60 Annales Universitatis Paedagogicae Cracoviensis Studia Naturae, 3 (supplement): 60–66, 2018, ISSN 2543-8832 DOI: 10.24917/25438832.3supp.8 Iwona Skomorucha*, Ewa Sosnówka-Czajka Department of Poultry Breeding, National Research Institute of Animal Production, 32-083 Balice n. Kraków, Poland, *iwona.skomorucha@izoo.krakow.pl The effect of mixed herb extracts on selected stress parameters in broiler chickens of three genetic lines Introduction Stress is present in every living animal organism as a result of stimulation from envi- ronmental factors. It is an integral part of life, vital to life, and the normal adaptation of the body (Selye, 1936). Stress most o�en occurs due to unfavourable living conditions or a persisting situation that causes it (stressor), where the problem cannot be solved. Although stress is a response assisting the body in adapting to ambient conditions, it reduces welfare and thus productivity of animals, and o�en has a negative e�ect on the quality of animal products (Puvadolpirod, �axton, 2000b). In states of stress, the sympathetic-adrenal-medullary, hypothalamo-pituitary-ad- renal, and hypothalamo-pituitary-thyroid systems are stimulated. �e levels of cat- echolamines (Post et al., 2003), corticosterone (Puvadolpirod, �axton, 2000a,b) and the heterophil to lymphocyte ratio increase in blood (Scanes, 2016). Intensive livestock production contributes to stress, behavioural and physiological abnormali- ties, as well as many injuries and diseases of animals. �at is why, in many countries, notably the European Union, e�orts are made to make extreme systems of livestock production more welfare friendly. Currently, particular interest is given to medicinal plants and herbal preparations, which show broad and diverse biological and health properties. Scienti�c literature reports many studies concerning the bene�cial e�ect of dried herbs as a biocomponent of concentrate feeds or as extracts and infusions on the gastrointestinal, endocrine, and immune systems (Nasir, Grashorn, 2010; Wallace et al., 2010). Furthermore, they also have bacteriostatic, antiviral, sedative, and oxida- tion-reduction action (Wang et al., 2008; Wallace et al., 2010; Voljč et al., 2013). �e increased interest in medicinal plants and their potential use in animal husbandry is also due to the fact that they are relatively safe and require no withdrawal period. Skomorucha and Sosnówka-Czajka (2013) and Zhang et al. (2013) reported that some The effect of m ixed herb extracts on selected stress param eters in broiler chickens of three genetic lines 61 herbs and herbal extracts may reduce the body’s physiological response to stress asso- ciated with adverse housing conditions. �erefore, the aim of the study was to determine the e�ect of adding a mixture of anti-stress herbs to drinking water on the level of stress hormones (noradrenaline, adrenaline, corticosterone) and the heterophil to lymphocyte ratio in the blood of intensively reared broiler chickens of three genetic lines. Material and methods �e experiments were performed at a poultry farm of the National Research Institute of Animal Production in Aleksandrowice in Poland with 360 broiler chickens of three ge- netic groups. On the �rst day of life, broiler chicks were weighed, tagged, and divided into group I – ‘Ross 308’, group II – ‘Cobb 500’, and group III – ‘Hubbard Flex’ broilers. Each group had three subgroups. Birds were kept for 42 days on litter at a stocking density less than 33 kg/m2. All groups had uniform environmental (temperature, air humidity, light regime) and feeding conditions. Birds were fed ad libitum starter (ME 3000 kcal, CP 21.0%), grower (ME 3100 kcal, CP 19.8%), and �nisher diets (ME 3100 kcal, CP 18.5%) based on concentrates at 1–3, 4–5 and 6 weeks of age, respectively. Birds had free access to water drinkers throughout the