Effect of two brooding systems and four stocking densities on immune response and stress indicators of broiler chicks during the brooding period

Abstract

This experiment investigated the effects of the brooding system (BS) and stocking density (SD) on the immune response of 1-14-day-old broiler chicks. A 3672 one-day-old Cobb 500 chicks were raised in two BSs (conventional floor brooding system (CFBS) and the newly developed multi-tier closed brooding system (NDBS)) at four different SDs (30, 60, 90, and 120 chicks/m²), resulting in a 2 × 4 factorial arrangement. Immunity data on lymphoid organ weights, maternal antibody titres to IBV, IBDV, and NDV, and stress indicators were collected on days 6 and 12. The results showed that CFBS and NDBS had similar effects on the relative development of chick lymphoid organs (Bursa, spleen, and thymus), the durability of the maternal humoral immune response against IBDV, and the maintenance of normal levels of stress indicators. Birds in CFBS outperformed NDBS in terms of the durability of maternal immunity to IBV and NDV. Birds in NDBS had a higher liver weight percentage than CFBS. Maternal immunity, lymphoid organ weights %, glucose and cholesterol concentrations were not altered by SD. In contrast to relative weights, differences in absolute lymphoid weights (g) were attributed to differences in live weights. An increase in SD per area unit resulted in an increase in heterophils and a decrease in lymphocyte percentage resulted in an increase in the ratio of heterophils to lymphocytes(H:L). In conclusion, birds in the higher SD had lower live weights and higher H:L ratios. Smarter brooding systems are needed to usher in a new era for the poultry industry.

Highlights

• The brooding system and/or stocking density accounted for the stress factors of the birds.

• Both brooding systems increased the relative development of chick lymphoid organs and the durability of the maternal humoral immune response to IBDV.

• Smarter brooding systems are needed to apply the critical control point standard and usher in a new era for the poultry industry.

• As stocking density per unit area increased, live and liver weights decreased, and the lymphocyte ratio increased.

Keywords:

• Broilers
• brooding system
• immune status
• stress indicators
• stocking density

Introduction

Poultry production has been increasing faster than other livestock worldwide in the agricultural sector for decades (Relić et al. 2019). Since the immune system of newly hatched chicks is not yet functionally developed, and they have limited ability to synthesise antibodies endogenously, the brooding period was chosen for the study. Accordingly, the early brooding period is critical for the proper protection and development of chicks against invading pathogens (Jha et al. 2019). Therefore, any mismanagement during this period is likely to result in suboptimal performance that often cannot be compensated for in the remaining production cycles (Lin et al. 2006; Demeke, 2007; Jha et al. 2019).

Rearing systems are a critical factor affecting chick comfort, welfare, health, and production and reproductive performance (Willis et al. 2002; Chen et al. 2013). Poultry were kept either indoors or outdoors (Yan et al. 2021). Many researchers assessed the effects of various rearing systems on broiler performance and health (Al-Bahouh et al. 2012; Leinonen et al. 2014; Vanan et al. 2014; Ghanima et al. 2020). The outcomes of these studies on various rearing systems are inconclusive in terms of bird health and performance. For example, Al-Bahouh et al. (2012), rearing broilers in cages may result in higher production efficiency than rearing broilers on the ground. Ghanima et al. (2020) found that litter housing systems had higher serum, liver, and muscle cholesterol content, lower numbers of eosinophils, lymphocytes, basophils, and monocytes, and lower phagocyte activity. In addition, Ghanima et al. (2020) observed no differences between the three housing systems (cages, litter, and perforated plastic slate) in terms of the humoral immune response against the Newcastle disease virus and avian influenza. Many underlying factors, such as litter materials, coccidiosis, and others, influence bird health and contribute to the differences (Choct, 2009). Although most scientific publications investigated broiler rearing in free-range, battery cage, and intensive systems (Ahmad et al. 2019a, b), the current trial was the first of its kind to evaluate a newly developed, multi-tier closed brooding system (Innovative Control System Company).

This closed system is automated, fully isolated, and equipped with strict biosecurity. It has a conveyor belt for cleaning droppings and is equipped with humidity and CO₂ temperature sensors to control the critical control point (CCPs), i.e. ventilation of the entire, temperature, and relative humidity, as close as possible to the bird's needs. The system can be used to raise chicks on the farm or in the hatchery and allows for higher stocking densities (more birds per square meter). Thereafter, birds can be moved to rearing houses at the age of 7–10 days of age, to grow out of houses when they are more developed and less susceptible to variations in house climate and infection with pathogens. Most important is the number of birds, or kg/m², as it is hypothetically possible to develop improved designs that can control all CCPs. If such a system proves successful, the impact will be remarkable. As an example, two new brooding systems (2 × 25 m with 10 tiers) can produce about 150,000 chicks every 10 days, more than enough to be housed in a broiler farm with 6 houses. Therefore, this research is conducted to test this concept and investigate its advantages/disadvantages in practice.

