British Poultry Science
ISSN: 0007-1668 (Print) 1466-1799 (Online) Journal homepage: http://www.tandfonline.com/loi/cbps20
Effects of a blend of essential oils and overcrowding stress on the growth performance, meat quality, and heat shock protein gene expression of broilers S. M. Hosseini, H. Farhangfar & R. Nourmohammadi To cite this article: S. M. Hosseini, H. Farhangfar & R. Nourmohammadi (2017): Effects of a blend of essential oils and overcrowding stress on the growth performance, meat quality, and heat shock protein gene expression of broilers, British Poultry Science, DOI: 10.1080/00071668.2017.1390209 To link to this article: http://dx.doi.org/10.1080/00071668.2017.1390209
Accepted author version posted online: 09 Oct 2017.
Submit your article to this journal
Article views: 1
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=cbps20 Download by: [UNIVERSITY OF ADELAIDE LIBRARIES]
Date: 11 October 2017, At: 00:41
CBPS-2016-515
Publisher: Taylor & Francis & British Poultry Science Ltd
Ed. Kjaer, September 2017;
Journal: British Poultry Science
ip t cr
us
Effects of a blend of essential oils and overcrowding stress on the growth performance,
an
meat quality, and heat shock protein gene expression of broilers
M
S. M. HOSSEINI1, H. FARHANGFAR1 AND R. NOURMOHAMMADI2 Running Head: Essential oils and overcrowding stress
Department of Animal Sciences, College of Agriculture, University of Birjand,
ed
1
Birjand 97175 and 2Young Researchers and Elites Club, Birjand Branch, Islamic Azad
ce pt
University, Birjand, Iran
Ac
Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 00:41 11 October 2017
DOI: 10.1080/00071668.2017.1390209
Accepted for publication 28th August 2017
Correspondence to: Dr S. M. Hosseini, Department of Animal Sciences, College of Agriculture, University of Birjand, P.O. Box 331/97175, Birjand, Iran. E-mail: [email protected]
Abstract. 1. Overcrowding stress is common in the poultry industry. Chickens exposed to long-term stressful situations are characterised by welfare impairment and immunosuppression. 2. The present study evaluated the effects of a blend of essential oils (EOB; cinnamaldehyde and thymol) and stocking density on the performance, gut microflora, meat quality, and
ip t
3. One-day-old Ross 308 male broiler chickens (n = 360) were allocated to 4 experimental
cr
groups from d 22 to 42. Each treatment had 6 replicates of 15 chicks. Two groups were
at a low stocking density (LSD) of 10 birds/m2.
us
subjected to a high stocking density (HSD) of 20 birds/m2 and the other two groups were kept
an
4. The results of this study indicate that overcrowding stress decreased growth performance parameters, blood IgG and heterophil:lymphocyte ratio (H:L) but increased IgA and IgM
ed
the breast muscle.
M
levels. High stocking density reduced water-loss rate and pH decline at 45 min post mortem in
5. Essential oils supplementation elevated H:L ratio but decreased breast meat redness and
ce pt
pH24.
6. Significant interactions between EOB and stocking density were observed for corticosterone level (CS) and mRNA levels of heat shock protein 70 (HSP70) in brain and heart. Although HSD increased CS and HSP70 when compared to LSD, the effects of the former were inconsistent with EOB supplemented diets.
Ac
Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 00:41 11 October 2017
physiological stress markers of broilers.
7. In conclusion, dietary EOB supplementation could improve some of the biomarkers associated with overcrowding stress in broiler chickens.
Keywords: stress, broilers, gene expression, growth, immunity
INTRODUCTION
Optimal nutrition and environment must be provided for improved performance of broilers. In the contemporary broiler chicken production systems, different environmental factors can result in elevated levels of stress. Overcrowding stress is considered one of the most important stressors in broiler production due to its effects on growth performance, health, welfare status, and immune system (Gomes et al., 2014). In addition, mortality, susceptibility to diseases,
ip t
were related to stress conditions caused by overcrowding (Tong et al., 2012). Depending on
cr
the country and the production system, different stocking densities are applied in poultry
us
production. However, a high stocking density (HSD) can be stressful and can have deleterious effects on profitability and physiological parameters of broilers (Houshmand et al., 2012).