study. In each group from 21 to 35 days of rearing, water drinkers were supplemented for 5 h/day (08:00–13:00 hours) with a tincture from mixed herbs at 2 ml/l water. �e herb mixture contained 30% chamomile (Matricaria chamomilla L.), 10% oregano (Origanum vulgare L.), 10% yarrow (Achillea millefolium L.), 10% knotgrass (Poligonum aviculare L.), 10% valerian (Valeriana o�cinalis L.), and 20% in�orescence of large-leaved lime (Tilia platyphyllos L.). �is mixture was selected based on the present authors’ earlier research. �e tinc- ture from mixed herbs was made by a professional herbal company and has been cer- ti�ed to conform to the company’s quality standards. At 21, 28, 35, and 42 days of age, blood was collected from 10 birds per group to determine the levels of corticosterone (by enzyme immunoassay), noradrenaline, and adrenaline (by radioimmunoassay). Blood smears were made from the collected blood to count lymphocytes and heter- ophils and to calculate the heterophil to lymphocyte ratio (H:L). Lymphocytes and heterophils were counted under a Nikon YS 100 microscope a�er May-Grünwald and Giemsa staining. �e results were statistically analysed by an analysis of variance, and signi�cant di�erences were estimated with Duncan’s test. �e statistical calculations were per- formed with Statgraphics plus 6.0. E�ects were considered signi�cant at a probability of p ≤ 0.05 and p ≤ 0.01. Iw on a S ko m or uc ha , E w a S os nó w ka -C za jk a 62 Tab. 1. �e level of stress hormones in the blood of broiler chickens; values shown as di�erent letters within the line di�er signi�cantly according to the Duncan test at p ≤ 0.05; n = 10 Hormones [ng/ml] Days of rearing excrement Group Ross 308 Cobb 500 Hubbard Flex SEM Noradrenaline 21 121.56 132.16 146.68 27.94 28 64.54 70.58 118.34 18.83 35 87.94 101.49 122.68 16.12 42 99.93a 50.31b 98.78a 16.69 Adrenaline 21 53.18 146.44 133.34 39.66 28 78.96 60.67 127.39 25.93 35 78.38 154.85 164.72 30.50 42 76.19 34.03 64.17 17.90 Corticosterone 21 11.85 14.03 12.14 2.56 28 17.10 17.33 15.58 4.33 35 14.93 18.74 15.98 2.15 42 19.65 19.41 20.09 2.39 Note: group I – ‘Ross 308’, group II – ‘Cobb 500’, and group III – ‘Hubbard Flex’ broilers; SEM – standard error of the mean Results On day 42 of the experiment, the noradrenaline concentration was lowest in the blood of ‘Cobb 500’ compared to ‘Ross 308’ and ‘Hubbard Flex’ broilers (p ≤ 0.05) (Tab. 1). No statistically signi�cant di�erences in the adrenaline and corticosterone levels were found between the experimental groups. On day 21 of age, ‘Cobb 500’ broiler chick- ens were characterised by the highest heterophil percentage and lowest lymphocyte percentage compared to the other genetic groups (p ≤ 0.01), which translated into a di�erence in the H:L ratio between these groups (p ≤ 0.01) (Tab. 2). On day 42 of age, the di�erence in the percentage of heterophils and lymphocytes, and also in the H:L ratio became apparent between ‘Ross 308’ and ‘Cobb 500’ chickens (p ≤ 0.01). Discussion A decisive role in the stress mechanism, or the body’s response to environmental stim- uli, is played, among others, by the stress hormones corticosterone, adrenaline, and noradrenaline (Puvadolpirod, �axton, 2000a; Pohle, Cheng, 2009). Catecholamines (adrenaline and noradrenaline) are involved in numerous physiological and patho- logical processes, and their release into the bloodstream may increase blood pressure, heart beat, and muscle tension, in addition to increasing blood glucose levels by stim- ulating the conversion of hepatic glycogen to glucose (Virden et al., 2007; Kober et al., 2010). The effect of m ixed herb extracts on selected stress param eters in broiler chickens of three genetic lines 63 Tab. 2. Heterophil and lymphocyte levels (%) in the blood of broiler chickens; values shown as di�erent letters within the line di�er signi�cantly