Broilers must be raised under optimal environmental conditions to reach their genetic potential for growth and health, and any deviation from optimal conditions can result in lymphoid organs regression, immunosuppression, and altered physiological responses, all of which increase susceptibility to disease (Chandra and Newberne, 2012; Demeke, 2007; Hofmann, 2021; Lin et al. 2006). Immune stress induced by external factors such as intensive rearing (high stocking density) can negatively affect broiler health, immune homeostasis, immunity, lymphoid organs, circulating leukocytes, viability, well-being, physical activity, and product quality (Astaneh et al. 2018; Gholami et al. 2020; Nasr et al. 2021; Nawarathne et al. 2020). The Hetrophil: Lymphocyte ratio (H:L ratio) is a reliable indicator of high glucocorticoid levels (Astaneh et al. 2018; Nasr et al. 2021).

To our information, there are few, if any, publications on the stocking density (SD) of broilers raised to 14 days of age. Most publications target the final SD at the end of the production cycle (i.e. 20 birds/m² from beginning to end) (Atilgan and Koknaroglu, 2006; Gholami et al. 2020; Anderson et al. 2021). Density/m² depends on access to free space, age at slaughter, breed type, countries, etc. (Costantino et al. 2018; EFSA, 2022). Lack of available space for birds has been associated with behavioural and endocrinological changes indicative of stress and decreased well-being. Stress activates hypothalamic-pituitary-adrenal signalling pathways that play an important role in balancing physiological and immunological responses in birds (Tilbrook and Fisher, 2020; Konsman, 2023).

Farmers and researchers use a variety of SDs during the brooding period. However, the effects of these different SDs have not been studied. In addition, there are few studies on the selection of appropriate SD in different BS or on the combined effect of SD and BS on broiler immunity and stress indicators (Abo Ghanima et al. 2020), so this study was conducted to investigate this issue during the brooding period. The main objectives of this study were to evaluate the functional development of lymphoid organs, maternal immunity antibody titres in response to IBV, IBDV, and NDV, and stress indicators in broiler chicks raised in two different brooding systems (NDBS versus CFBS) with different SDs, and to determine the most appropriate SD in each system to raise the chicks under comfortable conditions. The results of the study will be useful for poultry farmers involved in poultry production and rearing.

Materials and methods

Ethical approval

The study was approved by the Ethics, Methodology, and Animal Welfare Studies Committee of King Saud University, Saudi Arabia (No. KSU-SE-23-17).

Housing, treatments, and experimental design with chicks

Three thousand six hundred seventy-two day-old chicks (Cobb 500) were obtained from a local hatchery. Upon arrival, the chicks were sexed, weighed, and distributed to 24 pens of equal weight. Based on Alqhtani et al. (2022) and England et al. (2023), feather sexing was performed as the initial test to determine the percentage of males and females (50%: 50% sex ratio for each pen). At the hatchery, all birds were immunised against infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), and Newcastle disease virus (NDV). The feeder, feeder spaces, water line, and the number of birds per water nipple were distributed according to our previous study by Qaid et al. (2016). Birds were fed a commercial crumbled starter feed and received feed and water ad libitum (Qaid et al. 2016). Critical control points were controlled as per breeder guidelines (Cobb, 2012) for chicks 0-14 days of age, except for the rate of SD per target weight.

The treatments consisted of two BS (CFBS and NDBS) and four SD (30, 60, 90, and 120 chicks/m²), corresponding to 2 × 4 factorial arrangement (8 treatments). Following Nawarathne et al. (2020), who studied the effects of three stocking densities (40, 80, and 120 chicks/m²) at the brooding stage on the performance and stress responses of Cobb 500 broiler chickens, four different SD were selected for this work. Each treatment was repeated three times in each of the two brooding systems. System A was conventional floor brooding in pens with fresh wood shavings similar to commercial facilities. System B was a new brooding system (a multi-tier closed system).

Measurements

Nine chicks per treatment of approximately average weight from each experimental unit were randomly selected on days 6 and 12. Sample selection was largely the same (4 male and 5 female chicks for each treatment). Blood samples were collected and left overnight at room temperature (25 °C) for the serum to separate. A drop of fresh blood was prepared and spread thinly on a pre-cleaned glass slide. The air-dried slides were stained with a Giemsa stain for 2 min. A total of 100 cells were randomly selected from objective fields for examination of the number of heterophils and lymphocytes, then observed and counted from each slide. The 100× oil immersion lens was in focus under a light microscope, and the H:L ratio was then calculated for determining stress in chicks. Giemsa dye and working solutions were prepared as described by Meirelles and Covas (2011). Giemsa stain stains erythrocytes pink, platelets very light pink, the nuclear chromatin of heterophils magenta and the cytoplasm of lymphocytes sky blue.

