIEWICZ M., BATOR- -dust litter to selected species and strains SKA M., NIEMIEC J. Infl uence of the of Steinernematidae and Heterorhabditi- system of rearing on cholesterol level dae under laboratory conditions 161 and its fraction in blood serum of slow- PRZYSUCHA T., SLÓSARZ J., GO- growing chickens 219 ŁĘBIEWSKI M., KUNOWSKA-SLÓ- WOJCIECHOWSKA I., KAMIO- SARZ M. Comparison of calving course NEK M., MORYTZ B. Studies on the of Limousine purebreds and their cross- potential use of entomopathogenic nem- breeds with Polish Holstein-Friesian atodes for biological control of cows 167 in the stables 227 REKIEL A., WIĘCEK J., PARUCH M., ZDANOWSKA-SĄSIADEK Ż., MI- PTAK J., BLICHARSKI T. Number of CHALCZUK M., RIEDEL J., ŁUKA- piglets born and reared by sows with dif- SIEWICZ M., DAMAZIAK K. Genotype ferent number of mammary teats 173 – factor infl uencing performance of RIEDEL J., MICHALCZUK M., ZDA- chicken production 237 NOWSKA-SĄSIADEK Ż. Assesment of ŻYCZYŃSKI A., NOWAK Z. Compara- slaughter value of three broiler chicken tive behaviour analysis of some colubrids genotypes 179 with reference to suitability of captive SOKOŁOWSKI G., STRZAŁKOW- bred for reintroduction to natural SKA A., ŚWIDEREK W., FISZDON K., habitat 243 GAJEWSKA M. Semen quality param- eters in outbred male mice from four dif- ferent selected lines 187 SERIES EDITORIAL BOARD SERIES EDITORIAL ADVISORY COUNCIL Editor-in-Chief Prof. DSc. Andrzej Chwalibóg () Anna Rekiel Prof. DSc. Konrad Dąbrowski (U.S.A.) Prof. Ewgienij Dobruk (Belarus) Animal Science series Secretary Dr hab. Robert J. Eckert (Poland) Katarzyna Góral-Radziszewska Prof. DSc. Sophie Ermidou-Pollet () Address of Editorial Offi ce Prof. dr hab. Grażyna Garbaczewska (Poland) Wydział Nauk o Zwierzętach SGGW Prof. DSc. Adrian Harrison (Denmark) ul. Ciszewskiego 8, 02-786 Warszawa Prof. dr hab. Jarosław O. Horbańczuk (Poland) Poland Prof. dr hab. Marta Kamionek (Poland) Prof. dr hab. Anna Rekiel (Poland) Prof. DSc. Francois K. Siebrits (R.S.A.) EDITORS Prof. dr hab. Jacek Skomiał (Poland) Statistics editor – Wojciech Hyb Prof. dr hab. Romuald Zabielski (Poland) English language consultant – Natalia Filipczak Polish language consultant – Agata Kropiwiec THEME EDITOR Genetics and animal breeding – Elżbieta Michalska Biology and ecology – Elżbieta Pezowicz Animal nutrition and feedstuffs – Iwona Kosieradzka Behaviour and welfare of animal – Tadeusz Kaleta Animal husbandry and production technology – Justyna Więcek

The Editorial Board (Offi ce) of “Annals of Warsaw University of Life Sciences – SGGW. Animal Science” informs that the printed version of the journal is the original version. Covered in: AGRO, Index Copernicus WARSAW UNIVERSITY OF LIFE SCIENCES PRESS e-mail: [email protected]

ISSN 1898-8830

EDITORIAL STAFF Anna Dołomisiewicz PRINT: Agencja Reklamowo-Wydawnicza A. Grzegorczyk Krystyna Piotrowska www.grzeg.com.pl List of Reviewers 2013

Adamczyk Krzysztof, Batkowska Justyna, Bernacka Henryka, Bielański Paweł, Biesiada-Drzazga Barbara, Bombik Elżbieta, Bombik Teresa, Borczyk Bartosz, Brodacki Antoni, Chwalibóg André, Cieśla Angelika, Czech Anna, Drozd Leszek, Dudek Mariusz, Fliszkiewicz Monika, Gabryszuk Mirosław, Gajewska Marta, Gburzyński Paweł, Giżejewski Zygmunt, Gruszecki Tomasz, Gugołek Andrzej, Janiszewski Paweł, Janocha Alina, Jaworska Magdalena, Kasprzyk Anna, Kowalska Dorota, Kozłowski Krzysztof, Krawczyk Józefa, Łukaszewicz Ewa, Matusevicius Paulius, Matuska-Łyżwa Joanna, Michalska Grażyna, Mitura Stanisław, Nogalski Zenon, Passini Anna, Pezowicz Elżbieta, Popowska-Nowak Elżbieta, Prusak Beata, Sakowski Tomasz, Seremak Beata, Skrzecz Iwona, Smalec Elżbieta, Stachurska Anna, Strachecka Aneta, Sura Piotr, Szczepkowski Mirosław, Szczerbińska Danuta, Szmidt Maciej, Tkaczuk Cezary, Tomek Andrzej, Traczykowski Adam, Wasilewska-Nascimento Beata, Winnicka Anna, Witeska Małgorzata, Wójtowski Jacek

Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 7–11 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Estimation the effectiveness of probiotics as a factor infl uencing the results of fattening rabbits MARIAN BRZOZOWSKI, PAWEŁ STRZEMECKI Department of Animal Breeding and Production, Warsaw University of Life Sciences – SGGW

Abstract: Estimation the effectiveness of probiot- organisms (Fuller 1989, Bielecka et al. ics as a factor infl uencing the results of fattening 2002). There are studies showing posi- rabbits. The aim of this study was to establish the tive effect of probiotics addition as a diet infl uence of addition Bacillus cereus var. toyoi, as probiotics factor, on young rabbits fattening re- supplement in and swine feeding sults. The level of 400 mg per 1 kg of probiotic (Barrow 1992, Jin et al. 1997, Jadamus preparation was used in experimental group (EG, et al. 2000, Jadamus et al. 2002). There n = 34) as factor affecting fattening results. The are also many studies show positive ef- control group (CG, n = 32) was fed commercial fect of probiotics using on young rabbits feed. Experiment started at weaning (35 days) and fi nished at 84 days. The following data were col- productivity – growth results and health lected: body weight at weaning; body weight ev- condition (Gippert et al. 1992, Maertens ery week up to 84 days of age; feed intake during et al. 1994, Kamra et al. 1996, Tachikawa fattening, health status fattened rabbits, dressing et al. 1998, Voros and Voros 1998, Mc percentage. The positive impact of used prepara- Nitt et al. 2000, Kustos et al. 2004, Ker- tion on health status and yield results of fattened fryers was observed. mauner and Struklec 2005, Brzozowski et al. 2007a, Brzozowski et al. 2007b, Key words: rabbits fattening, probiotics factor, Combes et al. 2012). Bacillus cereus var. toyoi The aim of this study was to establish the infl uence of addition Bacillus cereus INTRODUCTION var. toyoi as probiotics factor, on young rabbits fattening results. The digestive process is very com- plex and fragile in rabbits. Young rab- bits are especially exposed to negative MATERIAL AND METHODS impacts of pathogen bacteria. Breed- New Zealand White young rabbits were ers need any factor, that can to prevent used in the study. There were two groups pathogen bacteria growth in digestive of fattening rabbits: control group (CG, tract. Probiotics are the usual bacteria n = 32 kits) and the experimental group that all animals need for their digestive (EG, n = 34 kits). well-being. By probiotics using we can The rabbits from CG were fed by improve the growth and development of standard feed mixture produced by De the normal, desirable microbial popula- Heus Koudijs Hima. EG was fed by the tion in the gut, allowing them to main- same feed with addition Bacillus cereus tain domination over the undesirable var. toyoi, as probiotics factor. The use 8 M. Brzozowski, P. Strzemecki of Bacillus cereus var. toyoi is ideal be- RESULTS AND DISCUSSION cause it has an advantage due to its good heat stability that is important because The body weight changes during fatten- relatively high values of temperature oc- ing are presented in Table 1. cur during pelletizing of animals’ feed. Animals, that were randomized to the The vegetative form of B. toyoi spores experimental group (EG), were heavier quickly germinates in the intestinal tract. than those in the control group (CG). The The rapid germination rate of B. toyoi difference was statistically signifi cant and has been demonstrated in piglets (Thelen was maintained during the fi rst period of and Pallauf 1996). 1 g of used prepara- fattening (up to age of 56 days). The aver- tion contains 1·109 cfu (colony forming age weight of animals at the end of fatten- units) Bacillus cereus var. toyoi. The lev- ing proved to be similar in both groups. el 400 mg of preparation per 1 kg feed Weight gain during the fattening period was used in experimental group. were signifi cantly higher in the control Experiment started at weaning (35 group. This results indicates, that the used days) and fi nished at 84 days. preparation did not have positive impact The following data were collected: to the rabbits growth and body gain. 1. Body weight at weaning and than The fattening results of rabbits are every week up to 84 days of age. presented in Table 2. 2. Surviving rate from weaning to the On rabbit farms, it is estimated that end of fattening. the mortality of young weaned is about 3. Feed intake during fattening. 10–12% (Bielański et al. 2002). In the 4. Dressing percentage. experimental group this result was much The results were statistically evalu- more favorable (3%), which may result ated by analysis of one-way variance us- from the application of probiotics. It was ing SPSS 14.0 PL for Windows (SPSS also observed better health status of the 2006).

TABLE 1. Body weights and gain during fattening (x ±sd) Average body weight (g) Rabbits age (days) Control group Experimental group 35 771a ±120 869.30a ±165 42 1 016.32A ±152 1 138.94A ±180 49 1 218.84a ±229 1 350.73a ±256 56 1 395.68 ±283 1 468.06 ±268 63 1 629 ±278 1 680.76 ±232 70 1 874.64 ±277 1 891.09 ±247 77 2 119.40 ±290 2 125.55 ±259 84 2 387 ±310 2 342.55 ±266 Body gain from 35 to 84 1 616a ±256 1 473.25a ±203 days (g) A – data in rows differ statistically at p < 0.01; a – data in rows differ statistically at p < 0.05. Estimation the effectiveness of probiotics as a factor... 9

TABLE 2. Health status of fattening rabbits in control and experimental groups Estimated groups Items Control Experimental Number of weaned kits 32 (100%) 34 (100%) Number of kits with diarrhea 9 (28.12%) 8 (23.53%) Number of kits, which falls 4 (12.5%) 1 (2.94%) Number of fattened kits 28 (87.5%) 33 (97.06%) animals in the experimental group (less Dressing percentage (yield) was cal- cases of diarrhea). culated according to the formula: The results of feed intake during fat- weight of cooled carcass tening are presented in Table 3. 100 (%) body weight before slaughter TABLE 3. Feed intake Group Control Experimental The results are presented in Table 5.

Average feed intake TABLE 5. The results of average dressing per- during fattening 3.66 ±0.32 3.73 ±0.27 centage of fattening rabbits (kg per 1 kg of gain) Group Control Experimental 51.81a Average dressing (%) 52.94a ±24 There was no impact on the effi cien- ±1.8 cy of the preparation of the feed conver- a – data in rows differ statistically at p < 0.05. sion: in both groups the average feed consumption per 1 kg of gain did not dif- There was observed a higher dress- fer statistically. ing percentage in experimental group at The group of 21 animals in the CG the same body weight in both groups: it and 27 in the EG were slaughtered; for shows the higher share of edible parts in this group of animals were calculated av- the experimental group of rabbits. The erage fi nal mass and dissection’s indica- difference in slaughter effi ciency may tors (Table 4). also result in higher average body weight TABLE 4. Dissection results of fattening rabbits (average for groups, x ±sd) Estimated groups Items Control (n = 21) Experimental (n = 27) Body weight before slaughter (g) 2 298 ±260 2 295 ±258 Weight of the head (g) 152 ±17 146 ±13 Weight of the skin (g) 356 ±53 351 ±58 Weight of the gastrointestinal tract (g) 338 ± 26 334 ±49 Weight of meat offal (g) 94 ±13 92 ±15 Weight of carcass (g) 1 243 ±159 1 258 ±160 Weight of cooled carcass (g) 1 137 ±161 1 157 ±155 10 M. Brzozowski, P. Strzemecki at weaning of young rabbits in the ex- lus cereus var. toyoi (probiotic) use in fat- perimental group. tening of rabbits. Proc. of 15th Int. Symp. of Housing and Diseases of Rabbits, Fur- bearing Animals and Pet Animals, Celle, CONCLUSIONS 103–107. COMBES S., FORTUN-LAMOTHE L., CAUQUIL L., GIDENNE T., 2012: Con- The used preparation, containing Bacil- trolling the rabbit digestive ecosystem to lus cereus var. toyoi as probiotics factor, improve digestive health and effi cacy. improved: Proc. 10th World Rabbit Congress, Sharm – the health status rabbits from experi- El-Sheikh, 475–494. mental group, FULLER R., 1989: Probiotics in man and – the yield results fryers from experi- animals. J. Appl. Bacteriol. 66, 365–378. mental group. GIPPERT T., VIRAG G., NAGY I., 1992: Lacto-Sacc in rabbits nutrition. J. Appl. The used preparation did not infl uenced Rabbit Res. 15, 1101–1104. to: JADAMUS A., VAHJEN W., KUHN I., – fi nal weight of fattened rabbits, 2000: The effect of probiotic toyocerin in – feed intake. fattening poultry. Lohmann Information 23, 3–6. JADAMUS A., VAHJEN W., SCHAFER K., REFERENCES SIMON O., 2002: Infl uence of the pro- biotic strain Bacillus cereus var. toyoi BARROW P., 1992: Probiotics for chickens. on the development of enterobacterial In: Probiotics. Ed. E. Fuller Chapman growth and on selected parameters of and Hall, Dordrecht, 225–257. bacterial metabolism in digesta samples BIELAŃSKI P., NIEDŹWIADEK S., ZA- of piglets. J. Anim. Physiol. Anim. Nutr. JĄC J., 2002: Chów królików. Wyd. Fun- (Berl) 86, 42–54. dacji Rozwój SGGW, Warszawa. JIN L.Z., HO Y.W., ADULLAH N., JALA- BIELECKA M., BIEDRZYCKA E., MAJ- LUDIN S., 1997: Probiotics in poultry: KOWSKA A., WASILEWSKA E., modes of action. World’s Poultry Science ZDUŃCZYK Z., JUŚKIEWICZ J., JĘ- Journal 53, 351–368. DRYCHOWSKI L., WRÓBLEWSKA B., KAMRA D.N., CHAUDHARY L.C., ROTKIEWICZ T., ROTKIEWICZ Z., SINGH R., PATHAK N.N., 1996: In- 2002: Probiotyki, prebiotyki i synbiotyki fl uence of feeding probiotics on growth – wpływ na mikroekosystem przewodu performance and nutrition digestibility in pokarmowego zwierząt zdrowych i infe- rabbits. World Rabbit Sci. 4 (2), 85–88. kowanych bakteriami . Pedia- KERMAUNER A. STRUKLEC M., tria Współczesna 4/1, 94–94. 2005: Effect of feed additive “Kanne BRZOZOWSKI M., ROKICKA A., AN- Fermentgetreide”(FPB) on fattening and TUSZEWICZ W., 2007a: The effect of some digestive parameters of growing th Bacillus cereus var. toyoi (probiotic) rabbits. Proc. 14 Symp. on Housing and on rabbits growth and survivability up Diseases of Rabbits, Furbearing and Pet to weaning. Proc. of 15th Int. Symp. of Animals, Celle, 57–68. Housing and Diseases of Rabbits, Fur- KUSTOS K., KOVACS D., GODOR-SUR- bearing Animals and Pet Animals, Celle, MAN K., EIBEN C., 2004: Effect of pro- 158–162. biotic BioPlus 2B® on performance of th BRZOZOWSKI M., ANTUSZEWICZ W., growing rabbit. Proc. VIII World Rabbit ROKICKA A., 2007b: Results of Bacil- Congress, Puebla, 874–879. Estimation the effectiveness of probiotics as a factor... 11

MAERTENS L., RENTERGHEM Van R., Streszczenie: Ocena efektywności probiotyku GROOTE De G. 1994: Effects of di- jako czynnika poprawiającego wyniki tuczu kró- etary inclusion of Pacifl or® (Bacillus lików. Celem badań było określenie wpływu do- CIP 5832) on the milk composition and datku Bacillus cereus var. toyoi, jako czynnika performances of does and on caecal and probiotycznego, na wyniki tuczu młodych kró- growth parameters of their weanlings. lików. W doświadczeniu zastosowano poziom World Rabbit Sci. 2 (2), 67–73. 400 mg preparatu na 1 kg paszy w żywieniu króli- Mc NITT J., PATTON N.M., LUKE- ków z grupy doświadczalnej (EG, grupa liczyła 32 FAHR S.D., CHEEKE P.R., 2000: Rabbit osobniki). Grupa kontrolna (CG, licząca 34 osob- production. Interstate Publishers, Dan- niki) była żywiona mieszanką pełnoporcjową bez dodatku preparatu. Doświadczenie rozpoczęto ville, Il. u królików w wieku 35 dni wraz z odsadzeniem SPSS, 2006: 14.0 for Windows user’s guide. i zakończono w wieku 84 dni. Zbierano następu- SPSS Inc., USA. jące dane: masa ciała przy odsadzeniu; masa ciała TACHIKAWA T., SEO G., NAKAZAWA M., co tydzień do wieku 84 dni; spożycie paszy pod- SUEYOSHIM., OHISHI T., JOH K., czas tuczu; stan zdrowotny młodych w czasie tu- 1998: Estimation of probiotic by infec- czu, wydajność rzeźna. Stwierdzono pozytywny tion model of infant rabbit with entero- wpływ stosowanego preparatu na stan zdrowotny hemorrhagic Escherichia coli O 157:H7. i wydajność rzeźną. J. Japanese Association Infectious Dis- eases 72 (12), 1300–1305. MS. received November 2013 THELEN U., PALLAUF J., 1996: Effect of Bac. cereus on the composition of the gut fl ora in early weaned piglets. Proc. Soc. Author’s address: Nutr. Physiol. 5, 144. Marian Brzozowski VOROS G., VOROS A., 1998: Effect of Wydział Nauk o Zwierzętach SGGW ToyoCerin® on performance, mortality Katedra Szczegółowej Hodowli Zwierząt and caecum fl ora in growing rabbits (in ul. Ciszewskiego 8, 02-786 Warszawa Hung.). Proc. 10th Rabbit Day, Kaposvar, Poland 81–88. email: [email protected]

Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 13–22 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Comparison of the laying and egg weight of laying hens in two types of cages KRZYSZTOF DAMAZIAK1, JULIA RIEDEL1, MONIKA MICHALCZUK1, ANNA KUREK2 1Department of Poultry Breeding 2Department of Horses Breeding Warsaw University of Life Science – SGGW

Abstract: Comparison of the laying and egg mance after hens moving suggest that the layers weight of laying hens in two types of cages. This adapt more easily and faster to conditions of the C study was aimed at comparing production results type cages. The egg weight was, probably, more of laying hens kept in two types of cages: fur- dependent on general laying performance and age nished cages and conventional cages. It covered of the hens than on cage type. the period from the 36th till the 54th week of hens life that was divided into two experimental stages: Key words: laying hens, types of cage, laying per- stage I – from week 36 till week 44, and stage II formance, egg weight – from week 46 to week 54. Till week 44, 190 layers were reared in groups (10 hens each) in furnished cages (F) and 190 layers were kept in- INTRODUCTION dividually in conventional cages (C). In week 45, the hens from furnished cages (F) were moved to Rearing conditions of laying hens are conventional cages (FC), whereas these from con- recently arising much controversies, ventional cages (C) were randomly merged into groups of 10 hens and transferred to furnished which is due to the implementation of cages (CF). Egg laying (%) and egg weight (g) EC Directive 1999/74/EC that stipu- were controlled as well as percentage contribution lates rearing standards for layers in the of in standard egg weight classes was de- EU Member States (European Commis- termined in both experimental stages. The study sion 1999). This Directive obliges egg showed a signifi cant (P ≤ 0.01) effect of cage type on the laying performance of the hens but only producers to replace traditional battery in the second stage of the study, as well as a sig- cages by new furnished cages with an 2 nifi cant (P ≤ 0.01) effect of hens moving to dif- increased area (750 cm per 1 hen) and ferent cages. In both cases, higher egg laying was possessing additional equipment. With reported for the hens from the conventional cages. no explicit evidence that hens rearing Egg weight in the fi rst and the second stage of the in furnished cages contributes to their experiment was signifi cantly (P ≤ 0.01) higher in the groups housed in the . A higher improved welfare (Barnett et al. 2009, egg weight (P ≤ 0.01) was determined in the lay- Tactacan et al. 2009, Lay et al. 2011), ers in the second stage of the study. Both in the producers had to incur vast expenses to fi rst (P ≤ 0.01) and in the second (P ≤ 0.05) stage, modernize their hen houses according analyses showed a signifi cant effect of cage type to the Directive and in some cases were on the contribution (%) of eggs in particular egg weight classes. A higher percentage of eggs in forced to eliminate their fl ocks. Too lit- the L class was obtained from the hens housed in tle time has gone since the fi nal dead- the furnished cages. Differences in laying perfor- line of cages replacement (1.01.2012) to 14 K. Damaziak et al. conclude on any consequences of these provided by observations of ability changes to production farms that had ad- to adapt to altered rearing conditions. justed their production standards to Di- The aim of this study was to com- rective guidelines as well as to the laying pare two production parameters: laying hens themselves that were the focus of performance and egg weight, of laying interest in this fi ght for “rearing condi- hens reared in conventional cages and tions improvement” between producers furnished cage, as well as to compare the and animal rights defenders. Problems impact of a rapid change in rearing con- in reconciling these two sides may result ditions on these two parameters. from diffi culties in the unequivocal deter- mination of animal welfare (Rodenburg et al. 2008). In the case of laying hens, MATERIAL AND METHODS the level of welfare may be determined The experiment was conducted with based on observations of their behavior two types of three-store cages. Fur- (Appleby and Hughes 1991), changes nished cages (F), with area for 10 hens, in their plumage (Sherwin et al. 2010), equipped according to guidelines of Di- their ability to absorb calcium from feed- rective 1999/74/EC (European Commis- stuff and its further use in the calcifi ca- sion 1999) – Figure 1, and individual tion process (Nasr et al. 2012) as well as cages, i.e. conventional cages (C), ad- the incidence of cannibalism symptoms justed for individual housing of hens, (Gunnarsson et al. 1999). Also produc- with area of 1,196 cm2, height of 44 cm, tion performance may be indicative of and equipped only in one nipple drinker the birds adaptation to rearing conditions. and 26 cm long feeders (Fig. 2). Simultaneously, this performance is of The study included 380 ISA Brown the key signifi cance to producers as it de- hens: 190 layers kept in groups in fur- termines poultry production profi tability nished cages and 190 layers kept individ- (Sosnówka-Czajka et al. 2010). Another ually in conventional cages. The housing important information may as well be

FIGURE 1. Furnished cages for laying ISA Brown hens at the RZD Wilanów-Obory experimental farm, SGGW (photo J. Riedel) Comparison of the laying and egg weight of laying... 15

FIGURE 2. Conventional individual cages for laying ISA Brown hens at the RZD Wilanów-Obory experimental farm, SGGW (photo J. Riedel) conditions (light program, temperature The other housing conditions and feed and air humidity) were consistent with mixture remained unchanged. the ISA Brown Management Guide Observations were continued in two (www.hendrix-genetics.com 2008). All stages (9 weeks each). The fi rst stage birds were receiving the same powdered (from week 36 to week 44) covered the feed mixture in the quantity of 114 g per period when the hens were staying in the hen daily. The nutritive value of the feed same cage as at the beginning of pro- mixture was provided in Table 1. duction, whereas the second stage (from week 46 to week 54) covered the period TABLE 1. Nutritive value of basal diet applied in ISA Brown laying hens since hens transfer to different cages till the end of experiment. The week when Nutritive value Unit Content in diet the cages were changed (45th week) was EMN kcal 2 750.00 not included into any of the stages in EMN MJ 11.60 order to eliminate the impact of direct Total protein % 17.00 stress induced by hens transfer. Crude fi ber % 4.40 Since week 44 to week 54 of hens Crude fat % 3.90 life, laying performance and egg weight were controlled in hens from both types Ash % 12.10 of cages. The percentage of egg lay- ing was calculated on an everyday and In week 45 of hens life, their hous- weekly basis. Eggs were weighed 3 ing conditions were changed as follows: times a week, next day after laying. Data the laying hens from furnished cages (F) achieved enabled calculating: the mean were moved to conventional cages (FC) laying performance (%), the mean egg and housed individually, and the laying weight (g), and the percentage contri- hens from conventional cages (C) were bution of eggs in particular egg weight randomly merged into groups of 10 birds classes (F, C, FC, CF). and transferred to furnished cages (CF). 16 K. Damaziak et al.

The statistical analysis of results was RESULTS AND DISCUSSION carried out using the statistical pack- age SPSS 21.0 (SPSS 2010). Normality Laying performance of parameters distribution was verifi ed The laying performance of ISA Brown with the Kołmogorow-Smirnow test (of hens kept in both types of cages before all parameters examined only egg lay- the change of the housing system (36–44 ing had normal distribution). The effect week of life) was at a similar level of ca. of cage type, hens age and the effect of 89% (Table 2) and slightly lower than the cage change on laying performance was standard values of 91–94% (www.hen- examined with one-way analysis of vari- drix-genetics.com 2008). A signifi cantly ance. Differences in egg laying between higher (P ≤ 0.01) laying performance in groups in particular weeks were deter- group C was determined only in week 2, mined with the T-test. The impact of 5 and 6 of observations (Fig. 3), which cage type on egg weight was estimated was however insignifi cant to the total re- with the Mann-Whitney test, and the im- sult from this period. The higher laying pact of cage change – with the Kruskal- performance of hens kept in conventional -Wallis test. The contribution of eggs in cages compared to furnished cages was particular egg weight classes was com- confi rmed by Glatz and Barnett (1996), pared with the Chi-square test. The dif- whereas Appleby et al. (2002) as well ferences were considered signifi cant at as Guesdon and Faure (2004) achieved P ≤ 0.01 and P ≤ 0.05. The variability of a similar laying percentage in both types the investigated traits was expressed by of cages. Egg laying was found to depend the standard error of the mean (±SE). to the greatest extent on the rapid transfer to a different type of cage. In both groups analyses demonstrated a decrease in lay- ing performance between week 44 and 46 of hens life: by 8.0% in hens moved from group C to group CF, and by 1.0%

TABLE 2. Least squares means (LSM) and SE of the ISA Brown laying hens production (%) depending on cage type Hens age Effect of Laying production (%) (weeks) cage type Conventional cage (C) Furnished cage (F) 36–44 LSM ±SE LSM ±SE NS 89.5 0.4 88.9 0.3 Furnished cage (CF) Conventional cage (FC) 46–54 LSM ±SE LSM ±SE ** 85.7 0.4 90.4 0.5 Effect of cage ** ** × type conversion **difference signifi cant at P ≤ 0.01. Comparison of the laying and egg weight of laying... 17

% F / FC C / CF 95 A A A a 93 A A 91 A A a 89 B A B 87 B B

85 b B 83 b B 81 B B 79

77

75 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 age of hens a, b – difference signifi cant at P ≤ 0.05; A, B – difference signifi cant at P ≤ 0.01. FIGURE 3. Weekly laying production (%) of ISA Brown hens during experimental period. F/FC – hens kept in furnished cage and transferred to conventional ones; C/CF – hens kept in furnished conventional cage and transferred to furnished ones in hens moved from group F to group cage was due to the number of birds hav- FC (Fig. 3). The statistical analysis of ing both direct and visual contact. Hens the entire period after cage shifting dem- kept individually in groups C and FC had onstrated signifi cantly higher (P ≤ 0.01) only visual contact with two neighboring laying performance in FC group com- hens, whereas layers kept in group cag- pared to CF group. Also in the succes- es (F and FC) had a direct contact with sive weeks of the experiment the higher 9 hens housed in the same cage and visu- laying performance was observed in the al contact with 20 hens from neighboring hens from conventional cages – except cages. It may be speculated that the rapid for the 4th and 7th week after the change decrease in laying performance directly of cages (Fig. 1). after hens transfer from C to CF result- The laying performance of hens ed from a hierarchy being established moved from group C to group CF de- in a group of hens that have so far been creased signifi cantly (P ≤ 0.01) reaching kept individually (Fig. 3). Already after the maximum value of barely 88.8% in 4 weeks the performance returned to the week 50 of hens life and the minimal level determined before cage change. value of 81.3% in week 46 of hens life The mean egg production in this period (Fig. 3). Undoubtedly the signifi cant dif- was lower by 3.8% (P ≤ 0.01) compared ference in the laying performance of hens to the fi rst stage of the study (Table 2). after movement to a different type of In contrast, hens moved to cages with 18 K. Damaziak et al. a signifi cantly lesser area adapted to onstrate that hens are capable of recog- new housing conditions as early as af- nizing up to 27 other hens and treating ter a week. It may be speculated that the them as members of their fl ock. A higher diminished egg production was caused number of hens in a group induces stress rather by hens taking out of the cages and and predisposes to continuous fi ghts for their transfer than by the change of rear- dominance. It seems, therefore, that in ing conditions. As reported by Lay et al. our experiment the number of hens and (2011), the DEFRE (Department for En- behavioral interactions between them vironment, Food and Rural Affairs 2006) had a greater impact on changes in their study shows that the method of birds laying performance than the size and catching and taking out of cages affects equipment of cages they were kept in. their blood level of corticosterone. In ad- dition, in the successive weeks of the ex- Egg weight and egg weight classes periment analyses showed in this group Egg weight in the fi rst and second stage higher laying performance compared to of the experiment was signifi cantly the period when these birds were housed higher (P ≤ 0.01) in the groups kept in in the group furnished cages. The laying furnished cages (by 0.3 g and 0.4 g, re- performance of hens moved from group spectively, Table 3). The egg weight was F to group FC was higher after cage shift- also found to be signifi cantly (P ≤ 0.01) ing by 1.5% (P ≤ 0.01; Table 2), reaching affected by the change of cage type. In the maximum value of 92.3% in week 52 both variants of the change, higher egg (Fig. 3). It suggests that the direct contact weight was determined in hens from with other hens may be a more stressful the second stage of the study (Table 3). factor than the reduction of living space. When comparing laying performance Investigations by Douglis (1948) dem- of the investigated hens (Fig. 3) and

TABLE 3. Least squares means (LSM) and SE of the ISA Brown hens’ eggs weight (g) depending on cage type Hens age Effect of cage Eggs weight (g) (weeks) type Conventional cage (C) Furnished cage (F) (n = 4 738) (n = 4 608) 36–44 LSM ±SE LSM ±SE ** 61.0 0.07 61.3 0.07 Furnished cage (CF) Conventional cage (FC) (n = 4 109) (n = 4 669) 46–54 LSM ±SE LSM ±SE ** 62.5 0.08 62.1 0.07 Effect of cage type ** ** conversion × ** ** Effect of hens age (F = 37.86) (F = 15.76) **difference signifi cant at P ≤ 0.01; F – Fischer test. Comparison of the laying and egg weight of laying... 19 egg weight it may be concluded that the and a lower number of small eggs (S, M) higher level of laying performance cor- were determined after cage change. responded to lower weight of eggs, and The tendency for a lesser egg weight that lower egg production corresponded along with a higher egg production has to higher egg weight. A similar tendency been known for years. Even the study of was observed in the contribution of eggs Jully (1930) demonstrates that hens lay- in particular weight classes (Table 4). ing eggs with the weight higher than the Both in the fi rst (P ≤ 0.01) and in the sec- average for the fl ock were characterized ond (P ≤ 0.05) stage of the experiment by lower laying performance than the a signifi cant effect of cage type was hens laying lighter eggs than the average

TABLE 4. Share (%) of ISA Brown hens’ eggs in different weight classes (S, M, L, XL)

Effect Hens age Share of eggs in egg weight classes of cage (weeks) type Conventional cage (C) Furnished cage (F) ** (n = 4 738) (n = 4 608) 36–44 SMLXLSMLXL 3.4% 64.5% 31.0% 1.1% 2.0% 63.3% 33.6% 1.1% Furnished cage (CF) Conventional cage (FC) * (n = 4 109) (n = 4 669) 46–54 SML XL S M LXL 1.6% 59.7% 37.1% 1.6% 1.5% 63.3% 34.0% 1.2% Effect of cage type ** NS × conversion *difference signifi cant at P ≤ 0.05; **difference signifi cant at P ≤ 0.01; NS – difference not signifi - cant. Egg weight classes: S (48–53 g); M (54–63 g); L (64–73 g); XL (>74 g). demonstrated on this parameter. Better for the fl ock. It is diffi cult to conclude results were achieved in the case of hens explicitly whether the decline of laying kept in furnished cages: in the fi rst stage performance induced by stress after hens (F) they were characterized by a higher transfer to a different type of cage was number of large eggs (L) and a fewer due to disorders in the ovulation process number of small eggs (S), whereas in or to simply elongation of egg forma- the second stage (CF) – also by a higher tion period, which normally spans for ca. number of large eggs (L) but also of ex- 24 h (Hiramoto et al. 1990). The longer tra large eggs (XL). In turn, the effect of period of egg formation is usually linked cage change turned out to be signifi cant with its longer retention in the shell gland only in groups C–CF (P ≤ 0.01), where and formation of a thicker shell that may a higher number of large eggs (L, XL) affect its weight (Berg 1945). 20 K. Damaziak et al.

The second factor that could contrib- REFERENCES ute to differences in egg weight in the fi rst and second stage of the study was the age APPLEBY M.C., HUGHES B.O., 1991: of laying hens (Table 3). Baumgartner Welfare of laying hens in cages and alter- et al. (2007) and Zita et al. (2009) demon- native systems: environmental, physical and behavioural aspects. World’s Poult. strated a signifi cant impact of layers age Sci. J. 47, 109–128. on the weight of laid eggs. According to APPLEBY M.C., WALKERA.W., NICOL C.J., the ISA Brown Management Guide, the LINDBERG A.C., FREIRE R., HUGHES mean egg weight in weeks 36–44 of hen B.O., ELSON H.A., 2002: Development life reaches 64.6 g, whereas in weeks of furnished cages for laying hens. Br. 46–54 it accounts for 65.1 g (www.hen- Poult. Sci. 43, 489–500. drix-genetics.com 2008). Though the BARNETT J.L., TAUSON R., DOWNING J.A., JANARDHANA V., LOWENTHAL J.W., weight of eggs determined in our experi- BUTLER K.L., CRONIN G.M., 2009: The ment was lower than the standard values, effects of a perch, dust bath, and nest box, the differences in egg weight between either alone or in combination as used in the subsequent stages of the study could furnished cages, on the welfare of laying therefore result from various age of the hens. Poult. Sci. 88, 456–470. laying hens. BAUMGARTNER J., BENKOVÁ J., PEŚKOVICOVÁ D., 2007: Effect of line, age and individuality on yolk cholesterol content and some other egg quality traits CONCLUSIONS in leghorn type yolk cholesterol selected hens. XVIII European Symposium on the In summary, the type of cage the laying quality of poultry meat and XII European hens were kept in (conventional cages Symposium on the quality of eggs and and furnished cages) had no signifi cant egg products, September 2–5, Prague, effect on their laying performance in the 35–36. fi rst stage of the experiment. Differences BERG L.R., 1945: The relationship of clutch in egg production after birds transfer sug- position and time interval between eggs to gest that the layers were more easily and eggshell quality. Poult. Sci. 24, 555–563. DEFRA, 2006: The welfare effect of differ- faster adapting to conditions of the con- ent methods of depopulation on laying ventional cages, despite a smaller living hens. http://randd.defra.gov.uk/Default. space. The weight of laid eggs was, prob- aspx?Menu&Module=More&Location= ably, more dependent on the laying per- None&Project ID=11925&FromSearch= formance and age of hens than on cage Y&Publisher=1&SearchText=aw0231& type. No sound evidence was achieved SortString=ProjectCode&SortOrder=Ac from results of the analysis of two ex- s&Paging=10#Destription. DOUGLIS M., 1948: Social factors infl uenc- perimental factors (laying performance ing the hierarchies of the domestic hen: and egg weight) to declare the furnished interactions between resident and part- cages as better from the viewpoint of time members of organized fl ock. Physi- hens laying performance and thus cost- ol. Zool. 21, 147–182. -effectiveness of egg production. Comparison of the laying and egg weight of laying... 21

European Commission, 1999: Council Direc- SHERWIN C.M., RICHARDS G.J., NI- tive 1999/74/EC of 19 July 1999 laying COL C.J., 2010: Comparison of the wel- down minimum standards for the protec- fare of layer hens in 4 housing systems in tion of laying hens. Offi cial Journal of the the UK. Br. Poult. Sci. 51, 488–499. European Union L203, 0053–0057. SOSNÓWKA-CZAJKA E., HERBUT E., GLATZ P.C., BARNETT J.L., 1996: Effect SKOROMUCHA I., 2010: Effect of dif- of perches and solid sides on production, ferent housing systems on productivity plumage and foot condition of laying and welfare of laying hens. Ann. Anim. hens housed in conventional cages in nat- Sci. 10, 349–360. urally ventilated shed. Aust. J. Exp. Agr. SPSS, 2010: SPSS, 21.0 for Windows user’s 36, 269–275. guide, 2010. SPSS Inc., USA. GUESDON V., FAURE J.M., 2004: Laying TACTACAN G.B., GUENTER W., LE- performance and egg quality in hens kept WIS N.J., RODRIGUEZ-LECOMPTE in standard or furnished cages. Anim. J.C., HOUSE J.D., 2009: Performance and Res. 53, 45–57. welfare of laying hens in conventional and GUNNARSSON S., KEELING L.J., SVED- enriched cages. Poult. Sci. 88, 698–707. BERG J., 1999: Effect of rearing factors www.hendrix-genetics.com, 2008. Technical on the prevalence of fl oor eggs, cloacal Information, Commercial Layers, ISA cannibalism and in com- Brown. mercial fl ocks of loose housed laying ZITA L., TŮMOVÁ E., ŚTOLC L., 2009: hens. Br. Poult. Sci. 40, 12–18. Effect of genotype, age and their interac- HIRAMOTO K., MURAMATSU T., OKU- tion on egg quality in brown-egg laying MURA J., 1990: Protein synthesis in hens. Acta Vet. BRNO 78, 85–91. tissues and in the whole body of laying hens during egg formation. Poult. Sci. 62, Streszczenie: Porównanie nieśności i masy jaj 264–269. kur nieśnych utrzymywanych w dwóch typach JULLY M.A., 1930: Problems in egg weigh- klatek. W badaniach porównywano wyniki pro- ing in relation to production. Poult. Sci. dukcyjne kur niosek utrzymywanych w dwóch 9, 207–218. typach klatek: klatkach wzbogaconych i klatkach LAY D.C., FULTON R.M., HESTER konwencjonalnych. Badaniami objęto okres od P.Y., KARCHER D.M., KJAER J.B., 36. do 54. tygodnia życia kur podzielony na dwa MENCH J.A., MULLENS B.A., NEW- etapy: I – od 36. do 44. tygodnia, II – od 46. do 54. BERRY R.C., NICOL C.J., O’SULLIVAN tygodnia. Do 44. tygodnia 190 niosek utrzymy- P.O., PORTER R.E., 2011: Hen welfare wano grupowo (po 10 kur) w klatkach wzbogaco- in different housing systems. Poult. Sci. nych (F) i 190 niosek utrzymywano pojedynczo 90, 278–294. w klatkach konwencjonalnych (C). W 45. tygo- NASR M., MURRELL J., WILKINS L.J., dniu nioski z klatek wzbogaconych (F) przenie- NICOL C.J., 2012: The effect of keel siono do klatek konwencjonalnych (FC), a ptaki fractures on egg-production parameters, z klatek konwencjonalnych (C) połączono losowo mobility and behaviour in individual lay- po 10 i wprowadzono do klatek wzbogaconych (CF). W obu etapach badań kontrolowano nieś- ing hens. Anim. Welfare 21, 127–135. ność (%) i masę jaj (g), określono też procentowy RODENBURG T.B., TUYTTENS F.A.M., udział jaj w standardowych klasach wagowych. DE REU K., HERMAN L., ZOONS J., Wykazano istotny wpływ (P ≤ 0,01) typu klatki SONCK B., 2008: Welfare assessment of na nieśność kur, ale tylko w drugim etapie badań, laying hens in furnished cages and non- oraz istotny wpływ (P ≤ 0,01) przeniesienia kur. -cage systems: assimilating expert opin- W obu przypadkach większą nieśność wykazy- ion. Anim. Welfare 17, 355–361. wały kury w klatkach konwencjonalnych. Masa 22 K. Damaziak et al. jaj w pierwszym i drugim etapie doświadczenia MS. received in November 2013 była istotnie większa (P ≤ 0,01) w grupach utrzy- mywanych w klatkach wzbogaconych. Większą masę jaja (P ≤ 0,01) stwierdzono u kur w drugim etapie badań. Zarówno w pierwszym (P ≤ 0,01), jak i w drugim (P ≤ 0,05) etapie badań wykaza- no istotny wpływ typu klatek na udział (%) jaj Authors’ address: w poszczególnych klasach wagowych. Więcej jaj Julia Riedel w klasie L uzyskano u kur utrzymywanych w klat- Wydział Nauk o Zwierzętach SGGW kach wzbogaconych. Różnice w nieśności kur po Katedra Szczegółowej Hodowli Zwierząt przeniesieniu sugerują, że nioski o wiele łatwiej Zakład Hodowli Drobiu i szybciej adaptują się do warunków klatek C. ul. Ciszewskiego 8, 02-786 Warszawa Masa uzyskanych jaj była bardziej zależna od po- Poland ziomu nieśności i wieku kur niż rodzaju klatki. e-mail: [email protected] Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 23–28 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

The effect of dietary fat source on feed digestibility in chinchillas (Chinchilla lanigera) ROBERT GŁOGOWSKI, DANUTA DZIERŻANOWSKA-GÓRYŃ, KRZYSZTOF RAK Department of Animal Breeding and Production, Warsaw University of Life Sciences – SGGW

Abstract: The effect of dietary fat source on feed nous colon and caecum as it was showed digestibility in chinchillas (Chinchilla lanigera). also in other rodents (Langer 2002, Pé- The objective of this study was to determine the rez 2011). The colonic separation mech- effects of the inclusion of the vegetal and animal fat to the diet on the apparent digestibility in chin- anism, leading to the accumulation of chillas. 18 young chinchillas were assigned to microorganisms in caecum, results in the three groups and fed control diet or with the ad- formation of re-ingested caecotrophes dition of either linseed (VF) or lard (AF). The ap- (Holtenius and Björnhag 1985), rich in parent digestibility coeffi cient (ADC) was calcu- microbial derived protein that contribute lated for dry matter (DM), organic matter (OM), crude protein (CP), crude fi bre (CF), nitrogen free to overall nutritional balance (van Zyl extract (NFE) and ether extract (EE). The results and Delport 2010). showed that there was no signifi cant effects of fat Fat supplementation in animal diets is addition on most of the studied constituents ex- usually performed either to increase the cept for increased digestibility of EE. energy value of the feed or to improve Key words: chinchilla, digestion, fat, palatability the nutritional quality of products de- rived from animals (Doreau and Chiliard 1997). Interesting aspects of rabbit die- INTRODUCTION tary fat supplementation were discussed by Casado et al. (2012), who claimed Among companion mammals, small her- that (fi rst) it does not decrease the fi bre bivores constitute a substantial majority. content, resulting in a reduction of pro- One of them, chinchilla (Chinchilla lani- duction costs and (second) improves gera), originating from , feed palatability. The latter is a matter of can be regarded popular, yet scarcely de- great concern for chinchilla housing in scribed in scientifi c literature. The basic captivity due to their rather refi ned pref- specifi cs of its nutrition were reported by erences (unpublished observations). Wolf et al. (2008), who suggested that Little is known about the digestion the crude fi bre level in the chinchilla diet of supranutritional doses of fat in com- should not exceed 15%. Alike in guinea- panion rodents on the contrary to rats, -pigs, crude fi bre is digested more effi - probably the most common laboratory ciently by chinchillas than by rabbits or species (Wang et al. 2011). In the case rats (Sakaguchi 2003). of chinchillas and guinea-pigs, despite The ability to utilize fi brous feed in major resemblances, the considerable chinchillas is attributed to their volumi- 24 R. Głogowski, D. Dzierżanowska-Góryń, K. Rak differences were reported in their meta- (35 g per 1 chinchilla) were thoroughly bolic patterns (Holtenius and Björnhag mixed with the appropriate amount of 1985). Moreover, the nutrients diges- linseed oil. Similar procedure was per- tion in guinea-pigs, as compared to rab- formed for lard, but before mixing it was bits, also showed substantial differences slightly heated in the water bath (Table 2). (Franz et al. 2011). Therefore, regarding Drinking water was constantly avail- the above we found justifi able to study able. the possible effects of dietary addition of TABLE 2. The chemical composition of diets of- animal and vegetal fats on the feed con- fered to control and experimental animals (%) sumption and digestion in chinchillas. Item C VF AF DM 88.58 89.52 89.96 OM 81.50 82.63 82.92 MATERIALS AND METHODS CP 19.35 18.90 18.77 EE 3.58 6.61 6.22 Animal, diets anad mnagement CF 8.73 8.13 8.16 NFE 49.84 48.99 49.77 A total of 18 young chinchillas (±74 days old) were assigned to three groups C – control group; VF – vegetable fat group; AF – animal fat group; DM – dry matter; OM – organic (n = 6) and placed in metabolic cages for matter; CP – crude protein; EE – ether extract; 2 weeks. The environmental conditions CF – crude fi bre; NFE – nitrogen-free extract. were as follows: temperature 18–19°C and humidity 30–35%. Chinchillas in control group (C) were fed commercial Sample collection pelleted chinchilla feed (Table 1), and with the 3% (of feed DM) addition of ei- After 7 days of adaptation period, feces ther vegetable (linseed oil – VF group) and urine were collected daily for next or animal fats (lard – AF group). De- week. The feed and water intake were liberately measured amounts of pellets also recorded.

TABLE 1. Composition of the basal diet Sample analytical determinations Content (per 1 kg Ingredients Content analyses in collected material of feed) were performed by Weenden’s method, Protein 175 g a conventional laboratory procedure. Fibre 120 g Fat 40 g The apparent digestibility coeffi cients Ash 65 g (ADC) of nutrients were calculated as: Lysine 12 g Methionine + Cysteine 10 g intake excretion (g) Metabolizable energy 9.8 MJ ADC = 100 (%) Ca 8.5 g intake (g) P 6.5 g Na 2 g Statistical analysis Vitamin A 12 000 i.u. One-way ANOVA analysis of variance Vitamin D3 1 200 i.u. Vitamin E 72 mg was performed using Statistica 9 soft- Cu 10 mg ware (StatSoft Poland, Cracov). Dif- The effect of dietary fat source on feed digestibility... 25 ferences were considered signifi cant at a diet with quite low content of saturated p < 0.05. animal fat (5%) and signifi cantly lower Data are presented in tables as means than that for a corn oil (Mullen and Mar- ± standard deviation. tin 1990). These results were attribu- ted to the chemical composition of fats added, suggesting differential alterations RESULTS AND DISCUSSION in membrane composition and cellular function possibly occurring in central The addition of fat had an effect of the nervous system (e.g. brain). feed intake in chinchillas (Table 3). One plausible explanation of the re- Compared to control, animals offered versed relation between feed and wa- diet supplemented with linseed oil con- ter consumption in both experimental sumed lower amounts of feed and animal groups can be of a behavioral type. Pos- fat supplementation caused higher intake sibly, increased water drinking compen- in chinchillas. sated the decrease in feed ingestion as Interestingly, inverse effects were it was proposed by Wolf et al. (2008). noted for water intake. Animals receiv- Considering numerous reports on strong ing pellets with linseed oil drank sig- preference for diets supplemented with nifi cantly more water than those, who saturated/solid fats in rats, the claim that consumed lard enriched diet. However it the addition of linseed oil decreased the should be noted, that the water intake in palatability of feed in chinchillas is jus- VF group was substantially higher also tifi able (Mullen and Martin 1990, Wang than that in control group, which possi- et al. 2011). bly indicates that the dietary addition of The addition of linseed oil signifi - linseed oil had stronger effect on animals cantly increased water intake in chinchil- than that of lard. las, compared to C and AF groups. How- Wolf et al. (2003) reported higher ever, our results are more similar to those amounts of feed and water intake for recorded for animals fed diet mixed of chinchillas fed complete (pelleted) diet, native components (Wolf et al. 2003). In- but it has to be noted that the feed com- terestingly, chinchillas fed hay-only and position in our study was apparently dif- complete feed diets showed substantially ferent. Thus, neither the feed’s quality higher water intake. The most striking nor palatability effects on the intake can- difference was an enormously high wa- not be dismissed. In captive chinchillas ter consumption reported for chinchillas nutrition, the quality of the complete dry fed fresh grass. To our knowledge it is feed is essential. Poor quality of pelleted extremely unusual to offer fresh forage feed often implicates digestive disorders (grass) for captive chinchillas. The ex- and low palatability may cause starva- tensive feeding of greens and fresh fruits tion in chinchillas (unpublished observa- to chinchillas was reported a cause of tions). bloat, serious digestive system disease Early studies on rats showed that the (Richardson 2003). Therefore there is preference for diets with high level of a need for a gradual and sparingly serv- beef tallow (34%) was similar to that for ing of fresh feedstuffs in chinchillas. 26 R. Głogowski, D. Dzierżanowska-Góryń, K. Rak

The rearing conditions like tempera- chinchillas – 70.9 vs. 71.13 respectively ture and humidity are crucial not only (Sakaguchi et al. 1987). However, higher for the well-being but also for the repro- ADC values were recently reported for duction of chinchillas (Richardson 2003, greater cane rat, related to chinchillas Busso et al. 2012). Since such data are as well as to guinea-pigs (van Zyl and missing in Wolf et al. (2003) paper, the Delport 2010). It was suggested that the conception that above mentioned dis- coprophagy signifi cantly contributed to crepancies occurred due to the effect cane rats ability to utilize high fi brous of environmental factors cannot be dis- food. Although in our study we did not missed. measure coprophagy, it’s impact on There were no signifi cant differences ADC in chinchillas cannot be dismissed, in the amount of excreted feces observed regardless of dietary fat level or source. in our study but the volume of urea in In the present study we did not ob- VF group was signifi cantly higher than serve signifi cant differences in OM di- that in control, which likely refl ects the gestibility between groups. Overall value increased intake of water (Table 3). of ADC for OM in chinchillas resembled The DM digestibility coeffi cients that, reported for rabbits fed diets with of all diets were similar (Table 4). Our the intermediate level of fi bre (Gidenne results confi rmed that feeds with high et al. 2000). Interestingly, Sakaguchi DM content are relatively well digested et al. (1987) described lower OM digest- by chinchillas. Compared to other small ibility in guinea-pigs, rabbits, rats and rodent species, only guinea-pigs show hamsters fed diet containing different similarly high rate of DM digestion as levels of CP and CF than those used in

TABLE 3. Daily feed and water consumption with feces and urine excretion (g) Item C VF AF Feed intake 22.95 ±1.95a 21.35 ±1.99b 23.04 ±3.65 Feces excretion 11.30 ±3.70 10.81 ±2.31 11.71 ±2.86 Water intake 21.67 ±4.50a 26.90 ±8.44b 21.50 ±6.62 Urea excretion 12.33 ±4.48 a 17.57 ±6.56b 13.31 ±5.51 a–b – difference signifi cant at p < 0.05. C – control group; VF – vegetable fat group; AF – animal fat group.

TABLE 4. ADC of nutrients (%) Item C VF AF DM 71.13 ±4.87 71.02 ±2.86 71.20 ±2.65 OM 73.79 ±4.60 74.01 ±2.54 73.91 ±2.38 CP 71.18 ±9.87 70.85 ±7.22 69.49 ±6.59 CF 37.55 ±9.58 32.87 ±6.14 33.56 ±5.80 NFE 80.38 ±2.70 79.68 ±1.45 80.08 ±1.77 EE 84.51 ±5.19a 91.66 ±1.96b 90.80 ±1.55b a–b – difference signifi cant at p < 0.05. C – control group; VF – vegetable fat group; AF – animal fat group; DM – dry matter; OM – organic matter; CP – crude protein; CF – crude fi bre; NFE – nitrogen-free extract; EE – ether extract. The effect of dietary fat source on feed digestibility... 27 our study (196 and 126 vs. 175 and 120 g gestive effi ciency, regardless of the type per 1 kg of CP/CF, respectively). On of added fat (Table 4). It seems likely that the other hand, in guinea-pigs and rab- the the heating of lard just before adding bits fed hay only diet – 72 g per 1 kg of it to the feed, made it more accessible to CP (Franz et al. 2011), the digestibility the digestive enzymes, therefore improv- of OM was even lower than observed in ing its absorption (Wang et al. 2011). chinchillas. Therefore it seems reason- able to elucidate the differences in OM digestion in chinchillas with the effect of CONCLUSIONS the dietary CP level, a dependency con- The study showed that the addition of fat fi rmed by Rogier (1971). to chinchilla diet had a moderate effect The dietary addition of fat did not al- on the apparent digestion of most of the ter the CP digestibility. Interestingly, the constituents. Taken together with the feed ADC for protein previously reported for intake results it may be suggested, that the other rodent species (rat, hamster) was differences in chemical composition of substantially higher (Sakaguchi et al. dietary fats may contribute to their effects 1987). However, our results are in ac- on diet preference and consequently have cordance with those reported by Rogier an infl uence on the intake of protein and (1971), suggesting that typical CP di- carbohydrates in chinchillas. gestibility in chinchillas, regardless of coprophagy level, is about 70%. The digestibility of CF was lower in REFERENCES experimental groups as compared to con- trol. This effect can be most likely attrib- BUSSO J.M., PONZIO M.F., FIOL DE CU- NEO M., RUIZ R.D., 2012: Reproduc- uted to the detrimental effect of high fat tion in chinchilla (Chinchilla lanigera): diet on the number of cellulolytic bacte- Current status of environmental control ria, previously reported for ruminants as of gonadal activity and advances in re- well as for non-ruminants (Doreau and productive techniques. Theriogenology Chiliard 1997). 78, 1–11. Differences in NFE digestion, ob- CASADO C., MOYA V.J., FERNÁNDEZ C., served in our study, were negligible. The PASCUAL J.J., BLAS E., CERVERA C., apparent digestion of NFE in chinchillas 2012: Diet digestibility in growing rab- bits: effect of origin and oxidation level was similar to that, recorded for guinea- of dietary fat and vitamin E supplementa- -pigs and other non-ruminant species tion. World Rabbit Sci. 18, 57–63. (horses, ponies and rabbits) fed alfalfa- DOREAU M., CHILIARD Y., 1997: Diges- -grain diet in digestibility comparison tion and metabolism of dietary fat in farm trial (Slade and Hintz 1969). animals. Br. J. Nutr. 78, 15–35. The signifi cant effect of dietary fat ad- FRANZ R., KREUZER M., HUMMEL J., dition on digestibility was found in EE. HATT J.-M., CLAUSS M., 2011: Intake, selection, digesta retention, digestion and Chinchillas in both experimental groups gut fi ll of two coprophageous species, rab- digested EE more effectively than those bits (Oryctolagus cuniculus) and guinea in control. Noticeably, the animals in VF pigs (Cavia porcellus), on a hay-only diet. and AF groups revealed similar EE di- J. Anim. Phys. Anim. Nutr. 95, 564–570. 28 R. Głogowski, D. Dzierżanowska-Góryń, K. Rak

GIDENNE T., PINHEIRO V., FALCĂO E., WANG Y., CORWIN R., JARAMILLO D.P., CUNHA L., 2000: A comprehensive WOJNICKI F.J., COUPLAND J.N., approach of the rabbit digestion: conse- 2011: Acceptability and digestibility of quences of a reduction in dietary fi bre emulsions in a rat model: effects of sol- supply. Livest. Prod. Sci. 64, 225–237. id fat content and lipid type. J. Am. Oil HOLTENIUS K., BJÖRNHAG G., 1985: Chem. Soc. 88, 235–241. The colonic separation mechanism in WOLF P., SCHRÖDER A., WENGER A., the guinea-pig (Cavia porcellus) and the KAMPHUES J., 2003: The nutrition of chinchilla (Chinchilla laniger). Comp. the chinchilla as a companion animal Biochem. Physiol. 82A, 537–542. – basic data, infl uences and dependences. LANGER P., 2002: The digestive tract and J. Anim. Phys. Anim. Nutr. 87, 129–133. life history of small mammals. Mamm. WOLF P., BUCHER L., ZUMBROCK B., Rev. 32, 107–131. KAMPHUES J., 2008: Daten zur Was- MULLEN B.J., MARTIN J.R., 1990: Mac- seraufnahme bei Kleinsäugern und de- ronutrient selection in rats: effect of fat ren Bedeutung für die Heimtierhaltung. type and level. J. Nutr. 120, 1418–1425. Kleintierpraxis 53, 217–223. PÉREZ W., VAZQUEZ N., JERBI H., 2011: Gross anatomy of the intestine and their Streszczenie: Wpływ dodatku tłuszczu na straw- peritoneal folds in the chinchilla (Chin- ność paszy u szynszyli. Celem badań było okre- chilla lanigera). J. Morph. Sci. 28, ślenie wpływu dodatku tłuszczu roślinnego 180–183. i zwierzęcego na współczynnik strawności pozor- RICHARDSON V.C.G., 2003: Diseases of nej u szynszyli. 18 młodych osobników przypo- small domestic rodents. 2nd Ed., Black- rządkowano do trzech grup żywieniowych, które well Publishing, Oxford. otrzymywały paszę podstawową (grupa kontrol- ROGIER J.C., 1971: Effi ciency of protein na) bądź wzbogaconą o dodatek oleju lnianego utilization by growing chinchilla fed two (VF) lub łoju (AF). Poziom strawności pozornej levels of protein. MSc thesis, University (ADC) oznaczono dla suchej masy (DM), materii of British Columbia. organicznej (OM), białka surowego (CP), włókna SAKAGUCHI E., 2003: Digestive strategies surowego (CF), związków bezazotowych wycią- of small hindgut fermenters. Anim. Sci. J. gowych (NFE) oraz ekstraktu eterowego (EE). 74, 327–337. Wykazano brak istotnych różnic w wynikach SAKAGUCHI E., ITOH H., UCHIDA S., oceny strawności pomiędzy grupami, z wyjąt- HORIGOME T., 1987: Comparison of kiem EE. fi bre digestion and digesta retention time between rabbits, guinea-pigs, rats and MS. received in November 2013 hamsters. British J. Nutr. 58, 149–158. SLADE L.M., HINTZ H.F., 1969: Compari- son of digestion in horses, ponies, rab- bits and guinea-pigs. J. Anim. Sci. 28, 842–843. Authors’ address: Van ZYL A., DELPORT J.H., 2010: Digest- Robert Głogowski ibility of nutrients and aspects of the di- Wydział Nauk o Zwierzętach SGGW gestive physiology of the greater cane Katedra Szczegółowej Hodowli Zwierząt rat, Thryonomys swinderianus in two ul. Ciszewskiego 8, 02-786 Warszawa, Poland seasons. Afr. Zool. 45, 254–264. e-mail: [email protected] Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 29–35 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Changes in glioblastoma multiforme ultrastructure after diamond nanoparticles treatment. Experimental model in ovo MARTA GRODZIK Department of Animal Nutrition and Feed Science, Warsaw University of Life Sciences – SGGW

Abstract: Changes in glioblastoma multiforme general lack of the successful therapy. ultrastructure after diamond nanoparticles treat- Therefore, it is important to identify po- ment. Experimental model in ovo. Glioblastoma tential drugs and explore more effi cient multiforme (GBM) is the most common primary malignancy in the brain and confers a uniformly therapeutic strategies for the treatment poor prognosis. Despite decades of research on of malignant gliomas (Sathornsumetee the topic, limited progress has been made to im- and Rich 2008, Jain 2013). Animal mod- prove the poor survival associated with this dis- els for cancer experiments, such as chick ease, new therapeutic strategies are still needed. embryo chorioallantoic membrane, are The application of nanotechnology to disease treatment, diagnosis, monitoring, drug delivery successful and powerful tools to inves- platform and to the control of biological systems tigate therapeutic aspects of glioma (an- is promising, also in cancer therapy. Diamond na- giogenesis, metastasis) that cannot be noparticles (DN) are bioactive substance toward studied in 2D cell culture systems (Stroj- glioma tumour cultured on the chicken embryo nik et al. 2010). chorioallantoic membrane (CAM). DN reduce tumor mass and volume and inhibited new blood Increasing usage of nanomaterials vessel development in glioma tumor. In the pres- (also nanoparticles) in the past decades, ent experiment we additionally observed, that DN for various biological and medical ap- caused changes in the tumor ultrastructure testify plications (such as imaging, diagnosis, to the ongoing process of cell death, probably car- therapy and drug delivery), shows that ried out by autophagy. nanotechnology is the important tool for Key words: autophagy, diamond nanoparticles, developing modern life sciences (Roco glioma, in ovo culture, transmission electron mi- 2003). The unique potential of nanopar- croscope ticles is due to their size in nanoscale and physicochemical properties that allow INTRODUCTION to overcome the limitation of using the bulk materials of the same composition Malignant glioma cells like glioblastoma as a traditional therapeutic and diagnos- multiforme (GBM) have rapid growth tic agents (Zhang et al. 2008). Among rate and an aggressive nature. This can- various nanoparticles, diamond nano- cer is the one of the most common brain particles (DN) are the one with promis- tumors and is known for the relatively ing prospects in applications that require high morbidity. The short time progno- optical transparency, chemical inertness, sis for patients with GBM is associated hardness and low cytotoxicity toward with intratumoral heterogeneity on the living organism. Moreover, DN are the genomic and cytopathologic level and most bioactive nanoparticles among all 30 M. Grodzik the allotrope forms of carbon, including of nanoparticles were determined by C60, fullerenes, carbon black, and sin- JEM-2000EX transmission electron mi- gle and multi-walled carbon nanotubes croscope (TEM) at 200 kV (JEOL Ltd., (Huang et al. 2007, Liu et al. 2007) also Tokyo, ). Figure 1 shows the image towards gliomas. Diamond nanoparti- of the diamond nanoparticles from TEM. cles reduced tumor mass and volume The nanoparticles were 4–5 nm size and and inhibited new blood vessel develop- rounded shape. Zeta potential of nano- ment in GBM tumors cultured in ovo. It diamond hydrocolloid was examined was observed that DN signifi cantly de- with Zetasizer Nano-ZS90 (Malvern In- creased expression of angiogenic factors: struments Ltd., Malvern, UK) and was FGF-2 and VEGF (Grodzik et al. 2011). measured as –39 mV. To prevent parti- The effect of diamond nanoparticles on cles aggregation, soniphication was per- morphology of glioblastoma multiforme formed before every application. cells in tumor is still unclear. The objec- tive of the investigation was to evaluate Cells and chicken embryos changes in glioma tumor cells ultrastruc- U87MG glioblastoma multiforme ture after DN treatment being the effect (GBM) cells (HTB-14; American Type of metabolic changes in the cell. Culture Collection, Manassas, VA) were maintained in Dulbecco’s modifi ed Eagle MATERIALS AND METHODS medium (Sigma-Aldrich Corporation, St Louis, MO) with addition of 10% fetal Diamond nanoparticles bovine serum (Sigma-Aldrich) and 1% Antibiotic Antimycotic Solution (Sigma- Diamond nanoparticles (DN) were ob- -Aldrich). tained from SkySpring Nanomaterials The fertilized eggs (Gallus gallus) (Houston, TX, USA). In the experiment, were supplied by a commercial, local concentration 500 μg per 1 ml was used. (Marylka, Poland). The strains Physicochemical characteristics of DN used depending on availability, included were performed. The shape and the size various crosses among Ross, Cobb, and

FIGURE 1. TEM image of diamond nanoparticles. Scale bar – 100 nm Changes in glioblastoma multiforme ultrastructure after diamond... 31

Hubbard, kept for 4 days at 12°C. Chick- baked for another 24 h. The blocks were en embryos were incubated at 37°C with cut into ultrathin sections (50–80 nm) us- 60–70% of relative humidity in incuba- ing an ultramicrotome (LKB Ultratome tor (PHU Walenski, Gostyn, Poland). III, ) and transferred onto cop- Embryonic day 0 (E0) was designated as per grids, 200 mesh (Agar Scientifi c Ltd. the day when the eggs were placed into GB). Subsequently, the sections were the incubator. contrasted using uranyl acetate (uranyl acetate dehydrate, puriss. p.a., ACS rea- Glioma tumor culture gent, ≥98.0% (T) Fluka, Sigma-Aldrich) The eggs were divided into two groups and lead citrate (lead (II) citrate tribasic (2×40 eggs): group I (control) and group trihydrate – purum, for electron micros- II (tumor treatment diamond nanopar- copy, Sigma-Aldrich). The morphologi- ticles). The GMB culture suspension cal structure of each GBM was inspected (3–4 ·106 cells in 30 μl cell culture me- using a JEM-1220 transmission electron dium) was injected inside the silicon ring microscope (TEM) at 80 keV (JOEL, Ja- placed onto the CAM at day E6. Dia- pan) coupled with a digital camera (Mo- mond nanoparticles were injected into rada) and Olympus Soft Imaging Solu- the tumor (form II group) after 7 days tions software (Olymus, ). from start of the culture. The chicken em- bryos were killed by decapitation at day RESULTS AND DISCUSSION E18. The GBM tumors that grew inside or near the silicone ring were resectioned Diamond nanoparticles reduce mass, and passed for further investigations. volume and number of vessels in glio- blastoma multiforme tumor cultured Transmission Electron Microscopy on chicken’s embryo CAM (Grodzik The tumors were cut into pieces of about et al. 2011). In the present experiment we 1 mm3 and fi xed in the 2.5% solution of additionally observed, that DN caused glutaraldehyde (grade I, 25% in H2O, changes in the tumor ultrastructure. The purifi ed for use as an electron micros- electron microscopy images from both copy fi xative, Sigma-Aldrich) in 0.1 M groups (control group and group treated phosphate buffer (pH 6.9). Then, the with diamond nanoparticles) showed samples were rinsed in the same buffer a typical ultrastructure of the glioma and transferred to the 1% solution of tumors with glioblastoma multiforme osmium tetroxide (Sigma-Aldrich) in cells, epithelium cells and erythrocytes 0.1 M phosphate buffer (pH 6.9) for 1 h. (Fig. 2). In the control group (Fig. 2 Subsequently, the samples were rinsed in A, B, C; Fig. 3 A, B, C) glioblastoma distilled water, dehydrated in the ethanol multiforme cells with cell structures gradient and impregnated with epoxy (nucleus, mitochondria, Golgi appara- embedding resin (Epoxy – Embedding tus, rough endoplasmic reticulum with Kit, Fluka, Sigma-Aldrich). The next day, ribosomes, transport vesicles) were vis- the samples were embedded in the same ible. Cancer cells have characteristic and resin and baked for 24 h at 36°C. Then, unique metabolism, that is an adapta- the blocks were transferred to 60°C and tion to their microenvironment (low pH, 32 M. Grodzik

FIGURE 2. Glioblastoma multiforme ultrastructure from control group (A, B, C) and after diamond nanoparticles treatment (D, E, F). Scale bar: A, B, D, E – 5 μm, C, F – 2 μm; GC – glioblastoma multiforme cell, RBC – red blood cells, L – lumen of vessel, EC – endothelial cell, N – nucleus, A – autophagosome hypoxia, anaerobic glycolysis, intensive structure of eukaryotic cells, where lipid cell divisions). Well-developed rough synthesis, protein folding, and protein endoplasmic reticulum (ER) and numer- maturation take place. ER is the major ous secretory and endocytotic vesicles signal-transducing organelle that senses prove high secretory activity of GBM and responds to changes of homeostasis cells. Endoplasmic reticulum (ER) is the (Baumann and Walz 2001). In general, Changes in glioblastoma multiforme ultrastructure after diamond... 33

FIGURE 3. Glioblastoma multiforme ultrastructure from control group (A, B, C) and after diamond nanoparticles treatment (D, E, F). Scale bar: A, D – 5 μm, B, E – 2 μm, C, F – 500 nm; GC – glioblas- toma multiforme cell, RBC – red blood cells, L – lumen of vessel, EC – endothelial cell, N – nucleus, M – mitochondrion, ER – rough endoplasmic reticulum, E – endocytosis intense or persistent ER stress induces varying width and depth, transport vesi- apoptosis or autophagy, resulting in cell cles). Endocytosis is essential for all the death. Also the structure of glioma cells cells to internalize nutrients, antigens, proves intensive cellular metabolism. pathogens and cell surface receptors, Structures characteristic for endocytosis from the plasma membrane into mem- can be observed (membrane cavities of brane-bounded, endocytic vesicles, to 34 M. Grodzik regulate cell homeostasis, cell signal- and collapsed, vascular endothelial cells ing and development. There are multiple have lost their shape and elasticity, in- pathways for endocytic uptake into cell, side degenerated mitochondria, deprived depending on the size and kind of the of cristae, and numerous autophagocytic transported compound or substance, they vesicles (autophagosomes) can be seen. can be divided into 4 classes: clathrin- Changes visualized in glioblastoma -mediated endocytosis, caveolae, mac- multiforme ultrastructure testify to the ropinocytosis, and phagocytosis (Do- ongoing process of cell death, probably herty and McMahon 2009). Very impor- carried out by autophagy. Autophagy, tant and well visible cell structures are also known as programmed cell death the mitochondria, which key role is the type II, can be induced by various cel- energy metabolism and regulation of cell lular stress mediated signaling pathways death (Wen et al. 2013). The mitochon- involved in nutrient signaling, growth dria of glioblastoma mutliforme cells in factor status, energy sensing, hypoxia, control group have clearly defi ned folds oxidative and ER stress. The role of au- in the inner membrane, named cristae. tophagy is complicated and may have In the group treated with diamond na- diametrically opposite consequences for noparticles, despite the cells that can be the tumor cell, that is important for the identifi ed are the same as in the control regulation of cancer development and group, the morphology of these cells is maintain, as well as for the response of different (Fig. 2 D, E, F; Fig. 3 D, E, F). tumor cells to anticancer therapy (Liu First of all, the large spaces between the et al. 2013). Reduction of the number of few deformed cells of glioblastoma mul- GBM cells, and a strong degeneration of tiforme are visible. Primary tumor cells those which remained alive suggest that that previously existed in the extracel- autophagy activated in these cells leads lular matrix probably infl uenced by ND to their death. underwent cell death. Glioma cells have irregular shapes; look like they lost their elasticity and rigidity. Inside the cell, cell CONCLUSIONS structures have also a different morphol- ogy comparing to the control group. The Diamond nanoparticles administration to number of organelles essential for proper glioblastoma multiforme tumor grown metabolism is decreased. Additionally, on the CAM, not only inhibit the de- in the mitochondria crests were degen- velopment of the blood vessels but also erated, endoplasmic reticulum is less affect the metabolism of cancer cells. visible (smaller network of cisterns and Ultrastructure of the glioma cells after ribosomes), endocytosis is practically treatment with ND clearly suggests au- stopped. In the ultrastructural image of tophagy leading to the cell death. How- these cells appeared vesicles character- ever, the mechanism of initiation of this istic for autophagy – highly conserved process is still unclear. The results ob- cellular homeostatic process. Changes tained encourage further research aimed can be observed in the structure of blood at the anti-cancer application of diamond vessels as well. Their lumen is irregular nanoparticles. Changes in glioblastoma multiforme ultrastructure after diamond... 35

Acknowledgment STROJNIK T., KAVALAR R., BARONE T.A., PLUNKETT R.J., 2010: Experimental This work was supported by grants model and immunohistochemical com- from the National Science Centre (NCN parison of U87 human glioblastoma cell NN311540840). xenografts on the chicken chorioallantoic membrane and in rat brains. Anticancer Res. 30, 4851–860. REFERENCES WEN S., ZHU D., HUANG P., 2013: Target- ing cancer cell mitochondria as a thera- BAUMANN O.,WALZ B., 2001: Endoplas- peutic approach. Future Med. Chem. 5, mic reticulum of animal cells and its or- 53–67. ganization into structural and functional ZHANG L., GU F.X., CHAN J.M., domains. Int. Rev. Cytol. 205, 149–214. WANG A.Z., LANGER R.S., DOHERTY G.J., McMAHON H.T., 2009: FAROKHZAD O.C., 2008: Nanoparticles Mechanisms of endocytosis. Annu. Rev. in medicine: therapeutic applications and Biochem. 78, 857–902. developments. Clin. Pharmacol. Ther. 83, GRODZIK M., SAWOSZ E., WIERZBI- 761–769. CKI M., ORLOWSKI P., HOTOWY A., Streszczenie: Zmiany w ultrastrukturze glioblasto- NIEMIEC T., SZMIDT M., MITURA K., ma multiforme po zastosowaniu nanocząstek dia- CHWALIBÓG A., 2011: Nanoparticles mentu. Badania modelowe in ovo. Glioblastoma of carbon allotropes inhibit glioblastoma multiforme (GBM) jest najczęściej występującym multiforme angiogenesis in ovo. Int. J. złośliwym nowotworem pierwotnym mózgu o bar- Nanomedicine. 6, 3041–3048. dzo złych rokowaniach. Pomimo dekad lat badań HUANG H., PIERSTORFF E., OSAWA E., na tym problemem, niewielki postęp został uczy- HO D., 2007: Active nanodiamond hy- niony aby wydłużyć życie chorym, nowe strategie drogels for chemotherapeutic delivery. terapeutyczne są nadal poszukiwane. Zastosowanie Nano. Lett. 7, 3305. nanotechnologii w leczeniu chorób, diagnostyce, JAIN R.K., 2013: Normalizing tumor mi- monitornigu, platformach dostarczania substancji croenvironment to treat cancer: bench to aktywnych i kontroli systemów biologicznych daje bedside to biomarkers. J. Clin. Oncol. 31, nadzieję na poprawę aktualnej sytuacji, również 2205–2218. w terapii nowotworów. Nanocząstki diamentu LIU K.K., CHENG C.L., CHANG C.C., (DN) są bioaktywnymi substancjami w stosunku do guza mózgu hodowanego na błonie kosmówko- CHAO J.I., 2007: Biocompatible and wo-omoczniowej zarodka kury. DN redukuje masę detectable carboxylated nanodiamond i objętość guza oraz hamuje rozwój nowych naczyń on human cell. Nanotechnology 18, krwionośnych (angiogenezę). W prezentowanym 325102. doświadczeniu dodatkowo zaobserwowano zmia- LIU W.T., HUANG C.Y., LU I.C., ny w ultrastrukturze komórek guza pod wpływem GEAN P.W., 2013: Inhibition of glio- działania nanocząstek diamentu, które świadczą ma growth by minocycline is mediated o zachodzących procesach śmierci komórkowej, through endoplasmic reticulum stress- prawdopodobnie na drodze autofagii. induced apoptosis and autophagic cell death. Neuro. Oncol. 15, 1127–1141. MS. received in November 213 ROCO M.C., 2003: Nanotechnology: conver- Author’s address: gence with modern biology and medicine. Marta Grodzik Curr. Opin. Biotechnol. 14, 337–346. Wydział Nauk o Zwierzętach SGGW SATHORNSUMETEE S., RICH J.N., 2008: Katedra Żywienia Zwierząt i Gospodarki Designer therapies for glioblastoma multi- Paszowej forme Ann. NY Acad. Sci. 1142, 108–132. ul. Ciszewskiego 8, 02-786 Warszawa, Poland e-mail: [email protected]

Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 37–47 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

The occurrence of entomopathogenic fungi in the Chojnowski Landscape Park in Poland JOANNA JARMUŁ-PIETRASZCZYK1, KAMILA GÓRSKA1, MARTA KAMIONEK1, JAN ZAWITKOWSKI2 1Department of Animal Environment Biology 2Department of Environmental Improvement Warsaw University of Life Sciences – SGGW

Abstract: The occurrence of entomopathogenic INTRODUCTION fungi in the Chojnowski Landscape Park in Po- land. The study was aimed at estimating species Entomopathogenic fungi focussed re- composition and abundance of entomopathogenic searchers’ attention already in the 19th fungi in the Chojnowski Landscape park. The ef- century. Now, they are used in a small fect of site, season (spring, summer, autumn) and scale in organic farming in Western temperature on the frequency of isolation of ento- mopathogenic fungi was also analysed. The effect , USA, in the counties of Latin of the fi rst two factors was estimated based on the America and in Australia. The fungi analysis of soil samples taken from meadow 1, proved useful in replacing chemical in- forest 1 and orchard in spring, summer and au- secticides, particularly when controlling tumn 2010. Three species of entomopathogenic pests which move vertically in soil ac- fungi (Beauveria bassiana, Metarhizium aniso- pliae and Isaria fumosorosea) were isolated in cording to ground water changes and the the study area. Site and temperature affected the range of the root zone. Most promoted frequency of isolation of particular species. On are the bio-preparations based on local meadow 1 and in orchard M. anisopliae appeared strains of microorganisms. The use of to be the dominating species while forest 1 was imported ecotypes developed under dif- dominated by B. bassiana. From among meadow and forest sites sampled in the autumn, forest 3 ferent selective factors (e.g. temperature (nature reserve) was characterised by the highest ranges) does not often bring expected re- density of entomopathogenic fungi. M. anisopliae sults (Mierzejewska 2001). and B. bassiana were most often isolated from Entomopathogenic fungi are an en- meadow sites while B. bassiana and I. fumosoro- vironmental-friendly alternative to plant sea – from forest sites. B. bassiana and I. fumoso- rosea infected with signifi cantly higher protection chemicals. They are used to frequency at 20°C than at 25°C. control harmful insects and arachnids (mainly mites, Acarina) which make Key words: entomopathogenic fungi, biology, bio- insecticides, Beauveria bassiana, Metarhizium losses in agricultural and forest crops anisopliae, Isaria fumosorosea, the Chojnowski and transfer microorganisms that are Landscape Park pathogenic to humans, animals and plants (Bałazy 2004). Biological con- trol of pests does not result in the ac- quired resistance in contrast with the use of chemical pesticides (Paruch and 38 J. Jarmuł-Pietraszczyk et al.