an
Several parameters could be measured as stress biomarkers in broilers, including plasma levels of corticosterone (CS), glucose, cholesterol, nitric oxide; heterophil:lymphocyte (H:L)
M
ratio and lymphoid organs weight (Munck et al., 1984). It is considered that HSD increases
ed
aggressive behaviour among birds and causes stress, thereby resulting in higher CS and cholesterol levels in chicken plasma (Post et al., 2003). Reduction in the numbers of
ce pt
lymphocytes and monocytes and enhancement in the numbers of heterophils, which leads to a higher H:L ratio, have been reported for stressed broilers (Hosseini et al., 2016). It has been demonstrated that HSD causes an imbalance in the microbiota population (Feddes et al., 2002) and can negatively influence meat quality (Osman, 1993). Heat shock proteins (HSPs) are a group of proteins that are present in cells of all living organisms but are expressed at
Ac
Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 00:41 11 October 2017
physiological stress (such as oxidative stress and inflammation), and behavioural adjustments
high levels when cells are exposed to thermal and non-thermal stressors (Soleimani et al., 2012). Previous research in broilers showed that social isolation may elicit HSP70 expression (Soleimani et al., 2012). Stress conditions appear to affect the response of broilers to different feed additives, such as plant extracts. Essential oils (EOs) are derived from plants and have distinct biological functions, such as immunostimulating effects (Hosseini et al., 2016), antimicrobial,
antifungal or antioxidant activities (Silva et al., 2012). Due to their positive effects of EO on gut microflora and antioxidant effects, it is possible that dietary supplementation with EO can help the broilers overcome any deficiency and increase their tolerance to stressful conditions. The immune status is known to play an important role in protecting the organism from various infections, and phytogenic feed additives derived from plants may increase cellular and
ip t
important role in strengthening the defence system of birds against invasion by infectious
cr
organisms. However, mechanisms that interact with the EOs have not yet been thoroughly
us
studied in broilers.
The positive effects of EOs on broiler health have already been reported, but to the best
an
of our knowledge, no information is available concerning the potential effects of these compounds in HSD reared broilers. Considering the more beneficial effects of plant extracts
M
under stressful conditions, we hypothesised that supplementation with a blend of EOs, as a
ed
beneficial feed additive, could alleviate the detrimental effects of overcrowding stress. Therefore, the present study was conducted to evaluate the efficacy of a blend of EOs (EOB)
ce pt
on finishing broilers reared in crowded conditions.
MATERIALS AND METHODS
All procedures carried out in this experiment were approved by the Animal Ethics Committee of the University of Birjand.
Ac
Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 00:41 11 October 2017
humoral immune responses of birds (Botsoglou et al., 2002). Therefore, they could play an
Blend of EOs
Enviva® EO 101, a commercial feed additive containing EOB from cinnamon (Cinnamomum
verum) and thyme (Thymus vulgaris), was used in this study. Enviva® EO is supplied as a fine white agglomerated encapsulated powder, (Danisco Animal Nutrition, Marlborough, UK), and 100 g of this product provides a guaranteed minimum of 4.5 g cinnamaldehyde and 13.5 g thymol.
Table 1 near here
Birds, diets, and management
Three hundred and sixty 21-day-old Ross 308 male broiler chicks were used in this experiment. The chicks were raised up to 21 d of age on a typical commercial broiler starter mash diet (Table 1). On d 22, broilers were individually weighed (805 ± 3.0 g) and assigned
ip t
arrangement with two levels of EOB (0 or 100 g/tonne of diet as the manufacturer
cr
recommended) and two levels of stocking density (10 birds/m2 or 20 birds/m2). The Iranian
us
Animal Welfare Commission recommends a maximum stocking density of 37 kg/m2 (0.067 m2/bird) for broilers in mechanically ventilated sheds and water-based cooling. For non-
an
mechanically ventilated sheds, the maximum is 28 kg/m2 (0.084 m2/bird). These recommendations follow the guidelines published by Barnett et al. (2008). For this
M
experiment, the birds were placed at the stocking densities (high and low) just mentioned, according to practical management conditions. The chicks were randomly allotted to 4
ed
experimental groups with 6 replicates of 15 chicks for each experimental group. The pens had
ce pt
dimensions of 1.2 m length × 0.85 m width × 0.7 m height. There was no significant difference in the body weight among groups when the chickens received the experimental diets. The basal diets were formulated based on maize and soybean meal to meet nutrient requirements of broiler chickens (Table 1; NRC, 1994). Basal diet with or without the blend of EOB was offered in mash form to the birds from d 22 of age. The birds had free access to
Ac
Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 00:41 11 October 2017
to 1 of the 4 dietary treatments. The experimental treatments consisted of a 2 × 2 factorial
feed and water and were maintained on a 23L:1D lighting program. The floor space occupied by feeders and drinkers was not calculated in the floor space provided to the birds. The
number of feeders and drinkers in each pen was constant throughout the experiment. Nipple drinkers were used for drinking water and tube feeders were used for feeding the chicks throughout the study.