Sera were then collected from the remaining blood samples by centrifugation (5,000 x g) for 10 min at 4 °C and the resulting serum was stored at −20 °C until assayed for glucose and cholesterol by the spectrophotometer method and for maternal antibody titres against NDV, IBV, and IBDV by ELISA. Glucose and cholesterol levels were measured calorimetrically with a spectrophotometer (Randox Laboratories, UK), and commercial diagnostic kits were used to determine their concentrations in serum. IBV, IBDV, and NDV virus antibody titres were determined using specific commercial enzyme-linked immunosorbent assay (ELISA) kits from reagent manufacturers (Flockchek, IDEXX Laboratories, Inc., USA). Optical density values were determined at a wavelength of 650 nm using an ELISA reader (BioTek ELX800). The relative amount of antibodies in the sample was determined by calculating the sample-to-positive (S/P) ratio as described by Alqhtani et al. (2022). After bleeding, sampled chicks were killed by dislocating the neck and the liver and lymphoid organs were removed and weighed. Absolute (g) and relative (% body weight) weights were used.

Statistical analysis

For statistical analysis of the collected data, the two-way model ANOVA was used using the SAS procedure “General Liner Models procedure” (SAS-Institute, 2004) within a randomised complete block design (RCBD). The model used was as follows: $${\gamma }_{\mathit{ijk}}=\mathrm{\mu }+{BS}_i+{SD}_j{+{\mathit{BSSD}}_{ij}+e}_{\mathit{ijk}}$$

The components of this model are as follows:

Where Υ_ijk= a measured value of each observation; µ = mean; BS_i= effect of brooding system type; SD_j = effect of stocking density level; BSSD_ij = interaction between brooding system and stocking density, e_ijk = experimental error. Duncan’s multiple range test was used to compare means when significant (statistical significance was assumed at (P < 0.05). Before analysis, the per cent data were transformed with the arcsine function; however, the actual per cent data are reported.

Results

Absolute and relative weights of liver and lymphoid organs

As shown in Table 1, BS affected the live weights of slaughtered birds on days 6 and 12. Birds reared with CFBS performed better than birds reared with NDBS (p < 0.05). On day 12, SD affected the live weight of the birds slaughtered. On day 12, SD affected the live weight of slaughtered birds. As birds aged, body weight decreased as SD increased. There was no apparent interaction between BS and SD on the live weight of slaughtered birds (P > 0.05).

Table 1. Effect of the brooding system (BS) and stocking density (SD) on live body weights (g) of slaughtered birds at 6 and 12 days of age.

Treatment	BS^1	SD^2	Live body weight (g)
			Day 6	Day 12
1	CFBS	30	181	440
2	CFBS	60	177	435
3	CFBS	90	173	390
4	CFBS	120	182	356
5	NDBS	30	164	320
6	NDBS	60	155	339
7	NDBS	90	153	319
8	NDBS	120	145	292
SEM^3			5.02	12.46
Main effect BS
CFBS			178^a	405^a
NDBS			154^b	317^b
SEM			2.51	6.23
Main effect SD
30			172	380^a
60			166	387^a
90			163	355^b
120			163	324^c
SEM			3.55	8.81
Effect (P-value)
BS			<.0001	<.0001
SD			0.2341	<.0001
BS × SD			0.1671	0.1195

^a,b,c Means in the same column with no common letters differ significantly (p < 0.05). ¹BS brooding system ²SD Stocking density per m² (birds/m²). ³SEM: Standard error of the mean. n= nine samples per treatment. CFBS: Conventional floor brooding system, NDBS: Newly developed multi-tier closed brooding system.

The mean absolute and relative weights of liver and lymphoid organs at 6 and 12 days of age, respectively, are shown in Tables 2 and 3. As shown in Table 2, the absolute weight of the liver was affected by BS and SD (p < 0.05), but not by their interactions (P > 0.05). On days 6 and 12, birds reared with CFBS had higher liver weights than birds reared with NDBS. In addition, liver weights (g) on day 6 of life were significantly higher in 30 birds/m² than in 120 birds/m² (p < 0.05), with 60 and 90 birds/m² in between. On day 12 of life, liver weights (g) were significantly higher at SD with 30 and 60 birds/m² than at SD with 120 birds/m² (p < 0.05), with 90 birds/m² being an intermediate value. These results suggest that absolute liver weights during the breeding period are dependent on SD.

Table 2. Effect of the brooding system (BS) and stocking density (SD) on the absolute liver and lymphoid organ weights (g) of broiler chicks reared at two different systems and four levels of stocking densities collected at 6 and 12 days of age.

