47 Annales Universitatis Paedagogicae Cracoviensis Studia Naturae, 3 (supplement): 47–53, 2018, ISSN 2543-8832 DOI: 10.24917/25438832.3supp.6 Renata Muchacka1*, Ewa Sosnówka-Czajka2, Iwona Skomorucha2, Edyta Kapusta1, Agnieszka Greń1, Zofia Goc1 1Department of Animal Physiology and Toxicology, Institute of Biology, Pedagogical University of Cracow, Podbrzezie 3, 30-054 Kraków, Poland, *renata.muchacka@up.krakow.pl 2Department of Poultry Breeding, National Research Institute of Animal Production, Krakowska 1, 32-083 Balice n. Kraków, Poland Antioxidant enzymes activity, GSH and MDA level in eggs from heritage breeds Introduction In eggs, as in any raw food material, there are a number of di�erent quality chang- es. �e moment of laying the egg is a  speci�c beginning of these changes. From that moment, the eggs begin to undergo metabolic processes that contribute to the gradual deterioration of the quality of the raw material until the loss of consumer and technological usefulness (Jankowska, 2010). On the farm, there is a  group of factors that have an impact a�er the egg is laid and modi�es their quality. �ese include hygienic conditions, and the manner and conditions for the collection and distribution of raw egg product. �e temperature and distribution time between the producer and the consumer as well as the storage conditions are decisive here (Ca- lik, 2011). Another group are factors that a�ect the quality of eggs even before they are laid depend on the laying hens and, to a large extent, on the farmer. �e quality of eggs is in�uenced by the origin of laying hens (i.e. breed, line). It a�ects, among other things, the size of the egg, the colour of the eggshell, and the nutritional value (Singh et al., 2009; Calik, 2013). Moreover, the following factors are also important: the age of the laying hens (older hens laid larger eggs, but of inferior quality shells), the rearing system, their feeding (Batkowska, Brodacki, 2017), and veterinary pre- vention (Calik, 2013). �e aim of the study was to determine the di�erences in antioxidant enzyme activ- ities and di�erences in the level of reduced glutathione and malondialdehyde in eggs from three heritage breeds. R en at a M uc ha ck a, E w a S os nó w ka -C za jk a, Iw on a S ko m or uc ha , Ed yt a K ap us ta , A gn ie sz ka G re ń, Z of ia G oc 48 Material and methods Hens and experimental design �e experiment was carried out with 99 hens, approximately 48 weeks of age, of three heritage breeds, ‘Greenleg Partridge’ (Z-11), ‘Sussex’ (S-66), and ‘Rhode Island Red’ (R-11), which were assigned to Groups I to III (33 hens per group), respectively. Each group was subdivided into 3 subgroups, each having 11 birds. Hens were reared in the litter system at a stocking density of 9 birds · m-2 and had outdoor access with green paddocks (2.5 m2/hen). �e birds were fed ad libitum with standard diets based on concentrates for laying hens, which contained 17.5% protein and 11.6 MJ ME/ kg feed (Fig. 1). �e diet consisted of wheat, maize, soybean expeller, dried alfalfa, rapeseed oil, limestone, di-calcium phosphate, common salt, DL-methionine, and DJ pre�x. �e birds had free access to water throughout the experiment. All the groups were managed under uniform environmental (air humidity and temperature, lighting programme) and feeding conditions. A  lighting schedule of 16 h of light and 8 h of darkness (16L : 8D) was used. Sample collection and laboratory analyses At 48 weeks of age, 6 eggs from each group were collected for the next three days. Eggs were evaluated a�er 24 h of refrigerated storage at 4°C and 55% humidity. In the sam- ples of egg yolks and albumens, the concentration of glutathione (GSH) and malon- dialdehyde (MDA), and the activity of superoxide dismutase (SOD), glutathione per- oxidase (GPx), and catalase (CAT) were estimated. �e collected yolks and albumens were homogenised in 50 mM phosphate bu�er cooled to 4°C and containing K2HPO4 (dipotassium hydrogen phosphate) + KH2PO4 (potassium dihydrogen phosphate) + Fig. 1. Nutritive value of the diet 17.5 0.41 0.86 3.7 0.4 0 5 10 15 20 Crude protein Methionine Lysine Calcium Phosphorus [%] Components A ntioxidant enzym es activity, G S H