Broiler performance parameters Feed intake and body weight for each replicate were recorded from 22 to 42 d. Average daily gain (ADG), average daily feed intake (ADFI), and feed conversion rate (FCR) were further calculated.
ip t
At the end of the study, on d 42, two chicks were randomly chosen from each pen, weighed,
cr
and anaesthetised with carbon dioxide and killed by cervical dislocation. Spleen, thymus, and
us
bursa of Fabricius were collected and weighed. Relative weights of lymphoid organs were
an
calculated as percentage of live weight.
Immunoglobulin concentration and stress indicators
M
At 42 d of age (end of the experiment), a 4-ml blood sample was obtained from the wing vein of 2 broilers from each replicate (12 samples per treatment), and then was collected into 2
ed
tubes (2 ml in each tube). The first tube contained heparin as the anticoagulant. One drop of
ce pt
whole blood from each sample was used to make a thin smear on a clean microscope slide. The dried smear was stained with Wright’s stain. For each slide, a total of 60 cells were counted using a light microscope and the H:L ratio was calculated. The rest of the blood sample was centrifuged (5000 g) for 10 min at 4ºC. The collected plasma was stored at −20ºC until analysed. The plasma IgG, IgM and IgA concentrations were determined using an
Ac
Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 00:41 11 October 2017
Relative weights of lymphoid organs
enzyme-linked immunosorbent assay ELISA kit from Bethyl Laboratories (Montgomery, TX, USA). The ELISA procedure was carried out according to the protocol of the manufacturer and absorbance was measured at 450 nm. The serum of blood samples in the second tube was separated and used to measure concentrations of corticosterone (CS), nitric oxide (NO), glucose and cholesterol. Serum CS and NO levels were determined using an RIA kit (Cayman Chemical Company, MI). Serum
levels of glucose and cholesterol were measured using an automated chemistry analyser (Hitachi 902 Automatic Analyzer, Hitachi, Tokyo, Japan) using colorimetric methods and following the instructions of the manufacturer of the corresponding reagent kit (Zhongsheng Biochemical Co., Ltd., Beijing, China).
ip t
The contents of small intestine (12 samples per treatment) were carefully hand-stripped into
cr
sterile containers. The survival of Lactobacillus, Salmonella, and total aerobes was
us
determined according to the procedures described by Horn et al. (1996) with some
modifications (Cengiz et al., 2015). The cfu as log10 per g for each of these bacteria within
M
Meat quality
an
digesta was counted on the basis of colony morphology and characteristics.
Immediately after slaughter, the left side of the pectoralis major (white) muscle was quickly
ed
removed for meat quality evaluation. Physicochemical characteristics of breast muscle
ce pt
samples, such as water-loss rate, pH45, pH24, and meat colour, were evaluated. Water-loss rate was estimated by determining expressible fluid using a modification of the filter paper press method described by Wiebicki and Deatherage (1958). The breast samples were exposed to the air at room temperature for 30 min and meat colour was measured (average value of 3 measurements was taken from the middle and 2 corners of the muscle samples) using a
Ac
Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 00:41 11 October 2017
Intestinal microbiota
Miniscan XE (Hunterlab Associates Laboratory Inc., Reston, VA) chromameter set on the L*
(lightness), a* (redness), and b* (yellowness) system (CIE, 1976). White and black tiles were used as standards. Muscle samples were stored at 4°C until analysed for the pH values. At 45 min and 24 h post mortem, pH was measured by insertion of pH meter electrode (Sentron 1001 pH System, Roden, the Netherlands) into the left Pectoralis major muscle. The pH meter was calibrated
by measuring buffer solutions at pH 4.0 and 7.0 (Merck - Darmstadt, Germany), at ambient temperature.