Treatment	BS^1	SD^2	Liver weight (g)		Bursa weight (g)		Spleen weight (g)		Thymus weight (g)
			6 d	12 d	6 d	12 d	6 d	12 d	6 d	12 d
1	CFBS	30	8.15	15.2	0.253	0.713	0.140	0.419	0.986	1.36
2	CFBS	60	8.12	14.7	0.280	0.666	0.140	0.356	0.890	1.35
3	CFBS	90	8.10	13.4	0.254	0.631	0.136	0.299	0.833	1.06
4	CFBS	120	7.92	12.0	0.270	0.483	0.134	0.297	0.880	0.972
5	NDBS	30	8.24	13.5	0.220	0.448	0.127	0.321	0.711	0.916
6	NDBS	60	7.75	13.9	0.234	0.472	0.132	0.310	0.602	1.09
7	NDBS	90	7.69	13.1	0.180	0.559	0.120	0.311	0.567	1.04
8	NDBS	120	7.11	12.1	0.237	0.464	0.141	0.242	0.587	0.886
SEM^3			0.176	0.265	0.019	0.028	0.013	0.023	0.031	0.036
Main effect BS
CFBS			8.07^a	13.8^a	0.264^a	0.623^a	0.137	0.343^a	0.897^a	1.19^a
NDBS			7.70^b	13.1^b	0.218^b	0.486^b	0.130	0.296^b	0.617^b	0.984^b
SEM			0.092	0.141	0.01	0.014	0.006	0.011	0.016	0.019
Main effect SD
30			8.20^a	14.3^a	0.237	0.581^a	0.133	0.370^a	0.848	1.14^a
60			7.94ab	14.3^a	0.257	0.569^a	0.136	0.333ab	0.746	1.22^a
90			7.89ab	13.3^b	0.217	0.595^a	0.128	0.305^b	0.700	1.05ab
120			7.51^b	12.0^c	0.253	0.474^b	0.137	0.269^b	0.733	0.929^b
SEM			0.128	0.2	0.013	0.019	0.008	0.016	0.023	0.027
Effect (P-value)
BS			0.038	0.023	0.034	<.0001	0.865	0.045	<.0001	0.006
SD			0.001	<.0001	0.542	0.044	0.490	0.020	0.106	0.032
BS × SD			0.082	0.160	0.889	0.162	0.635	0.402	0.989	0.147

^a,b,c Means in the same column with no common letters differ significantly (p < 0.05). ¹BS brooding system ²SD Stocking density per m² (birds/m²). ³SEM: Standard error of the mean. n= nine samples per treatment. CFBS: Conventional floor brooding system, NDBS: Newly developed multi-tier closed brooding system.

Table 3. Effect of the brooding system (BS) and stocking density (SD) on the relative liver and lymphoid organ weights (%) of broiler chicks reared at two different systems and four levels of stocking densities collected at 6 and 12 days of age.

Treatment	BS^1	SD^2	Liver weight: BW		Bursa weight: BW		Spleen weight: BW		Thymus weight: BW
			6 d	12 d	6 d	12 d	6 d	12 d	6 d	12 d
1	CFBS	30	4.50	3.47	0.14	0.16	0.08	0.10	0.55	0.31
2	CFBS	60	4.60	3.39	0.16	0.15	0.08	0.08	0.50	0.31
3	CFBS	90	4.73	3.46	0.15	0.16	0.08	0.08	0.48	0.27
4	CFBS	120	4.36	3.37	0.15	0.13	0.07	0.08	0.48	0.27
5	NDBS	30	5.03	4.26	0.13	0.14	0.08	0.10	0.43	0.29
6	NDBS	60	5.05	4.12	0.15	0.14	0.09	0.09	0.39	0.32
7	NDBS	90	5.03	4.15	0.12	0.18	0.08	0.10	0.37	0.32
8	NDBS	120	4.94	4.21	0.17	0.16	0.10	0.09	0.41	0.31
SEM^3			0.136	0.121	0.010	0.009	0.008	0.007	0.019	0.014
Main effect BS
CFBS			4.55^b	3.42^b	0.15	0.15	0.08	0.08	0.51^a	0.29
NDBS			5.01^a	4.19^a	0.14	0.15	0.08	0.09	0.40^b	0.31
SEM			0.072	0.086	0.010	0.006	0.004	0.005	0.013	0.013
Main effect SD
30			4.76	3.86	0.14	0.15	0.08	0.10	0.49	0.30
60			4.83	3.75	0.16	0.15	0.08	0.09	0.45	0.32
90			4.88	3.81	0.13	0.17	0.08	0.09	0.43	0.30
120			4.65	3.79	0.16	0.15	0.09	0.08	0.45	0.29
SEM			0.101	0.122	0.007	0.006	0.005	0.005	0.014	0.01
Effect (P-value)
BS			<.0001	<.0001	0.476	0.957	0.098	0.237	<.0001	0.345
SD			0.339	0.932	0.604	0.179	0.219	0.430	0.130	0.700
BS × SD			0.501	0.871	0.644	0.499	0.225	0.468	0.502	0.541

^a,b,c Means in the same column with no common letters differ significantly (p < 0.05). ¹BS brooding system ²SD Stocking density per m² (birds/m²). ³SEM: Standard error of the mean, n= nine samples per treatment. CFBS: Conventional floor brooding system, NDBS: Newly developed multi-tier closed brooding system.

