Prenatal auditory stimulation and impacts on physiological response to feed restriction in broiler chickens at market age

Abstract

Feed restriction could induce physiological stress in broiler chickens, leading to welfare issues. Prenatal stimulation could improve stress-coping mechanisms in poultry. The present study aimed to elucidate the effects of subjecting developing embryos to auditory stimulation on physiological stress response to feed restriction in broiler chickens at market age. A total of 423 hatching eggs of Cobb 500 (Gallus domesticus) were subjected to the following auditory treatments: 1) no additional sound treatment other than the background sound of the incubator's compressors at 40 dB (CONTROL), 2) exposure to pre-recorded traffic noise at 90 dB (NOISE), and 3) exposure to Mozart's Sonata for Two Pianos in D Major, K 488 at 90 dB) (MUSIC). The NOISE and MUSIC treatments were for 20 min/h for 24 h (a total of 8 h/d), starting from embryonic days (ED) 12 to hatching. On d 42, an equal number of birds from each prenatal auditory stimulation (PAS) group were subjected to either ad libitum feeding (AL) or 30-h of feed restriction (FR) in a completely randomised design. The FR chickens exhibited significantly higher serum levels of corticosterone (CORT), and heat shock protein (HSP) 70 compared to those of AL. Prenatal auditory stimulation, particularly NOISE, led to lower serum levels of CORT and alpha-1-acid glycoprotein (AGP) levels compared to the CONTROL group. Additionally, NOISE significantly increased brain mRNA glucocorticoid receptor and HSP70 gene expression. The cecal population of E. coli and Lactobacillus spp. was not significantly affected by prenatal auditory stimulation. In conclusion, our findings suggest that prenatal auditory stimulation, particularly NOISE, positively impacts broiler chickens' ability to cope with feed restriction.

INTRODUCTION

Feed restriction is commonly employed to manage obesity in broiler breeders (Hartcher and Lum, 2019; Tahamtani et al., 2020), as well as to induce moulting in laying hens (Santos et al., 2015; Mishra et al., 2022). Broiler chickens, however, are typically fed ad libitum during growth. However, during pre-slaughter operation, which includes fasting before harvesting, catching and loading, transportation, unloading and lairaging at the processing plant, broilers could be feed-deprived for up to 33 h (Nijdam et al., 2004). Broilers may also be subjected to feed deprivation to prevent metabolic disorders, enhance bird health (mainly to decrease skeletal issues), alter carcass composition, or reduce body fat content (Nielsen et al., 2003). Although feed restriction is beneficial in reducing carcass contamination and improving health and production, increasing evidence suggests that limiting feed intake can trigger physiological stress reactions and feelings of frustration, monotony, and hunger, resulting in repetitive object pecking, excessive drinking, and heightened activity levels in poultry (reviewed by de Jong and Jones, 2006). The requirement for feed is unyielding and essential for all livestock animals (Blundell, 2001). Given the possible detrimental effects of feed restriction on the well-being of broiler chickens, it is crucial to develop a strategy to enhance stress resilience and their ability to cope with feed restriction during rearing and pre-slaughter operations.

Life's prenatal and neonatal phases can shape an animal's phenotype. A wealth of research in mammals, fish, and birds highlights how differences in experiences during these critical periods can significantly influence the ability of individual animals to adapt to their postnatal environment and, consequently, health and well-being (reviewed by Rutherford et al., 2012; Dixon et al., 2016). Early environmental factors have the potential to impact developing biological systems, causing permanent changes in structure and function throughout life (Viltart et al., 2007). Welberg and Seckl (2001) suggested that prenatal stimulation can significantly influence the regulation of the hypothalamic-pituitary-adrenal (HPA) axis and an individual's ability to cope with stressful situations later in life. For example, during sensitive embryonic stages, exposing embryos to thermal manipulation, such as specific durations and degrees of heat or cold, has been shown to induce long-lasting thermotolerance in poultry during postnatal life (Al-Zghoul, 2018; Meteyake et al., 2023a, Meteyake et al., 2023b). There's been a growing interest in the impacts of prenatal auditory stimulation on poultry in recent years (Kesar, 2013; Sanyal et al., 2013a, 2013b; Tong et al., 2015; Donofre et al., 2020). Chaudhury et al., 2006, Chaudhury et al., 2009 demonstrated that prenatal auditory stimulation with species-specific sound and sitar music at 65 dB for 15 min per hour led to increased synaptic density in the hippocampus of chicks, resulting in improved synaptic plasticity and memory formation in day-old chicks. Similarly, Sanyal et al. (2013a) reported that prenatal exposure to patterned sound at 110 dB significantly increased neuronal nuclear area, number, and volume in specific brain regions, such as the brainstem auditory nuclei, auditory cortex analog, and hippocampus. In contrast, unpatterned noise exposure reduced these brain regions' volume and decreased neuron number in day-old chicks. These findings suggest that prenatal auditory stimulation can modulate poultry's longer-term adaptability.