Janowicz 2004). Moreover, fungal bio- 1993) used also to isolated entomopath- -preparations do not need obeying the ogenic nematodes. The method consists waiting periods or special protective in placing in soil some live insects e.g. measures (Mierzejewska 2001). caterpillars of various butterfl y species It is extremely important to create ap- which are attacked by isolated patho- propriate habitat conditions (e.g. refuges) gens. Soil with trap insects is incubated to enable survival of species susceptible in the lab at appropriate temperature to man-made changes in the landscape. (Górny and Grüm 1993). Such approach would allow for main- Caterpillars of the greater wax taining possibly richest gene pool of (Galleria mellonella L.) from own cul- potential entomopathogenic organisms ture of the Department of Zoology, War- (Bałazy 2004, Quesada et al. 2007). saw University of Life Sciences were The Chojnowski Landscape Park has used as trap insects in this study. Soil great landscape and natural values. It is samples were collected in 2010 from vari- part of the so-called green ring surround- ous meadow and forest habitats and from ing the city agglomeration of Warsaw. an orchard situated in the Chojnowski The park is localised on light sandy or Landscape Park (Masovian Province). sandy-loamy soils of relatively high fer- Sampling sites were characterised by the tility which supports many forest and following properties (names of sites are bog-meadow communities. Apart from further used throughout the text): rich fl ora, the park provides also favour- • meadow 1 – situated in the village able conditions for the development of Łoś near the Jeziorka River on peat abundant fauna (Walczak et al. 2001). soils and bordering forest 1, Practically no chemical control meas- • meadow 2 – wet meadow growing on ures are applied to protect tree stands in alluvial and peat soils situated by the forest areas of the park. This is favour- Jeziorka River in the village Gołków, able for the development of soil meso- • meadow 3 – several hundred me- fauna including insects and their natural tres SW of Wólka Pęcherska. Single enemies like entomopathogenic nema- young pine, and trees grow todes and fungi. Forest entomopatho- on this untypical meadow situated in genic fungi fi nd convenient conditions the close vicinity of forest 3, for the growth and development in for- • forest 1 – mixed forest (pine, oak, est litter and on the soil surface. For the birch, small-leaved linden and other year round they have a chance to contact tree species) situated in the village their hosts permanently dwelling forest Prace Duże. Soil samples were taken bottom or penetrating soil for pupation from sites covered by brown soils, or wintering (Tkaczuk 2008). • forest 2 – mixed forest (pine, oak, birch, small-leaved linden and other tree species) situated in the village MATERIAL AND METHODS Bogatki. Soil samples were taken Entomopathogenic fungi were isolated from sites covered by brown and from soil with the method of trap insects rusty soils, (Zimmerman, 1986 in Górny and Grüm The occurrence of entomopathogenic fungi... 39

• forest 3 – nature reserve Biele Choj- which soil samples were taken in autumn nowskie, soil samples were taken from 2010. Results obtained from the analysis sited overgrown by mixed coniferous of soil samples taken in spring and sum- forest Querco roboris-Pinetum (old- mer (forest 1, meadow 1 and orchard) growth forest of Scotch pine with a and in autumn (meadow 1, meadow 2, small admixture of the common oak meadow 3, forest 1, forest 2, forest 3 and and the common birch). Soil samples orchard) served for testing the effect of were taken from the forest outskirts, temperature on the frequency of isola- • orchard – a young apple orchard situ- tion of particular species of entomopath- ated on brown soils in a village. ogenic fungi. Obtained results were sta- Mixed soil samples were taken from tistically processed with the Statgraphics studied habitats with the Egner’s cane. Plus 4.1 software using simple and mul- Soil from each site was placed in a plastic ti-parametric ANOVA. Tukey’s test was container (V = 250 cm3) and 10 larvae of used to compare the means at the signifi - the greater wax moth (G. mellonella L.) cance level of α = 0.05. were added to each container. Caterpil- lars with soil were incubated at 20 and 25°C. Experiments were made in tripli- RESULTS AND DISCUSSION cate. In total, entomopathogenic fungi infected 508 trap larvae out of 1,753 Polish soils are characterised by a great isolated dead insects during the whole abundance of entomopathogenic fungi experiment. and, as shown in studies by Tkaczuk The fi rst observation of larval mortal- (2008), by rather diverse species com- ity was made after 4 days of contact of position. This author found at least two caterpillars with soil, the next were made species of entomopathogenic fungi in every 3 days until the 40th day of experi- more than 80% of soil and litter sam- ment. Dead insects were removed and ples from various habitats in Poland (one containers with soil were supplemented species in 18.2% samples, two species with live caterpillars. Dead insects when in almost 40% samples, three species soft suggested infection by nematodes in 34.3% samples and four or more spe- while tough insects with the symptoms cies in 7.9% samples). For comparison, of mummifi cation were superfi cially in more than 40% of soil samples from sterilised with disinfectant 1% sodium abroad the author found the presence of hydroside solution and rinsed in dis- only one species of entomopathogenic tilled water. Individuals with a visible fungi, in more than 30% samples – two mycelium did not undergo sterilisation. species and in 22.5% and 5.6% samples Then, the caterpillars were transferred – the presence of three and four or more to Petri dishes lined with fi lter paper to species, respectively. In each of these ex- achieve sporifi cation of fungi necessary amples the respective fi gures are smaller for their determination. than those from Polish soils. The effect of habitat was determined Despite the fact that entomopatho- for 6 sites (meadow 1, meadow 2, mead- genic mitosporic fungi are widespread, ow 3, forest 1, forest 2, forest 3), from especially in the soil habitat, the 40 J. Jarmuł-Pietraszczyk et al. knowledge of the factors affecting their occurrence, population structure, mecha- nisms of their persistence and virulence against potential hosts is still scarce. Defi nitely better studied in this aspect are fungi of the order Entomophthorales (Tkaczuk 2008). Three species of entomopathogenic fungi: B. bassiana, M. anisopliae and I. fumosorosea were found in studied sites. They were isolated from 96, 77 and 73% of collected soil samples, respec- tively. B. bassiana was the most often isolated species. It was present in every site in a given season. M. anisopliae was not found in summer in forest 1 and I. fumosorosea was absent from meadow 1 in spring and from meadow 1 and mea- dow 2 in autumn (Table 1). Spring Common occurrence of these fungi in Summer soils from various country habitats was confi rmed in many studies (Miętkiewski et al. 1991, 1991–1992 and 1998, Miętkiewski and Kloczarek 1995, Bajan and Kmitowa 1997, Marjańska-Cichoń et al. 2005, Sapieha-Waszkiewicz et al. 2006). Clear dominance of mentioned species in Polish soils, without the dis- tinction of particular habitats, was also noted by Tkaczuk (2008). According to this author, the species less frequently recorded in Polish soils are I. farinosa, OrchardOrchard Meadow 1 Meadow 1 Forest 1 Forest 1 L. lecanii and M. fl avoviride which were not found in the study sites. Entomopathogenic fungi caused 3.033.03 8.33 11.116.06 5.68 7.07* 13.89 20.69 3.45 9.97 5.56 50.00 6.90 13.12 26.72 2.78 12.90 0.00 4.84 18.18 6.82 15.54 1.61 3.41* 9.09 5.35 20°C 25°C mean 20°C20°C 25°C 25°C mean mean 20°C 20°C 25°C 25°C mean mean 20°C 25°C mean death of 15 to 53% (mean 29%) of trap 12.1218.18 0.00 19.44 6.06 18.81 0.00 24.14 55.56 0.00 39.85 0.00 25.81 12.90 34.09 9.09 29.95 11.00 caterpillars depending on site and sea- son. The lowest mortality caused by these microorganisms was found in soil taken from forest 1 in summer and the highest – in that taken from for- est 3 in autumn. A high fungal activity was also observed in soil sampled from Mortality factor M. anisopliae I. fumosorosea fungi Total Other factors Mortality factor B. bassiana TABLE 1. Mortality of trap insects placed in soil relation to season and site (%) TABLE B. bassiana 7.14 2.63 4.89 3.80 3.45 3.62 15.89 6.90 11.39 M. anisopliae 11.22 6.58 8.90 31.65 29.31 30.48* 0.00 0.00 0.00 I. fumosorosea 9.18 2.63 5.91 10.13 1.72 5.93 3.74 3.45 3.59 Total fungi 27.55 11.84 19.70 45.57 34.48 40.03 19.63 10.34 14.99 Other factors 5.10 11.84 8.47 10.13 18.97 14.55 3.74 8.62 6.18 Autumn Mortality factor Orchard Meadow 1 Forest 1 20°C 25°C mean 20°C 25°C mean 20°C 25°C mean B. bassiana 4.82 0.00 2.41 18.68 2.94 10.81 7.59 21.74 14.67 M. anisopliae 19.28 14.44 16.86* 13.19 11.76 12.48 0.00 6.52 3.26 I. fumosorosea 13.25 2.22 7.74 0.00 0.00 0.00 12.66 26.09 19.37* Total fungi 37.35 16.67 27.01 31.87 14.71 23.29 20.25 54.35 37.30 Other factors 3.61 2.22 2.92 2.20 1.47 1.83 2.53 2.17 2.35 Autumn Mortality factor Meadow 2 Meadow 3 Forest 2 Forest 3 20°C 25°C mean 20°C 25°C mean 20°C 25°C mean 20°C 25°C mean B. bassiana 47.92 1.59 24.75* 25.00 21.05 23.03 6.36 6.52 6.44 20.00 44.59 32.30* M. anisopliae 6.25 3.17 4.71 4.69 7.89 6.29 0.00 6.52 3.26 0.00 4.05 2.03 I. fumosorosea 0.00 0.00 0.00 10.94 26.32 18.63* 7.27 6.52 6.90 30.67 6.76 18.71 Total fungi 54.17 4.76 29.46 40.63 55.26 47.94 13.64 19.57 16.60 50.67 55.41 53.04 Other factors 4.17 11.11 7.64 3.13 2.63 2.88 0.00 2.17 1.09 1.33 8.11 4.72 *Tukey’s test α < 0.05. 42 J. Jarmuł-Pietraszczyk et al. meadow 1 in summer and in soil from lars in meadow 2 than in forest habitats meadow 3 taken in autumn (Table 1). but showed much smaller activity there Most trap larvae infected by ento- as compared with that in meadow 1. mopathogenic nematodes, which caused Miętkiewski et al. (1991–1992 and death of from 45 to 79% of the larvae of 1998) also reported abundant occur- G. mellonella, were found in soil taken rence of B. bassiana in meadow soil but from meadow 1, forest 1 and orchard in M. anisopliae was the dominating spe- spring, summer and autumn. In soil tak- cies in their studies. B. bassiana is known en from meadow 2, meadow 3 and for- to grow better in habitats rich in organic est 2 entomopathogenic nematodes were substances which is probably associated also the reason of the highest mortality with its ability to develop in the sapro- of trap larvae (from 49 to 82%). Only phytic phase. Maybe this was the reason in soil samples taken from forest 3 the of the dominating role of B. bassiana in number of G. mellonella larvae infected infecting trap insects from soil of mead- by nematodes (42%) was lower than ow 2 since the meadow was situated on those infected by entomopathogenic fun- river alluvia and peat soils. gi (53% – Table 1). The death of caterpil- M. anisopliae showed relatively high lars was also caused by other factors like activity in soil sampled from orchard be- saprophytic fungi, mites, non-fruiting ing the dominating species there. The mycelium, bacteria or other unidentifi ed prevalence of M. anisopliae compared factors. with other species could result from its Not all species of entomopathogenic resistance to agricultural factors like fungi are equally characteristic for vari- plant protection chemicals, mineral fer- ous ecosystems – forests, meadows, crop- tilisation, intensive soil cultivation (Tka- lands and other (Bajan and Kmitowa czuk 2008). Some studies (Janowicz 1997, Miętkiewski et al. 1998, Tkaczuk et al. 2004, Paruch et al. 2004) indicate, 2008). As shown by Tkaczuk (2008), however, quite opposite that M. anisop- M. anisopliae is the dominating species liae is a species particularly sensitive to in the country meadow and pasture soils. plant protection chemicals. The species According to other studies (Miętkiewski can best stand the absence of potential et al. 1991–1992 and 1998), M. anisop- hosts from among other species of ento- liae predominated over other species of mopathogenic fungi. Its conidia are able fungi in meadow soil. This species ap- to survive on a primary host for a long peared also characteristic for such type period of time (Miętkiewski 1992). of soils in the Chojnowski Landscape Three species of fungi: M. anisop- Park, especially for meadow 1, where it liae, I. fumosorosea and B. bassiana dominated in every season and was the were isolated from the soil from orchard. reason of remarkable mortality of trap The presence of the same species of en- insects. tomopathogenic fungi in orchard soils B. bassiana defi nitely dominated was noted by many authors (Marjańska- in meadow 2, from where soil samples -Cichoń et al. 2003, 2005, Janowicz et al. were taken only in autumn. M. anisopliae 2004, Paruch et al. 2004). Paruch et al. caused higher mortality of trap caterpil- (2004) reported higher mortality of cat- The occurrence of entomopathogenic fungi... 43 erpillars caused by fungi from orchards other species of fungi was noted by without chemical protection measures Miętkiewski et al. (1992). The authors compared with the soil from a control found also an abundant presence of orchard that was subjected to chemical B. bassiana. In my study B. bassiana protection and nursing practices. As seen was much less frequent but the second from their studies, frequent application numerous species after M. anisopliae of plant protection chemicals reduced the was I. fumosorosea, the species not re- populations of benefi cial, entomopatho- corded by Miętkiewski et al. (1992). genic soil fungi. Studies carried out by Miętkiewski et al. Pesticides were used moderately in (1992) differed from mine in that they the studied orchard. Observed occur- compared the occurrence of entomopath- rence of entomopathogenic fungi, main- ogenic fungi in soils from herbicide fal- ly of M. anisopliae and I. fumosorosea, low and orchard sward. They found that differed from that presented by Paruch M. anisopliae infected larvae more of- et al. (2004). In the studied orchard ten in soils from herbicide fallow than M. anisopliae was dominating and its in soils from orchard sward, B. bassiana activity was highest in autumn. In the showed the reverse pattern. control orchard of Paruch et al. (2004), Two species of entomopathogenic M. anisopliae was not found while in fungi: B. bassiana and I. fumosorosea orchards, where pesticides were not dominated in studied forest habitats. used, the species was most active only in In each of the three studied forests, spring. I. fumosorosea dominated in or- M. anisopliae was less frequent. Ac- chards studied by Paruch et al. (2004). cording to Tkaczuk (2008), the domi- Statistical analysis showed signifi cant nating species in Poland, in both forest site-specifi c occurrence of I. fumoroso- soil and litter, is B. bassiana. Similarily sea. This fungus was isolated more fre- Głowacka and Świeżyńska (1993) and quently from forest than from meadow Bajan et al. (1995) pointed to B. bassi- habitats. The least frequent in studied ana as the most frequent species in for- sites was M. anisopliae. This species est habitats. The species dominated in was isolated more often from meadow studied forest 1 and forest 3, from which and orchard than from forest habitats. In soil samples were taken only in autumn. autumn it dominated in meadow 1 and I. fumosorosea was also numerous in in spring and summer – in orchard (Ta- studied forests being the most frequently ble 1). In the soil from meadow 1, the isolated species in forest 1 and 2 in au- mean contribution of M. anisopliae to all tumn. As shown by Tkaczuk (2008) I. fa- entomopathogenic fungi isolated there rinosa, I. fumosorosea and M. anisopliae was 66% while in the soil from orchard are almost equally frequent in soil-litter it amounted 50%. The frequency of iso- samples from various forests in Poland lation of M. anisopliae in forest 1 was and their abundance is nearly equal. In signifi cantly lower than that in meadow my studies I. farinosa was not found at and orchard. all and I. fumosorosea was isolated more Numerous occurrence of M. anisop- often than M. anisopliae. liae in orchard soil as compared with 44 J. Jarmuł-Pietraszczyk et al.

Studied habitats were characterised of trap larvae by M. anisopliae at dif- by different abundance of entomopatho- ferent values of temperature. The spe- genic fungi. More of these microorgan- cies was slightly more frequent at 25°C. isms were found in meadow 1 than in Miętkiewski et al. (1994) obtained dif- orchard and forest 1. Various factors like ferent results in this aspect. They con- favourable location (close to the river fi rmed lower thermal requirements of and forest) that provided appropriate I. fumosorosea but found that B. bassiana soil moisture and enhanced the develop- infected most often at 25°C. However, in ment of diverse fl ora and fauna could be the study by Sapieha-Waszkiewicz et al. the reason of observed pattern. Greatest (2006) B. bassiana and I. fumosorosea richness of meadow soil in entomopath- infected trap larvae more frequently at ogenic fungi compared with orchard soil 20°C than at 25°C, similarly as they did might be also associated with sporadic in my experiments. Both species domi- application of pesticides and less inten- nated also at lower (18°C) but not at sive cultivation. higher (28°C) temperature in the study From among meadow and forest of Tkaczuk and Miętkiewski (1996). habitats sampled in autumn particularly This phenomenon may be explained by great abundance of entomopathogenic a marked differentiation of features of the fungi was noted in the nature reserve strains originating from various habitats. – forest 3. This confi rms earlier fi ndings M. anisopliae infected more trap lar- (Miętkiewski et al. 1991–1992, Bałazy vae at 25°C but the difference was not 2006, Tkaczuk 2008) that more diverse statistically signifi cant. High thermal re- ecosystems with richer fl ora and fauna quirements of this species were also ob- (including entomofauna) are more attrac- served by various authors (Miętkiewski tive for entomopathogenic fungi, which et al. 1993 and 1994, Tkaczuk and may fi nd their potential hosts there. Miętkiewski 1996, Sapieha-Waszkie- In general, a greater activity of en- wicz et al. 2006). tomopathogenic fungi is observed in Entomopathogenic fungi have a great autumn due to the translocation of in- biocoenotic value being a factor con- sects to soil for wintering. Insects dy- trolling population density of insects. ing of mycosis substantially enrich the Therefore, some species may be used for soil in the infection material (Tkaczuk biological control of plant pests. That is 2008). The number of conidia produced why the understanding of species com- by B. bassiana on insects in the for- position and ecology of this group of est litter in autumn varies from 109 to fungi and protection of their habitats is 1,010 m2 while in late spring and sum- so important. Protected forests, nature mer it is 107–108 m2 (Bałazy 2006). reserves and national parks should play Temperature at which trap insects a role of shelters and refuges for these contacted the soil signifi cantly affected fungi (Bałazy 1981). Semi-natural habi- the activity of B. bassiana and I. fumoso- tats play similar role for the maintenance rosea. Both species infected more lar- and species diversity of entomopatho- vae at 20°C than at 25°C. No signifi cant genic fungi in agricultural landscapes differences were noted in the infection (Tkaczuk 2008). The occurrence of entomopathogenic fungi... 45

CONCLUSIONS BAJAN C., KMITOWA K., 1997: Thirty years studies on entomopathogenic fungi 1. The following species of ento- in the institute of ecology, PAS. Polish mopathogenic fungi were isolated in Ecological Studies 23, 3–4: 133–154. BAJAN C., KMITOWA K., MIERZEJEW- the study area: SKA E., POPOWSKA-NOWAK E., – Beauveria bassiana, MIĘTKIEWSKI R., GÓRSKI R., MIĘT- – Metarhizium anisopliae, KIEWSKA Z., GŁOWACKA B., 1995: – Isaria fumosorosea. Występowanie grzybów owadobójczych 2. Habitat and temperature affected the w ściółce i glebie borów sosnowych frequency of isolation of particular w gradiencie skażenia środowiska leśne- species of entomopathogenic fungi. go. Prace IBL, Seria B, 24: 87–97. Such a relationship was not found for GŁOWACKA B., ŚWIEŻYŃSKA H., 1993: Grzyby owadobójcze występujące na season. owadach leśnych. Prace IBL, Seria A, 3. B. bassiana and I. fumosorosea in- 767: 117–136. fected insects signifi cantly more of- GÓRNY M., GRÜM L., 1993: Methods in ten at 20°C than at 25°C. soil zoology. PWN, Warszawa: 188–196. 4. The species most often isolated JANOWICZ K., DZIĘGIELEWSKA M., in autumn were: B. bassiana and KAUP G., PARUCH K., 2004: Wybrane M. anisopliae in meadow habitats mikroorganizmy glebowe w biologicz- nym zwalczaniu szkodników roślin. Fo- and B. bassiana and I. fumosorosea lia Universitatis Agriculturae Stetinensis in forest habitats. 234(93): 131–138. MARJAŃSKA-CICHOŃ B., MIĘTKIEW- Acknowledgements SKI R., SAPIEHA-WASZKIEWICZ A., 2003: Występowanie grzybów owado- Thanks are due to Directors of the Choj- bójczych w glebie z sadów jabłoniowych nowski Landscape Park for a permit to o odmianach wrażliwych na parcha i par- take soil samples at the outskirts of the choodpornych. Postępy w Ochronie Ro- Bile Chojnowskie Nature Reserve and ślin 43(2): 799–802. from other parts of the park. MARJAŃSKA-CICHOŃ B., MIĘTKIEW- SKI R., SAPIEHA-WASZKIEWICZ A., 2005: Występowanie i skład gatunko- REFERENCES wy grzybów owadobójczych w glebach z sadów jabłoniowych. Acta Agrobotani- BAŁAZY S., 1981: Ściółka leśna ostoją grzy- ca 58(1): 113–124. bów owadobójczych. Las Polski 3: 14–15. MIERZEJEWSKA E., 2001: Wykorzystanie BAŁAZY S., 2004: Znaczenie obszarów miejscowych szczepów strzępczaków chronionych dla zachowania zasobów owadobójczych do biologicznego zwal- grzybów entomopatogenicznych. Kos- czania szkodników. Postępy Nauk Roln. mos 53(262): 5–16. 5: 85–93. BAŁAZY S., 2006: Rozpoznanie i próby MIĘTKIEWSKI R., 1992: Doglebowe stoso- oszacowania roli grzybów entomopa- wanie grzybów owadobójczych w zwal- togenicznych w drzewostanach. Studia czaniu szkodników roślin. Ochrona Ro- i Materiały Centrum Edukacji Przyrodni- ślin 36(11): 7–8. czo-Leśnej 4(14): 154–165. 46 J. Jarmuł-Pietraszczyk et al.

MIĘTKIEWSKI R., 1994: Owady jako „pu- SAPIEHA-WASZKIEWICZ A., WOLIŃ- łapki” do wychwytywania entomopato- SKA J., WOLIŃSKI J., 2006: Występo- gennych grzybów z gleby. Ochrona Roś- wanie grzybów owadobójczych w glebie lin 38(11): 12–14. z upraw gryki. Fragmenta Agronomica MIĘTKIEWSKI R., DZIĘGIELEWSKA M., 23, 1(89): 174–181. JANOWICZ K., 1998: Entomopathogen- TKACZUK C., 2008: Występowanie i poten- ic fungi isolated in the vicinity of Szcze- cjał infekcyjny grzybów owadobójczych cin. Acta Mycologica 33(1): 123–130. w glebach agrocenoz i środowisk semi- MIĘTKIEWSKI R., KLOCZAREK R., naturalnych w krajobrazie rolniczym. 1995: Grzyby owadobójcze izolowane Rozprawa Naukowa. Wyd. Akademii z gleby pobranej z pól spod koniczyny Podlaskiej, Siedlce. czerwonej i ziemniaków. Zesz. Nauk. TKACZUK C., MIĘTKIEWSKI R., 1996: WSR-P., Siedlce, 39: 91–95. Occurrence of entomopathogenic fungi MIĘTKIEWSKI R., MIĘTKIEWSKA Z., in different kinds of soil. Roczn. Nauk JANKOWSKI K., 1993: Grzyby wyizo- Roln., Seria E, 25(1/2): 43–48. lowane z humusu, kompostu ze śmieci QUESADA-MORAGA E., NAVAS-COR- i osadu ze ścieków miejskich na owa- TÉS J. A., MARANHAO E.A.A., OR- dy pułapkowe. Acta Mycologica 28(2): TIZ-URQUIZA A., SANTIAGO-AL- 161–169 VAREZ C., 2007: Factors affecting the MIĘTKIEWSKI R., MIĘTKIEWSKA Z., occurrence and distribution of entomo- SAPIEHA A., 1992: Występowanie grzy- pathogenic fungi in natural and culti- bów owadobójczych w glebie pochodzą- vated soils. Mycological Research 111: cej z sadu. Zesz. Nauk. WSR-P., Siedlce, 947–966. 31: 209–219. WALCZAK M., RADZIEJOWSKI J., SMO- MIĘTKIEWSKI R., TKACZUK C., ZA- GORZEWSKA M., SIENKIEWICZ J., SADA L., 1991–1992: Występowanie GACKA-GRZESIKIEWCZ E., PISAR- grzybów entomopatogennych w glebie SKI Z., 2001: Obszary chronione w Pol- ornej i łąkowej. Acta Mycologica 27(2): sce. Instytut Ochrony Roślin, Warszawa: 197–203. 100–101. MIĘTKIEWSKI R., TKACZUK C., ŻU- REK M., Van Der GEEST L.P.S., 1994: Streszczenie: Występowanie grzybów entomo- Temperature requirements of four ento- patogenicznych na terenie Chojnowskiego Parku mopathogenic fungi. Acta Mycologica Krajobrazowego. Celem podjętych badań było 29(1): 109–120. określenie składu gatunkowego i nasilenia wy- MIĘTKIEWSKI R., ŻUREK M., TKA- stępowania grzybów entomopatogenicznych na CZUK C., BAŁAZY S., 1991: Wystę- wybranych siedliskach w Chojnowskim Parku powanie entomopatogennych grzybów Krajobrazowym. Zbadano także, czy na częstość w glebie ornej, leśnej oraz ściółce. Rocz. izolowania grzybów entomopatogenicznych wy- Nauk Roln., seria E, 21(1/2): 61–68. wierają wpływ następujące czynniki: siedlisko, PARUCH K., JANOWICZ K., KA- pora roku (wiosna, lato, jesień) oraz temperatura. PUCH G., 2004: Wykorzystanie owado- Na podstawie badań prób glebowych pobranych bójczych grzybów glebowych do walki z łąki 1, lasu 1 i sadu w okresie wiosny, lata i je- ze szkodnikami sadów. W: Ogólnopolska sieni 2010 roku został oceniony wpływ siedliska naukowa konferencja ochrony roślin sa- i pory roku na występowanie grzybów entomopa- downiczych, Skierniewice 25–26 lutego togenicznych. Na badanym terenie wyizolowa- 2004, L: 122–124. no trzy gatunki grzybów entomopatogenicznych The occurrence of entomopathogenic fungi... 47

(Beauveria bassiana, Metarhizium anisopliae MS. received in November 2013 i Isaria fumosorosea). Stwierdzono wpływ siedli- ska i temperatury na częstość izolacji poszczegól- nych gatunków. Na łące 1 i w sadzie gatunkiem do- minującym okazał się M. anisopliae, zaś w lesie 1 B. bassiana. Z siedlisk łąkowych i leśnych, z któ- rych glebę pobrano jesienią, największe nasilenie grzybów entomopatogenicznych obserwowano Authors’ address: w lesie 3 (rezerwacie). Na siedliskach łąkowych Joanna Jarmuł-Pietraszczyk najczęściej izolowanymi gatunkami były M. Wydział Nauk o Zwierzętach SGGW anisopliae i B. bassiana, na siedliskach leśnych Katedra Biologii Środowiska Zwierząt B. bassiana i I. fumosorosea. B. bassiana i I. fu- ul. Ciszewskiego 8, 02-787 Warszawa mosorosea istotnie częściej infekowały owady Poland w 20°C niż 25°C. e-mail: [email protected]

Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 49–57 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

The effect of feeding on aminopeptidase and non-specifi c esterase activity in the digestive system of pike-perch (Sander lucioperca L.) MACIEJ KAMASZEWSKI, TERESA OSTASZEWSKA Department of Ichthyobiology and Fisheries, Warsaw University of Life Sciences – SGGW

Abstract: The effect of feeding on aminopeptidase INTRODUCTION and non-specifi c esterase activity in the digestive system of the pike-perch (Sander lucioperca L.). Considering a dynamic development The pike-perch (Sander lucioperca L.) at the age of 18 days were fed for 21 days using three differ- of aquaculture in the world, it becomes ent diets: Aglo Norse (An), casein-gelatin (Cas), necessary to increase the production of cod meal with gelatin (Mac) and nauplius Arte- fi sh fodder, to improve its quality and mia salina (Art – control diet). On the last day to look for cheaper, alternative protein of the experiment, fi sh fed Art and An diets had sources (Hardy 1996). At present, the the statistically signifi cant highest body mass, length, and survival. On the last day, the highest most common and the best source of pro- aminopeptidase activity in the anterior intestine tein for fodder production is fi sh meal. and posterior intestine was registered in fi sh fed However, the prices getting higher and nauplius Artemia salina. The lowest activity of interest growing bigger makes us search this enzyme in the anterior intestine was to be for alternative protein sources to substi- found in fi sh fed with Cas diet, while there was no difference between among groups in the posterior tute fi sh meal in fodder (Hardy 1996). intestine. The non-specifi c esterase activity was The most frequent sources are vegetable registered in the stomach, liver, anterior intestine products and various food industry by- as well as in the posterior intestine. The lowest ac- -products. This is why research is done tivity of this enzyme in the stomach was observed on introducing different protein sources in the pike-perch fed with the Cas diet. In the an- to fodder, sources like soybean meal, terior intestine, the highest activity was registered in fi sh fed with Art, and the lowest – on the Cas casein, gluten and others (Ostaszewska and Mac diet. The results of the current research et al. 2005a, Kamaszewski et al. 2010, prove that feeding the pike-perch An diet has Kamaszewski et al. 2013). However, al- a positive effect of the survival of the pike-perch, ternative protein sources often have an their growth rate and the activity of the enzymes. unbalanced amino acid pattern and have By contrast, feeding pike-perch Cas and Mac diets did not satisfy nutritional needs of fi sh, resulting endogenous dietary factors, which may in their low survival, growth rate and low activity lead to a decrease in growth rates as well of the enzymes examined. as lesions in the digestive system of fi sh Key words: pike-perch, Sander lucioperca, amino- (Ostaszewska et al. 2005a, Ostaszewska peptidase, non-specifi c esterase, histochemistry et al. 2010). 50 M. Kamaszewski, T. Ostaszewska

The Percidae, among them the pike- lation system equipped with a biological -perch (Sander lucioperca) are popular fi lter and UV lamps. The water tempera- in aquaculture. Notwithstanding this, ture was 20.6 ±0.9°C, pH 7.5–8.1, and cannibalism, high death rate or poor up- the content of total ammonia nitrogen take of artifi cial diets constitute a serious did not exceed the level of 0.1 mg/L, problem in breeding the pike-perch in and the nitrite – 0.01 mg/L. The 14 h recirculating aquafarming systems (Os- light: 10 h dark photoperiod was ap- taszewska et al. 2005b, Szczepkowski plied. Aquariums were illuminated with et al. 2011). Therefore research is done poor light (100 lx). The experiment was to optimize feeding especially the earli- conducted in four nutritional groups, fi ve est life stages of the pike-perch and to repetitions each. Fish were fed every two examine the physiology of the fi sh diges- hours using following diets: commercial tive system. To this end, studies are on- diet Aglo Norse – An (Larvae Feed Ewos going where digestive enzymes during – Bergen, ), two experimental ontogeny are observed, their occurrence, Casein-gelatin diets – Cas (Ostaszewska location and activity. This information et al. 2005a) and cod meal-gelatin diet can be helpful when time indications are – Mac (Kamaszewski and Ostaszewska made when to launch a diet feeding the 2013). The control group was fed Ar- species and whether the diet meets nutri- temia salina nauplii ad libidum. The pro- tional requirements of the species given tein and lipid content is given in the Ta- (Kamaszewski et al. 2010). ble 1. In the fi rst week of the experiment, The aim of this research was to de- fi sh were given a feed ration represent- fi ne the infl uence of various diets on the ing 50% of their biomass, in the second survival, growth rate and distribution as week 15% and in the third – 10%. well as the activity of aminopeptidase M, exopeptidase from intestine, and non- TABLE 1. The content of protein (%) and lipids (%) in diets used in the experiment (according to -specifi c esterase, enzyme related in li- the manufacturer’s data) pid metabolism, in the digestive system Protein Lipid of juvenile pike-perch. Diet (%) (%) Artemia salina (Art) 50 10 MATERIAL AND METHODS Aglo Norse (An) 59 21 Casein-gelatin (Cas) 48 11 The experiment was conducted in the Cod meal-gelatin (Mac) 64 8 Department of Ichthyobiology and Fish- eries, Warsaw University of Life Scienc- To conduct histochemical examina- es. Juvenile stages of pike-perch, at the tions, 10 fi sh were sampled from each age of 18-day post hatching (total body group on the fi rst and on the last day lenght 18.59 ±1.68 mm; body weight of the examination. Fish were anesthe- 0.05 ±0.01 g) were breeding for three tized using MS-222 preparation (tricaine th th weeks (from 18 to 39 day after hatch- methanesulphonate, Sigma-Aldrich, ing). Fish were stocked at a density of Munich, Germany), weighed to the near- 5 individuals per litre in water recircu- est 0.01 g (body weight, BW), measured The effect of feeding on aminopeptidase and non-specifi c... 51 in total length (LT) to 0.02 cm and fro- a microscope Nikon Eclipse 90i and zen with liquid nitrogen. The research a co-operating camera Nikon Digital material was stored at –80°C until it was Sight DS-U1 (Nikon Corporation, To- analyzed. Samples were cut to a thick- kyo, Japan). ness of 10 μm using a cryostat (Leica CH The average as well as the standard 1900, Leica Microsystems, Nussloch, deviation for the survival, total length Germany). The activity of aminopepti- and weight of the fi sh were calculated dase M (membrane alanyl aminopepti- using a program Statistica ver. 10.0. Dif- dase, EC 3.4.11.2) was detected using ferences between the groups were tested a method according to Nachlas et al. us- using one-way ANOVA and Tukey’s ing a substrate L-LeucineβNaphthyl-am- (HSD) post hoc test (P ≤ 0.05). ide (Sigma) in 0.1 M phosphate buffer of pH 7.0 (Lojda et al. 1979). The activity of non-specifi c esterase (EC 3.1.1) was RESULTS AND DISCUSSION detected using a method according to Go- mori using a substrate 1-Naphthyl acetate The highest survival rate in the experi- (Sigma) in 0.1 M phosphate buffer of pH ment was observed in fi sh fed with nau- 7.4 (Lojda et al. 1979). The activity of plii Artemia salina (65.5 ±14.8%), while enzymes on histochemical preparations the lowest rate was shown in the fi sh fed was marked on the grounds of the intes- with Mac (35.4 ±16.4%), and the differ- ity of coloration. In research was using ences were statistically signifi cant (Ta- scale, where (+++) means a very strong ble 2). On the twenty fi rst day of the ex- histochemical reaction (90–100% area of periment fi sh fed with Artemia salina cell with a positive reaction), (++) means and An diet had a statistically signifi cant a strong histochemical reaction (60–90% higher total body length and body weight area of cell with a positive reaction), (+) in comparison to fi sh fed experimental means a moderate histochemical reac- diets Cas and Mac (Table 2). tion (40–60% area of cell with a positive When breeding a pike-perch in reaction), (+/-) means a weak reaction aquaculture, it is a common practice to (0–40% area of cell with a positive reac- feed the earliest stages of this species tion) and (-) means no reaction on prepa- with nauplii Artemia salina, and only rations examined. Microscopic examina- 17–19 days after hatching feed them ar- tions and photographs were made using tifi cial diets. It is a period when the di- gestive system of a pike-perch is fully TABLE 2. Survival (%), total length, and body weight of pike-perch fed with experimental diets on twenty fi rst day of the experiment (mean ±SD, n = 10) Diets Growth parameters Art An Cas Mac Survival (%) 65.5 ±14.8a 62.3 ±7.7a 43.9 ±18.1ab 35.4 ±16.4b Total body length (mm) 43.2 ±1.9a 42.6 ±2.7a 26.2 ±2.5b 28.5 ±2.6b Body weight (g) 0.61 ±0.08a 0.58 ±0.11a 0.11 ±0.03b 0.14 ±0.04b Means with different letter superscripts in the same row are signifi cantly different (P ≤ 0.05). 52 M. Kamaszewski, T. Ostaszewska differentiated and becomes capable of activity of the enzyme experienced no digesting and absorbing nutrient con- difference and showed the same level as tents from diets (Ostaszewska 2005). on day one (Table 3). Infl uence the feed- According to Ljunggren et al. (2003) ing had on the aminopeptidase M activ- and Ostaszewska et al. (2005b), a fodder ity in juvenile pike-perch stages was ob- that meets the nutritional requirements served in the brush border of the anterior of juvenile pike-perch stages is the Aglo intestine. High activity of this enzyme Norse diet (An). It was also proven in the was present in specimen fed with nau- current research that fi sh fed the An diet plii Artemia salina. As in Segner et al. had the quickest growth rate as well as (1989) it was proven that the larvae of the greatest survival rate among fi sh fed European whitefi sh (Coregonus lavare- on experimental diets. tus) fed with zooplankton showed higher Histochemical analysis revealed the aminopeptidase M activity than fi sh fed presence of aminopeptidase M in the with commercial fodder. The lowest ac- brush border and in the supranuclear tivity of aminopeptidase M in the brush enterocyte area of the anterior and pos- border of the anterior intestine was to terior intestine in the pike-perch of all be observed in the pike-perch fed with nutritional groups on the fi rst and the last Cas diet. The low activity of the enzyme day of the experiment (Fig. 1). As with can be a proof that the Cas diet does not the examined fi sh, the presence of ami- meet nutrititional requirements of the de- nopeptidase in the anterior and posterior veloping pike-perch (Kamaszewski and intestine was observed in other species Ostaszewska 2013). of fi sh (Segner et al. 1989, Tengjaroenkul The presence of non-specifi c este- et al. 2000). On the last day of the ex- rase was reported in the gastric epithe- periment an elevated aminopeptidase M lium, gastric glands, anterior intestine, grade was reported in all the nutritional posterior intestine and liver of the pike- groups in comparison to the fi rst day of -perch. A similar location of this enzyme the experiment (Table 3), as in Segner in various fi sh species was described by et al. (1989) and Gisbert et al. (1999). On many authors (Hirji and Courtney 1983, the last day of the experiment there was Gawlicka et al. 1995, Gisbert et al. 1999, a very strong reaction of aminopeptidase Kozarić et al. 2006). M in the brush border of the anterior in- On the fi rst day of the experiment testine in fi sh fed with nauplii Artemia the gastric epithelium was reported to salina, while the weakest reaction was show a non-specifi c esterase activity on registered in fi sh fed Cas diet (Table 3). a modest level, and the gastric glands In the posterior intestine the activ- – on a weak level (Fig. 1; Table 3). The ity of aminopeptidase M was weaker activity expression of non-specifi c es- comparing to the anterior intestine (Ta- terase in gastric epithelium maintained ble 3). On the last day of the experiment a steady level in all the nutritional groups a strong reaction of aminopeptidase M in excluding the Cas group, where the ac- the brush border of the anterior intestine tivity of the enzyme declined on the last in fi sh fed with nauplii Artemia salina day of the experiment (Table 3). On the was observed, while in other groups the last day, the activity level of the enzyme The effect of feeding on aminopeptidase and non-specifi c... 53

FIGURE 1. Localization of the digestive enzymes in the cross-section of pike-perch gastrointestinal tract. Aminopeptidase M location: in the striated border (white arrows) and in the supranuclear area enterocyte (grey arrows) of the anterior intestine (A) and posterior intestine (B). Non-specifi c esterase location: in the gastric epithelium (white arrows) and in gastric glands (grey arrows) (C), in the entero- cyte cytoplasm (white arrows) and in the striated border (grey arrows) of the anterior intestine (D) and posterior intestine (E). Location of non-specifi c esterase in hepatocyte cytoplasm (F) 54 M. Kamaszewski, T. Ostaszewska

TABLE 3. Location and activity of aminopeptidase M and non-specifi c esterase in gastric epithelium and gastric glands, anterior and posterior intestines and liver hepatocydes of the pike-perch fed with: nauplii Artemia salina (Art), Aglo Norse (An), casein-gelatin (Cas), cod meal with gelatin (Mac) on the fi rst and the last day of the experiment 1st 21st day of experiment day of Enzyme Distribution of enzymes experi- Art An Cas Mac ment Brush border + +++ ++ + ++ Anterior Supranuclear area intestine +/- ++ + + + of enterocyte Aminopeptidase M Brush border +/- ++ +/- +/- +/- Posterior Supranuclear area intestine +/-++++ of enterocyte Epithelium + + + +/- + Stomach Gastric glands +/- ++++ Brush border ++ ++ + +/- +/- Anterior Supranuclear area intestine +++ ++ ++ ++ ++ Non-specifi c esterase of enterocyte Brush border + + ++ + + Posterior Supranuclear area intestine ++ ++ ++ ++ ++ of enterocyte Liver +++ +++ +++ +++ +++ was higher in gastric glands in all the nu- in the pike-perch fed with An diet, and tritional groups in comparison to the fi rst a low activity in the specimen fed with day (Table 3). The non-specifi c esterase Cas and Mac (Table 3). On the last day activity increase in gastric glands can be of the experiment, the non-specifi c este- a sign of the stomach being properly de- rase activity in the supranuclear area of veloped and carrying out all physiologi- enterocyte declined slightly in all nutri- cal functions (Gawlicka et al. 1995). tional groups (Table 3). The non-specifi c In the anterior and posterior intes- esterase activity in the enterocyte brush tine, the non-specifi c esterase was lo- border of the anterior intestine in fi sh fed cated in the brush border of the anterior with nauplii Artemia salina stayed high intestine and in the supranuclear area of from the fi rst to the last day of the ex- enterocyte (Fig. 1). On the fi rst day of periment (Table 3). In all the other nu- the experiment a weaker reaction of non- tritional groups the activity decreased -specifi c esterase in the brush border was (Table 3). In the brush border of the pos- reported, while the activity was higher terior intestine, the non-specifi c esterase in the supranuclear area of enterocyte of activity on the last day of the experiment the anterior and posterior intestine (Ta- was low, as it was on the fi rst day ex- ble 3). In comparison to the fi rst day of cept for fi sh fed with An diet, where the the experiment, there was a moderate ac- activity was slightly higher (Table 3). tivity of the enzyme in the brush border On the last day of the experiment the The effect of feeding on aminopeptidase and non-specifi c... 55 non-specifi c esterase activity in the su- and enzyme activity. By contrast, casein- pranuclear area of enterocyte of the pos- -gelatin and cod meal-gelatin diets did terior intestine was on a similar level not meet the nutritional requirements of in all the nutritional groups and did not the pike-perch, resulting in low survival change compared to the fi rst day of the rate, slow growth rates and low activity experiment (Table 3). The non-specifi c of the enzymes examined. esterase in the pike-perch showed a high- er activity in the supranuclear area of en- terocyte compared to other fi sh species REFERENCES (Segner et al. 1989, Baglole et al. 1998, Tengjaroenkul et al. 2000, Kozarić et al. BAGLOLE C.J., GOFF G.P., WRIGHT G.M., 1998: Distribution and ontogeny of di- 2006). The non-specifi c esterase induces gestive enzymes in larval yellowtail and and supports the process of pinocytosis winter fl ounder. J. Fish Biol. 53, 4, 767– (Ribeiro et al. 1999), a mechanism of di- 784. gesting and absorbing nutrients in the in- GAWLICKA A., TEH S.J., HUNG S.S.O., testine. The non-specifi c esterase showed HINTON D.E., de la NOÜE J., 1995: signs of lower activity in the anterior and Histological and histochemical changes midgut intestine of starving fi sh (Baglole in digestive tract of white sturgeon larvae et al. 1998). The pike-perch fed with Cas during ontogeny. Fish Physiol. Bioch. 14, 5, 357–371. and Mac diets were reported to experi- GISBERT E., SARASQUETE M.C., WIL- ence a lower non-specifi c esterase ac- LIOT P., CASTELLÓ-ORVAY F., 1999: tivity in the brush border of the anterior Histochemistry of the development of intestine compared to specimen fed with the digestive system of Siberian sturgeon An and Art. The decrease in the activity during early ontogeny. J. Fish Biol. 55, was probably due to the fact that Cas and 3, 595–616. Mac diets do not meet the nutritional re- HARDY R.W., 1996: Alternate protein sources for salmon and trout diets. Anim. quirements of the developing fi sh. Feed Technol. 59, 1, 71–80. The non-specifi c esterase activity in HIRJI K.N., COURTNEY W.A.M., 1983: liver was on a similar level on the fi rst Non-specifi c carboxylic esterase activity and on the last day of the experiment in the digestive tract of the perch, Perca (Table 3). High enzyme expression oc- fl uviatilis L. J. Fish Biol. 22, 1, 1–7. cured in hepatocyde cytoplasm (Fig. 1). KAMASZEWSKI M., OSTASZEWSKA T., The high activity of the enzyme is asso- 2013: The effect of feeding on morpho- logical changes in intestine of pike-perch ciated with intensive lipid and carbohy- (Sander lucioperca L.). Aquacult. Int. drate metabolism ongoing in the hepato- DOI 10.1007/s10499-013-9693-y. cyde cytoplasm (Gawlicka et al. 1995, KAMASZEWSKI M., NAPORA-RUT- Kozarić et al. 2006). KOWSKI Ł., OSTASZEWSKA T., 2010: Effect of feeding on digestive enzyme ac- tivity and morphological changes in the CONCLUSIONS liver and pancreas of pike-perch (Sander lucioperca). Isr. J. Aquacult. – Bamidgeh. The results of present studies prove that 62, 4, 225–236. feeding the pike-perch An diet has posi- tive effects on their survival, growth rates 56 M. Kamaszewski, T. Ostaszewska

KAMASZEWSKI M., PRASEK M., OSTA- PepT2 expression and digestive tract SZEWSKA T., DABROWSKI K., 2013: morphology in common carp (Cyprinus The infl uence of feeding diet containing carpio L.). Comp. Bioch. Physiol. Part A. wheat gluten supplemented with dipep- 157, 2, 158–169. tides or free amino acids on structure RIBEIRO L., SARASQUETE C., DI- and development of the skeletal muscle NIS M.T., 1999: Histological and histo- of carp (Cyprinus carpio). Aquacult. Int. chemical development of the digestive DOI 10.1007/s10499-013-9683-0. system of Solea senegalensis (Kaup, KOZARIĆ Z., KUŽIR S., PETRINEC Z., 1858) larvae. Aquaculture 171, 3–4, GJURČENIĆ E., OPAČAK A., 2006: 293–308. Histochemical distribution of digestive SEGNER H., RÖSCH R., SCHMIDT H., enzymes in intestine of goldline, Sarpa von POEPPINGHAUSEN K.J., 1989: salpa L. 1758. J. Appl. Ichthyol. 22, 1, Digestive enzymes in larval Coregonus 43–48. lavaretus L. J. Fish Biol. 35, 2, 249–263. LJUNGGREN L., STAFFAN F., FALK S., SZCZEPKOWSKI M., ZAKĘŚ Z., SZCZEP- LINDEN B., MENDERS J., 2003: Wean- KOWSKA B., PIOTROWSKA I., 2011: ing of juvenile pikeperch, Stizostedion Effect of size sorting on the survival, lucioperca L., and perch, Perca fl uviatilis growth and cannibalism in pikeperch L., to formulated feed. Aquacult. Res. 34, (Sander lucioperca L.) larvae during in- 4, 281–287. tensive culture in RAS. Czech J. Anim. LOJDA Z., GOSSRAN R., SCHEB- Sc. 56, 11, 483–489. LER T.H., 1979: Enzyme histochemistry. TENGJAROENKUL B., SMITH B.J., A laboratory manual. Springer-Verlag, CACECI T., SMITH S.A., 2000: Dis- Berlin, Heidelberg, New York. tribution of intestinal enzyme activities OSTASZEWSKA T., 2005: Developmental along the intestinal tract of cultured Nile changes of digestive system structure in tilapia, Oreochromis niloticus L. Aqua- pike-perch (Sander lucioperca L.). Elec- culture 182, 3–4, 317–327. tr. J. Ichthyol. 2, 65–78. OSTASZEWSKA T., DABROWSKI K., Streszczenie: Wpływ żywienia na aktywność ami- PALACIOS M.E., OLEJNICZAK M., nopeptydazy i niespecyfi cznej esterazy w układzie WIECZOREK M., 2005a: Growth and pokarmowym sandacza (Sander lucioperca L.). morphological changes in the digestive Sandacze (Sander lucioperca L.) w wieku 18 dni tract of rainbow trout (Oncorhynchus były żywione przez 21 dni trzema dietami: Aglo mykiss) and pacu (Piaractus mesopo- Norse (An), kazeina-żelatyna (Cas), mączka tamicus) due to casein replacement with z dorsza z żelatyną (Mac) i naupliusami Artemia soybean proteins. Aquaculture 245, 1–4, salina (Art – dieta kontrolna). Ostatniego dnia 273–286. doświadczenia ryby żywione Art i An miały sta- OSTASZEWSKA T., DABROWSKI K., tystycznie istotnie większą masę i długość ciała CZUMIŃSKA K., OLECH W., OLEJ- oraz przeżywalność. Ostatniego dnia doświad- NICZAK M., 2005b: Rearing of pike- czenia najwyższą aktywność aminopetydazy -perch larvae using formulated diets – w jelicie przednim i tylnym stwierdzono u ryb fi rst success with starter feeds. Aquacult. żywionych naupliusami Artemia salina. Najniż- Res. 36, 12, 1167–1176. szą aktywność tego enzymu w jelicie przednim OSTASZEWSKA T., DABROWSKI K., stwierdzono u ryb żywionych dietą Cas, w jelicie tylnym zaś nie stwierdzono różnic między grupa- KAMASZEWSKI M., GROCHOW- mi doświadczalnymi. Aktywność niespecyfi cznej SKI P., VERRI T., RZEPKOWSKA M., esterazy stwierdzono w żołądku, wątrobie, jelicie WOLNICKI J., 2010: The effect of plant przednim i tylnym. Najniższą aktywność tego protein based diet, supplemented with enzymu w żołądku obserwowano u sandaczy ży- dipeptide or free amino acids on PepT1, wionych dietą Cas. W jelicie przednim najwyż- The effect of feeding on aminopeptidase and non-specifi c... 57 szą aktywność stwierdzono u ryb żywionych Art, MS. received in November 2013 natomiast najniższą u ryb żywionych Cas i Mac. Wyniki obecnych badań potwierdzają, że ży- wienie sandaczy dietą An korzystnie wpływa na Authors’ address: przeżywalność, tempo wzrostu ryb i aktywność Maciej Kamaszewski enzymów. Żywienie sandaczy dietami Cas i Mac Wydział Nauk o Zwierzętach SGGW powodowało natomiast niską przeżywalność ryb Pracownia Ichtiobiologii i Rybactwa oraz tempo wzrostu, a także niską aktywność ba- ul. Ciszewskiego 8, 02-786 Warszawa, Poland danych enzymów. e-mail: [email protected]

Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 59–66 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Changes in the size of population of the European wild boar Sus scrofa L. in the selected voivodeships in Poland during the years 2000–2011 WIOLETA KNIŻEWSKA, ANNA REKIEL Department of Pigs Breeding, Warsaw University of Live Sciences – SGGW

Abstract. Changes in the size of population of the discussed species is observed (Bom- the European wild boar Sus scrofa L. in the se- bik et al. 2007). It results from its high lected voivodeships in Poland during the years adaptative capacities in relation to vary- 2000–2011. The aim of the work was to analyze the changes in the population of Sus scrofa L. in ing living conditions, change of broad- the selected voivodeships in Poland in the years leaved and mixed forests into coniferous 2000–2011 and to determine their direction, with monocultures and from urbanization of consideration of hunting and level of utilization. natural habitats of wild boars (Podgór- The data for analysis were obtained from Re- ski et al. 2013). Wild boars play a posi- search Station of the Polish Hunting Association (PZŁ) in Czempiń. There was found the increase tive role in forest environment. The ani- in population of wild boar in hunting season mals of the mentioned species limit the 2011/2012 vs. 2000/2011, including the highest number of harmful insects, which consti- one in the Świętokrzyskie Province (256%) and tute a threat to particular tree species and Małopolska Province (264%) with the simultane- when burrowing, they prepare a surface ous high rise in hunting (250% and 413%, respec- tively). The concentration of wild boar population of earth to natural renewal of the forest. in 6 examined voivodeships, irrespectively of ba- The wild boars remove dead and sick sic size of the population (level of population in individuals what decreases the scale of season 2000/2001 in the voivodeships from group incidence of new sources of diseases and I (>10 thousand heads) and group II (<1.5 thou- a risk of their spread out (Haber 1969). sand heads) was increased. A moderate level of population utilization occurred to be insuffi cient They may also cause various damages. what caused a constant progression of the popu- During digging of the land (burrowing), lation number in the studies voivodeships in the they dig out small tree seedlings and years 2000–2011. self-sown plants, destroy forest nursery Key words: wild boar, population, changes due to digging out the seeds, they tram- ple forest cultivations and burrow inner forest meadows. The sounders of wild INTRODUCTION boars case the anxiety in hunting site and scare the animals of other species, The dynamics of changes in the popula- destroy nests, built on the ground, tion of wild boar is determined by envi- eat their hatches and eggs. They con- ronmental factors (Bieber and Ruf 2005, sume also weak progeny of other game Geisser and Reyer 2005). On the territo- animals (Haber 1969). The wild boars ry of Europe, including also Poland, the constitute reservoir of various diseas- increase of the number of the animals of es what creates a threat to human and 60 W. Kniżewska, A. Rekiel animals health (Tropiło 1996, Lipowski discussed populations were the small- 2003, Flis 2011). Sounders of wild boar est ones (in respect of number) in the destroy cultivated fi elds and grasslands scale of the country (<1,500 heads), i.e. and the urbanized ones on the peripher- Świętokrzyskie and Małopolskie. In the ies of urban agglomerations (Sondej and compared seasons, i.e. 2011/2012 and Jaroszewicz 2010, Matysek 2012) what 2000/2001 for the mentioned above is connected with the necessity to pay the voivodeships, the analysis of the popu- indemnities (GUS 2013). Environment lation size was performed and the re- protection, including population of many sults were referred to forest area (head species of wild animals, also boars, is per 1,000 ha) and the scale of hunting. connected with the need of creating the Level of obtaining of wild boars was passages for animals via quick traffi c determined as a sum of animals, be- roads and combining of forest complex- ing shot during the hunting season, i.e. es with the aim to create ecological runs in the period since 1st April, until 31st (Bobek et al. 2009). March of the next year. Index of the level The aim of the work was to analyze of utilization of wild boar population in the changes in population of wild boar a given season was calculated from the in the selected voivodeships in Poland ratio of mean number of the obtained in the years 2000–2011 and to determine wild boars and the mean number of wild their direction, including hunting and boar population according to the state on utilization level. 31st March and was expressed in per- centage. MATERIAL AND METHODS RESULTS AND DISCUSSION Materials for research were obtained form the Research Station of PZŁ in In hunting season of 2011/2012 vs. Czempiń. The results were gained from 2000/2001, there was recorded the in- hunting regions of the Polish Hunt- crease of the number of wild boar popula- ing Association. The evaluation of the tion in Poland (Fig. 1). Progression con- number of wild boars was carried out by cerned also the analyzed voivodeships the method of tracking and the whole- (Fig. 2) where the population in seasons -year observations (Błaszczyk 2006). 2000/2001 was as follows: group I: West The basis for detailed analysis was Pomerania – 18,255 heads; Warmia and constituted by the number of wild boars Mazury – 10,729 heads; Wielkopolskie – in six voivodeships. The fi rst group in- 12,985 heads; Lubuskie – 10,030 heads; cluded the voivodeships which possessed group II: Świętokrzyskie – 1,314 heads the greatest populations (in respect of and Małopolskie – 1,314 heads. Dur- number) of wild boars (>10,000 heads) ing the research period, a small decline in the hunting season of 2000/2001. in production was recorded only twice There were the following voivodeships: in the scale of the country. It occurred West Pomerania, Warmia and Mazury, during hunting season of 2003/2004 Wielkopolskie and Lubuskie. Group II and 2011/2012. In opinion of Haber covered two voivodeships where the (1969) it is a natural phenomenon for the Changes in the size of population of the European... 61

300 000

250 000 Size

200 000

150 000

100 000

50 000

0 0 11 12 /201 /20 /20 03/200404/200505/200606/200707/200808/2009 000/2001001/2002002/20030 0 0 0 0 0 009 010 011 2 2 2 2 2 2 2 2 2 2 2 2 Hunting seasons FIGURE 1. Size of wild boar Sus scrofa population in Poland in the years 2000–2011 (hunting seasons 2000/2001 and 2001/2012

40 000 35 000 2000/2001 30 000 2011/2012 25 000 20 000 15 000 10 000 5 000 0

e ie kie azury ski M zys d Lubuskie opol an Mał a więtokr i Wielkopolsk Ś West Pomerania arm W FIGURE 2. Size of wild boar Sus scrofa population in the voivodeships from group I and II in hunt- ing seasons 2000/2001 and 2011/2012. Group I – hunting seasons of 2000/2001 – size of population >10,000: Voivodeships: West Pomerania, Warmia and Mazury, Wielkopolskie, Lubuskie; Group II – hunting season of 2000/2001 – size of population <1,500: Voivodeships Świętokrzyskie and Małopolskie discussed species because during 1–3 the annual rate of the population growth, years, the number of the population may changes in forest economy and cli- decline twice or thrice or to become in- matic changes. The highest increase of creased without infl ow of individuals the population in the scale of the coun- from other populations. It results from try had place in the hunting seasons of a degree of survivability of squeakers 2008/2009 and amounted to 37,678 heads (piglets), intensity of shooting, level of as compared to the previous season. 62 W. Kniżewska, A. Rekiel

The progressive changes (heads) in the of population. When the population voivodeships from group I were as fol- stays in poor conditions, the juvenile lows: West Pomerania – 5,077 (16.45%), individuals do not receive the possibil- Warmia and Mazury – 3,855 (19.99%), ity of reproduction and the older animals Wielkopolskie – 4,754 (26.42%), Lu- do not loose it. Adult females constitute buskie – 1,511 (10.23%) and from group a reproduction reservoir for the popula- II: Świętokrzyskie – 795 (27.49%) and tion while the increase of the population Małopolskie – 1,137 (39.53%). is found on the normal level. When the After 12 hunting seasons, the number conditions are very good, a considerable of wild boars in the West Pomerania participation of the youngest group in Voivodeship increased by 90% and in the reproduction causes that the increase Warmia and Mazury – by 110%; how- of the population may be doubled (Bie- ever, the population stayed smaller ber and Ruf 2005). than in the former voivodeship (i.e. The increasing big-area cultivations West Pomerania). The population of of cereals, potatoes and maize consti- the discussed animal species was also tute the supplementation of natural but increased in the Lubuskie and Wielko- constantly decreasing feeding base be- polskie voivodeships by 63 and 68%, ing favourable for improvement of in- respectively. In the voivodeships from dividual condition of animals. Addition- group II, after 12 hunting seasons, the ally, the occurring climate changes, the signifi cant increases of the population effect of which brings mild winters with size were recorded: Świętokrzyskie – by a small snow cover, make the survival 256% and in Małopolska – by 264%, of weaker individuals easier as their en- what indicates more dynamic changes ergy expense connected with seeking for than in the voivodeships from group I. feed is smaller (Haber 1969, Servanty Such quick increase of the population et al. 2009). In the opinion of Chojnowski fi nds the source in accelerated sexual (2005), the mean temperature in winter maturity, high fertility of the species and period (December–February) amounting high natural birth rate (Haber 1969). The to ca. 0°C is favourable for increase of wild boars are characterized by a high the population of wild boar. adaptability to varying living conditions Changes in agriculture alter the re- and specifi c reproduction predisposi- production capacities of the species. tions (Bieber and Ruf 2005, Podgórski Frequent penetration of maize cultiva- et al. 2013). Moment of obtaining sex- tions and its high participation in diet ual maturity by females is determined increase the number of ovulating ovary by threshold body weight amounting to cells in sows of wild boar. The average 30 kg. When possessing abundant feed number of ripened cells per one ovary in their hunting place, the females may amounts to 4–5. When the diet is rich in reach this state before completing the insulinogenic maize, the number of the fi rst year of life. Simultaneously, the cells increases up to 6–7. Additionally, better are the living conditions, the bet- the reproduction potential is increased ter is the survivability of young animals owing to a good abundance of the soils what causes the increase of the number in bio-elements (Bieber and Ruf 2005, Chojnowski 2005). Changes in the size of population of the European... 63

During 12 seasons, hunting of the ani- According to the Supreme Coun- mals of the discussed species has been cil of the Polish Hunting Association subject to changes (Fig. 3); in group I it (Uchwała 1999), in the matter of the increased as follows: West Pomerania principles of management of wild boar – by 108%, Warmia and Mazury – by population in Poland, the correct density 83%, Wielkopolskie – by 93% and Lu- of the animals of the discussed species buskie – by 68%. The increase of hunt- is equal to 20 heads per 1,000 ha of for- ing material of wild boars by shooting in est area. In hunting season of 2000/2001, the voivodeships from group II was con- the degree of population density in three siderably higher: in the Świętokrzyskie voivodeships may be recognized as cor- – by 250% and in Małopolskie – as much rect – Warmia and Mazury, Wielkopol- as by 413%. skie and Lubuskie (Table 1). After twelve

35 000 30 000 2000/2001  2011/2012

20 000 15 000 10 000 5 000 0

ia e ury an z lskie r olski o e p m Ma Lubuskie d tokrzyskie ałop Po ielko ę M st W ia an Świ We rm Wa FIGURE 3. Obtaining hunting material of wild boar Sus scrofa in the voivodeships from group I and II in hunting seasons 2000/2001 and 2011/2012. Explanations the same as in Figure 2

TABLE 1. Mean density of wild boar population in the voivodeships from group I and II in hunting seasons of 2000/2001 and 2011/2012, in heads per 1,000 ha. Group I – hunting season 2000/2001 – size of population >10,000 heads in the voivodeship; Group II – hunting season 2000/2001 – size of popula- tion <1,500 heads in the voivodeship

Hunting season Changes in season 2011/2012 Group Voivodeship 2000/2001 2011/2012 vs. 2000/2001 West Pomerania 27.87 52.61 +88.77 Warmia and Mazury 18.96 37.79 +99.31 I Wielkopolskie 20.54 36.30 +76.73 Lubuskie 19.98 30.08 +50.55 Świętokrzyskie 4.64 14.77 +218.32 II Małopolskie 3.92 13.57 +246.17 64 W. Kniżewska, A. Rekiel seasons, the density in the voivodeships discussed species and humidity in the from group I was increased and was by forests, therefore, the phenomenon of 1.5–2 times higher than it results from intensity of hunting damages is foresee- the recommendations of SHC – Supreme able. Hunting Council (Uchwała 1999). The The level of utilization of wild boar greatest differences in the size of forest population has been given in Table 2. In area per one head in the compared sea- the compared seasons, it was similar in sons were recorded in the Małopolskie the voivodeships form group, including Voivodeship; it was by 3.5-fold decrease the fact that in seasons of 2011/2012 in of forest area. the West Pomerania Voivodeship, it oc- Burrowing of wild boar in fi eld and curred to be the highest one and exceed- forest cultivations increases the aeration ed the level of 100%; in the Warmia and of the soil what accelerates the degra- Mazury Voivodeship, it was the smallest dation of humus particles and enriches one. The level of utilization of the popu- the soil in nitrogen. It facilitates, inter lation in group II vs. I was clearly low- alia, quicker natural renewal of forest er; the differences amounted to ca. 40– cultures. On peat territories, burrowing –60%. Flis (2011) expresses the opinion causes, however, oxidation of unique that hunting on the mean level of 85% substances present in the peat what dis- is not suffi cient and results in further turbs incidence of natural species of increase of the population in the coun- plants and creates the conditions for liv- try. It intensifi es the consequences, con- ing of invasive species. The wild boars, nected with the discussed phenomenon which live outside the limits of natural i.e. hunting damages and penetration of habitats of their incidence, may be the urbanized areas. Under the present en- reason for degradation of species versa- vironmental conditions, in which good tility of plants (Sondej and Jaroszewicz feeding conditions are accessible, hunt- 2010). The degree of penetration of ag- ing size should be established on the ricultural cultivation areas is determined level of ca. 100% of the spring number by the seasons of the year, access of feed of animals. In exceptionally good habi- in forest habitats, being natural for the tats for the discussed animal species, the