and M D A level in eggs from heritage breeds 49 0.1 mM EDTA (ethylenediaminetetraacetic acid disodium salt) (pH = 7.0) + 0.1% BSA (bovine serum albumin) and then centrifuged. �e obtained supernatants of the yolks and albumens were used for further investigations. �e activity of SOD in the supernatants of homogenates of yolks and albumens was determined according to spectrophotometric cytochrome method by Diplock et al. (1991). �e activity of CAT was determined according to the spectrophotometric method by Aebi (1983), and the activity of GPx was estimated by the modi�ed method of Lück (1963). �e activity of the studied antioxidative enzymes in yolk and albumen extracts was expressed in U · mg-1 of protein. MDA reacts with thiobarbituric acid (TBA) in an acid medium and the produced coloured TBA-complex that could be measured colorimetrically according to the method of Ohkawa et al. (1979). MDA concentration was expressed in nM · mg-1 protein. Total protein concentration in the supernatants of homogenates of the yolk and albumen was determined according to the Bradford method (1976) with bovine serum albumin as a standard. In the supernatants of yolks and albumens, obtained a�er deproteinisation and centrifugation, GSH was determined according to the method of Ellman (1959). �e concentration of GSH was estimated in the yolk and albumen in μM · g-1 of protein. Statistical analysis All data was analysed by the analysis of variance procedures (ANOVA) and Duncan’s multiple-range test using the computer program Statistica version 10.0 PL. �e data was expressed as means ± SD and was considered as signi�cant when P values were less than 0.05. Tab. 1. Antioxidant enzymes activity and the level of MDA and GSH in yolk; values shown as di�erent letters within the line di�er signi�cantly according to the Duncan test at p ≤ 0.01 Item Group SD I II III SOD [U/mg] 3.0240a 7.5730b 3.0130a 1.0810 GPx [U/mg] 0.0044 0.0065 0.0048 0.0034 CAT [U/mg] 0.0470 0.0650 0.0890 0.0620 GSH [µM/g] 0.3410b 0.3850b 0.1250a 0.0170 MDA [µg/g] 2.4490ab 2.2500a 2.4680b 0.0460 Results �e highest activity of SOD, both in yolks (Tab. 1) and albumens (Tab. 2), was found in eggs of Sussex hens (p ≤ 0.01). GPx and CAT activity was in all eggs at a similar level. �e highest level of GSH was in egg yolks of ‘Greenleg Partridge’ and ‘Sussex’ hens (p ≤ 0.01) R en at a M uc ha ck a, E w a S os nó w ka -C za jk a, Iw on a S ko m or uc ha , Ed yt a K ap us ta , A gn ie sz ka G re ń, Z of ia G oc 50 (Tab. 1) and in egg albumens of ‘Greenleg Partridge’ hens (p ≤ 0.01) (Tab. 2). �e lowest level of MDA was found in egg yolk and egg albumen of ‘Sussex’ hens (p ≤ 0.05) (Tab. 1–2). Tab. 2. Antioxidant enzymes activity and the level of MDA and GSH in albumen; values shown as di�er- ent letters within the line di�er signi�cantly according to the Duncan test at p ≤ 0.01 Item Group SD I II III SOD [U/mg] 1.1320a 3.1870b 1.1290a 0.4580 GPx [U/mg] 0.0049 0.0062 0.0079 0.0032 CAT [U/mg] 0.0640 0.0650 0.0940 0.0660 GSH [µM/g] 0.3250b 0.1500a 0.1460a 0.0160 mDA [µg/g] 1.8790ab 1.6080a 1.9130b 0.0220 Discussion In optimal conditions, there is a  balance between oxidants and antioxidants in the body. �e increase in free radical production or decrease of antioxidant activity causes a  disturbance of balance in the oxidation reaction, which is called oxidative stress. During oxidative stress, stationary concentrations of reactive oxygen species (ROS) increase signi�cantly. Free radicals oxidize row fatty acids (lipids), structural proteins, and enzymatic proteins (Surai, 2015). �erefore, it is important that the body’s antiox- idant system functions properly. �e quality characteristics of eggs and also the anti- oxidant enzymes and GSH found in the eggs are synthesised and formed in the hen’s body during eggs formation. According to our research, the activity of antioxidant enzymes (mainly SOD) and the levels of GSH and MDA found in eggs di�er depend- ing on the breed of hens. �e highest activity of SOD, the highest level of GSH, and the lowest level of MDA were characterised by eggs from Sussex hens, which may indicate they have the best quality. Similar results were obtained in the previous studies by