BS also influenced the absolute weights of lymphoid organs on days 6 and 12. At days 6 and 12, birds reared with CFBS had higher lymphoid organ weights than birds reared with NDBS (except for the spleen at day 6, which was not affected). The SD had a significant effect on the absolute lymphoid organs weights n day 12, but not on day 6. At 12 days of age, the weights of lymphoid organs (g) were significantly higher in SD with 30 and 60 birds/m² than in SD with 120 birds/m² (p < 0.05), with 90 birds/m² representing an intermediate value. These results suggest that the absolute weights of the lymphoid organs during the brooding period depend on SD. There was no discernible interaction between BS and SD on the absolute weights of the birds’ lymphoid organs (P > 0.05).

As shown in Table 3, there were no main or interaction effects between BS and SD for the relative weights of liver and lymphoid organs. However, birds reared with NDBS had greater liver-to-live weight ratios (P < 0.05) than birds reared with CFBS at days 6 and 12, in contrast to the relative weights of the thymus at days 6.

Antibody titres of maternal immunity against IBV, IBDV, and NDV

In this study, maternal immunity of broiler chicks against IBV, IBDV, and NDV on days 6 and 12 of life was not affected by BS or by increasing the number of chicks per unit area during a brooding period or their interaction (p > 0.05) (Table 4). However, birds in CFBS had advantages in the durability of the maternal humoral immunity response, which lasted longer against IBV and NDV during the experiment. Birds in NDBS, on the other hand, had a higher maternal anti-IBV titre and a higher rate of degradation. In addition, significant interactions between BS and SD at day 6 were the result of differences in antibody titres against IBV (p < 0.039) and NDV (p < 0.018) at different SD and BS. Antibody titres against IBV of broiler chicks at day 6 raised under CFBS in all SDs was significantly lower than those of birds raised under the NDBS. Thus, antibody titres against IBV of broiler chicks reared under NDBS (3.36, 3.24, 3.37, and 3.40 at SDs, 60, 90, and 120 chicks/m², respectively) was significantly higher than those of birds reared under CFBS (3.28, 3.20, 3.32, and 3.01 at SDs of 30, 60, 90, and 120 chicks/m², respectively), resulting in a significant interaction between system and density (p < 0.01). In addition, antibody titre against NDV was significantly higher (3.74) in broiler chicks raised under NDBS at (SD 60 chicks/m²) than in birds raised under CFBS (3.54) resulting in a significant interaction between system and density (p < 0.01).

Table 4. Effect of the brooding system and stocking density on maternal antibody titre against IBV, IBDV and NDV of broiler chicks reared at two different systems and four levels of stocking densities from 0 to 14 days of age and collected at 6 and 12 days of age.

			Anti-maternal antibody titre (Log)
Treatment	BS^1	SD^2	Anti-IBV		Anti-IBDV		Anti-NDV
			6 d	12 d	6 d	12 d	6 d	12 d
1	CFBS	30	3.28	2.49	3.69	3.31	3.65	3.04
2	CFBS	60	3.2	2.58	3.72	3.33	3.54	3.17
3	CFBS	90	3.32	2.33	3.67	3.23	3.66	3.00
4	CFBS	120	3.01	2.49	3.67	3.39	3.66	3.17
5	NDBS	30	3.36	2.37	3.71	3.27	3.63	2.88
6	NDBS	60	3.24	2.33	3.73	3.28	3.74	3.01
7	NDBS	90	3.37	2.28	3.70	3.35	3.50	2.96
8	NDBS	120	3.40	2.46	3.67	3.41	3.68	3.06
SEM^3			0.068	0.068	0.044	0.054	0.054	0.063
Main effect BS
CFBS			3.20^b	2.47^a	3.69	3.32	3.63	3.10^a
NDBS			3.34^a	2.36^b	3.70	3.33	3.64	2.98^b
SEM			0.033	0.038	0.022	0.029	0.028	0.033
Main effect SD
30			3.32	2.43	3.7	3.29	3.64	2.96
60			3.22	2.46	3.73	3.31	3.64	3.09
90			3.35	2.31	3.69	3.29	3.58	2.98
120			3.21	2.48	3.67	3.4	3.67	3.12
SEM			0.047	0.052	0.034	0.037	0.038	0.046
Effect (P-value)
BS			0.005	0.041	0.586	0.796	0.835	0.014
SD			0.089	0.123	0.674	0.193	0.399	0.051
BS × SD			0.039	0.43	0.986	0.384	0.018	0.78

^a,b Means in the same column with no common letters differ significantly (p < 0.05). ¹BS: brooding system ²SD: Stocking density per m² (birds/m²). ³SEM: Standard error of the mean; n= nine samples per treatment. CFBS: Conventional floor brooding system; NDBS: Newly developed multi-tier closed brooding system.

Chick stress indicators

The effects of BS and SD on the stress indicators of broiler chicks during the rearing period are shown in Tables 5 and 6.

Table 5. Effect of the brooding system and stocking density on serum glucose and cholesterol concentrations of broiler chicks reared at two different systems and four levels of stocking densities from 0 to 14 days of age and collected at 6 and 12 days of age.

