Effects of stocking density and environmental conditions on performance, immunity, carcase characteristics, blood constitutes, and economical parameters of cobb 500 strain broiler chickens

Abstract

The effects of stocking density and environmental conditions on performance, economic profit, carcase characteristics, immunity, and blood plasma parameters of Cobb 500 strain of broiler chickens were evaluated. The 4 climate regions (mild and humid, semi-arid, alpine, hot and dry) and 4 densities (10, 15, 17 and 20 chicks/m²) were carried out as a completely randomised design with 4 × 4 factorial arrangement of treatments. The corn and soybean based diets were formulated according to the nutrition requirements guide of the Cobb 500 for starter and grower periods. The amount and the composition of the diet were the same for all experimental groups. Growth performance and feed intake were measured weekly. After the injection of sheep red blood cell at 15 and 35 days, blood samples were taken at 24 and 42 days, respectively, to study the immunity of broiler chickens. At the end of the experiment, 3 birds were slaughtered from each experimental unit for measuring the weights of carcase, abdominal fat, gastrointestinal organs (the total weight of gastrointestinal tract and caeca, and liver) and blood parameters. The interaction effects of stocking density and climate region on feed conversion ratio (in starter period), survival rate, cost, profit, cholesterol, triglyceride, high density lipoproteins (HDL), HDL/low density lipoproteins (LDL) ratio, and liver enzymes aspartate aminotranferases (AST), also known as serum glutamic oxaloacetic transaminase (SGOT), and alanine aminotranferase (ALT) were significant (p < .05). Climate and density each had a significant effect on production index (p < .05). The highest production index was obtained in alpine climate. Moreover, the density of 10 chicks/m² showed the highest production index. The results showed that rearing Cobb strain chickens in hot and dry climate and at the density of 17 chicks/m² had the most economic benefit. Based on the results of this study to achieve the highest profit in different climates, a density of 17 chicks/m² (0.633 ft² per bird) is recommended for Cobb 500 strain chickens.

Highlights

• Nutritional efficiency changes by environmental factors such as climate and stocking density.

• Obtain sufficient information to combined effect of stocking density and climate on the performance of Cobb 500 broiler chickens.

• The most reached profit was in the dry climate at a density of 17 chicks/m² compared to other climates and densities.

Keywords:

• Antibody
• cholesterol
• climate
• economic
• environment

Introduction

Nowadays, the increase in meat consumption and its high price has led to a substitute product (i.e. poultry), it is more efficient from an economic point of view (Costantino et al. 2018).

To reduce the costs, apparently, feeding methods play an important role in poultry operation. For instance, the cost of providing a ration in poultry can vary from 55% to 75% of the total cost of breeding. However, to be more efficient (i.e. reducing the cost), factors such as genetics, nutrition, and environmental conditions can play a substantial role in poultry farming (Alltane et al. 2018; Namakparvar et al. 2018).

Chicken meat production has undertaken a great alteration over the past 50 years, with productivity increases due to genetics, nutrition, and management, to that extent that broiler chickens have the most efficiency among the meat-producing animals (Costantino et al. 2018).

As a result of genetic selection, nutritional advances, and managerial developments, the production and supply of broiler meat to consumers have significantly increased over the past decade (Alltane et al. 2018; Namakparvar et al. 2018). The need for continued improvements in growth and feed utilisation is addressed in broiler breeding (Jahanpour et al. 2015). The correct method of feeding, strain selection, stocking density, flock sensitivity to pathogens and metabolic disorders, proper breeding capacity and appropriate slaughter age are factors that can affect economic performance (Baeza et al. 2012; Berg and Yngvesson 2012; Hughes 2012; Kryeziu et al. 2018). Environmental stressors such as stocking density, temperature, and humidity are among the most important factors affecting the performance of the broiler industry (Pompeu et al. 2018).

Many variables such as environmental conditions (climate and stocking density) can influence nutritional efficiency (Gharaghani et al. 2015). The most usual stocking densities are 33–42 kg live weight/m² kg (at least in Europe) according to the EU regulation 43/2077 and 30 kg live weight/m² in warm climate (Qaid et al. 2016).

Attia et al. (2016) conducted a study with Arbour Acres and Hubbard strains in warm climate, and they observed significant improvements in body weight gain, feed conversion ratio, carcase performance, and meat quality with Arbour Acres chicks compared to the Hubbard chicks (p < .05).

The slaughter age, building size, strain of broiler chicks, and other factors can influence stocking density selection (Horne and Bondt 2014). Economic conditions and market demand for chicken meat can affect the stocking density and the average body weight of each bird produced (Van Horne 2013).

Providing appropriate growing conditions can be a costly way for improving the welfare and performance of birds in different climates (Costantino et al. 2018). Feeding broiler chickens with enriched diets in controlled warm climate enhanced the performance, blood parameters, and liver weight, but increased abdominal fat (Awad et al. 2017).

Bouyeh et al. (2017) showed that warm and dry, as well as temperate and humid climates have better effects on performance (body weight gain and feed conversion ratio) in ostrich compared to alpine climate, although the rate of egg production was not significantly different in the three studied climate regions.

The benefits of increasing stocking density include improving productivity, making full use of limited available area, and increasing income. However, it is unclear what climates are better for future development or which stocking density is better in any climate.

Because of insufficient researches on the selection of suitable stocking density in different climates and very rare studies of combined effect of stocking density and climate on the performance of Cobb 500 broiler chickens, this experiment was carried out to provide further information. The most important objectives of this experiment were to study growth performance, economic efficiency, carcase quality, blood parameters, and immune responses of Cobb 500 broiler chickens in four different climatic conditions with 4 different densities. The goal of this study was to determine the most suitable stocking density in each climate in order to maximise profits in different climatic conditions.