Muchacka et al. (2016; 2018). It is known that the origin of the laying hens (i.e. breed, line) a�ects the quality of eggs. It a�ects egg size, shell colour, and the nutritional value (Singh et al., 2009; Calik, 2013) and probably the activity of antioxidant enzymes and levels of GSH (Muchacka et al., 2016; 2018). Di�erences in the activity of antioxidant enzymes and GSH levels may be caused by di�erences in the metabolism of the tested hens. It may be important because these substances can a�ect the quality of the poul- try products, e.g., eggs and protect the germ cell and growing embryo against ROS in the �rst days a�er the egg is laid. Based on the obtained results, it can be concluded that the activity of antioxidant enzymes (mainly SOD) and the levels of GSH and MDA which occur in eggs may depend on the breed of hens and may indicate their quality; however, further studies on this topic are necessary. A ntioxidant enzym es activity, G S H and M D A level in eggs from heritage breeds 51 References Aebi, H.E. (1983). Catalase. In: H.U. Bergmeyer (ed.), Methods of Enzymatic Analysis. New York: Aca- demic Press, pp. 273–286. Batkowska, J., Brodacki, A. (2017). Selected quality traits of eggs and the productivity of newly-created laying hens dedicated to extensive system of rearing. Archives of Animal Breeding, 60, 87–93. Bradford, M.M. (1976). A  rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle for protein-dye binding. Analytical Biochemistry, 72, 248–254. Calik, J. (2011). Ocena jakości jaj sześciu rodów kur nieśnych w zależności od ich wieku. Żywność Nauka Technologia Jakość, 5(78), 85–93. [In Polish] Calik, J. (2013). Zmiany cech jakościowych jaj pochodzących od kur nieśnych Żółtonóżka Kuropatwiana (Ż-33), w zależności od warunków ich przechowywania. Żywność Nauka Technologia Jakość, 2(87), 73–79. [In Polish] Diplock, A.T., Symons, M.C.R., Rice-Evans, C.A. (1991). Techniques in free radical research. London: Elsevier. Ellman, G.L. (1959). Tissue sul�ydryl groups. Archives of Biochemistry and Biophysics, 82, 70–77. DOI: 10.1016/0003-9861(59)90090-6 Jankowska, K. (2010). Od zniesienia do konsumpcji – jaja kurze (spożywcze). Zachowanie higieny w pro- dukcji jaj. Polskie Drobiarstwo, 2, 52–54. [In Polish] Lück, H. (1963). Peroxidases. In: H.U. Bergmeyer (ed.), Methods of enzymatic analysis. New York: Aca- demic Press, pp. 895–897. Muchacka, R., Sosnówka-Czajka, E., Skomorucha, I. (2018). Antioxidant enzymes activity, GSH and MDA level in eggs from heritage breeds reared under organic conditions. 8th International Scienti�c Symposium „Poultry Days 2018“, 21–22. June 2018, Brno, Czech Republic, 30. Muchacka, R., Sosnówka-Czajka, E., Skomorucha, I., Kapusta, E., Greń, A., Goc, Z. (2016). Lipid peroxi- dation, antioxidant enzymes activity and glutathione level in eggs from heritage breed and commer- cial crosses hens. Proceedings of 12th International Scienti�c Conference “Animal Physiology 2016”, May 13th–15th 2016, Bořetice, Czech Republic, 191–196. Ohkawa, H., Ohishi, N., Yagi, K. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry, 95, 351–358. Singh, R., Cheng, K.M., Silversides, F.G. (2009). Production performance and egg quality of four strains of laying hens kept in conventional cages and �oor pens. Poultry Science, 88(2), 256–264. DOI: 10.3382/ps.2008-00237 Surai, P. (2015). Antioxidant systems in poultry biology: superoxide dismutase. Journal of Animal Re- search and Nutrition, 1(1), 1–8. DOI: 10.21767/2572-5459.100008 Abstract �e objective of the study was to determine the activity of antioxidant enzymes and the level of GSH and MDA in the yolk and albumen of hen’s eggs. �e experiment was carried out with 99 hens of three heritage breeds (‘Greenleg Partridge’ – group I, ‘Sussex’ – group II, ‘Rhode Island Red’ – group III). Layers were reared in the litter system and had outdoor access. Birds were fed ad libitum standard diets based on concen- trates for laying hens and had free access to water throughout the experiment. All the groups were managed under uniform environmental (air humidity and temperature, lighting programme) and feeding conditions. At 48 weeks of age, 6 eggs from each group were collected. In the samples