72 Annales Universitatis Paedagogicae Cracoviensis Studia Naturae, 3 (suplement): 72–82, 2018, ISSN 2543-8832 DOI:10.24917/25438832.3supp.10 Ewa Sosnówka-Czajka*, Iwona Skomorucha Department of Poultry Breeding, National Research Institute of Animal Production, Kraków, Poland, *ewa.sosnowka@izoo.krakow.pl Selected blood parameters in organically raised hens fed with a purple coneflower supplemented diet** **Carried out based on Ministry of Agriculture and Rural Development decision no. HOR.re.027.5.2017 Introduction Organic farming requires continuous care for a high health status of birds to make poultry production pro�table and to obtain quality products, which is of direct inter- est to consumers. Impaired immune function leads to deterioration in productivity, e.g., poorer laying performance, lower hatchability, or increased mortality (Yunis et al., 2000; Sivaraman et al., 2005). On the other hand, by stimulating the immune sys- tem through modi�ed nutrition, it is possible to reduce the negative e�ects of com- promised immunity in poultry (Truchliński et al., 2006; Salim et al., 2018). Environ- mental stress associated with the used rearing system may, in general, interfere with antibody production and cellular immune response, making poultry more vulnerable to vital infections (Bartlett, Smith, 2003). At present, organic farmers are focused on increasing their use of herbs and herbal mixtures, which have positive e�ects on the animals’ metabolism, health, productivity, feed conversion, and reproduction. A large number of biologically active therapeutic substances are found in purple cone�ower, including polysac- charides, glycoproteins, and alkaloids (Dalby-Brown et al., 2005). Many of these substances are used to improve the body’s immune potential and to enhance mac- rophage activity (Goel et al., 2002). Allen (2003) reported that purple cone�ower extract can be used to increase resistance to coccidiosis in birds kept on litter. Ac- cording to Najafzadeh et al. (2011), purple cone�ower can be an e�ective means to prevent avian in�uenza in birds. �erefore, the aim of this study was to determine the e�ect of a dietary addition of dried purple cone�ower (Echinacea purpurea (L.) Moench) on selected blood param- eters of chicks and hens of the native ‘Greenleg Partridge’ breed (Z-11), maintained according to organic farming principles. 73 S elected blood param eters in organically raised hens fed w ith a purple coneflow er supplem ented diet Material and methods Animals and experimental design �e experiment was carried out on 252 ‘Greenleg Partridge’ hens (R-11) aged 20 weeks (wks) with 126 birds in each group with 6 subgroups per group. In each experimental subgroup, there was 1 rooster to 7 layers. Hens were maintained on a certi�ed poultry farm according to organic farming principles. Birds were divided into two groups ac- cording to diet: organic layer diet (group 1), and the same diet supplemented with cer- ti�ed purple cone�ower (Echinacea purpurea (L.) Moench) from 20 wks of age at 10 g/ kg feed (group 2). Layers were fed the certi�ed layer diet (17% protein and 11.1 MJ ME/ kg diet) following organic farming principles. �e diets contained organic components such as maize, wheat, soybean expeller, sun�ower cake, triticale, dehulled heat-treated lupin, peas, broad bean, soybean, maize gruel, dried lucerne, soybean oil, molasses, and monocalcium phosphate. �roughout the study, hens were fed ad libitum (optimal) and had constant access to water. Hens were kept on litter (6 birds/m2 of �oor area) and were allowed access to grassy yards (4 m2/bird). �e yards were equipped with roofs and drinkers. �e microclimate conditions and the light programme were adjusted to the ages of the birds in keeping with pullet rearing and layer management standards. At 34 weeks of rearing, eggs were collected for experimental incubations (100 eggs from each group). Eggs were incubated for 21 days under standard conditions. Sample collection and laboratory analyses Blood was collected from laying hens (from 15 birds per group – 2.5 ml from each hen) at 34 wks and from newly hatched chicks at one day of age (from 15 birds per group – 1 ml from each chick) to determine selected blood parameters. �e circulat- ing immunoglobulin complex was determined by the procedure