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Evaluation of a stress model induced by dietary corticosterone supplementation in broiler breeders: effects on egg yolk corticosterone concentration and biochemical blood parameters a

a

E. Babacanoğlu , S. Yalçin & S. Uysal

b

a

Faculty of Agriculture, Department of Animal Science, Ege University, Bornova İzmir, Turkey b

Department of Biochemistry, Dokuz Eylül University, İzmir, Turkey Accepted author version posted online: 26 Sep 2013.Published online: 08 Jan 2014.

To cite this article: E. Babacanoğlu, S. Yalçin & S. Uysal (2013) Evaluation of a stress model induced by dietary corticosterone supplementation in broiler breeders: effects on egg yolk corticosterone concentration and biochemical blood parameters, British Poultry Science, 54:6, 677-685, DOI: 10.1080/00071668.2013.847901 To link to this article: http://dx.doi.org/10.1080/00071668.2013.847901

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British Poultry Science, 2013 Vol. 54, No. 6, 677–685, http://dx.doi.org/10.1080/00071668.2013.847901

Evaluation of a stress model induced by dietary corticosterone supplementation in broiler breeders: effects on egg yolk corticosterone concentration and biochemical blood parameters E. BABACANOĞLU, S. YALÇIN

AND

S. UYSAL1

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Faculty of Agriculture, Department of Animal Science, Ege University, Bornova İzmir, Turkey, and 1Department of Biochemistry, Dokuz Eylül University, İzmir, Turkey

Abstract 1. This study aimed to evaluate a stress model induced by corticosterone (CORT) supplementation in the diet of broiler breeder hens. 2. A total of 60 Ross broiler breeder hens at 29 weeks of age were randomly divided into 4 groups with 15 hens each. The first group served as the control. The rest of the hens were given 1, 1.5 or 2 mg of CORT/ hen/d (CORT1, CORT1.5 and CORT2, respectively) for 7 d. Concentrations of yolk CORT, plasma uric acid, glucose, cholesterol, creatine kinase, heterophil (H):lymphocyte (L) ratio and duration of tonic immobility (TI) were measured at d 3, 5 and 7 of CORT supplementation. The same measurements were repeated at 3, 5 and 7 d after CORT was withdrawn from the diet. 3. There were no significant CORT dose effect on yolk CORT and plasma glucose concentrations. Higher plasma uric acid and H:L ratio was obtained for CORT1.5 and CORT2 than for CORT1. From 3 to 7 d of dietary CORT supplementation, yolk CORT and plasma uric acid concentrations and H:L ratio increased whereas plasma glucose concentration decreased. After CORT was withdrawn from the diet, the H:L ratio remained elevated. The duration of TI and plasma creatine kinase concentration did not change during and after CORT supplementation. 4. Yolk CORT concentration was correlated with plasma uric acid concentration during CORT supplementation. 5. The results suggest that dietary CORT supplementation could be used as a stress model and to evaluate hormone-mediated maternal effects in broiler breeder hens.

INTRODUCTION Organisms re-establish homeostatic conditions when confronted by stressors. Glucocorticoids participate in the control of whole-body homeostasis and response to stress (Lin et al., 2004). Plasma corticosterone (CORT), which is the main glucocorticoid in birds (Romero et al., 1998), increases rapidly from baseline concentration in response to stress stimuli (Rosales, 1994; Veenema et al., 2003; Romero and Reed, 2005, 2008). Stress also induces several physiological responses, such as increases in plasma glucose, uric acid and

heterophil (H) to lymphocyte (L) ratio (Maxwell, 1993; Puvadolpirod et al., 2000b; Lin et al., 2004). Furthermore, tonic immobility (TI) duration, which is an indicator of fearfulness, increases when plasma CORT is elevated (Jones et al., 1988). Groothuis and Schwabl (2008) reported that maternal hormones (CORT, progesterone, androstenedione, testosterone and 17 ß-estradiol) are transferred into the egg. Egg CORT has received more attention because higher concentrations of CORT affect the behaviour and immunity of the offspring (Rubolini et al., 2005) and

Correspondence to: S. Yalçın, Faculty of Agriculture, Department of Animal Science, Ege University, 35100 Bornova, İzmir-Turkey. E-mail: servet. [email protected] Present address for E. Babacanoğlu: Faculty of Agriculture, Department of Animal Science, Yüzüncü Yıl University, 65080 Van, Turkey. Accepted for publication 23 July 2013.