TABLE 2. Level of utilization of wild boar population in voivodeships from group I and II in hunting seasons of 2000/2001 and 2011/2012. Explanations the same as in Table 1 Hunting season Group Voivodeship 2000/2001 2011/2012 West Pomerania 82.07 101.7 Warmia and Mazury 84.90 73.95 I Wielkopolskie 84.68 88.02 Lubuskie 82.62 85.26 Świętokrzyskie 39.88 39.31 II Małopolskie 44.52 62.70 Changes in the size of population of the European... 65 increase of the population may reach to CONCLUSION 150%, therefore, hunting size should be established on the level of the imple- There was recorded an increase in wild mented gain (Flis 2011). boar population in the hunting season As it was given by Bieber and Ruf of 2011/2012 vs. 2000/2001, including (2005), one of the solutions of the prob- the highest one in the Świętokrzyskie lem of the increase of wild boar number Voivodeship (256%) and in the Mało- in Poland includes management of the polskie Voivodeship (264%), with the population depending on the habitat con- simultaneous high increase of hunting ditions of animals; it should be diversi- result (250 and 413%, respectively). The fi ed. When the population lives in worse density of wild boar population increased environmental conditions, it should be in six examined voivodeships, irrespec- managed via increase of the pressure to tively of the basic level of the popula- hunt sows. In case of correct manage- tion (size of the population in 2000/2001 ment, the increase of the population is in the voivodeships from group I equal to 130–160%. It is supplemented >10 thousand heads and group II by structural shooting, as recommend- <1.5 thousand heads). The moderate ed by the Supreme Hunting Council level of utilization of the population oc- (Uchwała 1999). The goal of the struc- curred to be insuffi cient what caused tural shooting of wild boars is to make a constant progression of the number the population older via directing of of the population in the examined hunting pressure to the youngest animals voivodeships in the years 2000–2011. who, at early sexual maturation, cause a double increase of the population; an- other aim includes improvement of gen- REFERENCES der structure (females : males) as 1 : 1.1 in favour of male individuals. Shooting BIEBER C., RUF T., 2005: Population dy- namics in wild boar Sus scrofa: ecology, of squeakers should reach to 60% of all elasticity of growth rate and implications hunted heads during the hunting season, for the management of pulsed resource and it should constitute 2–3 times more consumers. J. Appl. Ecol. 42, 1203–1213. than shooting of young animals (piglets). BŁASZCZYK J., 2006: Inwentaryzacja zwie- Shooting of the young individuals should rzyny. Łowiec Polski 2, 18–20. amount to 30% and that of the oldest ani- BOBEK B., MIKOŚ J., WASILEWSKI R., mals – 10% of the plan of hunting size 2009: Gospodarka łowiecka i ochrona dzikich zwierząt na Pomorzu Gdańskim. (Uchwała 1999). Structural shooting will Polskie Towarzystwo Leśne: Regionalna result in limitation of negative effects of Dyrekcja Lasów Państwowych, Gdańsk. the increase of the wild boar population BOMBIK E., WYSOKIŃSKA A., KON- in Poland, inter alia, decrease of the DRACKI S., GÓRSKI K., 2007: Zmiany level of hunting damages in agricultural liczebności i poziom eksploatacji popu- cultivations, and betterment of biotope lacji dzika (Sus scrofa L.) w okręgach ło- utilization by the wild boars. It will have wieckich województwa mazowieckiego. Rocz. Nauk. PTZ 3, 1, 125–132. also an infl uence on the increase on the CHOJNOWSKI A., 2005: Dzicza eksplozja. number of obtained animals. Łowiec Polski 3, 22–24. 66 W. Kniżewska, A. Rekiel

FLIS M., 2011: Groźna włośnica. Łowiec Uchwała Nr 106/99 Naczelnej Rady Łowiec- Polski 9, 52–53. kiej z dnia 27 kwietnia 1999 roku w spra- GEISSER H., REYER H.U., 2005: The in- wie zasad gospodarowania populacjami fl uence of food and temperature on popu- dzika. lation density of wild boar Sus scrofa in the Thurgau (). J. Zool. 267, Streszczenie: Zmiany liczebności populacji dzika 89–96. europejskiego (Sus scrofa L.) w wybranych woje- GUS, 2013: Leśnictwo. Warszawa. wództwach w Polsce w latach 2000–2011. Celem HABER A., 1969: Dzik. PWRiL, Warszawa. pracy była analiza zmian populacji dzika europej- LIPOWSKI A., 2003: Dzik europejski (Sus skiego w wybranych województwach w Polsce scrofa L.) jako rezerwuar zakaźnych w latach 2000–2011 oraz określenie ich kierunku chorób świń. Medycyna Wet. 59, 10, z uwzględnieniem pozyskania łowieckiego i po- 861–863. ziomu eksploatacji. Dane do analizy udostępniła MATYSEK W., 2012: Zdziczałe miasta. Stacja Badawcza PZŁ w Czempiniu. Stwierdzo- Łowiec Polski 1, 34–37. no wzrost populacji dzika w sezonie łowieckim SERVANTY S., GILLARD J.M., TOI- 2011/2012 vs. 2000/2001, w tym największy w województwach świętokrzyskim (256%) i ma- GO C., BRANDT S., BAUBET E., 2009: łopolskim (264%), przy równocześnie znacznym Pulsed resources and climate-induced wzroście pozyskania łowieckiego (250 i 413%). variation in the reproductive traits of Zagęszczenie populacji dzika wzrosło w sześciu wild boar under high hunting pressure. badanych województwach, niezależnie od bazo- J. Anim. Ecol. 78, 1278–1290. wej wielkości populacji (liczebność populacji SONDEJ I., JAROSZEWICZ B., 2010: w sezonie 2000/2001 w województwach z grupy Konsekwencje działalności dzików (Sus I (>10 tys. szt.) i grupy II (<1,5 tys. szt.). Umiar- scrofa L.) dla roślin. Wiad. Ekol. 1, 56, kowany poziom eksploatacji populacji okazał 3–11. się być niewystarczający, co spowodowało stałą TROPIŁO J., 1996: Choroby dzików. Łowiec progresję liczebności populacji w badanych woje- Polski 3, 18–19. wództwach w latach 2000–2011. PODGÓRSKI T., BAŚ G., JĘDRZEJEW- SKA B., SONNICHSEN L., ŚNIEŻKO MS. received in November 2013 S., JĘDRZEJEWSKI W., OKARMA H., 2013: Spatiotemporal behavioral plastic- Authors’ address: Wydział Nauk o Zwierzętach SGGW ity of wild boar (Sus scrofa) under con- Katedra Szczegółowej Hodowli Zwierząt trasting conditions of human pressure: ul. Ciszewskiego 8, 02-786 Warszawa primeval forest and metropolitan area. Poland J. Mammal. 94, 1, 109–119. e-mail: [email protected] Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 67–75 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

The welfare of horses assessed by the investigations of chosen parameters of the stable microclimate TADEUSZ KOŚLA, AGNIESZKA POROWSKA Department of Animal Environment Biology, Warsaw University of Life Sciences – SGGW

Abstract: The welfare of horses assessed by the opment on the minimum conditions for investigations of chosen parameters of the sta- the maintenance of livestock for which ble microclimate. The performed investigations protection norms are not defi ned in the aimed at assessing the welfare of horses on the basis of the examination of chosen parameters of UE regulations which has been in effect the stable microclimate. The investigations were since 30 June 2010 (Rozporządzenie carried out at the Wolica horse riding complex of 2010). Warsaw University of Life Sciences. The objects The main principle of animal welfare of the investigations were two buildings with is to ensure that animals are hungry or breeding environments. The investigations were carried out during three seasons: summer, autumn thirsty, free from pain, traumas and dis- and winter. The investigated basic parameters of eases as well as from fear, stress and dis- the microclimate were: air temperature, relative comfort and are able to behave in their humidity, air cooling force and movement as well normal way (Kołacz and Bodak 1999). as the type and intensity of lighting. The results While assessing the welfare of animals were compared with the binding norms. The ob- tained results show that the parameters agree with we should remember the inventive act the recommendations of animal hygiene exceed- of March 1928, signed by the President ing the norms only sporadically. The welfare was of Poland I. Mościcki (Obwieszczenie maintained. 1932) concerning the protection of ani- Key words: welfare of horses, stable, microcli- mals and forbidding their ill-treatment. mate At present one can observe the division of the welfare indexes into four groups: physiological, behavioural, INTRODUCTION health and productive (Moberg 1985, Broom and Johnson 1993). There are Since 1997 The Law on the Protection of also some supplementary indicators such Animals (Ustawa 2003) has been in ef- as barn parameters including its micro- fect in Poland. It regulates the legal situ- climate (Kołacz and Bodak 1999). ation of animals, point 1 of the Law says The barn should maintain the op- that animal as a living creature, capable timum microclimate for the animals of suffering is not a thing. The human which ensure their welfare, productiv- being should respect, protect and provide ity and effective use. Badly designed or care to it as well as humane treatment. constructed appliances of the building Detailed laws, regulating horse breed- do not allow obtaining satisfactory pro- ing can be found in the Regulation of the ductive effects, the animals show health Minister of Agriculture and Rural Devel- 68 T. Kośla, A. Porowska problems and their welfare cannot be ful- measured in the buildings with natural ly ensured (Lewandowski 1997, Kołacz and artifi cial lighting and outside for the 2000, Fiedorowicz et al. 2004). comparison. The performed investigations aimed Temperature (°C), relative humidity at assessing the welfare of horses on the (%), dew-point (°C) and atmospheric basis of chosen parameters of the stable pressure (hPa) were measured with a ther- microclimate. mo-higrometer LB-707B with a built-in barometric modulus (produced by LAB- -EL). In the stables the panel was in- MATERIAL AND METHODS stalled in the middle of the corridor and The investigations were carried out at the probe was tested at the height of Wolica stables of Warsaw University of 80 cm from the fl oor. The measurements Life Sciences from 21 June 2006 to 16 of the air cooling force were done with February 2007. The investigated objects the Hill Katathermometer and the results were two stables (new and old) built in were also used for calculating the air the same area where the measurements movement velocity (Kośla 2011). At the were also taken. The objects comprised same time the air movement velocity was physically separate breeding environ- measured with the help of the vane an- ments. The evaluation concerned the mi- emometer with digital readout. Illumina- croclimate of the buildings. An attempt at tion (lx) was measured with a light meter. assessing the conformity of zoohygienic The measurements were taken inside the parameters with the binding standards new and old stable with attention paid to (Rozporządzenie 2010). The current the reading place, i.e. the middle of the order concerning horse management is corridor, in a sunny box (considered as very general, so other zoohygienic stand- light) and in a dark box. Also two alter- ards accepted for stables were also used natives were considered: measurements (Rozporządzenie 2003, Fiedorowicz taken with a natural light source (func- et al. 2004, Kołacz and Dobrzański 2006, tion “S”) and artifi cial light source (func- Kośla 2011). tion “F”). The photocell was installed in The examined period was divided six positions: paralell to the ceiling and into three measuring seasons: summer, fl oor and to four walls/barriers and the autumn and winter. In each season, at a mean result was calculated (Janowski week’s interval, measurements were tak- 1978, Kośla 2011). On the basis of the en for 5 days with three series of meas- results of light intensity measurements in urements every day (at 7 a.m., 1 and the buildings and outside, the room light- 7 p.m.). The measurements were taken ing coeffi cient was calculated (Kołacz in both neighbouring stables and outside and Dobrzański 2006, Kośla 2011). between the buildings. Statistical analysis was done using The measurements included the air the Statistica 5.0™ programme, ANOVA temperature, relative humidity, tempera- module. The signifi cance of differences ture of the dew-point, atmospheric pres- between the experimental groups was sure, air cooling force and movement. calculated using the LSD test (least sig- In each season the light intensity was nifi cant differences) or Tukey’s test. The welfare of horses assessed by the investigations of chosen... 69

RESULTS AND DISCUSSION training). Each window is of a rectangle shape with the glass surface of 0.68 m2. The new stable was built in 2003 together There are two windows in each box. with the adjacent riding school. A shorter The building interior lightening was wall of the school adjoins the long stable calculated from the ratio of the window wall from the north-east side. Due to that surface to the fl oor and it amounted to there are no windows on that side of the 1 : 21.9. Lightening standards for breed- stable so one row of boxes is lighted only ing horses and for competitive horses is with artifi cial light. Hopper windows 1 : 10 (Jodkowska 2007) or 1 : 12 (Kośla (18) are only on the south-west side at 2011) and for other horses it is 1 : 15 the height of 2.15 m from the fl oor. It is (Jodkowska 2007). Only for working a safe height, so the window panels do horses which most of the day spend out- not need any extra protection. However, side the stable the accepted ratio can be their location near the roof limits the nat- 1 : 20 (Kośla 2011). ural light infl ow (Janowski 1978, Kołacz The old stable is an adapted farm and Dobrzański 2006, Kośla 2011). building. Windows are on both long The location of windows only on one walls and on a short south-west wall. wall caused the uneven lighting with natu- Their shape is a standing rectangular of ral light of the entire stable. Boxes un- 122 cm over the fl oor on the short wall der the windows are clearly lighter than (7 windows) and 172 cm on the long those situated on the other side. Results walls (12 windows). Due to a low loca- of the intensity of lightening confi rm that tion of windows they are protected with phenomenon (Table 1). Artifi cial lighten- metal bars. The surface of glass panels ing decreases light defi cit, however, it re- is 0.42 m2 (7 windows) and 0.62 m2 (12 sults from the observations that artifi cial windows). The degree of stable lighten- light was turned on only when the light ing calculated as a ratio of window to was needed by the users of horses (for the fl oor surface amounted to 1 : 46.7. grooming or saddling a horse before the It shows that the window surface is too

TABLE 1. Seasonal average light intensity (lx) Corridor Dark box Sunny box Light the new the old the new the old the new the old Outside stable stable stable stable stable stable The summer season Natural 37.0 114.0 4.2 75.0 68.8 98.3 8 850.0 Artifi cial 138.8 106.7 13.8 49.8 61.3 95.8 8 850.0 The autumn season Natural 20.5 10.7 2.5 34.5 103.7 57.3 1 360.0 Artifi cial 138.3 41.3 13.0 40.7 118.2 71.0 1 360.0 The winter season Natural 1.4 11.0 0.5 30.3 5.6 36.8 780.0 Artifi cial 13.8 46.8 1.7 67.2 7.3 80.0 780.0 70 T. Kośla, A. Porowska small in relation to the recommended ference in temperature between the new standards (Kołacz and Dobrzański 2006, and old stable at 7 a.m. in autumn is sig- Jodkowska 2007, Pirkelmann et al. 2010, nifi cant at p ≤ 0.05. The remaining values Kośla 2011). Table 1 shows a compari- of temperature in the new and old stable son of results describing light conditions did not show any signifi cant differences. inside and outside the stables depending In winter the temperature drop below the on the site of the measurements taken required minimum value was observed distinguishing between natural and arti- a few times but it was more frequent in fi cial light. The obtained results confi rm the old stable. On the other hand, the ten- the division into the, so-called, light and dency to exceed the maximum value in dark side of the stable (Fig. 1). summer was revealed by the results of

FIGURE 1. Room lighting index (%) Good stable conditions ensuring ani- the measurements taken in the new sta- mal welfare include a complex of phys- ble. The optimum scope of temperatures ico-chemical parameters of the building recommended for horses which do not microclimate. The most important of cause any welfare disturbances is from 5 them from the animal hygiene point of to 28°C (Rozporządzenie 2003, Kołacz view are: air temperature and humidity, and Dobrzański 2006). air movement and its cooling force, at- Horses tolerate lower values of tem- mospheric pressure and lightening. perature much better than higher both The averaging results of the tempera- while resting, moving or during activi- ture measurements in the new and old sta- ties. In the moderate values of tempe- ble and outside are presented in Table 2. rature the mechanisms and processes Statistical calculations show that the dif- responsible for the thermal balance are The welfare of horses assessed by the investigations of chosen... 71

TABLE 2. Seasonal values of air temperature (°C) at 7 a.m., 1 and 7 p.m. Air tem- 07:00 13:00 19:00 perature the new the old the new the old the new the old outside outside outside (°C) stable stable stable stable stable stable The summer season Mini- 21.8 20.7 23.7 23.8 25.2 33.1 24.7 23.9 21.2 mum Average 23.0 22.2 26.0 26.0 22.5 37.0 27.0 26.1 25.9 Maxi- 25.2 23.3 29.8 28.5 29.25 40.1 28.6 28.1 28.6 mum The autumn season Mini- 12.5 4.5 1.0 3.9 4.0 1.6 7.6 5.5 0.9 mum Average 13.9 8.7 4.6 12.6 11.6 11.0 12.4 10.8 7.7 Maxi- 15.9 11.8 9.2 16.8 16.5 16.2 16.0 16.0 13.3 mum The winter season Mini- 4.6 2.9 –0.2 5.3 2.6 0.2 2.5 3.5 –1.3 mum Average 8.3 5.6 1.5 6.3 4.9 2.5 5.8 5.1 1.4 Maxi- 11.3 7.4 2.9 7.5 7.0 4.1 9.6 7.5 3.7 mum able to maintain the organism tempera- the elements of its construction may ture within a safe range (Wolski 1987, cause biodegradation of the building ma- Golachowski 2005). terials (Wiśniewska 2005). Air humidity and temperature in the Mean relative humidity values for farm buildings are particularly impor- stables and seasons are compared in Ta- tant for the welfare and health state of ble 3. No signifi cant differences were horses (Kołacz and Dobrzański 2006, noted in the air relative humidity values Kośla 2011). In case of the air tempera- in the new and old stable. Air humidity ture drop its relative humidity increases. showed an increasing tendency in the af- A very thorough measure of air humid- ternoons and evenings reaching in one of ity is the comparison of air temperature the measurements the acceptable value and dew-point temperature (Kołacz of 80% – Figure 2 (Fedorski 2003, Fie- and Dobrzański 2006, Kośla 2011). derowicz 2006, Kołacz and Dobrzański The determined upper limit of the rela- 2006). tive humidity for horses in Poland is The dew-point phenomenon affects 80% (Lewandowski 1997, Kołacz and in a signifi cant way the humidity condi- Dobrzański 2006, Kośla 2011). tions in the farm building and thus the A too high humidity level affects un- animal welfare. In the case when cold at- favourably the microclimatic conditions mospheric air gets into the stable through in the building and affecting negatively the ventilators then the dew-point 72 T. Kośla, A. Porowska

TABLE 3. The mean values for relative humidity (%) in different seasons at 7 a.m., 1 and 7 p.m. 07:00 13:00 19:00 Season the new the old the new the old the new the old outside outside outside stable stable stable stable stable stable Summer 68.79 64.94 52.60 46.35 48.38 26.32 48.48 50.16 45.13 Autumn 73.58 68.80 86.01 65.82 67.92 66.81 64.82 69.25 75.54 Winter 70.60 72.00 86.76 68.46 73.44 80.34 70.40 71.36 82.20

90.0 80.0 70.0 60.0 07:00 50.0 13:00 40.0 19:00 30.0 20.0 acceptable 10.0 0.0 12345 FIGURE 2. The values of relative humidity (%) in the new stable in the winter season temperature may be exceeded, especial- 7 a.m. In the winter season no statistical- ly when the horse density is high (Kośla ly signifi cant differences were observed. 2011). It leads to the cooling of their or- While comparing the air movement ve- ganisms and their susceptibility to cold locity in the new and old stable (Table 6), increases. A signifi cant difference of the the obtained results proved to be statisti- dew-point temperature (Table 4) in the cally signifi cant at 7 a.m. in the summer new and old stable was observed in the season at p ≤ 0.01 and in the autumn sea- autumn season at 7 a.m. (p ≤ 0.05). son at the signifi cance level p ≤ 0.05. The results of the LSD test confi rm Mean values of atmospheric pres- the statistically signifi cant differences in sure for a given season formed a char- the air cooling force (Table 5). A highly acteristic pattern. The highest readings signifi cant difference (p ≤ 0.01) between were obtained in the summer season, the cooling force in the new and old sta- a bit lower in the autumn season and the ble was noted in the summer season at lowest in winter. 7 a.m. and in the autumn season also at The welfare of horses assessed by the investigations of chosen... 73

TABLE 4. Seasonal values the temperature of dew point (°C) at 7 a.m., 1 and 7 p.m. 07:00 13:00 19:00 Tempera- the new the old the new the old the new the old ture (°C) outside outside outside stable stable stable stable stable stable The summer season Minimum 15.3 12.7 12.7 9.2 12.0 7.7 11.5 12.0 7.6 Average 17.2 15.3 15.3 14.0 14.7 13.1 14.8 14.5 12.3 Maximum 19.5 18.2 17.5 19.8 20.3 19.6 18.2 18.3 17.2 The autumn season Minimum 6.8 –1.4 –1.9 –0.4 0.0 –0.5 0.4 –1.1 –3.5 Average 9.3 3.3 2.6 6.3 4.8 4.7 5.9 5.3 3.9 Maximum 12.1 7.2 6.8 11.6 10.9 10.3 11.0 11.5 10.5 The winter season Minimum –0.6 –1.6 –2.2 0.0 –1.1 –1.9 –3.3 –0.9 –4.2 Average 2.7 0.5 –0.7 0.7 0.3 –0.7 0.1 0.1 –1.7 Maximum 6.7 4.1 2.0 3.2 3.5 2.1 3.6 2.3 2.2

TABLE 5. The average values of cooling from katathermometer (W·dm–2) at 7 a.m., 1 and 7 p.m. in different seasons Cooling (W·dm–2)

Season 07:00 13:00 19:00 the new the old the new the old the new the old outside outside outside stable stable stable stable stable stable Summer 1.82 2.52 2.14 1.99 1.53 1.57 2.12 1.95 2.68 Autumn 3.22 4.80 6.87 4.75 3.88 6.02 4.07 4.03 6.61 Winter 3.76 4.67 8.96 4.45 4.85 8.43 4.71 4.94 7.43

TABELA 6. The average and maximum values air speed (m·s–1) at 7 a.m., 1 and 7 p.m. Air 07:00 13:00 19:00 speed the new the old the new the old the new the old –1 outside outside outside (m·s ) stable stable stable stable stable stable The autumn season Average 0.13 0.29 0.71 0.54 0.22 0.79 0.27 0.20 0.80 Maxi- 0.28 0.46 1.58 1.42 0.62 1.28 0.46 0.41 1.32 mum The winter season Average 0.10 0.17 1.13 0.16 0.18 1.01 0.19 0.22 0.65 Maxi- 0.25 0.32 2.09 0.33 0.28 2.03 0.46 0.56 1.68 mum 74 T. Kośla, A. Porowska

CONCLUSIONS KOŁACZ R., 2000: Zasady wentylacji bu- dynków dla trzody chlewnej. Trzoda 1. Thermal conditions in both stables do Chlewna 8: 9–11. not always agree with the standards. KOŁACZ R., BODAK E., 1999: Dobrostan zwierząt i kryteria jego oceny. Medycyna In the winter season a temperature Weterynaryjna 55 (3): 147–154. drop below the required minimum KOŁACZ R., DOBRZAŃSKI Z., 2006: value was observed a few times, more Higiena i dobrostan zwierząt gospodar- frequently in the old stable. On the skich. Wydawnictwo AR, Wrocław. other hand, in summer a tendency to KOŚLA T., 2011: Metodyka badań z higieny exceed the maximum value was ob- zwierząt i prewencji weterynaryjnej. Wy- served in the new stable. dawnictwo SGGW, Warszawa. 2. Low intensity of natural light caused LEWANDOWSKI J., 1997: Mikroklimat w budynkach inwentarskich dla trzo- by architectural causes should be dy chlewnej i bydła. Poradnik. IBMER, compensated with artifi cial light. Un- Warszawa. fortunately most of the day horses MOBERG G.P., 1985: Animal Stress. APS, were kept in the stable with the lights Bethesda, Maryland, 195. turned off. Especially in the new sta- Obwieszczenie Ministra Spraw Wewnętrz- ble boxes without windows should be nych z dnia 23 kwietnia 1932 r. w spra- additionally lighted. wie ogłoszenia jednolitego tekstu rozpo- rządzenia Prezydenta Rzeczypospolitej o ochronie zwierząt. Dz.U. z 1932 r. nr 42, poz. 417. REFERENCES PIRKELMANN H., AHLSWEDE L., ZEIT- LER-FEICHT M., 2010: Hodowla koni. BROOM D.M., JOHNSON K.G., 1993: Organizacja stajni i żywienie. Wydawnic- Stress and Animal Welfare. Chapman and two RM, Warszawa. Hall, London. Rozporządzenie Ministra Rolnictwa i Rozwo- FEDORSKI J., 2003: Poradnik dla hodow- ju Wsi z dnia 2 września 2003 r. w spra- ców i miłośników koni. PWRiL, Poznań. wie minimalnych warunków utrzymywa- FIEDOROWICZ G., ŁOJEK J., CLAU- nia poszczególnych gatunków zwierząt SEM E., 2004: Budowa nowych stajni i gospodarskich. Dz.U. z 2003 r. nr 167, modernizacja budynków inwentarskich poz.1629 z nowelizacją z dnia 8 marca dla koni. Przegląd hodowlany 12: 17–20. 2004 r. Dz.U. z 2004 r. nr 47, poz. 456. FIEDOROWICZ G., 2006: Budowa nowych Rozporządzenie Ministra Rolnictwa i Rozwo- stajni i adaptacja budynków inwentar- ju Wsi z dnia 28 czerwca 2010 r. w sprawie skich na potrzeby chowu koni. Budow- minimalnych warunków utrzymywania nictwo wiejskie 7: 16–19. gatunków zwierząt gospodarskich innych GOLACHOWSKI A., 2005: Temperatura niż te, dla których normy ochrony zostały a zdolność konia do wysiłku. Świat Koni określone w przepisach Unii Europejskiej. 20 (12): 16–19. Dz.U. z 2010 r. nr 116, poz. 778. JANOWSKI T.M., 1978: Zoohigiena, higie- Ustawa o ochronie zwierząt z dnia 21 sierp- na środowiska i siedliska hodowlanego nia 1997 roku. Dz.U. z 2003 r. nr 106, oraz zwierząt gospodarskich. PWN, War- poz. 1002. szawa, Kraków. WIŚNIEWSKA J., 2005: Mikroklimat pa- JODKOWSKA E., 2007: Wskazania przed nujący w pomieszczeniu inwentarskim. rozpoczęciem budowy ośrodka hippicz- Acta Scientiarum Polonorum. Architek- nego. Hodowca i Jeździec 1 (12): 30–37. tura 4 (2): 59–72. The welfare of horses assessed by the investigations of chosen... 75

WOLSKI L., 1987: Fizyka obiektów rolni- wilgotność względną, prędkość i siłę oziębiającą czych. Wydawnictwo Politechniki War- powietrza, rodzaj i intensywność oświetlenia, szawskiej, Warszawa. a otrzymane wyniki zestawiono z obowiązujący- mi normami. Uzyskane wyniki wskazują, iż para- metry są zgodne z zaleceniami zoohigieny i tylko Streszczenie: Dobrostan koni oceniony z wyko- sporadycznie przekraczają normy. Dobrostan był rzystaniem badań wybranych parametrów mikro- zachowany. klimatu stajni. Celem przeprowadzonych badań była ocena dobrostanu koni na podstawie badania wybranych parametrów mikroklimatu stajni. Ba- MS. received in November 2013 dania i pomiary przeprowadzono na terenie kom- pleksu hippicznego Wolica SGGW. Obiektami Authors’ address: badań były dwa budynki stajni wykazujące róż- Tadeusz Kośla nice pod względem pierwotnego przeznaczenia, Wydział Nauk o Zwierzętach SGGW położenia, wymiarów, oświetlenia, warunków Katedra Biologii Środowiska Zwierząt mikroklimatycznych. Badania zostały przeprowa- ul. Ciszewskiego 8 dzone w okresie trzech sezonów roku: letnim, je- 02-786 Warszawa siennym i zimowym. Oceniono czynniki kształtu- Poland jące mikroklimat stajni, tj. temperaturę powietrza, e-mail: [email protected]

Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 77–83 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

The welfare of horses assessed by the zoohygienic inventory method TADEUSZ KOŚLA, AGNIESZKA POROWSKA Department of Animal Environment Biology, Warsaw University of Life Sciences – SGGW

Abstract: The welfare of horses assessed by the stable should be kept on the bedding zoohygienic inventory method. The performed in boxes, tying stalls or loose housing investigations aimed at assessing the welfare of without tying. The surface of the box for horses in accordance with the rules and methods of zoohygienic inventory. The investigations were adult horse with the height at withers of performed and the measurements taken at the over 1.47 m should amount to at least Wolica riding complex of Warsaw University of 9 m2 and for the mare with foal – 12 m2. Life Sciences. The basic parameters creating the The air cycle, degree of dust loading, and microclimate were evaluated in the research de- concentration of gases is maintained at scribed in the paper “The welfare of horses as- sessed by the investigations of chosen parameters the level harmless for animals. of the stable microclimate” published in the pres- At present the assessment of the ent issue of Animal Science. Using the rules and animal welfare includes the, so-called, methods of zoohygienic inventory the current supplementary indexes, namely para- paper presents the farm buildings, their equip- meters of the farm building, effi ciency ment, ventilation system and closest surroundings in which the horses are kept. The investigations of the ventilation system and the way included the concentration of ammonia in the of the movement restriction of animals stables, to demonstrate the effectiveness of drains (Kołacz and Bodak 1999). The possibil- and ventilation. The obtained results were com- ity of ensuring the proper management pared with the binding standards. The obtained re- conditions for the animals including the sults show that the parameters agree with zoohy- gienic recommendations and exceed the standards minimized content of ammonia additives only sporadically. The welfare of animals was is a criterium of a proper structure of the maintained. farm building (Lewandowski 1997). In Key words: welfare of horses, stable, zoohygienic order to prevent mechanical injury, the inventory, ventilation, ammonia concentration stables as well as paddocks should met the safety rules preventing mutilations, injuries and other factors dangerous for INTRODUCTION the health and life of horses (Fiedoro- wicz et al. 2004). Since 28 June 2010, horse management The aim of the investigations was the has been regulated in Poland by the Reg- assessment of the welfare of horses kept ulation of the Minister of Agriculture in the Wolica stable in accordance to the and Rural Development on the minimum rules and methods of zoohygienic in- conditions for the maintenance of live- ventory. The zoohygienic inventory was stock for which the protection norms performed and the ventilation function- are not defi ned in the UE regulations ing and ammonia concentrations were (Rozporządzenie 2010). Horses in the evaluated. 78 T. Kośla, A. Porowska

MATERIAL AND METHODS Statistical analysis of ammonia con- centration was done using the programme The investigations were performed in Statistica 5.0™, ANOVA modulus. The the Wolica stables of Warsaw University signifi cance of differences between ex- of Life Sciences from 21 June 2006 to perimental groups was calculated with 16 February 2007. The investigation ob- the help of the LSD or Tukey’s tests. jects comprised two stables in the same area. An attempt to assess the compat- ibility of zoohygienic and zootechnical RESULTS AND DISCUSSION parameters in relation to the binding standards (Kołacz and Dobrzański 2006, Stables, riding schools, paddocks and Rozporządzenie 2010, Kośla 2011). The additional buildings form the didactic- basic dimensions, location of the stables, -breeding complex of Warsaw Univer- paddocks, stud accomodations (riding sity of Life Sciences situated at Wolica, school, lungeing space), their position near Warsaw. relative to each other and cardinal points The complex of stables and paddocks were defi ned. In order to assess the is situated on the escarpment, in a rela- buildings the measurements of the doors, tively level area. The driveway is hard- windows, fl oors, boxes, partitions, ven- -surfaced. The entire area is fenced and tilation openings, the width of passages secured. Horses which are kept in the and corridors were taken. It was checked new and old stable are mainly taken care whether the mangers and water-bowls of by students who are interested in horse are placed at the proper height. It was as- riding. The staff has the basic knowledge sessed whether the applied solutions are necessary for proper performing the sta- functional and meet the safety require- ments and whether they do not upset the ble work, feeding the horses, cleaning horse state of welfare. The method and boxes, turning out horses to the pad- frequency of the change of bedding and docks and everyday grooming. In the general state of cleanness in the stables stable one can notice that everything is were evaluated. The paddocks were also kept in order, feeding times are observed assessed. It was checked whether the and any improprieties or happenings are horses had access to water both in boxes immediately taken care of. During the and paddocks and the hygiene of man- performed investigations no cases of gers and water-bowls was also exam- aggression or harassment were noted. ined. The functioning of the gravity ven- Horses were well treated and the work tilation was estimated. To demonstrate load connected with riding is rationally the effectiveness of drains and ventila- planned. Animals showing any alarming tion was conducted measurement of the health signs are immediately examined ammonia concentration. The measure- by a veterinary surgeon. Physical and ment of the ammonia concentration was psychological condition of horses does taken in both stables in the middle of the not arouse suspicion as to the correctness corridor at 7 a.m. using the gas detector of looking after them (Pirkelmann et al. WG-2 and gauge tubes (Kośla 2011). 2010). The welfare of horses assessed by the zoohygienic inventory method 79

The new stable was built in 2003 to- Every other partition is built of bricks up gether with the adjoining riding school. to 120 cm and has a heater installed. The stable is made of brick, with a tiled Through the verticale bars between roof and the attic is used for farm pur- boxes the horses can see each other poses. The building is located with its and air circulation in the stable is easier longitudinal axis in the south-east direc- which agrees with the principle of the tion, it is of 10.5 m in width and 38.0 m horse welfare (Kołacz and Dobrzański in length. The height of the building 2006). The boxes have sliding doors of measured inside is 3.0 m. The building 1.1 m in width. Kołacz and Dobrzański can be entered through a double door (2006) report that they cannot be narrow- with the total measurement of 2.2 m in er than 1.3 m. Their upper barred part has width and 2.9 m in height. Additionally a hinge allowing opening it in such a way the door is barred which allows open- that the horse may lean its head out to the ing the door which protects the horses corridor. Inside each box there are a ro- against the unfavourable temperature tating manger, an automatic water-bowl increase. Behind the door there are the and a rock-salt installed about 100 cm special rooms: saddle-room, scrubbing over the fl oor which allows their welfare room, locker room and utility room. In- (Kołacz and Dobrzański 2006). Rotating manger allows quick and effi cient feed- side the stable there two rows of loose- ing the horses with bulky food without -boxes for horses. Between them there is the necessity of entering each box. Au- a corridor of 2.5 m in width. The fl oor in tomatic water-bowl assures permanent the corridor and driveway is paved with access to fresh water. Horses are kept on the concrete blocks and wooden blocks the bedding which is cleaned every day, are used in the boxes. Between the corri- i.e. excrements and wet bedding are re- dor and the line of boxes, there is a dung moved systematically. Hay is delivered channel of 14 cm in width protected with directly on the bedding in the corner of a grid from above. a box. Feeding the concentrate is done Boxes are situated in two rows: 9 three times a day. Halter is screwed to boxes on the north-west side and 8 boxes each box as well as the folding (for safe- on the other side. The boxes are of the ty reason) saddle rack. same size – 2.9 m in width and 3.9 m A system of gravity ventilation was in length which makes the total surface installed in the stables – there are square 2 of 11.31 m per horse. It agrees with the ventilatory openings in the ceilings standards (Kołacz and Dobrzański 2006, whose side is 14 cm. They are located Rozporządzenie 2010). Box structure perpendicular to the long walls 4 open- is based on the steel-wooden construc- ings in a row situated over every second tion which can be easily disassembled. partition in such a way that they collect Partitions and doors up to 120 cm in the air from two neighbouring boxes. height are made of wooden boards and There are 4 rows of the exhausting venti- above them there is an openwork part of lation openings. 105 cm in height which agrees with The old stable is a farm build- standards (Kołacz and Dobrzański 2006). ing which was rebuilt and adapted for 80 T. Kośla, A. Porowska a stable in the 1970s. The long axis of 2006, Rozporządzenie 2010). Parti- the stable runs from north-east to south- tions between boxes are made of a solid -west. wood up to the height of 117 cm and the The object is made of bricks with the barred part is of 135 cm in height over attic used as store-room for bulky feed the solid part which disagrees with the (hay) and bedding (straw). The roof is standards because the solid partitions made of wood covered with roofi ng pa- should be of 140 cm in height (Kołacz per. The stable is 55.0 m in length and and Dobrzański 2006, Pirkelmann et al. 9.5 m in width. The height from the 2010). Front walls of the boxes from the fl oor to the ceiling is 2.95 m. The sta- side of the corridor are of the same meas- ble can be entered from three sides: the urements, i.e. the solid part up to 142 cm main doors on the edges of the building and the barred part over it up to 113 cm. are 2.1 m in width and 2.4 in height and In each box there are a rotating manger, a double door in the middle of the build- an automatic water-bowl and a rock-salt ing on the south-east side. Similarly as installed about 90 cm over the fl oor. The in the new stable the doors have an addi- ventilation in the building is of a gravity tional grating installed. On the north-east type. There are four exhaust shafts with end of the stable the utility and social the surface of 65 × 55 cm. These shafts rooms are located as well as the saddle end with a roof ventilator. room and sanitation facilities which take The results of the measurements of up about 10.8 m of the entire building. the air movement in the ventilation shafts There are two doors to the utility room, in the new and old stable are compared one from the outside and one from the in Table 1. In the summer season the inside. There is a comfortable driveway gravity ventilation did not work in both for a tractor with a trailer. Hay and straw stables, it worked better in the autumn are systematically threw down manually and winter seasons when the difference onto the middle of the corridor through in temperature values inside and outside the chute opening in the roof. Inside the the buildings was bigger than in summer. stable there was a feeding and dunging More often the air movement was noted passage of 2.26 m in width and two rows in the exhaust shaft in the old stable than of boxes located on its both sides. On the in the new one. junction of corridor and boxes there is In the old stable a treatment surface of a shallow (3 cm in depth) sewage groove. the 3.6 × 4.1 m is separated. It is parallel Floor in the corridor is paved with the to the corridor and it is used for shoeing concrete blocks and boxes have concrete of horses, grooming, saddling and veteri- fl oors. Horses are kept on the bedding nary treatments. with the same method of cleaning them as Between the old and new stables there in the new stable. Boxes are also located is a paddock of 1,440 m2 in size (60 × in two rows along the long walls. Their × 24 m). The fencing is made of a metal measurements are not the same varying tube whose upper border is at the height from 8.5 to 14.1 m2 which means that in of 100 cm. Directly behind that paddock the case of the smallest boxes the stand- there is another, bigger paddock of the ards are not met (Kołacz and Dobrzański 1,875 m2 (75 × 25 m). Fencing is made The welfare of horses assessed by the zoohygienic inventory method 81

TABLE 1. The air movement in the ventilation shafts in autumn and winter season at 7 a.m., 1 and 7 p.m. Air movement Date of 07:00 13:00 19:00 measurement new stable old stable new stable old stable new stable old stable Autumn season 12/10/2006 0.00 0.00 0.04 0.00 0.00 0.00 19/10/2006 0.00 0.00 0.00 0.00 0.00 0.00 26/10/2006 0.00 0.00 0.08 0.00 0.00 0.00 02/11/2006 0.00 0.00 0.00 0.00 0.00 0.00 09/11/2006 0.00 0.00 0.00 0.44 0.04 0.08 Winter season 01/02/2007 0.04 0.41 0.62 0.22 0.04 0.27 07/02/2007 0.00 0.22 0.00 0.08 0.00 0.27 09/02/2007 0.00 0.22 0.22 0.27 0.04 0.04 14/02/2007 0.04 0.04 0.00 0.00 0.00 0.00 16/02/2007 0.00 0.22 0.00 0.41 0.00 0.27 of the same material but the height was 2004) and their rotation is frequent and 130 cm with an additional tube at the time consuming. There are no green pas- 70 cm in height (Sasimowski 1984). The tures for horses. two level fencing does not allow the hors- Behind the new stable there is a dung es to get out (Zwoliński 1983). Corners of pit. It has a concrete bottom and a meter the paddocks are cut. It prevents the hors- high walls in accordance with the rules es to bunch together in the corners which of environmental protection (Runowski could be dangerous during establishing et al. 2006). Next to both stables, on the social hierarchy or during the horse the paved surface the washing stand is panic (Jodkowska 2007). While building situated. A corridor connects the riding the new stable and the riding school, the school with the stable. It has a separate paddock surface was improved and cov- gallery, a mirror on a part of the long ered with coarse sand mixed with gravel. wall, oblique bands and elastic surface Unfortunately in the ground there are made of sand fraction and sawdust. The also some stones of several centimeters riding complex also include a horse ex- in diameter which increase the danger erciser and a roofed lunging ring. of crippling the horse’s foot. Also the The results of the investigations of smaller stones of the gravel are danger- ammonia concentration in the stable are ous because they may become stuck be- presented in Table 2. The obtained val- tween the shoe and the hoof, inducing ues do not show the overstepping of the a pressure. During warm days plastic accepted standards for that compound portable water-bowls kept full with wa- in the stable air. The norm amounts to ter are placed on the paddock. All horses 20 ppm (Kołacz and Dobrzański 2006, use the accessible paddocks (Hansen Kośla 2011). 82 T. Kośla, A. Porowska

TABLE 2. Seasonal maximum values of ammonia concentration in the stable air at 7 a.m. Ammonia concentration Season new stable old stable mg·m–3 ppm mg·m–3 Summer 5.0 7.1 0.0 Autumn 3.1 4.4 0.0 Winter 5.1 7.2 0.0

CONCLUSIONS REFERENCES

1. The conditions of horse keeping in the FIEDOROWICZ G., ŁOJEK J., CLAU- stables of Warsaw University of Life SEM E., 2004: Budowa nowych stajni Sciences – Wolica are correct and do i modernizacja budynków inwentarskich dla koni. Przegl. Hod. 12: 17–20. not disturb in a signifi cant way their HANSEN M., 2004: Oczywiście – wypusz- welfare either in the physical or psy- czać! Koń Polski 10 (245): 70–72. chical state. On the basis of the per- JODKOWSKA E., 2007: Wskazania przed formed investigations it can be stated rozpoczęciem budowy ośrodka hippicz- that the horses are kept at a good level nego. Hodowca i Jeździec 1 (12): 30–37. of their welfare. KOŁACZ R., BODAK E., 1999: Dobrostan 2. The building a new stable, despite its zwierząt i kryteria jego oceny. Medycyna high aesthetic qualities, is not func- Wet. 55 (3): 147–154. KOŁACZ R., DOBRZAŃSKI Z., 2006: tional in respect to its feed storage. Higiena i dobrostan zwierząt gospodar- There is no utility attic. skich. Wydawnictwo AR, Wrocław. 3. The system of gravity ventilation in KOŚLA T., 2011: Metodyka badań z higieny the new stable did not function prop- zwierząt i prewencji weterynaryjnej. Wy- erly or effectively. dawnictwo SGGW, Warszawa. 4. The presence of ammonia in the of LEWANDOWSKI J., 1997: Mikroklimat the air new stable was detected, how- w budynkach inwentarskich dla trzo- dy chlewnej i bydła. Poradnik. IBMER, ever, its level did not exceed the per- Warszawa. missible standards. PIRKELMANN H., AHLSWEDE L., ZEI- 5. The total surface of the paddocks is TLER-FEICHT M., 2010: Hodowla koni. too small in relation to the number of Organizacja stajni i żywienie. Wydaw- horses kept in both stables. Horses nictwo RM, Warszawa. use them for a short time because Rozporządzenie Ministra Rolnictwa i Roz- the rotation is high. The gravel on woju Wsi z dnia 28 czerwca 2010 r. w sprawie minimalnych warunków the paddock ground is too coarse and utrzymywania gatunków zwierząt gospo- contains unwanted stones. darskich innych niż te, dla których normy 6. Horses have no access to pastures ochrony zostały określone w przepisach which is improper in view of their Unii Europejskiej. Dz.U. z 2010 r. nr 116, ethological needs poz. 778. The welfare of horses assessed by the zoohygienic inventory method 83

RUNOWSKI H., RADZIMIERSKI M., cy, postępując zgodnie z zasadami i metodyką SOBCZYK T., WOJCIECHOWSKA M., inwentaryzacji zoohigienicznej, opisano budynki 2006: Kodeks Dobrej Praktyki Rolniczej. inwentarskie, ich wyposażenie, system wentyla- Wydawnictwo SGGW, Warszawa. cji i najbliższe otoczenie, w którym utrzymywane SASIMOWSKI E., 1984: Przewodnik do były konie. Zbadano stężenie amoniaku w po- ćwiczeń z hodowli i użytkowania koni. mieszczeniach, a otrzymane wyniki zestawiono Wydawnictwo AR, Lublin. z obowiązującymi normami. Uzyskane wyniki ZWOLIŃSKI J., 1983: Hodowla koni. wskazują, iż badane elementy środowiska koni PWRiL, Warszawa. były zgodne z zaleceniami zoohigieny i tylko sporadycznie przekraczają normy. Dobrostan pod tym względem był zachowany. Streszczenie: Dobrostan koni oceniony metodą inwentaryzacji zoohigienicznej. Celem przepro- wadzonych badań była ocena dobrostanu koni MS. received in November 2013 zgodnie z zasadami i metodyką inwentaryzacji Authors’s address: zoohigienicznej. Badania i pomiary przeprowa- Tadeusz Kośla dzono na terenie kompleksu hippicznego Wolica Wydział Nauk o Zwierzętach SGGW SGGW. Podstawowe parametry tworzące mikro- Katedra Biologii Środowiska Zwierząt klimat oceniono w „Dobrostan koni oceniony ul. Ciszewskiego 8 z wykorzystaniem badań wybranych parametrów 02-786 Warszawa mikroklimatu stajni” opublikowanym w aktual- Poland nym numerze Animal Science. W niniejszej pra- e-mail: [email protected]

Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 85–90 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Effect of cow’s breed and feeding season on the content of bioactive whey protein of milk produced according to principles of the biodynamic farming BEATA A. KUCZYŃSKA1, KAMILA S. PUPPEL1, EWA METERA2, TOMASZ SAKOWSKI2, ALEKSANDRA KAPUSTA1, ARKADIUSZ BUDZIŃSKI1, HENRYK GRODZKI1 1Cattle Breeding Department, Warsaw University of Life Sciences – SGGW 2Animal Science Department, Institute of Genetics and Animal Breeding, Polish Academy of Science, Jastrzębiec

Abstract: Effect of cow’s breed and feeding sea- feeding was 5.38 g/l, however reduced to the level son on the content of bioactive whey protein of herself by the winter 4.62 g/l. The similar trend milk produced according to principles of the bio- was shown in the milk of HF cows in the sum- dynamic farming. The aim of this study was to mer season the concentration of this white was determine genetic effect of cow breed and feed- 4.60 g/l, and in the winter season underwent low- ing season on content of bioactive whey protein ering up to 4.41 g/l. The experiment demonstrated of cow’s milk. In the selected biodynamic dairy statistically signifi cant differences for the breed farm, cows of two different breeds, i.e. Brown and feeding season for the content of β-lacto- Swiss (BS) and Holstein-Friesian black and white globuline in cow’s milk. (HF) were kept under the same environment con- ditions. The samples of milk were collected twice Key words: whey protein, β-lactoglobuline, (in summer and winter) from 30 cows (in the RP-HPLC, breed, season same for number from BS and HF). Content of following whey protein: β-lactoglobuline (β-LG), α-lactoalbumine (α-LA), lactoferrin (Lf), lacto- INTRODUCTION peroxidase (Lp), lisozyme (Lz) and bovine serum albumine (BSA) were determined used RP-HPLC The main purpose of the biodynamic technique. The studies showed a signifi cant ef- farming is production functional food, fect of breed and feeding season on the content of whey proteins in milk. The average content of rich in bioactive components important whey proteins in milk of cows ranged from 0.73% to humans health. Whey proteins have for the HF breed in winter to 0.84% for the BS unique properties, beyond their impor- breed in summer. Important differences weren’t tance in nutrition; they exhibit chemi- stated in the content of lactoferrin and lactopero- cal, physical, physiological, functional xidase in the milk, and for her the content fl uctu- ated from 0.201 to 0.259 g/l and from 0.111 to and technological usefulness (McIntosh 0.154 g/l appropriately at examined breeds. There et al. 1998). The term of whey proteins were no statistically signifi cant differences in the has been used to describe the group of level of α-lactoalbumine in the milk of cows. Its milk proteins that remain soluble in milk content was ranged from 1.663 to 1.994 g/l. Sta- serum or whey after precipitation of ca- tistically important differences were shown in conducted experience for the breeds and the feed- sein at pH 4.6 and temperature 40°C. ing season (P ≤ 0.01) for the content of β-lacto- Traditionally, β-LG, α-LA, bovine serum globuline from BS cows in the period of summer albumine (BSA), immunoglobuline (Ig), 86 B.A. Kuczyńska et al. and proteose-peptone fractions have been TABLE 1. Components of the treatments in ex- considered the major characterized com- periment ponents of this fraction. Probably many Treatment factors affect the whey protein compo- Composition Summer Winter sition of bovine milk, including genetic feeding feeding and environmental (Metera et al. 2010). season season In the literature available no infor- Ingredient (kg/d) mation was found about whey protein Hay meadow 8.68 – content of milk from Brown Swiss (BR) Yellow lupine and pea 0.50 – and Holstein-Friesian (HF) cows kept in Oats and fi eld pea 2.0 – a certifi ed organic farm in Poland. This Forage grass with red 50.00 – is a new solution and makes it possible clover to analyze changes occurring in content Spelt 2.0 – whey protein in cow’s milk caused by Corn meal – 1.0 the biodynamic farming. Oats with fi eld pea meal – 4.0 Rye meal – 2.0 Grass silage with red – 24.0 MATERIALS AND METHODS clover Grass hay with red – 6.0 The experiment was conducted at the clover biodynamic farm of Juchowo, which is JPM 18.37 17.21 located in a small village of Juchowo BTJE 1 381.9 1 370.7 (north-western Poland) – about 90 km BTJN 1 950.7 1 734.6 from the Baltic Sea. The owner of the farm is a public benefi t foundation – the Stanisław Karłowski Foun dation, found- ed in 2001. From a herd of about 300 Sampling animals, maintained in a free-stall dairy shed, two groups of 15 were selected by The cows were milked daily at 05:30 and the analogue method: BS and HF cows, 17:30 and milk yield was recorded at taking into consideration the stage of each milking. Representative milk sam- lactation (45 ±9 days), daily milk yield ples were collected from each cow dur- and the age of cows (primiparous). Two ing milking by means of a milk autosam- dietary treatments were applied: in the pler in the milking parlour. Milk samples summer feeding season: hay meadow; were collected once: in the 45 ±9 days of yellow lupine, pea; oats and fi eld pea; lactation. Combined milk from morning forage grass with red clover and spelled. and evening milking was placed in ster- In the winter season: corn meal; oats with fi eld pea meal, rye meal, grass silage with ile bottles, preserved with Mlekostat CC red clover and grass hay with red clover and immediately submitted to the Cattle (Table 1). The diets were formulated us- Breeding Division (Milk Testing Labo- ing French National Institute for Agri- ratory of Warsaw University of Life Sci- cultural Research (INRA) system. ences) for composition analysis. Effect of cow’s breed and feeding season... 87

Milk analyses Chromatographic separation was per- Total protein concentration of the milk formed using the following eluents: A – was determined by automated infrared 0.1% TFA in the solution of acetonitrile : analysis with a MilkoScan FT – 120 in- : water (5 : 95), B – 0.1% TFA in the strument (Foss Electric). The samples acetonitrile. were heated up to 40°C in a water bath, the number of pump strokes was set to Statistical analyses 22, and the pipette shake was set to 5. The data obtained were analyzed statis- Evaluation of hygienic status of the tically using a multi-factor analysis of milk was based on somatic cell count on variance (least squares) by means of the Somacount – 150 (Bentley). SPSS 12.0 packet software. Only inter- Whey proteins: β-lactoglobuline actions between factors whose infl uence (β-LG), α-lactoalbumine (α-LA), bovine was statistically signifi cant (P ≤ 0.001, serum albumine (BSA), lactoferrin (Lf), P ≤ 0.01 or P ≤ 0.05) were considered in lysozyme (Lz) and lactoperoxidase (Lp) the study. The level of signifi cance was were examined using: high-performance determined after performing preliminary liquid chromatography (Agilent 1100 se- statistical analyses. ries; Agilent Technologies) with a set-up The model used was: Yijk = μ + Ai + consisting of a quaternary pump, an UV + B + (A × B ) + e detector, an auto-sampler and Chemsta- j i j ijk tion software. The analytical standards where: were purifi ed proteins, i.e. β-lactoglob- Yijk – dependent variable, uline (β-LG; 90%), α-lactoalbumine Ai – breed effect (i = 1 – 2), (α-LA; ≥85%), lactoferrin (Lf; 90%), ly- Bj – season of the feeding (j = 1 – 2), sozyme (Lz; 95% from hen egg whites) (Ai × Bj) – fi xed interaction effect and lactoperoxidase (Lp), which were between breed and season of the feeding. purchased from Sigma Aldrich (Germa- ny). Acetonitrile super gradient and tri- fl uoroacetic (TFA) were obtained from RESULTS AND DISCUSSION Merck and Sigma Aldrich; membrane fi lters 0.45 μm were manufactured by Content of total protein and composition Pall Laboratory. The chromatographic of protein fraction of cow’s milk is very conditions were as follows: important for the dairy industry. It is also – chromatographic columns with Su- fundamental for its nutritive value as well pelcosilTM LC-318 Supelco and Su- as processability (Barłowska et al. 2011). pelguard, with particle size 5 μm, For the fi rst time in Poland Reklewska pore size 300 Å; et al. (2003) showed, that content of – conditions for linear gradient elution: biological active components in milk of 0% B – 32% for 10 min, 32–52% B cows, especially from protein fraction, for 25 min, 52–80% for 3 min; depend on system and season of feed- – fl ow rate of mobile phase 1.2 ml per ing, that is environmental conditioning. min; Considering environmental ones, nutri- – ambient temperature of separation. tion is the most important determinant TABLE 2. The composition of whey protein in milk of cows independent of breed and of feeding season Season Signifi cant Summer feeding Winter feeding Parameters BS HF BS HF season breed season × breed LSM SE LSM SE LSM SE LSM SE Milk yield (k/d) * NS NS 26.09 5.21 23.84 7.54 21.03 3.70 21.75 5.97 SCC (thou./cm3) NS NS NS 162 297.04 126 235.74 189 235.54 135 159.92 Protein (%) NS NS NS 3.93 0.89 3.65 1.07 3.75 0.59 3.59 0.53 Whey protein (%) ** * NS 0.84 0.09 0.77 0.11 0.75 0.87 0.73 0.05 Lz (μg/l) NS * NS 34.35 20.16 44.39 20.10 29.65 17.69 44.30 23.28 Lf (g/l) NS NS NS 0.200 0.11 0.250 0.21 0.260 0.15 0.204 0.10 Lp (mg/l) NS NS NS 0.154 0.12 0.111 0.25 0.149 0.10 0.154 0.23 BSA (g/l) NS NS NS 0.412 0.87 0.351 0.81 0.348 0.10 0.363 0.12 α-LA (g/l) NS NS NS 1.994 0.45 1.897 0.63 1.906 0.48 1.663 0.25 β-LG (g/l) ** ** NS 5.385 0.65 4.603 0.69 4.621 0.58 4.411 0.58 SCC – somatic cell count, SE – standard error of the mean; NS – no signifi cant, *P ≤ 0.05, **P ≤ 0.01. Effect of cow’s breed and feeding season... 89 of milk chemical composition, followed The average content of whey proteins by production system, health status, as in milk of cows ranged from 0.73% for well as the effect of herd and labour. the HF breed in winter to 0.84% for the Within genetic factors, breed of the cow BS breed in summer. maintained in our country seems to have The content of β-lactoglobuline from substantial infl uence on milk compounds BS cows in the period of summer feed- (Brodziak et al. 2012). Comparison study ing was 5.38 g/l, however reduced to the quality of milk from BS and HF breeds level herself by the winter 4.62 g/l. found a higher content of protein and of fat in the milk (Kuczyńska et al. 2011). Acknowledgement The studies showed a signifi cant effect Research was realized within the of selected breed and feeding season project “BIOFOOD – innovative, func- on the content of whey proteins in milk tional products of animal origin” no. (Table 2). Similar results were obtained POIG.01.01.02-014-090/09 co-fi nanced in (De Marchi et al. 2008). by the European Union from the Euro- The average content of whey pro- pean Regional Development Fund with- teins in milk of cows ranged from 0.73% in the Innovative Economy Operational for the HF breed in winter to 0.84% Program 2007–2013. for the BS breed in summer. The level of functional whey proteins in milk is REFERENCES strongly infl uenced by the breed of cows (Brodziak et al. 2012). Breed of cows sig- BARŁOWSKA J., SZWAJKOWSKA M., nifi cantly affect the content of lysozyme LITWIŃCZUK Z., KRÓL J., 2011: Nu- in cow’s milk. No signifi cant differences tritional value and technological suitabil- in the content of lactoferrin and lactoper- ity of milk from various animal species used for dairy production. Comp. Rev. oxidase in the milk was recorded. There Food Sci. Food Saf. 10 (6), 291–302. were no statistically signifi cant differ- BRODZIAK A., BARŁOWSKA J., KRÓL J., ences in the level of α-lactoalbumine in LITWIŃCZUK Z., 2012: Effect of breed the milk of cows. Its content was ranged and feeding system on content of selected from 1.663 to 1.994 g/l. The experiment whey protein in cow’s milk in spring- demonstrated statistically signifi cant dif- summer and autumn-winter seasons. ferences for the breed and feeding sea- Ann. Anim. Sci. 12, 2, 261–269. De MARCHI M., BITTANTE G., DAL son for the content of β-lactoglobuline in ZOTTO R., DALVIT C., CASSANDRO cow’s milk. M., 2008: Effect of Holstein Friesian and Brown Swiss breeds on quality of milk and cheese. J. Dairy Sci. 91, 4092–4102. CONCLUSIONS KUCZYŃSKA B., PUPPEL K., METERA E., PIEŚNIAK J., GRODZKA A., SA- To sum up season and breed had signifi - KOWSKI T., 2011: Technological use- cant infl uence in created of content of fulness of milk from Brown Swiss and Holstein-Friesian black and white cows bioactive components of whey protein of kept in a certifi ed organic farm. Ann. cow’s milk produced according to prin- Warsaw Univ. of Life Sci. – SGGW, ciples of the biodynamic farming. Anim. Sci. 49, 69–76. 90 B.A. Kuczyńska et al.

McINTOSH G.H., ROYLE P.J., Le LEU R.K., serwatkowych w mleku. Wykazano większą ogól- REGESTER G.O., JOHNSON M.A., ną zawartość białek serwatkowych w mleku obu GRINSTED R.L., KENWARD R.S., ras bydła w okresie żywienia letniego w porówna- SMITHERS G.W., 1998: Whey proteins niu z żywieniem alkierzowym. Średnia zawartość as functional food ingredients? Int. Dairy białek serwatkowych w mleku krów wahała się od J. 8, 425–434. 0,73% dla rasy PHF zimą do 0,84% dla rasy BS METERA E., SAKOWSKI T., SŁONIEW- latem. Nie stwierdzono istotnych różnic w kształ- SKI K., REMBIAŁKOWSKA E., KU- towaniu się zawartości laktoferyny i laktopero- CZYŃSKA B., 2010: Wpływ systemu ksydazy w mleku, a ich zawartość wahała się od produkcji na zawartość substancji bio- 0,201 do 0,259 g/l i od 0,111 do 0,154 g/l odpo- wiednio w mleku badanych ras. Nie stwierdzono aktywnych w mleku krów – przegląd pi- również statystycznie istotnych różnic w pozio- śmiennictwa. Przeg. Hod. 2, 1−4. mie α-laktoalbuminy w mleku badanych krów. Jej REKLEWSKA B., BERNATOWICZ E., zawartość kształtowała się na poziomie wartości REKLEWSKI Z., NAŁĘCZ- TARWA- referencyjnych w zakresie od 1,663 do 1,994 g/l. CKA T., KUCZYŃSKA B., ZDZIAR- W przeprowadzonym doświadczeniu wykazano SKI K., OPRZĄDEK A., 2003: Content statystycznie istotne różnice dla rasy i sezonu ży- of biological active components in milk wienia (P ≤ 0,01) dla zawartości β-laktoglobuli- of cows depending on system and season ny. Koncentracja β-laktoglobuliny w mleku krów of feeding. Zesz. Nauk. Przegl. Hod. 68, rasy BS w okresie żywienia letniego wynosiła 1, 85–98. 5,38 g/l, jednakże zimą obniżyła się do poziomu 4,62 g/l. Podobną tendencję wykazano w mleku Streszczenie: Wpływ rasy krów i sezonu żywie- krów rasy PHF. W sezonie letnim stężenie tego nia na zawartość bioaktywnych białek serwat- białka wynosiło 4,60 g/l, a w sezonie zimowym kowych w mleku produkowanym według zasad uległo obniżeniu do 4,41 g/l. biodynamicznego rolnictwa. Celem pracy było określenie zawartości białek serwatkowych MS. received in November 2013 w mleku w zależności od rasy i sezonu żywienia. W wybranym gospodarstwie specjalizującym się Authors’ addresses: w produkcji mleka według zasad rolnictwa bio- Beata Anna Kuczyńska, Kamila Sylwia Puppel dynamicznego utrzymywane są w tych samych Aleksandra Kapusta, Arkadiusz Budziński warunkach środowiskowych dwie rasy krów, tj. Henryk Grodzki Brown Swiss (BS) i Polska Holsztyńsko-Fryzyj- Wydział Nauk o Zwierzętach SGGW ska (PHF). Próbki mleka od 30 krów (w tej samej Katedra Szczegółowej Hodowli Zwierząt liczbie od BS i PHF), były pobierane dwukrotnie ul. Ciszewskiego 8, 02-786 Warszawa w sezonie żywienia letniego i zimowego. Przy Poland e-mail: beata_kuczyń[email protected] wykorzystaniu wysokosprawnej chromatogra- fi i cieczowej w odwróconym układzie faz RP- Ewa Metera, Tomasz Sakowski -HPLC oznaczono następujące białka serwatkowe: Instytut Genetyki i Hodowli Zwierząt β-laktoglobulinę (β-LG), α-laktoalbuminę (α-LA), Polskiej Akademii Nauk laktoferynę (Lf), laktoperoksydazę (Lp), lizozym Jastrzębiec (Lz) i bydlęcą albuminę serum (BSA). W bada- ul. Postępu 1 niach wykazano istotny wpływ rasy (P ≤ 0,05) 05-552 Wólka Kosowska i sezonu żywienia (P ≤ 0,01) na zawartość białek Poland Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 91–94 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

The quality of camel wool held in the Tunisian Sahara Desert EWA KUŹNICKA, ANNA GRONDKOWSKA Department of Animal Breeding and Production, Warsaw University of Life Sciences – SGGW

Abstract: The quality of camel wool held in the valuable thin fi bers are soft and provide Tunisian Sahara Desert. The goal of present an attractive material for scarves and work was to investigate wool quality of camels sweaters of the highest quality. The fi b- breeding in the Tunisian part of Sahara Desert. The study was carried out with 10 camel males at ers of medium thickness may be used the age of three years. The samples of wool were for the production of outerwear. Thick taken from the left mid-side of a shoulder. Due to wool fi nds its way in the manufacture of the low regrowth rate of wool, the samples were tents, carpets, blankets, ropes and halters not split into the external and internal fractions as well as insulating material (Chand before a measurement. The individual fi bers were classifi ed into three groups: 1) fi bers with continu- et al. 2011). The quality of camel’s wool ous medulla, 2) fi bers with intermittent medulla, also depends on the part of body it is ob- 3) fi bers without medulla. Then the percentage tained from. Thinner wool, taken from of each type of fi bers was evaluated. Thickness a side of the body or from the abdominal, measurement was made using the microprojec- is best suited for spinning. This is the fi rst tion method according to Polish Standard PN-72/ /P-04900. At least 600 fi bers were measured in shearing wool fi ber of the diameter from each sample. The high fraction of hair from the 20 to 23 microns and is obtained from core constant rate of 78% wool with a thickness camels under the age of 3 years. Long- of up to 48 μm, and the hair thickness variation er (and also rougher) wool comes from of 47% be attributed to the roughness of the wool. the front of the neck and the top of the The wool of the camels under study should be used for the production of carpets and handicrafts. humps. The color of camel wool is from The occurrence of continuous core both in the thin cream to almost black and is easy to dye and thick fi bers confi rmed the adaptation of cam- (Huebscher 2008, Mathias 2010). els to a large diurnal temperature variation, as in Wool is harvested by combing as llamas and alpacas. well as picking molting fi ber or clipping, Key words: camel, wool, quality which takes place once a year in spring. The average yield for an individual in stocks is 5 kg. The fi bers of wool ob- INTRODUCTION tained from adult male dromedary have a diameter of 31–35 microns. Thinner The camel population is around 26 mil- wool is produced by yearlings and its lion (FAO statistics from 2011). Most of yield is from 1 to 4 kg (Khan et al. 2003, them are dromedaries, whose habitat are Huebscher 2008, Mathias 2010). dry, semi-arid, or desert areas of . A typical farm consisting of 46 cam- Camel’s wool is a valuable and sought- els generates about 12% of its annual -after product. Its value depends mainly income from the sale of greasy wool on the thickness and length. The most (Mathias 2010). 92 E. Kuźnicka, A. Grondkowska

The goal of present work was to inves- RESULTS AND DISCUSSION tigate wool quality of camels breeding in the Tunisian part of Sahara Desert. The average thickness of the wool was 40.26 μm (Table 1) and ranged from 28.80 to 48.13 μm. The average devia- MATERIALS AND METHODS tion was 18.33 μm, with 47.61% vari- ance which demonstrates a high diversi- The study was carried out with 10 camel ty of wool thickness in Tunisian camels. males at the age of three years. The wool in our samples appeared thin- The samples of wool were taken from ner compared to the study of Rozbicka the left mid-side of a shoulder. Due to the (2006). Other authors reported that 80% low regrowth of wool, the samples were of wool derived from adult camel had not split into the external and internal a thickness of 17–20 μm. This difference fractions before a measurement. The in- could be due to the quality of feed. The dividual fi bers were classifi ed into three best fi bers of inner fractions have been groups: 1) fi bers with continuous me- derived in central , and their dulla, 2) fi bers with intermittent medulla thickness ranges from 19 to 24 μm, while 3) fi bers without medulla. Then the the thickness of fi bers in the outer frac- percentage of each type of fi bers was tion can vary between 20 and 120 μm evaluated. Thickness measurement was (Petrice 1995, Huebscher 2008). made using the microprojection method Most of the tested samples consisted according to Polish Standard PN-72/ of continuous and discontinuous hair /P-04900. At least 600 fi bers were meas- core (Fig. 1), and their fractions were, ured in each sample. respectively, 78 and 17%. Coreless hair was only 5% but their fraction was

TABLE 1. Mean thickness of fi ber per wool sample Fiber diameter Number of sample mean thickness standard deviation variation (μm) (μm) (%) 1 37.01 22.65 61.19 2 40.00 17.21 43.03 3 28.80 24.57 85.31 4 46.98 25.95 55.24 5 47.18 15.96 33.82 6 48.13 10.70 22.23 7 46.20 17.71 38.33 8 41.78 15.22 36.43 9 34.87 16.51 47.35 10 31.66 16.84 53.19 Mean 40.26 18.33 47.61 The quality of camel wool held in the Tunisian Sahara Desert 93

4,71% 16,91%

fibres with continuous core fibres with intermittent core fibres without core

78,39% FIGURE 1. The percentage of fi bres with continuous core, intermittent core, and without core in all samples under test greater than that reported by Rozbicka temperature variation, as in llamas and (2006). The fraction of continuous-core alpacas (Kujaszewska and Kuźnicka hair in a sample varied between 50 and 2012). 100%; the share of intermittent-core hair was from 0.2 to 33%, and the coreless hair comprised 1 to 18% of the sample. CONCLUSIONS In three samples, there was no hair with- out a core, and their share in the remain- The high fraction of hair with continu- ing samples was very small (Fig. 2). ous core amounting to 78% wool with The occurrence of continuous core a thickness of up to 48 microns, and hair both in the thin and thick fi bers results thickness variation of 47% is a proof of from the adaptation to a large diurnal high roughness of the wool. The wool of

100

80

60 Fibers with intermittent core 40 Fibers with continuous core 20 Fibers without core 0 1 6 7 e e 5 e e pl mpl mpl Sa SampleSample 2 Sample 3 Sam 4 Sa SamplSampleSample 8 9 Sample 10 FIGURE 2. The participation of fi bers without core, with continuous core, and intermittent core in each trial 94 E. Kuźnicka, A. Grondkowska the camels under study should be used Streszczenie: Jakość wełny wielbłądów utrzymy- for the production of carpets and handi- wanych na obszarze tunezyjskiej Sahary. Analizie crafts. The occurrence of continuous core poddano wełnę pochodzącą od 10 wielbłądów utrzymywanych na obszarze tunezyjskiej Saha- both in the thin and thick fi bers confi rms ry. Próby pobrano z boku za łopatką od samców the adaptation of camels to a large diur- w wieku 3 lat. Ze względu na mały odrost wełny nal temperature variation, as in llamas pomiary zostały przeprowadzone na całym zespo- and alpacas. le włosowym, nie rozdzielano włókien na frakcję zewnętrzną i wewnętrzną. W trakcie pomiarów grubości poszczególne włókna klasyfi kowano do 3 grup: 1) włókna o rdzeniu ciągłym; 2) włókna REFERENCES o rdzeniu przerywanym; 3) włókna bez rdzenia. Pomiar grubości dokonywany był metodą pro- CHAND K., JANGID B., ROHILLA P., jekcyjną za pomocą lanametru na wyodrębnionej 2011: Traditional of knowledge of pro- frakcji (pęczku). Obliczono procentowy udziału cessing and value addition to dromedary włosów rdzeniowych, bezrdzeniowych i z rdze- camel wool. Indian Journal of Traditional niem przerywanym występujących w badanych Knowledge 10(2), 316–318. próbkach. Bardzo duży udział włosów z rdzeniem HUEBSCHER C., 2008: Bactrian Camel. ciągłym, wynoszący 78% przy grubości wełny Spin-off Magazine 3, 50–51. dochodzącej do 48 μm i zmienności grubości wło- KHAN B., IQBAL A., RIAZ M., 2003: Pro- sów wynoszącej 47%, może świadczyć o szorst- duction and management of camels. De- kości wełny. Wełna badanych wielbłądów powin- partment of Livestock Management, Uni- na być wykorzystywana do produkcji dywanów versity of Agriculture, Faisalabad. i rękodzieła ludowego. Występowanie rdzenia KUJASZEWSKA J., KUŹNICKA E., 2012: ciągłego zarówno we włosach cienkich, jak i gru- Alpaki – warunki chowu, użytkowanie, bych wskazuje na przystosowanie do dużej dobo- zabiegi pielęgnacyjne i profi laktyka. Ży- wej zmienności temperatury, podobnie jak u lamy cie Wet. 7, 591. i alpaki. MATHIAS E., 2010: Adding value to live- stock diversity. Rome. MS. received in November 2013 PETRICE O., 1995: Harvesting of textile animal fi bres. FAO Agri. Services Bul- letin 122. PN-72/P-04900. Metody badań surowców Authors’ address: włókienniczych, wełna. Ewa Kuźnicka, Anna Grondkowska Wydział Nauk o Zwierzętach SGGW ROZBICKA A., 2006: Charakterystyka Katedra Szczegółowej Hodowli Zwierząt okrywy włosowej udomowionych wiel- ul. Ciszewskiego 8 błądowatych. Praca magisterska SGGW, 02-786 Warszawa Warszawa. Poland Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 95–103 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Canine von Willebrand’s disease – knowledge and awareness among dog breeders and owners in Poland AGNIESZKA ŁOBODZIŃSKA, JOANNA GRUSZCZYŃSKA Department of Genetics and Animal Breeding, Warsaw University of Life Sciences – SGGW

Abstract: Canine von Willebrand’s disease breeds (Latimer 2011), with a high preva- – knowledge and awareness among dog breed- lence (from 15 to 60%) in 10 breeds, e.g. ers and owners in Poland. Von Willebrand’s dis- Dobermann and Airedale Terrier, (Gins- ease (vWD), one of the most common congenital bleeding diatheses caused by a defi cient or de- burg and Bowie 1992), whereas mixed fective von Willebrand’s factor (vWF), is found breed dogs are rarely affected (Brooks in different breeds of dogs. The objective of the 1999). study was to perform a preliminary analysis of the This blood coagulation disorder is as- knowledge and awareness of von Willebrand’s sociated with hereditary platelet disfunc- disease among dog breeders and owners in Po- land. The online survey was addressed to dog tion caused by presence of defected von breeders and owners, comprised 25 questions and Willebrand’s factor (vWF) in plasma, was voluntary and anonymous. It was placed on which is necessary for platelet adhesion. 12 thematic Internet forums dedicated to differ- This leads to defects of primary haemo- ent breeds of dogs. The respondents provided 231 stasis (Mischke 2012). Three types of answers, 76 of which were complete. The results of the survey indicate that the dog breeders and the disease with different space of muta- owners are highly interested in genetic tests and tions and different modes of inheritance want more general access to scientifi c informa- are distinguished depending on whether tion. By their own admission, the dog owners vWF defi ciency is qualitative or quantita- and breeders have partly insuffi cient knowledge tive (Mischke 2012). vWD variants differ about vWD. Considerable gaps in their knowl- edge about other issues (including breeding work) in inheritance pattern, heterogeneity of were also evidenced. Being a contribution to the gene defects, and variation in expression discussion about canine vWD, this article high- of the mutated gene, thus resulting in dif- lighted the need to improve the education of dog ferent levels of vWF : Ag concentration breeders and owners, and to make them aware of (Brooks et al. 2001) and heterogeneity in their role in canine health care. the clinical signs of the disease (Brooks Key words: von Willebrand’s disease, coagulation et al. 1992). Furthermore, the heteroge- disorders, Canis lupus familiaris neity of the disease forms can also be infl uenced by a number of factors that regulate vWF levels, e.g. the level of INTRODUCTION thyroid hormones and adrenaline (Me- yers et al. 1990). Antithrombotic drugs Von Willebrand’s disease (vWD) is one or the age of puppies have little effect on of the most common haemorrhagic dia- vWF concentrations (Moser et al. 1996). theses in dogs (Denis and Wagner 1999). It has been identifi ed in over 50 canine 96 A. Łobodzińska, J. Gruszczyńska