Materials and methods

The experimental protocol was ratified by the Animal Ethic Committee of the Science and Research Branch, Islamic Azad University, Tehran, Iran, and the experiment performed with respect to the International Guidelines for research involving animals.

A total of 79,360 newly hatched, straight-run Cobb 500 broiler chicks were used under 4 climate regions (mild and humid, semi-arid, alpine, hot and dry) with 4 separate broiler chicken houses (farms) for each climate, 4 stocking densities within each house (10, 15, 17, and 20 chicks per m²), and 4 replicate pens within each density. Each replicate was included 20 m², hence, there were 200 chicks for density 10 chicks/m², 300 chicks for density 15 chicks/m², 340 chicks for density 17 chicks/m², and 400 chicks for density 20 chicks/m² (totally 200 + 300 + 340 + 400 = 1240 chicks for replicate 1 and so on). So, there were 4 × 4 × 4 × 1240 = 79,360 chicks totally (Tables 1 and 2). Table 2 summarises studied geographic climate conditions. Per the definition of rainfall per year, it is divided into four categories (Kaviani and Alijani 2001). More than 400 mm/year is labelled as mild and humid climate; between 250 and 400 mm/year is called alpine climate; between 150 and 250 mm/year is known as semi-arid climate; and finally, less than 150 mm/year is defined as dry climate. The experimental period was 6 weeks (42 days). Diets were prepared according to the Cobb recommendations (Cobb 500, 2012), and during the whole period of study the chicks had ad libitum access to the water and feed. The experimental diets were without any commercial growth promoter additives. The chemical composition (calculated analysis) of each diet is given in Table 3. Same commercial and conventional farms used as broiler farming houses. The humidity, temperature, vaccination programme, litter, type, lighting, ventilation, nebuliser, and other management conditions were regulated based on standard instructions for Cobb 500 strain (Cobb 500, 2012).

Table 1. Experimental treatments and arrangement.

Treatment	Climate region	Density, chicks/m^2
1	Mild and humid	10
2	Mild and humid	15
3	Mild and humid	17
4	Mild and humid	20
5	Semi-arid	10
6	Semi-arid	15
7	Semi-arid	17
8	Semi-arid	20
9	Alpine	10
10	Alpine	15
11	Alpine	17
12	Alpine	20
13	Hot and dry	10
14	Hot and dry	15
15	Hot and dry	17
16	Hot and dry	20
Density, chicks/m^2	Chicks per replication (pen)	Chicks per treatment or farm (4 replications)	Chicks per climate (4 farms)	Chicks per 4 climates
10	200	800	3200	12,800
15	300	1200	4800	19,200
17	340	1360	5440	21,760
20	400	1600	6400	25,600
Total	1240	4960	19,840	79,360

Table 2. Geographical characteristics of four studied climates.

Climate	Rainfall, mm/year	Geographical coordinates		Average annual temperature, ˚C	Average annual rainfall, mm	Altitude from the sea, m
		Latitude	Longitude
Mild and humid	More than 400	31.32	53.30	17.9	789.2	48
Semi-arid	Between 250 and 400	36.39	59.38	14.3	250	1077
Alpine	Between 150 and250	38.22	48.12	9.2	295.5	1294
Hot and dry	Less than 150	32.48	51.33	16.3	125	1600

Table 3. Percentage and calculated composition of experimental diets during the starter (1–21 days of age) and grower (22–42 days of age) periods.

Ingredient, g/kg or %	Starter period (1–21 days of age)	Grower period (22–42 days of age)
Corn	62.17	64.27
Soybean meal	31.2	28.50
Soybean oil	2.90	3.40
Dicalcium phosphate	1.45	1.35
Calcium carbonate	1.20	1.10
Salt	0.15	0.13
dl-methionine	0.17	0.15
Vitamin and mineral premix*	0.60	1.00
Lysine HCl	0.16	0.10
Calculated analysis
Energy (kcal ME/kg	3083	3176
Protein (%)	19.0	18.0
Calcium (%)	0.96	0.90
Available phosphorus (%)	0.48	0.45
Methionine (%)	0.40	0.39
Methionine + cysteine (%)	0.84	0.82
Lysine (%)	1.10	1.05

* Calcium pantothenate, 4 mg/g; niacin, 15 mg/g; vitamin B6 (pyridoxine), 13 mg/g; vitamin A, 5000 U/g; vitamin D3, 500 U/g; vitamin E, 1000 mg/g; vitamin K3, 1.5 mg/g; vitamin B2, 1 mg/g; Cu, 3 mg/g; Zn, 15 mg/g; Mn, 20 mg/g; Fe, 10 mg/g; K (potassium), 0.3 mg/g.

The performance traits included body weight gain, feed intake and feed conversion ratio for the starter (1–21 days), the growth (22–42 days) and the whole (1–42 days) periods. Economic traits included the meat production of live chick/m², feed and chick costs, total cost, income/m², profit/m², final body weight, survival rate and production index (Mousavi et al. 2015).

To determine blood plasma components, 3 chicks from each experimental unit were selected randomly, at the end of experiment (42 day of age). After blood sampling from the wing vein, 3 blood samples were mixed together and a pooled sample was immediately sent to the laboratory to measure the biochemical parameters including fat, glucose, enzyme, protein, and uric acid. The kits of Pars azmoun Company (Iran) were used to measure blood parameters (Poorghasemi et al. 2015). The basis of all mentioned measurements was colorimetric method. The calculated total globulin concentration for each serum sample was obtained by subtracting total albumin concentration (measured) from total protein concentration (measured) for the same sample (Jahanpour et al. 2013).