HSP70 mRNA expression The level of HSP70 protein expression was determined as previously described by Hosseini et
ip t
and brain (whole cerebrum) were disrupted, minced and homogenised in a tissue homogeniser
cr
(Powermax AHS 250, ProScientific Inc., Oxford, CT). The HSP70 mRNA levels in the
us
samples were analysed by quantitative real-time polymerase chain reaction (qRT-PCR, Hao et al., 2012). The chicken HSP70 gene sequence deposited in GenBank (NM001006685.1)
M
Statistical analysis
an
under the accession number J02579 was used as nucleotide sequence in the current research.
In this experiment, performance data were subjected to repeated measures analysis and pen
ed
considered as experimental unit. All data were analysed as a completely randomised design
ce pt
with 2 × 2 factorial treatment arrangements using the GLM procedures of SAS (SAS, 2000). The model included the main effects of EOB and stocking density, and associated two-way interactions. Level of significance was set at 5%, and when a significant effect was indicated, treatment means were separated using Tukey-Kramer’s test.
Ac
Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 00:41 11 October 2017
al. (2016). From all the birds killed at d 42 (12 per treatment), tissues of heart, liver, kidney,
RESULTS
The effects of EOB and stocking density on the growth performance of broilers are shown in Table 2. As expected, stocking density had significant effects on broiler performance. Average daily gain was significantly higher (P < 0.001) in birds at LSD than those at HSD during the experimental period (22 to 42 d of age). Average daily feed intake was significantly decreased in birds at HSD (P < 0.001) in comparison to those at LSD. As
stocking density decreased, the FCR was reduced (P < 0.001). No significant effect of EOB supplementation was observed on any of the measured performance parameters. However, the EOB supplementation tended (P = 0.078) to increase FCR during the experiment. Throughout the experimentation period, only one bird died in the control treatment. Because of this limited number of mortality, no statistical analysis was performed (data not shown).
ip t
the lymphoid organs (% of BW) of broilers are presented in Table 3. Neither stocking density
cr
(LSD and HSD) nor EOB supplementation had a significant effect (P > 0.05) on the relative
us
weights of the spleen, thymus, and bursa of Fabricius. In addition, no interaction effect was observed between EOB and stocking density (P > 0.05).
Table 2 and 3 near here
an
As shown in Table 3, stocking density had a significant effect on plasma immunoglobulin levels. The high stocking density significantly increased the plasma IgA (P
ISSN: 0007-1668 (Print) 1466-1799 (Online) Journal homepage: http://www.tandfonline.com/loi/cbps20
Effects of a blend of essential oils and overcrowding stress on the growth performance, meat quality, and heat shock protein gene expression of broilers S. M. Hosseini, H. Farhangfar & R. Nourmohammadi To cite this article: S. M. Hosseini, H. Farhangfar & R. Nourmohammadi (2017): Effects of a blend of essential oils and overcrowding stress on the growth performance, meat quality, and heat shock protein gene expression of broilers, British Poultry Science, DOI: 10.1080/00071668.2017.1390209 To link to this article: http://dx.doi.org/10.1080/00071668.2017.1390209
Accepted author version posted online: 09 Oct 2017.
Submit your article to this journal
Article views: 1
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=cbps20 Download by: [UNIVERSITY OF ADELAIDE LIBRARIES]
Date: 11 October 2017, At: 00:41
CBPS-2016-515
Publisher: Taylor & Francis & British Poultry Science Ltd
Ed. Kjaer, September 2017;
Journal: British Poultry Science
ip t cr
us
Effects of a blend of essential oils and overcrowding stress on the growth performance,
an
meat quality, and heat shock protein gene expression of broilers
M
S. M. HOSSEINI1, H. FARHANGFAR1 AND R. NOURMOHAMMADI2 Running Head: Essential oils and overcrowding stress
Department of Animal Sciences, College of Agriculture, University of Birjand,
ed
1
Birjand 97175 and 2Young Researchers and Elites Club, Birjand Branch, Islamic Azad
ce pt
University, Birjand, Iran
Ac
Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 00:41 11 October 2017
DOI: 10.1080/00071668.2017.1390209
Accepted for publication 28th August 2017
Correspondence to: Dr S. M. Hosseini, Department of Animal Sciences, College of Agriculture, University of Birjand, P.O. Box 331/97175, Birjand, Iran. E-mail: [email protected]
Abstract. 1. Overcrowding stress is common in the poultry industry. Chickens exposed to long-term stressful situations are characterised by welfare impairment and immunosuppression. 2. The present study evaluated the effects of a blend of essential oils (EOB; cinnamaldehyde and thymol) and stocking density on the performance, gut microflora, meat quality, and
ip t
3. One-day-old Ross 308 male broiler chickens (n = 360) were allocated to 4 experimental
cr
groups from d 22 to 42. Each treatment had 6 replicates of 15 chicks. Two groups were
at a low stocking density (LSD) of 10 birds/m2.
us
subjected to a high stocking density (HSD) of 20 birds/m2 and the other two groups were kept
an
4. The results of this study indicate that overcrowding stress decreased growth performance parameters, blood IgG and heterophil:lymphocyte ratio (H:L) but increased IgA and IgM
ed
the breast muscle.