Treatment	BS^d	SD^e	Glucose (mg/dL)		Cholesterol (mg/dL)
			6	12	6	12
1	CFBS	30	208	214	135	119
2	CFBS	60	225	205	138	128
3	CFBS	90	234	207	139	128
4	CFBS	120	217	215	143	129
5	NDBS	30	217	196	135	111
6	NDBS	60	233	205	136	113
7	NDBS	90	228	212	140	115
8	NDBS	120	225	212	143	128
SEM^f			8.68	8.75	4.25	4.67
Main effect BS
CFBS			221	210	139	126
NDBS			226	206	139	117
SEM			4.73	4.64	2.46	2.39
Main effect SD
30			213	205	135	115^b
60			229	205	137	121ab
90			231	210	140	122ab
120			221	214	143	129^a
SEM			6.35	6.44	3.21	3.33
Effect (P-value)
BS			0.52	0.52	0.89	0.01
SD			0.24	0.74	0.43	0.07
BS × SD			0.84	0.62	0.99	0.49

^a,b,c Means in the same column with no common letters differ significantly (p < 0.05). ^dBS brooding system ^eSD Stocking density per m² (birds/m²). ^fSEM: Standard error of the mean. n= nine samples per treatment. CFBS: Conventional floor brooding system; NDBS: Newly developed multi-tier closed brooding system.

Table 6. Effect of the brooding system and stocking density on serum heterophil, lymphocyte, and heterophil: lymphocyte ratio of broiler chicks reared at two different systems and four levels of stocking densities from 0 to 14 days of age and collected at 6 and 12 days of age.

Treatment	BS^1	SD^2	Heterophil (%)		Lymphocyte (%)		H:L ratio
			6	12	6	12	6	12
1	CFBS	30	25.9	35.1	58.3	57.7	0.45	0.61
2	CFBS	60	28.6	38.3	55.6	53.9	0.51	0.72
3	CFBS	90	31.5	39.9	55.5	51.4	0.57	0.78
4	CFBS	120	33.4	41.4	53	49.1	0.63	0.84
5	NDBS	30	27.1	37.3	57.1	54.9	0.48	0.69
6	NDBS	60	31	38	55.7	53.7	0.56	0.71
7	NDBS	90	31.8	40	52.8	49.7	0.61	0.81
8	NDBS	120	33.7	42.1	51.7	48	0.66	0.87
SEM^3			0.875	1.23	0.963	1.21	0.002	0.026
Main effect BS
CFBS			29.9	38.7	55.6	53	0.54	0.74
NDBS			30.9	39.4	54.3	51.6	0.58	0.77
SEM			0.493	0.632	0.517	0.615	0.001	0.001
Main effect SD
30			26.5^c	36.2^c	57.7^a	56.3^a	0.47^d	0.65^b
60			29.8^b	38.2bc	55.7ab	53.8^a	0.54^c	0.72^b
90			31.7ab	40.0ab	54.2bc	50.6^b	0.59^b	0.80^a
120			33.6^a	41.8^a	52.4^c	48.6^b	0.65^a	0.86^a
SEM			0.662	0.868	0.705	0.856	0.001	0.002
Effect (P-value)
BS			0.135	0.439	0.102	0.106	0.064	0.282
SD			<0.0001	0.0004	<0.0001	<0.0001	<0.0001	<0.0001
BS × SD			0.684	0.769	0.633	0.761	0.983	0.662

^a,b,c Means in the same column with no common letters differ significantly (p < 0.05). ¹BS brooding system ²SD Stocking density per m² (birds/m²). ³SEM: Standard error of the mean. n= nine samples per treatment. H:L ratio = heterophil: lymphocyte ratio. CFBS: Conventional floor brooding system, NDBS: Newly developed multi-tier closed brooding system.

There were no significant effects (p > 0.05) of BS or the interaction between BS and SD on serum glucose and cholesterol concentrations, heterophil percentage, lymphocyte percentage, and H:L ratio of the chicks on days 6 and 12. However, BS was found to have a significant effect on serum cholesterol levels on day 12 (p < 0.05), which were higher in CFBS than in NDBS. In addition, the H:L ratio at day 6 tended to be higher (p = 0.64) in birds raised in the NDBS than in birds raised in the CFBS (0.58 vs. 0.54).

Serum cholesterol levels tended to be dependent (p = 0.07) on SD at day 12 (115, 121, 122, and 129 mg/dl at of SDs 30, 60, 90, and 120 chicks/m², respectively) (Table 5). Cholesterol concentration in chicks was lowest at an SD of 30 chicks/m² and highest at an SD of 120 chicks/m². Chicks with SD of 60 and 90 chicks/m² had intermediate cholesterol concentrations. SD significantly (p > 0.001) affected heterophily, lymphocyte percentage, and H:L ratio of broiler chicks during the experiment. However, there was no significant effect (p > 0.05) on the serum glucose concentration and serum cholesterol concentration of the chicks.