The antibody titre against the sheep red blood cell (SRBC) was used to study the immune system of experimental birds (Shabani et al. 2015). At 15 and 35 days of age, 2 birds were randomly chosen from each experimental unit, and 0.5 mL of red blood cell suspension (2%) (3 times washed with a physiological serum) was injected into the wing vein (prepared by Razi Institute, Karaj, Iran). Seven days after injection (at 24 and 42 days of age), the blood samples were collected from the birds. The antibody titre changes were evaluated by injection sheep red blood cell (2%) as a non-pathogenic antigen, in 2 phases of blood sampling, in which the same same 2 birds used. Blood samples were kept in the laboratory for 1 day and afterwards the blood serum was separated in a centrifuge at 1000 xg for 10 minutes. In the beginning, serum samples were put in a warm oven for 30 minutes at 55° C, to neutralise the complement and avoid its interference with anti-SRBC antibody. To specify the antibody titre, microtiter hemagglutination was used. When measuring samples, the logarithm to base 2 of the last dilution in which hemagglutination was seen, was recorded as antibody titre. To determine components of response to SRBC (IgG and IgM), the IgM antibody which is sensitive to mercaptoethanol and therefore could be calculated by isolating the resistant antibody to mercaptoethanol (IgG), and deducting this amount from total response (total response – IgG = IgM).

At 42 days of age, 3 broiler chickens were collected with +/− 5% of the average body weight of the box from each pen, starved for 4 h before slaughter to evaluate carcase components. The chickens were weighed before slaughter. Data from carcase components were measured with a digital scale (0.001 precision) and the ratio of each weighted component/defeathered body weight was recorded (Saraei et al. 2014).

Statistical analysis

The experiment was performed as a two-factor factorial design, consisting of 4 climates (mild and humid, semi-arid, alpine, hot and dry), 4 broiler chicken houses (farms) for each climate, 4 densities (10, 15, 17 and 20 chicks per m²) for each farm, and 4 replicate pens for each treatment based on a completely randomised (CRD) balanced design. Data were arranged using Excel (2010) software and analysed by SAS (2009) software with Proc GLM procedure. To compare the mean of different treatments, Duncan’s multi-domain test was used. The statistical model of design was as follows: $${\mathrm{X}}_{\text{ijk}}=\mu +{\mathrm{A}}_{\mathrm{j}}+\text{Bk}+{\text{AB}}_{\text{ij}}+{\mathrm{e}}_{\text{ijk}}$$

(X_ijk) = the record of each observation, (µ) = the mean, (A_j) = climate effect, (B_k) = effect of stocking density, (AB_jk) = the interaction effect of climate and density, (E_ijk) = error effect

Results

The results of performance (Tables 4–6), blood plasma parameters (Tables 6–8), immunity (Table 9), and carcase components traits (Tables 10–12) are provided below.

Table 4. Effect of environmental conditions and stocking density on body weight gain (BWG) (g/chick/day), feed intake (FI) (g/chick/day), and feed conversion ratio (FCR) (g/g) of Cobb 500 broilers at starter (1–21 days of age), grower (22–42 days of age) and whole (1–42 days of age) periods*.

Treatments	Starter			Grower			Whole
	BWG	FI	FCR	BWG	FI	FCR	BWG	FI	FCR
Climate
Mild and humid	40.03^b	52.53^a	1.31	80.33	169.15^b	2.11	60.18	110.84^b	1.84
Semi-arid	39.98^b	51.48^b	1.29	81.36	172.68^a	2.13	60.67	112.08^a	1.84
Alpine	41.25^a	52.97^a	1.29	80.67	171.33^a	2.13	60.96	112.15^a	1.84
Hot and dry	40.16^b	52.98^a	1.32	80.37	171.42^a	2.14	60.26	112.20^a	1.86
SEM	0.27	0.35	0.01	0.56	0.64	0.01	0.25	0.26	0.00
p Value	.003	.0097	.16	.55	.001	.65	.11	.0006	.19
Mean	40.35	52.49	1.30	80.68	171.15	2.12	60.52	111.82	1.84
Density, chick/m^2
10	40.70	52.05	1.28^b	83.65^a	172.06	2.06^b	62.17^a	112.06	1.80^d
15	40.11	52.48	1.31^ab	81.71^b	170.75	2.09^b	60.91^b	111.62	1.83^c
17	40.73	52.17	1.28^b	79.50^c	171.27	2.16^a	60.11^c	111.72	1.86^b
20	39.88	53.25	1.33^a	77.87^d	170.49	2.19^a	58.88^d	111.87	1.90^a
SEM	0.27	0.35	0.01	0.56	0.64	0.01	0.25	0.26	0.00
p Value	.07	.08	.007	<.0001	.33	<.0001	<.0001	.67	<.0001
Mean	40.35	52.49	1.30	80.68	171.15	2.12	60.52	111.82	1.84

* Means within each comparison column with no common superscript differ significantly at p < .05.

Table 5. Effect of environmental conditions and stocking density on performance and economical parameters of Cobb 500 broilers*.