The composition of gut microflora is critical in shaping animals' health and disease status. The microorganisms residing in the gastrointestinal tracts are essential for the host's physiological, immunological, and protective functions. Previous research has suggested that thermally manipulated broiler embryos from embryonic day (ED) 10 to 18 exhibited altered mRNA expression of digestive enzymes and nutrient transporter genes on d 7 post-hatch, indicating a long-lasting impact of prenatal stimulation and supplementation on gut microflora in broiler chickens. There is growing evidence that the gut microbiota interacts with the brain via the neurological, immunological, and endocrine systems to control brain activity and behavior (Suganya and Koo, 2020). Recently, Mitin et al. (2022) reported that dietary supplementation with probiotics (B. subtilis) was beneficial in dampening stress and fear responses in ducks subjected to catching and crating.

Given the growing evidence that prenatal stimulation could better prepare animals for challenges in later life, the present study aimed to elucidate the effects of subjecting developing embryos to noise and music on physiological stress response to feed restriction in broiler chickens at market age. We measured serum levels of corticosterone, heat shock protein 70, ceruloplasmin, and alpha-1-acid glycoprotein as stress biomarkers. The present study also ascertained the impacts of prenatal auditory stimulation on the expression of glucocorticoid receptors and heat shock protein 70 genes in the brain and gut microflora.

MATERIALS AND METHODS

Ethical Statement

The experiment was conducted following the guidelines of the Research Policy of Universiti Putra Malaysia, as outlined in the Guide for the Care and Use of Experimental Animals (UPM/IACUC/AUP-R058/2019).

Experimental Incubators

A total of three incubators (Ova-easy 180 Advance Series II Cabinet Incubator, Brinsea Products Ltd, Sandford, UK) with a carrying capacity of 180 eggs per incubator were used in this study. These incubators were equipped with electronic temperature and humidity control with three trays on tilted platforms, ensuring that the eggs could be turned as preset. The preset incubation temperature was 37.8°C for each incubator (precision of 0.5°C), and the relative humidity ranged between 55% on embryonic day (ED) 1 to 17 to 65% on ED 18- hatching. Eggs were turned automatically every 2 h from ED 1 to ED 18 through an angle of 90°. On ED 18, eggs were placed into hatching trays and were not turned for the remainder of the incubation process. All incubators were located in the same ventilated room with a controlled environmental temperature of 24 to 25°C (accuracy of 2°C). The incubators were installed with double-walled, soundproof medium-density fiberboard (MDF) insulators to prevent interference with sound treatments. Sound pressure levels (SPL) were measured inside and outside the operating incubators using the sound meter level. The SPL outside the operating incubators dropped below 50 dB, while inside, the operating incubator was maintained at 90 dB. A total of 423 hatching eggs of Cobb 500 (Gallus domesticus) weighing 65 to 72 g were incubated in experimental incubators (Ova-easy 180 Advance Series II Cabinet Incubator, Brinsea Products Ltd, Sandford, UK) with a carrying capacity of 180 eggs that were placed in double insulated, soundproof acoustic padding foam at 37.8°C (±1°C) with relative humidity (RH) of 55-60% from ED 1 to ED 18 and 37.2°C (±1°C) with a 75 % RH from ED 19 to hatching.

Auditory Stimulation Protocols

The auditory stimulation protocol in the current study was based on recently published work (Hanafi et al., 2023). The specific details and adjustments made to the auditory protocols were in accordance with our prior work to ensure consistency and comparability.

Experimental Design

Prenatal Stage

The study involved 3 prenatal auditory stimulation treatments. The prenatal auditory stimulation groups are as follows:

• 1.No sound stimulation: The eggs were incubated and received no additional sound stimuli except the compressor's (40 dB) sound, which is unavoidable. This treatment was termed the "CONTROL" group.
• 2.Noise stimulation: The eggs were incubated under conditions similar to those of CONTROL. From ED 12 to ED 21, the eggs were exposed to pre-recorded traffic noise at 90 dB (± 2 dB) for 20 min/h over 24 h (a total of 8 h/d). This treatment was termed the "NOISE" group. The last cycle of noise stimulation was approximately at 09:00.
• 3.Music stimulation: The eggs were incubated under conditions similar to those of the CONTROL and NOISE groups. From ED 12 to ED 21, the eggs were exposed to low-level classical music (Mozart's Sonata for Two Pianos in D Major, K 488) at 90 dB (± 2 dB) for 20 min/h over 24 h (a total of 8 h/d). This treatment was termed the "MUSIC" group. The last cycle of music stimulation was approximately at 09:00.