of Lowry (Lowry et al., 1951) modi�ed by Ślebodziński et al. (1982), and IgG was analysed by quantitation tests (Chicken IgG ELISA Kit, Alpha Diagnostic Intl., Inc.). �e results were read us- ing a Tecan Spectra Classic reader based on KCJunior so�ware (Bio-Tek Instruments, Inc.). Blood for haematocrit determinations was centrifuged in haematocrit capillaries with an MPW-52 centrifuge, and the result was read from a reader placed on the centri- fuge rotor. Blood smears were stained with a May-Grünwald-Giemsa (MGG) protocol for calculation H:L. �e number of red and white blood cells was counted in a Bürker chamber under a Nikon YS 100 microscope. Statistical analysis �e results were statistically analysed by one-way analysis of variance and estimated with Duncan’s test. �e statistical calculations were made with Statistica ver. 12 so�- ware. Ew a S os nó w ka -C za jk a, Iw on a S ko m or uc ha 74 Results Appendix 1 – �gure 1 shows the levels of the circulating immunoglobulin complex in 35-week-old organic ‘Greenleg Partridge’ hens and in day-old chicks derived from these hens. �ere was only a tendency for higher immunoglobulin complex in birds fed the purple cone�ower diet. Similar relationships were observed for IgG, and the lack of statistically signi�cant di�erences results from a large scatter of data (Appendix 1 – Fig. 2). Z-11 hens fed the purple cone�ower diet and chicks originating from them were characterised by the lowest H:L ratio at p ≤ 0.05 (Appendix 1 – Fig. 3). No sig- ni�cant di�erences were observed between the experimental groups of Z-11 hens and chicks in the level of leukocytes (Appendix 1 – Fig. 4), erythrocytes (Appendix 1 – Fig. 5) and haematocrit (Appendix 1 – Fig. 6). Discussion �e body’s immunity depends on the maintenance of immune homeostasis which is associated with systemic homeostasis. �e e�ciency of the immune system is o�en determined from the level of immunoglobulins (Sivaraman et al., 2005). According to Wang et al. (2000), total Ig level may be indicative of the potential e�ciency of the humoral system, whereas the IgG level does not always re�ect the actual activity of the immune system. Singh et al. (2006) concluded that circulatory immune complexes in broiler chickens average 2.46 g/dl and increase to 2.99 g/dl a�er stimulating the immune system with dietary vitamin E and selenium. In our study, the circulatory immune complexes tended to increase by about 15% in the blood serum of ‘Greenleg Partridge’ hens fed a purple cone�ower supplemented diet compared to the hens fed a standard organic diet. One of the indicators of chick quality is the body’s immunity, which allows the chicks to counteract the development of pathogens, thus having a positive e�ect on their survival during rearing (Kogut, Klasing, 2009; Korver, 2012). In birds, maternal antibodies are transferred from layer’s serum to egg yolk and later to the yolk sac and blood circulatory system, which provides the embryos and newly hatched chicks with passive immunity (West et al., 2004; Bencina et al., 2005). Because the level of immunity in laying hens determines the immunity of the chicks, in our study, we found a similar relationship for the chicks from Z-11 hens supplemented with purple cone�ower as in adult birds, namely, a tendency for the circulating immune complexes to increase by 9%. Our study showed very large individual di�erences in the level of circulating immune complexes; therefore, they were not signi�cant, despite high dif- ferences between the groups. 75 Serum IgG level in chickens re�ects the body’s humoral immunity status (Juul-Madsen, Sørensen, 2004). In our study, we found the level of immunoglobulins to vary widely between individual birds. �erefore, although between-group di�er- ences in IgG concentration were high and reached 23%, they were not statistically signi�cant. Similar correlations were also observed by Wang et al. (2000). Carlander et al. (2003) showed high individual di�erences in IgY levels between egg yolks. Ac- cording to Najafzadeh et al. (2011), purple cone�ower can be e�ective in improving poultry immunity. Yamada et al. (2011) found purple cone�ower extract to increase IgA, IgG, and IgM levels. In our study, we did not �nd a conclusive e�ect of adding purple cone�ower to the feed of ‘Greenleg Partridge’ hens