Treatments	Body weight at 42 days of age, g/chick	Survival rate, %	European production index	Produced body weight in m^2, kg/m^2	Cost, $ (kg/m^2)	Income, $ (kg/m^2)	Profit, $ (kg/m^2)
Climate
Mild and humid	2570	96.52^a	321.2^ab	38.22^ab	37.76^a	40.96^ab	3.19^b
Semi-arid	2590	95.75^c	320.0^ab	38.29^ab	37.87^a	41.02^ab	3.15^b
Alpine	2602	96.09^b	323.9^a	38.57^a	38.00^a	41.33^a	3.32^b
Hot and dry	2573	95.24^d	314.0^b	37.82^b	35.94^b	40.52^b	4.57^a
SEM	10.74	0.09	2.61	0.16	0.12	0.17	0.19
p Value	.12	<.0001	.05	.01	<.0001	.01	<.0001
Mean	2583.57	95.90	319.77	38.23	37.39	40.96	3.56
Density, chick/m^2
10	2653^a	95.52^c	335.0^a	25.33^d	24.38^d	27.14^d	2.75^b
15	2600^b	95.97^b	324.5^b	37.41^c	36.18^c	40.08^c	3.90^a
17	2566^c	96.49^a	317.8^b	42.08^b	41.11^b	45.09^b	3.98^a
20	2515^d	95.62^c	301.7^c	48.09^a	47.91^a	51.52^a	3.61^a
SEM	10.74	0.09	2.61	0.16	0.12	0.17	0.19
p Value	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001
Mean	2583.57	95.90	319.75	38.22	37.39	40.96	3.56

* Means within each comparison column with no common superscript differ significantly at p < .05.

Table 6. Significant interaction effects of environmental conditions and stocking density on body weight gain (BWG), feed conversion ratio (FCR) at starter period (1–21 days of age), economical indices, and plasma constitutes of Cobb 500 broilers*.

Climate	Density, chick/m^2	BWG, g/chick/day	FCR, g/g	Survival rate, %	Cost, $ (kg/m^2)	Profit, $ (kg/m^2)	Total Cholesterol, mg/dL	Triglycerides, mg/dL	VLDL, mg/dL	HDL Cholesterol, mg/dL	LDL/HDL	AST, U/L	ALT, U/L
Mild and humid	10	40.38^bc	1.29^ab	96.43^a–d	24.48^g	2.95^b	159.3^a–d	59.64^b	11.92^b	73.39^a–d	1.21^abc	293.7^abc	534.6^de
	15	40.61^bc	1.29^ab	96.64^ab	36.51^e	3.19^b	164.2^a	54.85^bcd	10.97^bcd	71.57^a–e	1.24^abc	294.7^abc	559.2^abc
	17	39.30^c	1.34^a	96.85^a	41.41^c	3.33^b	163.9^a	59.12^bc	11.82^bc	72.63^a–d	1.19^bc	296.6^ab	548.3^a–e
	20	39.85^c	1.32^a	96.15^b–e	48.62^a	3.31^b	164.4^a	52.72^d	10.54^d	69.19^de	1.26^ab	292.2^bc	526.5^e
Semi-arid	10	39.96^c	1.29^ab	95.12^gh	24.36^g	2.64^b	161.8^ab	56.39^bcd	11.27^bcd	73.91^ab	1.19^bc	296.2^ab	541.8^b–e
	15	39.43^c	1.31^ab	96.02^cde	36.67^e	3.70^b	164.4^a	59.59^b	11.92^b	71.09^b–e	1.22^abc	294.6^abc	535.2^cde
	17	40.78^bc	1.23^b	96.61^abc	41.81^c	3.34^b	159.5^abc	52.89^cd	10.57^d	75.60^a	1.13^c	293.6^abc	524.3^e
	20	39.73^c	1.31^ab	95.26^fg	48.64^a	2.93^b	161.4^ab	66.07^a	13.21^a	69.66^cde	1.24^abc	297.8^a	528.8^e
Alpine	10	41.87^ab	1.23^b	95.87^def	24.49^g	2.85^b	157.7^bcd	56.42^bcd	11.28^bcd	69.82^b–e	1.28^ab	293.2^abc	557.5^b–e
	15	39.97^c	1.33^a	96.10^cde	36.75^e	3.71^b	161.9^ab	52.67^d	10.53^d	71.63^a–e	1.23^abc	292.7^bc	543.3^a–e
	17	43.13^a	1.23^b	96.70^ab	41.84^c	3.78^b	161.8^ab	59.60^b	11.92^b	71.74^a–e	1.26^ab	295.7^ab	545.1^a–e
	20	40.01^c	1.35^a	95.67^efg	48.94^a	2.93^b	154.9^cd	60.15^b	12.03^b	73.42^abc	1.19^bc	292.0^bc	557.4^a–d
Hot and dry	10	40.59^bc	1.30^ab	94.65^h	24.18^g	2.58^b	162.7^ab	59.80^b	11.96^b	68.31^e	1.31^a	296.2^ab	525.1^e
	15	40.42^bc	1.30^ab	95.12^gh	34.79^f	4.98^a	153.9^d	58.81^bcd	11.76^bcd	71.64^a–e	1.19^bc	290.6^c	539.7^b–e
	17	39.69^c	1.33^a	95.80^ef	39.36^d	5.45^a	160.4^abc	58.31^bcd	11.66^bcd	73.13^a–d	1.22^abc	295.1^abc	560.0^ab
	20	39.95^c	1.35^a	95.39^fg	45.43^b	5.27^a	157.1^bcd	56.32^bcd	11.26^bcd	71.14^b–e	1.21^abc	296.4^ab	567.2^a
SEM		0.54	0.02	0.19	0.24	0.39	1.77	1.94	0.38	1.25	0.03	1.46	7.38
p Value		.004	.04	.04	<.0001	.02	.0004	<.0001	<.0001	.006	.03	.03	<.0001
Mean		40.35	1.30	95.90	37.39	3.56	160.59	57.71	11.54	71.74	1.22	294.44	543.40

* Means within each comparison column with no common superscript differ significantly at p < .05; VLDL: Very low density lipoprotein; HDL: High Density Lipoproteins; AST: Aspartate amino-transferase; ALT: Alanine aminotranferase.

Table 7. Effect of environmental conditions and stocking density on plasma constitutes (lipids and glucose) of Cobb 500 broilers at 42nd day of age*.