Postnatal Stage

At hatch, 210 chicks from each prenatal treatment group were assigned to 42 cages in groups of 5. Hence, there were 14 cages of birds for each prenatal treatment group. The cages were in three environmentally controlled rooms. The length, width and height of each cage were 90, 56 and 50 cm, respectively (a total area of 5,040 cm² per cage). Thus, the floor space provided was 0.1m² per bird. The ambient temperature on d 1 was set at 32°C and then gradually reduced to 24°C by d 21. The birds were fed starter (2,950 kcal ME/kg; 21% crude protein) and finisher (3050 kcal ME/kg; 19% crude protein) diets from d 1 to 21 and d 22 to 42, respectively. Chicks were administered live Newcastle vaccine (SunVac ND Clone, Sunzen Biotech, Shah Alam, Selangor, Malaysia) intraocularly on d 7 and d 21. On d 42, an equal number of birds from each prenatal auditory stimulation (PAS) group were subjected to either ad libitum feeding (AL) or feed restriction for 30 h (FR). Drinking water was always available. There were seven cages of birds for each prenatal auditory stimulation-feeding regimen subgroup (PAS-FR).

Blood Sampling

On d 43, following feed restriction, 14 chicks (2 birds per cage) from each prenatal auditory stimulation-feeding regimen (PAS-FR) subgroup were randomly selected. Their blood samples (3 mL) were collected via the wing vein for the determination of serum levels of corticosterone (CORT), ceruloplasmin (CPN), alpha-1-acid glycoprotein (AGP), and heat shock protein (HSP) 70. At approximately 09:00 h, each bird was caught and sampled, one immediately after another. The time elapsed from catching to obtaining a blood sample was less than 50 s. This procedure should not influence circulating corticosterone levels (Craig, 1985; Lagadic et al., 1990; Romero and Reed, 2005). Blood samples were collected into blood collection tubes (BDVacutainer, NJ) containing the anticoagulant EDTA and stored on ice. Blood samples were centrifuged at 3,500 × g at 4°C for 15 min. Plasma samples were transferred into 1.5 mL microcentrifuge tubes and stored at −80°C until further analysis.

Determination of Serum Levels of CORT, CPN, AGP, and HSP70

CORT Assay The serum levels of CORT were measured by a commercial high-sensitivity enzyme-linked immunoassay (EIA) kit (AC- 15F1, IDS, Boldon, UK). The intra- and inter-assay variability were less than 6.7% and 7.8%, respectively, and the detection limit was 27 ng/ml. The protocol of analysis was according to the manufacturer's recommendations (Zulkifli et al., 2014; Najafi et al., 2015a).

HSP70 Assay Blood samples were collected, and serum levels of HSP70 were analyzed using a commercially available ELISA Kit (Sun Red Bio, Shanghai, China). The serum levels of HSP70 were determined according to the manufacturer's recommendation.

CPN and AGP Assay The serum levels of CPN were determined, as previously explained (Zulkifli et al., 2014). About 20.375 g of sodium acetate trihydrate was dissolved in 250 mL of distilled water and adjusted to pH 6.2 using glacial acetic acid. Subsequently, 0.615 g of 1,4-phenylenediamine dihydrochloride (Sigma P1519; Sigma Aldrich, St. Louis, MO) was added to the prepared buffer and kept in the dark for at least 45 min. Then, 100 mL of the described buffer was mixed with 10 mL of samples or standards and added to each microplate well, gently shaken and kept in the dark for 20 min. After 20 min, the plate was read using a microplate reader at 550 nm (Multiskan FC Microplate Photometer; Waltham). Serial dilution of standards was prepared using a known CPN concentration against purified CPN (Sigma Chemical Co., St. Louis, MO). To reach 12.75 mg/mL of CPN concentrations, 20 mL of pig serum was added with 60 mL of saline buffer. Serial dilutions were prepared accordingly to reach concentrations of 6.375, 3.1875, 1.59375, 0.79608, 0.39804, 0.199, and 0.099 mg/mL of CPN.

