Gamma-amino butyric acid (GABA) supplementation alleviates dexamethasone treatment-induced oxidative stress and inflammation response in broiler chickens

Abstract

This experiment was conducted to investigate the effect of Gamma-amino butyric acid (GABA) on growth performance, serum and liver antioxidant status, inflammation response and hematological changes, in male broiler chickens under experimentally induced stress via in-feed dexamethasone (DEX). A total of 300 male chicks (Ross 308) on day 7 after hatching, were randomly selected into four groups which were positive control group (PC, without any treatment), negative control (NC, with 1 mg/kg DEX), a third group received 1 mg/kg DEX and 100 mg/kg GABA (DG ⁺) and the last one was (DG ⁺⁺) which received 1 mg/kg DEX and 200 mg/kg GABA. Each group has five replicates (15 birds/replicate). Dietary GABA modulated DEX-induced adverse effects on body weight, feed intake, and feed conversion ratio. The DEX-induced effect of serum levels of IL-6 and IL-10 was reduced by dietary GABA supplementation. The activity of serum and liver superoxide dismutase, catalase, glutathione peroxidase were enhanced and malondialdehyde was reduced by GABA supplementation. The serum levels of total cholesterol & triglyceride were higher while low-density lipoprotein & high-density lipoprotein were lower in GABA groups than NC group. GABA supplementation also significantly decreased the heterophil, heterophil/lymphocyte ratio and elevated the activities of aspartate aminotransferase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) than NC group. In conclusion, dietary GABA supplementation can alleviate DEX stress-induced oxidative stress and inflammation response.

Keywords:

• Chickens
• gamma-amino butyric acid
• oxidative stress
• dexamethasone
• inflammation response
• stress

Introduction

Intensive rearing of domestic animals in the livestock industry, especially in the chicken industry, causes a lot of stress in animals including immunological challenges, oxidative stress, and transportation (Jeong et al., 2020). Stress has many detrimental effects on broiler chickens; including a reduction growth performance, higher susceptibility to disease, and impaired immune function (Lin et al., 2006a, 2006b). Moreover, stress can influence the turkey’s susceptibility to viral infections, including influenza viruses (Ali et al., 2013). It has been reported that animal exposure to any stress activates the hypothalamus-pituitary adrenal axis; thus stimulating the secretion of glucocorticoid from the adrenal gland. A rise in glucocorticoids level is a hallmark of stress (Osho and Adeola, 2020). Glucocorticoids maintain carbohydrates metabolism and natural growth and also play an important role in dealing with stressful attitudes (Kuo et al., 2018). High concentrations of glucocorticoids have a catabolic effect on skeletal muscles by inhibiting protein production and increasing the breakdown of muscle fibers which leads to a decrease in muscle weight and ultimately a decrease in growth (Furukawa et al., 2016; Wang et al., 2016). Glucocorticoids release is accompanied by the production of free radicals, which is the basis for the oxidation of membrane lipids, the loss of meat quality, the weakening of the immune system, and the reduction of production (Lin et al., 2004).

Previously showed that dexamethasone treatment of turkey, reduced viral infections, including swine influenza virus infections (Ali et al., 2013). However, studies have shown that the use of dexamethasone mimics the adverse effects of increased corticosterone in broiler chickens and causes an increase in free radicals and induction of oxidative stress (Eid et al., 2003). Dexamethasone (DEX) is a synthetic glucocorticoid used as an immunosuppressive agent. It has been used to induce oxidative stress and to investigate stress responses in poultry species (Njagi et al., 2012; Osho and Adeola, 2020).

Dietary supplements could have positive effects on decreasing stress and relieving the immunosuppression induced by DEX (Eladl et al., 2019; Osho and Adeola, 2020). Multiple approaches have been undertaken to reduce the deleterious effects of stressors in chickens, including supplementation with various types of fees additives (Haldar et al., 2011) for example, selenium (Huang et al., 2021), black seed (Eladl et al., 2019), and lycopene (Fathi et al., 2022).

Gamma-aminobutyric acid (GABA), a non-protein amino acid is commonly distributed in the tissues of animals and plants (El-Naggar et al., 2019; Li et al., 2010; Park and Kim, 2015).