Reduced vWF levels increase the of affected dogs. It was open to active tendency towards spontaneous and pro- members and occasional users of Inter- longed bleeding from mucous mem- net forums. The survey was anonymous branes during the exchange of teeth, in- and completely voluntary. jury or surgery (Stokol 2012). In animals affected with vWD there is bleeding on Survey design the surface of the skin and mucous mem- The respondents were asked to complete branes. The duration of bleeding due to a survey containing 25 questions. Eight tissue trauma is prolonged and out of of them were general questions that fo- proportion to the type of injury sustained cused on the respondents’ dogs (e.g. breed (Brooks 1992). Owners should be aware and origin) and tested their knowledge of that some diseases concomitant with breeding. Thirteen questions pertained to vWD, as well as certain drugs, infections various aspects of von Willebrand’s dis- and endocrinological disorders may ag- ease (vWD) in dogs: predispositions to gravate the bleeding (Brassard and Mey- disease, knowledge about genetic back- ers 1991, Latimer 2011). ground, diagnostic methods and treat- Especially dog owners and breeders ments. Four questions concerned the re- should have a knowledge about vWD, be- spondents’ subjective assessment of their cause it is essential not only to eliminate knowledge and awareness regarding affected animals and vWD carriers, but vWD and dog breeding methods in their also to mate animals properly based on broadest sense. The study was performed genetic tests, after determining possible online and was generated and conducted risk of the disease in puppies. However, through website www.moje-ankiety.pl. access to relevant information is insuf- The survey was made available, between fi cient and there are no reports about the May and October 2012, at 12 internet fo- knowledge and awareness of dog breed- rums for breeders of dog breeds in which ers and owners about vWD. Moreover, in the disease was detected and at forums Poland the literature regarding vWD dis- dedicated to the breeding of purebred ease is scarce (Wessely-Szponder 1999, dogs, in thematic sections on canine 2001 and 2003, Wessely-Szponder and health. Once the web survey was acces- Szponder 2001). Therefore, the aim of sible (http://moje-ankiety.pl/respond- this study was to determine the level of 24155.html), the respondents were able knowledge and awareness about canine to complete it and their answers were vWD among dog breeders and owners in automatically sent to the platform, while Poland. the authors could monitor, correct and gather the data for later processing. Most MATERIAL AND METHODS questions were closed-ended (18) and the participants were allowed to choose one Respondents of the suggested answers, most of which were Yes or No. Open questions were in The survey was addressed to dog own- the minority (7 out of 25). Some semi- ers and breeders, interested in von Wil- -open questions provided space to allow lebrand’s disease as well as to the owners participants to choose their own answers Canine von Willebrand’s disease – knowledge and awareness... 97 or to justify their choice. Some questions and breeders were less willing to answer were of the multiple-choice type and al- open and semi-open questions, which lowed the respondents to select several required more of their time and effort, answers they felt were appropriate. The especially with regard to the questions respondents were also permitted to give concerning vWD. However, many more no answer to a question. In case the sur- respondents answered multiple-choice vey question was unclear, the respon- and closed-ended questions. dents could leave a comment or note in Below we present a summary of the the designated area. All participants were survey results, paying special attention asked the same questions. to the answers concerning vWD. The In the survey the names of the breeds answers obtained from the questionnaire followed the FCI Nomenclature of Dog survey show that it is not possible to de- Breeds, taking into account Polish no- termine the incidence of canine vWD in menclature of breeds (http://www.zkwp. Poland or to make defi nitive conclusions pl/zg/regulaminy/ Systematyka_ras_ about the attitudes of dog owners and FCI.pdf, 2012). breeders. Out of 94 respondents, 11% had Shet- Data analysis land Sheepdogs while the others owned Data were analysed based on the re- other breeds (82%) or mixed breed dogs spondents’ answers. Surveys sent to the (7%). Seventy-three percent out of 107 designated area on the platform were respondents had dogs from breeders. taken into account, even if they were in- Ninety-three percent of the respond- complete. Because all the answers from ents (out of 71 people) agreed that infor- all the surveys were analysed, a percent- mation obtained from molecular diagnos- age result for the total number of an- tic tests and provided in the pedigree can swers obtained was given for some of the be of benefi t to dog breeders or owners. questions. The results of the survey were According to the International Breeding used not only to estimate the knowledge Rules of the FCI, a stud book can be pub- and awareness levels, but also to gather lished by the Polish Kennel Club or an- maximum information based on the in- other national kennel club recognized by dividual experiences of the respondents. the FCI, and although it does not guar- Basic statistical parameters, the propor- antee the quality of the dog, it states its tion of particular answers and fi gures origin (provides information about the were prepared using Microsoft Offi ce dog’s ancestors), which prevents buy- Excel 2007. ing a dog from a puppy mill. By buying a puppy with a pedigree certifi cate, we know that it meets the breed standard. RESULTS AND DISCUSSION What is more, litter information sheet contains all notes about possible defects A total of 231 respondents (breeders and and imperfections. dog owners) fi lled in the survey. Unfor- All the respondents knew that dogs tunately, only 76 people (33%) returned are affl icted with genetic diseases, completely fi lled surveys. Dog owners but almost half of them considered 98 A. Łobodzińska, J. Gruszczyńska their knowledge on that subject to be cide to buy or intentionally mate dogs insuffi cient, especially with regard to affected with vWD or dogs carrying the vWD. The available literature provides vWD gene. The fear of genetic defects information about the dog breeds in (including von Willebrand’s disease), which a given disease was noted (La- which eliminate potential sires from timer 2011) and which breeds of dogs breeding, discourages buying and mat- are predisposed to certain types of vWD ing a dog carrying the vWD gene. and which breeds are sporadically af- The results obtained showed that fected (Ackerman 2011). The majority knowledge in this area is insuffi cient of respondents knew that some breeds and suggested that more attention should of dogs are predisposed to particular be devoted to these issues, because half genetic conditions. Sixty-one percent of the respondents had no knowledge of the respondents (out of 76 people) heard the frequency of vWD in certain breeds that genetic molecular tests (DNA analy- of dogs. Yet, the respondents were cor- ses) for canine genetic diseases can be rect in indicating the most vulnerable performed in Poland, 13% of which con- breeds (Fig. 1). The largest percentage cerned vWD analyses. The respondents of answers pointed to Dobermann as the were aware of the benefi ts that breeders breed genetically predisposed to vWD. have using genetic test in breeding prac- Johnstone and Crane (1981), Ginsburg tice as well as their increasing popular- and Bowie (1992), Riehl et al. (2000) ity and usefulness in diagnosing many and Mattoso et al. (2010) estimated the serious conditions, and in identifying the incidence of vWD in this breed to ex- carriers and affected dogs early in life. ceed 60%. Bell (2011) showed that in- However, they pointed out that the tests breeding affects more than 60% of the are expensive, there are no accredited Dobermann population due to deliberate laboratories that perform these tests, and increase in homozygosity. Most of the 76 some veterinary doctors have incomplete respondents (86.84%) knew that inbred knowledge about the disease. Further- mating, which increases homozygosity more, unlike in Great Britain, for exam- (inbreeding) in offspring, can be a delib- ple, dog owners and breeders in Poland erate breeding practice, while 92.11% of are not obliged to perform genetic profi l- the 76 respondents realized that this car- ing of particular animals. Unfortunately, ries the risk of genetic defects. In fact, the respondents mistakenly believe that the genetic tests and the screening pro- genetic tests are universal regardless of gramme developed for Dobermann dogs species or breed. The respondents’ an- make it possible to identify affected dogs swers demonstrate the need to prepare and carriers so as to replace these dogs training programmes for breeders and with healthy individuals through proper veterinary doctors, and to make avail- selection of offspring for further breed- able regularly updated information to the ing. The incidence of vWD in Dober- public for educational purposes. mann dogs in Poland was found to be It follows from the questions con- similar to that in other countries (Wesse- cerning vWD that most respondents ly-Szponder and Szponder 2001). The (70% out of 76 people) would not de- diagnostic tests used in Dobermann dogs Canine von Willebrand’s disease – knowledge and awareness... 99

FIGURE 1. Percentage of answers to the question “Do you know that some breeds are genetically more/less predisposed to vWD?” (a total of 177 respondents) enabled the identical mutations to be type 1 (the mildest and most common detected in Poodle and Manchester Ter- form of the disease; depending on muta- rier dogs (Venta et al. 2004, Boudreaux tion type, it may have either recessive or 2012). Along with Scottish Terrier, Ger- dominant inheritance), one case of type 2 man Shepherd Dog, Shetland Sheepdog (bleeding is much more serious; associ- and Bernese Mountain Dog, these breeds ated with an autosomal recessive gene) were indicated by the respondents to be and two cases of type 3 vWD (the most particularly predisposed to vWD (Fig. 1). acute and life-threatening form; associ- This is in agreement with the results of ated with an autosomal recessive gene). Johnstone et al. (1993) for Scottish Ter- Most respondents admitted that no vWD rier, Raymond et al. (1990) and Pathak was diagnosed in their dogs, but the pos- (2004) for Shetland Sheepdog, Arnold sible reason was that the dogs were not et al. (1997) for Bernese Mountain Dog, tested for these conditions. This was and http://fallingbranch.com/library/ probably due to the fact that the respond- vonwill.htm for German Shepherd Dog. ents were unaware of vWD, and did not The answers sent by the 76 respon- need and had no possibility of conduct- dents show that the disease was diagnosed ing such tests in their breeding prac- in 6 dogs, which included three cases of tice. Only one person performed tests at 100 A. Łobodzińska, J. Gruszczyńska a commercial laboratory, and the more ing breeding work) were also revealed. active group of breeders declared that The Internet defi nitely serves as one of they only choose properly tested animals the main sources of information about for breeding and refuse to mate dogs if the possible symptoms of vWD, the di- they have no test results. agnostic methods and curative therapies, Most out of the 76 respondents (72%) but it is not always the appropriate route admitted that their current knowledge to reach potential dog owners and breed- of vWD is inadequate (Fig. 2). In addi- ers. Because the level of respondents’ tion, 39% of them have diffi cult access knowledge is low, it might be necessary to information of interest and 71% of re- to use other methods of disseminating spondents would be interested in expand- the knowledge of vWD. Perhaps the ing their knowledge of vWD. Therefore, introduction of mandatory routine tests the respondents’ answers show that dog identifying different types of vWD and owners and breeders realize that access distinguishing this syndrome from other to information about vWD is diffi cult. bleeding diseases, especially in the rep- The results of the present survey show resentatives of genetically predisposed that the dog owners and breeders have, breeds, would make the breeders aware by their own admission, little knowledge of the scale of the problem (Wessely- about vWD. Considerable gaps in their -Szponder 2001). knowledge about other issues (includ-

FIGURE 2. Percentage of answers to the question “Please summarize your observations and determine the degree that refl ects your opinion on this subject. Use space under the table for possible comments” (a total of 76 respondents = 100%; percentages do not add up to 100 because respondents were allowed to select 4 answers) Canine von Willebrand’s disease – knowledge and awareness... 101

To date, studies on canine vWD in 3. In the analysed group, the respon- Poland have been very rare (Wessely- dents with low knowledge and aware- -Szponder and Szponder 2001). If con- ness regarding vWD showed their ducted, they usually covered a small willingness to expand their knowl- group of patients and failed to approach edge in this area. the problem comprehensively, often dis- 4. It was considered necessary to create regarding the key aspects of the disease. a platform for exchanging data and In contrast, international studies are observations between dog owners, conducted on a much greater scale and dog breeders and veterinary doctors, represent higher standards. Tightness of and for researchers to provide updat- funds is only one of the factors limiting ed information about the availability the development of scientifi c research of diagnostic tests. and new technologies in Poland. Many laboratories are very well equipped and can perform such genetic tests provided REFERENCES that there is demand and interest from breeders. The results of studies conduc- ACKERMAN L., 2011: Hemolymphatic Disorders. In: Genetic Connection: A ted in foreign centres are used to create Guide to Health Problems in Purebred databases, which are valuable sources of Dogs. 2nd ed. American Animal Hospital information for breeders. Being a con- Association, Lakewood: 94. tribution to the discussion about canine ARNOLD S., MÜLLER A., BINDER H., vWD, the present article highlighted the MEYERS K., GIGER U., 1997: Von Wil- need to improve the education of dog lebrand factor concentrations in blood plas- breeders and owners, and to make them ma of Bernese mountain dogs. Schweiz. aware of their role in canine health care. Arch. Tierheilkd. 139 (4): 177–182. BELL J., 2011: The Ins and Outs of Pedigree Analysis, Genetic Diversity, and Genetic Disease Control. http://siriusdog.com/ CONCLUSIONS bell-pedigree-analysis-genetic-diversity. htm (Accessed 30.12.2012). Analysis of the results enabled us to BOUDREAUX M.K., 2012: Inherited plate- make the following conclusions: let disorder. J.Vet. Emergency Crit. Care 1. The respondents stated that pedigree 22 (1): 30–41. certifi cate is considered an important BRASSARD J.A., MEYERS K.M., 1991: document which guarantees the dog’s Evaluation of the buccal bleeding time and platelet glass bead retention as as- origin, while the results of molecular says of hemostasis in the dog. Thromb. tests included in the certifi cate pro- Haemost. 65: 191–195. vide valuable information for dog BROOKS M., 1992: Management of Canine breeders and owners. von Willebrand’s Disease. Problems in 2. The respondents mistakenly believe Veterinary Medicine 4 (4): 636–646. that genetic tests are universal regard- BROOKS M., 1999: A Review of Canine less of species or breed. Inherited Bleeding Disorders: Biochemi- cal and Molecular Strategies for Disease Characterization and Carrier Detection. J. Heredity 90 (1): 112–118. 102 A. Łobodzińska, J. Gruszczyńska

BROOKS M., DODDS W.J., RAY- MISCHKE R., 2012: Overview of haemo- MOND S.L., 1992: Epidemiologic fea- stasis. In: BSAVA Manual of Canine and tures of von Willebrand’s disease in Do- Feline Haematology and Transfusion berman pinschers, Scottish terriers, and Medicine. Ed. M.J. Day, B. Kohn. 2nd ed. Shetland sheepdogs: 260 cases (1984- British Small Animal Veterinary Associa- -1988). J. Am. Vet. Med. Assoc. 200 (8): tion, Gloucester: 182–183. 1123–1127. MOSER J., MEYERS K.M., MEIN- BROOKS M.B., ERB H.N., FOURE- KOTH J.H., BRASSARD J.A., 1996: MAN P.A., RAY K., 2001: von Will- Temporal variation and factors affecting ebrand disease phenotype and von Wil- measurement of canine von Willebrand lebrand factor marker genotype in Dober- factor. Am. J. Vet. Res. 57: 1288–1293. man Pinschers. Am. J. Vet. Res. 62 (3): PATHAK E.J., 2004. Type 3 von Willebrand’s 364–369. disease in a Shetland sheepdog. Can. Vet. DENIS C.V., WAGNER D.D., 1999: Insights J. 45: 685–687. from von Willebrand disease animal RAYMOND S.L., JONES D.W., BRO- models. CMLS. Cell. Mol. Life Sci. 56: OKS M.B., DODDS W.J., 1990: Clinical 977–990. and laboratory features of a severe form GINSBURG D., BOWIE E. J., 1992: Mole- of von Willebrand disease in Shetland cular genetics of von Willebrand disease. sheepdogs. J. Am. Vet. Med. Assoc. 197 Blood 79 (10): 2507–2519. (10): 1342–1346. JOHNSTONE I.B., CRANE S., 1981: Von RIEHL J., OKURA M., MIGNOT E., Willebrand’s Disease in Two Families of NISHINO S., 2000: Inheritance of von Doberman Pinschers. Can. Vet. J. 22 (8): Willebrand’s disease in a colony of Do- 239–243. berman Pinschers. Am. J. Vet. Res. 61: JOHNSTONE I.B., NORRIS A.M., HIR- 115–120. ZER L., 1993: Type III von Willebrand’s STOKOL T., 2012: Von Willebrand’s dis- disease in Scottish terriers: A report of ease. In: BSAVA Manual of Canine and two cases. Can. Vet. J. 34: 679–681. Feline Haematology and Transfusion LATIMER K.S., 2011: Duncan & Prasse’s Medicine. Ed. M.J. Day, B. Kohn. 2nd ed. Veterinary Laboratory Medicine and Clin- British Small Animal Veterinary Associa- ical Pathology. 5th ed. Blackwell Publish- tion, Gloucester: 246–251. ing, Ames: 111–112, 115–118, 133. VENTA P.J., BREWER G.J., YUZBA- MATTOSO C.R.S., TAKAHIRA R.K., BEI- SLYAN-GURKAN V., SCHALL W.D., ER S.L., ARAÚJO J.P., Jr., CORREN- 2004: DNA encoding canine von Will- TE J.E., 2010: Prevalence of von Wille- ebrand factor and methods of use. Unites brand disease in dogs from Săo Paulo States Patent No. US 6,780,583 B1. State, Brazil. J. Vet. Diagn. Invest. 22: WESSELY-SZPONDER J., 1999: Choroba 55–60. von Willebranda u psów. Medycyna Wet. MEYERS K.M., WARDROP K.J., 55 (5): 288–291. DODDS W.J., BRASSARD J., 1990: Ef- WESSELY-SZPONDER J., 2001: Choroba fect of exercise, DDAVP, and epinephrine von Willebranda u psów – diagnostyka on the factor VIII:C/von Willebrand factor i postępowanie. Magazyn Wet. 10 (6): complex in normal dogs and von Wille- 52–54. brand factor defi cient Doberman pinscher WESSELY-SZPONDER J., 2003: Skazy dogs. Thromb. Res. 57 (1): 97–108. krwotoczne u psów. Magazyn Wet. 12 (5): 5–12. Canine von Willebrand’s disease – knowledge and awareness... 103

WESSELY-SZPONDER J., SZPONDER T., czona na 12 wybranych tematycznych forach 2001: Wstępne badania nad występowa- internetowych, dotyczących różnych ras psów. niem choroby von Willebranda u psów Od respondentów otrzymano 231 odpowiedzi, w Polsce. Życie Weterynaryjne 76 (1): z czego 76 pełnych. Wyniki ankiety wskazują, iż 26–28. hodowcy oraz właściciele psów są niezwykle za- Związek Kynologiczny w Polsce – Zarząd interesowani problematyką testów genetycznych, Główny, 2012: Nomenclature of dog pragną bardziej powszechnego dostępu do infor- breeds FCI (Systematyka ras wg FCI macji naukowej. W pewnym stopniu właściciele z uwzględnieniem polskiego nazew- i hodowcy psów posiadają niewystarczający zasób nictwa ras) 21: 1–28. http://www.zkwp. wiedzy dotyczący vWD, co sami przyznali w an- kiecie. Także w zakresie innych zagadnień (m.in. pl/zg/regulaminy/Systematyka_ras_FCI. pracy hodowlanej) stwierdzić można znaczne jej pdf (Accessed 19.10.2012). braki. Niniejszy artykuł jest głosem w dyskusji na temat vWD u psów, który jednocześnie ujawnił Streszczenie: Choroba von Willebranda u psów potrzebę szerszej edukacji hodowców i właścicie- – stan wiedzy i poziom świadomości hodowców li psów, a także konieczność uświadomienia im i właścicieli psów w Polsce. Choroba von Wil- roli, jaką pełnią w ochronie zdrowia psów. lebranda (vWD) jest jedną z najczęściej wy- stępujących wrodzonych skaz krwotocznych, spowodowaną niedoborem bądź zaburzeniem MS. received in November 2013 czynnościowym czynnika von Willebranda, odno- towaną wśród psów różnych ras. Celem pracy była wstępna analiza stanu wiedzy i poziomu świado- mości na temat choroby von Willebranda wśród Authors’ address: hodowców i właścicieli psów w Polsce. Dlatego Joanna Gruszczyńska też w jej realizacji przeprowadzono ankietę skie- Wydział Nauk o Zwierzętach SGGW rowaną do hodowców i właścicieli psów. Ankieta Katedra Genetyki i Ogólnej Hodowli Zwierząt on-line zawierała 25 pytań, wypełnienie jej było ul. Ciszewskiego 8, 02-786 Warszawa, Poland dobrowolne i anonimowe. Została ona umiesz- e-mail: [email protected]

Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 105–111 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Effect of classic massage therapy on the heart rate of horses working in hippotherapy. Case study JACEK ŁOJEK, ANNA ŁOJEK, JOANNA SOBORSKA Department of Animal Breeding, Warsaw University of Life Sciences – SGGW

Abstract: Effect of classic massage therapy on used therapeutically to reduce muscle the heart rate of horses working in hippotherapy. tone, restore muscle fl exibility, improve Case study. The objective of this study was deter- joint range of motion and, as a result, has mine the effect of classical massage therapy on heart rate in horses working in hippotherapy. We the potential to improve the horse’s loco- hypothesised that massage can be used to improve motor function. Thus it allows the horse the horse’s welfare, because it mentally relaxes to move and perform in its full move- the horse. The relationship between variables ment potential. Hill and Crook (2010) were examined to determine whether the selected explore the effects of massage to the factors (use or non-use of massage, the method of securing the patient on the horse, way of get- caudal muscles (retractors) of the equine ting on a horse, place of treatment) infl uenced the hindlimb on active and passive limb pro- heart rate of two horses working in hippotherapy. traction and stride length and found that The results showed signifi cant but very different massage signifi cantly increased passive effects on heart rate of particular horses subjected (P = 0.01) and active limb (P = 0.01) to a massage. It was also shown that heart rate of horses working in hippotherapy is highly infl u- protraction. In human patients massage enced by environmental factors. therapy reduces feelings of anxiety and improves self-esteem (Hernandez-Reif Key words: massage, horses, hippotherapy et al. 1998). It is likely emotions also play an important role in the horse, par- INTRODUCTION ticularly with respect to his work. The horse may not function optimally unless Massage is defi ned as the systematic it is psychologically comfortable and therapeutic stroking or kneading of the confi dent (Denoix and Pailloux 1996) body or one of its parts (Blood and Stud- and massage may be able to promote this dert 1999). It has a wide spectrum of ef- feeling of well-being in work. This study fects. It allows to relieve the body from is one of the fi rst attempt to utilise ob- pain and stress, reduces pressure, helps to jective outcome measures to determine achieve relaxation. Massage is currently the effectiveness of classical massage on widely used in veterinary physiotherapy welfare of hippotherapy horses. for both its physiological and psycho- The objective of this study was to de- logical effects (Valberg 1996, Roetting termine the effect of classical massage 1999). Horses have a very large muscle therapy on heart rate of horses working in mass compared to their body structure. hippotherapy. We hypothesised that mas- Massage is a manual therapy that can be sage have positive psychological effect on hippotherapy horses reducing their 106 J. Łojek, A. Łojek, J. Soborska heart rate and mentally relaxing horse, etic nervous system and the vagus nerve and may therefore play a valuable role connected to disorders, in research on in the development of strategies used to psychological and environmental stres- improve a horse’s welfare. sors (Rietmann et al. 2004, Łojek and Poszepczyńska 2006), behavioural dis- functions (Bachmann et al. 2003), train- MATERIAL AND METHODS ing methods or for individual evaluation of the animals for temperament (Visser The research was carried out in the fa- et al. 2002), emotional status and the cility for blind and low-vision people strategies of coping with environmental in Laski, Poland, where hippotherepeu- conditions. tic activities for pre-school and school The examination of the heart beat was children are organized. The facility in carried out with the Heart Rate Monitor Laski has been under the patronage of model S 810 manufactured by POLAR the Polish Hippotherapeutic Association ELECTRO OY. The analysis and cor- since 2006. rection at the level of 6.0 (average level The subjects comprised two geldings of correction) was performed with Po- with different records in hippotherapy lar Precision Performance for Windows work: which allows to record and analyze the – Horse A: hucul, 15 years old, withers data in graphical form. height 138 cm, calm and composed; The research lasted from the end of working for 9 years as hippotherapy April till the end of June 2012, during horse. hippotherapeutic activities with children – Horse B: fjord, 13 years old, withers at pre-school age with various degrees height 130 cm, lazy but courageous; of vision disability. Heart rate of both working for 2 years as hippotherapy horses was measured during their parti- horse. cipation in the hippotherapeutic activi- All horses were in regular work in ties during the following periods: hippotherapy and identically managed at – in the last week of April, when the the same hippotherapy center. Both geld- horses were not massaged, ings received the massage treatment. We – in May, when the horses were mas- attempted to ensure that the experimen- saged, tal conditions were the same for both – in the fi rst two weeks of June, when horses. The intention was to minimize the horses were massaged. any physical or emotional stress in the The organization of the research com- subjects, which may have affected the prised the following procedures: results of heart rate measurements. – from 8:30 a.m. to about 9:40 a.m. The trait under examination in the the horses were groomed and conse- horses was the heart rate measured by quently taken for a ride in the coun- the constant heart rate monitor (beats tryside; per minute – bpm). The analysis of this – after returning the horses had some feature is usually applied in research on rest and subsequently they started changes in the balance of the sympath- activities with children at the pre- Effect of classic massage therapy on the heart rate of horses... 107

-school, which lasted from 10:15 a.m. the data did not result in normalization of to 12 a.m. During these activities the the distribution. heart rate was measured; The Pearson Chi-square test and the – After returning from the therapy precise Fischer tests were used to ex- around 12 a.m. the fi rst horse was let amine the interdependence between the out on a paddock and the second one features in order to decide whether the was massaged for about 1 hour. Then chosen factors resulted in the change in the horses were changed. heartrate of the two horses working in The heart rate measurements (bpm) hippotherapy. of both horses working in hippotherapy The small sample size limited our sta- were taken with reference to the condi- tistical analysis to nonparametric tests: tions of the conducted therapy. In the Kruskal–Wallis test and Mann–Whitney analyses the SPSS Statistical Package U test. ver. 19.0 (2012) was used. The four cho- sen factors of the analysis were as fol- lows: RESULTS AND DISCUSSION 1. Month of therapy – so the use or non- -use of massage: One of the most important and most • April – no massage applied, popular types of massage is the classical • May – massage applied, massage. Depending on its intensity it • the fi rst two weeks of June – mas- can be healing, stimulating or relaxing. sage applied. Massage is a common method for “well- 2. The method of securing the patient on ness” purposes, its effi cacy for several the horse: indications is still under debate. The psy- • from the horseback, chological effects of massage in humans • from the ground. are well documented (Hernandez-Reif 3. The method of mounting the horse: et al. 1998, Hemmings et al. 2000). How- • from a ramp, ever, effects on behavior or stress pa- • from the ground. rameters described for man (Hemmings 4. The place where the therapy were 2001, Moyer et al. 2004) have been conducted: found also in horses, where grooming or • in the forest, massage reduced heart rate and caused • in the lime-tree alley. positive behavioral responses, when The data were checked for normality performed on preferred sites of allo- of distribution with Kolmogorov–Smir- grooming (Feh and de Mazieres 1993, nov Test (K-S test) for one sample. Due McBride et al. 2004). to the fact, that the value of the test prob- This study demonstrates that massage ability turned out to be lower than the ac- of the horses had signifi cant infl uence cepted level of signifi cance (P ≤ 0.05) the on their heart rate during hippotherapy. hypothesis that the analysed distribution The results of the Kruskal–Wallis test is identical with normal distribution was allowed to reject the null hypothesis rejected. Logarithmic transformation of on lack of infl uence of massage on the heart rate in both horses used for hippo- 108 J. Łojek, A. Łojek, J. Soborska therapy. The results of the Mann–Whit- est, in May, when massage was applied ney U test showed signifi cant differences throughout the month, it clearly increased (P ≤ 0.01) between the heart rates de- (Table 1 – The month of conducting ther- pending on the month when the therapy apy). The heart rate decreased slightly in was performed (and thus on applying June – the month when massage was ap- or not applying massage), the method plied only for the fi rst two weeks. Dif- of securing the patient on the horse, the ferent reaction to applying massage as method of getting on the horse and the a relaxing factor was observed in Horse place of therapy (Table 1). B. The horse’s heart rate signifi cantly de- The results indicate signifi cant differ- creased in May compared to April. This ences between the horses. This is shown tendency continued in June, when the by the total average heart rate of Horse horse was massaged only for half of the A (56.2 bpm) and Horse B (43.5 bpm), month. This could result from the indi- as well as the horses individual reac- vidual preferences of the horses, as far as tion to the conditions of conducting of the sense of touch is concerned, although the therapy, including applying massage Horse A has never showed negative re- (Table 1). In the case of Horse A, no action to touch (e.g. during grooming), relaxing effect of massage on the heart but the research shows that the horse’s beat during hippotherapeutic activities heart rate increased as a result of mas- with children was observed. In April saging. The horses’ reaction to massage the heart rate of this horse was low- indicates, that different horses may react

TABLE 1. The results of the analysis of the heart rate (bpm) of two horses working in hippotherapy depending on factors connected with the conditions of the conducted therapy Horse A Horse B Factors nxSEM P nxSEM P Month of therapy (use or non-use of massage) April – no massage applied 953 41.2 0.289 <0.001 1273 53.2 0.535 <0.001 May – massage applied 1963 62.8 0.197 1057 35.9 0.151 June – massage applied 649 58.5 0.198 585 35.8 0.179 Method of securing the patient on the horse From the horseback 726 65.1 0.335 <0.001 585 35.8 0.179 <0.001 From the ground 2839 54.0 0.227 2330 45.4 0.349 Method of mounting the horse From a ramp 2842 54.0 0.226 <0.001 1198 37.8 0.257 <0.001 From the ground 723 65.1 0.336 1717 47.4 0.435 The place where the therapy were conducted Forest 1015 53.3 0.469 <0.001 1056 35.4 0.129 <0.001 Lime-tree alley 2550 57.4 0.217 1859 48.1 0.413 Total 3565 56.2 0.207 2915 43.5 0.290 The level of signifi cance assumed as highly signifi cant: P ≤ 0.01; SEM – standard error of the mean. Effect of classic massage therapy on the heart rate of horses... 109 differently to this procedure. It should be tion of this factor into the research aimed pointed out that the massage does not al- at defi ning, whether the moment of ways cause the positive result. Massage putting the patient on the horse is percep- increases endorphin release, which may tible for the animal. Horses were more explain the reported sensation of well- tolerant to mounting from a ramp rather -being post massage (Kaada and Torste- than from the ground (Table 1) which can imbo 1987). Massage may also have had be explained by the fact, that mounting a benefi cial psychological effect on the from a ramp is defi nitely less strenuous horses used in this study, aiding relaxa- for the horse’s back and is more comfort- tion and improving their sense of well- able for the animal. The average heart being. rates measured at this method of mount- Both horses reacted differently to ing were lower than the total average of the method of securing the patient horses A and B. This could also be due to (Table 1). Horse A reacted with increas- the fact that the horse waited a while for ing heart rate in the case of securing from the patient while standing near the ramp the horse’s back and Horse B showed the and only then was he starting the work. same increase in heart rate while secur- When mounting from the ground, the ing the patient from the ground. This work started immediately. may be due to the fact, that Horse B was Lower average heart rates were re- working as hippotherapy horse for only corded in both horses during work in the 2 years and may not be accustomed to forest compared to the lime-tree alley the securing person, walking at his side. (Table 1). This result may be contributed Moreover, the presence of two people to fact, that the forest is a much calmer – the person leading the horse and the environment for work. It is a natural en- person securing the patient – may have vironment, isolated from external infl u- caused his anxiety. On the other hand, ences and from wind. The conditions of Horse A has been used in hippotherapy work in the lime-tree alley were totally for a long time and has already been ac- different. Very often strong wind oc- customed to greater number of persons curred, and on adjacent areas fi eld works around him during the therapy. It must were carried out, which was connected be also taken into consideration, that the with noise from the tractors and other activities with securing the patient from machines. Both the results of this study, the horseback have been conducted very and results of studies performed by other rarely and this method of securing the Authors indicate, that the environment patient might have been something un- plays a crucial role in conducting hip- expected for the horse, thus causing his potherapy, which directly infl uences the heart rate to rise. main condition of successful therapy The reactions of both horses to the – the patient’s safety. method of mounting and the location The results of our work should be of performing the therapeutic activities viewed as a preliminary insight into the were similar. The method of putting the effectiveness of classical massage on patient on the horse depends on the type heart rate of horses working in hippo- of illness and its advancement. Introduc- therapy. It is recommended that further 110 J. Łojek, A. Łojek, J. Soborska experiments be undertaken using a large DENOIX J.M., PAILLOUX J.P., 1996: Phys- number of animals to confi rm the fi nd- ical Therapy and Massage for the Horse. ings of this work. Manson Publishing, London: 103–123. FEH C., de MAZIERES J., 1993: Grooming at a preferred site reduces heart rate in horses. Anim. Behav. 46: 1191–1194. CONCLUSIONS HEMMINGS B.J., 2001: Physiological, psychological and performance effects 1. Signifi cant infl uence of massage on of massage therapy in sports: a review the heart rate during the hippothera- of the literature. Phys. Therap. Sport 2: peutic activities was observed. 165–170. 2. The reaction of the horses to massage HEMMINGS B., SMITH N., GRAYDON J., indicates, that different horses may DYSON R., 2000: Effects of massage on have a totally different reaction to this physiological restoration, perceived re- covery, and repeated sports performance. procedure and one has to bear in mind Br. J. Sports Med. 34: 109–115. that this procedure will not bring the HERNANDEZ-REIF M., FIELD T., FIELD T., expected result in any case. THEAKSTON H., 1998: Multiple sclero- 3. A crucial role of the environmental sis patients benefi t from massage therapy. conditions of therapy was demon- J. Bodywork Mov. Ther. 2: 168–174. strated. The conditions of conducting HILL C., CROOK T., 2010: The relationship the therapy, i.e. the method of secur- between massage to the equine caudal ing the patient, the method of mount- hindlimb muscles and hindlimb protraction. Equine Vet. J. 42 (Suppl. 38): 683–687. ing the horse and the location of the KAADA B., TORSTEIMBO O., 1987: Vaso- therapeutic activities had a signifi cant active intestinal polypeptides in connec- infl uence on the heart rates of the tive tissue massage. Gen. Pharmacol. 18: horses at work. 379–384. 4. The highly signifi cant individual dif- ŁOJEK J., POSZEPCZYŃSKA A., 2006: ferences between horses in terms of Ocena stresu u koni arabskich w trak- their reaction to the same conditions cie treningu do pokazów hodowlanych. Doniesienie wygłoszone na LXXI of conducting the therapy emphasize Zjeździe PTZ, Bydgoszcz. the importance of proper selection of McBRIDE S.D., HEMMINGS A., ROBIN- horses for hippotherapy. SON K., 2004: A preliminary study on the effect of massage to reduce stress in the horse. J. Equine Vet. Sci. 24: 76-81. REFERENCES MOYER C.A., ROUNDS J., HAN- NUM J.W., 2004: A meta-analysis of BACHMANN I., BERNASCONI P., massage therapy research. Psycholog. HERMANN R., WEISHAUPT M.A., Bull. 130: 3–18. STAUFFACHER M., 2003: Behavioural RIETMANN T.R., STUART A.E.A., BER- and physiological responses to an acute NASCONI P., STAUFFACHER M., stressor in crib biting and control horses. AUER J.A., WEISHAUPT M.A., 2004: Appl. Anim. Behav. Sci. 82: 297–311. Assessment of mental stress in warm- BLOOD D.C., STUDDERT V.P., 1999: blood horses: heart rate variability in Saunders Comprehensive Veterinary comparison to heart rate and selected Dictionary. 2nd edn. Eds: C. Byres, behavioural parameters. Appl. Anim. Be- W.B. Saunders, London: 699. hav. Sci. 88: 121–36. Effect of classic massage therapy on the heart rate of horses... 111

ROETTING A.K., 1999: Manual lymphatic na konia, miejsce prowadzenia terapii, wpływają drainage. Treatment of the horse’s leg. na częstotliwość tętna koni pracujących w hipo- http://www.diss.fu-berlin.de/diss/re- terapii. Masaż wpłynął odmiennie na każdego ceive/FUDISS_thesis_000000000152 z badanych koni. U jednego z nich nie stwierdzo- (Accessed 4.08.2010). no rozluźniającego wpływu masażu na pracę serca VALBERG S.J., 1996: Muscular causes of w czasie zajęć hipoterapeutycznych z dziećmi, zaś exercise intolerance in horses. Vet. Clin. u drugiego zastosowanie masażu spowodowało N. Am.: Equine Pract. 12: 495–515. znaczący spadek tętna. Wskazuje to, że różne ko- VISSER E.K., van REENEN C.G., van der nie mogą zupełnie odmiennie reagować na zabieg WERF J.T.N., SCHILDER M.B.H., masażu i należy się liczyć z tym, że nie w każdym przypadku przyniesie on spodziewany efekt. Tak- KNAAP J.H., BARNEVELD A., 2002: że warunki realizowania terapii (sposób asekura- Heart rate and heart rate variability dur- cji pacjenta, sposób wsiadania na konia i miejsca ing a novel object test and a handling prowadzenia terapii) w sposób istotny wpływały test in young horses. Physiol. Behav. 76: na częstotliwość tętna pracujących koni. 289–296. MS. received in November 2013 Streszczenie: Wpływ masażu klasycznego na parametry pracy serca koni pracujących w hi- poterapii. Studium przypadku. Celem badań była Authors’ address: ocena wpływu masażu na częstotliwość pracy Jacek Łojek, Anna Łojek, Joanna Soborska serca koni pracujących w hipoterapii. Zakłada- Wydział Nauk o Zwierzętach SGGW no, że masaż, mający pozytywny wpływ na stan Katedra Szczegółowej Hodowli Zwierząt psychiczny koni, może służyć jako zabieg relak- ul. Ciszewskiego 8 sujący konie w ich trudnej i monotonnej pracy 02-786 Warszawa z pacjentami. Określano też jak wybrane czynni- Poland ki: sposób asekuracji pacjenta, sposób wsiadania e-mail: [email protected]

Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 113–120 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Histological profi le of breast and leg muscles of Silkies chickens and of slow-growing Hubbard JA 957 broilers MONIKA ŁUKASIEWICZ1, NATALIA MROCZEK-SOSNOWSKA1, AGNIESZKA WNUK1, MACIEJ KAMASZEWSKI2, DOBROCHNA ADAMEK2, LEON TARASEWICZ3, PETER ŽUFFA4, JAN NIEMIEC1 1Department of Poultry Breeding, Warsaw University of Life Sciences – SGGW 2Department of Ichthyobiology and Fisheries, Warsaw University of Life Sciences – SGGW 3Faculty Media Art and Stage Design 4Czech and Slovak Breeders Association (race pigeons, chickens original, rabbits, exotic birds)

Abstract: Histological profi le of breast and leg (P ≤ 0.01). It was also demonstrated that the mean muscles of Silkies chickens and of slow-growing diameter of muscle fi bers of Silkies chickens was Hubbard JA 957 broilers. The experiment was more than two times smaller compared to the conducted with 60 slow-growing Hubbard JA Hubbard JA 957 chickens. 957 broilers and with 60 Silkies chickens reared Key words: Silkies and Hubbard chicken, meat, until the 63rd day of life, in three replications, 20 birds each, in a closed building on litter. A three- histology, morphometry of muscle fi bers -stage feeding program was applied in the rear- ing period. Feed mixtures used in the experiment did not differ in contents of nutrients. In the fi rst INTRODUCTION stage of rearing (till 21 days of life) the chickens were fed a standard starter feed mixture contain- Contemporarily only highly-selected hy- ing: 20.98% of total protein, 2,845 kcal/kg of me- brids of slaughter chickens and turkeys tabolizable energy, 1.17% of lysine and 0.98% of met. + cys. In the period from 22 to 56 days of are used in the production of poultry life they were receiving a grower type mixture meat in Poland. White meat of chick- containing: 20.0% of total protein, 2,900 kcal/kg ens, compared to red meat, is deemed as of energy, 1.04% of lysine and 0.88% of met. + basic in a healthy diet of man owing to, + cys., whereas contents of these components among other things, relatively low con- in the fi nisher type mixture administered till the end of the rearing period were as follows: 18% tents of fat and cholesterol. In addition, of protein, 2,965 kcal/kg of EM, 0.90% of lysine consumers are convinced by a relatively and 0.78% of met. + cys. On day 63 of rearing, low price and usually convenient por- 12 cockerels and 12 hens were selected random tions of this meat as well as by a lack of from each group. The birds were slaughtered in religious restrictions in its consumption a poultry slaughter house, and specimens of their (Jaturasitha 2004). major breast muscle (m. pectoralis superfi cialis) and thigh muscle (biceps femoris) were sampled Quality traits of poultry meat are de- for analyses. The breast and leg muscles of the termined by many factors, including spe- birds were characterized by diversifi ed diameters cies, breed or genetic line of birds, rear- of muscle fi bers depending on bird genotype. ing conditions, pre-slaughter handling The greatest diameters were found in breast and and meat processing procedures. The leg muscles of the slow-growing Hubbard JA 957 broilers, which was confi rmed statistically key one include the origin of birds and 114 M. Łukasiewicz et al. their age at slaughter. These two factors “skeletal muscles and adherent adipose, infl uence mainly the organoleptic traits connective and bone tissues, derived of meat, but also affect its technological from carcasses, half carcasses or quarter properties. A signifi cant factor determin- carcasses of particular types of animals ing meat quality is the rearing system. for slaughter”. One of the most valuable, In the intensive production of slaughter not only from the dietetic point of view, chickens, constituting the main source is poultry meat being a source of bal- of poultry meat in Poland, use is mainly anced protein, amino acids (e.g. leucine, made of hybrids with a high genetically- isoleucine, valine or arginine), fatty ac- -determined production potential. Con- ids and minerals (Wangang et al. 2010). sumers of poultry meat are increasingly High digestibility of poultry meat, its more interested in the welfare of animals assimilability, availability and low price as well as in the quality and safety of fi - make it very popular amongst consumers nal products. Many of them pay attention (Nowak and Trziszka 2010). The histo- to housing conditions, rearing system logical structure of muscles is subject to and color of carcass. Today also Polish some changes depending on their func- consumers are considering the purchase tions (Klont et al. 1998). Factors that of meat originating from chickens reared affect changes in the character of mus- in less intensive systems and addition- cle fi bers include: gender (Ozawa et al. ally having access to free ranges, as they 2000), age (Candek-Potokar et al. 1998), believe that this meat is characterized by breed (Ryu et al. 2008) and physical ac- higher quality. Furthermore, they search tivity (Karlsson et al. 1999). The quality for meat with good fl avor and health- of meat is determined by the content of -promoting values. Meat of slow-grow- three tissues: crosswise striated muscle ing chickens is characterized by a higher tissue, connective tissue and fatty tissue. content of protein and a low fat con- Mutual proportions and type of particu- tent desired by consumers (Khantaprab lar muscle fi bers may lead to changes in et al. 1997, Połtowicz et al. 2003). The the sensory assessment of meat (Damez elongation of rearing period affects the and Clerjon 2008). concentration of chemical compounds Meat of Silkies chickens has been for in breast and leg muscles, their more at- years appreciated in the Asian cuisine ow- tractive aroma, taste and, thus, sensory ing to its health-promoting values. Meat properties (Fujimura et al. 1994, Qing- of these chickens is characterized by dark hua 1994). A faster rate of these birds (blue) color of skin and tissue (Siriwan body weight gain, at unequal develop- et al. 2004). Nowadays, they are also be- ment of their entire bodies, often leads coming popular in America where their to metabolic disorders, exerts an adverse meat is deemed to be “super food” and effect on their health status and – most of “exclusive meat”. They owe this status all – on meat quality, which is especially to, among other things, high contents of signifi cant to consumers. antioxidants, proteins, vitamins (mainly Meat and animal products are essen- B-group vitamins and fat-soluble vita- tial components of a man diet. Accord- mins) as well as macro- and microele- ing to PN-65/A-82000, meat constitutes ments (calcium, iron, phosphorus). Histological profi le of breast and leg muscles of Silkies... 115

The Silkies chickens came to Europe protein, 2,900 kcal/kg of metabolizable from or from eastern , where- energy (EM), 1.04% of lysine and 0.88% as old literate says also about Singapore. of met. + cys., whereas contents of these Amateur breeders keep these hens main- components in the fi nisher type mixture ly due to their strong hatching instinct administered till the end of the rearing and great care over chickens. The Silkies period were as follows: 18% of protein, hens are trustful and are reared for their 2,965 kcal/kg of EM, 0.90% of lysine atypical fl uffy plumage (silky). They are and 0.78% of met. + cys. characterized by a dark mulberry color of One-day chicks, after weighing and and wattles turquoise color of ear- tagging, were allocated to two groups lobes, dark eyes and exceptional crop of (each of 60 birds), in three replications hair. Worthy of special attention is dark (20 birds each). On day 63 of rearing, (blue) color of their skin as well as fi ve from each group 12 cockerels and 12 toes in each foot. Today, this ornamen- hens were selected random from each tal hen in several colors is also reared in group. The birds were slaughtered in Europe. a poultry slaughter house, and specimens The goal of this study was to deter- of their major breast muscle (m. pecto- mine differences in the histological and ralis superfi cialis) and thigh muscle (bi- morphological structure of fi bers of ceps femoris) were samples for analyses. breast and leg muscles of Silkies chick- The samples (0.5×0.5×1.0 cm) were col- ens and slow-growing Hubbard JA 957 lected within 15 minutes since slaughter chickens. after appropriate chicks bleeding, and subjected to 24-hour fi xing. Next, the samples were rinsed in ethyl alcohol to METHODS AND MATERIALS remove the fi xing agent and dehydrated using a series of ethyl alcohols with in- The experiment was conducted with 60 creasing concentration. The dehydrated slow-growing Hubbard JA 957 broilers preparation were saturated with paraffi n. and with 60 Silkies chickens reared un- The saturation process was conducted in rd til the 63 day of life, in three replica- an incubator, at a melting point of paraf- tions 20 birds each, in a closed building fi n. Its duration was adjusted to the sam- on litter. A three-stage feeding program pled specimens of muscles and lasted was applied in the rearing period. Feed a few hours. In result, paraffi n blocks mixtures used in the experiment did not were formed. Paraffi n preparations were differ in contents of nutrients. In the sectioned using a Leica RM 2265 rotary fi rst stage of rearing (till 21 days of life) microtome (Leica Microsystems, Nuss- the chickens were fed a standard starter loch, Germany) by cutting the muscles feed mixture containing: 20.98% of total crosswise into 6 μm thick sections that protein, 2,845 kcal/kg of metabolizable were next stained with a standard H+E energy, 1.17% of lysine and 0.98% of method (Ostaszewska et al. 2008). In met. + cys. In the period since 22 to 56 each preparation, diameters of 300 mus- days of life they were receiving a grower cle fi bers were measured with a Nikon type mixture containing: 20.0% of total Ellipse E200 light microscope equipped 116 M. Łukasiewicz et al. in a Nikon DS-Fi2 camera and COOL fi bers improve its juiciness. In the group view 2.7 software. of Hubbard JA 957 chickens, diameters Results were developed statistically of breast muscle fi bers were relatively using the variance analysis, computed equal, 75–90% of the fi bers in particu- with the method of the least squares us- lar bundles had diameters in the range ing statistical software SPSS 19.0 PL of 70–80 μm, and 10–30% of the fi bers for Windows (SPSS Inc., Chicago, IL, had diameters in the range of 25–40 μm. USA). Differences were found signifi - In the group of Silkies chickens, dia- cant at p ≤ 0.05 and p ≤ 0.01. meters of breast muscle fi bers were more equalized and reached 25–35 μm in ca. 80–90% of the fi bers and less than 25 μm RESULTS AND DISCUSSION in 10–15% of the fi bers. The same tendency was observed in Results of microstructural analysis of case of leg muscles – larger diameters the major breast muscle (m. pectoralis were demonstrated in Hubbard JA 957 superfi cialis) and thigh muscle (biceps birds. Diameters of leg muscle fi bers in femoris) of chickens were presented the group of Hubbard JA 957 chickens in Table 1. Both the breast (Figures 1 were relatively equalized, i.e. 70–90% of and 2) and leg muscles of the experimen- fi bers in particular bundles had diameters tal birds were characterized by diversifi ed between 60 and 70 μm, and 10–30% of diameters of muscle fi bers depending on fi bers had diameters in the range of 25– genotype. Cross sections showed a dis- –50 μm. In the group of Silkies chickens, tinct shelf structure in most of the fi bers. the diameters of leg muscle fi bers, like- The largest diameter was determined in wise these of breast muscles, were more the breast muscle of slow-growing Hub- equalized compared to the Hubbard JA bard JA 957 birds, which was confi rmed 957 chickens because 80–90% of fi bers statistically (P ≤ 0.01) – Table 1. The in particular bundles had diameters in the diameter of breast muscle fi bers in the range of 20–35 μm, and in 10% of the fi b- group of Hubbard JA 957 birds reached ers diameters were lesser than 20 μm. 75.61 μm, whereas in the group of Silk- According to Dransfi eld and Sośnicki ies chickens it was more than two times (1999), the fast-growing chickens are smaller (33.23 μm). More advantageous characterized by a larger diameter of to consumers is fi ne-fi ber meat as fi ne muscle fi bers compared to the chickens TABLE 1. Fiber diameter of breast and leg mus- with a slow growth rate. As reported by cles of chickens (♀ + ♂) other authors, increased diameter and length of muscle fi bers may be due to Fiber diameter (μm) Group intensive selection (Brocka et al. 1998, breast muscles leg muscles Guernec et al. 2003) and changes ap- Silkies 33.23B 28.73B pearing in the size and shape of muscle Hubbard JA 957 75.61A 62.51A fi bers (Bogucka and Kapelański 2004). SEM 1.311 1.262 Skeletal muscles are a heterogenous tissue that is characterized by multi- A, B – statistically signifi cant differences at P ≤ 0.01. ple types of muscle fi bers that affect Histological profi le of breast and leg muscles of Silkies... 117

breast muscle – Silkies chickens leg muscle – Silkies chickens

breast muscle – Hubbard JA957 chickens leg muscle – Hubbard JA957 chickens FIGURE 1. Cross-section of breast and leg muscles of Silkies and Hubbard JA 957 chickens diversifi ed characteristics of particular 1999). Dankowiakowska et al. (2012) muscles (Bottinelli and Reggiani 2000). showed that an increased temperature of There are many factors that contribute to hatching to 38.5 or 39.0°C resulted in an changes in the character of muscle fi bers, increased number of fi bers with a larger these including: gender (Ozawa et al. diameter in the breast muscle of broiler 2000), age (Candek-Potokar et al. 1998), chickens. The experiment conducted by breed (Ryu et al. 2008), and physical ac- Sobolewska et al. (2011) indicates that tivity (Jurie et al. 1999). Investigations in the period of 35-day rearing of broiler conducted by Castellini et al. (2002a, chickens, a more intensive increase of 2002b) and Branciari et al. (2009) dem- muscle fi ber diameters was observed be- onstrate that the access to free runs may tween days 8 and 21. The breast muscle also infl uence the character of muscle fi bers of chickens are fully developed al- fi bers. ready around the 8th week of birds life. The number and type of muscle fi b- The impact of muscle fi bers character ers are determined already in the fetal on meat quality has been investigated for life (Petersen et al. 1998, Karlsson et al. years. The character of muscle fi bers is 118 M. Łukasiewicz et al. determined genetically and is typical of The end product may be especially ap- particular hybrids and breeds. Histologi- preciated by a taste-loving consumer, cal and biochemical properties of mus- a connoisseur or a dietitian owing to its cles, including the type, number, propor- originality and to the fact of being a val- tions and diameter of muscle fi bers as uable source of animal protein. well as their metabolic character affect the pH value and water absorbability of meat, whereas these traits infl uence meat REFERENCES quality (Bereta and Eckert 2010). From a consumer’s point of view, proportions BERETA A., ECKERT R., 2010: Profi l hi- and diameters of particular types of fi b- stologiczny mięśni ma ścisły związek z jakością mięsa wieprzowego. Wiad. ers may lead to changes in the sensory as- Zoot. 43, 4, 65–70. sessment. A higher number of fi bers with BOGUCKA J., KAPELAŃSKI W., 2004: small and medium diameters improves Histopathological changes in longissi- meat quality (Choi and Kim 2009). mus lumborum muscle of Polish Land- Additional increase in the thickness of race and crossbred Stamboek and Torhyb white muscle fi bers has a positive impact pigs. Anim. Sci. Pap. Rep. 22, Supp. 3, on tenderness but a negative impact on 67–72. BOTTINELLI R., REGGIANI C., 2000: juiciness of meat. In case of red fi bers, Human skeletal muscle fi bres: molecular this dependency is opposite (Cameron and functional diversity. Prog. Biophys. et al. 1998, Migdał et al. 2005). In hens, Mol. Biol. 73, 195–262. almost entire breast muscle is constituted BRANCIARI R., MUGNAI C., MAM- by white fi bers. MOLI R., MIRAGLIA D., RANUCCI D., DAL BOSCO A., CASTELLINI C., 2009: Effect of genotype and rearing CONCLUSION system on chicken behavior and muscle fi ber characteristics. J. Anim. Sci. 87, Results of histomorphometric analyses 4109–4117. BROCKA L., HULSEDGGE B., MER- demonstrate that breast and leg muscles KUS G., 1998: Histochemical character- of slow-growing Hubbard JA 957 chick- istic in relation to meat quality properties ens were characterized by a signifi cantly in the longissimus lumborum of fast and greater diameter of muscle fi bers com- lean growing lines of large white pigs. pared to muscles of Silkies chickens. Meat Sci. 50 (4), 441–420. These differences were most of all due CAMERON N.D., OKSBJERG N., HENCK- to genetic factors. Though slowly grow- EL P., NUTE G., BROWN S., WO- ODN J.D., 1998: Relationships between ing, the Hubbard JA 957 chickens are se- muscule fi bre traits with meat eating in lected for body weight gains. The Silkies pigs, BSAS Annual Meeting, 123. chickens are a native breed of Chinese CANDEK-POTOKAR M., ZLENDER B., hens that have for years been valued LEFAUCHEUR L., BONNEAU M., for the health-promoting properties of 1998: Effects of age and/or weight at their meat (higher content of carnosine). slaughter on longissimus dorsi muscle: Chickens of this breed could be success- biochemical traits and sensory quality in pigs. Meat Sci. 48, 287–300. fully used for small-scale production. Histological profi le of breast and leg muscles of Silkies... 119

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PETERSEN J.S., HENCKEL P., OKS- dzono na 60 brojlerach wolno rosnących Hub- BJERG N., SØRENSEN M.T., 1998: bard JA 957 oraz 60 kurczętach Jedwabistych Adaptations in muscle fi bre characteris- odchowywanych do 63. dnia życia, w trzech tics induced by physical activity in pigs. powtórzeniach po 20 szt., w zamkniętym budyn- J. Anim. Sci. 66, 733–740. ku na ściółce. W okresie odchowu zastosowano PN-65/A-82000. Mięso i podroby zwierząt trzyfazowy program żywienia. Mieszanki użyte rzeźnych. Wspólne wymagania i bada- w doświadczeniu nie różniły się pod względem nia. zawartości składników pokarmowych. Kurczęta POŁTOWICZ K., WĘŻYK S., CYWA-BEN- w pierwszym okresie odchowu do 21. dnia ży- KO K., 2003: Wykorzystanie rodzimych cia żywiono standardową mieszanką typu starter: o zawartości 20,98% białka ogólnego, ras kur w produkcji mięsa bezpiecznego 2845 kcal na kg energii metabolicznej, 1,17% li- dla zdrowia konsumenta. Praca zbioro- zyny i 0,98% met. + cys. W okresie od 22. do 56. wa. Zakrzewo, 21–32. dnia stosowano mieszankę typu grower, zawierają- QINGHUA Z., 1994: Palace chicken supe- cą 20,0% białka ogólnego, 2900 kcal na kg energii rior Chinese breed. Misset World Poultry metabolicznej (EM), 1,04% lizyny i 0,88% met. + 10, 47. cys. Zawartość wcześniej wymienionych składni- RYU Y.C., CHOI Y.M., LEE S.H., SHIN H.G., ków w mieszance fi niszer, stosowanej do końca CHOE J.H., KIM J.M., HONG K.C., odchowu, wynosiła odpowiednio: 18% białka, KIM B.C., 2008: Comparing the histo- 2965 kcal na kg EM, 0,90% lizyny i 0,78% met. + chemical characteristics and meat quality cys. W 63. dniu odchowu wybrano z każdej grupy traits of different pig breeds. Meat Sci. po 12 kogutów i 12 kur o masie ciała zbliżonej 80, 363–369. do średniej w grupie. Kurczęta ubito w ubojni SIRIWAN P., RATTANAWARAHA A., drobiu i pobrano wycinki mięśnia piersiowego PIMSARN S., SONLOY W., NAGKIT- powierzchniowego (m. pectoralis superfi cialis) SET S., 2004: A preliminary study in or- oraz dwugłowego uda (biceps femoris). Mięśnie der to develop and improve black-boned piersiowe i nóg ptaków doświadczalnych charak- chickens. J. Anim. Husbandry 68, 44–53 teryzowała zróżnicowana średnica włókien mięś- (In Thai). niowych w zależności od genotypu. Największą SOBOLEWSKA A., ELMINOWSKA- średnicą charakteryzowały się mięśnie piersiowe -WENDA G., WALASIK K., BOGUC- i nóg ptaków wolno rosnących Hubbard JA 957, KA J., SŁAWIŃSKA A., SZCZERBA A., co zostało potwierdzone statystycznie (P ≤ 0,01). Stwierdzono, że średnica włókien mięśniowych ŻMUDA-TRZEBIATOWSKA M., 2011: kurcząt Jedwabistych była o ponad połowę mniej- Increase in thickness of the pectoral mus- sza w porównaniu do kurcząt Hubbard JA 957. cle fi bers in meat-type chicken beetween 1st and 35th day of fattening. Proc. XXIII International Poultry Symposium PB MS. received in November 2013 WPSA, Poznań. WANGANG Z., XIAO S., SAMARAWEE- RA H., LEE E. J., AHN D., 2010: Improv- Authors’ address: ing functional value of meat products. Monika Łukasiewicz Meat Sci. 86, 15–31. Wydział Nauk o Zwierzętach SGGW Katedra Szczegółowej Hodowli Zwierząt Zakład Hodowli Drobiu Streszczenie: Profi l histologiczny mięśni pier- ul. Ciszewskiego 8, 02-786 Warszawa siowych i nóg kurcząt Jedwabistych oraz wolno Poland rosnących Hubbard JA 957. Badanie przeprowa- e-mail: [email protected] Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 121–127 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Comparison of components and number of Nosema sp. spores of wintering Carniolan and Italian bees debris BEATA MADRAS-MAJEWSKA1, LUIZA OCHNIO2, MACIEJ OCHNIO1, JOANNA ŚCIEGOSZ1 1Department of Animal Breeding 2Departament of Informatics Warsaw University of Life Sciences – SGGW

Abstract: Comparison of components and num- INTRODUCTION ber of Nosema sp. spores of wintering Carniolan and Italian bees debris. The aim of the experi- During winter bees do not leave the nest ment is to investigate and compare the compo- nents of the winter debris of two breeds of bees: and accumulate feces in the rectum wait- Carniolan and Italian and microscopic tests of the ing for warmer days and for an opportu- bees samples from the experimental debris for the nity to leave the hive and to empty bow- presence of Nosema sp. The study was carried els (Muszyńska and Bornus 1981). When on 20 wintering bee colonies in the apiary of the the colonies in the autumn and winter Apiculture Division of Warsaw University of Life Sciences – SGGW. time create a cluster, the activity of bees The research material was debris falling dur- is reduced. They move at the small space ing the winter at the bottom of the hive that was within the comb and collect only a small bees, wax cappings, eggs, wax, pollen pellets, amount of food needed to maintain the dark hardened fungal clods and fecal stains on the proper conduct of the necessary proc- pads. The examination was conducted during the overwintering 2008/2009. In the spring of 2009 esses of life. Flights of bees are rare, but microscopic examination was conducted (samples only during the transient (usually fl eet- of bees from the experimental debris for the pres- ing) warming. Sometimes at the end of ence of Nosema sp.). Pollen pellets are originated February in our climatic conditions, also from bee bread, which bees use to feed the brood happen that temperature rises above and was collected from two different breeds of 10°C, which bees use for the fi rst fl ights bees. The presence of eggs in debris testifi es to the fact that in winter queen of two studied breeds for purifi cation and water supply of hive of bees are also lying eggs. The presence of fecal (Muszyńska and Bornus 1981). spots in debris is sporadic but it is the sign of no- Bees do not clean their nests and semosis. Microscopic examination confi rmed the do not throw away trash during winter. assumption that the appearance of the fecal spots Falling to the bottom of the hives dead was a symptom of Nosema sp. presence. Nosema sp. infects bees of both breeds. Carniolan breed insects, food debris (pollen, honey, sug- bees tend to self-medicate from nosemosis. Lack ar), wax cappings, dried crumbs, pieces of feces does not indicate the lack of nosemosis in of propolis and other various organic the case of both mentioned breeds of bees. molecules and inorganic contaminants Key words: honeybee, winter debris, nosemosis, (sand) accumulate in the form of de- breeds of bees bris (Chmielewski 1992). The practice 122 B. Madras-Majewska et al. of wintering bees of different races in The inner and outer walls of the areas with harsh winters shows that the hive contaminated by feces indicate the fi rst debris is made up of bees that have presence of nosemosis in the colony. It fallen to the bottom and froze. The high- should be done a study to determine the est daily debris is observed during the debris infection of bees with Nosema sp. fi rst two months of overwintering hives, spores. when there is the peak concentration of Nosemosis of bees is well-known for bees in the cluster and there is continu- beekeepers and veterinarians as a chronic ous replenishment of outer shell by the of the honey bee (Apis mellifera) caused bees from a center of the comb. Debris by intraspecifi c parasite N. apis specie in the second half of wintering consist – Eukaryota, Fungi, Microsporea (Gajda of the bees died due to excessive physi- 2010). The parasite locates and develops ological overload, with overfi lled gut. in the cells of the midgut epithelium of Next appears the stability of thermoregu- workers, queens, and drones (Gliński lation violation in comb, increased food and Rzedzicki 1983). Nosema spores are consumption, death of older bees in the ingested along with food and water by colony, which is an important evalu- adult honey bees. Symptoms of the dis- ation index of wintering colonies and ease include dysentery, increased winter their possible start to the new beekeep- loss of bees, reduced honey production, ing season (Skubida 1994). According and shortened life span of the worker to Chmielewski (1991, 1992), on the ba- bees. These symptoms are most notice- sis of qualitative and quantitative debris able in the spring. The disease usually composition can be conducted observa- fades away in the summer and briefl y re- tions on the state of health of the colony. turns again in the fall of the year (Reed Small debris refl ects good overwintering 2010). Worker bees and queen bees have of bees, and large and moist one, the im- been found to become infected at simi- proper overwintering. Annual examina- lar rates when inoculated with Nosema tion of debris allows the early detection spores (Thomas et al. 2004). The clinical of certain diseases and the possibility of symptoms appear only in severe course potential therapeutic interventions such of disease (Table 1). as (Chmielewski 1992): From a clinical point of view, nose- • Debris – dead bees and the mentioned mosis is a disease of older bees working earlier ingredients are organic matter, outside the hive. Young bees, up to 15 which is the breeding ground and the days of age, were infected less often. The substrate for the development of dif- greatest severity of disease occurs in ear- ferent species of mites as house dust ly spring – April, May (Glinski and Rze- mite, dried fruit mite, brown mite, dzicki 1983). The disease spreads easily storage mite, narrow mite, fl our mite; in a bee colony as bees licking the feces • The presence of fungal clods indi- of sick bees infect themselves. Nosemo- cates the occurrence of ; sis causes death of bees (Marcinkowski • Traces of feces are a sign of diarrhea 1994). in the colony, which is caused by In the case of notice the symptoms of nosemosis infection. the disease it is proved it can be treated Comparison of components and number of Nosema sp. spores of wintering... 123

TABLE 1. Summary of symptoms and changes occurring in nosemosis Etiology Disease Bees age Clinical symptoms Post-mortem lesions and occurrence – loss of fl y ability, abdominal Nosema apis older distension, diarrhea midgut strongly in- Nosemosis winter, early bees – feces yellow loam color, grit- fl ated, pearly white spring, spring -like consistency with many different preparations (Topol- The study was carried on 20 wintering ska et al. 2008, Nanetti 2009, Thrasyvoulou bee colonies (10 of Carniolan bees and et al. 2009, Gajda 2010). At the low level 10 of Italian bees) in the apiary of the of colonies contamination disease resolves Apiculture Division of Warsaw Univer- spontaneously. In a time of generous food sity of Life Sciences – SGGW. There base and of radical replacement of source were selected colonies of similar force, of infection – alive infected bees, it disap- in the same types of hives (Wielkopol- pears (Tomaszewska 1995). ska bee hive), with the same number of Results of apiary wintering is deter- frames and with a similar number of bees mined by the number of dead colonies dur- in colonies measured accordingly to the ing this period of time, as well as the state method described by Aimdorf and Gerig of health of those colonies which survived (1999). the wintertime. Taking care of health con- The bottom boards of examined 20 trol and condition status of colonies during hives were checked eight times during the overwintering it is recommended a de- the wintering season. The measurements bris composition and number of Nosema were taken at two-week intervals start- sp. spores inspection. Each year a number ing from 19 November 2008. Therefore, of colonies in the apiary cannot stand the plastic pads were used, which have pre- wintering what is resulting in their weak- viously been properly fi tted to the shape ening or death. On the strength of the colo- and dimensions of the bottom board of ny during the wintering signifi cant impact the hive so that they could be easily re- has breed of bees. moved and replaced to and from hives The aim of the experiment was to in- (600 × 375 mm). Pads were placed in vestigate and compare the components hives on bottom boards under the frames of the winter debris of two breeds of (5.11.2008) and all debris could fall free- bees: Carniolan and Italian and micro- ly directly to them. After each debris col- scopic tests of bees samples from the lection plastic pads were cleaned out and experimental debris for the presence of again placed on the bottom of the hive. Nosema sp. Winter debris was collected and the description of the components (such as bees, wax cappings, eggs, wax, pol- MATERIAL AND METHODS len pellets, dark hardened fungal clods and fecal stains on the pads) measure- The experiment was conducted on honey ment was made. Each test accurately bee (Apis mellifera) Carniolan and Ital- determined the quantitative and qualita- ian during the overwintering 2008/2009. tive composition of debris. To test the 124 B. Madras-Majewska et al. signifi cance of differences of the breeds 10 squares 2 mm3 of bees in each group of debris compo- 1 square 0.2 mm3 nents (such as number of bees, number 1/4 squares (4 – small squares) 0.05mm3 of eggs and number of wax cappings mean values) Student’s t-test was used. calculated number of spores 10,000 This test applies to the statistics made on X 0.05 studied samples and is suffi cient for used in the research groups size. Differences (1) between the means in all groups were It should be noted that the tested col- calculated at the 0.05 signifi cance level onies were not treated for disease (Olech and Wieczorek 2012). . There were also observed all abnor- mal changes that could be symptoms RESULTS AND DISCUSSION of diseases such as fecal spots or dark hardened fungal clods. Then, the tested In Carniolan bees hives on the bottom material was packed into separate enve- board was found on average less bees lopes with pre-marked number of hives, and wax cappings but more eggs than the date and the information about the in Italian bee hives. Because of the wide debris. Samples were stored in a freezer variation in results (some hives had re- (at –20°C). ally small amounts of debris, but some In the spring of 2009 microscopic hives were heavily littered) the standard examination was conducted (samples of deviations were high. There were not bees from the experimental debris for found signifi cant differences between the presence of Nosema sp.). Nosema the means in any group (at the 0.05 sig- detection method used in the study re- nifi cance level) of tested components lied mainly on the procedures developed between Carniolan and Italian race by Wilson and Ellis (1966), Cantwell (Table 2). (1970), Fingler et al. (1982), Kauko et al. In debris there was generally very (2002), Rogers et al. (2002), OIE (2004), small amount of wax itself, but pol- Topolska and Hartwig (2005). len pellets was very common in almost Examination was carried out as fol- every hive and in almost whole season lows: to each sample were collected 30 (from November 2008 till March 2009). bees to test it for the presence of Nosema Especially in February when pollen pel- sp. Abdomens were detached with tweez- lets were found in all examined hives. ers and were homogenized with distilled Feces were observed only in three hives water in a ratio of 1 ml per bee. A drop with various intensity and in one case the of the homogenate was transferred to a trace of faces did not indicated nosemo- chamber used for counting blood cells sis infection (which was verifi ed later by (Fusch Rosenthal) and calculated the the test for Nosema sp. presence). number of possible Nosema sp. spores Fungal clods were found in January, in four small squares. In the case of in- in February and in March in four hives, fection number of Nosema sp. was cal- but not too many of them. Generally it culated in a single bee according to the was more often observed in Italian bees following formula: hives (Table 3). Comparison of components and number of Nosema sp. spores of wintering... 125

TABLE 2. The average number of some debris components in the whole wintering season in Carniolan and Italian bees hives Apis mellifera breed Value Number of bees Number of eggs Number of wax cappings mean 236.40 48.00 59.60 Carniolan SD 23.02 94.65 39.30 mean 326.50 33.00 61.75 Italian SD 110.41 34.30 19.00

TABLE 3. The results of the investigation of some debris components in Carniolan and Italian bees hives Bee breed Wax Pollen pellets Feces Fungal clods Carniolan + ++++ +++ + Italian + ++++ – ++ Designations: (–) lack of component, (+) single presence, (++) double or few times presence, (+++) numerous, (++++) high intensity.

TABLE 4. Results of the dead bees samples tested for Nosema sp. in 2008/2009 season The level of infection Bee breed Number of observations – (N) + (S) ++ (M) +++ (H) Carniolan 80 69 3 2 6 Italian 80 76 0 1 3 Designations: (–) no spores, (+) single spores not in each fi eld of vision, (++) single spores in each fi eld of vision, (+++) numerous spores in each fi eld of vision, (N) – not infected, (S) – slightly infected, (M) – moderately infected, (H) – heavily infected.