The serum levels of AGP were determined using a commercial ELISA kit specific to chicken (Cat. No. AGP-5, Life Diagnostics Inc., West Chester, PA).

Determination of Brain mRNA Gene Expressions of GR and HSP70

The brain mRNA gene expressions of glucocorticoid receptor (GR) and heat shock protein (HSP) 70 were determined using real-time polymerase chain reaction (RT-PCR). On d 43, seven birds from each subgroup were randomly selected and sacrificed by decapitation. The entire brain samples were removed, frozen quickly in liquid nitrogen, and stored at -80°C for gene expression analysis. According to the manufacturer's instructions, total RNA was briefly isolated from individual brain tissues using the RNeasy Mini kit (cat. 74104, Qiagen Inc, Valencia, CA). Purified total RNA (30 mg) was reverse transcribed using a QuantiNova reverse transcription kit (cat. no. 205413, Qiagen Inc, Valencia, CA) following the manufacturer's recommended protocols. The purity of extracted RNA was quantified by measuring the optical density ratio of 260/280 nm. The acceptable ratio between 1.8 and 2.0 was considered for further analysis. RT-PCR was performed using a QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, CA) on a CFX 96 Real-time system (Biorad, Hercules, CA). Briefly, the 20 μL reaction mix was prepared from 10 μL of master mix; 2 μL forward primer (10 pmol); 2 μL reverse primer (10 pmol); 2 μL cDNA of the sample; and 6.5 μL of nuclease-free water. Cycling parameters were 95°C for 5 min, 40 cycles of 95°C for 5 s, followed by 30 s at 55°C, and 72°C for 10 s with final melting at 95°C for 20 s. Post- PCR melting curves confirmed the specificity of the single-target amplification, and fold changes in gene expression were normalized to the housekeeping gene β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). β-actin displayed low stability and GAPDH displayed higher stability, hence GAPDH was chosen as the housekeeping gene. The sequences of specific primers for GR, HSP70, β-actin, and GAPDH were used, as reported by Ramiah et al. (2020). Primer sequences and accession number are provided in Table 1. All reactions were performed in triplicate. As for the gene expression calculations to assign the cycle threshold (CT) values. The ^ΔCT values were calculated by subtracting experimental CT values from the CT values for β-actin and GAPDH, which act as housekeeping genes. The group with the highest mean ^ΔCT value (lowest gene expression) per amplified gene target was set to 0, and the mean ^ΔCT values of the other groups were set relative to this calibrator (^ΔΔCT). The ddCT values were calculated as powers of 2 (2^ΔΔCT) to account for the exponential doubling of the PCR.

Table 1.

The primer sequences of HSP 70, GR, β-actin and GAPDH genes used for RT-PCR.

Target gene	Primer sequences 5′- 3′	Accession numbers	Amplicon sizes (bp)	References
HSP70	F-5′-CGTCAGTGCTGTGGACAAGAGTA-3′ R-5′-CCTATCTCTGTTGGCTTCATCCT-3′	NM-001006685	145	Uerlings et al., 2018
GR	F-5’CATGAACCTCGAAGCTCGCAAGA-3’ R- 5’ACCTCCAGCAGTGACACCAG-3’	DQ-227738	126	Liu et al., 2014
β-actin	F-5′TGTGCAAGGCCGGTTTCG-3′ R-5′CATCACCAACGTAGCTGTCTTTC-3′	NM-205518	126	Ebrahimi et al., 2015
GAPDH	F-5′GATCCCTTCATCGACCTGAA-3′ R-5′ACAGTGCCCTTGAAGTGTCC-3′	GQ184819.1	77	Ebrahimi et al., 2015

Abbreviations: F, forward; R, reverse; HSP70, heat shock protein 70; GR, glucocorticoid receptor; GAPDH, glyceraldehyde -3-phosphate dehydrogenase.