It represents about 30% of the neurotransmitters in the central nervous system that is generally found in nature as the brain, heart, and kidneys as well fetuses, rice in plants, bacteria and yeast (Hashimoto 2011). GABA is one of the primary inhibitors in the central nervous system (Jonaidi et al., 2012) that has been used to reduce stress intensity (Chen et al., 2014; Wang et al., 2011;). Studies indicated that GABA has an important role to reduce the heat production as well as the fever and has positive effects on the growth performance of the broiler (Dai at el. 2011), as well as the immune activity and anti-oxidative function in chicken’s subject to heat stress (Zhang et al., 2012 ; Chen et al., 2013, 2014).

In addition to its function of neurotransmission, it performs a variety of other physiological functions, such as enhancing memory and enhancing immunity against stress, and it is also sedative and low blood pressure (Hayakawa et al., 2004). The lack of GABA in the body leads to the appearance of symptoms that similar to epilepsy as it is considered anti-depressants in humans and works to remove tension (Oh and Choi, 2000; Omori et al., 1987). In the current experiment, DEX was administered to broiler chickens' diet to induce stress. This finding may provide a useful evidence for the application of GABA in diets to mitigate immunological stress and improve antioxidant activity in broiler chickens. Our objective was to investigate the effect of dietary GABA supplementation on anti-oxidative function and immune response in broiler chickens induced stress via in-feed in-feed DEX supplementation.

Materials and methods

All experimental procedures used in this study were approved by the Animal Ethics Committee of Payam Noor University, Tehran, Iran (Ethics ID: IR.PNU.REC.1401.147).

Birds, diets and experimental design

A total of 300 male broiler chicks (Ross 308) were used in this study. On day 7 after hatching, 300 birds were individually tagged and weighed. All birds were randomly assigned to 4 experimental diets including positive control (PC) without any treatments, negative control (NC) supplemented with 1 mg/kg dexamethasone, 1 mg/kg dexamethasone and 100 mg/kg GABA (DG ⁺¹) and 1 mg/kg dexamethasone and 200 mg/kg GABA (DG ⁺⁺²). The optimum dietary concentration of DEX for broiler chickens was determined to be 1 mg/kg (Osho and Adeola, 2020). Each group had five replicates (15 birds/replicate). GABA used in the present experiment was purchased from Tiancheng Biotechnology, Jinan, China. Dexamethasone was purchased from Alfa Aesar (Tewksbury, MA). Both materials were in powdered form. The chicks were housed (10 birds/m²) in an environmentally controlled cages at the poultry farm, Payam Noor University, Iran. The house was maintained at a temperature based on the age of the birds. It was initially 32 °C then was reduced by 3 °C each week, to be 21 °C at the fifth week of age. This was controlled by an air conditioner.

All birds received a basal diet consisting of corn-soybean meal-based diet meeting the nutritional requirements of poultry recommended by the National Research Council (1994) (Table 1). A starter diet and water were available ad libitum for the first 21 days then changed to a grower diet till the end of the experiment. The chicks were allocated into 4 groups of 5 replicates (15 chicks each) in a completely randomized design. Newcastle disease vaccination program was done on days 7, 17, and 28 of age in drinking water. The infectious bursal disease (Gumboro) vaccination was done on day 14 in drinking water.

Table 1. Diet chemical composition.

Ingredient (%)	Starter diet (1–21 d)	Grower diet (22–42 d)
Yellow corn	53.85	57.60
Corn gluten meal	6.9	6.0
Soybean oil meal	33.2	30.3
Soybean oil	2.0	2.0
DCP^a	1.8	1.8
Ground limestone^b	1.4	1.4
Mineral and vitamin premix^c	0.3	0.3
Salt	0.3	0.3
DL-methionine^d	0.1	0.1
Lysine^e	0.1	0.15
Choline chloride	0.05	0.05
Total	100	100
Diet chemical composition
Moisture (%)	9	9
Dry matter (%)	91	91
Crude protein (%)	22.9	21.8
ME (Kcal/kg)	3048	3081
Crude fat (%)	4.01	4.39
Crude fiber (%)	3.67	3.78
Ash (%)	6.23	5.59
Calcium (%)	0.98	0.97
Available phosphorus (%)	0.48	0.48
Lysine (%)	1.20	1.17
Methionine (%)	0.50	0.48

^aDCP: dicalcium phosphate (contain 18% Phosphorus and 21% calcium).