© 2013 British Poultry Science Ltd

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impair offspring phenotype and viability (Saino et al., 2005). It was noted that 80% of the total amount of CORT in the egg was in the yolk (Royo et al., 2008). A correlation between plasma CORT and egg yolk CORT concentrations has also been reported (Downing and Bryden, 2002; Love et al., 2005; Rettenbacher et al., 2005; Downing and Bryden, 2008; Love and Williams, 2008) indicating that the hen’s condition during egg formation affects embryo development (Henriksen et al., 2011a). Experimental elevation of yolk CORT was demonstrated under stress conditions in both domestic and wild avian species (Hayward and Wingfield, 2004; Saino et al., 2005; Singh et al., 2009; Okuliarova et al., 2010). Yolk CORT accumulates from maternal plasma preferentially in the early stages of follicular development in low-stress environments and additionally in the last stages of egg formation in high-stress environments (Okuliarova et al., 2010). Singh et al. (2009) suggested that egg yolk CORT, but not albumen, can be used to measure stress in a fashion similar to H:L ratio. Broiler breeders are confronted by environmental stressors such as high-stocking density, quantitative feed and water restriction and poor litter quality (Rosales, 1994; Mench, 2002). Moreover, genetic selection, which is applied to increase the efficiency of feed and meat yield of progeny, has also increased the sensitivity of broiler breeders to stress (Rauwa et al., 1998; de Jong et al., 2002). Although stress models for broilers (Puvadolpirod et al., 2000b; Post et al., 2003b) and laying hens (Mumma et al., 2006) were studied by infusion of adrenocorticotropin, or dietary CORT, a stress model for broiler breeders does not exist. Therefore, this study aimed to evaluate a stress model induced by CORT supplementation in the broiler breeder diet. For this purpose, egg yolk CORT concentration was determined, blood physiological parameters were measured to investigate if the diet resulted in marked changes, and TI was used to measure fear behaviour during CORT supplementation. Measurements were repeated after CORT was withdrawn from the diet to estimate the duration of effect of the dietary supplementation with CORT.

MATERIALS AND METHODS This study was approved by the Ege University Animal Use and Care Committee (permit number 2007–41). Husbandry and feeding The experiment was conducted using 60 Ross broiler breeder hens at 29 weeks of age that were placed into individual cages

(48 × 40 × 45 cm). Breeder hens were given 145 g/d of a diet containing 165 g of crude protein/kg and 10.68 MJ ME/kg. Water was provided ad libitum and the lighting regime was 16 light:8 dark during the experiment. Broiler breeder hens were divided into 4 treatment groups with 15 replicate hens in each treatment group. The first group served as the control. The daily feed ration fed to the treatment groups of hens were supplemented with 1, 1.5 or 2 mg CORT/hen/d (CORT1, CORT1.5 and CORT2, respectively) for 7 d. For each hen, the dose of CORT (Sigma C2505 corticosterone, 92%, Sigma Aldrich, Inc., USA) was dissolved in 1 ml of 99% ethanol (Post et al., 2003a, 2003b) and mixed into the feed ration. One ml of ethanol was added daily to the feed ration of hens in the control group. Experimental design did not include a control group without ethanol, because harmful effects of low concentrations of ethanol (it was 0.227 ml/kg of hen per day) for short periods have not been reported (Peebles et al., 1996). Traits measurement Eggs and blood were sampled from CORT dose groups on 3, 5 and 7 d of CORT supplementation while samples from the control were taken on d 3 only. Samples were collected after TI measurement from 9 hens which laid eggs between 09:00 and 11:00 on the sampling day. The yolk was separated from the egg, homogenised and a 30 µl yolk sample was transferred to a 1.5 ml tube after 1 ml of pure water was added and stored at −20°C until CORT was assayed. Blood was taken via lithium tubes, within 3 min after handling to avoid handling stress, from the brachial vein of 9 hens. Blood was centrifuged (3–18 V Sigma) for 10 min at 4°C (4500 × g) and plasma was stored at −20°C for further analyses. One drop of blood was stained using WrightGiemsa staining solution, approximately 4 h after methyl alcohol fixation to identify leucocyte differentiation. The H and L were determined by counting 100 cells by light microscopy (Gross and Siegel, 1983) and the H:L ratio was calculated. TI reaction was carried out in a separate room. The hen was placed on its back with the head hanging in a U-shaped wooden cradle and restrained with one hand on its sternum for 10 s. If the hen remained immobile for 10 s after removal of the hand, a stopwatch was started to record latencies until the hen righted itself. If the hen righted itself in less than 10 s it was considered that TI had not been induced, and the restraint procedure was repeated. If the hen did not show a righting response over the 10 min test period, a maximum score of 600 s was given for duration (Jones, 1986; Campo and Davila, 2002).

A STRESS MODEL IN BROILER BREEDERS

After CORT was withdrawn from the feed (after CORT), the same procedures were repeated at 3, 5 and 7 d to collect yolk and blood samples and to measure TI duration of hens. Samples from control hens were also taken on d 3 after CORT was withdrawn from the feed.