Treatments	Glucose, mg/dL	Total cholesterol, mg/dL	Triglycerides, mg/dL	VLDL (Very low density lipoprotein), mg/dL	HDL Cholesterol (High Density Lipoproteins), mg/dL	LDL Cholesterol (Low Density Lipoproteins), mg/dL	LDL/HDL
Climate
Mild and humid	195.3^b	163.0^a	56.58	11.31	71.70	87.48	1.22
Semi-arid	192.1^c	161.8^a	58.74	11.74	72.57	86.63	1.20
Alpine	198.0^a	159.1^b	57.21	11.44	71.65	88.51	1.24
Hot and dry	194.0^bc	158.5^b	58.31	11.66	71.06	87.34	1.23
SEM	0.83	0.88	0.97	0.19	0.62	0.65	0.01
p Value	<.0001	.0008	.37	.3789	.40	.24	.24
Mean	194.85	160.59	57.71	11.54	71.74	87.49	1.22
Density, chick/m^2
10	193.6^b	160.4	58.06	11.61	71.36^b	88.67	1.25
15	194.6^b	161.1	56.48	11.59	71.48^b	86.93	1.22
17	194.0^b	161.4	57.48	11.49	73.28^a	87.74	1.20
20	197.3^a	159.5	58.82	11.46	70.85^b	86.62	1.22
SEM	0.83	0.88	0.97	0.19	0.62	0.65	0.01
p Value	.006	.41	.38	.38	.03	.12	.19
Mean	194.87	160.59	57.71	11.54	71.74	87.49	1.22

* Means within each comparison column with no common superscript differ significantly at p < .05.

Table 8. Effect of environmental conditions and stocking density on plasma constitutes (enzymes, proteins and uric acid) of Cobb 500 broilers at 42nd day of age*.

Treatments	Uric acid, mg/dL	Aspartate amino- transferase (AST), U/L	Alanine amino- transferase (ALT), U/L)	Total protein, g/dL	Albumin, g/dL	Globulin, g/dL
Climate
Mild and humid	4.44	294.3	542.1^ab	3.92	1.10^b	1.47^a
Semi-arid	4.69	295.5	532.6^b	3.90	1.15^a	1.40^ab
Alpine	4.69	293.4	550.8^a	3.97	1.15^ab	1.36^b
Hot and dry	4.64	294.5	548.0^a	3.93	1.17^a	1.35^b
SEM	0.12	0.73	3.69	0.03	0.01	0.02
p Value	.45	.23	.003	.53	.03	.005
Mean	4.61	294.44	543.38	3.93	1.14	1.40
Density, chick/m^2
10	4.80^a	294.8	539.8	3.94^ab	1.16	1.37
15	4.39^b	293.1	544.4	3.90^b	1.11	1.37
17	4.44^b	295.3	544.4	4.03^a	1.16	1.45
20	4.83^a	294.6	545.0	3.86^b	1.14	1.40
SEM	0.12	0.73	3.69	0.03	0.01	0.02
p Value	.02	.20	.72	.004	.11	.07
Mean	4.61	294.44	543.40	3.93	1.14	1.40

* Means within each comparison column with no common superscript differ significantly at p < .05.

Table 9. Effect of environmental conditions and stocking density on immunity of Cobb 500 broilers at 28th and 42nd day of age (when inoculated with sheep red blood cells on days 15 and 35, respectively)*.

Treatments	28 day of age			42 day of age
	Immunoglobulin G antibody (IgG)	Immunoglobulin M antibody (IgM)	Total antibody (IgT)	Immunoglobulin G antibody (IgG)	Immunoglobulin M antibody (IgM)	Total antibody (IgT)
Climate
Mild and humid	1.81^b	0.81^b	2.62^b	3.37	2.65^c	6.03^b
Semi-arid	2.10^a	1.21^a	3.32^a	3.50	3.46^a	6.96^a
Alpine	2.15^a	1.20^a	3.35^a	3.48	3.09^b	6.57^a
Hot and dry	2.25^a	1.12^a	3.37^a	3.43	3.07^b	6.51^ab
SEM	0.10	0.09	0.19	0.10	0.09	0.17
p Value	.02	.01	.01	.83	<.0001	.003
Mean	2.08	1.08	3.17	3.44	3.07	6.52
Density, chick/m^2
10	2.03	1.00	3.03	3.42	2.96	6.39
15	2.09	1.10	3.20	3.50	3.12	6.62
17	2.07	1.10	3.18	3.40	3.09	6.50
20	2.12	1.14	3.26	3.46	3.10	6.57
SEM	0.10	0.09	0.19	0.10	0.09	0.17
p Value	.93	.75	.85	.91	.65	.80
Mean	2.08	1.08	3.17	3.44	3.07	6.52

* Means within each comparison column with no common superscript differ significantly at p < .05.

Table 10. Effect of environmental conditions and stocking density on carcase components of Cobb 500 broilers at 42nd day of age*.

Treatments	Live body weight, g	Defeathered body weight, g	Full GIT carcase weight, g	Empty GIT carcase weight, g	Eviscerated carcase, %
Climate
Mild and humid	2545	2322	2107^b	1587^b	75.42^b
Semi-arid	2541	2327	2127^a	1599^b	75.42^b
Alpine	2545	2328	2120^ab	1623^a	76.62^a
Hot and dry	2530	2317	2107^b	1588^b	75.42^b
SEM	6.78	6.36	6.36	7.30	0.32
p Value	.39	.57	.05	.0015	.01
Mean	2540.15	2323.43	2115.34	1599.04	75.73
Density, chick/m^2
10	2551	2333	2123^a	1604	75.62
15	2545	2330	2124^a	1602	75.47
17	2531	2315	2104^b	1600	76.15
20	2533	2315	2110^ab	1590	75.46
SEM	6.78	6.36	6.36	7.30	0.32
p Value	.10	.06	.05	.57	.40
Mean	2540.00	2323.25	2115.25	1599.00	75.74

* Means within each comparison column with no common superscript differ significantly at p < .05. GIT: gastrointestinal tract.