^bLimestone (contain 35% calcium).

^cMineral and vitamin premix produced by Heropharm and composed (per 3 kg) of vitamin A 1,20,00,000 IU, vitamin D 32,500,000 IU, vitamin E 10,000 mg, vitamin K3 2000 mg, vitamin B1 1000 mg, vitamin B2 5000 mg, vitamin B6 1500 mg, vitamin B₁₂10 mg, niacin 30,000 mg, biotin 50 mg, folic acid 1000 mg, pantothenic acid 10,000 mg, manganese 60,000 mg, zinc 50,000 mg, iron 30,000 mg, copper 4000 mg, iodine 300 mg, selenium 100 mg and cobalt 100 mg.

^deli-methionine (produced by Evonic Co. and contain 99% methionine).

^eLysine: lysine hydrochloride (contain 98% lysine).

1 g/kg of DEX premix. DEX premix: 0.01 g of dexamethasone (DEX) (Alfa Aesar (Tewksbury, MA) added to 9.9 g of ground corn. Dexamethasone premix was added at the expense of corn to supply 1 mg DEX/kg diet.

1, 2 g/kg of GABA premix. GABA premix: Prepared as 1 g of GABA (Tiancheng Biotechnology, Jinan, China) added to 9 g of ground corn. GABA premix was added at the expense of corn to supply 100, 200 mg GABA/kg diet.

Growth performance and Mortality

Average BW gain (ABWG) and average feed intake (AFI) were measured weekly, and the average feed conversion ratio (AFCR) was calculated. Mortality was recorded as it occurred during daytime and used to calculate the mortality-adjusted feed conversion ratio.

Hematological and biochemical examination

On day 42, after 8-hour fasting (Wang et al., 2011), 5 broilers per group randomly selected. Two blood samples were taken by wing vein puncture, under gentle restraint without anesthesia. One of the blood samples (3 mL) was taken into a tube containing an anticoagulant (disodium EDTA) and kept on ice for hematological examination (Al Wakeel et al., 2017). The second blood sample (3 mL) was taken without anticoagulant to measure biochemical parameters in serum. Serum was separated by centrifugation for 10 min at 2,500 rpm and stored at −20 °C until assayed.

Hematological parameters including white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), heterophil (H) and lymphocytes (L) in whole blood were analyzed using an automatic blood analyzer (ADVIA 120, Bayer, NY, USA). For, differential leukocyte counts (H/L ratio), a drop of blood was smeared on a glass slide, left to dry, and then stained with Giemsa stain. One hundred leucocytes were counted on each slide including heterophils and lymphocytes. The H/L ratio was calculated by dividing the number of heterophils by the number of lymphocytes. Two slides were counted and the means were calculated for each bird (Al Wakeel et al., 2017; Gross and Siegel, 1983).

The activities of aspartate amino transferase (AST), alanine amino transferase (ALT), and alkaline phosphatase (ALP) in serum were measured with appropriate laboratory kits (Pars Azmoon, Tehran, Iran). GPx activity was determined with a commercially available enzyme kit (Ransel, RANDOX/RS-504 supplied by Randox Laboratories, Crumlin, UK), CAT and SOD activities were determined with the commercially available enzyme kit (Ransod, RANDOX/SD-125 supplied by Randox Laboratories) and autoanalyzer (Alcyon 300, USA) according to the manufacturers’ protocols.

The level of malondialdehyde (MDA) concentration in serum was measured with the tiobarbituric-acid reaction by the method of Fathi et al. (2022). Inflammatory parameters such, IL-6, IL-8 and IL-10 concentrations were determined with ELISA kits (Pars Azmoon, Tehran, Iran) according to the manufacturer’s instructions. Serum lipid profiles including Cholesterol, Triglyceride, High-Density Lipoprotein (HDL) and High-Density Lipoprotein (LDL) were measured by spectrophotometer with standard commercial kits (Pars Azmoon, Tehran, Iran) according to the manufactures instructions.