M
levels. High stocking density reduced water-loss rate and pH decline at 45 min post mortem in
5. Essential oils supplementation elevated H:L ratio but decreased breast meat redness and
ce pt
pH24.
6. Significant interactions between EOB and stocking density were observed for corticosterone level (CS) and mRNA levels of heat shock protein 70 (HSP70) in brain and heart. Although HSD increased CS and HSP70 when compared to LSD, the effects of the former were inconsistent with EOB supplemented diets.
Ac
Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 00:41 11 October 2017
physiological stress markers of broilers.
7. In conclusion, dietary EOB supplementation could improve some of the biomarkers associated with overcrowding stress in broiler chickens.
Keywords: stress, broilers, gene expression, growth, immunity
INTRODUCTION
Optimal nutrition and environment must be provided for improved performance of broilers. In the contemporary broiler chicken production systems, different environmental factors can result in elevated levels of stress. Overcrowding stress is considered one of the most important stressors in broiler production due to its effects on growth performance, health, welfare status, and immune system (Gomes et al., 2014). In addition, mortality, susceptibility to diseases,
ip t
were related to stress conditions caused by overcrowding (Tong et al., 2012). Depending on
cr
the country and the production system, different stocking densities are applied in poultry
us
production. However, a high stocking density (HSD) can be stressful and can have deleterious effects on profitability and physiological parameters of broilers (Houshmand et al., 2012).
an
Several parameters could be measured as stress biomarkers in broilers, including plasma levels of corticosterone (CS), glucose, cholesterol, nitric oxide; heterophil:lymphocyte (H:L)
M
ratio and lymphoid organs weight (Munck et al., 1984). It is considered that HSD increases
ed
aggressive behaviour among birds and causes stress, thereby resulting in higher CS and cholesterol levels in chicken plasma (Post et al., 2003). Reduction in the numbers of
ce pt
lymphocytes and monocytes and enhancement in the numbers of heterophils, which leads to a higher H:L ratio, have been reported for stressed broilers (Hosseini et al., 2016). It has been demonstrated that HSD causes an imbalance in the microbiota population (Feddes et al., 2002) and can negatively influence meat quality (Osman, 1993). Heat shock proteins (HSPs) are a group of proteins that are present in cells of all living organisms but are expressed at
Ac
Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 00:41 11 October 2017
physiological stress (such as oxidative stress and inflammation), and behavioural adjustments
high levels when cells are exposed to thermal and non-thermal stressors (Soleimani et al., 2012). Previous research in broilers showed that social isolation may elicit HSP70 expression (Soleimani et al., 2012). Stress conditions appear to affect the response of broilers to different feed additives, such as plant extracts. Essential oils (EOs) are derived from plants and have distinct biological functions, such as immunostimulating effects (Hosseini et al., 2016), antimicrobial,
antifungal or antioxidant activities (Silva et al., 2012). Due to their positive effects of EO on gut microflora and antioxidant effects, it is possible that dietary supplementation with EO can help the broilers overcome any deficiency and increase their tolerance to stressful conditions. The immune status is known to play an important role in protecting the organism from various infections, and phytogenic feed additives derived from plants may increase cellular and
ip t
important role in strengthening the defence system of birds against invasion by infectious
cr
organisms. However, mechanisms that interact with the EOs have not yet been thoroughly
us
studied in broilers.
The positive effects of EOs on broiler health have already been reported, but to the best
an
of our knowledge, no information is available concerning the potential effects of these compounds in HSD reared broilers. Considering the more beneficial effects of plant extracts
M
under stressful conditions, we hypothesised that supplementation with a blend of EOs, as a
ed
beneficial feed additive, could alleviate the detrimental effects of overcrowding stress. Therefore, the present study was conducted to evaluate the efficacy of a blend of EOs (EOB)
ce pt
on finishing broilers reared in crowded conditions.