kept in accordance with the rules of organic farming. Likewise, Gurbuz et al. (2010) reported no unequivocal e�ect of purple cone�ower extract on the level of speci�c antibodies in 60-day-old Hy- Line pullets. In contrast, Chaves et al. (2007) and Ma et al. (2009) showed the number of speci�c antibodies to increase following purple cone�ower supplementation. Immune system e�ciency is determined, among others, from the level of lym- phocytes (Yurong et al., 2005). According to Wang et al. (2000), lymphocytes play a very signi�cant role in the body’s immune response, and a change in the proportion of lymphocyte subclasses alters the immune function. Yamada et al. (2011) reported purple cone�ower extract to increase the level of lymphocytes. Jurkstiene et al. (2004) found that purple cone�ower supplement increased the leukocyte and lymphocyte counts. According to Bany et al. (2003), purple cone�ower extract in�uences the level of granulocytes and lymphocytes. Our study showed the e�ect of supplemental purple cone�ower on the level of blood heterophils and lymphocytes in Z-11 hens. One of the parameters indicative of avian immunity is the heterophil to lymphocyte ratio (H:L). �e purple cone�ower supplement used in our study reduced the H:L ratio in layers, which may suggest that they were less stressed, more resistant, and showed better welfare compared to control hens. Similarly, the H:L ratio was lower in chicks hatched from eggs of hens receiving purple cone�ower diets. During the production process, poultry is constantly exposed to stress factors, in- cluding those related to the rearing system. Hangalapura et al. (2004a,b) hold that the cellular immune response becomes activated under chronic stress. Stress in laying hens decreased the number of white blood cells (Mashaly et al., 2004), but this can be counteracted through dietary modi�cation, e.g., the addition of herbal immunostim- ulants. Goel et al. (2002) concluded that biologically active substances found in purple cone�ower can be used to improve the body’s immune potential and to increase mac- rophage activity. However, our study showed no e�ect of dietary purple cone�ower on selected blood count parameters of organically raised ‘Greenleg Partridge’ hens. In summary, the organically raised ‘Greenleg Partridge’ hens and chicks responded to the purple cone�ower supplement by changes in blood picture, which are impor- S elected blood param eters in organically raised hens fed w ith a purple coneflow er supplem ented diet Ew a S os nó w ka -C za jk a, Iw on a S ko m or uc ha 76 tant indicators of avian health and condition. 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S elected blood param eters in organically raised hens fed w ith a purple coneflow er supplem ented diet Ew a S os nó w ka -C za jk a, Iw on a S ko m or uc ha 78 Yamada, K., Hung, P., Park, T.K., Park, P.J., Lim, B.O. (2011). A comparison of the immunostimulatory e�ects of the medicinal herbs Echinacea, Ashwagandha and Brahmi. Journal of Ethnopharmacology, 137(1), 231–235. DOI: 10.1016/j.jep.2011.05.017. Yunis, R., Ben-David, A., Heller, E.D., Cahaner, A. (2000). Immunocompetence and viability under com- mercial conditions of broiler groups di�ering in growth rate and in antibody response to Escherichia coli vaccine. Poultry Science, 79, 810–816. Yurong, Y., Ruiping, S., Shimin, Z., Yibao, J. (2005). E�ect of probiotics on intestinal mucosal immunity and ultrastructure of cecal tonsils of chickens. Archives of Animal Nutrition, 59(4), 237–246. Abstract �e aim of the study was to determine the e�ect of dietary addition of dried purple cone�ower (Echinacea purpurea (L.) Moench) on selected blood parameters of chicks and hens of the native ‘Greenleg Partridge’ breed (Z-11), maintained on a certi�ed poultry farm according to organic farming principles. Birds were divided into two groups according to diet: organic layer diet (group 1), and the same diet supplemented with certi�ed purple cone�ower from 20 weeks of age at 10 g/kg feed (group 2). Layers were fed the certi- �ed layer diet (17% protein and 11.1 MJ ME/kg diet) following organic farming principles. Experimental incubations were performed at 34 weeks of age. Blood was collected from laying hens at 34 weeks and from newly hatched chicks at one day of age to determine