Liver parameters

Five birds from each group were randomly selected and killed by cervical dislocation, without anesthesia. Liver specimens, from the left lobe, of each bird, were collected in phosphate buffer saline (PBS), pH 7.5 and kept at -20 °C for analysis of activity of antioxidant and interleukins tests. Approximately 2 g of chicken liver was homogenized with 9 mL of 0.9% sodium chloride buffer (w/v, 1:9) on ice, and then centrifuged at 1000 g at 4 °C for 10 min to obtain the supernatant. The supernatant was stored at -80 °C until further analysis. Total superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) activities and malondialdehyde (MDA) concentration in the supernatant of the liver homogenate were determined with commercially available assay kits (Pars Azmoon, Tehran, Iran) via an automated spectrophotometric analyzer (Alcyon 300, USA). All procedures were performed according to the manufacturer’s instructions.

Statistical analysis

Data from all response variables were subjected to a one-way analysis of variance by applying the SAS program (SAS 2012) based on a completely randomized design (CRD) with four treatments and five replicates per treatment using a general linear model (GLM). Significant differences among groups means were separated using Tukey’s test at 5% probability.

Results

GABA supplementation significantly modulated effects of DEX on growth performance and mortality

The effects of in-feed DEX and GABA supplementation on growth performance and mortality were summarized in Table 2. DEX supplemented group (NC) has decreased average BW, FI and increased FCR compared with the control positive group (PC, without treatment) (P < 0.01). However, both levels of GABA significantly increased the FI, BW and decreased the FCR compared to the birds of the NC group (P < 0.01).

Table 2. Growth performance and mortality of broiler chickens fed diets containing Gamma-amino butyric acid (GABA) concentration at 100 or 200 mg/kg with or without dexamethasone (DEX).

Treatments	BW (g)	FI (g)	FCR	Mortality
PC	3050^a	5429^a	1.78^b	0.03^b
NC	2287^c	4459^c	1.95^a	0.06^a
DG+	2700^b	4725^b	1.75^b	0.03^b
DG++	2730^b	4722^b	1.73^b	0.03^b
SEM	110	150	0.09	0.005
P-value	< 0.01	< 0.01	< 0.01	< 0.01

^a–dMeans with different superscripts in the same columns are significantly different (p < 0.05).

BW: Body weight; FI: Feed intake; FCR: Feed conversion ratio; PC; positive control (Without any treatment); NC: negative control (with 1 mg/kg DEX); DG+:1 mg/kg DEX and 100 mg/kg GABA; DG++: 1 mg/kg DEX and 200 mg/kg GABA.

GABA supplementation modulated antioxidant activities and MDA concentration in serum and liver

There was a significant difference between experimental treatments in the SOD, CAT, GPx activities and the concentration of MDA in the plasma and liver of birds (Table 3). Dietary GABA supplementation mitigated the DEX-induced effect on the activity of serum and liver SOD, CAT, and GPx activities (P < 0.01). In addition, Dietary GABA supplementation significantly decreased MDA content in serum and liver compared with the NC group (p < 0.01).

Table 3. Serum and liver antioxidant function of broiler chickens fed diets containing Gamma-amino butyric acid (GABA) concentration at 100 or 200 mg/kg with or without dexamethasone (DEX).

Parameters		Treatments				SEM	p Value
		PC	NC	DG + DG	DG++DG
Serum	GPx (Mu/mL)	8326.1^a	1121.9^d	2019.42^c	2370.28^b	98.50	< 0.01
	SOD (U /mL)	364.90^b	270.25^c	380.93^b	405.79^a	29.50	< 0.01
	CAT (N mol/min/mL)	81.94^a	26.60^d	54.18^c	75.53^b	3.50	< 0.01
	MDA (n mol/ml)	11.57^c	14.85^a	12.70^b	12.24^b	0.98	< 0.01
Liver	GPx (U /mg protein)	19.01^a	14.00^b	17.25^a	18.50^a	2.10	< 0.01
	SOD (U/ mg protein)	750^a	630^b	751^a	770^a	15.1	< 0.01
	CAT (U /mg protein)	45.1^a	26.2^d	38.2^c	41.2^b	2.5	< 0.01
	MDA (nmol/mg protein)	0.34^bc	0.48^a	0.36^b	0.33^c	0.02	< 0.01

^{a, b, c}Mean values in the same row with different superscript letters were significantly different (p < 0.05).