according to the Duncan test at p ≤ 0.01; n = 10 Item Days of rearing Group Ross 308 Cobb 500 Hubbard Flex SEM heterophils 21 33.90a 47.30b 34.70a 2.23 lymphocytes 61.00a 46.30b 60.80a 1.85 H:L 0.57a 1.04b 0.58a 0.06 heterophils 28 29.20 31.80 34.80 2.61 lymphocytes 64.90 60.50 58.70 3.18 H:L 0.47 0.56 0.64 0.07 heterophils 35 41.90 37.70 35.30 3.99 lymphocytes 52.80 55.00 58.50 4.11 H:L 1.35 0.71 0.65 0.37 heterophils 42 37.00a 21.10b 27.90 3.17 lymphocytes 56.10a 71.10b 62.60 3.47 H:L 0.74a 0.31b 0.49 0.09 Note: group I – ‘Ross 308’, group II – ‘Cobb 500’, and group III – ‘Hubbard Flex’ broilers; SEM – standard error of the mean Cheng et al. (2001) and Pohle and Cheng (2009) report that changes in adrena- line and noradrenaline blood levels may serve as an indicator of welfare and coping with stress. In our study, ‘Cobb 500’ broiler chickens responded most favourably to drinking water with a mixed herb extract, which was re�ected in the lowest level of noradrenaline and a tendency for lower blood adrenaline compared to ‘Ross 308’ and ‘Hubbard Flex’ chickens. �e bene�cial e�ect of adding mixed herbs to water on ‘Cobb 500’ chickens was also re�ected in the lower H:L ratio, especially when compared with ‘Ross 308’ chickens. Likewise, Naja� and Torki (2010) found a signi�cant decrease in the amount of heterophils and an increase in the amount of lymphocytes in the blood of broiler chickens in response to a diet with herbs. In turn, Yesilbag et al. (2012) showed increased heterophil percentage as well as increased H:L percentage ratio in the blood of birds fed a diet supplemented with an extract from oregano and rosemary essential oils compared to the control group. Long-term stress results in the corticos- terone stress hormone being released into the blood of birds (Virden et al., 2007). A  high level of this hormone adversely a�ects the body and may induce cardiovascu- lar diseases, hypercholesterolemia, damage to the digestive tract, changes in immune function, as well as metabolic changes (Puvadolpirod, �axton, 2000c; Virden et al., 2007). Kannan et al. (1997) report that a lower blood corticosterone concentration is generally indicative of the body’s lower susceptibility to stressors. However, in our study, we did not observe any e�ect of supplementing the herb mixture on a lower lev- el of this hormone in the blood in any of the chickens from the experimental groups. In summary, the herb extract had the most favourable e�ect on relieving the body’s physiological response to stress, and thus on improving the welfare of ‘Cobb 500’ com- Iw on a S ko m or uc ha , E w a S os nó w ka -C za jk a 64 pared to ‘Ross 308’ and ‘Hubbard Flex’ broilers. It can therefore be considered that active substances in herbs have varying e�ects on the body of broiler chickens of dif- ferent origin. References Cheng, H.W., Dillworth, G., Singleton, P., Chen, Y., Muir, W.M. (2001). E�ects of group selection for productivity and longevity on blood concentrations of serotonin, catecholamines, and corticosterone of laying hens. Poultry Science, 80, 1278–1285. DOI: 10.1093/ps/80.9.1278 Kannan, G., Heath, J.L., Wabeck, C.J., Souza, M.C.P., Howe, J.C., Mench, J.A. (1997). E�ects of crating and transport on stress and meat quality characteristics in broilers. Poultry Science, 76, 523–529. DOI: 10.1093/ps/76.3.523 Kober, A.K.M.H., Aoyama, M., Sugita, S. (2010). Immunohistochemical localization of catecholamine biosynthetic enzymes in the adrenal gland of the domestic fowl (Gallus domesticus). Poultry Science, 89, 1709–1715 . DOI: 10.3382/ps.2009-00588 Naja�, P., Torki, M. (2010). 