From that number only few hives CONCLUSIONS showed Nosema sp. appearance. More often and more heavily infected were 1. Wax debris consisted of bees, eggs, Carniolan bees (Table 4). wax cappings, crumbs of wax, pollen It was very interesting that at the be- pellets, fecal spots and fungal clods ginning of the wintering the number of of both bee breeds. spores was high and from November till 2. The presence of eggs in debris of December even rose to the number of both breeds testifi es to the fact that in 16.6 millions of spores, it seemed to at winter some queen bees are also ly- the same level for some time but later on ing eggs. it seemed to fall and in March it reached 3. The presence of fecal spots in debris about half a million of spores (Fig. 1). is sporadic in studied breeds but it is the sign of nosemosis. 126 B. Madras-Majewska et al. Number of spores (in millions)

FIGURE 1. Number of spores of Nosema sp. in one of the Carniolan bees colony examined in 2008/2009 season

4. Microscopic examination confi rmed FINGLER E.G., NASH W.T., SZABO T.I., the assumption that the appearance 1982: A Comparison of To Techniques of the fecal spots was a symptom of for the Measurement of Nosema Disease Nosema sp. presence. in Honey Bee Colonies Wintered in Al- berta, Canada. Am. Bee J. 122, 369. 5. Nosema sp. infects bees of both GAJDA A., 2010: Nosema ceranae w rodzinach breeds. pszczoły miodnej. Życie Wet.: 140–143. 6. Lack of feces does not indicate the GLIŃSKI Z.F., RZEDZICKI J., 1983: Cho- lack of nosemosis. roby pszczół. PWN, Warszawa. KAUKO L., HOŃKO S., VARTIAINEN H., 2002: Winter mortality and Nosema apis REFERENCES Z. The diagnostic value of nosema spore counts – a clinical aproach. Ann UMCS AIMDORF A., GERIG L., 1999: Lehrgang 57 (19), 199–204. zur Erfassung der Volksstärke. Informa- MARCINKOWSKI J., 1994: W dążeniu do cje szwajcarskiego Instytutu Pszczelar- sukcesu. Pszczelarz Polski 5(3), 3–8. skiego Liebefeld, 9. MUSZYŃSKA J., BORNUS L., 1981: Nie- CANTWELL G.E., 1970: Standard methods które zmiany w organizmie pszczół ro- for counting Nosema spores. Am. Bee J. botnic z zimowego kłębu. Pszczeln. Zesz. 110(6), 222–233. Nauk. 25, 15–30. CHMIELEWSKI W., 1991: Stosowanie NANETTI A., 2009: Api Herb as an alterna- wkładek na osyp zimowy walca z roz- tive product to treat Nosema infection. kruszkami. Pszczearstwo 8, 10–19. COLOSS Workshop: Nosema disease: CHMIELEWSKI W., 1992: Osyp zimo- Lack of knowledge and work standard- wy w ulach siedliskiem rozkruszków. ization. Guadala-jara, 19–22 October. Pszczelarstwo 4, 2–11. Comparison of components and number of Nosema sp. spores of wintering... 127

OIE, 2004: Manual of Diagnostic Tests and Streszczenie: Porównanie części składowych Vaccines for Terrestial Animals. 5 ed., osypu zimowego oraz ilości spor Nosema sp. Paris. pszczół rasy kraińskiej i włoskiej. Celem do- OLECH W., WIECZOREK M., 2012: Zasto- świadczenia jest zbadanie i porównanie części sowanie metod statystyki w doświadczal- składowych osypu zimowego pszczół dwóch nictwie zootechnicznym. Wydawnictwo ras kraińskiej i włoskiej oraz przeprowadzenie SGGW, Warszawa. badań mikroskopowych prób pszczół z osypów REED F., 2010: Effects of Queen Source doświadczalnych pod kontem występowania and Age of Colony on Nosema (Nosema Nosema sp. Doświadczenie przeprowadzono apis) Spore Load in Honey Bees (Apis na pszczole miodnej (Apis mellifera). Do badań użyto pszczół z 20 zimujących rodzin pszczelich mellifera). JOE 48 (4). w pasiece Pracowni Hodowli Owadów Użytko- ROGERS R.E.L., BISHOP B., MACKEN- wych SGGW w Warszawie. Materiałem badaw- ZIE K., 2002: Nosema detection. Wild- czym był osyp spadający w czasie zimy na dno ula, wood Labs Inc. tzn. pszczoły, łuseczki woskowe, jaja, wosk, ob- SKUBIDA P., 1994: Termoregulacja w okre- nóża pyłkowe, ciemne stwardniałe grudki grzybi- sie zimowania. Pszczelarstwo 2, 16–17. cze oraz plamy kału na podkładkach. Doświadcze- TOMASZEWSKA B., 1995: Leczenie nose- nie przeprowadzono podczas zimowli 2008/2009. mozy. Pszczelarstwo 4, 7–8. W okresie wiosennym 2009 roku przeprowadzono THOMAS C.W., POMPER K.W., HUNT G., badania mikroskopowe (prób pszczół z osypów THACKER E.M., JONES S.C., 2004: doświadczalnych na występowanie Nosema sp.). Nosema apis infection in worker and Obecność obnóży pyłkowych świadczy o obec- queen Apis melifera. Apidologie 35(1), ności czerwiu w rodzinach obydwu ras. Obnóża 49–54. pyłkowe pochodzą z pierzgi, którą pszczoły wy- THRASYVOULOU A., GEIRGIOS G., korzystują do karmienia czerwiu. Obecność jaj CHRYSOULA T., ELISAVET L., 2009: w osypie obydwu ras pszczół świadczy o tym, że Attempts to control Nosema ceranae in w zimie niektóre matki też czerwią. Kał wystę- puje sporadycznie i jest symptomem nosemy. Ba- Greece. 41 Kongres Apimondii, Mont- pellier. dania mikroskopowe potwierdziły przypuszcze- TOPOLSKA G., HARTWIG A., 2005: Di- nie, że pojawienie się kału świadczy o obecności agnosis of Nosema apis infection by Nosema sp. Nosema sp. poraża pszczoły obydwu investigations of two kinds of samples: ras. Pszczoły rasy kraińskiej wykazują tendencje do samoleczenia. Brak kału u dwu ras pszczół nie dead bees and live bees. J. apic. Sci. 49, wskazuje na brak nosemozy. 75–80. TOPOLSKA G., GAJDA A., HARTWIG A., 2008: Polish honey bee colony-loss dur- MS. received in November 2013 ing the winter of 2007/2008. J. apic. Authors’ address: Sci. 52, 95–104. Beata Madras-Majewska WILSON C.A., ELLIS L.L., 1966: A new Wydział Nauk o Zwierzętach SGGW technique for the detection of nosema in Pracownia Pszczelarska apiaries. Am. Bee J. 106, 131. ul. Nowoursynowska 166, 02-787 Warszawa Poland e-mail: [email protected]

Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 129–133 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

New sites of the rare Microlepidoptera species on Warsaw basin ANNA MAZURKIEWICZ, DOROTA TUMIALIS, ELŻBIETA PEZOWICZ Department of Animal Environment Biology, Warsaw University of Life Sciences – SGGW

Abstract: New sites of the rare Microlepidoptera typically anthropogenic Warsaw habitat species on Warsaw basin. An inventory studies (Pola Mokotowskie). on Microlepidoptera were performed in 2008 on Study area involved grounds neigh- meadows neighbouring the then planned War- saw-Modlin Airport. The studies revealed the bouring the then planned Warsaw-Mod- occurrence of 165 species representing 26 fami- lin Airport covered mainly by xeric lies in the area including 37 species fi rst noted in meadow communities with single dwarf Masovian Province. Species that deserve special specimens of trees and shrubs (mainly attention due to a low number of sites in Poland hawthorn and ). The communities include: pimpinellae (Zeller, 1839), Scythris seliniella (Zeller, 1839), Schiffermuelle- were extensively used (mown twice ria schaefferella (Linnaeus, 1758), Pseudateme- a year without undersowing and fertilisa- lia fl avifrontella (Denis et Schiffermüller, 1775), tion). verbascella (Denis et Schiffermüller, 1775), inopella (Zeller, 1839), Epi- notia rubiginosana (Herrich-Schäffer, 1851), MATERIAL AND METHODS Lobesia artemisiana (Zeller, 1847), pulvillana (Herrich-Schäffer, 1851), Studies were carried out since March till verbascalis (Denis et Schiffermüller, 1775), Ep- ascestria pustulalis (Hübner, 1823). the end of August 2008. Basic methods applied included: sweeping with Key words: Microlepidoptera, new records, War- net over herb vegetation, catching speci- saw basin mens by a single stroke, fl ushing out the moths by shaking them off from tree and INTRODUCTION shrub branches. Moreover, 6 times (once a month) a light trap was used in places Mazowiecka Lowland is a region poorly most interesting with respect to plant recognised in terms of the occurrence cover. 250 W mercury lamp used for this and distribution of species from many purpose was powered by 750 W Honda families of tiny moths (Microlepido- EX7 generator. Pre-imaginal stages were ptera); moreover, many data have only also searched for to breed them until ob- an historical importance. The only com- taining imagines. plex study that has recently appeared from this region pertains to Gracillari- RESULTS AND DISCUSSION idae in the Skarpa Ursynowska Reserve (Jaworski 2009), several rare species of Performed fi eld studies in the Warsaw- Microlepidoptera were reported by Ma- -Modlin Airport revealed the occurrence zurkiewicz and Wrzesień (2008) from of 165 species of Microlepidoptera, 130 A. Mazurkiewicz, D. Tumialis, E. Pezowicz which is ca. 8.5% of this group in Poland. Systematic order and nomencla- Caught species belonged to 26 families. ture is presented based on a study “The From among caught species 37 were new of Poland. A distributional for Masovian Province (Table 1). Eleven checklist” (Buszko and Nowacki 2000). species described below deserve special attention – they are most rare from among 1. Depressaria pimpinellae (Zeller, species found in the area and known from 1839). Caterpillars of this species devel- a few sites in Poland. The description of op on umbels of the burnet saxifrage and some species is supplemented by a short the greater burnet saxifrage (Pimpinella diagnose of the state of their population in saxifraga and P. major Toll, 1964). In the study area. the last 50 years, the species has been

TABLE 1. A list of recorded species of Microlepidoptera new for Masovian Province No. Species 12 1. semipurpurella (Stephens, 1835) 2. Eriocrania sparrmannella (Bosc, 1791) 3. Eriocrania subpurpurella (Haworth, 1828) Tischeriidae 4. Emmetia gaunacella (Duponchel, 1843) 5. Emmetia marginea (Haworth, 1828) 6. monachella (Hübner, 1796) 7. Tinea semifulvella (Haworth, 1828) Yponomeutidae 8. trifasciata (Staudinger, 1871) 9. messingiella (Fischer v. Röslerstamm, 1840) 10. bipunctella (Fabricius, 1775) Depressariidae 11. Agonopterix alstromeriana (Clerck, 1759) 12. Depressaria pimpinellae (Zeller, 1839) 13. lobella (Denis et Schiffermüller, 1775) 14. oculella (Thunberg, 1794) 15. Semioscopis steinkellneriana (Denis et Schiffermüller, 1775) 16. albidella (Nylander, 1848) 17. Elachista argentella (Clerck, 1759) New sites of the rare Microlepidoptera species... 131

Table 1 cont. 12 18. Elachista poae (Stainton, 1855) 19. Elachista pollinariella (Zeller, 1839) Scythrididae 20. Scythris seliniella (Zeller, 1839) 21. Schiffermuelleria schaefferella (Linnaeus, 1758) 22. colutella (Fabricius, 1794) Amphisbatidae 23. Pseudatemelia fl avifrontella (Denis et Schiffermüller, 1775) 24. brizella (Treitschke, 1833) 25. derasella (Denis et Schiffermüller, 1775) 26. infernella (Herrich-Schäffer, 1851) 27. (Denis et Schiffermüller, 1775) 28. scalella (Scopoli, 1763) 29. (Zeller, 1839) 30. Epinotia rubiginosana (Herrich-Schäffer, 1851) 31. metzneriana (Treitschke, 1830) 32. Lobesia artemisiana (Zeller, 1847) 33. (Herrich-Schäffer, 1851) 34. fabriciana (Linnaeus, 1767) 35. Anania verbascalis (Denis et Schiffermüller, 1775) 36. Epascestria pustulalis (Hübner, 1823) 37. fumella (Eversmann, 1844) reported from several sites in various ated gypsophila Gypsophila fastigiata. parts of Poland (Buszko and Nowacki This is a one-generation species (moths 2000). Several specimens were caught appear since the middle of May till July). with the butterfl y net. Population of It occurs locally but in sites of occur- this species is probably numerous in the rence is relatively abundant. Sites of this study area. species known so far in Poland are situ- Scythrididae ated mainly in the south of the country 2. Scythris seliniella (Zeller, 1839). (Baran 2005). Several specimens were Larvae develop on the fi eld wormwood caught with butterfl y net in few places, campestris and on the fastigi- so population is probably numerous. 132 A. Mazurkiewicz, D. Tumialis, E. Pezowicz

Oecophoridae in Poland (Buszko and Nowacki 2000). 3. Schiffermuelleria schaefferella One specimen was caught with the light (Linnaeus, 1758). Caterpillars of this trap. species live in rotten wood and under the 8. Lobesia artemisiana (Zeller, 1847). bark of various dead trees (Toll 1964). It Host plants for the larvae are mainly the was reported from single stands in vari- common bugloss Anchusa offi cinalis, the ous parts of Poland (Buszko and Nowa- viper’s bugloss vulgare and fi eld cki 2000). One specimen was caught wormwoods Artemisia spp. The species with the light trap. is known in Poland from single stands. Amphisbatidae Several specimens were caught with the 4. Pseudatemelia fl avifrontella (Denis light trap. et Schiffermüller, 1775). Caterpillars of 9. Phtheochroa pulvillana (Herrich- P. fl avifrontella feed on dead leaves (Toll -Schäffer, 1851). Caterpillars feed on 1964). In the last 50 years, the species milkvetch plants spp. The has been found in only two stands in Po- species was noted in single dispersed land (Buszko and Nowacki 2000). One sites in Poland. Single specimens of this specimen was caught with the light trap. species were caught with the light trap. Gelechiidae Pyralidae 5. Nothris verbascella (Denis et 10. Anania verbascalis (Denis et Schiffermüller, 1775). The species is tro- Schiffermüller, 1775). Host plants of the phically associated with various species larvae are germanders Teucrium spp. and of mullein Verbascum spp. Known in Po- mullein Verbascum spp. The species has land from single dispersed sites (Buszko one generation per year – adults appear and Nowacki 2000). Several specimens in June and July. Several specimens were were caught with the light trap and a net caught with the light trap. One may as- at dusk. It may be assumed that the po- sume that the species is quite numerous pulation of this species is quite numer- in the study area. ous in the study area. 11. Epascestria pustulalis (Hübner, 1823). Larvae feed (mine) in parenchy- 6. Ptocheuusa inopella (Zeller, 1839). ma of leaves of the common bugloss An- Larvae develop on the dwarf everlast chusa offi cinalis. The species is known plants (Elsner in Poland from a few dispersed stands. et al. 1999). The species in known in Po- Numerous mines with larvae were ob- land from several sites in various parts of served in many sites of the study area, the country (Buszko and Nowacki 2000). moreover, several specimens were at- One specimen was caught with the light tracted by light and several specimens trap. were caught with net during the day. The Tortricidae study area is probably inhabited by one 7. Epinotia rubiginosana (Herrich- of more numerous populations of this -Schäffer, 1851). Host plants for this spe- species in Poland. cies are pine Pinus silvestris and spruce Most of the described species are Picea excelsa. In the last 50 years the trophically associated with plants char- species has been found in only two sites acteristic for dry and open meadow com- New sites of the rare Microlepidoptera species... 133 munities which covered a larger part of Streszczenie: Nowe stanowisko rzadkich the study area. These habitats developed gatunków Microlepidoptera w Kotlinie War- there as a result of specifi c way of man- szawskiej. W 2008 roku przeprowadzono badania inwentaryzacyjne dotyczące Micro- agement (mowing twice a year) which is lepidoptera na obszarze łąk sąsiadujących recommended in order to maintain them. z planowanym wówczas portem lotniczym It would be also recommended to leave Warszawa-Modlin. W wyniku badań stwier- trees and shrubs, mainly oaks, trembling dzono na tym terenie występowanie 165 ga- poplar, blackthorn and hawthorn, at the tunków reprezentujących 26 rodzin. Wśród nich było 37 gatunków po raz pierwszy wy- edges of meadows (at least in part of kazanych z województwa mazowieckiego. the area). These are the host plants for Do gatunków zasługujących na szczególną caterpillars of several relatively rare spe- uwagę ze względu na niewielką liczbę zna- cies in Poland (e.g. Emmetia gaunacella, nych w Polsce stanowisk zaliczono: Depres- Acrocercops brongniardella or Luquetia saria pimpinellae (Zeller, 1839), Scythris seliniella (Zeller, 1839), Schiffermuelleria lobella). schaefferella (Linnaeus, 1758), Pseudate- melia fl avifrontella (Denis et Schiffermül- ler, 1775), Nothris verbascella (Denis et Schiffermüller, 1775), Ptocheuusa inopella REFERENCES (Zeller, 1839), Epinotia rubiginosana (Her- rich-Schäffer, 1851), Lobesia artemisiana BARAN T., 2005: The Scythrididae (Lepi- (Zeller, 1847), Phtheochroa pulvillana (Her- doptera: ) of Poland. rich-Schäffer, 1851), Anania verbascalis Poznań, Toruń. (Denis et Schiffermüller, 1775), Epascestria BUSZKO J., NOWACKI J., 2000: The Lepi- pustulalis (Hübner, 1823). doptera of Poland. A distributional check- MS. received in November 2013 list. Poznań, Toruń. ELSNER G., HUEMER P., TOKAR Z., 1999: Die Palpenmotten (Lepidoptera, Gelechiidae) Mitteleuropas. Bratislava. JAWORSKI T., 2009: Kibitnikowate (Lepi- doptera: Gracillariidae) rezerwatu Skarpa Ursynowska w Warszawie. Wiad. Ento- mol. 28, 1, 53–60. MAZURKIEWICZ A., WRZESIEŃ R., 2008: Rzadkie gatunki Microlepidoptera złowione w Warszawie. Wiad. Entomol. Authors’ address: 27, 3, 490. Anna Mazurkiewicz TOLL S., 1964: Klucze do oznaczania owa- Wydział Nauk o Zwierzętach SGGW dów Polski. Cz. XXVIII Motyle – Lepi- Katedra Biologii Środowiska Zwierzat doptera, Zeszyt 35. Oecophoridae. War- ul. Ciszewskiego 8, 02-786 Warszawa Poland szawa. e-mail: [email protected]

Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 135–139 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

The effect of density on the breeding optimization of the tropical house cricket Gryllodes sigillatus (Walker) (Orthoptera: Gryllidae) ANNA MAZURKIEWICZ1, DOROTA TUMIALIS1, ELŻBIETA PEZOWICZ1, JAKUB URBAŃSKI2, PAWEŁ GALEWSKI1, KATARZYNA GÓRAL3 1Department of Animal Environment Biology, Warsaw University of Life Sciences – SGGW 2Department of Genetics and Animal Breeding, Warsaw University of Life Sciences – SGGW 3Insect Factory

Abstract: The effect of density on the breeding an additive to animals’ diet. Moreover, optimization of the tropical house cricket Gryl- insects are more and more often con- lodes sigillatus (Walker) (Orthoptera: Gryllidae). sidered food for humans. This is because The study was aimed at testing the density effect in the tropical house cricket breeding on its sur- of the need to search for new protein vival and growth rate when fed ad libitum. The sources for growing human population tropical house crickets were kept in nine contain- and of a curiosity and desire to learn ers of a volume of 81 litres each. Three experi- new tastes and to break nutritional stere- mental variants were used: 7.5 ml of crickets were otypes. For the second mentioned reason placed in the fi rst container, 15 ml in the second and 30 ml in the third. Temperature in contain- the fi rst in Poland restaurant “Co To To ers was 29°C, the experiment lasted 25 days. Ob- Je”, that serves exclusively dishes, tained results showed that survival did not depend has recently been open in Warsaw. on the initial density in culture containers while Above mentioned circumstances indi- crickets kept at a high density had smaller body cate that the market for food insects has length. The results may affect the optimization of house cricket breeding. a great potential but needs some improve- ment. Insects imported are usually weak Key words: the tropical house cricket, Gryllodes and soon die of exhaustion and conditions sigillatus, survival, population density, growth rate, optimization of breeding of transportation. Country breeding still needs to be developed since its effi ciency may be higher and there is a defi cit of in- INTRODUCTION sects from local production. Food insects most often bred include: Terraristics enjoys increasing popularity. crickets, cockroaches, locusts, larvae More and more and of wood-eaters, mealworms and wax are kept by both experienced and novice moths. Crickets are bred to the larg- breeders. The animals require mainly est extent. They have low requirements live food. The nutritive requirements of compared with other food insects. The reptiles, amphibians, but also of popular tropical house cricket Gryllodes sigilla- African pigmy hedgehogs Atelerix albi- tus (Walker) has relatively high protein ventris (Wagner, 1841) and many bird content from among bred insect species species, are fulfi lled by feeding them with (Table 1). insects. They are the main component or 136 A. Mazurkiewicz et al.

The tropical house cricket is the productivity and the quality of animal smallest of bred crickets (18–22 mm products in food invertebrates. of length). It has two transverse black bands on its abdomen and resembles the house cricket. Females are larger than MATERIAL AND METHODS males and wingless. Males have partly reduced wings, hence they contain less Experimental crickets were provided chitin and are more easily digested. The with basic elements of welfare. Having tropical house crickets are distinguished ad libitum access to food and water they by low aggression towards other crick- were free from hunger and thirst. Crickets ets and cannibalism is rare among them. were free from discomfort – their resting Biting animals that feed on them is also area was made of cardboard egg trays rare. The insects are very fast, which is which increased living area and offered an advantage for animals actively hunt- a shelter. The animals were free from ing for their prey. They also show a high pain, injuries, diseases and stress. Stud- fertility and resistance to viruses (like ies were carried out in the food insect e.g. Acheta domesticus Densovirus) and farm “Fabryka owadów” in Warsaw. fungi, which makes them ideal animals Before setting up the experiment, for mass production. The development freshly hatched (maximum 24 h after from hatching till imago lasts 8–15 hatching) cricket larvae were mixed to weeks depending on temperature. obtain uniform sample. The experiment

TABLE 1. Nutritive value of crickets (http://www.livefoodsdirect.co.uk/Category/Banded-Crickets) Specifi cation Gryllodes sigillatus Gryllus assimilis Gryllus bimaculatis Energy (kcal/kJ per 100 g) 164/687 130/546 153/640 Protein (%) 21.4 15.2 14.7 Fat (%) 6.9 5.7 8.3 Carbohydrates (%) 4.0 4.5 4.9 Water (%) 66.1 73.3 70.8 Ash (%) 1.6 1.3 1.3

Performed studies had to estimate the lasted 25 days. The initial number of effect of initial density on survival and crickets in 1 ml (mean from three sam- body length in crickets. This information ples) was 424.7. is useful to increase the profi tability of The tropical house crickets were kept breeding. in nine containers of a volume of 81 li- The main aim of performed studies tres each (60×40×34 cm). Temperature was to intensify breeding. This requires, in containers was 29°C. All containers however, providing animal welfare and had the same ventilation and were placed hygienic conditions. Like in farm ani- at the same height on shelves to avoid mals, habitat conditions affect health and differences in temperature between cul- tures. The effect of density on the breeding optimization... 137

Ten pieces of cardboard egg trays calculated by multiplying the fi nal vol- glued together were placed in each con- ume of crickets by the number of crick- tainer. They served as the main living ets in 5 ml. Mean body length of crickets area for crickets. was calculated from measurements of 90 There was a small waterer (a bowl randomly selected specimens from each with hydrogel which prevented larvae experimental variant. from drowning) in each container to pro- To calculate the survival of crickets’ vide crickets with permanent free access larvae, their number was counted and to water. Dry food consisted of a mix- calculated per 1 ml before experiment ture of wheat bran and fi shmeal (a pro- in the same way as in the end of experi- tein additive that inhibits cannibalism) ment. in the ratio 2 : 1 and trays with carrot – ANOVA, non-parametric Kruskal- a base of diet during the experiment. The -Wallis rank test and non-paramet- access to food and water was provided ric Mann-Whitney U test were used to ad libitum to exclude food as the growth check the statistical signifi cance of dif- limiting factor. ferences.

TABLE 2. The relationship between survival and fi nal number of crickets and the initial sample volume after 25 days of experiments Variant (initial Standard N Mean Minimum Maximum Χ2 p volume) deviation emp 7.5 3 27.6000 2.12838 25.30 29.50 Survival 15 3 16.7667 1.70098 15.10 18.50 5.600 0.061 30 3 18.4000 3.96863 15.40 22.90 7.5 3 879.0000 67.10440 806.00 938.00 Final 15 3 1070.0000 109.05503 963.00 1181.00 7.200 0.027 30 3 2343.6667 505.55349 1966.00 2918.00

Three experimental variants were RESULTS AND DISCUSSION used: 7.5 ml of crickets were placed in the fi rst container, 15 ml in the second, Performed experiments showed that the and 30 ml in the third. Each variant was initial volume of crickets used in each triplicated. experimental variant did not affect insect On 25th day of experiment crickets survival (p = 0.061) – Table 2. were taken off from culture containers The effect of initial volume of crickets and their volume measured with a cylin- on body length after 25 days of experi- der with millilitre scale. Than two random ment was highly signifi cant (p < 0.001). 5 ml samples were taken from each cul- The longest body had the crickets from ture, transferred to containers and photo- experimental variant with the initial vol- graphed. The photos were used to count ume of 7.5 ml, the shortest – those from individuals with the Adobe Photoshop the variant with initial volume of 30 ml software. Final number of crickets was (Table 3). 138 A. Mazurkiewicz et al.

TABLE 3. The relationship between body length and initial volume of the sample after 25 days of experiments Initial Standard N Mean Minimum Maximum Femp p volume deviation 7.5 90 7.2744 0.91216 5.70 10.10 15 90 6.9544 0.95637 5.10 10.00 38.247 <0.001 30 90 6.1344 0.83305 4.50 8.30

Initial volume signifi cantly affected kept at 27°C achieved higher survival at the fi nal number of crickets in experi- high densities but their growth rate de- mental variants (p = 0.027). The high- clined (Lyimo et al. 1992). est number of insects was obtained In order to obtain the greatest number from culture of initial volume of 30 ml of adult crickets in the shortest possible (Table 2). time one may set up a culture of rela- Many studies (pertaining, however, tively high density but crickets will not mostly to vertebrates) on density-de- achieve as large body size as they would pendent survival indicate that young in- at lower densities. dividuals may die more often at a high density (Sheperd and Cushing 1980, Van der Veer 1986, Myers and Cadigan 1993, REFERENCES Ohman and Hirche 2001, Holbrook and BARNES A.I., SIVA-JOTHY M.T., 2000: Schmitt 2002). In view of the breeding Density-dependent prophylaxis in the effi ciency an important fi nding of our mealworm beetle, Tenebrio molitor L. study is that the initial volume does not (Coleoptera: Tenebrionidae): cuticular affect crickets’ survival. One may expect melanization is an indicator of invest- that similar mechanisms were involved ment in immunity. Proc. R. Soc. Lond. here as in mealworms which at high den- B22, 267, 177–182. GIMNIG J.E., OMBOK M., OTIENO S., sities acquired higher resistance to vi- KAUFMAN M.G., VULULE J.M., ruses and fungi (Barnes and Siva-Jothy WALKER E.D., 2002: Density-depen- 2000). Survival of mosquito larvae bred dent development of Anopheles gambiae at medium temperature also increased at (Diptera: Culicidae) larvae in artifi cial high densities (Lyimo et al. 1992). Note- habitats. J. Med. Entomol., 39, 162–72. worthy, no symptoms of viral or fungal HAWLEY W.A., 1985: The effect of larval diseases were observed during our ex- density on adult longevity of a mosquito, Aedes sierrensis: epidemiological conse- periments. quences. J. Anim. Ecol. 54, 955–964. Performed studies showed that an in- HOLBROOK S.J., SCHMITT R.J., 2002: crease of density resulted in decreased Competition for shelter space causes body length. Slower growth rate of lar- density-dependent predation mortality in vae and smaller mass of imagines was damselfishes. Ecology 83, 2855–2868. noted e.g. in mosquitoes (Gimnig et al. LYIMO E.O., TAKKEN W., KOELLA J.C., 2002). Body mass of female pupae of 1992: Effect of rearing temperature and lar- val density on larval survival, age at pupa- this species was conversely proportional tion and adult size of Anopheles gambiae. to density (Hawley 1985). Mosquitoes Entomol. Exp. Appl. 63, 265–271. The effect of density on the breeding optimization... 139

MYERS R.A., CADIGAN N.G., 1993: Den- dostępie do pokarmu ad libitum. Świerszcze ba- sity-dependent juvenile mortality in ma- nanowe w czasie eksperymentu były hodowane rine demersal fi sh. Can. J. Fish. Aquat. łącznie w dziewięciu pojemnikach o pojemności Sci. 50, 1576–1590. 81 l każdy. Zastosowano trzy warianty doświad- OHMAN M.D., HIRCHE H.J., 2001: Densi- czeń: w pierwszym pojemniku umieszczono ty-dependent mortality in an oceanic co- 7,5 ml świeżo wylęgłych świerszczy, w drugim pepod population. Nature 412, 638–641. 15 ml, a w trzecim 30 ml. Każdy wariant do- SHEPERD J.G., CUSHING D.H., 1980: świadczenia powtórzono trzykrotnie. Temperatu- A mechanism for density-dependent ra w pomieszczeniu wynosiła 29°C. Eksperyment survival of larval fish as the basis for trwał 25 dni. W wyniku przeprowadzonych badań stwierdzono, że objętość początkowa świersz- a stock-recruitment relationship. J. Cons. czy użytych w każdym wariancie doświadczenia Int. Explor. Mer. 39, 160–67. nie wpływa na przeżywalność owadów, jednak Van Der VEER H.W., 1986: Immigration, świerszcze hodowane w większym zagęszczeniu settlement, and density-dependent mor- miały mniejszą długość ciała. Uzyskane wyniki tality of a larval and early postlarval mogą przyczynić się do zoptymalizowania ho- 0-group plaice (Pleuronectes platessa) dowli tego gatunku świerszcza. population in the western Wadden Sea. Mar. Ecol. Prog. Ser. 29, 223–236. http://www.livefoodsdirect.co.uk/Category/ MS. received in November 2013 Banded-Crickets Authors’ address: Streszczenie: Wpływ zagęszczenia na optymali- Anna Mazurkiewicz zację hodowli świerszcza bananowego Gryllodes Wydział Nauk o Zwierzętach SGGW sigillatus (Walker) (Orthoptera: Gryllidae). Praca Katedra Biologii Środowiska Zwierząt miała na celu zbadanie wpływu zagęszczenia po- ul. Ciszewskiego 8, 02-786 Warszawa czątkowego w hodowli świerszczy bananowych Poland na ich przeżywalność oraz szybkość wzrostu przy e-mail: [email protected]

Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 141–149 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Effect of nanoparticles of copper and copper sulfate administered in ovo on hematological and biochemical blood markers of broiler chickens NATALIA MROCZEK-SOSNOWSKA1, MARTYNA BATORSKA1, MONIKA ŁUKASIEWICZ1, AGNIESZKA WNUK1, EWA SAWOSZ2, SŁAWOMIR JAWORSKI2, JAN NIEMIEC1 1Department of Animal Breeding and Production 2Department of Animal Nutrition and Feed Science Warsaw University of Life Sciences – SGGW

Abstract: Effect of nanoparticles of copper and concentrations of glucose and cholesterol and copper sulfate administered in ovo on hemato- increased levels of selected microelements: cal- logical and biochemical blood markers of broiler cium, phosphorus and iron. chickens. At the fi rst stage of the study the experi- Key words: in ovo, nanoparticles, colloid, hema- mental material included 300 clutching eggs of tological and biochemical markers in chicken Hubbard Flex chickens. The eggs were divided blood into three groups: control (without injection in ovo), Nano50 (in ovo injection of colloidal copper nanoparticles) and NanoCuSO4 (in ovo injection of colloidal nanoparticles of copper sulphate). INTRODUCTION Experimental solutions were administered by in ovo injection using a sterile needle 0.3 mm as fol- Copper is an element commonly occur- lows: Nano50 group – colloid of copper nanopar- ring in organisms of plants and animals. ticles (concentration: 50 ppm), and NanoCuSO4 group – colloid of copper sulfate (concentration: As a microelement, it is a constituent of 50 ppm), to the air cell of the egg. The eggs were active centers of many enzymes being of incubated under standard conditions. After hatch- key signifi cance to metabolic processes. ing, 50 chicks were selected from each group for In addition, it is a component and acti- 42-day rearing. Birds were fed standard complete vator of enzymes in many redox reac- feed mixtures for broilers. On the last day of rear- ing (day 42), 12 females and 12 males were se- tions, however its major roles are seen lected from each group and their blood was sam- in the synthesis of red blood cells and as- pled for assays of hematological and biochemical sistance in connective tissue formation. markers. Hematological analyses included deter- Also, this element plays an important minations of: WBC, RBC, Hb, heterophils, lym- role in iron metabolism – it facilitates phocytes, monocytes, eosinophils and basophils, its absorption and binding to transfer- whereas biochemical analyses included assays of the following markers in blood serum: glu- rin, which transfers iron to erythrocytes, cose, cholesterol, triglycerides, HDL-cholesterol, where hemoglobin is being synthesized. urea, calcium, magnesium, phosphorus, iron, AS- Copper present in ceruloplasmin (serum PAT, and ALAT. The use of copper nanoparticles protein) is one of the most active forms evoked an increase in blood levels of RBC, HGB, of this element in organisms and in this HTC, heterophils, monocytes and basophils. In addition, in blood serum in contributed to reduced form it regulates iron metabolism and 142 N. Mroczek-Sosnowska et al. transport (Witkiewicz 2008). By activat- as reproduction disorders. Copper defi - ing the enzyme indispensable of erythro- cit deteriorates keratin synthesis and, as cytes synthesis, it infl uences the proper a result, causes more frequent incidence functioning of the erythropoietic system. of lameness and increased incidence of Defi ciency of copper and iron leads to mastitis. Poultry demand for copper is anemia and low utilization of vitamin C. not that big, but necessary for the proper Signifi cant is also its impact on the re- development of the organism. In poultry, generation of connective tissue by, e.g., is from 5 to 25 mg in 1 kg of the mix- synthesis of collagen and elastin and on ture (Smulikowska 1996), depending the development of the nervous system on the species, age and the production by synthesis of dopamine. Furthermore, direction. Why or copper in 1924 was copper and zinc prevent damages in- included in the essential micronutrients. duced by free oxygen radicals. This ele- Furthermore, it is one of two major tran- ment plays also a signifi cant role in the sition metals present in the body. regulation of glucose and cholesterol Copper is an important microele- metabolism. It is a constituent and an ac- ment, indispensible for vital processes tivator of a numerous group of enzymes, and for the proper development of living particularly of oxidases: cytochrome, organisms as it plays signifi cant meta- lysyl, ascorbic, ketocholic superoxide bolic functions. For instance, it facili- dismutase and tyrosinase (Makarski and tates iron absorption from the gut, takes Zadura 2006). Copper defi ciency induc- part in hemoglobin synthesis and there- es osteoporosis, reduced body immunity, fore has a great impact on red blood cells increases blood level of cholesterol and production (erythropoiesis), it is also an anemia. In chickens it mainly contrib- indispensable element of many enzymes utes to: growth retardation, disorders of (Brzozowski 2007). Both its defi ciency the nervous and cardiovascular systems, and excess may evoke adverse and toxic anemia, disorders in plumage pigmenta- effects. However, information is lacking tion, disorders in ossifi cation, and myo- on the boundary between its necessary cardial fi brosis. When copper defi ciency and toxic concentrations in a body. It is is also a reduced production of eggs, but common knowledge that intracellular the larger mass. Moreover, the eggs are copper occurs mainly in mitochondria often without the shell (in the membrane and in the nucleus. It is capable of estab- of the egg) or with a thin shell, or distort- lishing links with nucleic acids where- ed. Defi ciency revealed disturbances in in it may induce permanent structural reproduction and development of sperm changes. It forms links especially easily and high mortality of embryos during with various sulfur-containing proteins, hatching. In addition, results in poor particularly with low-molecular metal- pigmentation colorful feathers feath- lothionein. ered breeds, slow growth, reducing body Nanotechnology – which is one of weight, anemia and ultimately death. the most intensively developing sci- Symptoms of copper defi ciency include ences – has entered into many research poor production results in dairy cattle, disciplines including: chemistry, phys- loss of hair color and hair loss as well ics, bioengineering and biomedicine. Effect of nanoparticles of copper and copper sulfate... 143

Nanotechnological processes have ena- bled structural modifi cations of many simple and complex substances, owing to which they can be transformed into submicroscopic objects. What is more, it has been quite recently discovered that the submicroscopic fragments of the matter are characterized by excep- tional biochemical traits. Examples of these substances include nanoparticles constituted by atoms of silver, chro- mium or copper. Nanoparticles are mi- FIGURE 1. Copper nanoparticles visible under electron microscope croscopic molecules the size of which is measured in nanometers (nm). They of the droplets in dry air, the grids were are defi ned as particles sized less than inserted into the TEM. The test was per- 100 nm. A nanometer equals one mil- formed in triplicate. The zeta potential lionth of a millimeter or the width of in water was measured using a Zetasizer three or four atoms. By comparison, a Nano ZS model ZEN3500 (Malvern In- human hair is ca. 10–50 nm in width, struments, Malvern, UK). whereas red blood cells are 2–6 nm and DNA is 2–12 nm in diameter. In ovo injection and incubation The objective of this study was to de- conditions of clutching eggs termine the effect of copper and copper sulfate nanoparticles injected in ovo on At the fi rst stage of the study, the experi- hematological and biochemical blood mental material included 300 clutching markers of broiler chickens. eggs of Hubbard Flex chickens (average weight 62.25 ±2.2g), that were stored for 4 days at a temperature of 12°C MATERIAL AND METHODS and humidity of 73%. The eggs were weighed and divided into three groups: Characterization of copper C (control), NanoCuSO4, and Nano50 nanoparticles (each of 100 eggs). Experimental solu- tions were administered by in ovo injec- The shape and size of the copper nano- tion using a sterile needle 0.3 ml as fol- particles were inspected (Fig. 1) using lows: Nano50 group – colloid of copper a JEM-1220 (JEOL, Tokyo, Japan) trans- nanoparticles (concentration: 50 ppm), mission electron microscope (TEM) at and NanoCuSO4 group – colloid of cop- 80 KeV, with a Morada eleven-mega- per sulfate (concentration: 50 ppm), to pixel camera (Olympus Soft Imaging the air cell of the egg. Injection orifi ces Solutions, Münster, Germany). Samples were tight-sealed and eggs were fi xed for the TEM were prepared by placing in an incubator under standard condi- droplets of hydrocolloids onto Formvar- tions (temperature 37.8°C, humidity -coated copper grids (Agar Scientifi c, 60%, egg revolution by 90° once a day Stansted, UK). Immediately after drying 144 N. Mroczek-Sosnowska et al. for 18 days). The eggs were incubated in ad libitum. Body weight of the birds (on a hatching apparatus by Heka company day: 1, 14, 35, 42 day), their mortality equipped in a temperature, air humidity and feed intake were monitored in the and egg rotation control module. During rearing period. incubation, the eggs were light-exposed twice (in day 6 and 18 of incubation) Determinations of hematological and weighed in order to determine egg and biochemical blood markers weight loss. On day 19 of incubation, the In week 6 of rearing, 12 hens and 12 eggs were transferred to a hatcher with cocks with body weight similar to the a temperature of 37.0–37.5°C and rela- average body weight in a group were tive air humidity of 75–80%. After hatch- selected from each group for slaughter. ing, one-day chicks were evaluated and Blood was sampled from their brachial healthy birds with healed navels were vein to 3-ml-heparinized test tubes. He- selected for further rearing. matological analyses were conducted in full blood for: WBC (white blood Rearing, housing conditions cells), RBC (red blood cells), Hb (hemo- and feeding globin), HCT (hematocrit), heterophils, A further stage of the study included 150 lymphocytes, monocytes, eosinophils, Hubbard Flex chickens (50 birds from and basophils. The following biochemi- each group) that were kept on litter till cal markers were assayed in blood serum 42 days of age under typical zoohygienic (30 min after sampling 3 ml of blood were conditions, in a room without daylight. centrifuged at 3,000 rpm for 10 min): One-day chickens after weighing and glucose, cholesterol, triglycerides, HDL- tagging were divided into three groups -cholesterol, urea, calcium, magnesium, (control – C, Nano50, and NanoCuSO4) phosphorus, iron, ASPAT, and ALAT us- in two replications, 25 birds each. Stock ing a CORMAY kit. density in a henhouse reached 11 birds Analytical results were subjected to per 1 m2. Immediately after moving to a statistical analysis by computing mean a production hall, the chickens from all values and standard deviation using the groups were vaccinated against Marek’s analysis of variance calculated with the disease, infectious bronchitis and coc- least squares method in a statistical soft- cidiosis. ware SPSS 19.0 PL (SPSS Inc., Chicago, The mean air temperature in the room II, USA). accounted for 24°C, and under radiators – for 34.5°C. The following parameters of the microclimate were measured since RESULTS AND DISCUSSION the 1st week to the end of rearing: hu- midity, temperature and contents of toxic Right after mercury, cadmium and cop- gases: ammonia, hydrogen sulfi de and per represent the most toxic heavy metals carbon dioxide. A three-stage feeding (Dojlido 1995). Even their minute quan- program was applied in the rearing pe- tities affect the course of biological proc- riod: starter (crumb), grower and fi nisher esses (Swędrzyńska and Sawicka 2010). (granulate) – Table 1. The birds were fed Results of assays of hematological blood Effect of nanoparticles of copper and copper sulfate... 145

TABLE 1. Composition and nutritive value of feed mixtures Starter Grower Finisher Specifi cation (1–14 day) (15–35 day) (36–42 day) Content (%) Corn 10.0 11.4 10.0 Wheat 53.0 55.0 59.6 Soybean meal 30.6 27.4 23.2 Feeding chalk 1.19 1.20 1.11 Acidic sodium carbonate 0.20 0.14 0.14 NaCl 0.24 0.28 0.28 Stimulant 0.01 0.01 0.01 FOSF 2-Ca 1.18 0.78 0.70 Soybean oil 2.10 2.40 3.60 Methionine 0.48 0.42 0.36 Lysine 0.36 0.34 0.36 Threonine 0.14 0.13 0.14 Premix 0.50 0.50 0.50 Nutritive value Metabolizable energy (ME) (MJ/kg) 12.52 12.76 13.20 Fat (%) 3.67 4.00 5.14 CP (%) 21.99 20.78 19.26 Methionine (%) 0.70 0.63 0.57 Methionine + Cysteine (%) 1.08 1.01 0.92 Lysine (%) 1.38 1.28 1.19 Ash (%) 5.83 5.35 4.96 markers are presented in Table 2. The in the number of erythrocytes (RBC) in the ovo injection of a copper sulfate colloid peripheral blood of the birds exposed increased counts of WBC, lymphocytes to copper. An increased percentage of and eosinophils compared to the other RBC was noted in the group Nano50. groups. In turn, the Nano50 group was The copper-exposed chickens were char- characterized by a decreased number of acterized by an increased frequency of leucocytes compared to the remaining changed erythrocytes in the peripheral groups. A similar effect was observed by blood that were effectively compensated Dmoch and Polonis (2007), who in their for by the increasing number of juvenile study with turkeys were adding copper erythrocytes in blood, owing to which chelate with lysine to drinking water for the number of red blood cells remained birds. unchanged (C, NanoCuSO4) or even in- During the experiment, signifi cant creased (Nano50). Analyses showed also (P ≤ 0.05) changes were determined in increased concentration of hemoglobin 146 N. Mroczek-Sosnowska et al.

TABLE 2. Hematological blood markers of chickens (♀ + ♂) Group Specifi cation C Nano50 NanoCuSO4 SE WBC – white blood cells (g/l) 13.77 12.00 14.52 1.62 RBC – red blood cells (t/l) 2.84b 3.44a 2.82b 0.12 Hb – hemoglobin (g/l) 134.66b 154.75a 148.0b 7.74 HCT – hematocrit (l/l) 0.36 0.43 0.40 0.02 Heterophils (%) 6.33B 13.75A 7.75B 2.32 Lymphocytes (%) 84.00 75.25 86.50 4.90 Monocytes (%) 7.33A 8.00A 2.00B 2.12 Eosinophils (%) 1.00 1.50 2.50 1.09 Basophils (%) 1.33 1.50 1.25 0.37 a, b – statistically signifi cant differences at P ≤ 0.05, A,B – statistically signifi cant differences at P ≤ 0.01. Conversion factor: g/dl×10 = g/l, g/l×0.1 = g/dl, g/dl×0.6206 = mmol, mmol×1.611 = g/dl; l/l×100 = %, %×0.01 = l/l; 109/l = thou./mm3, thou./mm3×1 = 109/l, 109/l = G/l, thou./mm3 = 103/mm3 = = 103/μl; t/l = 10–15; Tl = 1012; p/g = 10–12g. which allowed the birds to keep oxygen iron absorption into the body and its in- transport at an appropriate level. This corporation to hemoglobin (Fox 2003, group was additionally characterized by Mullally et al. 2004). Results reported increased numbers of heterophils, mono- by Dmoch and Polonis (2007), demon- cytes and basophils. strated a reducing tendency in the levels Changes in erythropoietic param- of hematocrit and hemoglobin after the eters in the chickens from experimental application of copper chelate. groups referred mainly to an increased The analysis of other studies on the concentration of hemoglobin in peri- impact of metals on erythropoietic pa- pheral blood, which was usually accom- rameters of fi sh demonstrated a decreas- panied by an increased number of RBC ing tendency in the number of erythro- (Table 2). The increased level of he- cytes, hematocrit value and hemoglobin moglobin could be due to its continued concentration in blood. Reduced values synthesis by erythrocytes already circu- of these parameters upon the infl uence of lating in the peripheral blood. Speckner cadmium were reported by Vincent et al. et al. (1989) emphasized the capability (1996) and upon the infl uence of copper of mature erythrocytes of fi sh for hemo- – by Ates et al. (2008). Decreased values globin synthesis that may continue even of these parameters were consequently for some time after cells release from the leading to anemia development in fi sh erythropoietic tissue. It is also likely that (Ruperelia et al. 1990). the increased level of hemoglobin in the Furthermore, analyses demonstrated group Nano50 could be linked with the a reduced percentage of monocytes in the homeopoetic function of copper. This NanoCuSO4 group, which was accompa- element was shown to directly stimulate nied by a suppressed metabolic activity of erythrocytes synthesis, as it determines these cells. Reduction was also observed Effect of nanoparticles of copper and copper sulfate... 147 in the phagocytic activity in the group of NanoCuSO4 group, whereas the high- chickens characterized by WBC number est concentration of HDL was assayed in increase. This indicates that NanoCuSO4 the control group. The administration of could contribute to accelerated damage copper nanoparticles caused an increase of phagocytes and to inhibited metabolic in the blood level of uric acid. A simi- activity of these cells. lar effect on increased levels of glucose Results of analyses of biochemical and uric acid was observed by Dmoch blood markers are provided in Table 3. and Polonis (2007), applying a copper- The in ovo injection of copper nanopar- -lysine chelate to chickens. The addition ticles resulted in decreased blood levels of copper contributed also to increased of glucose and cholesterol. The reduced serum levels of calcium, phosphorus and concentration of glucose confi rms fi nd- iron compared to the remaining groups. ings of Makarski and Zadura (2006) The increased levels of these elements who were administering copper chelate may be refl ected in production effective- to turkeys with drinking water. The ap- ness as they contribute to improved bone plied element had some effect on lipid mineralization. Upon the use of Cu-Lys and cholesterol metabolism as well as on chelate Dmoch and Polonis (2007) noted properties of myelin sheaths of nervous an increase in the level of calcium and to, fi bers. Turnlund et al. (1988) and Bakalii a lesser extent, in the level of phospho- et al. (1995) claim that copper addition rus. In the case of hepatic enzymes, a re- contributes to a decreased level of tri- duced level of ALAT was observed in the glycerides and reduced cholesterol syn- groups subjected to in ovo injection with thesis in blood plasma and tissues of ani- copper and copper sulfate colloids. An mals. In our experiment, the lowest level opposite effect was reported for ASPAT of triglycerides was determined in the concentration which in the NanoCuSO4

TABLE 3. Biochemical blood markers of chickens (♀ + ♂) Group Specifi cation C Nano50 NanoCuSO4 SE Glucose (mmol/l) 13.29 12.85 12.47 1.03 Cholesterol (mmol/l) 3.82 3.43 3.50 0.39 Triglycerides (mmol/l) 0.59 0.62 0.51 0.55 HDL (mmol/l) 2.39 1.95 2.11 0.44 Urea (mmol/l) 2.70 3.10 2.80 0.12 Calcium (mmol/l) 2.27 2.72 2.29 0.33 Magnesium (mmol/l) 0.80 0.85 0.98 0.14 Phosphorus (mmol/l) 1.93 2.27 1.87 0.23 Iron (mmol/l) 24.40 30.50 20.50 3.17 ALAT (mmol/l) 16.00 12.00 12.00 4.96 ASPAT (mmol/l) 368.50B 498.50B 911.00A 100.78 A,B – statistically signifi cant differences at P ≤ 0.01. 148 N. Mroczek-Sosnowska et al. group was almost threefold higher than DOJLIDO J.R., 1995: Chemia wód po- in the control group. Such a result may wierzchniowych. Wydawnictwo Ekono- be indicative of liver lesions, however mia i Środowisko, Białystok. FOX P.L., 2003: The copper-iron chronic- histopathological examination of liver les. The story of an intimate relationship. excluded the pathological effect of cop- BioMetals. 16, 9–40. per on this organ. MAKARSKI B., ZADURA A., 2006: Wpływ chelatu miedzi z lizyną na po- ziom składników hematologicznych CONCLUSION i biochemicznych krwi indyków. Annales Results obtained in this study demon- Universitatis Mariae Curie-Skłodowska, strate that the erythropoietic system of Lublin-Polonia 24 (48), 357–363. MULLALLY A.M., VOGELSANG G.B., chickens is susceptible to effects of met- MOLITERNO A.R., 2004: Warted sheep als, including copper (nanoparticles), and premature infants the role of trace which shows that changes in blood may metals in hematopoiesis. Blood Rev. 18, be used as an indicator of the impact of 227–234. toxic substances on chickens. Simulta- RUPERELIA S.G., VERMA Y., SAI- neously, the analysis of changes in the YED S.R., RAWALL U.M., 1990: Effect of cadmium on blood of tilapia, Oreo- peripheral blood enables more precise chromis mossambicus (Peters), during evaluation and interpretation of the ef- prolonged exposure. Bull. Environ. Con- fect a given factor has on bird body. tam. Toxicol. 45, 305–312. SMULIKOWSKA S. (red.), 1996: Normy Żywienia drobiu. Zalecenie Żywieniowe REFERENCES i wartość pokarmowa pasz. IFiŻZ PAN, ATES B., ORUN I., TALAS Z.S., DUR- Jabłonna. MAZ G., YILMAZ I., 2008: Effects of SPECKNER W., SCHINDER J.F., AL- sodium selenite on some biochemical BERS C., 1989: Age-dependent changes and hematological parameters of rainbow in volume and haemoglobin content of trout (Oncorhynchus mykiss Walbbaum, erythrocytes in the carp (Cyprinus carpio 1792) exposed to Pb+2 and Cu+2. Fish L.). J. Exp. Biol. 141, 133–149. Physiol. Biochem. 34, 53–59. SWĘDRZYŃSKA D., SAWICKA A., 2010: BAKALLI R.I., PESTI G.M., RAG- Wpływ miedzi na bakterie z rodzaju Azo- LAND W.L., KONJUFCA V., 1995: Di- spirillum występujące w ryzosferze sie- etary Copper in Excess of Nutritional wek kukurydzy i pszenicy. Woda – Śro- Requirement Reduces Plasma and Breast dowisko – Obszary Wiejskie 10, 2 (30), 167–178. Muscle Cholesterol of Chickens. Poult. TURNLUND J.R., 1988: Copper nutritive Sci. 74 (2), 360–365. bioavailability and the infl uence of di- BRZOZOWSKI T., 2007: Krew. W: Fizjolo- etary factors. J. Am. Diet. Assoc. 88, gia człowieka. Podręcznik dla studentów 303–310. medycyny. Red. S.J. Konturek, Elsevier VINCENT S., AMBROSE T., KU- Urban & Partner, Wrocław. MAR L.C.A., SELVANAYAGAM M., DMOCH M., POLONIS A., 2007: Wpływ 1996: Heavy metal cadmium infl uenced tiokompleksu miedziowego na wybrane anemia in the riverine major carp, Catla wskaźniki hematologiczne, biochemicz- catla. J. Environ. Biol. 17, 81–84. ne i zawartość składników mineralnych WITKIEWICZ K., 2008: Rola miedzi w orga- we krwi kurcząt brojlerów. Acta Sci. Pol., nizmie ptaków. Hodowca Drobiu 12, 80. Zootechnica 6 (3), 11–18. Effect of nanoparticles of copper and copper sulfate... 149

Streszczenie: Wpływ nanocząstek miedzi i siar- heterofi le, limfocyty, monocyty, eozynofi le, bazo- czanu miedzi podawanych in ovo na wskaźni- fi le. Do oznaczenia wskaźników biochemicznych ki hematologiczne i biochemiczne krwi kurcząt pobrano krew na surowicę, w której oznaczono brojlerów. Materiał doświadczalny w pierwszym wskaźniki biochemiczne: glukoza, cholesterol, etapie stanowiło 300 jaj lęgowych kurcząt Hub- triglicerydy, HDL-cholesterol, mocznik, wapń, bard Flex. Jaja podzielono na trzy grupy: kontro- magnez, fosfor, żelazo, ASPAT, ALAT. Zastoso- la, Nano50 i NanoCuSO4, z czego jaja z grupy wanie nanocząstek miedzi wpłynęło na wzrost Nano50 i NanoCuSO4 poddane zostały zabiegowi poziomu RBC, HGB, HTC, heterofi li, monocy- iniekcji in ovo. Eksperymentalne roztwory po- tów i bazofi li. Dodatkowo w surowicy stwierdzo- dano poprzez wstrzyknięcie in ovo, przy użyciu no obniżenie stężenia glukozy i cholesterolu przy sterylnej igły 0,3 mm kolejno do grup: Nano50 jednoczesnym wzroście poziomu wybranych mi- (koloid nanocząstek miedzi, stężenie 50 ppm), kroelementów: wapnia, fosforu i żelaza. NanoCuSO4 (koloid siarczanu miedzi, stężenie 50 ppm) do komory powietrznej jaja. Jaja inkubo- MS. received in November 2013 wano w standardowych warunkach. Po wykluciu z każdej grupy wybrano po 50 piskląt do odchowu trwającego 42 dni. Ptaki żywiono standardowy- Authors’ addresses: mi mieszankami pełnoporcjowymi dla brojlerów. Natalia Mroczek-Sosnowska W ostatnim 42. dniu odchowu z każdej grupy Wydział Nauk o Zwierzętach SGGW wybrano po 12 samic i 12 samców, od których Katedra Szczegółowej Hodowli Zwierząt pobrano krew celem określenia wskaźników he- ul. Ciszewskiego 8, 02-786 Warszawa matologicznych i biochemicznych. Wykonano Poland oznaczenia hematologiczne, tj.: WBC, RBC, Hb, e-mail: [email protected]

Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 151–156 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Carbon synthetized by RF PACVD method enhances the activity of antioxidants TOMASZ NIEMIEC1 , MACIEJ SZMIDT2, ANNA KRUK-ROSZKOWSKA1, EWA SAWOSZ-CHWALIBÓG1, KATARZYNA MITURA3 1Department of Animal Nutrition and Feed Science, Warsaw University of Life Sciences – SGGW 2Department of Morphologic Sciences, Warsaw University of Life Sciences – SGGW 3Department of Radiology and Radiotherapy Fundamentals, Koszalin University of Technology

Abstract: Carbon synthetized by RF PACVD the serious antioxidant defi ciencies and method enhances the activity of antioxidants. The they may initiate tumor processes or cell aim of the presented study was to evaluate the re- apoptosis. The limited amount of reduc- ductive activity of antioxidants in the presence of carbon manufactured by Radio Frequency Plasma ers in the organic matter leads to the Activated Chemical Vapor Deposition method (RF unstable redox homeostasis. Growing PACVD). 2,2-diphenyl-1-picrylhydrazyl (DPPH) oxidative strength of the environmental method was employed to estimate antioxidants factors causes the rapid decrease of the reductive potential. Based on the experimental antioxidant potential. The strength of the results, the antioxidant activity of carbon colloid was not confi rmed. Nevertheless the reductive ac- reducers in living organisms is limited by tivity of antioxidants measured in time manner, in the outside factors and endogenous bio- the presence of carbon, was signifi cantly elevated synthesis. That is why, the organism diet comparing to the antioxidants alone. The present- rich with the natural antioxidants (AA ed results suggest that the surface of carbon syn- and GSH) is necessary for the regular or- thetized by RF PACVD method suspended in wa- ter, creates more friendly reductive environment ganism functioning. The organic matter for antioxidants with hydroxyl groups (ascorbic should be protected by synthesized anti- acid – AA and butylated hydroxyanisole – BHA) oxidants (BHA) to extend their time of then with sulfhydryl groups (glutathione – GSH). stability. The environment may also be Key words: carbon powder particles, RF PACVD protected by slowing the oxidation reac- method, antioxidant, ascorbic acid, BHA, gluta- tions. In the living organism this may be thione, DPPH achieved by the establishment of proper ratio between the lipid antioxidants and unsaturated acids. In the organic matter INTRODUCTION it maybe achieved by proper storage con- ditions like the controlled temperature The external environmental factors like or the light protection. There is a large UV, optical radiation, temperature, or- demand for the research on the factors ganic and inorganic oxidants as well as which possess both the characteristics of internal cell oxidant factors are the po- the antioxidant and the ability of sustain- tential reasons of the oxidation stress, ing the proper environment for reduction which interfere the cell redox homeo- reactions. stasis. Such interferences may cause 152 T. Niemiec et al.

The recent study presents the addi- Carbon particles was suspended in water. tional biological benefi ts of carbon par- Investigated antioxidants (AOX): AA, ticles which are commonly considered GSH were dissolved in water. BHA was as the potential carrier of the molecules. dissolved in ethanol. In the solution of There are also a few studies evaluating 0.5 m·mol–1 AA, 0.7 m·mol–1 BHA and the specifi c characteristics of RF PACVD 100 m·mol–1 GSH, carbon particles (RF carbon particles which could be used in PACVD) were suspended in the concen- biomedicine or pharmaceutical industry. tration of 50 mg·l–1. The experiment was Czerniak-Reczulska et al. (2010) noted also performed with the incubation of all the infl uence of RF PACVD carbon on the solutions in 100°C for 10 min. the process of the proliferation of en- dothelial cells. Bakowicz-Mitura et al. 2,2-diphenyl-1-picrylhydrazyl (DPPH) (2007) observed that RF PACVD carbon assay may act as an antioxidant or anti-infl am- The free radical scavenging activity of matory factor. Catalitic properties of this the mix sample was measured by the type of carbon in organic environment, decrease in absorbance of methanolic have not been precisely studied yet. Per- DPPH solution at 517 nm (Krings and haps, as in the case of diamond obtained Berger 2001) in time manner (after 0.25; by detonation method used for proteo- 0.5; 1; 2; 3; 4; 5 min). The antioxidant lytic enzyme immobilization, the immo- activity was expressed as: % disappear- bilization reducers may modulate redox ance = [(A control – A sample)/A control] × 100% reaction in simple in vitro systems. We (A – absorbance). suspect that carbon synthetized by RF The results were subjected to statis- PACVD method may actively transfer tical analysis by two-way ANOVA and electrons in oxidoreductive reactions in Duncan’s range test. the organic matter. Therefore we pre- sume that RF PACVD carbon can protect antioxidant activity from environmental RESULTS AND DISCUSSION stress. The hypothesis was tested by de- termining the effect of RF PACVD car- According to Mitura (1987) and Bako- bon colloid on ascorbic acid, glutathione wicz (2003), RF PACVD particles ex- and tert-butyl-4-hydroxyanisole activity hibits the reducing activity. In our study, by measurements of reducing power of the analytical methods did not identifi ed antioxidants on 2,2-diphenyl-1-picrylhy- reducing strength of the RF PACVD car- drazyl (DPPH) and the iron ions. bon. AA possesses two active hydroxyl groups that participate in the redox reac- tions (Niemiec et al. 2005). RF PACVD MATERIAL AND METHODS carbon catalyzes reaction with AA in the DPPH test. Carbon elevates AA activity Manufacturing parameters of RF after 15 s incubation and signifi cantly in- PACVD carbon powder were described creases the vitamin activity after 3 min by Czerniak-Reczulska et al. (2010). The incubation. High temperature decreased samples were divided into three groups. the activity of AA from 50 to 10%. How- Carbon synthetized by RF PACVD method enhances... 153 ever in the presence of carbon powder, the activity of ascorbic acid decreased in

about 30% (Table 1). inter- -action Carbon particles did not affect the change in activity of glutathione in any of the experiments. BHA synthetic anti- oxidant strength reduction depends on the active –OH groups. Carbon signifi - cantly elevates antioxidant activity of

BHA from 30 s to 2 min incubation. High uence fl ANOVA temperature decreased activity of BHA in as it was expected. However within fi rst minute, reduction reaction was elevated comparing to the group without carbon particles (Table 2). GSH is a multifunctional thiol group containing tripeptide that is a powerful 2.0330.426 0.01910.203 0.00040.359 0.798 0.0000 0.195 0.0033 0.0000 0.329 0.0000 0.316 0.0000 0.0000 0.0000 0.0017 0.0000 0.0079 0.02320.0241 0.00630.0229 0.0021 0.500 0.6231 0.060 0.0011 0.047 0.1380 0.0235 0.1217 0.3045 0.3246

antioxidant (Table 3), found in most aero- CARBONRF PACVD temperature bic organisms (Banhegyi et al. 1997). c c c c The living organisms are exposed to b b b 2.83 1.86 0.76 the external environmental factors which 0.28 0.34 29.56 is the source of oxidative stress. This is the one of the harmful principal issues in the c c c c b b healthcare, where excess of ROS (chem- RF PACVD CARBON + AA CARBON + 0.10 0.29 4.72 6.02 5.08 0.32 0.17 ical species with unpaired electrons on 59.11 the molecular orbitals) generated in vari- b ous pathogenic processes are recognized b b b b b b as an indicator in cytotoxicity and the 0.18 0.15 0.16 0.08 0.10 cellular disorder. At the intracellular lev- 10.47

el, ROS are balancing between the bio- (AA) b a a chemical antioxidants such as ascorbic b b b ascorbic acid 0.05 0.15 0.11 0.12 1.90 0.38 0.38 acid or glutathione. In the organic mat- 52.04

ter (food) redox homeostasis is remains Reducting activity (%) of unstable due to the limited of amount of a a a a a a a reducers. Growing oxidative strength of the environmental factors leads to rapid 0.40 0.27 0.09 0.05 0.13 0.12 decrease in the antioxidant potential. The strength of the reducers in living organ- a a a a a a isms is limited by delivered factors from – 100°C – 100°C – 100°C SEM p SEM p p the organism diet and possibility of en- CARBON RF PACVD dogenous biosynthesis. Simultaneously, the known strategies for the preservation of the organic matter (food products) are 0.25 0.13 TIME (min) 0.5123 0.14 4 0.16 5 0.16 0.14 0.13 0.17 0.11 TABLE 1. Reducing activity of AA on DPPH radical in time manner AA 1. Reducing activity of TABLE a,b,c – RF PACVD CARBON + AA > RF PACVD CARBON and AA (p < 0.05). AA CARBON and > RF PACVD AA CARBON + a,b,c – RF PACVD TABLE 2. Reducing activity of BHA on DPPH radical in time manner ANOVA Reducting activity (%) of infl uence TIME RF PACVD CARBON inter- (min) RF PACVD CARBON BHA RF PACVD CARBON temperature + BHA -action – 100°C – 100°C – 100°C SEM p SEM p p 0.25 0.10a 0.197a 4.94b 3.751b 5.60b 8.941c 0.306 0.0000 0.306 0.0000 0.0000 0.5 0.00a 0.097a 5.26b 5.092b 6.74c 6.843c 0.178 0.0000 0.178 0.0000 0.0000 1 0.01a 0.060a 7.02b 7.134b 8.66c 8.246c 0.154 0.0000 0.154 0.0000 0.0000 2 0.16a 0.222a 12.12b 9.509b 13.82c 9.523b 0.189 0.0000 0.189 0.0000 0.0000 3 0.03a 0.272a 12.32b 7.461b 12.81b 6.970b 0.148 0.3110 0.148 0.3110 0.2235 a,b,c – RF PACVD CARBON + BHA > RF PACVD CARBON and BHA (p < 0.05).

TABLE 3. Reducing activity of GSH on DPPH radical in time manner Reducting avtivity (%) of ANOVA TIME glutathione RF PACVD (min) RF PACVD CARBON SEM p (GSH) CARBON + GSH 0.25 0.013a 45.33b 43.07b 1.832 0.0211 0.5 0a 12.87b 13.65b 1.945 0.0000 10a 10.46b 12.57b 1.734 0.0000 20a 10.80b 13.21c 0.956 0.0014 30a 4.05b 3.44b 0.531 0.0088 a, b, c – means with different superscripts are signifi cantly different (p < 0.05). Carbon synthetized by RF PACVD method enhances... 155 the supplemented synthetic antioxidants character of carbon allows for applying such BHA. The effectiveness of anti- its electrochemical properties from the oxidants depends on the accompanied donor to the acceptor state (Frąckowiak carriers employed to reach the target. and Beguin 2001). Number of studies presents the improve- ment of the antioxidant effi ciency, in the way of binding them to the nano-carriers CONCLUSIONS consisting of different carbon allotropes which exhibit reducing properties. Ac- According to the present results, carbon cording to Kato et al. (2009), water-solu- colloid increases the activity of antioxi- ble derivative of fullerene (C60) exhibit dants, especially in the environment that antioxidant activity. Flavonoids bind to affect their activity (high temperature). hydroxyl nanotubes revealed signifi cant Moreover, the effect of RF PACVD car- increase in the antioxidant properties in bon powder depends on the biochemical vitro (Nichit and Stamatin 2013). structure of the antioxidant. The pre- We observed the signifi cant increase sented results suggest that the surface of in the reducing force of the ascorbic acid carbon particles suspended in water, cre- and BHA in the RF PACVD colloidal en- ates friendly reductive environment for vironment. Probably the high reactivity antioxidants with hydroxyl group (AA of the obtained complexes is the result of and BHA), but not GSH. molecular self-assembly into supramo- lecular structures. Self-assembly begins at the level of atoms, where it relies on REFERENCES the chemical complementarity. On the BAKOWICZ K., 2003: Bioactivity of Dia- molecular level, it allows the materi- mond. Ph.D. Thesis, Technical Univer- als to precipitate and form highly orga- sity of Lodz (in Polish). nized structures. We presume that non- BAKOWICZ-MITURA K., BARTOSZ G., -covalent interactions (such as hydrogen MITURA S., 2007: Infl uence of diamond bonding and ionic interactions, hydro- powder particles on human gene expres- phobic interactions) between carbon and sion. Surface & Coatings Technology antioxidants, enhance the thermal stabil- 201: 6131–6135. BANHEGYI G., BRAUN L., CSALA M., ity of the new formed new complexes. PUSKAS F., MANDL J., 1997: Ascor- This happens in the process of stabiliz- bate metabolism and its regulation in ani- ing the engaged electrons on the carbox- mals. Free Radical Biology and Medicine ylic acid groups by electronic properties 23: 793–803 of RF PACVD carbon structure, espe- CZERNIAK-RECZULSKA M., NIEDZIEL- cially as the active interaction occurs SKI P., BALCERCZYK A., BAR- between the experimental factors. Many TOSZ G., KAROWICZ-BILINSKA A., MITURA K., 2010: Biological properties allotropes of carbon (graphite, diamond, of different type carbon particles in vitro fullerenes, nanotubes or graphen) are study on primary culture of endothelial presented as the interesting materials for cells. Journal for Nanoscience and Nano- electrochemical applications, like the en- technology 10: 1065–1071. ergy storage. Moreover, the amphoteric 156 T. Niemiec et al.

FRĄCKOWIAK E., BEGUIN F., 2001: Car- przeciwutleniaczem a wolnymi rodnikami. Im bon material for the electrochemical stor- skuteczniejszy przeciwutleniacz, tym stała szyb- age of energy in capacitors. Carbon 39: kości powstawania zredukowanej formy difeny- 937–950. lopikrylohydrazyny (DPPH-H) jest większa. Jak- KATO S., AOSHIMA H., SAITOH Y., MIWA kolwiek nie stwierdzono aktywności redukującej N., 2009: Highly hydroxylated or gamma hydrokoloidu węgla RF PACVD to jednak roz- cyclodextrin-bicapped water-soluble of puszczone w nim antyoksydanty istotnie zwięk- fullerene: antioxidant ability assessed by szyły swój potencjał redukujący w porównaniu electron spin resonance method and beta- do próbek zawierających czysty antyoksydant. catotene bleaching assay. Bioorganic & Uzyskane wyniki sugerują, że cząstki węgla RF PACVD zawieszone w wodzie stwarzają korzyst- medicinal chemistry letters. ne środowisko dla aktywności związków redu- KRINGS U., BERGER R.G., 2001: Antioxi- kujących z aktywną grupą hydroksylową (AA dant activity of some roasted foods. Food i BHA), ale nie dla związków z grupą sulfhydry- Chemistry 72: 223–229. lową (GSH). MITURA S., 1987: Nucleation of diamond powder particles in an RF methane MS. received in November 2013 plasma. Journal of Crystal Growth, 80: 417–424. Authors’ addresses: NICHIT C., STAMATIN I., 2013: The anti- Tomasz Niemiec, Anna Kruk-Roszkowska, oxidant activity of the biohybrides based Ewa Sawosz-Chwalibóg on carboxylated/hydroxylated carbon Wydział Nauk o Zwierzętach SGGW Katedra Żywienia Zwierząt i Gospodarki nanotubes-fl avonoid compounds. Digest Paszowej Journal of Nanomaterials and Biostruc- ul. Ciszewskiego 8, 02-786 Warszawa tures 8: 445–455. Poland NIEMIEC T., SAWOSZ E., CHWALI- e-mail: [email protected] BÓG E., 2005: Effect of high doses of l- ascorbic acid on the antioxidative/oxida- Katarzyna Mitura Instytut Technologii i Edukacji tive state in the rats. Journal of Animal Politechniki Koszalińskiej and Feed Sciences 14: 553–556. Katedra Radiologii i Radioterapii Materiałowej ul. Śniadeckich 2, 75-453 Koszalin Streszczenie: Węgiel wytworzony metodą RF e-mail: [email protected] PACVD zwiększa aktywność antyoksydantów. Maciej Szmidt Celem doświadczenia było zbadanie aktywności Wydział Medycyny Weterynaryjnej SGGW redukującej wybranych antyoksydantów (kwasu Katedra Nauk Morfologicznych askorbinowego, butylowanego hydroksyanizolu ul. Nowoursynowska 159 i glutationu) w obecności cząstek węgla wytwo- 02-776 Warszawa rzonego metodą RF PACVD testem DPPH. Test Poland ten polega na pomiarze kinetyki reakcji między e-mail: [email protected] Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 157–160 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Polymorphism of insulin-like growth factor IGF-1 in position 211 in national sheep breeds with carped wool compared to Polish Merino and European Mufl on (Ovis aries musimon)* ROMAN NIŻNIKOWSKI, GRZEGORZ CZUB, KRZYSZTOF GŁOWACZ, MARCIN ŚWIĄTEK, MAGDALENA ŚLĘZAK Department of Animal Breeding and Production, Warsaw University of Life Sciences – SGGW

Abstract: Polymorphism of insulin-like growth matomedin e.g. IGF-1 and stimulates the factor IGF-1 in position 211 in national sheep secretion of hypothalamic somatostatin breeds with carped wool compared to Polish Me- inhibits secretion of growth hormone rino and European Moufl on (Ovis aries musimon). Research was carried out on 1,684 sheep (1,093 ♀ (Krzymowski et al. 1998). Insulin factor and 591 ♂) originating from European Moufl on gene is exchanged among the conditions (Ovis aries musimon), its hybrids with Polish heat that allow the identifi cation of races, as sheep and four sheep breeds that are character- demonstrated in the Mediterranean coun- ized by a mixed wool compared to Polish Merino. tries (Pariset et al. 2006). This indicates All animals were subjected to gene identifi cation factor insulin-IGF-1, in the assessment of allele the possibility of using the factors in the C and T. In conclusion it should be noted that in study of animal origin, what has signaled the 7 examined breed groups sheep showed no Heindleder et al. (2001). Accordingly, tak- polymorphism alleles and genotypes of insulin- ing into consideration the fact that report- -like factor gene IGF-1, limiting its scope to de- ed in the cited work conditions infl uence termine the C allele and genotype CC. This result indicates the need for further research in this area frequency of insuline-like factor IGF-1, in “culture” sheep imported and adapted to Polish decided to examine the distributions of conditions and the production environment.* the presence of domestic sheep breeds Key words: sheep, IGF-1, distribution of alleles (PZO, 2013). Compared to the ancestor – and genotypes European Moufl on (Ovis aries musimon) and Polish Merino, and the sheep breeds of mixed wool. In addition, conditioning INTRODUCTION it can assist in the work of research into the origins of the sheep (Heindleder et al. Protein IGF-1 is one of the key compo- 2001), which also begins to raise more nents of the pathway of growth hormone and more interest. (Franco et al. 2005). IGF-1 is produced in the liver and is responsible for cell growth and body treatments. It is believed that MATERIAL AND METHODS the effect of growth hormone occurs in the tissue in cooperation with local so- The study Polish Merino sheep fl ock (two herds) of Wielkopolska voivode- * Work done as part of the international project no ship, sheep of mixed wool from 625/N-WĘGRY/2009/0. 158 R. Niżnikowski et al.