selected blood parameters. �e organically raised ‘Greenleg Partridge’ hens and chicks responded to the purple cone�ower supplement by changes in their blood picture, which are important indicators of avian health and condition. As in Z-11 chicks, purple cone- �ower supplemented to the layer diet reduced the H:L ratio and induced a tendency for a higher level of the circulating immunoglobulin complex and IgG in blood, which shows their better living comfort and higher immunity. Dietary purple cone�ower was observed to have no e�ect on the level of haematocrit, erythro- cytes, and leukocytes. High individual variation occurred for the circulating immunoglobulin complex and IgG in the organically raised ‘Greenleg Partridge’ hens. Key words: blood parameters, chicks, hens, immunity, organic production, purple cone�ower Received: [2018.07.12] Accepted: [2018.12.20] Kształtowanie się wybranych parametrów krwi kur utrzymywanych zgodnie z założeniami rolnictwa ekologicznego i żywionych paszą z dodatkiem jeżówki purpurowej Streszczenie Celem badań była ocena wpływu dodatku do paszy suszonej jeżówki purpurowej (Echinacea purpurea (L.) Moench) na kształtowanie się wybranych parametrów krwi piskląt oraz kur rasy rodzimej ‘Zielononóżka Kuropatwiana’ (Z-11) utrzymywanych zgodnie z założeniami rolnictwa ekologicznego na certy�kowanej fermie drobiu. Ptaki przydzielono do dwóch grup zróżnicowanych pod względem diety: nioski żywiono ekologiczną mieszanką paszową dla kur nieśnych (grupa 1), a w grupie 2 zastosowano od 20 tygodnia życia dodatek do paszy certy�kowanej jeżówki purpurowej w ilości 10 g/kg paszy. Nioski żywiono certy�kowaną mieszanką paszową dla kur nieśnych zgodnie z założeniami rolnictwa ekologicznego o zawartości białka 17% i energii metabolicznej wynoszącej 11,1 MJ/kg paszy. W 34 tygodniu odchowu przeprowadzono lęgi doświadczalne. Również w 34 tygodniu odchowu pobrano krew od kur nieśnych oraz w pierwszym dniu życia nowowyklutych piskląt w celu oznaczenia wybranych parametrów krwi. Reakcją na podanie jeżówki purpurowej były zmiany w obrazie krwi kur oraz piskląt rasy ‘Zielononóżka Kuropatwiana’, utrzymywanych zgodnie z założeniami rolnictwa ekologicznego, będące ważnymi wskaźnikami zdrowotności i kondycji zwierząt. Dodatek do paszy dla kur nieśnych jeżówki purpurowej wpłynął podobnie jak w przypadku pi- skląt Z-11 na zawężenie stosunku H:L, a także na wystąpienie tendencji do wyższego poziomu kompleksu immunoglobulinowego oraz IgG we krwi, co wskazuje na ich lepszy komfort bytowy i większą odporność. 79 Nie wykazano natomiast wpływu jeżówki purpurowej w paszy na kształtowanie się poziomu hematokrytu, erytrocytów oraz leukocytów. Stwierdzono występowanie bardzo dużej zmienności osobniczej w przypad- ku kompleksu immunoglobulinowego oraz IgG u kur rasy ‘Zielononóżka Kuropatwiana’ w chowie ekolo- gicznym. Słowa kluczowe: parametry krwi, pisklęta, kury, odporność, produkcja ekologiczna, jeżówka purpurowa Information on the authors 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 focuses also on poultry welfare and the technology of poultry production. S elected blood param eters in organically raised hens fed w ith a purple coneflow er supplem ented diet Ew a S os nó w ka -C za jk a, Iw on a S ko m or uc ha 80 Appendix 1 Fig. 1. �e level of the circulating immunoglobulin complex [g/dl]; the error bars denote the standard deviation (± SD) of the mean value; n = 15 Fig. 2. �e level of IgG [mg/ml]; the error bars denote the standard deviation (± SD) of the mean value; n = 15 81 Fig. 3. Heterophil to lymphocyte ratio – H:L; signi�cant di�erences between the treatments in groups in hens and chicks are indicated with di�erent letters (a, b – p < 0.05, Duncan test); the error bars denote the standard deviation (± SD) of the mean value; n = 15 Fig. 4. �e level of the leukocytes [thous/µl]; the error bars denote the standard deviation (± SD) of the mean value; n = 15 S elected blood param eters in organically raised hens fed w ith a purple coneflow er supplem ented diet Ew a S os nó w ka -C za jk a, Iw on a S ko m or uc ha 82 Fig. 5. �e level of the erythrocytes [M/µl]; the error bars denote the standard deviation (± SD) of the mean value; n = 15 Fig. 6. �e level of the haemoglobin [%]; the error bars denote the standard deviation (± SD) of the mean value; n = 15