PC: positive control (Without any treatment); NC: negative control (with 1 mg/kg DEX); DG+:1 mg/kg DEX and 100 mg/kg GABA; DG++: 1 mg/kg DEX and 200 mg/kg GABA; GPx: Glutathione peroxidase; SOD: Superoxide dismutase; CAT: Catalase; MDA: Malondialdehyde.

Effects of DEX and GABA supplementation on inflammatory cytokines in serum and liver

The serum and liver of cytokines of broiler chickens in experimental groups are presented in Table 4. Dexamethasone-induced effect on IL-6 and IL-10 was ameliorated by dietary GABA supplementation (p < 0.01).

Table 4. Serum and liver cytokines of broiler chickens fed diets containing Gamma-amino butyric acid (GABA) concentration at 100 or 200 mg/kg with or without dexamethasone (DEX).

Parameters		Treatments				SEM	p Value
		PC	NC	DG+	DG++
Serum	IL-6 (ug/mL)	10.50^d	17.50^a	15.10^b	12.90^c	0.75	0.01>
	IL-8 (ug/mL)	11.20	10.50	10.30	10.20	1.90	0.15
	IL-10 (ug/mL)	12.12^d	25.50^a	20.25^b	17.21^c	1.95	0.01>
Liver	IL-6 (ug/mL)	0.46^d	1.60^a	0.92^b	0.75^c	0.03	0.01>
	IL-8(ug/mL)	1.20	1.35	1.50	1.30	0.75	0.08
	IL-10 (ug/mL)	1.30^d	3.60^a	2.61^b	2.15^c	0.05	0.01>

^{a, b, c}Mean values in the same row with different superscript letters were significantly different (P < 0.05).

PC: positive control (Without any treatment); NC: negative control (with 1 mg/kg DEX); DG+:1 mg/kg DEX and 100 mg/kg GABA; DG++: 1 mg/kg DEX and 200 mg/kg GABA.

IL-6, interleukin-6; IL-8, interleukin-8; IL-10, interleukin-10.

Effects of DEX and GABA supplementation on hematological parameters

Although the experimental treatments did not have a significant effect on RBC, HCT or HGB values, GABA decreased the heterophil (H), lymphocyte (L) numbers, and heterophil/lymphocyte (H:L) ratio compared with DEX supplemented group (p < 0.01) (Table 5).

Table 5. Hematological parameters of broiler chickens fed diets containing Gamma-amino butyric acid (GABA) concentration at 100 or 200 mg/kg with or without dexamethasone (DEX).

Treatments	WBC (×10^3/µl)	RBC (×10^3/µl)	HGB (g/dl)	HCT (%)	Heterophile (/µl)	Lymphocyte (/µl)	Heterophile/ Lymphocyte
PC	181275.0	2.61	12.80	32.08	34060.5^b	141433^b	0.24^b
NC	183500.0	2.71	13.35	32.68	56749.5^a	119283^c	0.47^a
DG+	191325.0	2.73	14.60	35.13	26355.0^c	154190^a	0.17^c
DG++	186725.0	2.66	14.40	34.65	28151.5^c	158523^a	0.17^c
SEM	7044	0.82	2.70	2.54	4500	5142	0.02
p-value	0.53	0.49	0.14	0.54	0.00	0.00	0.00

^a–dMeans with different superscripts in the same columns are significantly different (p < 0.05).

PC: positive control (Without any treatment); NC: negative control (with 1 mg/kg DEX); DG+:1 mg/kg DEX and 100 mg/kg GABA; DG++: 1 mg/kg DEX and 200 mg/kg GABA; WBC: White blood cell; RBC: Red blood cell; HGB: Hemoglobin; HCT: Hematocrit.