Determination of Gut Microflora Population

On d 43, the caecum content of all birds that were sampled for brain tissue (seven birds per PAS-FR subgroup) was quickly collected, frozen in liquid nitrogen, and stored at -80°C for bacterial quantification analysis. The populations of Lactobacillus spp. and Escherichia coli (E. coli) in the cecum were done according to the real-time polymerase chain reaction (RT-PCR) method described by (Navidshad et al., 2016). DNA was extracted from the caecal content samples using QIAamp DNA Stool Mini kits (Qiagen, Hilden, Germany). The DNA purification was done using a QIAamp spin column (Qiagen, Hilden, Germany) based on the kit instructions. Finally, the DNA was preserved at -20°C until RT-PCR analysis. The DNA concentration and purity were determined by Nanodrop 2000 spectrophotometer (ThermoScientific, Wilmington, DE). Standard curves were formed using serial dilution of PCR products from pure cultures of Lactobacillus spp. and E. coli. The primer sequences for the targeted bacteria are shown in Table 1. The RT-PCR was performed using BioRad CFX96 Touch (BioRad) using optical grade plates. The PCR reaction was carried out in 20 μL total volumes using the QuantiNova SYBR Green PCR kit (Qiagen, Hilden, Germany). Each reaction consisted of 10 μL SYBR Green Supermix, 1 μL of each Primer, 2 μL of DNA sample and 6 μL molecular H₂O. The reaction conditions for amplification of DNA were 94°C for 5 min, followed by 40 cycles of 94°C for 20 s, 58°C (Lactobacillus spp.) or 60°C (E. coli) for 30 s, and 72°C for 20 s. To confirm the specificity of amplification, melting curve analysis was carried out after the last cycle of each amplification.

Statistical Analysis

All data are expressed as mean ± SEM. Data, except for gut microflora counts, were analyzed by 2-way ANOVA using SAS's general linear model (GLM) procedures (SAS Institute Inc., 2022). Before analyses, the Shapiro–Wilk test was used to test the normal distribution of the data. The experiment was performed as a 2-factor factorial design, consisting of 3 prenatal auditory stimulations (PAS) (CONTROL, NOISE, and MUSIC), 2 feeding regimen (FR) (ad libitum and feed restricted), and their interactions. Each PAS-FR subgroup treatment had seven replicate cages based on a completely randomized (CRD) balanced design. When significant effects were found, Duncan's multiple range test compared multiple means. To analyze effects on gut microbiota, non-parametric tests were used to evaluate the differences between the subgroups. The cecal E. coli and Lactobacillus spp. population data were analyzed using the nonparametric Kruskal–Wallis test. The statistical significance is considered as P ≤ 0.05.

RESULTS

The prenatal auditory stimulation had a negligible effect on the hatchability (CONTROL, 88.6%; MUSIC, 90.7%; NOISE, 92.2%). The effects of prenatal auditory stimulation (PAS) and feeding regimen (FR) on serum levels of CORT, CPN, AGP, and HSP70 are presented in Table 2. No significant PAS x FR interactions existed for serum levels of CORT (P = 0.4958), serum levels of CPN (P = 0.2569), serum levels of AGP (P = 0.7086), and serum levels of HSP70 (P = 0.1066). The NOISE birds had the lowest serum levels of CORT, followed by MUSIC and CONTROL. The highest serum levels of CPN were noted in the CONTROL chickens, followed by NOISE and MUSIC. The CONTROL and MUSIC chickens had significantly (P < 0.001) higher serum levels of AGP than the NOISE group. Exposing embryos to NOISE resulted in significantly (P < 0.0001) higher serum levels of HSP70 than those of CONTROL and MUSIC. Following feed restriction, CORT (P = 0.0104) and HSP70 (P = 0.0524) were significantly elevated. The feeding regimen had no significant effect on CPN (P = 0.0573) and AGP (P = 0.7074).

Table 2.

The primer sequences of cecal targeting total bacteria, E. coli and Lactobacillus spp.

Target Microbe	Primer Sequence 5′- 3′	Accession Numbers	Product Size (bp)	References
E. coli	F- 5’-GTGTGATATCTACCCGCTTCGC -3’ F-5’-AGAACGCTTTGTGGTTAATCAGGA-3’	RM13322	82	Jahromi et al., 2016
Lactobacillus spp	F- 5’-CATCCAGTGCAAACCTAAGAG -3’ F- 5’-GATCCGCTTGCCTTCGCA-3’	ID 1099	341	Jahromi et al., 2016

Abbreviations: F, forward; R, reverse; E. coli, Escherichia coli; Lactobacillus spp, Lactobacillus species.

Table 3 shows the effects of PAS and FR on brain mRNA gene expression of GR and HSP70. No significant PAS x FR interactions were noted for mRNA gene expression of GR (0.0019) and HSP70 (P < 0.0001). The NOISE birds showed significantly (P = 0.0019) higher mRNA gene expression of HSP70 than their CONTROL and MUSIC counterparts. The highest mRNA gene expression of GR was noted in the NOISE chickens, followed by MUSIC and CONTROL. Neither PAS nor FR significantly affected mRNA gene expression of HSP70 (P = 0.1407) and GR (P = 0.1251).