MATERIALS AND METHODS
All procedures carried out in this experiment were approved by the Animal Ethics Committee of the University of Birjand.
Ac
Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 00:41 11 October 2017
humoral immune responses of birds (Botsoglou et al., 2002). Therefore, they could play an
Blend of EOs
Enviva® EO 101, a commercial feed additive containing EOB from cinnamon (Cinnamomum
verum) and thyme (Thymus vulgaris), was used in this study. Enviva® EO is supplied as a fine white agglomerated encapsulated powder, (Danisco Animal Nutrition, Marlborough, UK), and 100 g of this product provides a guaranteed minimum of 4.5 g cinnamaldehyde and 13.5 g thymol.
Table 1 near here
Birds, diets, and management
Three hundred and sixty 21-day-old Ross 308 male broiler chicks were used in this experiment. The chicks were raised up to 21 d of age on a typical commercial broiler starter mash diet (Table 1). On d 22, broilers were individually weighed (805 ± 3.0 g) and assigned
ip t
arrangement with two levels of EOB (0 or 100 g/tonne of diet as the manufacturer
cr
recommended) and two levels of stocking density (10 birds/m2 or 20 birds/m2). The Iranian
us
Animal Welfare Commission recommends a maximum stocking density of 37 kg/m2 (0.067 m2/bird) for broilers in mechanically ventilated sheds and water-based cooling. For non-
an
mechanically ventilated sheds, the maximum is 28 kg/m2 (0.084 m2/bird). These recommendations follow the guidelines published by Barnett et al. (2008). For this
M
experiment, the birds were placed at the stocking densities (high and low) just mentioned, according to practical management conditions. The chicks were randomly allotted to 4
ed
experimental groups with 6 replicates of 15 chicks for each experimental group. The pens had
ce pt
dimensions of 1.2 m length × 0.85 m width × 0.7 m height. There was no significant difference in the body weight among groups when the chickens received the experimental diets. The basal diets were formulated based on maize and soybean meal to meet nutrient requirements of broiler chickens (Table 1; NRC, 1994). Basal diet with or without the blend of EOB was offered in mash form to the birds from d 22 of age. The birds had free access to
Ac
Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 00:41 11 October 2017
to 1 of the 4 dietary treatments. The experimental treatments consisted of a 2 × 2 factorial
feed and water and were maintained on a 23L:1D lighting program. The floor space occupied by feeders and drinkers was not calculated in the floor space provided to the birds. The
number of feeders and drinkers in each pen was constant throughout the experiment. Nipple drinkers were used for drinking water and tube feeders were used for feeding the chicks throughout the study.
Broiler performance parameters Feed intake and body weight for each replicate were recorded from 22 to 42 d. Average daily gain (ADG), average daily feed intake (ADFI), and feed conversion rate (FCR) were further calculated.
ip t
At the end of the study, on d 42, two chicks were randomly chosen from each pen, weighed,
cr
and anaesthetised with carbon dioxide and killed by cervical dislocation. Spleen, thymus, and
us
bursa of Fabricius were collected and weighed. Relative weights of lymphoid organs were
an
calculated as percentage of live weight.
Immunoglobulin concentration and stress indicators
M
At 42 d of age (end of the experiment), a 4-ml blood sample was obtained from the wing vein of 2 broilers from each replicate (12 samples per treatment), and then was collected into 2
ed
tubes (2 ml in each tube). The first tube contained heparin as the anticoagulant. One drop of
ce pt
whole blood from each sample was used to make a thin smear on a clean microscope slide. The dried smear was stained with Wright’s stain. For each slide, a total of 60 cells were counted using a light microscope and the H:L ratio was calculated. The rest of the blood sample was centrifuged (5000 g) for 10 min at 4ºC. The collected plasma was stored at −20ºC until analysed. The plasma IgG, IgM and IgA concentrations were determined using an
Ac
Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 00:41 11 October 2017
Relative weights of lymphoid organs
enzyme-linked immunosorbent assay ELISA kit from Bethyl Laboratories (Montgomery, TX, USA). The ELISA procedure was carried out according to the protocol of the manufacturer and absorbance was measured at 450 nm. The serum of blood samples in the second tube was separated and used to measure concentrations of corticosterone (CS), nitric oxide (NO), glucose and cholesterol. Serum CS and NO levels were determined using an RIA kit (Cayman Chemical Company, MI). Serum
levels of glucose and cholesterol were measured using an automated chemistry analyser (Hitachi 902 Automatic Analyzer, Hitachi, Tokyo, Japan) using colorimetric methods and following the instructions of the manufacturer of the corresponding reagent kit (Zhongsheng Biochemical Co., Ltd., Beijing, China).