Effects of DEX and GABA supplementation on serum enzyme activities

we found increased serum ALT, AST, and ALP levels in birds that were fed diets supplemented with DEX (Table 6) and dietary GABA effects are similar to DEX (p < 0.01).

Table 6. Activity of enzymes in serum of broiler chickens fed diets containing Gamma-amino butyric acid (GABA) concentration at 100 or 200 mg/kg with or without dexamethasone (DEX).

Treatments	ALT (U/L)	AST (U/L)	ALP (IU/l)
PC	10.37^d	148.80^d	359.34^c
NC	27.32^c	182.05^b	368.65^b
DG+	62.43^a	154.79^c	362.01^c
DG++	39.69^b	185.12^a	385.59^a
SEM	3.84	5.40	2.61
p-value	0.00	0.00	0.00

^a-dMeans with different superscripts in the same columns are significantly different (p < 0.05).

PC: positive control (Without any treatment); NC: negative control (with 1 mg/kg DEX); DG+:1 mg/kg DEX and 100 mg/kg GABA; DG++: 1 mg/kg DEX and 200 mg/kg GABA; ALT: Alanine transaminase; AST: Aspartate transaminase; ALP: alkaline phosphatase; GABA: gamma amino butyric.

Effects of DEX and GABA supplementation on serum lipid profiles

Table 7 presented the effects of DEX and GABA supplementation on the serum lipid profile of broiler chickens. Significant effects were observed for NC and GABA supplementation. So that stress induced by DEX significantly increases cholesterol, triglycerides, and LDL and decreases HDL in the serum of chickens under stress (p < 0.01). Meanwhile, the administration of both levels of GABA, without affecting serum cholesterol, significantly decreased LDL, HDL, and increased serum triglyceride in birds under induced stress.

Table 7. Serum lipid profile of broiler chickens fed diets containing Gamma-amino butyric acid (GABA) concentration at 100 or 200 mg/kg with or without dexamethasone (DEX).

Treatments	Cholesterol (mg/dl)	Triglyceride (mg/dl)	HDL (mg/dl)	LDL (mg/dl)
PC	180.80^d	182.37^d	97.35^a	31.34^d
NC	260.05^a	270.32^c	84.40^b	87.65^a
DG+	253.79^a	349.43^b	73.99^c	52.01^c
DG++	246.12^a	365.69^a	64.67^d	63.59^b
SEM	5.40	3.84	1.76	2.61
p-value	0.01	0.00	0.00	0.00

^a-dMeans with different superscripts in the same columns are significantly different (p < 0.05).

PC: positive control (Without any treatment); NC: negative control (with 1 mg/kg DEX); DG+:1 mg/kg DEX and 100 mg/kg GABA; DG++: 1 mg/kg DEX and 200 mg/kg GABA; HDL: High-density lipoprotein; LDL: Low-density lipoprotein.

Discussion

Today’s broiler chickens, which are raised in intensive farms, are usually subjected to several potentially immunosuppressive stimuli (including oxidative stress, high stocking density, and transportation) during their lifetime (Galha et al., 2008, Osho and Adeola, 2020). Dexamethasone is an analog of the glucocorticoid secreted when animals are under stress. Glucocorticoids act by decreasing the population of both B and T lymphocytes, promoting considerable immunosuppression. In the present study, broiler chickens were visibly depressed and less active during the immunological stress mimicked by in-feed DEX, in accordance with previous reports (Gao et al., 2008; Lin et al., 2004; Osho and Adeola, 2020). Decreased feed intake is a primary cause of reduced growth rate in broiler chickens (Osho and Adeola, 2020). The result of reduced feed intake agrees with the report from Sapolsky et al. (2000), who observed that DEX reduced appetite. In broiler chickens, one of the most recognizable effects of glucocorticoid treatment on growth performance is a drastic reduction in BW gain (Puvadolpirod and Thaxton, 2000a), which was observed in the current experiment.