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DOI: 10.3382/ps.2013-03081 Abstract �e aim of the study was to determine the e�ect of adding a mixture of anti-stress herbs to drinking water on stress hormone levels and the heterophil to lymphocyte ratio in the blood of intensively reared broiler chickens of three genetic lines. �e experiment was performed with ‘Ross 308’, ‘Cobb 500’, and ‘Hubbard Flex’ broiler chickens, which were divided into three experimental groups. Birds were maintained on litter for 42 days at a stocking density less than 33 kg/m2. All groups were provided with the same environmental and feeding conditions. In each group from 21 to 35 days of rearing, water drinkers were supplemented for 5 h/day (08:00–13:00 hours) with an alcoholic extract from mixed herbs (30% chamomile, 10% oregano, 10% yarrow, 10% knotgrass, 10% valerian, 20% in�orescence of large-leaved lime) at 2 ml/l water. At 21, 28, 35, and 42 days of rearing, blood was collected from 10 birds per group to determine corticosterone, noradren- aline, and adrenaline levels. �e heterophil to lymphocyte ratio (H:L) was also calculated. On day 42 of the study, 500 broiler chickens were characterised by the lowest noradrenaline concentration and a lower H:L ratio compared to ‘Ross 308’ and ‘Hubbard Flex’ (p ≤ 0.05) and ‘Ross 308’ chickens (p ≤ 0.01), respectively. It was concluded from the study that the herb extract had the most favourable e�ect on relieving the body’s physiological response to stress, and thus on improving welfare in ‘Cobb 500’ compared to ‘Ross 308’ and ‘Hubbard Flex’ broilers. It can therefore be considered that active substances in herbs have varying e�ects on the body of broiler chickens of di�erent origin. Key words: adrenaline, broiler chickens, corticosterone, H:L, mixed herb extract, noradrenaline Received: [2018.06.26] Accepted: [2018.12.12] Wpływ ekstraktu z mieszanki ziół na wybrane parametry stresu kurcząt brojlerów trzech linii genetycznych Streszczenie Celem badań było określenie wpływu dodatku do wody pitnej ekstraktu z mieszanki ziół wykazujących wła- ściwości antystresowe na poziom hormonów stresu oraz stosunek hetero�lii do limfocytów we krwi kurcząt brojlerów trzech linii genetycznych utrzymywanych w systemie intensywnym. Doświadczenie przeprowa- dzono na kurczętach brojlerach: ‘Ross 308’, ‘Cobb 500’ oraz ‘Hubbard Flex’, które przydzielono do trzech grup doświadczalnych. Ptaki utrzymywano przez okres 42 dni, na ściółce o obsadzie nie przekraczającej 33 kg/m2. Wszystkie grupy miały ujednolicone warunki środowiskowe oraz żywieniowe. W każdej grupie od 21 do 35 dnia odchowu przez 5 h/dobę (od 8.00–13.00) dodawano do poideł z wodą ekstrakt spirytusowy z mieszanki ziół (30% rumianku pospolitego, 10% ziela lebiodki pospolitej, 10% ziela krwawnika pospolite- go, 10% ziela rdestu ptasiego, 10 % kozłka lekarskiego, 20% kwiatostanu lipy szerokolistnej) w ilości 2 ml/l wody. W 21, 28, 35 oraz 42 dniu odchowu pobrano krew od 10 ptaków z grupy i oznaczono poziom: korty- kosteronu, noradrenaliny i adrenaliny a także obliczono stosunek hetero�lii do limfocytów (H:L). Kurczęta brojlery ‘Cobb 500’ charakteryzowały się w 42 dniu doświadczenia najniższym poziomem noradrenaliny oraz węższym stosunkiem H:L w porównaniu odpowiednio z kurczętami ‘Ross 308’ i ‘Hubbard Flex’ przy Iw on a S ko m or uc ha , E w a S os nó w ka -C za jk a 66 p ≤ 0,05 i kurczętami ‘Ross 308’ przy p ≤ 0,01. Na podstawie badań stwierdzono, że najkorzystniejszy wpływ podawanego ekstraktu z mieszanki ziół na łagodzenie �zjologicznej reakcji organizmu na stres, a tym sa- mym na poprawę dobrostanu stwierdzono w przypadku kurcząt brojlerów ‘Cobb 500’ w porównaniu z kur- czętami ‘Ross 308’ i ‘Hubbard Flex’. Można uznać, że substancje czynne zawarte w ziołach mają zróżnicowa- ny wpływ na organizm kurcząt brojlerów różnego pochodzenia. Słowa kluczowe: adrenalina, ekstrakt z mieszanki ziół, H:L, kortykosteron, kurczęta brojlery, noradren- alina Information on the authors 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. 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.