Table 3.

The effects of prenatal auditory stimulation and feeding regimen on the mean (±SEM) serum levels of corticosterone (CORT) (ng/ml), ceruloplasmin (CPN) (mg/ml), alpha-1-acid glycoprotein (AGP) (mg/ml) and heat shock protein 70 (HSP70) (ng/ml) in broiler chickens on d 43

Treatment	Parameters
	CORT	CPN	AGP	HSP70
Prenatal Auditory stimulation (PAS)
Control	2.14 ± 0.24a	3.54 ± 0.14a	32.56 ± 3.75a	16.97 ± 1.13b
Music	1.75 ± 0.15b	2.56 ± 0.048c	32.66 ± 3.15a	17.03 ± 1.18b
Noise	1.01 ± 0.09c	2.97 ± 0.08b	3.86 ± 0.42b	43.62 ± 5.49a
Feeding regimen (FR)
Feed restricted	1.89 ± 0.13a	3.17 ± 00.1	22.07 ± 2.86	27.70 ± 3.46a
Ad libitum	1.34 ± 0.14b	2.90 ± 0.07	20.89 ± 3.64	18.2 0± 1.29b
Analysis of variance
PAS	0.0005	< 0.0001	< 0.0001	< 0.0001
FR	0.0104	0.0573	0.7074	0.0524
PAS x FR	0.4958	0.2569	0.7086	0.1066

^a-cMeans (±SEM) within column subgroup with no common superscripts differ at P ≤ 0.05. n= 14 samples per parameter/ group

Prenatal auditory stimulation had no significant effect on the cecal E. coli (P = 0.5323) (Figure 1) and Lactobacillus spp. (P = 0.0996) (Figure 2) counts of ad libitum fed and feed-restricted chickens.

Figure 1.

Effects of prenatal auditory stimulation on E. coli population under ad libitum (AD) or feed restricted (FR) feeding. Abbreviations: CT- AD = No prenatal auditory stimulation -ad libitum feeding, CT-FR = No prenatal auditory stimulation – 30h feed restriction, MS- AD = Exposed to prenatal music stimulation -ad libitum feeding, MS-FR = Exposed to prenatal music stimulation- 30h feed restriction, NS – AD = Exposed to prenatal noise stimulation- ad libitum feeding, NS-FR = Exposed to prenatal music stimulation- 30h feed restriction. Means (±SEM) within column subgroup with no common superscripts differ at P ≤ 0.05.

Figure 2.

Effects of prenatal auditory stimulation on Lactobacillus spp. population under ad libitum (AD) or feed restricted (FR) feeding. Abbreviations: CT- AD = No prenatal auditory stimulation -ad libitum feeding , CT-FR = No prenatal auditory stimulation – 30h feed restriction, MS- AD = Exposed to prenatal music stimulation -ad libitum feeding, MS-FR = Exposed to prenatal music stimulation- 30h feed restriction, NS – AD = Exposed to prenatal noise stimulation- ad libitum feeding , NS-FR = Exposed to prenatal music stimulation- 30h feed restriction. Means (±SEM) within column subgroup with no common superscripts differ at P ≤ 0.05.

DISCUSSION

As expected, we noted feed withdrawal for 30 h elevated serum levels of CORT in broilers. Similarly, Najafi et al. (2016) feed-deprived broilers for 0, 6, 12, 18, 24, and 30 h and showed a significant increase in corticosterone following 24 and 30 h fasting. Corticosterone is the main hormone associated with stress in the avian species, and its circulating level has been commonly used to monitor physiological responses to various stressors (Post et al., 2003; Blas, 2015; Tilbrook and Fisher, 2020), including feed restriction (Najafi et al., 2015b, 2016, 2018). Najafi et al., 2015a, Najafi et al., 2015b demonstrated a linear relationship between CORT and the level of feed restriction in broiler breeders, suggesting the physiological indicator as a reliable measure of feed restriction stress in poultry. The elevated corticosterone levels in feed-restricted chickens could be attributed to physiological and psychological stress (Zulkifli et al., 1995, 2006).