Table 11. Effect of environmental conditions and stocking density on relative weight (% of live weight) of carcase components of Cobb 500 broilers at 42nd day of age*.

Treatments	Breast	Drumsticks (thighs)	Wings	Abdominal fat weight	Gizzard (ventriculus)	Heart	Neck	Head
Climate
Mild and humid	33.44	30.51^b	4.44	2.55	2.86	0.62	2.36	2.92^b
Semi-arid	33.44	31.68^a	4.53	2.58	2.93	0.63	2.43	3.01^a
Alpine	33.27	31.69^a	4.53	2.46	2.89	0.62	2.41	3.00^a
Hot and dry	33.22	31.50^a	4.48	2.53	2.89	0.63	2.45	3.01^a
SEM	0.24	0.23	0.03	0.04	0.03	0.00	0.02	0.01
p Value	.85	.0008	.19	.27	.56	.46	.07	.0004
Mean	33.35	31.35	4.50	2.53	2.89	0.62	2.41	2.98
Density, chick/m^2
10	33.36	31.18	4.48	2.49	2.86	0.61	2.42	2.97
15	33.24	31.48	4.47	2.61	2.85	0.63	2.41	2.98
17	33.47	31.36	4.54	2.51	2.98	0.62	2.38	2.98
20	33.35	31.36	4.49	2.50	2.87	0.62	2.44	3.00
SEM	0.24	0.23	0.03	0.04	0.03	0.01	0.02	0.01
p Value	.93	.84	.43	.14	.07	.54	.52	.73
Mean	33.35	31.35	4.50	2.53	2.89	0.62	2.41	2.98

* Means within each comparison column with no common superscript differ significantly at p < .05.

Table 12. Effect of environmental conditions and stocking density on relative weight (% of live weight) of carcase components of Cobb 500 broilers at 42nd day of age*.

Treatments	Back thoracic vertebrae (notarium)	Crop	Proventriculus	Pancreas
Climate
Mild and humid	3.03	0.48	0.42	0.29
Semi-arid	3.06	0.47	0.43	0.28
Alpine	3.12	0.48	0.42	0.29
Hot and dry	3.10	0.47	0.43	0.28
SEM	0.03	0.01	0.00	0.00
p Value	.18	.68	.84	.20
Mean	3.08	0.48	0.43	0.29
Density, chick/m^2
10	3.03	0.49	0.43	0.29
15	3.08	0.47	0.42	0.29
17	3.14	0.48	0.43	0.28
20	3.06	0.47	0.43	0.28
SEM	0.03	0.01	0.00	0.00
p Value	.10	.30	.87	.60
Mean	3.08	0.48	0.43	0.29

* Means within each comparison column with no common superscript differ significantly at p < .05.

Average daily body weight gain

Results showed that the interaction effect between climate and stocking density on average body weight gain was significant in starter period (p < .05), but no significant differences were observed in the body weight gain over the grower and whole periods (p ≥ .05). Moreover, the stocking density had a significant effect on the average body weight gain during the grower and whole periods (p < .05).

Feed intake

The effect of climate on feed intake was significant during the starter, grower and whole experimental periods (p < .05). However the interaction effect between stocking density and climate, and also the effect of stocking density on feed intake were not significant during the starter, grower and whole periods (p ≥ .05).

Feed conversion ratio

In the current research, the interaction effect between stocking density and climate on feed conversion ratio in starter period and the single effect of stocking density on feed conversion ratio in the starter, grower, and whole periods were significant (p < .05). But the interaction effect between stocking density and climate on feed conversion ratio in the grower and whole periods was not significant (p ≥ .05).

Body weight at 42 days of age

The climate did not have any effect on average body weight (p ≥ .05), but the effect of stocking density on body weight was significant at the end of experimental period (p < .05). The interaction effect between stocking density and climate on average body weight was not significant (p ≥ .05). Broiler chickens under alpine climate with a density of 10 chicks/m² had the highest, and the group under dry climate with a density of 20 chicks/m² had the lowest, body weight.

Survival rate

The results showed that the interaction effect between stocking density and climate on survival rate of broiler chickens was significant (p < .05). The lowest mortality was observed under mild and humid climate with 17 chicks density and the highest was under hot and dry climate with 10 chicks density (p < .05).

Production index

According to the results, the separate effects of climate and density on production index were significant (p < .05), but the interaction effect of these two factors on the production index was not significant (p ≥ .05). The highest and lowest production indices were respectively in the alpine and hot and dry regions (p < .05). Moreover, the highest and lowest production indices were respectively in the 10 and 20 chicks/m² densities (p < .05).

Meat production per m²

The climate and density separately had a significant effect on meat production based on live weight per m² (p < .05). The interaction effect of climate and density was not significant on meat production.

Economic expense

In evaluations, the individual factor and interaction effects between climate and density were significant for expenses (costs) (p < .05). The highest expenses in the current study were in alpine region with a density of 20 chicks/m² (p < .05), and the lowest expenses in the current study were in hot and dry region with a density of 10 chicks/m² (p < .05).

Economic earnings

In this evaluation, the interaction between climate and density was not significant (p ≥ .05), but climate and density independently had a significant effect on economic income (p < .05). Between the 4 stocking densities, the highest earnings were density of 20 chicks/m² (p < .05). Moreover, between the 4 climates, the highest earnings were alpine region (p < .05).