ip t
The contents of small intestine (12 samples per treatment) were carefully hand-stripped into
cr
sterile containers. The survival of Lactobacillus, Salmonella, and total aerobes was
us
determined according to the procedures described by Horn et al. (1996) with some
modifications (Cengiz et al., 2015). The cfu as log10 per g for each of these bacteria within
M
Meat quality
an
digesta was counted on the basis of colony morphology and characteristics.
Immediately after slaughter, the left side of the pectoralis major (white) muscle was quickly
ed
removed for meat quality evaluation. Physicochemical characteristics of breast muscle
ce pt
samples, such as water-loss rate, pH45, pH24, and meat colour, were evaluated. Water-loss rate was estimated by determining expressible fluid using a modification of the filter paper press method described by Wiebicki and Deatherage (1958). The breast samples were exposed to the air at room temperature for 30 min and meat colour was measured (average value of 3 measurements was taken from the middle and 2 corners of the muscle samples) using a
Ac
Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 00:41 11 October 2017
Intestinal microbiota
Miniscan XE (Hunterlab Associates Laboratory Inc., Reston, VA) chromameter set on the L*
(lightness), a* (redness), and b* (yellowness) system (CIE, 1976). White and black tiles were used as standards. Muscle samples were stored at 4°C until analysed for the pH values. At 45 min and 24 h post mortem, pH was measured by insertion of pH meter electrode (Sentron 1001 pH System, Roden, the Netherlands) into the left Pectoralis major muscle. The pH meter was calibrated
by measuring buffer solutions at pH 4.0 and 7.0 (Merck - Darmstadt, Germany), at ambient temperature.
HSP70 mRNA expression The level of HSP70 protein expression was determined as previously described by Hosseini et
ip t
and brain (whole cerebrum) were disrupted, minced and homogenised in a tissue homogeniser
cr
(Powermax AHS 250, ProScientific Inc., Oxford, CT). The HSP70 mRNA levels in the
us
samples were analysed by quantitative real-time polymerase chain reaction (qRT-PCR, Hao et al., 2012). The chicken HSP70 gene sequence deposited in GenBank (NM001006685.1)
M
Statistical analysis
an
under the accession number J02579 was used as nucleotide sequence in the current research.
In this experiment, performance data were subjected to repeated measures analysis and pen
ed
considered as experimental unit. All data were analysed as a completely randomised design
ce pt
with 2 × 2 factorial treatment arrangements using the GLM procedures of SAS (SAS, 2000). The model included the main effects of EOB and stocking density, and associated two-way interactions. Level of significance was set at 5%, and when a significant effect was indicated, treatment means were separated using Tukey-Kramer’s test.
Ac
Downloaded by [UNIVERSITY OF ADELAIDE LIBRARIES] at 00:41 11 October 2017
al. (2016). From all the birds killed at d 42 (12 per treatment), tissues of heart, liver, kidney,
RESULTS
The effects of EOB and stocking density on the growth performance of broilers are shown in Table 2. As expected, stocking density had significant effects on broiler performance. Average daily gain was significantly higher (P < 0.001) in birds at LSD than those at HSD during the experimental period (22 to 42 d of age). Average daily feed intake was significantly decreased in birds at HSD (P < 0.001) in comparison to those at LSD. As
stocking density decreased, the FCR was reduced (P < 0.001). No significant effect of EOB supplementation was observed on any of the measured performance parameters. However, the EOB supplementation tended (P = 0.078) to increase FCR during the experiment. Throughout the experimentation period, only one bird died in the control treatment. Because of this limited number of mortality, no statistical analysis was performed (data not shown).
ip t
the lymphoid organs (% of BW) of broilers are presented in Table 3. Neither stocking density
cr
(LSD and HSD) nor EOB supplementation had a significant effect (P > 0.05) on the relative
us
weights of the spleen, thymus, and bursa of Fabricius. In addition, no interaction effect was observed between EOB and stocking density (P > 0.05).
Table 2 and 3 near here
an
As shown in Table 3, stocking density had a significant effect on plasma immunoglobulin levels. The high stocking density significantly increased the plasma IgA (P