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CORT analysis The extraction process was performed in accordance with the method used in the commercial ELISA kit (Schwabl, 1993; Verboven et al., 2003; Rettenbacher et al., 2005; Von Engelhardt and Groothuis, 2005). A yolk sample (1.5 µl) was transferred to a tube, 1 ml of distilled water was added and was stored at −20°C until extraction. Samples were thawed and placed in a 5 ml glass tube and 1 ml of ethyl-acetate was added. The solvent was dried down under nitrogen until approximately 2 ml of the solvent remained and was then transferred to a 12 × 75 mm glass assay tube and dried completely. After drying, 250 µl of Assay buffer (1 ml Assay buffer: 9 ml water) was added. The solvent was shaken for 30 s and rested for 5 min. Samples were transferred to tubes and stored at −80°C until CORT assay. Yolk CORT concentration was determined by ELISA as described by Hau et al. (2001), using a commercial kit (Correlate-EIA Corticosterone Enzyme Immunoassay Kit Assay Designs Inc. Ann Arbor, MI). The sensitivity of the assay was 0.0040 ng/ml; the intra-assay and inter-assay coefficients of variation were 11.2% and 14.1%, respectively. Plasma biochemical analyses Plasma glucose, creatine kinase, cholesterol and uric acid concentrations were measured spectrophotometrically with an autoanalyser (Arhitecth C160059 and C16001902, Abbott Clinical Chemistry Arhitecth and Aeroset System, Abbott Laboratories, USA) (Malheiros et al., 2003). Plasma biochemical analyses were performed using commercial kits (Arhitecth Aeroset 7D76 Uric Acid Reagent Kit, Arhitecth Aeroset 3L82-20 Glucose Reagent Kit, Arhitecth Aeroset 7D63 Creatine Kinase Reagent Kit and Arhitecth Aeroset 7D62 Cholesterol Reagent Kit). Statistical analysis Data of CORT supplemented groups for the periods during CORT supplementation and after CORT was withdrawn from the diet were analysed by repeated measures methods using MIXED Models Procedures of SAS (2013). The best model was selected based on the smallest AIC (Akaike’s information criterion) and BIC (Bayesian information criterion), values which

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were obtained from covariance pattern model including Toeplitz. Preliminary analyses showed that interaction between CORT dose and day was not significant, except for H:L ratio, and thus the model included main effects of CORT dose and day. The model for H:L ratio included main effects of CORT dose and day and the interaction between them. The significance of linear and quadratic responses of day was determined for all variables. Biochemical blood parameters and TI were transformed to Log10 before being subjected to analyses. However, actual means are presented in the tables and figures. A second analysis was performed to determine differences between CORT dose groups and control on 3 d of CORT supplementation using one-way ANOVA. This analysis was repeated for data obtained after CORT was withdrawn from the diet. Comparisons of means were performed by the Student’s t-test. Statistical significance was based on P < 0.05. Mean values obtained from each broiler breeder hen during CORT supplementation were used to calculate pairwise Pearson’s correlation coefficients to evaluate the relationships between yolk CORT and blood parameters.

RESULTS During CORT supplementation Yolk CORT concentration was similar among CORT dose groups during CORT supplementation (Table 1). Yolk CORT concentration increased quadratically, being lower on d 3 than on d 5 and 7. On d 3 of CORT supplementation, yolk CORT concentration of CORT dose groups

Table 1. Least squares means and standard errors (mean ± SEM) for yolk corticosterone (CORT) concentration during CORT supplementation and after CORT was withdrawn from the diet for CORT dose (CORT) and day (n = 9 hens/CORT dose/day) Yolk CORT, ng/ml Effect CORT, mg/hen/day

Days

During CORT 1 1.5 2 3 5 7

Statistical analysis (P values) CORT Days Linear Quadratic a,b

3.06 3.25 3.25 2.95 3.36 3.26

± ± ± ± ± ±

0.500 0.010 0.021 0.039

0.09 0.09 0.09 0.09b 0.09a 0.09a

CORT withdrawal 2.41 2.52 2.52 2.84 2.30 2.31

± ± ± ± ± ±

0.06 0.06 0.06 0.07a 0.07b 0.07b

0.569 0.01).

DISCUSSION It was already shown that the CORT administration via drinking water and in a broiler diet may provide a useful model to study the physiological effects of stress in broilers without the need for excessive handling of chickens (Post et al., 2003b; Lin et al., 2004). This study aimed to evaluate a stress model for broiler breeders induced by CORT supplementation in the diet. Yolk CORT accumulates from maternal plasma preferentially in the early stages of follicular development in a controlled low-stress environment and also in the later stages of egg formation under stress (Okuliarova et al., 2010). Yolk CORT concentration was reported as 0.97 ng/g in quails (Hayward and Wingfield, 2004) and 0.77 and 1.61 ng/g for caged brown and white laying

1 1.5 2 3 5 7

± ± ± ± ± ±

0.3b 0.3a 0.3a 0.3b 0.3a 0.3a

0.004

Evaluation of a stress model induced by dietary corticosterone supplementation in broiler breeders: effects on egg yolk corticosterone concentration and biochemical blood parameters.

1. This study aimed to evaluate a stress model induced by corticosterone (CORT) supplementation in the diet of broiler breeder hens. 2. A total of 60 ...
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