Małopolska, Łódzkie i Podkarpackie DNA modifi cation by removal of heme (29 herds), and the European Moufl on compounds lysis of erythrocytes prod- (3 herds) i Mufl onowrzosówek (2 herds) ucts. DNA is isolated from leukocytes by from Wielkopolskie and Lubuskie. Ewes chromatography on mini-columns of sil- and rams were aged 2 to 11 years (Ta- icate A&A Biotechnology. The fraction ble 1). Herds were randomly selected thus obtained was used as template DNA for sampling. From the jugular vein of for amplifi cation of polymorphic gene animals blood was obtained into tubes allele fragment. Genotyping of allele containing EDTA, for the isolation was performer with KASPar® system. of genomic DNA for the analysis of mo- This system (www.kbioscience.co.uk) lecular genetics. The study was carried method is to use a point SNP polymor- out assessment of genes and genotypes phism using primers listed in Table 2. frequency of insulin-like gene factor Based on reading the DNA samples IGF-1. were genotyped within the ewes and DNA was isolated from blood leuco- rams shows distributions of alleles and cytes using the conserved EDTA. In or- genotypes frequency separately for each der to receive high quality DNA suitable race. Allele and genotype frequencies after freezing and thawing of a reusable, were compared depending on the breed 2 blood is pretreated with the resulting using SPSS v.21 with Chi test was

TABLE 1. Summary of experimental material used in the study in 2009–2012 Sex Breed Herd sampling ♀♂ 2010 – 2 ♂; 2011 – 3 ♀, 7 ♂; 2012 – 120♀, European Moufl on 123 73 64 ♂ Mufl onowrzosówka 8 7 2010 – 4♀ , 4 ♂; 2011 – 4 ♀, 3 ♂ 2009 – 114 ♀, 128 ♂; 2010 – 115 ♀, 113 ♂; Polish Heat Sheep 334 355 2011 – 105 ♀, 114 ♂ Swiniarka Sheep 109 33 2009 – 28 ♀, 16 ♂; 2010 – 81♀, 17 ♂ Polish Mountain Sheep – white 141 15 2010 Polish Mountain Sheep – coloured 168 13 2011 2010 – 101♀, 40 ♂; 2011 – 94♀, 48 ♂; Polish Merino 210 95 2012 – 15 ♀, 7 ♂ Total within gender 1 093 591 × Total 1 684

TABLE 2. The primers and SNP genotyping of the locus of IGF-1 Starters 3’ to 5’ Locus Name SNP Localization (forward/reverse) CACACACCTTGTTGCACTCC/ AY737509: 211 IGF-1 insuline-like factor Ekson 3 /GCTGAGTTGGTTGGATGCTCT C > Ta aPariset et al. (2006). Polymorphism of insulin-like growth factor... 159 assessed range of alleles and genotypes this area in “culture” sheep imported and frequency between races, sexes, and the adapted to Polish conditions and the pro- differences between the sexes in terms duction environment. of individual alleles and genotypes. The results are presented in tables. REFERENCES

RESULTS AND DISCUSSION FRANCO M.M., ANTUNES R.C., SIL- VA H.D., GOULARDT L.R., 2005. As- Studies have shown that gene at position sociation of PIT1, GH and GHRH poly- morphisms with performance and carcass 211, factor IGF-1 were found to have traits in Landrace pigs. J. Appl. Genetics only the C allele. Analyses of 1,684 sam- 46 (2): 195–200. ples collected from the rated sheep did HEINDLEDER S., JANKE A., WASS- not show the presence of the T allele in MUTH R., 2001: Molecular data on wild any case. In comparison with the results sheep genetic resources and domestic of Pariset et al. (2006), mainly describing sheep evolution. Arch. Tierz. (Special is- sheep found in the region of the Mediter- sue) 44, 271–279. ranean, Black and Germany, sheep tested KRZYMOWSKI T. (Ed.), 1998: Fizjologia zwierząt. PWRiL, Warszawa: 143–200. in Poland showed no polymorphism here. PARISET L., CAPPUCIO I., AJMONE- Due to the fact that many of the races -MARSAN P., BRUFORD M., DUN- comes from European Moufl on (Hein- NER S., CORTES O., ERHARDT G., dleder et al. 2001), it is expected that the PRINZENBERG E-M., GUTSCHER K., distribution of the conditions occurring JOOST S., PINTO-JUMA G., NIJMAN in sheep breeds reared in Poland (includ- I.J., LENSTRA J.A., PEREZ T., VAL- ing European Moufl on) was characteris- ENTINI A., 2006: Characterization of 37 Breed-Specifi c Single-Nucleotide Poly- tic of this part of the world and different morphisms in Sheep. J. Hered. 97 (5): from the observed trends. Perhaps it has 531–534. to do with another course of life proc- PZO, 2013: Hodowla Owiec i Kóz w Polsce esses of growth and development, which w 2012 roku. Wyd. PZO, Warszawa. wrote Krzymowski et al. (1998). In this SPSS v. 21 for Windows, IBM Inc. situation, can be a useful series of studies on sheep imported into Polish, polymor- Streszczenie: Polimorfi zm genu czynnika insuli- phism alleles and genotypes of insulin- nopodobnego IGF-1 w pozycji 211 u krajowych owiec o wełnie mieszanej w porównaniu do me- -like factor gene subject to change due to rynosa polskiego i mufl ona europejskiego (Ovis processes of adaptation – which requires aries musimon). Badania przeprowadzono na ma- further work in this area. teriale 1684 owiec (1093 ♀ i 591♂) pochodzących Generally, it should be noted that the od mufl ona europejskiego (Ovis aries musimon), summing up of the 7 examined breed jego mieszańców z wrzosówką oraz czterech groups sheep showed no polymorphism ras owiec charakteryzujących się okrywą wełnistą mieszaną porównywanych do merynosa alleles and genotypes of factor IGF-1, polskiego. Wszystkie zwierzęta poddane były limiting its scope to determine is the C identyfi kacji genu czynnika insulinopodobnego allele and genotype CC. This result in- IGF-1, w zakresie oceny występowania alleli C dicates the need for further research in i T. Podsumowując, należy stwierdzić, iż u ba- danych 7 grup rasowych owiec nie wykazano 160 R. Niżnikowski et al. polimorfi zmu występowania alleli i genotypów MS. received in November 2013 genu czynnika insulinopodobnego IGF-1, ograni- czając jego zakres do ustalenie jedynie do allelu C Authors’ address: i genotypu CC. Wynik ten wskazuje na potrzeby Roman Niżnikowski przeprowadzenia dalszych badań z tego zakresu Wydział Nauk o Zwierzętach SGGW u owiec kulturalnych pochodzących z importu Katedra Szczegółowej Hodowli Zwierząt i adaptowanych w polskich warunkach środowi- ul. Ciszewskiego 8, 02-786 Warszawa ska produkcyjnego. Poland e-mail: [email protected] Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 161–166 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Sensitivity of imago and larvae of the lesser mealworm Alphitobius diaperinus (Panzer 1797) in a sawdust litter to selected species and strains of Steinernematidae and Heterorhabditidae under laboratory conditions ELŻBIETA PEZOWICZ, ANNA MAZURKIEWICZ, DOROTA TUMIALIS Department of Animal Environment Biology, Warsaw University of Life Sciences – SGGW

Abstract: Sensitivity of imago and larvae of the The lesser mealworm is dangerous as lesser mealworm Alphitobius diaperinus (Panzer a potential carrier of disease-causing 1797) in a sawdust litter to selected species and organisms like: bacteria Escherichia strains of Steinernematidae and Heterorhabditi- dae under laboratory conditions. Sensitivity of sp. (McAllister et al. 1996), Salmo- imagines and larvae of the lesser mealworm to nella typhimurium, Bacillus, Strepto- selected species and strains of entomopathogenic coccus (McAllister et al. 1994), viruses nematodes was studied in a pine sawdust litter on causing Marek’s, Gumboro, Newcastle which chicken were kept from one to six weeks ac- diseases and bird fl u, parasites of the cording to their production cycle (Niemiec 1998). The following nematode species and strains were Eimeria sp., the larvae of tapeworms Rai- used: S. feltiae from bioinsecticides Ovinema lietina sp. and Choanotaenia sp. (Eidson and Nemaplus, S. affi nis, S. carpocapsae and et al. 1965, Wilson et al. 1986, Avancini H. bacteriophora strain Brecon. S. feltiae from and Ueta 1990, Goodwin and Waltman biopreparation Ovinema appeared most invasive 1996, Steelman 1996). The pathogens to the larvae and imagines of A. diaperinus. are transmitted when birds eat infected Key words: lesser mealworm, pine sawdust, ento- insects. A broiler may eat 450 larvae mopathogenic nematodes, biological control a day. Moreover, the insects bring sub- stantial economic losses. They destroy polyurethane isolation of farm houses INTRODUCTION during their migration and pupation The lesser mealworm can be found world- (Despins 1991, Steelman 1996). wide in Europe, Asia North and South The control of the lesser mealworm America, Australia and Africa. The in- with insecticides does not bring expect- sect inhabits dark, wet and warm places ed results, moreover, the chemicals have (Arshad and Khattack 1984, Awaknavar a long waiting periods and insects and Rajasekhar 1999) feeding on groats, quickly become immune to insecticides. fl our, cereal grains (Lorenzo 1990) or Therefore, entomopathogenic nematodes milk powder (Basak et al. 1991). First (EPNs) seem to be an alternative as bio- information on the presence of the lesser insecticides in the population control of mealworm in broiler houses appeared in this pest (Geden et al. 1985, 1987a and the 1950s in the USA (Gould and Mo- b, Geden and Axtell 1988, Szczepanik ses 1951, Harding and Biasell 1958). 2000, Szalanski et al. 2004). 162 E. Pezowicz, A. Mazurkiewicz, D. Tumialis

MATERIAL AND METHODS the experiment was 1·106 per m2 in the variant with larvae and 1·106 per m2 in Steinernema feltiae from bioprepara- that with beetles. Nematodes were intro- tions Ovinema and Nemaplus, S. affi nis, duced in 1 ml of water supplemented to S. carpocapsae, and H. bacteriophora 5 ml with distilled water. Experiments (Brecon) were selected for experiments were carried out at 28°C in the SANYO on the sensitivity of growth stages of chamber. Moisture was controlled every the lesser mealworm in straw and lit- day. After 7 and 14 days insect mortality ter. These nematodes were grown in the was counted and checked whether nema- laboratory of the Department of Zoo- todes were the reason. Control variant logy, Warsaw University of Life Scienc- consisted of insects in clean, moist pine es – SGGW with the following method. sawdust sprayed with 5 ml of distilled Ten caterpillars of Galleria mellonella water. were placed in a Petri dish of a diameter Tukey-Freeman test was used for sta- of 9 cm lined with fi lter paper and then tistical processing of results. 1 ml of a suspension of 500 larvae of ap- propriate nematode species was poured over the dish. Caterpillar mortality was RESULTS AND DISCUSSION controlled every day. Dead insects were transferred onto small sponges immersed As seen in Table 1 the highest effi ciency in water. After 7 to 14 days invasive in all types of substratum was found in larvae of nematodes started to transfer S. feltiae from Ovinema biopreparation. into water. After two-week keeping in The extensity of infection of A. diaperi- a fridge the invasive larvae of nematodes nus larvae was 60% in clean pine saw- were ready for experiment. dust and decreased to 42% in one-week Experiments with the larvae and bee- pine sawdust and to 34% in two-week tles of the lesser mealworm were per- sawdust. The older was the litter the formed in plastic boxes 9.5 cm long, more hen faeces and ammonia it con- 7.5 cm wide and 6.0 cm high. The box- tained. Therefore, nematodes lost their es were fi lled with pine sawdust from invasiveness to the lesser mealworm lar- a broiler house to a height of 3.0 cm. Ten vae in such a habitat. In three-week litter adult insects or 10 larvae of the lesser the effect of nematodes was the same as mealworm were placed in the box to- in the control (Table 1). Entomopatho- gether with pine sawdust from substra- genic nematodes are the animals very tum on which chicken were bred. Broiler resistant to various unfavourable exter- production lasted 6 weeks. nal conditions. For example, most pesti- Litter used in experiments was taken cides are not harmful to invasive larvae one, two, three and four weeks after the of these nematodes (Kaya 1990, Kami- broiler house was stocked with chicken. onek 1992) even at doses larger than Since no effect of nematode activity was the recommended. Some pesticides may found in three- and four-week litter, the even stimulate nematodes’ movements experiments with older litter were ne- (Vainio 1993). Only fungicides and her- glected. The dose of nematodes used in bicides, including ureal ones, decrease Sensitivity of imago and larvae the lesser mealworm... 163

TABLE 1. Comparison of the extensity of infection (%) of the lesser mealworm larvae after their con- tact with selected species/strains of Steinernematidae and Heterorhabditidae nematodes in substratum made of pine sawdust Percentage of dead larvae of A. diaperinus in which nematodes were noted Nematode species Sawdust One-week Two-week Three-week without faeces sawdust sawdust sawdust S. feltiae (Ovinema) 60 d 42 c 34 bc 2 a S. feltiae (Nemaplus) 46 c 36 c 20 b 2 a S. affi nis 32 bc 30 bc 20 b 4 a H. bacteriophora (Brecon) 20 b 20 b 8 a 4 a S. carpocapsae 32 bc 28 bc 16 a 2 a Control 4 a 4 a 2 a 2 a Different letters denote signifi cant differences in the extensity at p < 0.05. the pathogenicity of nematodes (Das and effi cient against both growth stages of Divakar 1987, Kamionek 1992). The ef- the insect. Geden et al. (1985) obtained fect of organic fertilisation was studied 35.8% infection in larvae and 10.2% in- by Bednarek and Gaugler (1997) who fection in adult forms of A. diaperinus demonstrated the increased effectiveness with nematodes S. feltiae in a substratum of entomopathogenic nematodes after the composed of pine sawdust, food remains contact with that fertiliser. Poultry faeces and faeces. Szalanski et al. (2004) ob- are also organic fertilisers but apart from tained similar results using a pine-cedar N, P and K it contains large amounts of substratum and nematode strains S. car- ammonia which is slowly released to pocapsae Mexican and S. feltiae Pye at the environment (Oudedag and Luesink a dose corresponding to 200 invasive lar- 1998). This is probably the reason why vae per insect. Pezowicz (2006) studied most nematode species lose their patho- the invasiveness of entomopathogenic genic properties. nematodes in straw and in litter from Mortality of adult insects of the lesser a broiler house and obtained lower ex- mealworm in various substrata is pre- tensity than in this study. sented in Table 2. Most effi cient were Not until recently attempts have been S. feltiae from Ovinema biopreparation. undertaken to improve entomopathogen- These nematodes caused 20% mortality ic nematodes’ ability of fi nding hosts, to in adult insects after two weeks of ex- increase their invasiveness and in gener- periment, 24% mortality in one-week lit- al to increase their effectiveness in insect ter and 4% mortality in three-week litter. control. Gaugler (1987) was a promoter S. affi nis caused 26% mortality in adult of studies on artifi cial selection, genetic insects kept in clean sawdust and 20% engineering and strain hybridization. mortality in one-week litter. Comparison Nematodes from Ovinema bioprepa- of the Tables 1 and 2 shows that S. feltiae ration are an outcome of these studies from Ovinema biopreparation were most (Tomalak 1994 and 1998). 164 E. Pezowicz, A. Mazurkiewicz, D. Tumialis

TABLE 2. Comparison of the extensity of infection (%) of the lesser mealworm imagines after their contact with selected species/strains of Steinernematidae and Heterorhabditidae nematodes in substra- tum made of pine sawdust Percentage of dead imagines of A. diaperinus in which nematodes were noted Nematode species One-week Two-week Three-week Clean sawdust sawdust sawdust sawdust S. affi nis 26 c 20 bc 18 b 6 a S. feltiae (Ovinema) 36 d 24 c 20 bc 4 a S. feltiae (Nemaplus) 24 b 18 a 14 a 4 a H. bacteriophora (Brecon) 28 bc 20 b 16 a 2 a Control 4 a 2 a 2 a 4 a Different letters denote signifi cant differences in the extensity at p < 0.05. CONCLUSIONS BASAK P.K., CHOUDHURI D.K., SEN- 1. The invasiveness of entomopatho- GUPTA T., 1991: On the beetle (Insecta: genic nematodes decreases with in- Coleoptera) infestation of milk powder creasing concentration of ammonia at Calcutta warehouses. J. Bengal Natur. from chicken faeces. History Soc. 10, 50–55. 2. Steinernema feltiae from the bio- BEDNAREK A., GAUGLER R., 1997: preparation Ovinema appeared most Compatibility of soil amendments with effective in controlling larva and im- entomopathogenic nematodes. J. Nema- agines of the lesser mealworm. tol. 29, 220–227. DAS J.N., DIVAKAR B.J., 1987: Compat- 3. Pine sawdust is a better substratum ibility of certain pesticides with DD-136 than litter to control the lesser meal- nematode. Plant. Prot. Bull. 39, 20–22. worm in broiler houses. DESPINS J.L., TURNEREC., PFEIF- FER D.G., 1991. Evaluation of methods to protect poultry house insulation from REFERENCES infestation by lesser mealworm (Coleop- tera: Tenebrionidae). J. Agric. Entomol. ARSHAD M.J., KHATTACK A., 1984: In- 8, 209–217. sect fauna of birds nests of N.W.F.P. Bull. EIDSON C.S., SCHMITTLE S.C., LAL J.B., Zool. Pakistan 2, 1–7. GOODE R.B., 1965: The role of the AWAKNAVAR J.S., RAJASEKHAR D.W., darkling beetle, Alphitobius diaperinus, 1999: Bird nests: source of infestation in the transmission of acute leukosis in to storage? Integr. Pest Menag. Rev. 4, chickens. Poult. Sci. 44, 1366–1367. 53–73. GAUGLER R., 1987: Entomogenous nema- AVANCINI R.M., UETA M.T., 1990: Ma- todes and their prospects for genetic im- nure breeding insects (Diptera and Co- provement. In: Biotechnology in Inver- leoptera) responsible for cestoidosis in tebrate Pathology and Cell Culture. Ed. caged layer hens. J. Appl. Entomol. 110, K. Maramorosh. Acad. Press, New York, 307–312. 457–484. Sensitivity of imago and larvae the lesser mealworm... 165

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SZCZEPANIK M., 2000: Temperature ef- i szczepy nicieni Steinernematidae i Heterorhabdi- fects on the effi cacy of nematodes against tidae w warunkach laboratoryjnych. Celem badań the lesser mealworm Alphitobius diaperi- było określenie wpływu nicieni entomopatoge- nus (Coleoptera: Tenebrionidae). J. Pol. nicznych na śmiertelność larw i postaci imaginal- Entomol. 69, 483–490. nych pleśniakowca lśniącego Alphitobius diaperi- TOMALAK M., 1994: Selective breeding of nus (Panzer 1797) w ściółce z trocin sosnowych. Steinernema feltiae (Filipjev) (Nematoda: Zbadano przeżywalność stadiów rozwojowych Steinernematidae) for improved effi cacy owadów w cyklu produkcyjnym hodowli kurcząt. in control of a mushroom fl y, Lycoriella Stwierdzono, że ze wzrostem stężenia amoniaku solani Winnertz (Diptera: Sciaridae). pochodzącym z odchodów kurcząt maleje inwa- zyjność nicieni entomopatogenicznych. Nicienie Biol. Sci. Technol. 4, 187–198. żyły tylko do trzeciego tygodnia cyklu produkcyj- TOMALAK M., 1998: Selekcja i mutagene- nego kurcząt. Najbardziej skutecznym gatunkiem za w genetycznym doskonaleniu nicieni w stosunku do larw i imago pleśniakowca okazał owadobójczych dla celów biologiczne- się S. feltiae z biopreparatu Ovinema. Trociny so- go zwalczania szkodników. Prace Nauk. snowe są lepszym podłożem niż ściółka słomiana IOR w Poznaniu, Poznań. do zwalczania pleśniakowca lśniącego w brojler- VAINIO A., 1993: Effect of pesticides on long- niach. -term survivals of Steinernema feltiae in the fi eld. IOBC/ wprs Bulletin 17, 3, 70–76. MS. received in November 2013 WILSON D.D., SCHMIDTMANN E.T., RICHARD R.D., LEHMAN R.D., 1986: Isolation of avian infl uenza from insects. Authors’ address: Arbovir. Reser. 6, 221–223. Elżbieta Pezowicz Wydział Nauk o Zwierzętach SGGW Katedra Biologii Środowiska Zwierząt Streszczenie: Wrażliwość imago i larw pleśnia- ul. Ciszewskiego 8, 02-786, Warszawa kowca lśniącego Alphitobius diaperinus (Pan- Poland zer 1797) w ściółce z trocin na wybrane gatunki e-mail: [email protected] Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 167–171 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Comparison of calving course of Limousine purebreds and their crossbreeds with Polish Holstein-Friesian cows TOMASZ PRZYSUCHA, JAN SLÓSARZ, MARCIN GOŁĘBIEWSKI, MAŁGORZATA KUNOWSKA-SLÓSARZ Department of Animal Breeding and Production, Warsaw University of Life Sciences – SGGW

Abstract: Comparison of calving course of Lim- INTRODUCTION ousine purebreds and their crossbreeds with Pol- ish Holstein-Friesian cows. Calving course of 100 Beef cows are not milked, hence the eco- cows of Limousine breed covered by bulls of the same breed as well as 100 cows of Polish Hol- nomic results of this branch of beef pro- stein-Friesian breed inseminated with Limousine duction depend mainly on the number of bull’s semen was investigated. In purebred popu- healthy and vital calves born from cows lation the material was taken from the beef cattle without any negative infl uence on their recording scheme provided by the Polish Asso- health and future reproduction. ciation of Beef Cattle Breeders and Producers. In case of commercial crossing the material was Among many factors infl uencing the taken from the questionnaires collected by the proper calves development, beside of technicians representing Mazovian Centre of Ani- husbandry conditions, the calving course mal Breeding and Reproduction in Łowicz. The is mentioned by many authors (Nogalski data base covered calving course degree and calf 2004, Grodzki et al. 2009, Przysucha body weight at birth degree. It was proved, that the signifi cantly higher ratio of deliveries when 2009). human help was needed was stated in the pure- Calving course depends on many fac- bred Limousine population (14%), in comparison tors like: breed, body weight and condi- to crossbreeding where the above mentioned ratio tion of cow, calving number, body weight was only 7%. The most diffi cult calvings were ob- and sex of calf at delivery. Calving dif- served for the calves of the highest birth weight. fi culty, growth rate and calf muscularity The high ratio of assisted calvings in commercial crossing as well as in purebred population of Lim- are the main traits used to formulate the ousines suggest that all of the deliveries should be evaluation criteria of breeding indexes monitored by the breeders. It should be also stated for beef cattle in many countries. that the ratio of diffi cult calvings in the commer- According to many authors (Philipp- cial crossing is signifi cantly lower than that ob- son 1976, Philippson 1977, Burfen- served in purebred PHF population. Because of so low ratio of assisted calvings in commercial ing et al. 1978, Meijering 1984, Berger crossing with Limousine bulls it should be recom- 1994, Nogalski and Klupczyński 1999, mended to use the semen of bulls which pedigree Grodzki et al. 2010), calving course is show high body weight at birth and muscularity. infl uenced by many genetic and pheno- Key words: Limousine, calving course, commer- typic factors. On the base of the wide cial crossing literature review Nogalski (2004) and Przysucha (2009) agreed, that the main factors affecting calving course were: cow age (calving number), calf body 168 T. Przysucha et al. weight at birth, calf sex, cow caliber and The aim of the research was to deter- its pelvis area, parents breed, pregnancy mine the frequency of diffi cult calvings length, parents genotypes, cow condition in Limousine cows and dairy cows in- and feeding during pregnancy, calf shape seminated by Limousine bulls semen in and its position at delivery. All the above the commercial crossing. mentioned factors are strictly connected one to the other. Following the rules of the most breed- MATERIAL AND METHODS ing programs for beef breeds it is easy to recognize, that calving course, calf body The calving course of 100 purebred Lim- weight at delivery, calf body shape, daily ousine (2–6 years old) cows inseminated growth rate, feed conversion, cutting rate by the same breed bulls as well as 100 and carcass quality are the main items to Polish Holstein-Friesian (PHF) cows the breeding value formula (Nogalski serviced by Limousine bulls semen (com- and Klupczyński 1999, Przysucha et al. mercial crossbreeds) was monitored. 2005, Przysucha et al. 2007). Results of beef cattle recording scheme In the young Polish beef cattle pro- conducted by Polish Association of Beef duction the purebred female population Cattle Breeders and Producers constitut- consists of fourteen beef breeds, but Lim- ed the material for investigation. ousine cows and heifers number amounts In case of commercial crossing (dairy to about 70% and in commercial crossing cow × Limousine bull) the calving course to 89%. It means, that the Polish breed- was examined by technicians from Ma- ers have already made a decision about zovian Centre of Animal Breeding and the most useful beef breed. Limousine Reproduction in Łowicz. breed is predisposed for the intensive The data base covered calving course fattening with the very high daily gain degree and calf body weight at birth de- at the moderate feedstuffs use. Animals gree. Calving course was evaluated as: kept for slaughtering can be fattened to normal, without any assistance (1), help the high body weight without any risk of the breeder needed (2). of over fattening. The proper use of beef Depending on body weight at birth (kg) bulls (without regard of their breed) is calves were divided into the following crucial for the commercial crossing re- groups: up to 35 kg, 36–45 kg, >45 kg. sults. This type of crossbreeding is often Statistical analysis of the calving dif- identifi ed with calving diffi culties. Pa- fi culties frequencies was carried out by pers provided by many authors clearly Chi-square test using SPSS 12.0 (Statisti- proved, that the calving diffi culties with- cal Product and Service Solution 1998). in specialized beef breeds are the same or even lower (in case of commercial crossing) than those in case of purebred RESULTS AND DISCUSSION dairy herd (Hanset 1981, Nix et al. 1998, The ratio of calving course examination Nogalski 2002, Przysucha and Grodzki, for purebred and crossbred populations 2008, Przysucha et al. 2009). was presented in Table 1. Comparison of calving course of Limousine purebreds... 169

TABLE 1. The ratio of calving course examination for purebred and crossbred populations Calving course Factor Unit 1 2 Total N 86 14 100 purebred % 86 14 100 Population N 93 7 100 commercial crossing % 93 7 100

Obtained results show signifi cantly with Limousine bulls is much lower than higher ratio of diffi cult calvings in the those shown by the authors dealing with purebred Limousine population in com- the purebred PHF (Fouz et al. 2013). parison to commercial crossing. Because of the relatively small per- The calf body weight at birth had centage of diffi cult deliveries in com- signifi cant infl uence on calving course mercial crossing with Limousine bulls, ratio. The highest ratio of diffi cult calv- this breed should be recommended to ings were found, when calf body weight use for that purpose. The highest calv- was the highest (Table 2). Obtained re- ing diffi culty, compared with pure Hol- sults are the same as those presented by steins was for crosses with Belgian Blue practically all the authors dealing with followed by Limousine and Galician the problem. Blonde (Fouz et al. 2013). TABLE 2. The infl uence of calf body weight at birth on calving course Calf body weight at Calving course (%) Population birth 12 (kg) <35 100 0 Purebred 35–45 89 11 >45 79 21 <35 100 0 Commercial crossing 35–45 91 9 >45 73 27

Relatively high percentage of dif- Irrespectively to calves genotype fi cult calvings (with assistance needed) (purebreds or crossbreeds) the frequen- in purebred Limousines and crossbreds cies of diffi cult calvings increase with the from commercial crossing of PHF cows birth body weight of the calves (Table 2). with Limousine bulls suggests, that all Higher rate of dystocia were observed in of the deliveries should be monitored by Limousine crossbreds (38%) compared the breeder. to purebreds (32%). It should be stated, that the ratio of diffi cult calvings in commercial crossing 170 T. Przysucha et al.

REFERENCES NOGALSKI Z., 2004: Zootechniczne uwa- runkowania jakości porodu jałówek BERGER P.J., 1994: Genetic prediction for i krów czarno-białych. Rozpr. i Monogr. calving ease in the United States: Data, 101, UWM Olsztyn. models and use by the dairy industry. J. NOGALSKI Z., KLUPCZYŃSKI J., 1999: Dairy Sci. 77, 1146–1153. Przebieg wycieleń, wielkość i żywotność BURFENING P.J., KRESS D.D., FRIED- cieląt w stadzie bydła mięsnego w Miel- RICH R.L., VANIMAN D.D., 1978: nie. Zesz. Nauk. Prz. Hod. 44, 353–363. Phenotypic and genetic relationships PHILIPSSON J., 1976: Studies on calving between calving ease, gestation length, diffi culty, stillbirth and associated fac- birth weight and preweaning growth. J. tors in Swedish cattle breeds. III. Ge- Anim. Sci. 47 (3), 595–600. netic parameters. Acta Agric. Scand. 26, GRODZKI H., NAWROCKI L., PRZY- 211–220. SUCHA T., GRODZKI G., KONOP- PHILIPSSON J., 1977: Studies on calving KA B., 2009: Chów bydła mięsnego. diffi culty, stillbirth and associated fac- 2009. Wielkopolskie Wydawnictwo Rol- tors in Swedish cattle breeds. VI. Effects nicze, Poznań. of crossbreeding. Acta Agric. Scand. 27, FOUZ R., GANDOY F., SANJUÁN M.L., 58–64. YUS E., DIÉGUEZ F.J., 2013: The use PRZYSUCHA T., 2009: Osobnicze uwarun- of crossbreeding with beef bulls in dairy kowania przebiegu ocieleń krów oraz herds: effects on calving diffi culty and umięśnienia i żywotności cieląt pocho- gestation length. Animal. 7 (2), 211–215. dzących po buhajach rasy piemontese GRODZKI H., PRZYSUCHA T., SLÓ- użytkowanych w Polsce i we Włoszech. SARZ J., 2010: The infl uence of com- Rozprawa habilitacyjna. Wyd. SGGW, mercial crossbreeding of dairy cows Warszawa. with bulls of French breeds (Blonde PRZYSUCHA T., GRODZKI H., 2008: Re- d’Aquitaine, Charolaise, Limousinee) lationship between calving course and on calving course. Ann. Warsaw Univ. of calf body weight at birth and calf/cow Life Sci. – SGGW, Anim. Sci 47, 31–38. body weight ratio. Elec. J. of Polish Ag- HANSET R., 1981: Selection problems when ric. Universities, seria Animal Husbandry antagonistic effects exist between pro- 11(3), 1–7. duction characteristics and calving diffi - PRZYSUCHA T., GRODZKI H., BRZO- culties. Livest. Prod. Sci. 8, 291–305. ZOWSKI P., ZDZIARSKI K., 2005: MEIJERING A., 1984: Dystocia and still- Wpływ wybranych czynników na prze- births in cattle – A review of causes, rela- bieg porodów krów rasy Limousine. Me- tions and implications. Livest. Prod. Sci. dycyna Wet. 61 (9), 1036–1038. 11, 143. PRZYSUCHA T., GRODZKI H., SLÓ- NIX J.M., SPITZER J.C., GRIMES L.W., SARZ J., 2007: Wpływ masy ciała kro- PLYLER B.B., 1998: A retrospective wy, kolejności i sezonu ocielenia oraz analysis of factors contributing to calf płci i masy cielęcia przy urodzeniu na mortality and dystocia in beef cattle. The- wyniki odchowu cieląt rasy salers. Me- riogenology 49, 1515–1523. dycyna Wet. 63 (3), 357–359. NOGALSKI Z., 2002: Effect of selected fac- PRZYSUCHA T., GRODZKI H., SLÓ- tors on the course of parturition in hol- SARZ J., GOŁĘBIEWSKI M., KU- stein-friesian heifers. J. of Polish Agric. NOWSKA-SLÓSARZ M., 2009: Wpływ Universities, Animal Husbandry 5 (2), kondycji krów rasy Limousinee przed http://www.ejpau.media.pl/series/vol- ocieleniem na rodzaj porodu. Medycyna ume5/issue2/animal/art-03.html. Wet. 65 (12), 854–856. Comparison of calving course of Limousine purebreds... 171

Statistical product and service solution case ka stwierdzono w populacji czystorasowej bydła version 8.0 for Windows, User’s Guide, Limousine (14%), w porównaniu z krzyżowa- 1998, by SPSS Inc., USA. niem towarowym krów mlecznych z buhajami tej rasy, gdzie odsetek trudnych ocieleń wyniósł za- ledwie 7%. Analiza statystyczna wykazała istotny Streszczenie: Porównanie przebiegu porodu wpływ masy cielęcia na rozkład ocen przebiegu u krów rasy Limousine i jej mieszańców z bydłem porodu w obu badanych populacjach. Najwięcej rasy polskiej holsztyńsko-fryzyjskiej. W pracy oce- trudnych porodów zanotowano w grupie cieląt niono przebieg porodów 100 krów rasy Limousi- o największej masie przy urodzeniu. Stosunkowo ne krytych buhajami tej samej rasy oraz 100 krów duży odsetek porodów wymagających udziału rasy polskiej holsztyńsko-fryzyjskiej odmiany człowieka zarówno w krzyżowaniu towarowym, czarno-białej (PHF) krytych w krzyżowaniu to- jak i w populacji czystorasowej Limousine, su- warowym z buhajami mięsnej rasy Limousine. geruje potrzebę monitorowania przez hodowcę W hodowli czystorasowej materiałem do badań wszystkich porodów. Należy podkreślić, że odse- były wyniki oceny użytkowości bydła mięsnego tek przypadków trudnych ocieleń w krzyżowaniu prowadzonej przez Polski Związek Hodowców towarowym z rasą Limousine jest znacznie mniej- i Producentów Bydła Mięsnego. W przypadku szy z ich częstością, jaką notuje się u bydła PHF krzyżowania towarowego materiałem do badań utrzymywanego w czystości rasy. były dane zawarte w „Kartach przebiegu ocie- lenia krowy” prowadzonych przez specjalistów Mazowieckiego Centrum Hodowli i Rozrodu MS. received in November 2013 Zwierząt Sp. z o.o. w Łowiczu. Dane dotyczące 100 porodów krów rasy Limousine inseminowa- nych nasieniem buhajów tej samej rasy oraz 100 Authors’ address: porodów krów PHF inseminowanych nasieniem Wydział Nauk o Zwierzętach SGGW buhajów mięsnej rasy Limousine obejmowały Katedra Szczegółowej Hodowli Zwierząt ocenę przebiegu porodu oraz masę cielęcia przy ul. Ciszewskiego 8 urodzeniu. Uzyskane wyniki jednoznacznie poka- 02-786 Warszawa zują, że znacznie większy i statystycznie istotny Poland udział porodów wymagających pomocy człowie- e-mail: [email protected]

Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 173–178 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Number of piglets born and reared by sows with different number of mammary teats ANNA REKIEL1, JUSTYNA WIĘCEK1, MARTA PARUCH1, JAROSŁAW PTAK2, TADEUSZ BLICHARSKI2 1Department of Pigs Breeding, Warsaw University of Life Sciences – SGGW 2Polish Pig Breeders’ and Producers’ Association “POLSUS”

Abstract: Number of piglets born and reared by INTRODUCTION sows with different number of mammary teats. The purpose of the studies was to determine the num- The number of teats in Suidae re- ber of piglets born (NPB) and reared until 21 day (NPR) by the sows of Polish Landrace and Polish veals considerable species and breed Large White breeds, differing in the number of differentiation (Haley and Lee 1992, teats. For calculations, the results from breeding Komosińska and Podsiadło 2002 as cited pig houses of the Mazovian region, collected dur- by Paruch 2011). Heritability index (h2) ing the years 2004–2009, were employed. Three of the mentioned trait was determined groups of females were distinguished for the par- ticular breeds: those ones, possessing 14, 15 or for pigs of different breeds, inter alia for 16 teats. The number of records for Polish Large Polish Landrace and Polish Large White White was equal to 1,019 and for Polish Land- and their crossbreds as well as for Lan- race, it was 1,732. The statistically confi rmed dif- drace, Yorkshire and Hampshire swine. ferences were revealed in respect of NPB by the The differences in its value depending groups of females, possessing 14, 15 or 16 teats (P ≤ 0.01) and NPR until the age of 3 weeks by the on the breed (purebreds/hybrids) and sows of the same breed, possessing 14 or 15 teats the site of teats (front/rear) were found (P ≤ 0.05). The differences in NPB and NPR in (McKay and Rahnefeld 1990, Haley and the sows which possessed 14, 15 or 16 teats were Lee 1992, as cited by Paruch 2011) . The not statistically confi rmed (P ≤ 0.05). Any univo- calculated values of h2 for the number cal relationships between the deaths of the piglets of teats amounted to 0.226–0.45 (Mc until 21 day of life and the number of teats in the sows of the examined breeds were not found; the Kay and Rahnefeld 1990, Lewczuk level of losses varied from 10.60 to 12.65%. The et al. 1991, Haley and Lee 1992, cited obtained results indicate that the number of teats by Paruch 2011) and less than 0.1 (Le- in the sows may affect the results of rearing the chowska and Ruda 2000). The studies piglet until 21 day. The access to mother milk is confi rmed also the incidence of specifi c not, however, the only one indicators of success in rearing of the progeny. relationship between the position in the group hierarchy, body weight of the pig- Key words: sows, number of teats, born piglets, lets and the mother teats, suckled by her reared piglets progeny (Surdacki and Jóźwiakowska- Rekiel 1988, Valros et al. 2002). Addi- tionally, Lee and Wang (2001) found that the number of teats, their conformation and distribution were not only the traits 174 A. Rekiel et al. subject to control and selection but also revealing effect on development of the 0.01 ≤ piglets. Se The aim of the study was to determine the number of the piglets born (NPB) cance at p fi and reared until 21 day (NPR) by Polish until 21 day Landrace and Polish Large White sows X with different number of teats. Number of piglets reared Se MATERIAL AND METHODS – signi A A, 0.05; ≤ The analysis employed the results, ob- tained in breeding pig houses of the Ma- X cance at p zovian region, as collected in the years fi

2004–2009. The number of the records Number of piglets born for Polish Large White breed was equal to 1,019 and for Polish Landrace – 1,732. The number of the piglets born and N reared until 21st day was calculated sep- arately for the sows of each breed; three groups of the females were distinguished i.e. those ones having 14, 15 or 16 teats. Se In the calculations (one-factor variance analysis), the package SPSS (2006) was

applied. until 21 day X Number of piglets reared RESULTS AND DISCUSSION

Statistically confi rmed differences were Se revealed for NPB in the Polish Landrace sows possessing 14, 15 or 16 teats (P Polish Large White Polish Large Polish Landrace ≤ 0.01) and for NPR up to the age of 3 weeks in the sows of the same breed pos- X – mean number of piglets in the litter; SE – standard error, a, a – signi – mean number of piglets in the litter; SE standard error, X sessing 14 or 15 teats (P ≤ 0.05) – Table 1. Number of piglets born The differences in NPB and NPR in Polish Large White sows who possessed 14, 15 or 16 teats have not been statisti- N cally confi rmed (P > 0.05). Any univo- cal relationships between the deaths of the piglets until the 21st day of life and the number of teats in the sows of the – number of observations; 14151614–16 621 283 1019 115 11.48 11.53 11.56 11.66 0.054 0.079 0.052 0.124 10.46 10.54 10.56 10.67 0.047 0.070 0.046 0.110 931 559 1732 A 242 11.26 A 11.46 11.44 0.046 A 11.59 0.059 10.32 a 0.039 0.090 10.47 a 0.042 10.43 10.50 0.054 0.036 0.082 Number of teats N (in columns). TABLE 1. Number of piglets born and reared by the PLW and PL sows with a differentiated number of teats (14–16) sows with a differentiated and PL 1. Number of piglets born and reared by the PLW TABLE Number of piglets born and reared by sows with different... 175 examined breeds were not found; the the number of teats of the sows, their cor- level of the losses varied from 10.60 to rect conformation and distribution has a 12.65% (Table 2). signifi cant effect on development of the

TABLE 2. Losses of piglets in the litters until 21 day of rearing according to the number of teats of their mothers Polish Large White Polish Landrace Number of teats Losses in the litter heads % heads % 14 1.013 11.63 0.941 10.60 15 0.987 11.38 0.988 11.32 16 0.995 11.61 1.092 12.65

Mazaraki (1961) and Janiszewska piglets. They are important performance et al. (1991), as cited by Paruch (2011), traits, being subject to control and selec- have not recorded any statistically signif- tion, therefore the attempt to develop the icant effect of the number of teats in the effective models of selection, oriented sows on NPB and NPR. The results of towards the number of teats in breeding own studies are confi rmed by the results, sows have been undertaken. obtained by Janiszewska et al. (1991) Buds of mammary teats are gener- and Buczyński et al. (1996), as cited by ated as early as during fetal life period. Paruch (2011). They observed that when Morphogenesis of the mentioned glands the sows possessed 15 teats and more, may reveal a high interspecies as well as NPB and NPR revealed a growing ten- in-species, in-breed or in-line variability dency. They stated also that in case of (Zaks 1969, as cited by Paruch 2011). In the increased number of teats in the sows opinion of Hotchkiss et al. (2007), the in- (≥15), the level of piglets’ death during creased expression of androgens in fetal rearing by mothers was lower and the period may bring about to disturbances weight of the litter was higher; the dif- in correct development of female, includ- ferences were not, however, statistically ing inhibition of teats and reproduction signifi cant. Jungst and Kuhlers (1983), system development. The changes in the as cited by Paruch (2011), studied the ef- level of the mentioned hormones affect fect of all teats of Duroc and Landrace the phenotypic modifi cations in adult fe- sows (situated in front of navel, anal males (masculinization and defeminiza- teats and crater teats) on the number of tion). Slob et al. (1980) and Warren et al. piglets on 21st day of rearing and did (1973), as cited by Paruch (2011), state not fi nd any relations. Also, they did not a lack of evidence indicating the origin fi nd any effect of the sow’s teats, having of androgens from developing ovaries a correct conformation and of defective in female fetuses. Most probably, pla- (crater) teats on weight of the litter on centa (Baum et al. 1991 and Houtsmuller 21st and 42nd day of life. On the other et al. 1995, as cited by Paruch 2011) and hand, Lee and Wang (2001) stated that adrenal glands (Stahl et al. 1991, as cited 176 A. Rekiel et al. by Paruch 2011) are the source of these fect of cooperation of many genes simul- hormones in female fetuses. Androgens taneously, therefore, it is very diffi cult to may be also of maternal origin (vom Saal choose the best selection method and the 1999, as cited by Paruch 2011) or come progress in respect of the trait is revealed from male fetuses which are found in at a very long time and to a minimal de- a defi ned intrauterine position (Ryan and gree. High possibilities in this respect Vandenbergh 2002, as cited by Paruch are found in BLUP model, e.g. in French 2011). The mentioned phenomenon sup- model, the teats are considered (EPSPA plements and explains one-direction fl ow 2007). It seems to be justifi ed because of blood via uterus or fetal membranes. – as it was shown in own studies (unpub- It is considered that effect of intrauter- lished data) – their too small number in ine position of on reproduction abilities sows (13 and less) contributes to more in pigs is small. Proportion of gender of than 6% increase of index of piglets’ fetuses in uterus has a greater effect on deaths during rearing period. The results the number of teats than their mutual dis- of the studies of Kim et al. (2005) also tribution has (Clark et al. 1993 and Saala indicate the justness of leaving the gilts et al. 1999, as cited by Paruch 2011). with 14 teats and more for repairing of Drickammer et al. (1999) and Ryan the herd. Their greater number causes and Vandenbergh (2002) report that the the increase of the number of piglets greater participation of males in the litter born in the litter and of the piglets reared causes decrease of the number of teats in until 21st day. There is a possibility of females (masculinization of females) as regression of the discussed medium-her- compared to females, originating from itable trait in the progeny in relation to the litter with greater participation of the mean value of the trait in the popula- females (feminization of females). The tion, therefore the control of mating and studies of Orzechowska et al. (2002), improvement of the trait are important in Górecki (2003) and Rekiel et al. (2012) breeding work. In own studies, the sows, confi rm the relationship between the being evaluated in respect of NPB and number of males in the litter from which NPR possessed the required number of the sow derived, and her reproduction teats but also, their number higher than results. Górecki (2003) indicates also to minimum was connected with the im- the increased number of teats in the fe- provement of the studied reproduction males coming from the litters in which and rearing parameters what has been there was a domination of female indi- confi rmed in literature (Lee and Wang viduals vs. males. In own studies, such 2001, Kim et al. 2005). The increase of relationships were not analyzed and only breeding progress in respect of the traits the effect of the number of teats in sows connected with reproduction is possible on NPB and NPR, what was confi rmed owing to intensively developing molecu- in some cases. lar genetics. The traits differ in heritabil- The number of teats is hereditary ity; their expression may occur at later similarly as quantitative traits; it reveals age or only in one gender. Control of the a high variability and is probably the ef- discussed traits is possible via study of Number of piglets born and reared by sows with different... 177

DNA polymorphism and mapping of ge- DRICKAMMER L.C., ROSENTHAL T.L., nome, connected with the incidence of ARTHUR R.D., 1999: Factors affecting QTL – loci of economically signifi cant the number of teats in pigs. J. Reprod. quantitative traits, including the number Fertil. 115, 97–100. EPSPA – European Pig Selection and Pro- of active teats (Bidanel and Rothschild duction Association, Warszawa 2007. 2002, Dekkers 2004, Rodriguez et al. GÓRECKI M.T., 2003: Sex ratio in litters of 2005, Sato et al. 2006). Mc Kay and domestic pigs. Biol. Lett. 40, 2, 111–118. Rahnefeld (1990) determined the herit- HOTCHKISS A.K., LAMBRIGHT C.S., ability of front and rear teats in purebred OSTBY J.S., PARKS-SALDUTTI L., pigs of maternal and paternal breeds and VANDENBERGH J.G., GRAY.L.E. Jr, their crossbreds and demonstrated that 2007: Prenatal testosterone exposure permanently masculinizes anogenital the heritability coeffi cient for front vs. distance, nipple development and re- rear teats was lower in purebred pigs productive tract morphology in female (0.15–0.21 vs. 0.20–0.39) and crossbreds sprague-dawley rats. Toxicol. Sci. 96 (2), (0.03–0.18 vs. 0.08–0.29). The discussed 335–345. traits, as affected by long-lasting selec- KIM J.S., JIN D.I., LEE J.H., SON D.S., tion, reveal a low genetic variability LEE S.H., YI Y.J., PARK C.S., 2005. Ef- what causes that the heritability coeffi - fects of teat number on litter size in gilts. cient has low values; such values for the Anim. Repr. Sci. 90 (1–2), 111–116. LECHOWSKA J., RUDA M., 2000: Efekty number of teats were also given by Le- doskonalenia liczby sutków u loch rasy chowska and Ruda (2000). polskiej białej zwisłouchej urodzonych w latach 1960–1990. Biul. Nauk. ATR Olsztyn 7, 123–128. CONCLUSIONS LEE C., WANG C.D., 2001: Genetic param- eter estimation with Normal and Poisson The results of own studies indicate that terror Mied models for teat number of the number of teats in sows may affect swine. Asian-Aust. J. Anim. Sci. 14 (7), the results of rearing the piglets until 910–914. Mc KAY R.M., RAHNEFELD G.W., 1990: 21st day of life. The access to and the Heritability of teat number in swine. Can. utilization of mother’s milk is not, how- J. Anim. Sci. 70 (2), 425–430. ever, the only one indicator of success in ORZECHOWSKA B., TYRA M., MU- rearing of the progeny. CHA A., 2002: Reproductive perfor- mance of sows from litters of various sex proportion. Application of scientifi c REFERENCES achievements in genetics, reproduction and feeding in modern pig production. BIDANEL J.P., ROTHSCHILD M., 2002: Wyd. ATR, Bydgoszcz. Current status of quantitative trait locus PARUCH M.W., 2011: Wpływ liczby gru- mapping in pigs. Pigs News and Informa- czołów sutkowych u loch ras matecznych tion. CAB International Review Article użytkowanych w stadach zarodowych 23 (2), 39–53. województwa mazowieckiego w latach DEKKERS J.C.M., 2004: Commercial ap- 2004-2009 na liczbę prosiąt urodzonych plication of marker-and-gene association i odchowanych do 21. dnia. Praca magi- in livestock: Strategies and lessons. J. sterska, SGGW, Warszawa. Anim. Sci. 82, 313–328. 178 A. Rekiel et al.

REKIEL A., WIĘCEK J., WOJTASIK M., Streszczenie: Liczba prosiąt urodzonych i odcho- PTAK J., BLICHARSKI T., MROCZ- wanych przez lochy o różnej liczbie gruczołów KO L., 2012: Effect of sex ratio in the sutkowych. Celem badań było określenie liczby litter in which Polish Large White and prosiąt urodzonych (LPU) i odchowanych do 21. Polish Landrace sows were born on the dnia (LPO) przez lochy rasy PBZ i WBP, różniące number of piglets born and reared. Ann. się liczbą gruczołów sutkowych. Do obliczeń po- Anim. Sci. 12(2), 179–185. służyły wyniki z chlewni zarodowych rejonu ma- RODRIGUEZ C., TOMAS A., ALVES E., zowieckiego zgromadzone w latach 2004–2009. RAMIREZ O., ARQUE M., MUNOZ G., Wyróżniono dla ras po trzy grupy samic, tj. mają- cych 14, 15 lub 16 sutków. Liczba rekordów dla BARRAGAN C., VARONA L., SILIO L., rasy WBP wyniosła 1019, a dla rasy PBZ 1732. AMILLS M., 2005: QTL mapping for Różnice potwierdzone statystycznie wykazano teat number in an Iberian-by-Meishan pig w LPU przez grupy loch PBZ mające 14, 15 lub intercross. Anim. Genet. 36(6), 490–496. 16 sutków (P ≤ 0,01) i w LPO do wieku 3 tygodni RYAN B.C., VANDENBERGH J.G., 2002: przez lochy tej rasy mające 14 lub 15 sutków (P Intrauterine position effects. Neuroscience ≤ 0,05). Różnice w LPU i LPO przez lochy WBP, and Biobehavioral Rev. 26, 665–678. które miały 14, 15 lub 16 sutków nie zostały po- SATO S., ATSUJI K., SAITO N., OKI- twierdzone statystycznie (P > 0,05). Nie stwier- TSU M., KOMATSUDA A., MITSU- dzono jednoznacznych zależności między upad- HASHI T., NIRASAWA K., HAYASHI kami prosiąt do 21. dnia życia a liczbą sutków T., SUGIMOTO Y., KOBAYASHI E., u macior badanych ras; poziom strat wahał się od 2006: Identifi cation of quantitative trait 10,60 do 12,65%. Uzyskane wyniki wskazują, że loci affecting corpora lutea and number liczba sutków u loch może wpływać na wyniki of teats in a Meishan × Duroc F2 re- odchowu prosiąt do 21. dnia. Dostęp do pokarmu source population. J. Anim Sci. 84(11), matki nie jest jednak jedynym wyznacznikiem 2895–2901. powodzenia w odchowie potomstwa. SURDACKI Z., JÓŹWIAKOWSKA-RE- KIEL A., 1988: Etologia prosiąt ssących. MS. received in November 2013 Wzrost prosiąt w zależności od ich roz- mieszczenia przy sutkach podczas ssa- nia. Zesz. Prob. Post. Nauk Roln. 335, 155–161. VALROS A.E., RUNDGREN M., SPIN- Authors’ address: KA M., SALONIEMI H., RYDHMER Wydział Nauk o Zwierzętach SGGW L., ALGERS B., 2002: Nursing behav- Katedra Szczegółowej Hodowli Zwierząt iour of sows during 5 weeks lactation and ul. Ciszewskiego 8, 02-786 Warszawa effects on piglet growth. Appl. Anim. Be- Poland hav. Sci. 76, 93–104. [email protected] Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 179–185 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Assesment of slaughter value of three broiler chicken genotypes* JULIA RIEDEL, MONIKA MICHALCZUK, ŻANETA ZDANOWSKA-SĄSIADEK Department of Animal Breeding and Production, Warsaw University of Life Science – SGGW

Abstract: Assesment of slaughter value of three INTRODUCTION broiler chicken genotypes. The study was con- ducted to examine slaughter parameters of three The European market for live poultry genotypes of meat type chicken: F1 crossbred derived from crossing of light hen of indigenous production is dominated by the broiler breed Greenleg Partridge with heavy meat type production using fast growing genetic cocks, F2 crossbred being an effect of re-crossing material. But actually the demand for obtained crossbred C×GP with meat type males alternative product increases. Consumer and medium growing Hubbard JA 957, designed are more interesting in chicken meat from for a longer, 9-week production. Chicken were reared till 63rd day of age. Examined parameters longer fattening system, characterized were: dressing percentage, breast and leg meat by better physical and chemical proper- yield and fatness. F1 crossbred (C×GP) were char- ties and better taste, as compared to the acterized by rather low body weight, typical for classic broiler. These birds are often kept slow growing chicken and good musculature, es- in outdoor system with the access to pas- pecially breast. Re-crossing with meat type cocks affected signifi cant (P < 0.01) improvement of the ture, what increases their attractiveness to the consumers. For the longer, 9-week, slaughter parameters. F2 crossbred (C×(C×GP) reached high body weight, typical for medium fattening alternative genetic material is growing chicken. In comparison with Hubbard JA used, characterized by slower growing 957 F2 crossbred had lower body weight, the same rate and better adaptation to different dressing percentage, better breast and worse leg environmental conditions and feeding musculature, and less abdominal fat. High breast meat percentage in carcass and less fatness sug- them diets, which improve meat quality gested, that these chickens can be used in meat and taste and health-promoting qualities production, as a medium growing material de- (Fanatico et al. 2005, Chen et al. 2013). signed for a longer fattening period (9 weeks). * On the other hand increased interest Key words: meat chicken, genotype, carcass traits in using an indigenous breeds or their hybrids, which are often better adapted to local climatic and/or consumer re- quirements is observed (Jaturasitha et al. 2008, Zanetti et al. 2010, Zhao et al. 2011, Dal Bosco et al. 2012, Wang *This work was carried out as part of the project et al. 2013). Also in Poland scientists are “BIOFOOD – innovative, functional products interested in possibility of using local of animal origin”, co-fi nanced by the European Union within the European Regional Develop- heritage breeds in organic or ment Fund under the Innovative Economy Ope- meat production (Brodacki et al. 2011, rational Programme. Brodacki i Batkowska 2011, Gornowicz 180 J. Riedel, M. Michalczuk, Ż. Zdanowska-Sąsiadek

2011, Muchacka et al. 2011). In Poland JA 957 broilers, designed for a longer, actually the popularity of Greenleg Par- 9-week production. tridge breed grows. It is an indigenous Total 3,300 meat type chicken derived breed, designed for outdoor laying pro- from three genotypes: F1 crossbred de- duction, because of its low weight and rived from crossing Greenleg Partridge lightweight construction. However the hen with meat type cocks; F2 crossbred, some of the Polish farms succesfully an effect of re-crossing F1 crossbred use males of Greenleg Partridge to pro- with meat type males; commercial me- duce (http.kaplony.pl; http.www. dium growing Hubbard JA 957, were kaplon.eu). It suggests the possibility to used in the study. One-day chickens use this breed as a maternal material to were placed to 30 pens (110 birds per produce hybrids suitable for the meat pen), 10 pens of each genotype. Chick- production. These crossbreds obtained en were kept in the same chicken house by single or double crossing with other on litter under controlled environment chicken breeds or lines can be a good conditions till 63rd day of age. All birds material for the longer fattening. were provided the same diets included The purpose of this study was to ex- Starter – till 14th day, Grower 1–15– amine slaughter analysis parameters of –28th days, Grower 2 – 29–35th day, and crossbred chicken C×(C×GP) derived Finisher – from 36th day. Access to feed from double crossing of hen of Polish in- and water was freely available. At the digenous breed Greenleg Partridge with 63rd day, 96 males and 96 females from heavy meat type cocks and to compare each genotype were taken according to them with the maternal breed C×GP and mean body weight for the group and with commercial medium growing Hub- sex. After 4 hour fastening chicken were bard JA 957 chicken. weighted individually and slaughtered in a professional slaughterhouse. Obtained chilled carcasses were dissected after MATERIAL AND METHODS 12 hour storage in 4°C. Carcass dis- section was carried out by removing of Hens of the Polish indigenous Greenleg breast, thigh and drumstick meat and Partridge (GP) light breed were crossed abdominal fat pad. Carcass dressing per- with the heavy meat type cocks (C) and centage was expressed as percentage of the obtained F1 crossbreds were exam- live body weight. Abdominal fat and the ined for slaughter analysis parameters. breast and leg muscles were removed Hens from this crossing were used as and weighed to determine their percent- a maternal material in next crossing, age share in the carcass. again with the heavy meat type cocks The obtained results were analysed (C). The chicken from the second gen- statistically by one-way analysis of vari- eration (F2) of crossbred (C×(C×GP)) ance as least square means (GLM proce- were also examined for slaughter anal- dure, SPSS 14.0 PL Software for Win- ysis parameters and compared with dows), separately for each sex. commercial medium growing Hubbard Assesment of slaughter value of three broiler chicken genotypes 181

RESULTS Slaughter results show, that C×(C×GP) crossbred F2 are typical me- The F1 generation of crossbred (C×GP) dium growing chicken characterized by characterized by worse slaughter para- high body weight, and good musculature meters than other birds (Table 1). Chick- of breast and leg. They can be suitable, en had much lower body weight and like Hubbard JA 957, for meat produc- muscle weight and also lower dressing tion in 9-week fattening system. percentage, what limited their usefulness Data presented in Table 1 indicate for meat production. Live body weight signifi cant (P < 0.01) differences among and carcass weight C×GP chickens were genotypes in most of slaughter traits. F2 similar to medium growing Hubbard crossbred was characterized by lower ISA Red JA at the age of 56 days (Aksoy body weight and, in effect, also lower et al. 2010) and to slow growing chickens carcass weight. However, regardless of at 81st day of age (Fanatico et al. 2005), these differences, both Hubbard JA 957 and the dressing percentage – likewise and crossbred C×(C×GP) chicken had slow growing and medium growing the same dressing percentage, similar chickens from Fanatico et al. (2005) and to six-weeks Cobb 500 broilers (Beg Wang et al. (2009) studies. However et al. 2011). Higher breast meat weight meat content in carcass of F1 crossbred and breast meat yield was found in the was equall to Hubbard JA 957 commer- crossbred (P < 0.01). Similar high val- cial chicken. Obtained results allow to ue (25%) was obtained in fast growing consider C×GP crossbred as slow grow- broilers at 56th day of age in Aksoy et ing chickens. They can be used as mater- al. (2010) study, and also in Arbo Acres nal breed in commercial crossing rather chicken at the age of 42 days in Liu et than as chickens for broiler production. al. (2011) study. Romero at al. (2009) The usefulness this crossbreds hens in suggested maternal effect on a chicken a broiler parent stock was confi rmed by breast meat yield. On the contrary, leg the results obtained in F2 generation. meat weight and leg meat yield were sig- In comparison with maternal breed nifi cantly lower than in Hubbard JA 957 CxGP, double crossbred C×(C×GP) had chickens. C×(C×GP) crossbred chicken higher body weight (P < 0.01), and con- had lower abdominal fat weight and ab- sistently much higher carcass weight and dominal fat percentage. were better muscled (P < 0.01). There was improved dressing percentage, in- creased breast meat percentage in the DISCUSSION carcass (P < 0.01). In the contrary leg meat percentage was lower (P < 0.01), Studies of Fanatico et al. (2005) and Ak- although leg weight was much higher soy et al. (2010) indicated genotype effect (P < 0.01). It indicates positive effect of on dressing percentage – fast growing re-crossing with meat type cocks. A sim- chickens has higher dressing percentage ilar characteristic both crossbred types than slow growing and medium growing was much worse musculature of legs ones. Body weight of fast growing broil- than breast (Table 1). ers Cobb (Aksoy et al. 2010) at the age of TABLE 1. Comparison of slaughter analysis of the crossbred chickens C×(C×GP) with maternal breed C×GP and with Hubbard JA 957 commercial chicken Crossbred F Crossbred F 1 2 Hubbard JA 957 Trait Sex C×GP C×(C×GP) P1 P2 P3 N LSM SE N LSM SE N LSM SE males 96 2 142 6 96 3 131 21 96 3 346 16 0.001 0.001 0.001 Live body weight, g females 96 1 689 5 96 2 437 10 96 2 700 6 0.001 0.001 0.001 males 96 1 494 6 96 2 304 16 96 2 472 12 0.001 0.001 0.001 Carcass weight, g females 96 1 184 5 96 1 819 10 96 2 014 8 0.001 0.001 0.001 males 96 319 2.6 96 588 7.0 96 538 4.8 0.001 0.001 0.020 Breast meat, g females 96 267 2.5 96 476 4.2 96 459 3.8 0.001 0.001 0.036 males 96 305 2.2 96 455 5.7 96 510 4.1 0.001 0.001 0.001 Leg meat, g females 96 226 1.6 96 330 2.9 96 385 3.4 0.001 0.001 0.001 males 96 45.6 1.13 96 45.0 2.28 96 75.3 2.30 0.824 0.001 0.001 Abdominal fat, g females 96 45.3 1.12 96 52.6 2.02 96 84.9 2.79 0.002 0.001 0.001 males 96 69.7 0.19 96 73.6 0.26 96 73.9 0.27 0.001 0.001 0.402 Dressing percentage, % females 96 70.1 0.22 96 74.6 0.23 96 74.6 0.30 0.001 0.001 0.790 Breast meat, males 96 21.3 0.15 96 25.5 0.21 96 21.8 0.31 0.001 0.239 0.001 % of carcass females 96 22.5 0.18 96 26.2 0.18 96 22.8 0.21 0.001 0.404 0.001 Leg meat, males 96 20.4 0.14 96 19.7 0.17 96 20.6 0.18 0.001 0.590 0.001 % of carcass females 96 19.1 0.12 96 18.2 0.13 96 19.1 0.21 0.001 0.699 0.001 Abdominal fat, males 96 2.1 0.05 96 1.6 0.04 96 3.0 0.09 0.001 0.001 0.001 % of carcass females 96 2.7 0.06 96 2.3 0.05 96 4.2 0.13 0.001 0.001 0.001

P1 – signifi cance of differences between crossbreds of F1 and F2 generations, P2 – signifi cance of differences between crossbreds of F1 generations and Hubbard JA 957, P3 – signifi cance of differences between crossbreds of F2 generations and Hubbard JA 957. Assesment of slaughter value of three broiler chicken genotypes 183

56 days amounted 3.4 kg, while the slow ing of fat deposition contained 20 g of fat growing Hubbard ISA Red JA weighted while carcasses from fatty line – 115 g. only 2.2 kg. Body weight of chicken in Also Dal Bosco et al. (2012) found that Fanatico et al. (2005) study ranged from chicken fatness is related to their geno- 2.1–2.2 kg in medium growing chicken type and is higher in fast growing broil- at the age of 81 day to 2.4–2.5 kg in fast ers, because of the combination of age, growing ones at the age of 53 day. Also low kinetic activity, and the high feed Wang et al. (2013) comparing slaughter intake. Fat content in carcasses of slow yield and meat quality of fast growing growing chicken at the age of 112 days and slow growing chickens found, that ranged from 3 to 6.5% according to dif- genotype had the large effect on these ferent raising system (Wang et al. 2009). features. Gornowicz et al. (2009) pay at- Fat percentage in six-week Arbo Acres tention to signifi cant differences in the broilers amounted to 1.2–1.5% (Liu quality of carcass and meat among Cobb et al. 2011, Wu et al. 2011), Hubbard 500, Hybro G+ and Ross 308 broiler 1.3–1.8% (Mahmood et al. 2007), Cobb chickens, while Janisch et al. (2011) did 500 from 1.2–2.2% (Beg et al. 2011) to not found signifi cant differences in body 2–2.5% (Hajati et al. 2009). weight or meat yield of Ross 308, Ross 708 and Cobb 700 broilers. A very good breast meat yield charac- CONCLUSION terized fast growing broilers Cobb 308, whose muscles at 56 day of age weighed Crossbred of F1 generation derived from 648 g (Aksoy et al. 2010). In the same Greenleg Partridge had much lower body experiment breast muscles of Hubbard weight, compared to commercial broil- ISA JA weighted only 288 g. Breast ers, and consistently also lower carcass muscles of Ross 308 chicken weight was and meat. But slaughter analysis results 450–500 g, breast meat percent in car- were not different so much from values cass amounted to 22–23% (Berri et al. obtained for most slow growing and me- 2008, Janish et al. 2011), and leg meat dium growing chickens. Hens from this percent – 26% (Janish et al. 2011). Arbo breed can be used as maternal breed in Acres chickens slaughtered at 42nd day a broiler parent stock. of age characterized very good muscula- Crossbred of F2 generation ture, both of breast 25–26% and 27.7%, (C×(C×GP)), obtained after re-crossing and leg 23–24% and 20.6%, respectively F1 with meat type males, were charac- in Liu et al. (2011) and Wu et al. (2011) terized by very good slaughter results, studies. especially high percentage of breast Guo et al. (2011) indicates genotype meat and low content of abdominal fat and selection direct impact on fatness. in carcass. It suggests the possibility to Fat weight in Arbo Acres chickens dif- use these chickens for longer fattening fered signifi cantly according to line period (9 weeks) to produce lighter, but – carcasses of chicken selected on lower- good muscled carcasses. 184 J. Riedel, M. Michalczuk, Ż. Zdanowska-Sąsiadek

REFERENCES GORNOWICZ E., 2011: Poultry meat pro- duction effi ciency under organic condi- AKSOY T., NARINC D., CUREK D.I., tions. Proc. of XXII International Poultry ONENC A., YAPICI N., 2010: Compari- Symposium PB WPSA, Poznań: 169. son of fast and medium-growing broiler GORNOWICZ E., LEWKO L., PIET- genotype raised indoors: growth perfor- RZAK M., GORNOWICZ J., 2009: The mance, slaughter results and carcass parts. effect of broiler chicken origin on carcase J. Anim. Vet. Advances 9: 1485–1490. and muscle yield and quality. J. Central BEG M.A.H., BAQUI M.A., SAR- European Agricult. 10: 193–200. KER N.R., HOSSAIN M.M., 2011: Ef- GUO L., SUN B., LENG L., WANG Y., fect on stocking density and feeding re- WANG N., LI H., 2011: Comparison of gime on performance of broiler chicken adipose tissue cellurarity in chicken lines in summer seasons. International J. Poult. divergently selected fot fatness. Poult. Sci. 10: 365–375. Sci. 90: 2024–2034. BERRI C., BESNARD J., RELANDEAU C., HAJATI, H., REZAEI M., SAYYAHZA- 2008: Increasing dietary lysine increases DEH H., 2009: The effect of enzyme fi nal ph and decreases drip loss of broiler supplementation on performance, car- breast meat. Poult. Sci. 87: 480–484. cass characteristics and some blood pa- BRODACKI A., BATKOWSKA J., ZIĘ- rameters of broilers fed on corn-soybean BA G., 2011: The infl uence of the genetic mael-wheat diets. International J. Poult. line and rearing system on dissection re- Sci. 8: 1199–1205. sults of slow growing slaughter chicken. JANISCH S., KRISCHEK C., WICKE M., Proc. of XXII International Poultry Sym- 2011: Color values and other meat quality posium PB WPSA, Poznań: 93. characteristics of breast muscles collected BRODACKI A., BATKOWSKA J., 2011: from 3 broiler genetic lines slaughtered at Physico-chemical meat traits of slow 2 ages. Poult. Sci. 90: 1774–1981. growing slaughter chicken. Proc. of 5th JATURASITHA S., SRIKANCHAI T., International Conference on the Quality KREUZER M., WICKE M., 2008: Differ- and Safety in Food Production Chain, ences in carcass and meat characteristics Wrocław: 20–21. between chicken indigenous to Northern CHEN X., JIANG W., TAN H.Z., XU G.F., Thailand (Black-Boned and Thai Native) ZHANG X.B., WEI S., WANG X.Q., and imported extensive breeds (Bresse 2013: Effects of outdoor access on growth and Rhode Island Red). Poult. Sci. 87: performance, carcass composition, and 160–169. meat characteristics of broiler chickens. LIU Z.H., LU L., LI S.F., ZHANG L.Y., Poult. Sci. 92: 435–443. ZHANG K.Y., LUO X.G., 2011: Effects Dal BOSCO A., MUGNAI C., RUGGERI S., of supplemental zinc source and level on MATTIOLI S., AND CASTELLINI C., growth performance, carcass traits, and 2012: Fatty acid composition of meat and meat quality of broilers. Poult. Sci. 90: estimated indices of lipid metabolism in 1782–1790. different poultry genotypes reared under MAHMOOD S., MEHMOOD S., AH- organic system. Poult. Sci. 91: 2039–2045. MAD F., MASOOD A., KAUSAR R., FANATICO A.C., PILLAI L.C., CA- 2007: Effects of feed restriction during VITT L.C., OWENS C.M., EMMERT starter phase on subsequent growth per- J.L., 2005: Evaluation of slower-growing formance, dressing percentage, relative broiler genotypes growth with and without organ weights and imune response of outdoor access: growth performance and broilers. Pakistan Vet. J. 27: 137–141. carcass yield. Poult. Sci. 84: 1321–1327. Assesment of slaughter value of three broiler chicken genotypes 185

MUCHACKA R., SOSNÓWKA-CZAJKA E., Streszczenie: Ocena wartości rzeźnej kurcząt SKOROMUCHA I., 2011: Effect of z trzech materiałów genetycznych. W badaniach housing system on productivity of broiler porównano kurczęta z trzech grup genetycznych: chickens. Proc. of XXII International mieszańce F1 powstałe ze skrzyżowania lekkich Poultry Symposium PB WPSA, Poznań: kur rasy Zielononóżka kuropatwiana z ciężkimi 177. kogutami typu mięsnego, mieszańce F2, efekt ROMERO L.F., ZUIDHOF M.J., RENE- powtórnego krzyżowania powstałego mieszańca MA R.A., NAEIMA A.N., ROBINSON Cobb×Zk z kogutami typu mięsnego oraz Hub- F., 2009: Effects of maternal energetic ef- bard JA 957, przeznaczone do dłuższego chowu fi ciency on egg traits, chick traits, broiler (9 tygodni). Kurczęta odchowywano do wieku 63 dni. Oceniano wydajność rzeźną, umięśnie- growth, yield, and meat quality. Poult. nie i otłuszczenie tuszek. Mieszańce F1 (C×Zk) Sci. 88: 236–245. charakteryzowała dość mała masa ciała, typowa WANG X.Q., CHEN X., TAN H.Z., ZHANG dla kurcząt wolno rosnących, oraz dobre umięś- D.X., ZHANG H.J., WEI S., YAN H.C., nienie, zwłaszcza piersi. Ponowne skrzyżowanie 2013: Nutrient density and slaughter age z kogutami typu mięsnego wpłynęło na istotną have differential effects on carcase per- (P < 0,01) poprawę parametrów oceny poubojo- formance, muscle and meat quality in wej. Mieszańce F2 (C×(C×Zk)) uzyskały dużą fast and slow growing broiler genotypes. masę ciała, typową dla kurcząt średniorosnących. Br. Poult. Sci. 54: 50–61. W porównaniu do kurcząt Hubbard JA 957 mie- WANG K.H., SHI S.R., DOU T.C., SUN H.J., szańce F2 miały małą masę ciała, taką samą wy- 2009: Effect of a free-raising system on dajność rzeźną, lepsze umięśnienie piersi, gorsze growth performance, carcass yield, and nóg i mniejsze otłuszczenie. Duży udział mięśni meat quality of slow-growing chicken. piersiowych w tuszce oraz małe otłuszczenie Poult. Sci. 88: 2219–2223. wskazują na przydatność tych kurcząt w użyt- WU H., GONG L.M., GUO L., ZHANG L.Y., kowaniu mięsnym jako materiał średnio rosną- LI J. T., 2011: Metabolism and Nutrition cy, przeznaczony do dłuższego, 9-tygodniowego Effects of the free fatty acid content in chowu. yellow grease on performance, carcass characteristics, and serum lipids in broil- MS. received in November 2013 ers. Poult. Sci. 90: 1992–1998. ZANETTI E., De MARCHI M., DALVIT C., MOLETTE C., REMIGNON H., CAS- SANDRO M., 2010: Carcase character- istics and qualitative meat traits of three Authors’ address: Italian local chicken breeds. Br. Poult. Julia Riedel Sci. 51: 629–634. Wydział Nauk o Zwierzętach SGGW ZHAO G.P., CUI H.X., LIU R.R., ZHENG Katedra Szczegółowej Hodowli Zwierząt M.Q., CHEN J.L., WEN J., 2011: Com- ul. Ciszewskiego 8, 02-786 Warszawa parisom of brest muscle meat quality in 2 Poland broiler breeds. Poult. Sci. 90: 2355–2359. e-mail: [email protected]

Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 187–193 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Semen quality parameters in outbred male mice from four different selected lines GRZEGORZ SOKOŁOWSKI1, ADRIANA STRZAŁKOWSKA2, WIESŁAW ŚWIDEREK1, KATARZYNA FISZDON1, MARTA GAJEWSKA2 1The Department of Genetics and Animal Breeding, Warsaw University of Life Sciences – SGGW 2The Department of Genetics and Laboratory Animal Breeding, The Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology in Warsaw

Abstract: Semen quality parameters in outbred INTRODUCTION male mice from four different selected lines. Breeding of (outbred) selective lines of labora- Breeding of selected lines of laboratory tory mouse was initiated in Warsaw University of Life Sciences about 40 years ago. It bred Heavy mouse was initiated in The Department (C) and Light (L) mice selected opposite for body of Genetics and Animal Breeding, War- weight at weaning (21st day of life), S mice line saw University of Life Sciences 43 years selected for higher testes weight, and control (K) ago (Sławiński 1974). It bred Heavy (C) mice without selection. All lines have identical and Light (L) mice selected opposite genetic background, but different directions of selections caused diversifi cation of specifi c phe- for body weight, S mice line selected notypic traits between them. The purpose of this for higher testes weight and control (K) study was to compare semen quantity and quality mice without selection. Next, lines were parameters in outbred C, K, L and S male mice in bred also in The Department of Genetics, the context of measurements of average body and The Maria Skłodowska-Curie Memorial testes weight for each line. Research materials were seminal fl uids squeezed Cancer Center and Institute of Oncology out of the vas deferens from 20 outbred C, K, L in Warsaw, where it started getting inbred and S male mice (5 males per group). Animals had stocks from them, but until now none of been euthanized, and necropsy was performed. selective lines is homozygotic inbred Body and testes weight was measured. Also sperm strain (G8-G13, depending on line). concentration, viability (by Eosin test), cytoplas- All lines have identical genetic back- mic membrane integrity degree (HOS test), sperm head morphology and maturity were estimated. ground, but different directions of selec- It was shown that S male mice, which have much tions caused diversifi cation of specifi c higher testes weight, also have a signifi cant in- phenotypic traits between them (Wirth- crease of viable spermatozoa according to control -Dzięciołowska et al. 2005). It was showed line. Moreover, sperm concentration from S males differences in reproduction traits such as is at least two times higher than in other selective lines. the ovulation rate, prenatal mortality and embryo number (Wirth-Dzięciołowska Key words: semen quality parameters, HOS, se- 1973). It was also correlation between lective lines, Eosin test, mouse body weight and lifetime reproduction 188 G. Sokołowski et al. rate as well as time of maturation (Wirth- Sperm quality parameters -Dzięciołowska et al. 1996). Moreover Tests were performed per 200 sperma- L mice lived longer than C mice (Wirth- tozoa (Krzanowska 1962), and analyzed -Dzięciołowska and Czumińska 2000). under light microscope – Olympus, type B091 and B201. Percentage of sperm MATERIAL AND METHODS without cytoplasmic droplet (mature spermatozoa), sperm with proximal (im- The purpose of this study was to com- mature spermatozoa) and distal droplet pare sperm quantity and quality pa- (during maturation, but normal) were rameters in outbred C, K, L and S male counted from fresh semen suspension mice in the context of measurements of (400× magnifi cation). average body and weight for each line First portion of suspension (10 μl) according to previous studies (Wirth- was diluted (1 : 5, v/v) with sterile wa- -Dzięciołowska 1973). ter (50 μl) and integrity of cytoplasmic Tests were carried out in The Depart- membrane of sperm tails was carried out ment of Genetics, The Maria Skłodowska- by Hypoosmotic Swelling Test (HOS). -Curie Memorial Cancer Center and In- Percentage of spermatozoa with integral stitute of Oncology in Warsaw. membrane (swollen tails) and without integral cytoplasmic membrane (broken Animals and straight tails) was counted (400× magnifi cation). Five adult (3–6 months old) males from Second portion of semen suspen- each of the C, K, L and S lines were used sion (10 μl) and 0,2% eosin Y (10 μl) in the study. Animals were maintained was mixed (1 : 1, v/v) and incubated for in a barrier facility (constant light cy- 10 min. Sperm viability was elucidated cle – 12L/12D, room temperature about (Eosin Test), by counting of percentage 23°C, water and food (Labofeed H) were of dead (red) and alive (green) sperm available ad libitum). It were mice post- head (400× magnifi cation). selected, obtained from the herd. Next, 10 μl of the sperm suspen- sion was extended (1 : 5, v/v) with PBS Necropsy and morphometrical (50 μl) and sperm number was evalu- parameters ated per at least 5 large squares and at Male mice been euthanized by cervi- least 200 spermatozoa were counted in cal dislocation, and necropsy was per- a haemocytometer. formed. Body and testes weight was Sperm head morphology was analyzed measured (±0,001 g). Research materials under 1,000× magnifi cation. Smears of were seminal fl uids squeezed out of the sperm suspension was fi xed in a mix- vas deferens into 100 μl of M2 Medium ture of 99.8% ethanol and acetic acid – common medium used for in vitro cul- (3 : 1, v/v) and stained with 1% eosin Y for ture of preimplantation stage embryos. It 15 min. Percentage of normal sperm can be used for collecting viable gametes head, and abnormal spermatozoa was outside a CO2 incubator and it also ex- counted with classifi cation of sperm head tend sperm fl uids. abnormalities by Krzanowska (1976). Semen quality parameters in outbred male mice from... 189

The statistical signifi cance of differ- The highest testes weight (Fig. 2) was ences between the analyzed lines were recorded in S males (0.56 g) in the main calculated by ANOVA (IBM SPSS Sta- direction of this line selection, which tistics 21). was more than 2 times higher than in C line and more than 3 times higher than in the K and L male mice. RESULTS AND DISCUSSION S male mice was characterized by 4 times higher sperm concentration Figure 1 shows the mean C, K, L, S male 162.19·106/ml (Fig. 3) in compari- mice body weight. It was noted signifi - son with C (37.86·106/ml) and K line cant differences between average body (39.55·106/ml). Level of this parameter weight of all lines. C (43.87 g) and S in L mice was about 1.5 times higher than (35.96 g) lines were characterized by in control and C males (68.31·106/ml), the highest body weight as well as L line but it was more than 2 times lower in re- (25.45 g) had the lowest body weight. lation to S males.