Economic benefit

The results of the analysis of variance indicated that the interaction effect of climate and density on the average profit of the production period were significant (p < .05). At the end of research, the highest and lowest economical benefits were perceived in the hot and dry climate with 17 and 10 chicks/m², respectively (p < .05).

Blood parameters

The results showed that the interaction effects of density and climate on the content of total cholesterol, triglycerides, very low density lipoproteins (VLDL), HDL, LDL/HDL ratio, AST and ALT were significant (p < .05). Moreover, the effects of climate on glucose, albumin and globulin levels were significant (p < .05). The stocking density had significant effects on glucose, uric acid and protein levels (p < .05). In the present study with varying stocking densities (chicks/m²), semi-arid climate with 15 chick density and mild and humid climate with 20 chick density treatments had the highest cholesterol, semi–arid climate with 20 chick density treatment had the highest triglycerides, semi-arid climate with 17 chick density treatment had the highest HDL, semi-arid climate with 20 chick density treatment had the highest AST, and hot and dry climate with 20 chick density treatment had the highest ALT (p < .05).

Immunity

At 28 and 42 days of age, the interaction effect of climate and density on IgG, IgM, and total antibody was not significant (p ≥ .05). Different climates significantly affected IgM and total antibody at 28 and 42 d of age (p < .05). At 28 and 42 d of age, the highest levels of total antibody was detected respectively in hot and dry and semi-arid climates (p < .05).

Carcase

Based on the results of this study, there were no significant interaction effect between climate and density on carcase components (p ≥ .05), but different climate regions significantly affected on full intestinal tract carcase weight, empty intestinal tract carcase weight, eviscerated carcase, thighs, and head, relative weights (p < .05). Also, stocking density had a significant effect on full intestinal tract carcase weight (p < .05).

Discussion

Alltane et al. (2018) investigated the effect of density at 3 levels (22, 18 and 14 chicks/m²) on performance traits, and they reported that the best performance parameters were achieved with a medium density (18 chicks/m²) and lower bird numbers. However, they stated that at the end of experiment the body weight gain of chickens with a moderate-low density was not significant. The results of this study, in agreement with Alltane et al. (2018), showed that 17 chicks/m² density in alpine climate had the greatest impact on daily growth in the starter period. Simitzis et al. (2012) found that reducing the density lowers the temperature and increases the airflow at the surface of the bird and this phenomenon causes the yield to increase. Furthermore, per their study, the temperature of the ambient plays a critical role in growth and feed consumption. For instance, more feed is consumed in a colder environment because the extra consumption causes to create more energy to warm up the bird’s body. However, at the starter level, there is less surface-to-bird density and this itself requires less energy to be used to heat the body of the bird. Instead, most of the energy from the feed at this level is used to grow the body of the bird. Hence, the bird’s growth and conversion rates improve. The final body weight in all experimental treatments with a density of 10 chicks/m² was higher than other density treatments in the different climates. Because the amount of space and feeder allocated to each bird in the density of 10 chicks/m² was higher than other groups, chicks had better growth by providing more space for easier access and less stress to obtain feed and water. In the current research, the maximum percentage of survival, production index and meat production/m² were achieved in the mild and humid climate, and in addition, the highest production efficiency index was found at 10 chicks/m² density. Esmail (2013) indicated that many factors (such as flock size, density, temperature, lighting, feed, water, etc.) influence the growth rate, feed intake, and mortality in broiler chickens and consequently the production index. The results of this study are consistent with the results of Esmail (2013).

Ike (2011) expressed that under heat stress condition and high stocking density, birds consume less feed and severe feed deficiency leads to reduces absorption of amino acids and other essential nutrients. In this study, maybe the overcrowding and inadequate feed intake are the reasons for reduced production index of broiler chickens in treatments with 20 chicks/m² density. Farhadi and Hosseini (2016) and also Heidari and Toghyani (2018) found stock density affect on broiler productivity significantly.

According to my research, the highest amount of meat density was 20 pieces in cold regions. This is consistent with the findings of the NCC (2017) and Vanhonacker et al. (2008). They stated that the amount of meat production in a density of 20 pieces compared with the lower density was as a result of an increase in the number of poultry in the experimental area not to the production efficiency. Also broiler growth is a quantitative property influenced by the genotype, and the environment. Since the habits of broiler chicks are more comfortable with cold weather, therefore, due to the constant diet and the difference in climatic conditions, one can conclude that different outcome is attributed to the interaction between the genotype and the environment (FAO (Food and Agriculture Organization of the United Nations) 2014).

According to the present research, a significant effect of climate (environment) and stocking density on profit earned at the end of experiment. The experimental treatment with 17 chicks/m² under hot and dry climate had the highest profit compared to other treatments, but at 10 chicks/m² density group showed a lower economic benefit at the end of experiment compared to other groups.

Due to its significantly better feed conversion ratio in the starter period and its effect on the amount of feed consumption in the whole period, density treatment of 17 chicks/m² in hot and dry climate had higher profit than other groups.

By altering density from 10 to 17 chicks/m², more profit was obtained ($based on kg/m²) $2.87 in hot and dry climate, $0.93 in alpine climate, $0.7 in semi-arid climate and $0.38 in mild and humid climate. Therefore, for example in a broiler house with 2000 m² area and under hot and dry climate, increasing the density from 10 to 17 chicks/m², will make $5740 more benefit in the whole period.

Begum et al. (2010) reported that broiler meat production per unit of floor space is an effective measure of profitability, and regarding to economic situations and market demand, changing stocking density can affect the average body weight of each bird.