The percentage of heterophils in the chicks was significantly increased by increasing SD (26.5, 29.8, 31.7, and 33.6% on day 6 and 36.2, 38.2, 40.0, and 41.8% on day 12 at SDs of 30, 60, 90, and 120 chicks/m², respectively) (p < 0.0001).). The chicks with an SD of 120 chicks/m² had the highest heterophil percentage, while the chicks with an SD of 30 chicks/m² had the lowest heterophil percentage. The chicks with an SD of 60 and 90 chicks/m² had an intermediate heterophil percentage.

In contrast to heterophile, the lymphocyte percentage of the chicks decreased significantly (p < 0.0001) with increasing SD (57.7, 55.7, 54.2, and 52.4% on day 6 and 56.3, 53.8, 50.6, and 48.6% on day 12 at SDs of 30, 60, 90, and 120 chicks/m², respectively). On day 6, chicks housed at 30 chicks/m² had the highest lymphocyte percentage (57.7%), whereas birds housed at SD of 120 chicks/m² had the lowest lymphocyte percentage (52.4%). Chicks housed at SDs of 60 and 90 chicks/m² had intermediate lymphocyte percentages. On day 9, chicks housed at SDs of 30 and 60 chicks/m² had a significantly higher lymphocyte percentage than chicks housed at 90 and 120 chicks/m².

Consequently, the heterophil: lymphocyte ratio of the chicks increased significantly (p < 0.0001) with increasing SD (47, 0.54, 0.59, and 0.65 on day 6 and 0.65, 0.72, 0.80, and 0.82 on day 12 at SDs of 30, 60, 90, and 120 chicks/m², respectively). Chicks with an SD of 30 were significantly higher than chicks with an SD of 60 chicks/m² on day 6 but did not differ on day 12. However, the H:L ratio was lower than that of chicks maintained at 90 and 120 chicks/m². In addition, the H:L ratio was highest for chicks housed at 120 chicks/m² (Table 6).

Discussion

Both brooding systemLigand of pertinence to thiss had similar effects on the relative development of chick lymphoid organs. Differences between BS in absolute weights of the liver (g) and absolute lymphoid organs (g) of birds were attributed to possible greater differences in the live weight of chick between systems and stocking densities. Although it is unlikely that sex affects lymphoid organs because the sex ratio in the barns was balanced and the sample selection between the sexes was approximately equal, further studies are needed on the effects of sex in association with BS and SD on selected parameters of broiler chicks during the brooding period. In partial agreement with Jin et al. (2019), they found that the rearing system affected absolute and relative thymus weight. They observed that absolute thymus weight and thymus-to-body weight ratios were significantly higher in birds from free-range rearing systems than in birds from conventional cage-rearing systems. Compared with birds from CFBS, birds from NDBS had significantly higher relative liver weight (%). In agreement with Ghanima et al. (2020), all carcase characteristics were found to be unaffected by the different housing systems (cage, litter, and perforated plastic slate) except for the percentage of the liver and breast, which was higher in caged reared birds.

As far as we are aware, this is one of the first studies of its kind on SD broiler chicks raised to 14 days of age, whereas most scientific publications in the literature focus on the last SD at the end of the rearing cycle. Therefore, there are very few, if any, immunity data with which to compare our work. According to the results of this study, SD had no significant effect on the relative weights of liver and lymphoid organs during the brooding phase. High SD can be stressful and have a detrimental effect on broiler immunity (Heckert et al. 2002; Houshmand et al. 2012; Thaxton et al. 2006). This means that birds suffer more under stressful conditions, such as a higher SD, resulting in less developed lymphoid organs. However, the SD of the brooding period does not significantly affect the relative weight of liver and lymphoid organs. In agreement with Nawarathne et al. (2020), they found that SD has no significant effect on the relative weights of lymphoid organs of chicks reared at different SDs during the brooding period. Previous studies have shown that the relative weights of lymphoid organs at market age are not affected by SD (Buijs et al. 2009; Heckert et al. 2002; Houshmand et al. 2012; Nawarathne et al. 2020; Thaxton et al. 2006). In contrast, Ravindran et al. (2006) found that the SD has effects on market-age lymphoid organs.

Maternal antibody levels increase with yolk uptake in the first 2 to 4 days, then decrease and are depleted by offspring in the first 2 weeks after hatching (Ahmed and Akhter, 2003; Ravindran and Abdollahi, 2021). Birds in CFBS had advantages in terms of the durability of the maternal humoral immune response, which lasted longer against IBV and NDV during the experiment. While birds in NDBS had a higher maternal anti-IBV titre with a higher decay rate. This suggests that maternal anti-IBV antibodies may be catabolized more rapidly with age in NDBS. SD has no effect on the humoral immune response. In agreement with Astaneh et al. (2018), it was found that Newcastle’ titre was not affected by treatments. There were no significant interactions between system and density for all parameters except antibody titre of maternal immunity to IBV (p < 0.039) and NDV (p < 0.018) at day 6 of life. BS by SD interaction suggests that a possible two-way interaction affects antibody titre of maternal immunity to IBV and NDV at day 6 of life by system and density together.