50 ABC 40 CEF

30 AdE BdF

20

10

0 CKL S body weight (g) 43.87 29.95 25.45 35.96

FIGURE 1. Average body weight (g) of selected mice lines: Heavy (C), Control (K), Light (L) and Testes Weight (S). ABCEF at P ≤ 0.01, d at P ≤ 0.05

0.60 BCD 0.50

0.40

0.30 aB 0200.20 C aD 0.10

0.00 CKL S testes weight (g) 0.21 0.17 0.15 0.56 FIGURE 2. Average testes weight (g) of selected mice lines: Heavy (C), Control (K), Light (L) and Testes Weight (S). BCD at P ≤ 0.01, a at P ≤ 0.05 190 G. Sokołowski et al.

ABC 160 140 120 100 80 C 60 40 A B 20 0 CKL S sperm concentraƟon (× 106/ml) 37.86 39.55 68,31 162.19 FIGURE 3. Sperm concentration [×106/ml] of selected mice lines: Heavy (C), Control (K), Light (L) and Testes Weight (S). ABC at P ≤ 0.01

The study of sperm head morphology The results of HOS test and Eosin test (Fig. 4) shows the highest percentage of (Fig. 5) showed that the largest sperm morphologically normal S male mice viability (alive, swollen tails) were ob- sperm heads (89%). This parameter in C, served in S male mice (alive – 59.70%, K and L lines oscillated between 79.9– swollen tails – 71.70%). The differences –83.3%. Higher completely mature sperm among other lines, C (55.15%; 66.65%), (Fig. 4) percentage was detected mainly K (50.85%; 61.20%) and L (55.90%; in the semen fl uids of K and S male mice 71.70%), were smaller and not statisti- (60.9–62.35%), than in L and C male cally signifi cant. (respectively 47.07 and 47.9%), but ob- Reproduction is one of the most served differences were not statistically important functions of the free-living signifi cant. and breeding organisms. Thus regular assessment of fertility parameters in

100 Ab 90 b A 80 70 60 50 40 30 20 10 0 CKL S morphology normal 79.90 81.50 83.30 89.00 (%) mature sperm (%) 47.9060.9047.0562.35 FIGURE 4. Sperm head morphology and tail membrane integrity of selected mice lines: Heavy (C), Control (K), Light (L) and Testes Weight (S). A at P ≤ 0.01, b at P ≤ 0.05 Semen quality parameters in outbred male mice from... 191

80 a 70 a 60 50 40 30 20 10 0 CKL S alive sperm (%) 55.15 50.85 55.90 59.70 swollen tails (%) 66.65 61.20 65.40 71.70 FIGURE 5. Sperm viability (Eosin test and HOS test) of selected mice lines: Heavy (C), Control (K), Light (L) and Testes Weight (S). a at P ≤ 0.05 animals seems to be necessary to maintain 2.5 times more spermatozoa is produced, high-quality reproductive herd (Bakker which is also alive and motile with nor- et al. 1978). Assessing reproductive pa- mal morphology. It should also be noted rameters is also of utmost importance in that testis weight could be correlated genetic control of outbred herds. (r = 0.3) with a body weight of lines ana- Sperm quality parameters were ana- lyzed. However, in other publications lyzed to determine viability, motility and there found no correlation between these maturity of spermatozoa, cytoplasmic morphologically parameters (Hill et al. membrane integrity of tails (Krzanow- 1990, Le Roy et al. 2001). C and L line, ska 1962, Gołas et al. 2011), sperm head which is selected for high and low body morphology (Krzanowska 1976) as well weight, respectively have also higher and as sperm concentration was counted. lower testes weight, but probably these Our study reveals that K and C male results are only the line effect and false mice have rather “basal” level of sperm correlation is caused by small number of parameters, nearing inbred strains pa- animals in analyzed groups. rameters (Gołas et al. 2011). L males Probably long-term selection (more fertility parameters are gently higher in than 100 generations) can not affect sperm relation with C and K results, which can quality directly (Wirth-Dzięciołowska make them effective males in reproduc- 1992). L males have smaller testes than tion. S male mice have the highest sperm C and K males, but L male mice semen quality parameters and sperm concentra- quality parameters are higher than C and tion. K average level. Testes size may affects sperm pro- Nevertheless, S males selected for duction, so in bigger testes much higher high testes weight have also higher body number of spermatozoa is produced (Le weight and fertility levels which can Roy et al. 2001, Gołas et al. 2011). Our be close to the maximum. Signifi cantly tests shows that in S males, which are better sperm parameters of S males are selected for high testes weight, at least the effect of an increase of selection for 192 G. Sokołowski et al. testes weight, what was confi rmed by Hill SŁAWIŃSKI T., 1974: Infl uence of geno- et al. (1990) and Le Roy et al. (2001). type and maternal effects on body weight during postnatal growth (on laboratory mice) (in Polish). Zeszyt. Nauk. AR, War- CONCLUSIONS saw: 46. WIRTH-DZIĘCIOŁOWSKA E., 1973: Ini- Increase of testes weight causes much tial research on reproduction mice se- higher sperm concentration and increase lected on hight and low body weight (in semen quality parameters in S males in Polish). Prace i Mat. Zoot. 4: 23–32. WIRTH-DZIĘCIOŁOWSKA E., 1992: The comparison with C, K and L mice. Ac- infl uence of long-term selection for body cording to our investigations, long-term weight on reproduction in laboratory selection for body weight seems not af- mice. Genetica Pol. 33: 57–62. fect directly qualitative and quantitative WIRTH-DZIĘCIOŁOWSKA E., CZU- parameters of semen in male mice. MIŃSKA K., 2000: Longevity and aging of mice from lines divergently selected for body weight for over 90 generations. REFERENCES Biogerontology 1: 169–176. WIRTH-DZIĘCIOŁOWSKA E., CZU- BAKKER H., WALLINGA J.H., POLI- MIŃSKA K., REKLEWSKA B., KAT- TIEK R.D., 1978: Reproduction and KIEWICZ M., 1996: Life time reproduc- Body Weight after Long-Term Selection tive performance and functional changes for Large Litter Size. Journal of Animal in reproductive organs of mice selected Sciences 46: 1572–1580. divergently for body weight over 90 gen- GOŁAS A., LECH T., JANUŁA M., BE- erations. Animals Science Papers and Re- DERSKA D., LENARTOWICZ M., STY- ports 14: 187–198. RNA J., 2011: Semen quality parameters WIRTH-DZIĘCIOŁOWSKA E., LIPSKA A., and embryo lethality in mice defi cient for WĘSIERSKA M., 2005: Selection for Trp 53 protein. Reproductive Biology 11 body weight induces differences in ex- (3): 250–263. ploratory behavior and learning in mice. HILL W.G., MARKS P.J., JENKINS J.C., Acta Neurobiol. Exp. 65: 243–253. LAND R.B., 1990: Selection on testis WIRTH-DZIĘCIOŁOWSKA E., SYSA P.S., size as an indicator of maturity in grow- PONIMASZ A., 1997: An attempt at ing animals. II Correlated responses in comparing external changes taking place reproductive rate. Genetic Sel. Evol. 22: turning maturation of male mice with the 247–255. development of the process of spermato- KRZANOWSKA H., 1962: Sperm quality genesis. Folia Biologica 45: 89–95. and quantity in inbred lines of mice and their crosses. Acta Biol. Cracov Ser Zool. Streszczenie: Parametry jakości plemników sam- 5: 279–290. ców myszy z czterech outbredowych linii selek- KRZANOWSKA H., 1976: Types of sperm- cyjnych. Celem przeprowadzonych badań było -head abnormalities in four inbred strains dokonanie oceny jakości parametrów plemników of mice. Acta Biologica Cracoviensa Se- nasieniowodowych, pobranych od 20 samców ries Zoologica 19: 79–85. (po 5 samców z linii) myszy z linii selekcjono- Le ROY I., TORDJMAN S., MIGLIORE- wanych przez wiele pokoleń: przeciwstawnie na -SAMOUR D., DEGRELLE H., ROU- masę ciała (C i L), masę jąder (S) oraz samców BERTOUX P.L., 2001: Genetic Architec- stanowiących linię kontrolną (K), w kontekście ture of testis and Seminal Vesicle Weight pomiarów średnich mas ciała i jąder dla po- in Mice. Genetics 158: 333–340. szczególnych linii. Materiał badawczy stanowiły Semen quality parameters in outbred male mice from... 193 plemniki pobrane z nasieniowodów od zwierząt MS. received in November 2013 poselekcyjnych. Oszacowano liczbę plemników w 1 ml pożywki (M2). Dokonano analizy para- Authors’ addresses: metrów jakości plemników, wykonując test oce- Grzegorz Sokołowski, Wiesław Świderek, ny żywotności plemników, test położenia kropli Katarzyna Fiszdon cytoplazmatycznej, który jest miarą dojrzałości Wydział Nauk o Zwierzętach SGGW plemników oraz test hipoosmotyczny (HOS) do Katedra Genetyki i Ogólnej Hodowli Zwierząt oceny integralności błony cytoplazmatycznej ul. Ciszewskiego 8, 02-786 Warszawa, Poland e-mail: [email protected] witek plemników. Ponadto dokonano oceny mor- [email protected] fologii główek plemników. Wykazano, że samce katarzyna_fi [email protected] linii S w porównaniu z osobnikami z linii kon- trolnej K oraz linii ciężkiej (C) i lekkiej (L) mają Adriana Strzałkowska, Marta Gajewska istotnie większą masę jąder, większy odsetek Centrum Onkologii – Instytut im. dojrzałych i żywotnych plemników, a także 2–4- M. Skłodowskiej-Curie w Warszawie -krotnie większą koncentrację plemników liczoną Zakład Genetyki i Hodowli Zwierząt w 1 ml medium. Laboratoryjnych ul. W.K. Roentgena 5, 02-785 Warszawa, Poland e-mail: astrzał[email protected] [email protected]

Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 195–201 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

The infl uence of nanodiamond particles on rat health status MACIEJ SZMIDT1, TOMASZ NIEMIEC2, EWA SAWOSZ-CHWALIBÓG2, KATARZYNA MITURA3 1Department of Morphologic Sciences, Warsaw University of Life Sciences – SGGW 2Department of Animal Nutrition and Feed Science, Warsaw University of Life Sciences – SGGW 3Department of Radiology and Radiotherapy Fundamentals, Koszalin University of Technology

Abstract: The infl uence of nanodiamond particles the biofunctional agent, nanodiamond on rat health status. The objective of the present should reveal the biocompatible charac- investigation was to evaluate the effect of nano- diamond (ND) particles on rats health status. teristics. It should exibit the lowest toxic 1 mg/kg b.w. of nanodiamond particles was ad- properties and it should remain neutral ministrated intravenously and intraperitoneum. for the organism parameters (Bakowicz- The presence of an adverse impact was examined. -Mitura et al. 2007). Accoridng to the re- The results show signifi cant changes in biochemi- cal (glucose and total protein level decrease) and cent studies, nanodiamonds present the hematological (elevated platelets count) param- high biological tolerance and they exhib- eters, only in case of intravenous injection. it biocompatibility with the blood com- Key words: nanodiamond particles, health status, ponents (Mitura et al. 2006). However, biochemical and hematological parameters, rats we observe the insuffi ciency of the in vivo studies characterizing the systemic organism response to diamond nanopar- INTRODUCTION ticles. The hematologic and biochemi- cal parameters of rats are published by The recent large interest in carbon na- Exotic Animal Companion Medicine nomaterials (fullerenes, nanotubes, na- Handbook for Veterinarians (Johnson- nodiamonds) is a consequence of their -Delaney 1996). unique mechanical, electrical and ther- Therefore, the objective of the study mical features. Numerous biological ap- was to evaluate the effect of intravenous plications suggested by several investi- gators include their potential application and intraperitoneal administration of na- as the drug delivery agents controlling nodiamond on biochemical and hemato- the release of genes, proteins or other logical parameters. Properly interpreted molecules (Bondar and Puzyr 2004). results of the blood chemistry values may Nanodiamonds present the high effec- provide the precise picture of the animal tiveness in drug delivering chemother- health status at the time of its sampling apy, presenting the low negative effects (i.e., nutritional status, disease condition associated with the known drug-delivery and stress after environmental changes). agents. It was proposed to apply nano- Therefore, using these data for diagnos- diamonds as a biochip or biosensor for tic purposes we compared the obtained a medical use (Puzyr et al. 2007). As results with reference or normal values. 196 M. Szmidt et al.

MATERIAL AND METHODS vidual cages for 10 days under standard conditions: temperature 22°C, humid- Diamond nanoparticles (ND) was pro- ity 50–70%, light/dark cycle 12/12h. duced by the method described by The animals had free access to water Danilenko (2003) with modifi cation of and feed. Diet was formulated in com- ampoule-free synthesis in the explosion pliance with NRC requirements (1995). chamber instead of ampoule synthesis. Rats were administrated by intravenous Graphite was placed directly into a cy- (group 1) and intraperitoneal injection lindrical charge consisting of a TNT- (group 2) of 0.5 ml of ND colloid (1 mg/ -hexogen mixture TG40. The charge was /kg body weight of ND particles). Clear enveloped in a water jacket to suppress deionized water was administrated by graphitization and reduce the unloading intravenously into the animals of control rate of the synthesized diamond. Shape group (group 3). At the end of the experi- and size of ultrananocrystalline diamond ment the rats were fasted for 12 h and (2–10 nm) were inspected by transmis- then sedated by intramuscular ketamine sion (TEM) and scanning (SEM) electron (Ketamini hydrochloricum 5%, Narka- microscope (Bakowicz 2003, Czerniak- mon, SPOFA, Praha, ). -Reczulska et al. 2010). Figure 1 shows Blood was sampled from the heart into that the grains of nanoparticles form- heparinized tubes and cooled to 4°C. The ing conglomerates (HR TEM). Figure 2 animals were euthanized by ketamine shows nanodiamond clusters (SEM). overdoses. The samples were collected ND at the concentration of 500 mg/l for further analysis. Blood morphology was suspended in deionized water then (red blood cell (RBC) count, hematocrit, the mixture was sterilized and sonifi - mean cell volume (MCV), hemoglobin cated. 21 male Wistar rats were divided concentration, white blood cell (WBC), into three equal groups and kept in indi- neutrophils, lymphocytes and platelets

FIGURE 1. Nanodiamond particles manufactured by detonation method — HR TEM. Reprinted with permission from Bakowicz (2003) The infl uence of nanodiamond... 197

FIGURE 2. View SEM of nanodiamond powder manufactured by detonation method. Reprinted with permission from Czerniak-Reczulska et al. (2010) count was determined using standard Platelets are assisting and modulat- methods and a Danam-510 analyser ing infl ammatory reactions and immune (). The following were assayed in responses. This is achieved by the regu- serum: total protein, albumin, aspartate lated expression of the adhesive and im- aminotransferase (AST), alanine ami- mune receptors on the platelet surface notransferase (ALT), glucose, triglycer- and also by the release of a multitude of ides using a Vitros DTII analyzer (John- secretory products including infl amma- son and Johnson). tory mediators and cytokines, which can mediate the interaction with leukocytes (Hundelshausen and Weber 2007). The RESULTS AND DISCUSSION primary regulator of platelet production is thrombopoietin (THPO). After binding The haematological parameters indicate to the cell receptor, it affects all stages of that hydrocolloid of detonation diamond development of megakaryocytes. THPO nanoparticles, regardless of the route of is synthetized mainly in the liver and it administration results in the elevation in is stimulated by IL-6 (Śliwińska-Staczyk platelet number in the rats, compared to 2005). Based on the presented results, we the control group. Other morphological hypothesize that the ND stimulates im- indicatives remained unchanged (Ta- mune system and it leads to the expres- ble 1). Additionally, hydrocolloidal ND sion of infl ammatory mediators (IL-6), intravenously injection decreased glu- which had consequences in the increased cose and total protein concentration in production of blood platelets. However the animal serum. Other biochemical pa- non-specific immune and hematologic rameters did not differentiate the experi- indicators (Niemiec et al. 2011), do mental groups and remained within the not prove any infl ammatory activity of reference values (Table 2). ND. However, the presented blood cell 198 M. Szmidt et al.

TABLE 1. Hematoloogical parameters in peripheral blood of control and experimental rats

Reference Injection SE Parameter * Control P-value values intravenous intraperitoneal pooled RBC** (1012 /l) 6.76–975 8.1 7.7 8.0 0.242 0.5561 Hematocrit (l/l) 37.6–50.6 41.8 41.9 42.6 0.99 0.207 MCV** (fl ) 50–80 52.0 54.0 53.0 0.88 0.207 Hemoglobin (g/dl) 11.5–16.1 14.0 14.0 14.2 0.26 0.861 WBC** (109/l) 6.6–12.6 8.6 8.3 7.6 1.197 0.8245 Neutrophils(%) 3–42 18.0 12.0 11.0 3.42 0.276 Lymphocytes (%) 50–95 81.0 88.0 88.5 3.46 0.285 Platelets (109/l) 150–460 487.3 b 645.5 a 715.2 a 45.70 0.009 *Exotic Animal Companion Medicine Handbook for Veterinarians, Johnson-Delaney 1996. **Abbreviations: RBC = red blood cells, MCV = mean corpuscular volume, WBC = white blood cells. a, b – signifi cant difference at P < 0.05.

TABLE 2. The level of blood serum parameter in control and experimental rats

Reference Injection SE Parameter * Control P-value values intravenous intraperitoneal pooled Glucose (mmol/l) 2.8–7.56 9.01 b 7.58 a 9.24 b 0.236 0.0002 Triglycerides (mmol/l) 0.35–1.4 1.1 0.97 1.39 0.119 0.0685 AST** (U/l) 39–111 124.6 119.4 112.0 8.33 0.573 ALT**(U/l) 20–61 57.7 54.28 51.42 5.414 0.7178 Albumin (g/l) 38–48 34.4 29.7 36.7 2.34 0.126 Total protein (g/l) 53–69 66.3 b 54.0 a 67.7 b 3.58 0.027 *Veterinary Reference Guide, Clinical Diagnostic Division Eastman Kodak Company. **Abbreviations: AST = aspartate aminotransferase, ALT = alanine aminotransferase. a, b – signifi cant difference at P < 0.05. parameters did not differ groups in the our study we demonstrated no signifi cant experiment. Puzyr et al. (2002) showed changes in the quantity of white and red that ND damages blood cells including blood cells. Histological studies also did erythrocytes by following mechanisms: not confi rm the negative impact of ND (1) direct binding of NDs to the cell on kidneys and liver cell structures (Nie- membrane proteins, which may cause miec et al. 2010). According to Puzyr et irreversible inhibition of their functions al. (2007), the modifi ed diamond nano- and (2) a imbalance of electrolytic and os- colloid administered for 6 months, did motic equilibrium caused by adsorption not affect the mortality of the tested mice. of blood plasma protein components to There were no abnormalities observed in ND particles (Puzyr et al. 2002). Degra- the process of organ growth and weight, dation of blood cells depends on the con- however the increased the level of leu- centration and ND surface properties. In kocytes in the blood was shown. ND The infl uence of nanodiamond... 199 administered subcutaneously did not tion of nanodiamond particles. However, cause any infl ammation nor damage of the histological assessment cannot rule the cells adjacent to applied nanopar- out the presence of microdamages in the ticles. On one hand, we expect certain examined organs. This was confi rmed by level of degradation of the treated cells Yu et al. (2005) who presented the cy- after diamond nanocolloid applied in vi- totoxicity of the diamond powder on the tro versus in vivo. This may be related kidney cells. to the numerous of extracellular factors (protein, lipoprotein, sugars) involved in the nanoparticles interactions. On the CONCLUSIONS other hand, diamond nanoparticles can The nanoscale diamond particles have affect the large number of the cells af- become an interesting innovative phar- ter in vivo injection. Puzyr et al. (2007) maceutical material. Due to particular observed no changes in the blood mor- size and surface structure, nanoparticles phological parameters after intravenous are well-suited for different biological injection of ND. However, the results of applications. Most of the toxicology in- that work were not signifi cant and indi- vestigations carried out on in vitro mod- cate the increase in the number of plate- els reveals positive results. The recent lets after nanocolloid injection. study presents wide biological benefi ts of The values of biochemical markers nanodiamond particles obtained by deto- of health in all experimental groups re- nation methods such as low cytotoxicity mained within the reference ranges. Glu- and biocompatibile (Bondar et al. 2005, cose and total protein concentration was Schrand et al. 2006, Bakowicz-Mitura signifi cantly lower in animals after in- et al. 2007). We have recently showed travenous injection of ND, compared to that intravenous and intraperitoneal in- the control group. Puzyr et al. (2007) has jection of 1 mg/kg b.w. detonation dia- documented that diamond nanoparticles mond powder increased platelet counts modulate concentration of biomarker on rats. Additionally, the intravenous of liver injury (bilirubin/transaminases) injection of nanoparticles decreased se- and lipid metabolism (cholesterol, low rum level of glucose and total protein. It density lipoprotein, triglycerides) indica- is hypothesized that ND stimulates the tors in the rabbit serum. We assumed that TPO synthesis pathway through the ac- ND affects the excretion of urine glucose tivation of pro-infl ammatory cytokines. and protein by mechanical damage of re- We also speculate that exposure to dia- nal corpuscles. Nephrotoxic compounds mond nanoparticles may lead to the ne- were shown to be a reason for pathologi- phrotoxicity characterized by hematuria cal changes such as proteinuria and gly- and proteinuria together with the reduced cosuria (Ascioglu et al. 2000). However, serum total protein and glucose level. Niemiec et al. (2008) has reported that Our results show the necessity of the de- histopathological examination of kidney termination of the minimum toxic dose and liver showed normal architecture, and the evaluation of the ND biological suggesting no morphological disturbanc- properties, especially in the experiments es in rats treated by intravenously injec- conducted in vivo. 200 M. Szmidt et al.

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Ponadto wlew dożylny hydrokoloidu ND wpłynął Tomasz Niemiec, Ewa Sawosz-Chwalibóg na zmniejszenie koncentracji glukozy i białka Wydział Nauk o Zwierzętach SGGW całkowitego we krwi badanych zwierząt. Analiza Katedra Żywienia Zwierząt i Gospodarki danych doświadczenia dowodzi pilnej potrzeby Paszowej ustalenia minimalnej dawki toksycznej nanoczą- ul. Ciszewskiego 8, 02-786 Warszawa stek ND oraz szczegółowej oceny biologicznych Poland właściwości nanodiamentów otrzymywanych e-mail: [email protected] metodą detonacyjną w badaniach na zwierzętach. [email protected] Katarzyna Mitura MS. received in November 2013 Instytut Technologii i Edukacji Politechniki Koszalińskiej Authors’ addresses: Katedra Radiologii i Radioterapii Materiałowej Maciej Szmidt, ul. Śniadeckich 2, 75-453 Koszalin Wydział Medycyny Weterynaryjnej Poland Katedra Nauk Morfologicznych e-mail: [email protected] ul. Nowoursynowska 159, 02-776 Warszawa Poland e-mail: [email protected]

Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 203–209 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Occurrence of entomopathogenic fungi in soil of Santiago and Fogo islands (Republic of Cape Verde) BEATA WASILEWSKA-NASCIMENTO Department of Science and Technology, University of Cape Verde

Abstract: Occurrence of entomopathogenic fungi biological control agents. In Europe, in in soil of Santiago and Fogo islands (Republic of the fi rst decade of the twenty-fi rst cen- Cape Verde). The soil is a habitat for many en- tury seven exotic species used in biologi- tomopathogenic fungi (EPF) all over the world. The Galleria bait method has been chosen to cal control were successfully replaced isolate the EPF from soil samples. The common by native species. In this period, a total occurrence of EPF in agricultural land was con- of 18 native species compared with six fi rmed by these studies, which concluded, that exotic ones were commercialized. The the soil of Cape Verde is a habitat for EPF, such situation was different in the African as Beauveria bassiana and Metarhizium aniso- pliae. The presence of B. bassiana was recorded continent, where from a total of 26 spe- in the cultivable land of the island of Fogo, while cies commercialized as natural enemies, M. anisopliae occurred in the soil of Fogo as well 25 were originate from material collect- as Santiago. There were no records of EPF in ed and initially bred in other continents woodlands. Statistically, more soil samples con- (Cock et al. 2009). taining B. bassiana were derived from perennial crops. The amount of sand in the soil determined The designing an effective biologi- the presence of the fungus. On the other hand, cal control in developing countries with M. anisopliae was present on less inclined tropical and subtropical climates is a pri- grounds. Fusarium spp. was present in half of the ority. In the regions, where government examined soil samples. The isolation of native programmes (e.g. Programme of the EPF and their subsequent application in develop- ing countries may signifi cantly contribute to re- Government of Cape Verde 2011–2016, duce pest populations in agriculture effectively. 2010) predict a growth in agricultural out- put, an effective plant protection should Key words: entomopathogenic fungi, Beauve- ria bassiana, Metarhizium anisopliae, Fusarium be provided. What is more, the abuse of spp., soil synthetic pesticides has been declared as being one of the environmental problems in these countries (Programme strategy INTRODUCTION of Cape Verde 2009–2012, 2010). The Cape Verde Islands are of volcan- The advantages of using biological meth- ic origin. The climate of this sub-sahe- ods to control crop pests were largely lian archipel is dry and semidry (de Faria discussed and documented in different 1970). The intensive rainfall between countries (Cock et al. 2009, Hussein August and October is responsible for et al. 2010). In the last few years there erosion, especially of steeply declined has been an increasing interest in native regions (between 25 and 45%). 204 B. Wasilewska-Nascimento

The islands of Santiago and Fogo tribute to a more effective pest control, both provide the climatic and ecological especially in cases, where exotic organ- conditions for the development of agri- isms have not yield any satisfying re- culture. sults. The agriculture of irrigated grounds is In these studies, the presence of EPF showing a growing tendency, each year in soil samples collected in agricultural increasing the areas where drop by drop and reforested areas of Santiago and irrigation is used instead of the still dom- Fogo was analyzed. inating traditional fl ooding irrigation. The most popular on irrigated grounds are orchards (Musa spp., Mangifera spp., MATERIAL AND METHODS citruses) and vineyards (Vitis sp.) Dryland farming, with its main crops Soil samples being corn and legumes such as Caja- The soil samples were collected in No- nus cajan, still occupy 95% of all arable vember and December 2011 from two grounds of Cape Verde (National Adap- islands of the Cape Verde archipelago. tation Programme of Action on Climate Seven localities were visited on the is- Change 2008–2012, 2007). lands of Santiago (63.6%) and four on Worldwide research indicates the ef- Fogo (36.4%). fectiveness of microorganisms in the 44 soil samples were collected (four pest control, in case of permanent tropi- samples in each location). In each loca- cal crops, such as citruses, bananas, co- tion, 2 kg of soil was collected at four conut palms, mango, guava, papaya and points around the plant and mixed to ob- pineapple (Dolinski and Lacey 2007). tain homogeneous sample. The samples The signifi cance of entomopathogen- were collected from 15 cm below the ic fungi (EPF) was mainly documented surface, previously having removed the as a key element of an integral defence plant waste. of plants against pests. There are 1,200 The soil was sampled in three dif- described species of EPF. 12–15 of these ferent types of habitat: reforested areas species are used throughout the world in (27.3% of localities), irrigated agricul- pest control (Kowalska and Pruszyński tural land (54.5%) and rainfed agricul- 2007), in the form of around 120 regis- ture (18.2%). tered biopesticides (Tkaczuk 2008). Reforested areas are highlands with Boczek and Lipa (1978) pointed out, a steep decline. The highlands of San- that entomopathogenic representatives tiago are mainly covered in Jacaranda of Ascomycetes occur mostly in tropical mimosifolia, Lantana camara and euca- and subtropical regions. lyptuses. In Fogo, however, Cupressus It has been proven, that the place of sp., Acacia sp., Pinus sp. and Eucalyptus origin predicts the behaviour of the fungi sp. stand out. tested afterwards in the biological pest Irrigated agricultural land is covered control (Bidochka et al. 2001). That is with perennial crops such as Musa spp., why the usage of local strains may con- Mangifera sp., Citrus sp., Vitis sp. Occurrence of entomopathogenic fungi in soil of Santiago... 205

Rainfed areas represent agricultural in the dark and identifi ed microscopi- land with perennial crop of pigeon pea cally using taxonomic keys described by Cajanus cajan. Barnett and Hunter (1987) and Watanabe The soil samples were transported (2009). in clear plastic bags to the laboratory of In order to statistically analyze the re- the National Institute for Research and sults, the SPSS programme was used. Agricultural Development (INIDA) lo- cated in Săo Jorge dos Órgăos (Santiago island) where they were sieved through RESULTS AND DISCUSSION a 3 mm mesh. The percentage of sand in the soil The mortality of larvae of G. mellonel- samples was determined by the Ana- la introduced into soil samples reached lytical Laboratory of Soil, Water and 40.5%. Plants (LASAP) located in Săo Jorge dos EPF were found in soil originate from Órgăos. Cape Verde. 57.8% of the collected soil samples contained EPF. Isolation and identifi cation of EPF Besides EPF, in 37.7% of samples The soil samples were studied in the the presence of other potential ento- laboratory for the presence of EPF using mopathogenic organisms such as rhab- the Galleria bait method (Zimmermann ditid nematodes was noted. In 4.5% of 1986). The last larval instar of Galleria soil samples no potential natural enemies mellonella (L., 1758) was used. were found. The larvae of G. mellonella (Lepidop- EPF were represented by isolates tera: Pyralidae) were originated in labo- of Beauveria bassiana (Hypocreales: ratory in INIDA. They were produced Cordycipitaceae) and Metarhizium ani- based on an artifi cial diet according to sopliae (Hypocreales: Clavicipitaceae). the recommendations of Dr. Charles Each species was isolated from 34.6% Waturu Nderito (2010). of soil samples (Fig. 1). The occurrence A total of 455 larvae of G. mellonella of Beauveria and Metarhizium in the soil was buried in 52 sterile closed plastic from the islands of Santiago and Fogo containers. Each box carried 400 g of soil and four larvae or 1,200 g of soil and confi rms the cosmopolitan character 12 larvae. The containers were incubated of the aforementioned species of fungi at 25°C ±1°C for 30 days. (Meyling and Eilenberg 2007, Sánchez- The mortality of larvae was recorded -Peńa et al. 2011). three times a week. The dead larvae were In addition, in 50.0% of samples sterilized on the surface with 5% sodium Fusarium spp. was found. 19.2% of soil hypochlorite and 70% alcohol followed samples contained fungi with unsporu- by two washes in sterile, distilled water lated mycelium (Fig. 1). and kept separately for four days in moist From some of the samples, two or chambers at 25°C. The fungi developed three different species of fungi were iso- on the cadavers were subsequently cul- lated. 7.7% of samples contained both tivated for seven days in PDA medium aforementioned species of EPF. 206 B. Wasilewska-Nascimento

FIGURE 1. Percent of soil samples from Cape Verde containing fungi (November and December 2011)

The fungus B. bassiana was found on of adult weevils Cosmopolites sordidus the island of Fogo (Table 1), in soil sam- (Coleoptera: Curculionidae) found on ples taken from perennial crops, repre- banana plantations in Santiago were sented by vineyards (75.0% of samples) infected by B. bassiana (Nascimento, and pigeon pea C. cajan cultivated in not published). This fact may indicate rainfed areas (85.7%). the presence of endophyte strains of

TABLE 1. Distribution of fungi in soil of different habitats on the Santiago and Fogo islands in Cape Verde, 2011 Percent of samples containing fungi Fungal species Reforestation Irrigated agricultural land Rainfed areas Santiago Fogo Santiago Fogo (Fogo) Beauveria bassiana 0.0 a 0.0 a 0.0 a 75.0 b 85.7 b Metarhizium anisopliae 0.0 a 0.0 a 40.0 ab 100.0 b 14.3 a Fusarium spp. 50.0 a 66.7 a 40.0 a 50.0 a 57.1 a Fungi with unsporula- 50.0 a 33.3 a 30.0 a 0.0 a 0.0 a ted mycelium Values in lines followed by the same letters are not signifi cantly different. According to Tkaczuk (2008), cul- B. bassiana associated with banana crops tivating plants from Fabaceae (Dolinski and Lacey 2007). favours the occurrence of EPF. But A signifi cant Pearson’s correlation Tkaczuk’s results obtained in Poland in- was shown between the presence of dicated a dominance of M. anisopliae in B. bassiana in soil samples and the pres- soils, where Fabaceae were cultivated. ence of sand (r = 0.647; p < 0.01). On There was no recorded presence of average, there was more sand in sam- B. bassiana in the soil of Santiago. Ear- ples originate from Fogo than there was lier studies, however, showed that 5% in samples from Santiago. Marjańska- Occurrence of entomopathogenic fungi in soil of Santiago... 207

-Cichoń et al. (2005) pointed out, that originate from wooded areas in Spain. In sandy soil is richer in entomopathogenic Poland, in the soil from forested habitats fungi than argillaceous soil. But in sandy the B. bassiana was defi nitely dominat- soil from Poland M. anisopliae was ing (Tkaczuk 2008). dominating. The presence of a Fusarium species The presence of the species Me- was frequently noted (40.0–66.7%) in tarhizium was confi rmed in all soil sam- soil samples collected on both islands ples collected on the island of Fogo on (Table 1). the vine plantation and in 14.3% of soil The common practice of isolating planted with pigeon pea. On the island of Fusarium from soil samples using the Santiago the presence of M. anisopliae Galleria bait method should be brought (40.0%) was related to banana planta- to attention. As Wenda-Piesik (2011) tions. All of these terrains had an incli- points out, Fusarium is an example of nation of no more than 25%. a phytopathogenic fungus also able to A signifi cant Pearson’s correlation induce lethal reaction in insects. The du- (r = –0.714; p < 0.01) was found between alistic properties of the various species the presence of M. anisopliae and the in- of Fusarium are the subject of studies in clination of the terrain. the biological control of mosquitoes and The EPF were isolated from soil nematodes Radopholus similis. Dolinski samples collected from lots occupied and Lacey (2007) assessed the potential by orchards, vine and C. cajan. These of Fusarium spp. in the biological con- perennial crops create benefi cial circum- trol of C. sordidus. stances for the survival of EPF, more so It is to be noted, that the survival and given the stability of the environmental pathogenicity of EPF depends on various conditions and, consequently, the pres- abiotic factors. The conidia of some of ence of potential hosts, as mentioned the isolates of B. bassiana lose their path- by Chandler et al. (1997) and Tkaczuk ogenicity after three hours of insolation (2008). (Kowalska and Pruszyński 2007). There- There were no EPF in the soil samples fore, the usage of native entomopatho- collected in reforested areas in Santiago gens in the biological control of different and Fogo (Table 1). The reforested ar- pests may be profi table. Native isolates eas of Cape Verde are located in steeply are adapted to the local climatic condi- inclined terrains and suffer from strong tions, as well as to the local entomofauna water erosion during the rainy season. (Tangchitsomkid and Sontirat 1998, Do- For the record, these studies were con- linski and Moino Jr. 2006), which is why ducted at the end of the rainy season. they are more effective against pests than The results of the studies are con- introduced isolates. sistent with the results of Sánchez-Peńa The EPF are used, on a large scale, all et al. (2011), who showed, that wooded over the world in the biological control areas are not always a source of natu- of various pests of crops, such as aphids, ral enemies. On the other hand, Que- locusts, trips, whitefl ies (Cavalcanti sada-Moraga et al. (2007) isolated both 2006). The development of plant protec- M. anisopliae and B. bassiana from soil tion in the Cape Verde should follow the 208 B. Wasilewska-Nascimento example and investigate the infl uence CHANDLER D., HAY D., REID A.P., 1997: of isolated strains of B. bassiana and Sampling and occurrence of entomo- M. anisopliae on pests occurring on the pathogenic fungi and nematodes in UK islands. soils. Appl. Soil. Ecol. 5: 133–141. COCK M.J.W., van LENTEREN J.C., BRODEUR J., BARRATT B.J.P., BIG- CONCLUSIONS LER F., BOLCKMANS K., CÔNSOLI F.L., HAAS F., MASON P.G., PARRA J.R.P., The occurrence of B. bassiana and 2009: The use and exchange of biological M. anisopliae in agricultural land of Cape control agents for food and agriculture. Verde was confi rmed. The presence of FAO. Backgroung study paper 47 (ftp:// B. bassiana was recorded in the soil with ftp.fao.org/docrep/fao/meeting/017/ higher sand content, while M. anisopliae ak569e.pdf; Accessed 11.05.2013). De FARIA F.X., 1970: Os solos da ilha de occurred on less inclined fi elds. Santiago (Arquipélago de Cabo Verde). The frequent presence of Fusarium Estudos, Ensaios e Documentos, No 124. spp. drew attention and should be stud- Junta de Investigações do Ultramar. Lis- ied more. boa. DOLINSKI C., MOINO Jr., A., 2006. Utiliza- Acknowledgment çăo de nematóides entomopatogęnicos The author wishes to thank Dr. Patri- nativos ou exóticos: o perigo das introdu- cia Stock for confi rming the identifi ca- ções. Nematol. Bras. 30: 139–149. DOLINSKI C., LACEY L.A., 2007: Micro- tion of nematodes and António Baptista bial Control of Pests of Tropi- Moreira who assisted in the collection cal Tree Fruits. Neotrop. Entomol. 36 (2): and processing of soil samples. 161–179. HUSSEIN K.A., ABDEL-RAHMAN M.A.A., REFERENCES ABDEL-MALLEK A.Y., EL-MARA- GHY S.S., JOO J.H., 2010: Climatic fac- BARNETT H.L., HUNTER B.B., 1987: Il- tors interference with the occurrence of lustrated Genera of Imperfect Fungi. Beauveria bassiana and Metarhizium an- 4 ed. Macmillan Publishing Company, isopliae in cultivated soil. Afr. J. 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Vuill. – Ecology of the entomopathogenic fungi Nematóides entomopatogęnicos (Rhab- Beauveria bassiana and Metarhizium ditida) – Orius insidiosus (Say) no con- anisopliae in temperate agroecosystems. trole de tripés (Thysanoptera) em cultivo Potential for conservation biological con- protegido. UFLA, Lavras. trol. Biol. Control 43: 145–155. Occurrence of entomopathogenic fungi in soil of Santiago... 209

National Adaptation Programme of Action WENDA-PIESIK A., 2011: Entomopatoge- on Climate Change 2008–2012, 2007: niczne grzyby z rodzaju Fusarium i ich Ministry of Environment and Agriculture znaczenie w regulacji liczebności szko- (http:// http://unfccc.int/resource/docs/ dliwych owadów. Entomopathogenic napa/cpv01.pdf; Accessed 12.09.2013) fungi of Fusarium sp. and their role in NDERITO C.W., 2010: Verbal communica- pest control. Adv. Agric. Sci. 4: 35–48. tion. Manuscript. ZIMMERMANN G., 1986: The Galleria Programme of the Government of Cape bait method for detection of entomo- Verde, 2011–2016, 2010 [Programa do pathogenic fungi in soil. J. Appl. Ento- Governo. VIII Legislatura 2011-2016. mol. 102: 213–215. Governo de Cabo Verde] (http://www. governo.cv/; Accessed 06.07.2012) Streszczenie: Występowanie grzybów owadobój- Program strategy of Cape Verde, 2009– czych w glebach wysp Santiago i Fogo (Republi- –2012, 2010 [Estratégia do programa de ka Zielonego Przylądka). Gleba jest siedliskiem Cabo Verde (2009–2012). Programa das wielu grzybów owadobójczych (EPF) na całym pequenas subvenções do Fundo Mundial świecie. Do izolowania EPF z prób glebowych do Ambiente às ONGs] (http://www.pla- zastosowano metodę owadów pułapkowych. tongs.org.cv; Accessed 06.07.2012). Powszechność występowania EPF na terenach QUESADA-MORAGA E., NAVAS-COR- rolnych została potwierdzona niniejszymi bada- TÉS J.A., MARANHAO E.A.A., OR- niami, w wyniku których stwierdzono, że gleby TIZ-URQUIZA A., SANTIAGO-ÁL- Republiki Zielonego Przylądka są siedliskiem VAREZ C., 2007: Factors affecting the EPF, takich jak Beauveria bassiana i Metarhizium occurrence and distribution of entomo- anisopliae. Obecność B. bassiana zarejestrowano pathogenic fungi in natural and cultivated w glebie wyspy Fogo a występowanie M. aniso- soils. Mycol. Res. III: 947–966. pliae zarówno na wyspie Fogo, jak i Santiago. Nie SÁNCHEZ-PEÑA, S.R., LARA J.S-J., ME- stwierdzono EPF na terenach zalesionych. Staty- DINA R.F., 2011: Occurrence of entomo- stycznie więcej prób glebowych zawierających B. bassiana pochodziło z terenów pokrytych upra- pathogenic fungi from agricultural and wami roślin wieloletnich, przy czym istotnym natural ecosystems in Saltillo, México, czynnikiem wpływającym na obecność grzyba and their virulence towards thrips and była zawartość piasku w glebie. M. anisopliae był whitefl ies. J. Insect. Sci. 11 (1): 1–10. natomiast obecny na terenach o mniejszym na- TANGCHITSOMKID N., SONTIRAT S., chyleniu terenu. Fusarium spp. był obecny w po- 1998: Occurrence of Entomopathogenic łowie przebadanych prób glebowych. Izolowanie Nematodes in Thailand. Kasetsart J. (Nat. EPF rodzimych z późniejszym ich zastosowaniem Sci.) 32: 347–354. w krajach rozwijających się może w istotny spo- TKACZUK C., 2008: Występowanie i poten- sób przyczynić się do skutecznego ograniczenia cjał infekcyjny grzybów owadobójczych liczebności populacji szkodników w uprawach. w glebach agrocenoz i środowisk semina- turalnych w krajobrazie rolniczym. Wy- MS. received in November 2013 dawnictwo Akademii Podlaskiej, Siedlce. WATANABE T., 2009: Pictorial atlas of soil Author’s address: and seed fungi: morphologies of cultured Beata Wasilewska-Nascimento fungi and key to species. 3 ed. Taylor & Department of Science and Technology Francis. University of Cape Verde Palmarejo, CP 279-Praia Republic of Cape Verde e-mail: [email protected]

Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 211–218 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Effect of rearing system and gender on histological profi le of chicken breast and leg muscles in hybrid (Cobb×Zk) AGNIESZKA WNUK1, NATALIA MROCZEK-SOSNOWSKA1, DOBROCHNA ADAMEK2, MACIEJ KAMASZEWSKI2, 1 1 MONIKA ŁUKASIEWICZ , JAN NIEMIEC 1Department of Animal Breeding and Production 2Department of Ichthyobiology and Fisheries Warsaw University of Life Sciences – SGGW

Abstract: Effect of rearing system and gender meat constitutes “skeletal muscles and on histological profi le of chicken breast and leg adherent adipose, connective and bone muscles in hybrid (Cobb×Zk). The experiment tissues, derived from carcasses, half car- was conducted on 930 slow-growing chickens from the crossing of a Cobb male and Greenleg casses or quarter carcasses of particu- Partridge female. The chicks were randomly as- lar types of animals for slaughter”. The signed to two groups: control (BW) which did main components of skeletal muscle tis- not have access to a free run and the experimental sue are muscle fi bers, which are highly group (W) using the grassy runs from 4 weeks of specialized. The number, size and type of age. The experiment showed a statistical effect of sex on breast (P ≤ 0.01) and leg (P < 0.05) muscle muscle fi bers, their biochemical, physi- fi ber diameter in Cobb×Zk hybrid roosters. There ological and histological characteristics were no signifi cant gender-dependent differences may lead to changes in meat quality and in the surface area of the muscles tested. No effect sensory evaluation (Damez and Cler- was either reported of the rearing system on the jon 2008). The chemical composition histological picture of breast and leg muscles. of skeletal muscle is 75% water, 19% Key words: rearing system, broiler chickens, mus- protein, 0.5–8% lipids and 1% glycogen cle fi ber diameter (Lefaucheur 2010). However, their main mass is formed by muscle fi bers, which diameters fall within the range of 10 to INTRODUCTION 100 μm, and largely depend on the type Animal products are an essential compo- of muscle fi bers (Tumova and Teimouri nent of a man diet. They are a source of 2009, Lefaucheur 2010). balanced protein, they contain low lev- Histological structure of muscles els of cholesterol and fat, which is at the slightly varies depending on their func- same time rich in unsaturated fatty acids tions (Klont et al. 1998). According to (Wangang et al. 2010). Poultry meat sur- Remignon et al. (1995), the diameter of passes meat of slaughter animals in terms muscle fi bers depends on their type and of nutritional value, because of a valu- to great extent on genetic background. able source of balanced animal protein Other factors important in this respect that is highly digestible and easy to as- include sex (Ozawa et al. 2000), age similate. According to PN-65/A-82000, (Candek-Potokar et al. 1998), breed (Ryu 212 A. Wnuk et al. et al. 2008), physical activity (Karlsson the possibility to use grassy free runs et al. 1999) and rearing system (Castel- from 4 weeks of age by the experimental lini et al. 2002a and 2002b, Branciari group. In each group the runs were 3×5 et al. 2009). m in size. Half of the run area was cano- Nowadays consumers are becoming pied. The run area was dry, sunny, with more aware of their needs and begin to permeable soil, covered with perennial pay attention to the welfare of the ani- ryegrass Lolium perenne L. (40%), red mals, making it one of the criteria when fescue L. (50%) and Ken- choosing products of animal origin. The tucky bluegrass L. (10%). research carried out on the British popu- The free run had an area with sand where lation clearly demonstrated that animal birds could use sand baths. A four-phase welfare was treated as a “very impor- feeding system was applied during the tant” or “important” factor, which indi- rearing with the use of starter, grower 1, rectly infl uences the suitability and sus- grower 2 and fi nisher feed mixtures (Ta- tainability of food (DEFERA 2011). For ble 1). Birds had unrestricted access to the most part it refers to both the health both feed and water. and food safety. According to Sundrum During the experiment after each (2001), attention is increasingly drawn to week an individual body weight, food the rearing system of birds, including the intake and death rate of chickens was density per m2 and access to free runs. checked. On the 63rd day of rearing, 24 The aim of this experiment was to de- females and 24 males were selected from termine the effect of the rearing system each group with body weight about aver- and gender on the histological profi le of age for each sex in a group and speci- breast and leg muscles in slow-growing mens of breast and leg muscles were Cobb×Zk chickens. collected for histology. Samples in size of 0.5×0.5×1 cm were collected within 15 min since slaughter after appropri- MATERIALS AND METHODS ate exsanguination of the chickens and subsequently subjected to 24-hours fi xa- The study was conducted at the Warsaw tion. The samples were then washed in University of Life Sciences – SGGW, ethanol to remove the fi xing agent and experimental fi eld station in Wilanów- dehydrated by a series of increasing -Obory. Experimental procedures were ethyl alcohol concentrations. Dehydrat- approved by the Ethics Commission (ap- ed samples were saturated with paraffi n. proval no. 27/2009 dated 16 April 2009). Paraffi n saturation was carried out in the The study was conducted on 930 chick- incubator at the melting point of paraffi n. ens from the cross of Cobb male and Saturation duration was adapted to mus- Greenleg Partridge female (Zk) reared cle samples collected and amounted to for 63 days. Day-old chicks were ran- a few hours. Paraffi n blocks were formed domly divided into two groups of 465 after completion of the saturation proc- chicks each: BW (control) and W (ex- ess. Microtome Leica RM 2265 (Leica perimental) in fi ve repetitions each of Microsystems, Nussloch, Germany) was 93 chicks. A differentiating factor was used to cut paraffi n sections. Muscle Effect of rearing system and gender on histological... 213

TABLE 1. Feed mixture composition and nutritional value according to producer’s Starter Grower I Grower 2 Finisher Specifi cation (1–11) (12–24) (25–37) (38–63) Content (%) Corn 10.00 11.40 10 10.00 Wheat 53.00 55.00 59.60 60.80 Soybean meal 30.60 27.40 23.20 21.60 Feeding limestone 1.19 1.20 1.11 0.97 Sodium bicarbonate 0.20 0.14 0.14 0.16 NaCl 0.24 0.28 0.28 0.26 Stimulator 0.01 0.01 0.01 0.01 Dicalcium phosphate 1.18 0.78 0.70 0.64 Soybean oil 2.10 2.40 3.60 4.40 Methionine 84% calcium salt 0.48 0.42 0.36 0.28 Lysine 0.36 0.34 0.36 0.28 Threonine 0.14 0.13 0.14 0.10 Premix C196 PX05802 0.5% 0.50 0.50 0.50 0.50 Nutritional value ME (kcal) 2990.20 3047.19 3125.72 3217.10 Fat 3.67 4.00 5.14 5.92 Protein 21.99 20.78 19.26 18.51 Fiber 3.60 2.55 2.45 2.41 Ash 5.83 5.35 4.96 4.67 Lysine 1.38 1.28 1.19 0.97 Methionine + cystine 1.08 1.01 0.92 0.76 Available phosphorus 0.45 0.38 0.36 0.35

Provided per kilogram of diet: STARTER: vitamin A 11.00 K UL; organic phosphorus 0.59%; cal- cium 0.98%; phosphorus available 0.45%; calcium chloride 0.24%; sodium 0.15%; chlorine 0.27%; potassium 0.90%; magnesium 0.17%; manganese 142.32 mg; copper 31.59 mg; selenium 0.41 mg; iron 191.51 mg; sulfur 0.34%; zinc 116.80 mg; lysin1.36%; methionine 0.31%; GROWER I vitamin A 11.00 K UL; organic phosphorus 0.51%; calcium 0.87%; phosphorus available 0.38%; calcium chloride 0.28%; sodium 0.15%; chlorine 0.29%; potassium 0.85%; magnesium 0.16%; manganese 141.84 mg; copper 30.82 mg; selenium 0.41 mg; iron 174.55 mg; sulfur 0.32%; zinc 115.03 mg; lysin1.26%; methionine 0.30%; GROWER II vitamin A 11.00 K UL; organic phosphorus 0.48%; cal- cium 0.81%; phosphorus available 0.36%; calcium chloride 0.28%; sodium 0.15%; chlorine 0.30%; potassium 0.77%; magnesium 0.16%; manganese 141.40 mg; copper 30.21 mg; selenium 0.40 mg; iron 165.98 mg; sulfur 0.30%; zinc 113.79 mg; lysin1.17%; methionine 0.28%; FINISHER vitamin A 11.00 K UL; vitamin D3 3.00 K UL; vitamin E 40.00 mg; organic phosphorus 0.73%; calcium 0.35%; calcium chloride 0.26%; sodium 0.15%; chlorine 0.27%; potassium 0.74%; magnesium 0.15%; manga- nese 140.80 mg; copper 29.92 mg; selenium 0.40 mg; iron 159.92 mg; sulfur 0.28%; zinc 113.14 mg; lysin1.06%; methionine 0.27%; 214 A. Wnuk et al. cross sections had the thickness of 5 μm. TABLE 2. The infl uence of gender on the dia- Standard H&E staining was performed. meter and area of chickens muscle fi bers in hybrid The diameter and area of 200 muscle (Cobb×Zk) fi bers was measured in each slide using Diameter (μm) Area (μm2) Gender a Nikon Ellipse E200 light microscope mp mn mp mn equipped with a Nikon DS-Fi2 camera Roosters 63.02A 50.82A 1401.54 1142.09 and COOL view 2.7 software. B B The results were analyzed statistically Hens 48.05 45.69 955.29 808.77 using the analysis of variance, calculated SE 3.44 3.66 119.71 113.93 by least squares method with statistical mp – breast muscle, mn – leg muscle; A,BMeans software SPSS 19.0 GB (SPSS Inc., Chi- with the different subscripts differ signifi cantly at cago, IL, USA). P ≤ 0.01.

TABLE 3. The infl uence of rearing system on the RESULTS AND DISCUSSION diameter and area of chickens muscle in hybrid (Cobb×Zk) Results of the analysis of breast muscle 2 Rearing Diameter (μm) Area (μm ) microstructure in chickens are shown in system mp mn mp mn Tables 2 and 3. The diverse diameters of muscle fi bers of breast muscles of the BW 54.09 52.32 1265.32 1100.50 birds were to a greater extent correlated W 56.98 44.18 1091.51 850.35 with gender than rearing system. The di- SE 4.76 3.38 150.92 123.70 ameter of muscle fi bers increases with mp – breast muscle, mn – leg muscle. the age of birds (Branciari et. al 2009). According to Sobolewska et al. (2011), individual giant fi bers could be distin- in broiler chickens reared to the 35th guished, the diameter of which, accord- day, the most intense increase in muscle ing to Dransfi eld and Sosnicki (1999), is fi ber diameter was between the 8th and generally about three times greater than 21st day of rearing. Breast muscle fi bers of the normal fi bers. The hypertrophy of fully developed not before the 56th day. muscle fi bers affects mainly fast-grow- According to Papinaho et al. (1996) and ing chickens and results from intense Geyukouku et al. (2005), the average fi b- selection (Brocka et al. 1998, Guernec er thickness of m. pectoralis major, m. et al. 2003, Lefaucheur et al. 2010). Study biceps femoris, m. extensor hallucis lon- by Miraglia et al. (2006) showed that the gus, and m. gastrocnemius is 60.0, 51.6, proportion of giant fi bers in the pecto- 59.8 and 60.45 μm, respectively. Fast- ralis major in slow-growing chickens -growing chickens have a much greater was 0.56%, in the fast-growing 3.17%, diameter of muscle fi bers as compared to while in ileotibialis lateralis it was 1.70 the slow-growing chickens (Khoshoii et and 3.18%, respectively. Intense muscle al. 2013). growth is associated with an increase in In the current experiment, the ma- thickness and the surface of muscle fi b- jority of muscle fi bers in the cross sec- ers, rather than their number. tions had a clear shelving structure, and Effect of rearing system and gender on histological... 215

Our experiment showed a statistical logical picture of breast and leg muscles effect of gender on breast (P ≤ 0.01) and (Table 3). However birds that could use leg (P < 0.05) muscle fi ber diameter in free runs had a larger diameter of breast male of Cobb×Zk hybrid (Table 2). They muscles (56.98 μm) and a lesser diameter had a larger diameter in both breast and of leg muscles (44.18 μm) compared to leg muscles (63.02 and 50.82 μm, respec- birds that did not benefi t from grassy free tively) compared to female (48.05 and runs (54.09 and 52.32 μm, respectively). 45.69 μm). Khoshoii et al. (2013) stud- Sowińska (2002) either did not identify ied the effect of gender on the diameter the infl uence of the rearing system on of breast muscle fi bers and demonstrated skeletal muscle morphology. Polak et al. that both slow-growing and fast-grow- (2010) compared m. pectoralis and m. ing male had a larger diameter of mus- gastroceneminus in 215 Anak Titan and cle fi bers. Biesiada-Drzazga et al. (2006) Isa chickens and showed that fi ber size demonstrated in geese that both breast was affected by the rearing system. In and leg muscles of females had a greater general, in birds of both lines, the fi ber diameter of muscle fi bers. However Mo- diameter of m. pectoralis was smaller in bini (2013) showed no effect of gender groups with outdoor access (P < 0.01). on the diameter of the muscle fi bers. Birds from the control group had There were no signifi cant gender-de- a greater surface area of muscle fi bers pendent differences in the surface area of in both breast and leg muscles (1,265.32 the muscles tested. However males had and 1,100.50 μm2, respectively) com- a greater surface area of muscle fi bers in pared to chickens from the experimental both breast and leg muscles (1,401.54 and group (955.29 and 808.77 μm2). The op- 1,142.09 μm2, respectively) compared to posite trend was shown in the study by females (955.29 and 808.77 μm2). The Branciari et al. (2009) where they com- largest area was observed in breast mus- pared an impact of the rearing system cles of males and the smallest in females and genotype on muscle fi ber area. These leg muscles. The study by Scheuermann authors showed that the fast-growing et al. (2004) clearly showed that gender chickens (Ross) reared under conven- had an infl uence on the surface area of tional conditions had the largest surface the breast muscle. This trend continued area of muscle fi bers. The opposite trend on 7th, 21st and 35th day of rearing at was observed in the case of slow-grow- a signifi cance level of P < 0.05. Accord- ing chickens where the fi ber surface area ing to Choi and Kim (2008), differences was larger than in the conducted ex- in fi ber number and size are primarily periment. In the conventional rearing it under the control of sex hormones. Dif- ranged from 955 to 1,610 μm2 and from ferences in fi bers of males and females 1,272 to 2,396 μm2, for breast and leg can arise from hormonal action if dif- muscles, respectively. Whereas in the ferences in androgen hormones are at organic rearing system, the fi ber surface a suffi ciently high level during periods area ranged from 908 to 1,699 μm2 and of prenatal fi ber formation. from 1,758 to 3,006 μm2 for breast and There was no signifi cant effect ob- leg muscles, respectively. served of the rearing system on the histo- 216 A. Wnuk et al.

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MIRAGLIA D., MAMMOLI R., BRAN- SCHEUERMANN G.N., BILGILI S.F., CIARI R., RANUCCI D., CENCI GOGA TUZUN S., MULVANEY D.R., 2004: B.T., 2006: Characterization of muscle Comparison of chicken genotypes: Mio- fi ber type and evaluation of presence of fi ber Number in Pectoralis Muscle and giant fi bers in two meat chicken hybrids. Myostatin Ontogeny. Poultry Sci. 83, Vet. Res. Commun. 30 (1), 357–360. 1404–1412. MOBINI B., 2013: Histological differences SOBOLEWSKA A., ELMINOWSKA- in intramuscular connective tissues com- -WENDA G., WALASIK K., BOGUC- position between dark and light colored KA J., SŁAWIŃSKA A., SZCZER- muscles in broiler chickens. Global Vet- BA A., ŻMUDA-TRZEBIATOWSKA erinaria 10 (3), 360–364. M., 2011: Increase in thickness of the OZAWA S., MITSUHASHI T., MITSU- pectoral muscle fi bers in meat-type MOTO M., MATSUMOTO S., ITOH N., chicken beetween 1st and 35th day of fat- ITAGAKI K., KOHNO Y., DOHGO T., tening. Proc. XXIII International Poultry 2000: The characteristics of muscle fi ber Symposium PB WPSA, Poznań. types of longissimus thoracis muscle and SOWIŃSKA J., 2002: Wpływ systemu utrzy- their infl uences on the quantity and qual- mania, typu użytkowego i obrotu pouboj- ity of meat from Japanese Black steers. owego na wybrane wskaźniki krwi, obraz Meat Sci. 54, 65–70. morfologiczny mięśnia piersiowego oraz PAPINAHO P. A., RUUSUNEN M.H., SU- jakość indyków rzeźnych. Wyd. UMW, UROMENT T., FLETCHER D.L., 1996: Olsztyn. Relationship between muscle biochemi- SUNDRUM A., 2001: Organic livestock cal and meat quality properties of early farming. A critical review. Livest Prod. deboned broiler beasts. J. Appl. Poultry Sci. 67, 207–215. Res. 5, 126–133. TUMOVA E., TEIMOURI A., 2009: Chick- PN-65/A-82000 Mięso i podroby zwierząt en muscle fi ber characteristics and meat rzeźnych. Wspólne wymagania i badania. quality: A review. Scienta Agriculturae POLAK M., PRZYBYLSKA-GORNO- Biochemica 40 (4): 253–258. WICZ B., FARUHA A., 2010: The effect WANGANG Z., XIAO S., SAMARAWEE- of different rearing conditions on muscle RA H., LEE E. J., AHN D. 2010: Improv- characteristic on muscle characteristics in ing functional vaule of meat products, broilers of two commercial lines- a light Meat Sci., 86:15–31. microscopic study. J. Poult. Sci. 47, 125– –132. Streszczenie: Wpływ systemu utrzymania oraz REMIGNON H., GARDAHAUT M.F., płci na profi l histologiczny mięśni piersiowych MARCHE G., RICARD F.H., 1995: Selec- i nóg kurcząt mieszańca Cobb×Zk. Doświadcze- tion for growth increases the number and nie przeprowadzono na 930 kurczętach wolno the size of muscle fi ber without changing rosnących pochodzących z krzyżowania koguta their typing in chickens. J. Muscle Res. Cobb oraz kury zielononóżki kuropatwianej. Pi- Cell Motil. 16, 95–102. sklęta losowo przydzielono do dwóch grup: kon- RYU Y.C., CHOI Y.M., LEE S.H., SHIN trolnej (BW) niemającej dostępu do wybiegu oraz H.G., CHOE J.H., KIM J.M., HONG grupy doświadczalnej (W), korzystającej z trawia- K.C., KIM B.C., 2008: Comparing the stych wybiegów począwszy od 4. tygodnia życia. histochemical characteristics and meat W przeprowadzonym doświadczeniu wykazano quality traits of different pig breeds. Meat statystyczny wpływ płci na średnicę włókien mię- Sci. 80, 363–369. śni piersiowych (P ≤ 0,01) oraz nóg (P < 0,05) 218 A. Wnuk et al. kogutów mieszańca Cobb×Zk. Nie stwierdzono Authors’ address: istotnych różnic dla wartości pola powierzchni Agnieszka Wnuk badanych mięśni w zależności od płci. Nie wyka- Wydział Nauk o Zwierzętach SGGW zano wpływu systemu utrzymania na wyniki ob- Katedra Szczegółowej Hodowli Zwierząt razu histologicznego mięśni piersiowych i nóg. ul. Ciszewskiego 8, 02-786 Warszawa Poland e-mail: [email protected] MS. received in November 2013 Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 219–225 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Infl uence of the system of rearing on cholesterol level and its fraction in blood serum of slow-growing chickens AGNIESZKA WNUK, NATALIA MROCZEK-SOSNOWSKA, MONIKA ŁUKASIEWICZ, MARTYNA BATORSKA , JAN NIEMIEC Department of Animal Breeding and Production, Warsaw University of Life Sciences – SGGW

Abstract: Infl uence of the system of rearing on more and more interested in conditions cholesterol level and its fraction in blood serum of in which domestic animals are kept. In slow-growing chickens. The experiment was car- highly developed countries for many ried out on 936 slow-growing chickens from the crossbreed of Cobb cock with Greenleg Partridge years a rising interest in a healthy diet hen. Chicks were randomly divided into two has been noticed, with paying special groups: controlled group (BW) with no access to attention to comfort of life of domestic the fi eld and experimental group (W) with access animals and high standards of animal to grass fi elds since their fourth week of life. In welfare in order to achieve high quality blood serum overall cholesterol, lipoproteins with high density (HDL), triacylglycerols (TG) level products (Sundrum 2001). Morphologi- was marked. The concentration of lipoproteins cal and biochemical blood tests are of with low density was calculated while using the great importance in animal treatment, it Friedewald et al. formula (1972). No statistically is one of basic measures used in condi- signifi cant infl uence of sex and system of breed- tion assessment, for example, of a cat ing on cholesterol and its fractions level in blood serum of crossbreed chickens was observed. or a dog (Kliszcz 2010). While looking at birds, unfortunately, there are only Key words: broiler chickens, slow-growing chick- some physiological norms available in en, blood the literature, insuffi cient to form a com- plete picture of biochemical and hema- INTRODUCTION tological indicators or the amount of li- pid compounds in blood serum of birds In Poland most poultry meat production (Krasnodębska-Depta and Koncicki is based on fast growing chickens kept in 2000, Mazurkiewicz 2005, Gryzińska an intensive system (Polak et al. 2010). et al. 2010). The fast growth rate while the whole A lot of various factors may infl uence body develops at different rates very levels of biochemical and hematological often results in numerous dysfunctions indicators in blood, among other things: such as: syndrome of sudden heart death, sex, age, species, race, diet, physiologi- ascites, limb dysfunctions – which, con- cal condition and rearing system (Mazur- sequently, have a bad infl uence on chick- kiewicz 2005). Blood plays a distinct en health and most of all – according to role in a proper function of animal or- consumers – on the meat quality (Julian ganisms. It takes part in transporting nu- 2005, Olkowski et al. 2008). Society is trients, thermoregulation, homeostasis, 220 A. Wnuk et al. defense reactions (Elagib et al. 2008). In meable soil, full of sunlight, with plants: blood, besides morphological elements, Lolium perenne L. (40%), Festuca rubra we can fi nd a lot of other chemical com- L. (50%) and Poa pratensis L. (10%). pounds which take part in a proper body There was also a sandy spot in the fi eld function. Cholesterol and triacylglycer- for the birds to take sand baths. During ol’s are transported in body fl uids as li- the period of breeding a four-step system poprotein particles which are classifi ed of feeding was implemented while using according to their density, chylomikro- food blends starter, grower 1, grower 2 ns, lipoproteins with very low density and fi nisher (Table 1). Birds had an un- (VLDL), lipoproteins with intermediate limited access to food and water. density (IDL), lipoproteins with low den- During the experiment after each week sity (LDL), lipoproteins with very high an individual body weight, food intake density (HDL). The basic function of li- and death rate of chickens was checked. poproteins is transporting lipids from the On the 63rd day of breeding there were place of their origin to their destination. chosen from each group 24 cocks and 24 The purpose of the conducted re- hens with body weight about average for search was to establish the infl uence of each sex in a group. Blood samples were breeding system on the level of choles- taken from wing veins using sterilized terol and its fractions in a blood serum of syringes and needles. After 30–60 min slowly growing chickens. since blood taking, the blood samples were spun for 10 min with 3,000 of spins per min. In the obtained blood serum MATERIAL AND METHODS the concentration of overall cholesterol, lipoproteins of high density (HDL), The research was conducted on an ex- triacylglycerol’s (TG) was marked. The perimental farm of University of Life concentration of lipoproteins with low Sciences RZD Wilanów-Obory in 2011. density was calculated while using the Procedures used in the research were in Friedewald et al. formula (1972). accordance with the ones established by the ethical commission 27/2009 on 16 CHOL TG LDL= (mmol/l) April 2009. The research was conduct- 2.2 HDL ed on 936 chickens from crossbreeding of Cobb male with Greenleg Partridge For marking overall cholesterol, rd kept until their 63 day of life. One-day HDL, triglycerides commercial sets by chickens were randomly divided into CORMAY Company were used. two groups: BW (controlled) and W (ex- The obtained results were worked out perimental) in fi ve repetitions each. An statistically while using variance analy- indicator which diversifi ed the groups sis of the smallest squares method in was the ability to use grass fi elds since a statistic program SPSS 19.0 (SPSS th the 4 week of life: the experimental Inc., Chicago, IL, USA). Values of the group (W). The fi elds were 3 m wide examined traits as given in the tables and and 5 m long. Half of the fi eld area was standard errors od of the means (SEM). under roof. The fi eld was dry with per- Infl uence of the system of rearing on cholesterol level... 221

TABLE 1. Feed mixture composition and nutritional value according to producer’s Starter Grower I Grower 2 Finisher Specifi cation (1–11) (12–24) (25–37) (38–63) Content (%) Corn 10.00 11.40 10.00 10.00 Wheat 53.00 55.00 59.60 60.80 Soybean meal 30.60 27.40 23.20 21.60 Feeding limestone 1.19 1.20 1.11 0.97 Sodium bicarbonate 0.20 0.14 0.14 0.16 NaCl 0.24 0.28 0.28 0.26 Stimulator 0.01 0.01 0.01 0.01 Dicalcium phosphate 1.18 0.78 0.70 0.64 Soybean oil 2.10 2.40 3.60 4.40 Methionine 84% calcium salt 0.48 0.42 0.36 0.28 Lysine 0.36 0.34 0.36 0.28 Threonine 0.14 0.13 0.14 0.10 Premix C196 PX05802 0.5% 0.50 0.50 0.50 0.50 Nutritional value ME (kcal) 2 990.20 3 047.19 3 125.72 3 217.10 Fat 3.67 4.00 5.14 5.92 Protein 21.99 20.78 19.26 18.51 Fiber 3.60 2.55 2.45 2.41 Ash 5.83 5.35 4.96 4.67 Lysine 1.38 1.28 1.19 0.97 Methionine + cystine 1.08 1.01 0.92 0.76 Available phosphorus 0.45 0.38 0.36 0.35 Provided per kilogram of diet: STARTER: vitamin A 11.00 K UL; organic phosphorus 0.59%; calcium 0.98%; phosphorus available 0.45%; calcium chloride 0.24%; sodium 0.15%; chlorine 0.27%; potas- sium 0.90%; magnesium 0.17%; manganese 142.32 mg; copper 31.59 mg; selenium 0,41 mg; iron 191.51 mg; sulfur 0.34%; zinc 116.80 mg; lysin 1.36%; methionine 0.31%; GROWER I vitamin A 11.00 K UL; organic phosphorus 0.51%; calcium 0.87%; phosphorus available 0.38%; calcium chloride 0.28%; sodium 0.15%; chlorine 0.29%; potassium 0.85%; magnesium 0.16%; manganese 141.84 mg; copper 30.82 mg; selenium 0.41 mg; iron 174.55 mg; sulfur 0.32%; zinc 115.03 mg; lysin 1.26%; methionine 0.30%; GROWER II vitamin A 11.00 K UL; organic phosphorus 0.48%; calci- um 0.81%; phosphorus available 0.36%; calcium chloride 0.28%; sodium 0.15%; chlorine 0.30%; potassium 0.77%; magnesium 0.16%; manganese 141.40 mg; copper 30.21 mg; selenium 0.40 mg; iron 165.98 mg; sulfur 0.30%; zinc 113.79 mg; lysin 1.17%; methionine 0.28%; FINISHER vitamin A 11.00 K UL; vitamin D3 3.00 K UL; vitamin E 40.00 mg; organic phosphorus 0.73%; calcium 0.35%; calcium chloride 0.26%; sodium 0.15%; chlorine 0.27%; potassium 0.74%; magnesium 0.15%; manga- nese 140.80 mg; copper 29.92 mg; selenium 0.40 mg; iron 159.92 mg; sulfur 0.28%; zinc 113.14 mg; lysin 1.06%; methionine 0.27%. 222 A. Wnuk et al.