It may seem that raising chickens at a density of 20 chicks/m² would be more beneficial, but in this research, the optimum density for maximum profit was 17 chicks/m². In the starter period, due to the fact that chicks are small, competition for the necessary space and access to feed is less, and therefore the effect of density on body weight gain was not significant. Because chicks are larger in grower period, and the competition for access to feed is greater, this factor made density treatment differences in meat production/m² significant. At a density of 20 chicks/m², chicken meat production was lower than for other treatments and thus the income of this treatment was less than other treatments, while treatments with a density of 17 chicks/m² had the highest profit.

The results exhibited that the density and climate had a significant on total cholesterol, triglyceride, VLDL, HDL, LDL/HDL ratio, AST, ALT levels (p < .05).

Moreover, broiler chickens under alpine climate with a density of 15 chicks/m² showed the lowest LDL and the highest HDL. It seems that climate impact led to decreased cholesterol, resulting in a decrease in LDL levels and an increase in HDL.

Nobakht and Hosseini Fard (2016) conducted a study on feeding bran to laying hens, diluting nutrients, which gave results consistent with those obtained in this study. Ojano-Dirain and Waldroup (2002) also confirmed the corrected protein and amino acid needs of broilers in ward weather.

Researchers claim that climate condition can influence on both blood fatty tissues as well as broiler chicken fatty body surface. Furthermore, it has been proven that in a cold climate energy released from the rationing of feed is used for the temperature and this itself causes no accumulation of fatty tissues. Therefore, this leads to a reduction of blood fatty tissues (Ezeh et al. 2012; Amini et al. 2015).

Aspartate aminotransferase is one of the most important enzymes in the aminotransferase group which catalyses alpha-keto acid to amino acids by transferring amine units. Evaluation of aspartate aminotransferase activity is a basic method for the diagnosis and assessment of liver and/or muscle damage. Generally, increasing aspartate aminotransferase has a high correlation with the amount and severity of cell damage (Nobakht and Hosseini Fard 2016). In the present study, broiler chickens with a density of 20 chicks/m² in dry and semi-arid climates had the highest levels of ALT and AST. Because at high densities, the competition of birds to consume more is high, so the possibility of muscular injury is also high and is the reason for increment these two liver enzymes in the blood serum. According to the results, the amount of Albumin is the highest in the warm and dry climate.

Increasing the temperature of the quail environment (heat stress) reduces total serum protein (Ozge et al. 2000; Ozbey et al. 2004). Zhang (2015) reported when broilers are exposed to heat stress condition, some changes (disruption of acid–base balance, increased blood pH and respiratory alkalosis) occur in the blood system, and the cardiovascular system is one of the systems that interferes with heat dissipation that this can affect the amount of albumin.

In the present study, the climate (ambient temperature and humidity) factor had a significant effect on IgT at 28 and 42 days of age, and broilers under humid climate had the highest level of IgT.

In the current experiment, the chickens grown in the density of 20 chicks/m² at 42 days of age had the lowest immune response.The high environmental temperature can weaken the immune system of broiler chickens (Niu et al. 2009).

One of the biggest problems in dry climate is a reduction in feed consumption. This in turn becomes a hindrance of body to grow and especially a smaller body weigh on lenphavi (Note: I think it should be lymphatic system. correct this word). Consequently, this negative chain of reaction from dry climate can result a weaker immune system (Amini et al. 2015; Gous, 2005).

Heckert et al. (2002) indicated that the high stocking density causes suppression of immunity in broiler chickens. Some researchers reported that the stress in poultry causes disruption of leukocyte function (Ozbey et al. 2004).

Lesson and Summers (2008) argued that during growth stages especially at the end of this period, as the body weight increases there is a competition among the birds for more feed consumption. Also, there is an increase in density. These two factors cause the stress level for the birds to increase. Consequently, higher stress level weakens immune system of the birds. However, increase in population density has a positive impact on micro-organism of the bird’s environment which becomes a breeding ground for microbes that can create a weaker immune system for the flock (Baghoyan 2006).

In the resent study, the effects of the different climate regions on the relative carcase, thigh, and head weights were significant (p < .05). The highest weight was for semi-arid and alpine climates. In addition, the weight of wings in the density of 17 chicks/m² was numerically heavier than other densities.

Based on the experiment of researchers, it is believed that poultry are animals with warm blood. As such, they (i.e. poultry) are comfortable only with a Limited Band of Temperature (LBT). Otherwise, any temperature either above or below the LBT, can have negative impact on the weight of bird. It worth noting that the ideal temperature for broiler chicken for the first 4-week ranges between 18 and 21 °C. Therefore, a comfortable temperature to improve the weight of the broiler chicken is more suitable in in a cold or semi-dry environment than warm climate. This is consistent with present research (Aengwanich and Simaraks 2004). In another report, increasing the stocking density caused increased litter moisture, resulting in the occurrence of dermatitis, foot pad lesions, breast wounds, and skin problems, and consequently, reduces the weight of carcases (Beg et al. 2016).

Conclusions

Different climates and density were affected on the flock’s economic performance. We tried to determine optimum stocking density in each climate region. The highest profit was obtained in hot and dry climate with a density of 17 chicks/m². Based on the results of this research, identification of optimum stocking density in different climate regions for broiler chicken meat production, provides information for developing in each climate and helps producers in different parts of the world to maximise product and profit.

Acknowledgments

This manuscript is prepared based on PhD thesis of first author at Science and Research Branch, Islamic Azad University, Tehran, Iran. The authors are grateful to the Science and Research Branch, Islamic Azad University, Tehran, Iran for supports. The authors thank the staff of the poultry office in the Livestock Department of Jihade-Agriculture Ministry; Cobb Vantress, Parastou Ghaem, Golestan Toyour; Peygir companies; the National Union of Poultry Farms, Iran for collaborating in conducting this research.

Disclosure statement

No potential conflict of interest was reported by the author(s).