Acute phase proteins (APPs) are a group of blood proteins primarily synthesized in the liver that change in concentration in response to tissue injury or infection and to non-inflammatory stimuli such as stress conditions in poultry (O'Reilly and Eckersall., 2014). According to Murata et al. (2004), APPs profoundly restore homeostasis in animals subjected to non-inflammatory, psychophysical stressors. Recently, APPs such as ceruloplasmin (CPN), alpha-1-acid glycoprotein (AGP), ovotransferrin (OVT), and have been used as indicators of physiological status in chickens (Shakeri et al., 2014; Najafi et al., 2015a, 2015b; Law et al., 2019). Najafi et al. (2016) reported that 30 h of feed deprivation increased serum levels of CPN and AGP in broilers. On the contrary, in the present study, serum levels of CPN and AGP were not elevated following feed withdrawal. There is no clear explanation for the findings, although the work of Najafi et al. (2016) involved 21-day-old broilers, while ours were 43-day-old. Work in pigs showed that age may affect the concentrations of some of the major APPs (Pineiro et al., 2009; Pomorska-Mól et al., 2012; Christoffersen et al., 2015).

The synthesis of heat shock protein (HSP) in response to stressors threatening the cell's survival is a crucial aspect of the cellular stress response. Heat shock protein act as molecular chaperones that play a vital role in maintaining protein homeostasis and preventing the irreversible aggregation of denatured proteins (Haslbeck and Vierling, 2015). These proteins are part of the stress core proteome and are considered universally conserved protective factors that help cells cope with various stress stimuli (Reyes and Casas-Tintó, 2022). Heat shock protein significantly alters the physiological response to thermal and nonthermal stressors and promotes stress resilience in poultry (Balakrishnan et al., 2023). The present study confirmed earlier findings (Zulkifli et al., 2000, 2002; Liew et al., 2003; Al-Aqil and Zulkifli, 2009; Najafi et al., 2016) that fasting may elicit HSP70 expression. Earlier work on the HSP70 in chickens involved brain samples, whereas the present study used serum samples to measure HSP70 regulations. Thus, the serum is a suitable sample to measure HSP70 in poultry in response to environmental insults relative to brain samples.

In their review, Dixon et al. (2016) presented evidence suggesting maternal conditions before lay or during incubation could profoundly impact poultry health, welfare, and productivity. However, previous work on the effect of prenatal stimulation or manipulation on stress resilience in poultry mainly focused on thermal challenges (Piestun et al., 2013; Al-Zghoul et al., 2019; Saleh et al., 2020). There is a dearth of information on prenatal environment and response to nonthermal stressors in poultry. The present findings, as measured by serum levels of CORT suggest that prenatal auditory stimulation has modified the ability of broiler chickens to cope with the stress attributed to 30 h of feed deprivation. The present findings indicate that although prenatal auditory stimulus was stressful to developing embryos (Hanafi et al., 2023), it could benefit broiler chickens during subsequent stressful episodes later in life. Thus, our results support the growing body of evidence that stress during development may alter animals' physiological responses throughout their lives (Cirulli et al., 2009; Henriksen et al., 2011; Dixon et al., 2016). The prenatal period is crucial as it is sensitive to external stimuli, which, when experienced at specific times during this stage, can profoundly influence the developmental process (Dobbing and Sands, 1979; Rice and Barone, 2000; Meteyake et al., 2023a, Meteyake et al., 2023b).

It is interesting to note that prenatal auditory stimulation had different effects on CORT, CPN, and AGP levels. Zulkifli et al. (2014) reported that exogenous corticosterone elevated AGP and CPN in chickens following three and seven days of treatment, respectively. The authors concluded that elevation in the levels of circulating corticosterone can induce an acute-phase response, and AGP was more sensitive to stressful stimuli than CPN. α1-Acid glycoprotein is well known as an immunoregulator affecting T-cell function and providing negative feedback on the APR, whereas ceruloplasmin is documented to have more protective effects by removing oxygen radicals, antihistamine activity, and reversing the hypoferremic state of the APR (Murata et al., 2004) Hence, it appears that the different functions of acute phase proteins could modulate their reaction to noxious stimuli.

The question of how prenatal stimulation can profoundly impact coping ability later in life remains elusive. Thermal manipulation during mid-stage or later-stage embryogenesis enhanced the capacity of chickens to express HSP70 and lead to concomitant heat tolerance later in life (Piestun et al., 2008; Zaboli et al., 2017; Al-Rukibat et al., 2017; Ramiah et al., 2022). Al-Amaz et al. (2024) reported that prenatal thermal manipulation increased the expression of some critical markers for heat shock protein-related genes in the brains of chickens. The present findings showed that prenatal exposure to NOISE from ED 12 to 21 enhanced the ability to induce HSP70 mRNA and protein expression in feed-deprived broilers at 43 d of age. According to Balakrishnan et al. (2023), heat shock proteins profoundly regulate the physiological stress response and promote stress tolerance in poultry. These essential proteins are synthesized in response to stressors threatening the cell's survival, exerting profound effects on the physiological stress response and bolstering stress tolerance (Barbe et al., 1988; Chen et al., 2018). These proteins function as molecular chaperones, orchestrating crucial tasks such as binding to other cellular proteins, facilitating intracellular transport, and ensuring proper folding into secondary structures. Doing so prevented protein aggregation during stress (Chirico et al., 1988).