In the present study, glucose levels at 6 and 12 days of life were not affected by BS. In contrast, Jin et al. (2019) found that the rearing system had significantly lower glucose. In agreement with Jin et al. (2019), who found that a conventional cage-rearing system increased glucose and cholesterol content compared to the free-range system, cholesterol content was increased at day 12 of age in CFBS compared to NDBS in this study.

Physiological stress indicators have been reported to be unaffected by stocking density (Dozier et al. 2006; Buijs et al. 2009). A similar result was reported by (Houshmand et al. 2012; Azzam and El-Gogary, 2015), who found that there was no relationship between blood cholesterol level and density stress in birds. In contrast to the results of the present study’, (Virden and Kidd, 2009; Nawarathne et al. 2020) indicated that blood glucose levels increased with increasing SD. Conversely, as stress increases, the demand for blood glucose increases, so adrenaline is released and glycogen reserves are mobilised from the body, which can lead to low blood glucose concentrations (Summers, 2006; Abuelgasim et al. 2021). However, serum cholesterol concentration at day 12 tended to be dependent (p = 0.07) on SD. Thus, cholesterol concentration in chicks was lowest at SD of 30 chicks/m² and highest at SD of 120 chicks/m². These results are in agreement with those of Dozier et al. (2006), according to which serum cholesterol concentration was significantly increased by higher SD. In agreement with Nawarathne et al. (2020), they found that a lower SD (40 chicks/m²) during the brooding period can reduce the stress response of birds compared to a higher SD. This finding is consistent with increased cholesterol and decreased glucose levels in chick's serum reported by other researchers (Vieira and Moran Jr, 1999; Peebles et al. 2004), as chicks switch from yolk-derived lipids to dietary carbohydrates as their primary energy source within the first two to three days after hatching. Vieira and Moran Jr (1999) found that the yolk sac is high in lipids and protein but very low in carbohydrates.

The lymphocyte, heterophil, and H/L ratios of the chicks were not significantly affected by the brooding system. The results showed that BS had no negative effects on the stress indicator, indicating that the birds were comfortable in both systems, especially in CFBS. Housing systems may affect the number of differentiated circulating leukocytes, and it was found that H/L ratio was significantly different in conventional battery cages, modified cages, and an intensive free-range system: 0.58, 0.43, and 0.38, respectively (Shini, 2003). Heterophilic birds are functionally equivalent to mammalian neutrophil granulocytes in that they have high phagocytic activity and are the first cells involved in inflammatory responses (Gross and Siegel, 1983; Genovese et al. 2013). Our results on H:L ratio are largely in agreement with most of the results published in the literature (Martrenchar et al. 1997; Feddes et al. 2002). Other studies showed that the H/L ratio is independent of SD at 15, 20, and 25 birds/m² (Beloor et al. 2010). High SD in broilers showed metabolic changes in blood biochemical parameters including a decrease in lymphocytes, an increase in heterophils with increased H/L ratio, an increase in blood stress hormones, a decrease in immune response, an increase in oxidative stress, increased susceptibility to infections as in Newcastle virus disease and necrotic enteritis, an increase in plasma levels of glucose, cholesterol, and corticosterone during the adaptive phase of stress (Astaneh et al. 2018; Nasr et al. 2021).

Conclusion

In conclusion, the results suggest that BS has similar effects in both CFBS and NDBS by increasing the relative development of chick lymphoid organ weights, the durability of the maternal humoral immune response against IBDV, the durability of the maternal humoral immune response against IBDV, the maintenance of normal glucose and cholesterol levels, and the stress indicator. Birds in CFBS outperformed NDBS in terms of the durability of maternal immunity against IBV and NDV, and further work is needed to make NDBS the most promising strategy.

SD did not alter maternal immunity, relative weights of lymphoid organs, or glucose and cholesterol concentrations. However, differences in absolute weights of liver and lymphoid organs (g) were due to differences in the live weights of chicks between BS and SD. An increase in SD per unit area resulted in an increase in heterophils and a decrease in lymphocyte content resulted in an increase in the ratio of heterophils to lymphocytes and consequently a decrease in live and liver weights.

Further studies are needed to investigate the effects of sex besides BS, SD on selected parameters of broiler chicks during the brooding period. Further efforts and intelligent changes are needed to apply the standard critical control point in the new system. Smarter rearing systems are needed to usher in a new era for the poultry industry.

Acknowledgments

The authors extend their appreciation to the Research Supporting Project number (RSPD2023R690), King Saud University, Riyadh, Saudi Arabia.

Disclosure statement

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

Data availability statement

The results and analyses presented in this paper are freely available upon request.

Additional information

Funding

Researchers Supporting Project number (RSPD2023R690), King Saud University, Riyadh, Saudi Arabia.