of egg yolks and albumens, the concentration of GSH and MDA, and activity of SOD, GPx, and CAT were estimated. �e results were sta- tistically analysed by one-way analysis of variance, and signi�cant di�erences were estimated with Duncan’s multiple range tests. �e highest activity of SOD, the highest level of GSH, and the lowest level of MDA were R en at a M uc ha ck a, E w a S os nó w ka -C za jk a, Iw on a S ko m or uc ha , Ed yt a K ap us ta , A gn ie sz ka G re ń, Z of ia G oc 52 characterised by eggs from ‘Sussex’ hens, which may indicate that they have the best quality. Based on the obtained results, it can be concluded that the activity of antioxidant enzymes (mainly SOD) and the levels of GSH and MDA that occur in eggs may depend on the breed of hens. Key words: antioxidant enzymes, eggs, GSH, hens, MDA Received: [2018.05.15] Accepted: [2018.07.30] Aktywność enzymów antyoksydacyjnych, poziom GSH i MDA w jajach pochodzących od kur ras zachowawczych Streszczenie Celem doświadczenia było zbadanie aktywności enzymów antyoksydacyjnych oraz poziomu GSH I MDA w żółtkach i białkach jaj kurzych. Materiał doświadczalny stanowiło 99 kur trzech ras zachowawczych (‘Zie- lononóżka Kuropatwiana’ – I grupa, ‘Sussex’ – II grupa i ‘Rhode Island Red’ – III grupa). Nioski utrzymy- wano w systemie ściółkowym z możliwością korzystania z trawiastych wybiegów. Ptaki karmiono ad libitum standardową paszą przygotowaną na bazie koncentratów dla kur niosek. W trakcie doświadczenia nioski miały swobodny dostęp do wody. Wszystkie grupy były utrzymywane w jednakowych warunkach środo- wiskowych (wilgotność powietrza i temperatura, program świetlny) i żywieniowych. W 48. tygodniu życia, z każdej grupy zebrano po 6 jaj. W próbkach żółtek i białek oznaczono poziom GSH i MDA oraz zbadano aktywność SOD, GPx i CAT. Wyniki zostały opracowane statystycznie za pomocą jednoczynnikowej anali- zy wariancji, szacując istotności różnic testem Duncana. Najwyższą aktywnością SOD, najwyższym pozio- mem GSH i najniższym poziomem MDA charakteryzowały się jaja od kur rasy ‘Sussex’, co może wskazywać na ich najlepszą jakość. Na podstawie uzyskanych wyników można stwierdzić, iż aktywność enzymów anty- oksydacyjnych (głównie SOD) oraz poziom GSH i MDA występujących w jajach mogą zależeć od rasy kur. Słowa kluczowe: enzymy antyoksydacyjne, jaja, GSH, kury, MDA Information on the authors Renata Muchacka https://orcid.org/0000-0002-8255-006X She is an assistant professor at the Department of Animal Physiology and Toxicology in the Institute of Biology, Pedagogical University of Cracow. She is interested mainly in animal physiology, oxidative stress in animals, and the quality of food products of animal origin. Ewa Sosnówka-Czajka https://orcid.org/0000-0003-3720-1685 She is an assistant professor at the Department of Poultry Breeding in the National Research Institute of Animal Production in Cracow. She is interested mainly in poultry welfare and the technology of poultry production. Iwona Skomorucha https://orcid.org/0000-0003-1241-7703 She is an assistant professor at the Department of Poultry Breeding in the National Research Institute of Animal Production in Cracow. She also focuses on poultry welfare and the technology of poultry production. Edyta Kapusta https://orcid.org/0000-0002-4350-5514 She is a laboratory specialist at the Department of Animal Physiology and Toxicology in the Institute of Biology, Pedagogical University of Cracow. She is interested in animal physiology, mainly in oxidative stress in animals. Agnieszka Greń https://orcid.org/0000-0003-2383-1096 She is a  professor at the Department of Animal Physiology and Toxicology in the Institute of Biology, Pedagogical University of Cracow. She is interested mainly in animal physiology, diabetes, and natural products, predominantly in diabetes prevention and therapy. A ntioxidant enzym es activity, G S H and M D A level in eggs from heritage breeds 53 Zo�a Goc https://orcid.org/0000-0001-9626-5895 She is an assistant professor at the Department of Animal Physiology and Toxicology in the Institute of Biology, Pedagogical University of Cracow. She is interested in research on animal cells in vitro and animal physiology.