The coordinated interplay between GR and mineralocorticoid receptor (MR) is demonstrated to regulate negative glucocorticoid feedback across different tiers of the hypothalamic-pituitary-adrenal (HPA) axis. The mineralocorticoid receptor governs the initiation or threshold of HPA axis activation, operating in a proactive mode, while the GR oversees termination following exposure to stressors during the recovery period, functioning in a reactive mode (De Kloet et al., 1998, 2005; Oitzl et al., 2010). Ahmed et al. (2016) reported that prenatal corticosterone treatment altered GR metabolic enzyme gene expression in the chicken ovarian follicles. In the present study, the effect of PAS on GR expression varied according to the type of auditory stimulation. The NOISE birds showed a higher expression of GR than their CONTROL and MUSIC counterparts. Changes in the number of receptors available for binding to glucocorticoids and such changes to the feedback system may significantly affect the glucocorticoid release concurrent with the magnitude of the expression levels. Modulating the negative feedback mechanism may occur through the increased GR levels, which could dampen HPA axis reactivity in the NOISE broilers. Hence, the enhanced GR expression in the NOISE chickens may have reduced their CORT following feed deprivation.

In their review, Hartman et al. (2018) suggested that prenatal stress may influence infant intestinal microbiota, resulting in heightened physiological and behavioral reactivity and postnatal developmental plasticity. Work in primates showed that prenatal stress resulted in lower gut Bifidobacteria and Lactobacillus counts 2 d after birth but increased to a peak between 8 and 16 wk of age (Bailey et al., 2004). Zheng et al. (2020) indicated that subjecting rats to cold stress during gestation improved the offspring's gut microbiota composition and increased the population of Bacteroides and Lactobacillus. On the contrary, our results suggest no clear link between prenatal auditory stimulation and caecal E. coli and Lactobacillus spp counts in broilers on d 43. Discrepancies in results could be associated with intrinsic and extrinsic factors (Sun et al., 2022). Intrinsic factors include host genetics, age, and sex, whereas extrinsic factors comprise dietary differences, behaviours, social relationships, and external environmental factors. Hinton et al. (2000) showed that feed withdrawal for 24 h had a negligible effect on cecal aerobes, Enterobacteriaceae, Salmonella typhimurium, and lactic acid bacteria counts. The present findings showed that the cecal E. coli and Lactobacillus spp. counts were not affected by 30 h of feed deprivation. Hinton et al. (2002) suggested that the changes caused by feed withdrawal in the digestive tract of broilers appear to manifest earlier in the crop than in the ceca.

Collectively, our data suggest that the NOISE birds were better able to cope with feed deprivation than their MUSIC counterparts. There appears to be no obvious explanation for the apparent better ability of the NOISE birds to acquire stress resilience than the MUSIC group, and only a speculative one can be offered at this stage. There is a possibility that the MUSIC treatment was not “sufficient” to evoke long-term modifications required to improve coping ability in chickens. Zulkifli et al. (2000) subjected chicks to 40%, 60%, and 80% feed restriction on d 4, 5, and 6 and heat challenge from d 21 to 41. The authors reported that only 60% of feed restriction improved heat tolerance later in life. The present findings suggested that the magnitude of physiological stress response experienced during the prenatal stage may profoundly influence the ability to cope with environmental stresses later in life. Thus, the magnitude of stress experienced by embryos may regulate the ability of chickens to cope with noxious stimuli at market age.

CONCLUSIONS

In conclusion, this study showed that prenatal auditory stimulation, particularly the NOISE treatment, could improve broiler chickens' capacity to cope with feed deprivation at market age. This suggests that prenatal auditory stimulation can affect the ability of broilers to cope with feed restriction and that the effect on the adaptive capacity lasts for a considerable time after cessation of the auditory stimulation. To maintain the health and reproduction of broiler breeders, severe feed restriction has to be applied to control obesity, and such practice is a major welfare problem in modern poultry production. Thus, further research is recommended to elucidate the potential of prenatal auditory stimulation to improve broiler breeder flock welfare.

DISCLOSURES

The authors declare no conflict of interest.