RESULTS AND DISCUSSION dicators. Moreover, we must pay atten- tion to individual differences in single Production results in both groups dur- bird populations. In some cases an indi- ing 63-day period of breeding are pre- vidual bird may be still placed within ref- sented in Table 2. The infl uence of sys- erence values even if it is sick (Piasecki tem rearing (with outdoor access) on and Krasoń 2012). the fi nal body weight of chickens was No statistically signifi cant infl uence observed. Rooster’s with access to the of sex or breeding system on cholesterol fi elds had a distinctly larger (P < 0.01) and its fractions level was observed in body weight when compared to rooster’s blood serum of slowly growing chickens the from the controlled group. On the (Table 3). The highest concentration of 63rd day of breeding cocks weighed on overall cholesterol was noticed in a blood average 2,134 g and hens 1,684 g. The serum of cocks with the ability to use the conducted statistic analysis did not show fi eld (2.317 mmol/l). The growth tenden- any signifi cant differences in food intake cy was observed for chickens from the or death rate. However, higher intake of experimental group (W) depending on food and higher death rate was noticed in their sex. The highest level of triacylg- the experimental group (W) – having the lycerol’s (0.867 mmol/l) was observed in ability to use the fi elds (Table 2). Similar the controlled group of chickens (BW). results were obtained in research by Cas- Brodacki et al. (2006) in their research tellini et al. (2002), Nielsen et al. (2003) did not observe any infl uence of a breed- and Fanatico et al. (2005). ing system on overall cholesterol and tri- The analysis of all known in literature acylglycerol’s level in a blood serum of biochemical tests of blood serum estab- turkeys. Kirkpinar et al. (2011) for broiler lished that birds in comparison to other chickens and Gryzińska et al. (2010) in animal species have different values for a blood serum of polar hens observed the those indicators. Only some physiologi- infl uence of sex on cholesterol and level. cal norms available in the literature are Mostly cholesterol and its fractions level insuffi cient to form a complete picture of is infl uenced by various food additives. both biochemical and hematological in- Research conducted by Dehkordi et al. (2010) proved signifi cant differences in TABLE 2. Infl uence of the system of rearing overall cholesterol and triacylglycerol’s on production results of broiler slow-growing level in a group of chickens fed with chicken a standard food blend. Toghyani et al. Specifi ca- FCR Mortality Sex BW (g) (2011) observed the infl uence of cinna- tion (%) (%) mon, Traesel et al. (2011) the mixture of ♂ 2 171A W 2.34 3.52 ethereal oils form oregano, salvia, rose- ♀ 1 688 mary and chili. Research conducted by ♂ 2 098B Gryzińska et al. (2010) proved that with BW 2.30 3.50 ♀ 1 680 age overall cholesterol, triacylglycer- BW – body weight; FCR – feed conversion ratio; ol’s and lipoproteins with low density A,B – means with the different subscripts differ (LDL) level decreases in blood serum. signifi cantly at P ≤ 0.01. Just the opposite is for lipoproteins with Infl uence of the system of rearing on cholesterol level... 223

TABLE 3. Cholesterol and its fractions and triglycerides in the blood serum of chickens slow-growing based on gender and rearing system (mmol/l) Specifi ca- Sex CHOL SEM TG SEM HDL SEM LDL SEM tion W 2.317 0.780 1.627 0.653 ♂ BW 2.140 0.867 1.433 0.667 0.214 0.060 0.165 0.071 W 2.307 0.737 1.607 0.667 ♀ BW 2.037 0.747 1.393 0.610 CHOL – cholesterol; TG – triglycerides; HDL – high-density lipoprotein; LDL – low-density lipopro- tein. high density (HDL) which concentration REFERENCES is the highest in blood of the youngest birds. Krasnodębska-Depta and Kon- BRODACKI A., BATKOWSKA J., ZADU- wicki (2002) studied the infl uence of a RA A., 2006: Wpływ systemu chowu na short-term heat stress on some chosen poziom wskaźników hematologicznych i biochemicznych krwi indyków rzeź- biochemical indicators in blood. nych. Annales Universitatis Mariae They observed that after 6, 26 and 50 Curie-Skłodowska Lublin-Polonia. Sec- h of heat stress in a group of turkeys, a tio EE 24 (45), 335–342. statistically signifi cant increase of tria- CASTELLINI C., MUGNAI C., Cal BOS- cylglycerol’s appeared. There was not CO A., 2002: Effect of organic produc- observed any infl uence on overall cho- tion system on broiler carcass and meat lesterol level. Wójcik et al. (2011) and quality. Meat Sci. 60, 219–225. CZECH A., OGNIK K., GRELA E.R., 2013: Czech et al. (2012) proved that a longer Effi cacy of a mixture of synthetic antiox- before-slaughter transport has a signifi - idant and proteinxanthophyll alfalfa con- cant infl uence on overall cholesterol con- centrate in Turkey hens feeding. Arch. centration in blood serum of chickens. Gefl ugelkd. 76 (2), 105–112. DEHKORDI S.H., MONGHAM A.Z., MAGHSOUDI N., AALI E., GERAMI CONCLUSIONS R., DEHSADEGHI E., 2010: The effects of fresh garlic on serum concentration of The infl uence of system rearing (with out- total cholesterol, total triglyceride and adipose tissues of broilers. Comp. Clin. door access) on the fi nal body weight of Pathol. 19, 363–365. chickens was observed. In the conducted ELAGIB H.A.A., MOHAMED H.E., ELZU- research no statistically signifi cant in- BEIR E.A., 2008: The effects of methio- fl uence of sex and system of breeding nine and energy levels on hematological on cholesterol and its fractions level in and biochemical indices in broiler under hot blood serum of crossbreed chickens was climate. Res. J. Poult. Sci. 2 (1), 15–20. observed. FANATICO A.C., PILLAI P.B., CA- VITT L.C., OWENS C.M., EMMERT J.L., 2005: Evaluation of slower-growing broiler genotypes grown with and without outdoor access: growth performance and carcass yield. Poult. Sci. 84, 1321–1327. 224 A. Wnuk et al.

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(BW) niemającej dostępu do wybiegu oraz gru- MS. received in November 2013 py doświadczalnej (W), korzystającej z trawia- stych wybiegów począwszy od 4. tygodnia życia. W surowicy oznaczono stężenie cholesterolu cał- kowitego, lipoprotein o dużej gęstości (HDL), Authors’ address: triacylogriceloli (TG). Zawartość lipoprotein Agnieszka Wnuk o małej gęstości (LDL) wyliczono na podstawie Wydział Nauk o Zwierzętach SGGW wzoru Friedewald et al. 1972. Nie wykazano sta- Katedra Szczegółowej Hodowli Zwierząt tystycznie istotnego wpływu płci oraz systemu ul. Ciszewskiego 8, 02-786 Warszawa utrzymania na zawartość cholesterolu i jego frak- Poland cji w surowicy krwi kurcząt mieszańca. e-mail: [email protected]

Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 227–236 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Studies on the potential use of entomopathogenic nematodes for biological control of animals in the stables IZABELA WOJCIECHOWSKA1, MARTA KAMIONEK1, BOLESŁAW MORYTZ2 1Department of Animal Environment Biology, Warsaw University of Life Science – SGGW 2Institute of Industrial Organic Chemistry in Warsaw

Abstract: Studies on the potential use of ento- The appearance of resistant pest races mopathogenic nematodes for biological control is an unfavourable effect of insecticide of animals in the stables. The study was aimed at application. Insects often develop the selecting species and strains of entomopathogenic nematodes to be used in practical control of the resistance to more than one type of in- housefl y in stables, which should provide welfare secticides. Overuse of insecticides and of bred animals. Test insect, the housefl y, and en- application of substances from the same tomopathogenic nematodes of the family Stein- chemical group decreases their effective- ernematidae and Heterorhabditidae were used in ness from year to year. A disadvantage experiments. Laboratory strains of nematodes and those commercially available in Poland and in Eu- of chemical means for insect control is rope were used in performed tests. Larvae, pupae their broad spectrum of activity. They and imagines of M. domestica were cultivated in kill both harmful and benefi cial organ- the Institute of Organic Industry in Warsaw. Four isms (Malinowski 2003). groups were created for each nematode species. Integrated method does not com- Not all nematode species and strains were equally pathogenic to housefl ies. pletely eliminate synthetic preparations. The term IPM was fi rst introduced in Key words: biopreparations, entomopathogenic 1959 by Stern. Chemical means should nematodes, housefl y, animal welfare be applied only if the need arises (Stern et al. 1959). INTRODUCTION The housefl y is a synanthropic insect which makes problems in farm houses. Excessive agricultural chemicalization The invasion of this insect stresses ani- leads to the degradation of the natural mals and causes economic losses. The environment. Insecticides accumulate in housefl y is a vector of viruses, bacteria, plants, soil and ground waters and their protozoans and fungi. Up to 6 million mi- remains are ingested by a consumer. croorganisms may be found on its body. They may pose a risk to human and ani- Its most intensive invasion takes place in mal life and health. Consequences after the summer month (Ignatowicz 2000). the contact with pesticides include the Preventing invasions of this insect is disturbances of nervous, respiratory and troublesome, particularly when the inte- alimentary systems, skin wounds and rior of farm houses is permanently open even death. to the external habitat. Therefore, breed- ers are advised to use several methods 228 I. Wojciechowska, M. Kamionek, B. Morytz of housefl y control in order to obtain ex- Methods pected effects. Housefl y control may be Laboratory tests performed in several ways using physi- Eleven species and strains of nematodes, cal, chemical and biological methods including fi ve commercial bioprepara- (Wojciechowska and Kamionek 2012). tions were used in experiments on the In this study entomopathogenic nema- housefl y control in stables. Tests were todes were used for this purpose. made on Petri dishes 10 cm in diameter Nematodes are used in the biological lined with a double layer of fi lter paper. control of the populations of harmful in- Twenty housefl y larvae were placed onto sects. Control group consisted of house- each dish and 5, 10, 20 or 50 invasive fl y larvae with distilled water. Mortality larvae of appropriate nematode spe- and the extensity of infection of housefl y cies were instilled onto dish. Tests were larvae by various species and strains of made in three repetitions. Mortality was Steinernematidae and Heterorhabdtidae checked every 24 h for 5 days. The con- nematodes were determined. trol consisted of larvae with 100 ml of water added instead of nematode sus- MATERIAL AND METHODS pension. After infection dead and live in- sects were counted and the former were Study material dissected to check whether nematodes were the cause of their death (Figures 1 Insects (M. domestica) and 2). Studies were carried out on the housefl y M. domestica, a species common in farm houses (stables). Experimental insects were cultivated in the Institute of Organ- ic Industry in Warsaw. Patented substra- tum prepared in the Institute was used to breed housefl ies. Entomopathogenic nematodes (EPNs) Steinernema affi nis, S. carpocapsae, S. feltiae, Heterorhabditis megidis: Is, Ic WNZ /2009, biopreparations based on nematodes S. feltiae: Owinema (Ovi- plant), Entonem (Koppert), Nemaplus FIGURE 1. Infected larvae of the housefl y (E-nema) and biopreparations based on (Wojciechowska 2013) nematodes Heterorhabditis bacteriopho- ra: Larvanem (Koppert) Nematop (E- STATISTICAL ANALYSIS nema) were used in this experiment. In the development of the results of the experiments used a using multivariate analysis of variance (ANOVA). It al- lows the inference the signifi cance of Studies on the potential use of entomopathogenic nematodes for biological... 229

nifi cant differences in average (p < 0.05). Calculations made using the computer program Statgraphic.

RESULTS AND DISCUSSION

The highest mortality in performed tests was noted with the use of S. feltiae (Owinema, Nemaplus) and S. carpo- capsae. The lowest mortality was noted FIGURE 2. Nematodes visible in a dead larva after application of S. riobrave, H. bac- (Wojciechowska 2013) teriophora (Larvanem and Nematop) and H. megidis Ic and Is. Most extensive the impact factor of the test to the test infection was recorded for S. carpocap- parameter (extensiveness of infestation, sae, S. feltiae (Owinema) and S. feltiae the number of outlets). When we found (Nemaplus). The lowest extensity was a signifi cant effect of a particular factor characteristic for S. affi ne, S. interme- on a particular trait, with LSD test was dia, S. riobrave and H. megoidis Ic and evaluated, between which there are sig- Is (Tables 1 and 2, Fig. 3). The reasons

TABLE 1. Mortality and the extensity of infection of four-day larvae of M. domestica after application of various species and strains of Steinernematidae (%). Initial dose of nematodes for insect – 50

Mortality Extensity of infection Nematode species (%) %SD S. affi ne 57b 30bc 3.67 S. intermedia 65c 37c 3.82 S. carpocapsae 80e 70ef 6.18 S. riobrave 55b 42bd 2.53 S. feltiae (Owinema) 90g 75g 6.58 S. feltiae (Nemaplus) 85f 73fh 6.26 S. feltiae (Entonem) 73d 57de 4.15

Different letters in columns denote signifi cant differences at p < 0.05.

TABLE 2. Mortality and the extensity of infection of four-day larvae of M. domestica after application of various species and strains of Heterorhabditidae (%). Initial dose of nematodes for insect – 50 Nematode species Mortality (%) Extensity of infection (%) SD H. megidis Ic 55b 24b 2.53 H. megidis Is 47a 21ab 1.17 H. bacteriophora (Nematop) 44a 18a 1.13 H. bacteriophora (Larvanem) 45a 15a 1.16 Different letters in columns denote signifi cant differences at p < 0.05. 230 I. Wojciechowska, M. Kamionek, B. Morytz

[%] 100 90 80 70 60 Mortality 50 Extensity of infection 40 30 20 10 0 ) e a ) c s di I I em ffin e ave is is n a br ema) nem) id id a in to g g matop) rv S. e e a (L S. rio (Ow (N (Nemaplus ra S. interm ae e H. m H. me o h S. carpocapsae lti ltia p e io . f r S. fe S. feltiae (En te S c a H. b H. bacteriophora FIGURE 3. Mortality and the extensity of infection of four-day larvae of Musca domestica of a higher extensity of infection in older The highest mortality was found in larvae should be sought in the anatomy the case of S. feltiae (Owinema, Nema- of natural openings, through which inva- plus) and S. carpocapsae, the lowest was sive larvae penetrate their body (Brzeski obtained after application of S. riobrave, and Sandner 1974). H. bacteriophora (Larvanem and The highest mortality was noted af- Nematop) and H. megidis. Mortality and ter application of S. feltiae (Owinema, the extensity of infection were signifi - Nemaplus) and S. carpocapsae, the low- cantly lower in pupae than in housefl y est – with S. riobrave, H. bacteriophora larvae. The former are immobile, which (Larvanem and Nematop) and H. megidis was probably the reason of their low Ic and Is (Tables 3 and 4, Fig. 4). mortality and extensity of infection (Ta- bles 5 and 6, Fig. 5).

TABLE 3. Mortality and the extensity of infection of two-day larvae of M. domestica after application of various species and strains of Steinernematidae (%). Initial dose of nematodes for insect – 50 Extensity of infection Nematode species Mortality (%) SD (%) S. affi ne 47b 29b 2.67 S. intermedia 39c 25c 2.82 S. carpocapsae 55e 45e 4.18 S. riobrave 45b 24b 2.53 S. feltiae (Owinema) 65f 53f 4.58 S. feltiae (Nemaplus) 60d 49de 4.26 S. feltiae (Entonem) 59c 40cd 3.15 Different letters in columns denote signifi cant differences at p < 0.05. Studies on the potential use of entomopathogenic nematodes for biological... 231

TABLE 4. Mortality and the extensity of infection of two-day larvae of M. domestica after application of various species and strains of Heterorhabditidae (%). Initial dose of nematodes for insect – 50 Nematode species Mortality (%) Extensity of infection (%) SD H. megidis Ic 20b 15b 2.53 H. megidis Is 16a 12a 1.17 H. bacteriophora (Nematop) 12a 10ab 1.13 H. bacteriophora 10a 7a 1.16 (Larvanem) Different letters in columns denote signifi cant differences at p < 0.05.

[%] 70 60 50 Mortality 40 Extensity of infection 30 20 10 0

e ) ) ) ) v s m Ic Is p ffine a a ne obr to mato S. ocapsae ri egidis p . Ne arvanem S (En m megidis ( L (Nemaplue . . a S. intermedia. car H H ora ( S ltia h fe iophor riop S. feltiae (Owinema) S. S. feltiae ter te c c a a . b H H. b FIGURE 4. Mortality and the extensity of infection (%) of two-day larvae of M. domestica

TABLE 5. Mortality and the extensity of infection of M. domestica pupae after application of various species and strains of Steinernematidae (%). Initial dose of nematodes for insect – 50 Nematode species Mortality (%) Extensity of infection (%) SD S. affi ne 25b 3ab 1.01 S. intermedia 37c 4bc 1.08 S. carpocapsae 45d 13de 1.16 S. riobrave 30c 9c 1.14 S. feltiae (Owinema) 60e 15e 2.63 S. feltiae (Nemaplus) 55e 12de 2.53 S. feltiae (Entonem) 48d 10d 1.17 Different letters in columns denote signifi cant differences at p < 0.05. 232 I. Wojciechowska, M. Kamionek, B. Morytz

TABLE 6. Mortality and the extensity of infection of M. domestica pupae after application of various species and strains of Heterorhabditidae (%). Initial dose of nematodes for insect – 50 Nematode species Mortality (%) Extensity of infection (%) SD H. megidis Ic 20b 4b 1.01 H. megidis Is 17a 3a 0.95 H. bacteriophora (Nematop) 14a 3a 0.95 H. bacteriophora (Larvanem) 11a 2a 0.95 Different letters in columns denote signifi cant differences at p < 0.05.

[%] 70 60 50 Mortality 40 Extensity of infection 30 20 10 0

e e e ) e a ve s Ic ffine s a ltia ma) ltia lu ia p e e e p elt a f in f f S. S. w S. S. egidis egidis Is arvanem) rpoca O (Entonem)m m ( L S. riobr ( (Nema (Nematop) S. intermediaca H. H. ra S. o h p riophora e ct a

. bacterioH. b H FIGURE 5. Mortality and the extensity of infection of M. domestica pupae

The highest mortality in performed of the families Sciaridae and Phoridae tests was demonstrated after application in mushroom-growing cellars. The high- of S. feltiae (Owinema and Nemaplus) est extensity of infection of Lycoriella and S. carpocapsae and the lowest with solani Winnertz was found after applica- the use of S. riobrave, H. bacteriophora tion of S. affi nis, S. feltiae (Nemaplus) (Larvanem and Nematop) and H. me- and S. feltiae (Owinema). In the case of gidis (Tables 7 and 8, Fig. 6). Megaselia halterata Wood the highest Nematodes of the families Stein- extensity was obtained with the use of ernematidae and Heterorhabditidae are S. affi nis, S. feltiae (Nemaplus) and effective in controlling many species of S. feltiae (Owinema) (Sznyk-Basałyga harmful insects. Studies have long been 2002). performed in many countries on nema- Commercial preparations based on todes as a means used in biological meth- entomopathogenic nematodes are pro- ods for pest control. They found practi- duced in many countries. In Poland the cal application in controlling dipterans production of biopreparation Owinema Studies on the potential use of entomopathogenic nematodes for biological... 233

TABLE 7. Mortality and the extensity of infection of M. domestica imagines after application of vari- ous species and strains of Steinernematidae (%). Initial dose of nematodes for insect – 50 Nematode species Mortality (%) Extensity of infection (%) SD S. affi ne 8b 0ab 0 S. intermedia 9c 0ac 0 S. carpocapsae 12d 1bd 0.55 S. riobrave 4a 0a 0 S. feltiae (Owinema) 13d 2bd 0.75 S. feltiae (Nemaplus) 11d 2bd 0.71 S. feltiae (Entonem) 10c 1bc 0.48 Different letters in columns denote signifi cant differences at p < 0.05.

TABLE 8. Mortality and the extensity of infection of M. domestica imagines after application of vari- ous species and strains of Heterorhabditidae (%). Initial dose of nematodes for insect – 50 Nematode species Mortality (%) Extensity of infection (%) SD H. megidis Ic 7b 0ab 0 H. megidis Is 5a 0a 0 H. bacteriophora (Nematop) 8b 0ab 0 H. bacteriophora (Larvanem) 3a 0a 0 Different letters in columns denote signifi cant differences at p < 0.05.

[%] 14 12 10 Mortality 8 Extensity of infection 6 4 2 0

) ) ia e a) s Ic Is d lu psa p is is nem) me id id a r inem matop rv S. affine te oca w ma eg e in S. riobraveO (N . ( (Ne (Entonem) (La carp e ra a S . e H. meg H. m o S ltia ltiae h e ltia p f fe S. fe riophor S. S. acterio b . . bacte H H FIGURE 4. Mortality and the extensity of infection of M. domestica imagines 234 I. Wojciechowska, M. Kamionek, B. Morytz is based on S. feltiae. Biopreparations Pezowicz (2005) in her studies on are considered safe in controlling pests the control of the lesser mealworm (Al- in agriculture and animal breeding (Poi- phitobius diaperinus Panzer) showed nar 1979, Pezowicz 2005). that nematodes infected both young and Unruh and Lacey (2001) used S. car- older larvae. The extensity of infection pocapsae to control a fruit fl y (Carpo- of older larvae was, however, signifi - capsa pomonella L.). Tomalak (2004) cantly higher. Higher extensity of infec- used H. megidis and S. feltiae against tion of older larvae is an effect of natural tree pests: the alder leaf beetle (Age- openings, of the size of stigmas, through lastica alni L.) and the oak fl ea beetle which the invasive larvae penetrate in- (Haltica quercetorum Foudras). This sect’s body (Koppenhöfer et al. 2000). study showed that prepupae and pu- My studies revealed the same regularity pae are sensitive to nematode infection. – nematodes infected two-day larvae to and that H. megidis was more effective a very small extent. According to Pezo- than S. feltiae. The western fl ower thrips wicz (2005), nematodes penetrated all (Frankliniella occidentalis, Pergande) growth stages of the insect. may also be controlled by nematodes Dipteran larvae were controlled more (Borgemeister et al. 2002). In this case effectively by nematodes of the fam- Heterorhabditis spp. was more patho- ily Steinernematidae than by those from genic than Steinernema spp. Bednarek Heterorhabditidae. The highest extensity et al. (2002) controlled grubs of a cock- of infection in laboratory tests was ob- chafer from the family Melolonthinae in tained after application of two nematode forests. Also the pea leaf weevil (Sitona species S. carpocapsae and S. feltiae lineatus L.) is sensitive to nematodes (Ovinema and Nemaplus). S. feltiae is (Jaworska and Ropek 1998). Mortality also most effective in the control of other of these insects was 70–80%. dipterans of the family Sciaridae in con- Insect species of different orders trast with S. carpocapsae which showed show differentiated susceptibility to much lower pathogenicity (Sznyk- nematodes. The easiest infected are the -Basałyga 2002). My studies do not con- butterfl y caterpillars. Beetles are resist- fi rm this observation since the extensity ant and the least mortality was found in of infection was 70%. dipteran populations (Dutky 1959). S. affi nis oraz H. megidis exerted the M. domestica is an undesirable insect weakest effect on M. domestica. in stables since it harasses animals and Performed studies demonstrated that carries diseases and parasites. Poland as S. feltiae is the most effective nematode a member of the EU should obey zoohy- species in controlling the number of the gienic standards and norms. This in- housefl y. Owinema and Nemaplus are the cludes also the control of housefl ies. biopreparations adapted to be applied in In presented study, the tested nema- practise. It is unfortunate that in Poland tode species and strains were more effec- abandoned production Owinema. tive for four-day larvae of M. domestica (55–90% mortality) than for small, two- -day larvae (39–65% mortality). Studies on the potential use of entomopathogenic nematodes for biological... 235

CONCLUSIONS KOPPENHÖFER A.M., FUZY E.M., BROWN I., KAYA H.K., GAUGLER R., 1. Most sensitive to nematodes were the 2000: Biological control agents for white larvae of M. domestica. Four-day lar- grups (Coleoptera: Scarabaeidae) in an- ticipate of the establishment of the Japa- vae were more sensitive than two-day nese beetle in California. J. Econ. Ento- ones. mol. 93 (1), 71–81. 2. Pupae and imagines were more resis- MALINOWSKI H., 2003: Odporność owa- tant to nematode infection. dów na insektycydy. Wydawnictwo Wieś 3. Larvae of the housefl y were more Jutra. Warszawa. effectively killed by nematodes of PEZOWICZ E., 2005: Nicienie owadobójcze the family Steinernematidae than by jako czynnik zmniejszający liczebność those of the family Heterorhabditi- populacji pleśniakowca lśniącego (Alphi- tobius diaperinus Panzer) w brojlerniach. dae. Rozprawy Naukowe i Monografi e. Wy- 4. The housefl y was most effectively dawnictwo SGGW, Warszawa. killed by nematodes S. feltiae from POINAR G.O., 1979: Nematode for biologi- biopreparations Owinema and Nema- cal control of insects. CRC Press. Hic., plus and by S. carpocapsae. Boca Raton, Florida. STERN V.M., SMITH R.F., Van Den BOSH R., HAGEN K.S., 1959: The integration REFERENCES of chemical and biological control of the spotted alfalfa aphid: the integrated con- BEDNAREK A., KAMIONEK M., PEZO- trol concept. Hilgardia 29 (2), 81–101. WICZ E., 2002: Zwalczanie pędraków SZNYK-BASAŁYGA A., 2002: Porówna- Melolonthinae jako przykład integro- nie biologicznej i integrowanej metody wanej metody zwalczania szkodników zwalczania szkodników upraw piecza- w lasach. Przegl. Hod. Zeszyty Naukowe rek Lycoriella solani (Dietera: Sciaridae) PTZ 60, 125–140. i Megaselia halterata (Dietera: Phoridae). BORGEMEISTER C., EBSSA L., PRE- Rozprawa doktorska. SGGW, Warszawa. MAHANDRA D., BERNDT O., 2002: TOMALAK M., 2004: Infectivity of ento- Biological control of soil-dwelling stages mopathogenic nematodes to soil dwell- of Western Flower Thrips Frankliniella ing developmental stages of the tree leaf occidentalis (Pergande) (Thysanophtera: beetles Altica quercetorum and Agelas- Tripidae) by entomopathogenic nema- tica alni. Entomol. Exper. Applic. 110, todes and Hypoaspis spp. (Acari: Laelpi- 125. dae). IOBS wprs Bull. 25, 29–32. UNRUH T.R., LACEY L.A., 2001. Control BRZESKI M.W., SANDNER H., 1974: Za- of codling moth, Cydia pomonella (Lepi- rys nematologii. PWN, Warszawa. doptera: Tortricidae), with Steinernema DUTKY S.R. 1959: Insect microbiology. carpocapsae: Effects of supplemental Adv. Appl. Microbiol. 1, 175–200. wetting and pupation site on infection IGNATOWICZ S., 2000: Wstrętne muchy. rate. Biol. Contr. 20, 48–56. Top Agrar. WOJCIECHOWSKA I., KAMIONEK M., JAWORSKA M., ROPEK D., 1998: Fungi 2012: Mała mucha – duży problem. Far- and nematodes interaction against S. lin- mer 7, 114–117. eatus. IOBC wprs Bull. 21, 113–116. 236 I. Wojciechowska, M. Kamionek, B. Morytz

Streszczenie: Biologiczne metody zwalczania MS. received in November 2013 muchy domowej w stajniach. Doświadczenia miały na celu wybór gatunków i szczepów ni- cieni owadobójczych, które zostaną wykorzysta- ne w praktycznym zwalczaniu muchy domowej w stajni, co powinno zapewnić dobrostan zwie- rzętom hodowlanym. Owad testowy – mucha Authors’ addresses: Izabela Wojciechowska, Marta Kamionek i owadobójcze nicienie z rodziny Steinernema- Wydział Nauk o Zwierzętach SGGW tidae i Heterorhabditidae zostały wykorzystane Katedra Biologii Środowiska Zwierząt w eksperymentach. Laboratoryjne szczepy ni- ul. Ciszewskiego 8, 02-787 Warszawa cieni i w Polsce i w Europie tych dostępnych na Poland rynku zostały wykorzystane w przeprowadzo- e-mail: [email protected] nych testach. Larwy, poczwarki i owady dorosłe [email protected] M. domestica były hodowane w Instytucie Prze- mysłu Organicznego w Warszawie. Cztery grupy Bolesław Morytz zostały stworzone dla każdego gatunku nicieni. Instytut Przemysłu Organicznego w Warszawie Nie wszystkie gatunki nicieni i szczepy były rów- ul. Annopol 6, 03-236 Warszawa nie zjadliwe dla much. Poland Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 237–242 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Genotype – factor infl uencing performance of chicken production ŻANETA ZDANOWSKA-SĄSIADEK, MONIKA MICHALCZUK, JULIA RIEDEL, MONIKA ŁUKASIEWICZ, KRZYSZTOF DAMAZIAK Department of Animal Breeding and Production, Warsaw University of Life Science – SGGW

Abstract: Genotype – factor infl uencing perfor- alternative methods consumers associate mance of chicken production. The aim of this also with the environment protection study was to establish the effect of the genotype (Bogosavljević-Bošković et al. 2012). on performance of chickens. The experimental material included 1,320 slow-growing chickens Latter-Dubois (2000) defi ned the rea- with two genotypes Hubbard JA 957 – available sons, why consumers choose alternative on the Polish market hybrid with a declared slow poultry housing systems as major con- growth and non-commercial hybrid C×GP, for tributors to better taste, higher nutrition- creation of which a light native breed Greenleg al value, higher welfare of the birds and Partridge hens were used. Chickens were main- tained up to 63 days of age. During the experiment greater safety of such food. body weight, feed intake and health of the birds In Poland, poultry meat from birds were under control. Based on the observations the kept in unconventional systems consti- growth rate and the feed conversion ratio (FCR) tutes a small part of the market and is were determined. It was found, that chickens available mainly as organic product. Ac- C×GP were characterized by lower body weight, slower rate of growth and higher feed conversion cording to the regulations of the Europe- ratio compared with the chickens Hubbard JA an Union (EC 1804/99) for organic pro- 957, but chickens C×GP had a higher health. duction, it is recommended to choose the right genetic material for such a system of farming. Currently in our country, the INTRODUCTION organic production is based on the fast- growing chickens, commercial hybrids Many people choose poultry as main and used in typical, intensive production. preferred brand of meat. However con- The only commercially available sumers frequently declare their willing- material of slow-growing chickens is ness to purchase the products obtained Hubbard JA 957. Using of birds of slow from chickens with less intensive farm- growth, better adapting to changing envi- ing, which provides a higher animal wel- ronmental conditions, showing a higher fare (Pavlovski et al. 2009). This trend health status is particularly important due is particularly evident among Western to the introduction of a number of limita- European and the United States socie- tions in respect of organic production. In ties, where consumers are interested in addition, rearing chickens in the alterna- the welfare standards and have a con- tive system should be reared longer and cern about the way the animals are kept be willing to prey (Branciari et al. 2009). in intensive poultry production (Vanho- Moreover the birds in non-conventional nacker et al. 2009). Raising chickens in system should reach slaughter weight in 238 Ż. Zdanowska-Sąsiadek et al. a period no longer than required for or- Hubbard JA 957 (660 birds in six repli- ganic production, which is 81st day. cation containing 110 birds in pen) and The aim of this study was to evalu- non-commercial hybrid C×GP (660 birds ate the results of production of hybrids in six replication containing 110 birds in with different genotypes in terms of their pen). Day-old chicks have been marked usefulness in non-conventional farming by chicken stamps and maintained up to systems. 63 days of age in compliance with the Regulation of the Minister of Agriculture MATERIAL AND METHODS and Rural Development dated 15 Febru- ary, 2010 on the requirements and man- The experiment was conducted in 2011 ner of maintaining livestock species, for in spring (March–May) at the experi- which protection standards are provisions mental station of the Warsaw University of the European Union. Stock density in 2 of Life Sciences (RZD Wilanów-Obory). the poultry house was 11 birds per m . Experimental procedures were approved In the experiment, 4-stage feeding was by the Ethical Commission (approval applied: the starter (0–14 days), grower no. 27/2009 of the 16 April 2009). The I (15–35 days), grower II (36–56 days) experimental material consisted of 1,320 and fi nisher (57–63 days). The compo- slow-growing chickens of two genotypes sition of components and the nutritional value of compound shown in Table 1. TABLE 1. Nutritive value of basal diet in broiler feeding Specifi cation Starter Grower I Grower II Finisher CONTENT (%) Maize 10 11.4 10 10 Wheat 53 55 59.6 60.8 Soybean meal 30.6 27.4 23.2 21.6 Limestone Ca39 1.165 1.175 1.085 0.945 Sodium bicarbonate 0.2 0.14 0.14 0.16 NaCl 0.24 0.28 0.28 0.26 Stimulator 0.01 0.01 0.01 0.01 Dicalcium phophate 1.18 0.78 0.7 0.64 Soybean oil 2.1 2.4 3.6 4.4 Methionine 0.48 0.42 0.36 0.28 Lysine 0.36 0.34 0.36 0.28 Threonine 0.14 0.13 0.14 0.1 Premix 0.525 0.525 0.525 0.525 NUTRITIVE VALUE EM (MJ) 12.52 12.76 13.20 13.47 Total protein (%) 21.99 20.78 19.26 18.51 Crude fat (%) 3.67 4 5.14 5.92 Crude ash (%) 5.83 5.35 4.96 4.67 Genotype – factor infl uencing performance of chicken production 239

During the experiment, the chickens also evident in the following weeks, were individually weighed at weekly in- chickens Hubbard JA 957 throughout the tervals. Feed intake and health of chick- rearing period had signifi cantly higher ens were also controlled. Based on the body weight compared with the chickens results of observations the growth rate, C×GP (P < 0.001). feed conversion ratio and birds’ mortal- The growth rate of chickens Hub- ity as a percentage of dead and culled to bard JA 957 at the beginning of rearing inserted were determined. was high and amounted to almost 120%. The results were statistically analyzed C×GP chickens had a lower rate of in- by one-way analysis of variance using crease during this period (94%). It should SPSS 21.0 PL for Windows software. be noted that in chickens C×GP growth rate declined steadily without clearly marked periods and a sudden drop in the RESULTS AND DISCUSSION fi nal period of rearing (8 and 9 weeks) was higher than in Hubbard. However, in In the experiment the body weight of chickens Hubbard JA 957, upon a high chickens Hubbard JA 957, and C×GP initial rate, its sudden decline and a very were monitored at weekly intervals. low level in the last week of life were Based on the obtained data growth rate observed. of birds was determined. Figure 1 shows Rachwał (2008) reports that in one the body weight changes between com- week old commercial hybrids growth pound and rate of growth of chickens. rate is about 140%, i.e. much higher than Signifi cant differences in body weight the results obtained in chickens C×GP were already noticed at the insertion, and slightly higher than that observed in Hubbard JA 957 chickens were heavier Hubbard JA 957. Chickens C×GP char- than chicken C×GP (P < 0.001). The dif- acterized by low body weight; the 63 ferences observed at the beginning were day old birds had a weight comparable

FIGURE 1. Body weight (g) and growth rate (%) in chickens depending of the genotype 240 Ż. Zdanowska-Sąsiadek et al. to that achieved by commercial broilers On the paddock practically do not move, at 35 day (Aviagen 2012). It should be spend most of their time lying down or noted, that the main purpose for creating standing. The slow growing birds show hybrid C×GP was not to maximize pro- totally different behavior. They more duction results, but to produce the ma- likely have a run, signifi cantly increase terial adapted to less intensive farming their activity, much less time are lying conditions. standing and eating, and signifi cantly Hubbard JA 957 chickens had signifi - more move and bury. cantly higher body weight, which oscil- Based on the measurements of body lated between 2,900 g. It is worth to note, weight and growth rate a preliminary that in poultry production the value of hypothesis, that chickens C×GP is the 2,000–2,500 g is given as a standard live genetic material better adapting to the weight of chickens for slaughter, then slow-range farming than Hubbard JA a carcass can get the weight mostly de- 957. However, it appears necessary to sired by consumers – about 1,300–1600 g conduct a distinct behavioral research (Fanatico et al. 2005). So Hubbard chicks and to determine the basis of their behav- reached too high body weight at 63 day ior in unconventional rearing system. of age, therefore slaughter of these birds Table 2 shows feed consumption (kg/ should have been carried out appropri- /kg) and mortality (%) in chickens de- ately earlier. If the aim was to determine pending on the genotype. the genotype of birds, that could be used Basing on these results, it is clear, that in alternative housing systems with ac- Hubbard JA 957 chickens use less feed cess to range, it is worth noting that the per 1 kg of body weight gain compared chickens with a slower rate of growth with the chickens C×GP. Such result can better adapt to such conditions. On the be explained by a signifi cantly higher basis of Branciari et al. (2009) it can be body weight of Hubbard chicks at slaugh- concluded, that the fast growing commer- ter. Feed consumption of 2.27 kg/kg (for cial hybrids behave very similarly in in- Hubbard) and 2.84 (for C×GP) obtained tensive production and organic farming. in the experiment was signifi cantly high- Chickens do not have the desire to prey er than that achieved in intensive produc- and do not display the typical behavioral tion. Commercial broilers slaughtered at poultry burying behavior. The birds are the age of 42 days use about 1.7 kg of unwilling to use runs and a large part of feed to gain 1 kg of body weight (Fed- time were staying in livestock buildings. des et al. 2002). However, with increas-

TABLE 2. Feed conversion ratio and mortality in chicken depending on the genotype Specifi cation Hubbard JA 957 C×GP Feed conversion ratio FCR (kg/kg) 2.27 2.84 Energy conversion (MJ/kg) 29.48 36.89 Protein conversion (kg/kg) 0,47 0,56 Mortality (%) 2.72A 0.30B A, B – difference signifi cant at P ≤ 0.01. Genotype – factor infl uencing performance of chicken production 241 ing time of rearing the birds an increase cess to open-air signifi cantly reduces of this parameter should be expected; mortality among them. These results fur- this is due to lowering rate of growth and ther confi rm the necessity of obtaining increasing feed consumption. Castellini slowly growing hybrids in the alternative et al. (2002) maintaining the chickens up housing systems. to 81 days of age received even higher The results obtained in this experi- feed consumption (3.0 kg/kg). ment as well as the ones of other authors Mortality of chickens in the experi- (Castellini et al. 2002, Fanatico et al. ment was generally low and amounted 2008, Branciari et al. 2009) indicate the to 2.72% for Hubbard JA 957 chickens need to adjust the growth rate of birds and 0.36% for C×GP. Particularly low to the chosen farming system. It should mortality for hybrid C×GP can prove the be noted, that due to the production re- high health of these birds, which is espe- sults, which are worse in comparison cially important when raising chickens with commercial broilers (lower body are reared in the conditions ensuring the weight, lower growth rate, higher con- access to open air. sumption of feed) producers of poultry Fanatico et al. (2008) pay special at- kept in the alternative housing systems tention to the genotype in shaping this must compensate for the loss resulting production factor. Slow-growing chick- in a correspondingly higher price for the ens have a lower mortality rate com- poultry product. pared to the fast-growing chickens. This is mainly due to a lower share of diseases associated with the fast pace of growth, REFERENCES among which tibial dyschondroplasia oc- curs. Castellini et al. (2002) found, how- AVIAGEN, 2012 : Broiler performance ever, that the main cause of falls in fast- objectives. http://en.aviagen.com/as- growing chickens is a ascites and sudden sets/Tech_Center/Ross_Broiler/Ross- 308BroilerPerfObj2012R1.pdf. death syndrome, which in slow-growing BOGOSAVLJEVIĆ-BOŠKOVIĆ S., RA- birds are much more rarer. KONJAC S., DOSKOVIĆ V., PETRO- Birds’ genotype may be particularly VIĆ M.D., 2012: Broiler rearing systems: important in shaping mortality in free a review of major fattening results and range or organic rearing conditions. meat quality traits. World’s Poult. Sci. J. Branciari et al. (2009) observed, that 68: 217–228. fast growing chickens characterized by BRANCIARI R., MUGNAI C., MAM- MOLI R., MIRAGLIA D., RANUCCI D., relatively low mortality when the rearing Dal BOSCO A., CASTELLINI C., 2009: is carried out in a closed breeding con- Effect of genotype and rearing system on ditions. Mortality in a fl ock of broilers chicken behavior and muscle fi ber char- considerably increase, when the birds acteristics. J. Anim. Sci. 87: 4109–4117. use the free run. However, exactly the CASTELLINI C., MUGNAI C., DAL BOS- opposite situation can be observed in CO A., 2002: Effect of organic produc- medium and slow-growing poultry, ac- tion system on broiler carcass and meat quality. Meat Sci. 60: 219–225. 242 Ż. Zdanowska-Sąsiadek et al.

EC 1804/99: Council Regulation of 19 July VANHONACKER F., VERBEKE W., 1999 supplementing. Regulation (EEC) Van POUCKE E., BUIJS S., TUYT- No 2092/91 on organic production of ag- TENS F.A.M., 2009: Societal concern ricultural products and indications refer- related to stocking density, pen size and ring thereto on agricultural products and group size in farm animal production. foodstuffs to include livestock produc- Livest. Sci. 123: 16–22. tion. FANATICO A.C., PILLAI P.B., CA- Streszczenie: Genotyp – czynnik warunkujący VITT L.C., OWENS C.M., EMMERT J.L., wyniki produkcyjne w stadach kurcząt rzeźnych. 2005: Evaluation of slower-growing Celem badania było określenie wpływu genotypu broiler genotypes grown with and without na wyniki produkcyjne kurcząt. Badania przepro- outdoor access: growth performance and wadzono na 1320 kurczętach o dwóch genotypach carcass yield. Poult. Sci. 84: 1321–1327. Hubbard JA 957 – dostępnym na rynku polskim FANATICO A.C., PILLAI P.B., HES- mieszaniec o deklarowanym wolniejszym tempie TER P.Y., FALCONE C., MENCH J.A., wzrostu oraz C×Zk – mieszaniec, do którego wy- OWENS C.M., EMMERT J.L., 2008: tworzenia wykorzystano kury rasy lekkiej zielono- Performance, livability, and carcass yield nóżki kuropatwianej. Kurczęta utrzymywano do of slow- and fast-growing chicken geno- 63. dnia życia. W czasie odchowu kontrolowano types fed low-nutrient or standard diets masę ciała, spożycie paszy i zdrowotność ptaków. and raised indoors or with outdoor ac- Na podstawie prowadzonych obserwacji określo- cess. Poult. Sci. 87: 1012–1021. no tempo wzrostu oraz wskaźnik zużycia paszy. FEDDES J.J.R., EMMANUEL E.J., ZUID- Stwierdzono, że kurczęta C×Zk charakteryzowała HOF M.J., 2002: Broiler performance, mniejsza masa ciała w porównaniu z kurczętami Hubbard JA 957 oraz wolniejsze tempo wzrostu. bodyweight variance, feed and water in- Mniejsze wykorzystanie paszy na przyrost masy take, and carcass quality at different stock- ciała stwierdzono dla kurcząt Hubbard JA 957. ing densities. Poult. Sci. 81: 774–779. Kurczęta C×Zk charakteryzowała natomiast wyż- LATTER-DOBOIS J., 2000: Poulets fer- sza zdrowotność. miers: leurs qualite’s nutritionnelle et organoleptiques et la perception du consommateur. Univ. Laval, Quebec. MS. received in November 2013 PAVLOVSKI Z., ŠKRBIĆ Z., LUKIĆ M., PETRIČEVIĆ V., TRENKOVSKI S., 2009: The effect of genotype and hous- ing system on production results of fat- tening chickens. Biotech. Anim. Hus. 25: 221–229. RACHWAŁ A., 2008: Wczesne ograniczenie spożycia paszy a wzrost kurcząt brojle- rów i skład ich tuszek. Hodowca Drobiu 1: 37–42. Authors’ address: Rozporządzenia Ministra Rolnictwa i Roz- Monika Michalczuk woju Wsi z dn. 15 lutego 2010 w sprawie Wydział Nauk o Zwierzętach SGGW wymagań i sposobu postępowania przy Katedra Szczegółowej Hodowli Zwierząt utrzymaniu gatunków zwierząt gospo- Zakład Hodowli Drobiu darskich, dla których normy ochrony zo- ul. Ciszewskiego 8, 02-786 Warszawa stały określone w przepisach Unii Euro- Poland pejskiej (Dz.U. 2010 nr 56, poz. 344). e-mail: [email protected] Annals of Warsaw University of Life Sciences – SGGW Animal Science No 52, 2013: 243–252 (Ann. Warsaw Univ. of Life Sci. – SGGW, Anim. Sci. 52, 2013)

Comparative behaviour analysis of some colubrids with reference to suitability of captive bred snakes for reintroduction to natural habitat ANDRZEJ ŻYCZYŃSKI, ZUZANNA NOWAK Department of Genetics and Animal Breeding, Warsaw University of Life Sciences – SGGW

Abstract: Comparative behaviour analysis of INTRODUCTION some colubrids with reference to suitability of captive bred snakes for reintroduction to natural Herpetofauna belongs to that most vul- habitat. A sample of newly hatched American colubrids, divided according to their level of nerable group of animals and those most domestication, understood herewith as the gen- endangered by anthropogenic pressure eration history in captivity, was tested in an open to their natural habitat. There have been fi eld test (OFT) for degree of activity and confi - many attempts of in situ conservation dence in open terrain and in confrontation with in- and some have proved quite effective dividuals of such species as Pantherophis guttatus and Lampropeltis getula splendida as well as L. g. – for more information on this, see the californiae, in order to compare their antagonistic Materials of VI World Congress of Her- and feeding behaviour reactions. The cluster com- petology – Manaus 2008. Despite these parison of defence reactions revealed no effect of efforts, more and more trials are under- domestication (many generation in captivity) in taken for ex situ strategies. Traditionally the face of potential danger. Even albino phase Kingsnakes, considered to be the form of this spe- this activity is the domain of universities cies most affected by captivity (altered genome), or ZOOs, which maintain certain species maintained their natural feeding response. The as gene banks – potential sources of ma- degree of activity in OFT conditions was seen to terial for reintroduction. In the case of decrease with the snakes’ age. Thus it was demon- snakes, there are quite a few examples strated that captive specimens bred in terrariums of attempts in this fi eld. During the years may be considered a gene bank for ex situ conser- vation strategy. However it is recommended that 1980–1986, some 34 specimens of cap- specimens as young as possible should be used tive bred Rocky Python Python sebae in reintroduction to natural habitat, as these will were released into the Great Fish River respond most faithfully to the pressure of natural Nature Reserve (Eastern Cape), but there selection, based on their behavioural variation not was no follow-up (Alexander and Marais supressed by apathy caused by captivity. 2007, Mattison 2013 – personal commu- Key words: Lampropeltis, Pantherophis, antago- nication). In the USA, fi ve AZA facilities nistic and feeding reactions, captive breeding, have created a consortium “Conservation ex situ conservation 244 A. Życzyński, Z. Nowak

Centre for Species Survival” undertak- though as early as 1975, John Coborn ing preparatory work for reintroduction organized the symposium “Conservation programs for various taxons, including and Captive Reptiles and Amphibians”, snakes. The species of choice are to be where the case was made by many her- the San Francisco garter Thamno- petologists for and against captivity as an phis sirtalis tetrataenia and the Louisi- aid to the preservation of wild species, ana Pine Snake Pituophis ruthveni (Mat- there is still much work to be done in tison 1995, Conway 2011, Mattison 2013 this fi eld. In any case, it must be admit- – personal communication). Godwin ted that the level of knowledge of her- et al. in 2008 presented a comprehen- petofauna husbandry is constantly grow- sive report on the captive propagation ing and additionally, the pure breeding of the critically endangered Eastern In- strategy according to locality of origin digo Snake Drymarchon couperi, which has become the ethical rule among the included suggestions concerning other most advanced keepers. As a result, in snake species. An initial scenario was many cases the numbers of certain spe- developed for supplementing the local cies in captivity exceed those ever to be population of Grey Banded Kingsnake seen in the wild and these can be treated Lampropeltis alterna by captive animals as pure gene banks. Their active popula- (Spanowicz and Życzyński 2002). Simi- tion, with a quite large effective number lar efforts were undertaken by Łódź Uni- (Ne) is much greater than could be as- versity, in cooperation with Łódź ZOO, sured by offi cial institutions limited by for a domestic (Polish) species – Smooth state budget conditions. But if we are to Snake Coronella austriaca (Zieliński treat captive animals as candidates for re- and Stanisławski 2001, Stanisławski stocking, we must be sure that these fulfi l 2003). All these attempts led to the re- the demands of gene bank purity, as well lease of captive snakes into the wild, but as the demands with regard to sanitarian were not continued after the fi rst trial. status, in order to prevent introduction of Another attempt worth mentioning exotic pathogens to the natural habitat. was the successful reintroduction of the The captive material must also exhibit Antiguan Racer Alsophis antiguae (Dal- the natural degree of fi tness that can be try 1999 and 2006). In this project, the subjected to natural selection. reintroduced snakes were fi eld collected The aim of the present study is a com- on Great Bird Island, tagged and released parative behavioural analysis of captive on small Rabbit Island. On the contrary bred snakes, which can be differentiated the results of reintroduction of Woma according to the level of their domestica- Python Aspidites ramsayi in Arid Recov- tion (understood herewith as the multi- ery Reserve in northern South Australia generation history in terrariums’ life). did not meet the short-term and medi- um-term success criteria mostly due to predator pressure (Moseby et al. 2011). MATERIALS AND METHODS Potential support for such work may The snakes used in this study were born come from private enthusiasts and keep- in Laboratory of Department of Genet- ers. This is a signifi cant new idea. Al- ics and Animal Breeding of WULS Comparative behaviour analysis of colubrids with... 245

– SGGW. They belonged to the genera The snakes were subjected to open Lampropeltis and Pantherophis. Au- fi eld test – OFT (Markowska 1979) scor- thors chose these taxons in view of their ing points for movement activity within popularity among keepers. Compared to the fi eld. Animals were placed within other genera, most of these snakes have a ring 90 × 90 cm, fenced with wooden a quite long generation history in captiv- walls. The surface of the ring consisted ity and thus constitute interesting materi- of squares 30 × 30 cm forming three al for an evaluation of the level of natural rows. The scores for activity were count- behaviour preserved. These may serve ed as follows: snake was getting one as a model for conclusions concerning point when slithered along one square other, potentially truly endangered spe- adjacent to the walls and 3 points for dis- cies, which are currently not stocked in tancing from the ring walls and entering collections. the square in the centre of the fi eld. Af- The material was divided into the fol- ter 5 min of observation the points were lowing groups: added up. 1. Ls × Lc – inter subspecies cross be- Later the snakes were confronted tween Sinaloan Lampro- with a young Corn Snake Pantherophis peltis triangulum sinaloe and Pueblan guttatus – a species preferring rodents as Milk Snake Lampropeltis triangulum the natural diet, and a Desert Kingsnake campbelli – (parents from multigenera- Lampropeltis getula splendida – liv- tional captive breeding) – here – captive ing (as the whole Lampropeltis) (domesticated) type – 10 specimens; on lizards and other snakes as its most 2. Lpp × Lpk – inter subspecies cross be- typical food. The points scored and body tween Arizona Mountain Kingsnake weights of each snake tested were sub- Lampropeltis pyromelana pyromelana jected to one-way ANOVA. When nec- and Chihuahua Mountain Kingsnake essary the F-test was followed by D-test Lampropeltis pyromelana knoblochi to verify the signifi cance of differences – second generation in captivity – in- among the groups. Additional analysis of termediate type – 3 specimens; covariance with body weight as an oper- 3. Ege – Great Plains Rat Snake Pan- and variable was done to check whether therophis emoryi – from fi eld collected the sizes of snakes could affect their con- parents – wild type – 8 specimens; fi dence and thus the results of OFT test. 4. Ege × Egg – interspecies cross be- During confrontations (lasting 5 min tween Great Plain Rat Snake Pan- each) with another snake, the following therophis emoryi (fi eld collected) and reactions were noted on a Yes or No basis: Corn Snake Pantherophis guttatus lack of any reaction, strong tail rattling (amelanistic form – fi xed mutation in (loud and lasting longer than 5 s), weak multigenerational captivity) – inter- tail rattling (lasting less than 5 s), tossing mediate type – 6 specimens; movements, sudden retreat, avoidance 5. Eggalb × Eggan – Pantherophis gut- and attack. tatus – a crossbreed of two fi xed mu- In authors’ experience, the chosen/ tations – snow albinism and anery- /described reactions refl ect well the emo- trystic (black albinism) – “domestic” tional status of the snake during the test. type – 7 specimens. The probabilities of each reaction in two 246 A. Życzyński, Z. Nowak separate trials, for confrontations with only. As the snakes aged, the differences Corn Snake P. guttata and Desert King- between groups disappeared. snake L. g. splendida and combined, The score in the II observation is gen- were subjected to cluster analysis (Nei erally smaller – vide Table 1. This can be 1972) permitting to build dendrograms explained by increased apathy caused by of behavioural distance. captive conditions. In an additional analysis (not in- The ANOVA results for snakes body cluded in all experiments), authors used weights were signifi cant P ≤ 0.05. As Sinaloan Milk Snake Lampropeltis tri- the activity in OFT could be affected by angulum sinaloe – 4 specimens, Grey- snakes’ sizes we made the additional anal- -banded Kingsnake Lampropeltis alterna yses, correcting the analysis for points – 2 specimens, and California Kingsnake scored in the fi rst observation by deviation Lampropeltis getula californiae (albino) from regression line for body weights. – 2 specimens. All of these were born The deviations from regression line a year later. These snakes were confront- (body weight as an operand variable) are ed with each other within their own spe- highly signifi cant (P ≤ 0.01) and confi rm cies and with albino Kingsnakes. that the signifi cant differences (P ≤ 0.05) in points scored in OFT (I observation – Table 1), were not caused by differences RESULTS in the sizes of specimens, but in reality The points scored in OFT in both obser- depended on group classifi cation. vations met the criteria of normal distri- Activity in the fi eld test was lower bution with mean values 7.7 and stand- during the II observation except in the ard deviation 5.4 and mean value 8.5 case of the group 5. Growth was differ- with standard deviation 5.5 for I and II ent in each group – in the pyromelana trial respectively. group, this was even a negative value. The differences between groups are The results for cluster analysis are signifi cant P ≤ 0.05 for the I observation shown in the dendrograms in Figures 1, 2, 3, 4 and 5. TABLE 1. Points scored in OFT and measurements of body weights during the I and II observations Scores in OFT Body weights [g] Groups N I Observation II Observation 1st Observation 2nd Observation x ± σ x ± σ x ± σ x ± σ Ls × Lc 10 7.9 ±0.9a 7.3 ±3.2 35.2 ±3.1A;E 36.4 ±3.3 Lpp × Lpk 3 3.3 ±1.8b;d 1.7 ±2.1 27.7 ±1.0B;F 26.3 ±3.3 Ege 8 10.9 ±2.5d;e 8.0 ±5.4 174.9 ±34.7A;B;C;D 185.1 ±37.0 Ege × Eggam 6 13.2 ±1.9a;b;c 10.3 ± 6.4 112.0 ±21.1C;E;F;G;H 141.9 ±21.3 Eggan × Eggalb 7 4.7 ±1.3c;e 7.6 ±1.9 40.5 ±2.9D;H 47.9 ±4.0 Letters (in pairs): a–e point out the signifi cant differences between means of groups for points scored during the fi rst trial – Duncan test; P < 0.05; Letters (in pairs): A–H show the signifi cant differences between means of groups for body weights dur- ing the fi rst trial – Duncan test; P < 0.05. Comparative behaviour analysis of colubrids with... 247

FIGURE 1. Comparison of group reactions during the I observation – confrontation with Pantherophis guttatus

FIGURE 2. Comparison of group reactions during the II observation – confrontation with Pantherophis guttatus

FIGURE 3. Comparison of group reactions during the I observation – confrontation with Lampropeltis getula splendida 248 A. Życzyński, Z. Nowak

FIGURE 4. Comparison of group reactions during the II observation – confrontation with Lampropeltis getula splendida

FIGURE 5. Comparison of group reactions – both types of confrontation grouped together

Confrontation with the relatively blood” as a factor differentiating the harmless Corn Snake Pantherophis gut- groups. tatus has not revealed any differences in During confrontation with their own reactions between groups, which could species (Greybanded Kingsnakes, Milk be attributed to either the level of domes- Snakes and California Kingsnakes), au- tication or the taxonomic position. thors observed some slight excitation In contrast to the I observation, dur- (avoidance, tail rattling) in the tested ing the II observation, played snakes. Confrontation with the albino an important part in sorting the snake Kingsnake resulted in an attack by the groups within clusters. This tendency re- latter showing the typical feeding re- mained constant during subsequent trials sponse. The Kingsnakes themselves did – vide Figures 3–5. not react to each other. Figures 3–5 show dendrograms that Following the attack, the trials were are practically identical with Figure 2. immediately halted, with no harm being There is no noticeable infl uence of do- caused to the snakes by the testing pro- mestication or the addition of “wild cedure. Comparative behaviour analysis of colubrids with... 249

DISCUSSION – probably due to apathy caused by cap- tive conditions. Analysis of the snakes’ activity in OFT Another source of information is pro- showed signifi cant differences only dur- vided by analysis of the dendrograms. ing the I observation (Table 1). Compari- The I confrontation, with the relatively son within genus Pantherophis revealed harmless Corn Snake (Fig. 1), showed that groups 3 and 4 did not differ signifi - a considerable variance in reactions be- cantly from each other but both deviate tween groups, which could not be attrib- from group 5 (the smallest specimen uted to either taxonomy or level of do- – Table 1) designated the most domes- mestication. The II confrontation, with ticated – the longest generation history a Corn Snake and the effect of exposing in captivity. Correction on regression the tested specimens to more danger- line for body weight showed that points ous stimuli i.e. Desert Kingsnake L. g. scored in OFT do not depend on body splendida, (Fig. 2–5) provided uniform weight. This result accords with the fact dendrograms arranged according to tax- that during the II observation, differences onomy and not the captivity status. between groups in terms of body weight Albino Kingsnakes tolerated each increased, but differences in activity other well, but in the presence of another (points scored in OFT) disappeared (vide species, manifested an immediate feed- Table 1). ing response by attempting to attack. Based on these fi ndings, we can con- Rodriguez-Robles and De Jesus-Es- clude that snakes kept for generations in cobar (1999) studied evo- captivity behave less actively than those lutional relationships by analyzing the fi eld collected or those with the addition mt DNA variability and adjusting their of “wild blood”. We may ask whether conclusions to the evolution of feed- decreased activity refl exes lower fi t- ing customs. According to the authors, ness ability. Each group of reptiles has a diet based on reptiles (lizards, snakes) its own survival strategy. Research car- is more primitive than one based on ro- ried on turtles (Mrosovsky and Gogfrey dents, which developed much later. They 1995) has shown that the more active presented their results in the form of young (1YO) specimens have a lower dendrograms, showing both the genetic survival rate than those less mobile, relations between genera of American whereas the higher mobility of young colubrids and the hypothetic evolution lizards (Van Damme et al. 1992) pro- of feeding behaviour. vides them a greater success rate in hunt- The results presented earlier corre- ing and a greater ability to avoid preda- spond with those quoted. Divergence tors. If greater activity and courage in in behaviour during confrontations with young snakes protects them in the wild, predators philogenetically approximates researchers may conclude that a longer feeding behaviour and differs by defi ni- generation stage in captivity debilitates tion from inner aggression. adaptation and fi tness. This is true for the The divergences noted above are very fi rst stages of life only, as during the II important from the point of view defi ned observation all groups behaved similarly in the title of this study. During the fi rst 250 A. Życzyński, Z. Nowak trials, we observed diversity in both the captivity as a function of potential, unde- fi tness of the specimens and their reac- sirable selection. tions during confrontation. These dif- Snakes play an important role in the ferences were reduced as the snakes got trophic chain. In the event of reintroduc- older. So any decision to reintroduce tion of captive material into the natural snakes should only consider very young habitat, the consequences of releasing specimens, i.e. hatchlings. Such mate- animals of genetically changed feeding rial may be characterized by greater fi t- customs could be at the least undesirable, ness – vide Table 1, although due to their even where the rules of taxonomic and smaller size, these could be more at risk population fi delity have been obeyed. It from predators. But this would allow for is worth mentioning that the team work- natural selection, a factor always present ing on the reintroduction of the Smooth in the natural habitat. Snake Coronella austriaca (Zieliński and The accordance of reactions with the Stanisławski 2001) were offering their dendrograms shown by Rodriguez-Rob- captive hatched snakes live small lizards les and De Jesus-Escobar revealed that (Lacerta agilis), which were preyed on. captive Lampropeltis snakes preserved This refl ected in terrarium conditions the their natural feeding behaviour. Thanks rules of natural selection for the snakes’ to their undisputed beauty, Kingsnakes survival. However, the results of this and Milk Snakes have gained enormous restocking are diffi cult to evaluate, as popularity among amateur snake keep- the snakes were not tagged for telemet- ers. In captive conditions, they are forced ric monitoring as they were in Daltry’s to adapt to a rodent based diet. Some (1999) project. specimens adapt to this easily; others Lack of proper documentation is al- are more reluctant. Nevertheless their ways the weakest point of any reintro- maintenance in captivity is increasingly duction project, as was pointed out by easy and the survival rate of hatchlings is Earnhardt (1999). This particularly con- gradually increasing. This fact is worthy cerns survival rates in the very fi rst pe- of attention. The most popular terrarium riod of release. species may have been subjected to un- The dendrograms and observations intentional selection for the ability to presented in this paper lead to conclution adapt to a diet that is atypical for them. that studied Lampropeltis snakes, despite Arnold (1981), working on garter snakes their history of captivity, preserved their Thamnophis elegans proved that feeding natural feeding habits, enabling them to preferences are genetically polymorphic play their natural role in the trophic chain and of a quantitative nature. Thus selec- when returned to their natural environ- tion for diet type could be possible and ment. Likewise the Pantherophis snakes, may also result in inherited changes in despite their level of domestication, did feeding customs, not caused by current not differ from one another in the general artifi cial captive conditions only. This set of reactions. problem is discussed by Conway 2011, focusing on the number of generations in Comparative behaviour analysis of colubrids with... 251

CONCLUSIONS RODRIGUEZ-ROBLES J.A., DE JESUS- -ESCOBAR J.M., 1999: Molecular sys- This model experiment shows that cap- tematics of New World Lampropeltinine tive snakes from multigeneration terra- Snakes (Colubridae): implications for rium colonies can be considered worthy biogeography and evolution habits. Bio- material for reintroduction, on condition logical Journal of the Linnean Society 68: 355–385. that the principles of genetic fi delity are MARKOWSKA A., 1979: Reakcja na zmianę observed. bodźca wzrokowego u szczurów z uszko- A planned release should be carried dzeniem kory przedczołowej, prążkowia out at a very young age, despite the ex- lub formacji hipokampa. Praca doktor- pected losses of very small and fragile ska. Instytut im. M. Nenckiego PAN. animals, in order to let them undergo MATTISON CH., 1995: The encyclopedia a process of natural selection based on of snakes. Blandford Press, New York. MOSEBY K.E., READ J.L., PATON D.C., fully exposed diversity. COPLEY P., HILL B.M., CRISP H.A., 2011: Predation determines the outcome of ten reintroduction attempts in arid REFERENCES South Australia. Biological Conservation ALEXANDER G., MARAIS J., 2007: A 144 (12): 2863–2872. guide to Reptiles of Southern Africa. MROSOVSKY N., GODFREY M.H., 1995: Struik Publishers, Cape Town. Manipulating sex ratios: turtle speed ARNOLD S.J., 1981: Behavioural variation ahead! Chelonian Conservation and Bi- in natural populations II. The inheritance ology 1 (3): 238–240. of a feeding response in crosses between NEI M., 1972: Genetic distance between geographic races of the garter snakes populations. American Naturalist 106: Thamnophis elegans. Evolution 35(3): 283–291. 510–515. SPANOWICZ M., ŻYCZYŃSKI A., 2002: CONWAY W.G., 2011: Buying time for wild Projekt zasilania lokalnej populacji Lam- animals with zoos. Zoo Biology 30: 1–8. propeltis alterna w rejonie Limpia Can- DALTRY J., 1999: Interim report – Re-intro- yon (Płd.-Wsch. Teksas). Przegląd Przy- duction of the Antiguan Racer (Alsophis rodniczy 13 (3): 147–150. antiguagae). Antiguan Racer Conserva- STANISŁAWSKI W., 2003: Hodowla kra- tion Project. www.eco-index.org/search/ jowych gadów w polskich ogrodach pdfs/1418report_6.pdf. zoologicznych. Materiały z I Spotkania DALTRY J., 2006: Reintroduction of the Herpetologów Polskich Ogrodów Zoolo- critically endangered Antiguan Racer Al- gicznych. Łódź 27–28 listopada 2003. sophis antiguagae to Rabbit Island Anti- Van DAMME R., BAUWENS D., BRA- gua. Conservation Evidence 3: 33–35. NA F., VERHEYEN R., 1992: Incubation EARNHARDT J.M., 1999: Reintroduction temperature differently affects hatching programmes: genetic trade-offs for the pop- time, egg survival and hatchling perfor- ulations. Animal Conservation 2: 279–286. mance in the lizard Podarcis muralis. GODWIN J., JOHNSON V., GUYER C., Herpetologica 48 (2): 220–226. RUSH M., 2008: Captive propagation ZIELIŃSKI P., STANISŁAWSKI W., 2001: of the Eastern Indigo Snake for reintro- Ochrona gniewosza plamistego w Polsce duction into Alabama. Report. Alabama Środkowej – wyniki wstępne. Materiały Dept. of Conservation and Natural Re- z konferencji „Metody czynnej ochrony sources Montgomery. Unpublished. herpetofauny”. Łagów Lubuski 17–18 listopada 2001. 252 A. Życzyński, Z. Nowak

Streszczenie: Porównawcza analiza zachowań naturalnej diecie, wystąpiły normalne reakcje węży z rodziny Colubridae pod kątem przydat- drapieżnicze. Stwierdzono zatem przydatność ności osobników hodowanych w niewoli do rein- populacji terraryjnej jako ewentualnego banku trodukcji do środowiska naturalnego. W modelo- genów dla strategii ochronnej ex situ, przy zalece- wym doświadczeniu przetestowano amerykańskie niu używania do reintrodukcji materiału możliwie węże z rodziny Colubridae podzielone według młodego, najwierniej reagującego na presję se- stopnia udomowienia (pokoleniowego stażu lekcji naturalnej, wymierzonej w wachlarz reakcji w warunkach niewoli). Zwierzęta zostały podda- niestłumionych warunkami niewoli. ne testowi otwartego pola (OFT), sprawdzającego ich aktywność i odwagę na otwartej przestrzeni MS. received in November 2013 oraz konfrontacji z innym wężem zbożowym Pantherophis guttatus oraz lancetogłowem kró- Authors’ address: lewskim Lampropeltis getula splendida i dodat- Andrzej Życzyński, Zuzanna Nowak kowo L.g. californiae, formą albinotyczną. Test Szkoła Główna Gospodarstwa Wiejskiego OFT wykazał ujemny wpływ wieku na aktywność w Warszawie terraryjnych zwierząt a konfrontacje z probanta- Wydział Nauk o Zwierzętach mi nie ujawniły wpływu udomowienia na reak- Katedra Genetyki i Ogólnej Hodowli Zwierząt cje obronne (klasterowa analiza skupień). Nawet ul. Ciszewskiego 8, 02-786 Warszawa u albinotycznych lancetogłowów, uznanych za Poland najbardziej udomowioną formę (zubożały, wsob- e-mail: [email protected] ny genotyp) utrzymywanych od pokoleń na nie- [email protected]