The negative effect of DEX on BW gain was explained by Puvadolpirod and Thaxton (2000b) who found that broiler chickens given adrenocorticotropic hormone had significantly lower nutrient digestibility than broilers in the non-stressed control group. These researchers concluded that the reduction in the digestion of nutrients was most likely due to an increase in feed passage rate in the presence of the stressor because birds treated with adrenocorticotropic hormone displayed polydipsia and polyuria during and after stress.

It is well known that glucocorticoid plays an important role in reduced anabolic and enhanced catabolic processes (Virden and Kidd, 2009). Perhaps, stressed broiler chickens used up more energy to adapt to the stress condition and less energy for growth. Meanwhile, supplementation with GABA relieved the inhibitory effect of in-feed DEX on growth performance of broiler chickens, suggesting a potentially important role for GABA to inhibit the adverse effects of stress in broiler chickens. This was similar to a previous report which showed that dietary GABA supplementation remarkably enhanced in FI, BW and reduced FCR caused by heat stress (Chand et al., 2016; El-Naggar et al., 2019; Zhong et al., 2020), and high stocking density stress (Jeong et al., 2020). In this study, dietary GABA supplementation improved the growth performance of birds reared under DEX-induced stress by increasing FI. This improvement maybe due to the increased FI in the GABA-supplemented groups, an effect that has also been documented by others (Keun-Tae et al., 2016; Tajalli et al., 2006). GABA seems not to have a direct effect on the digestion and metabolism of nutrients in birds but improves performance by increasing FI (Dai et al., 2011; Hu et al., 2016). Additionally, the increased body weight following GABA feeding could be due to increases in jejunal villus length, crypt depth, and mucous membrane thickness (Al Wakeel et al., 2017), which may in turn improve nutrient absorption. In addition, Zhong et al. (2020) found that the villus height of jejunum in GABA group broilers was higher than in the control group. They also showed trends toward longer villi and a greater villus/crypt ratio of ileum, and the villi were arranged more tightly in GABA group, which might increase the amount of villi or the area of absorption.

Furthermore, Zhang et al. (2012) revealed that dietary GABA supplementation increased the activities in the gastrointestinal tract of trypsin, amylase, and lipase enzymes. Moreover, Piqueras and Martinez (2004) demonstrated that GABA improved body weight in rats by enhancing gastrin release and digestive enzyme activities as well as by lowering cholecystokinin secretion, which prevents gastric distension and blocks its satiety effects.

Having demonstrated that glucocorticoids are involved in the altered redox balance in poultry as well as modulating immune function and enteric mucosal integrity (Gao et al., 2010; Lin et al., 2000; 2004; Mujahid et al., 2006; Osho and Adeola, 2020). The antioxidative systems, responsible for the deactivation, consist of an enzymatic system and non-enzymatic antioxidants. SOD, CAT, and GPx belong to the enzyme defense system (Jiao et al., 2019). SOD provides the efficient dismutation of superoxide radicals into less toxic hydrogen, whereas CAT and GPx convert (Ahmad et al., 2012). Indeed, GABA has shown beneficial effects in relieving oxidative stress induced by heat stress conditions. Furthermore, it induced higher hepatic glutathione peroxidase enzyme (GSH-Px) activities and decreased the malondialdehyde content. (Al Wakeel et al., 2017; Chand et al., 2016). GABA’s effects on reducing oxidation levels may be related to its ability to promote glutamate levels (Walls et al., 2015), thus enhancing antioxidant enzymes such as glutathione peroxidase activity (Al Wakeel et al., 2017; Chen et al., 2013). In poultry, glutathione peroxidase is one of the main enzymes responsible for the first line of antioxidant defense (Surai et al., 2019).

These findings prove the protective role of GABA supplementation in modulating the damage caused by the effects of stress. The increase of antioxidant enzyme activities has been shown to be a defensive response against oxidative stress (Devi et al., 2000). Additionally, the high activity of antioxidant enzymes decreases lipid peroxidation, thereby decreasing the MDA content (Ali et al., 2010). The reduced profile of MDA in the GABA treated chicks may be due to its antioxidant effect (Zhu et al., 2015). It had reported similar results that GABA significantly reduced MDA content in duodenum, jejunum, and ileal mucosa in heat stressed broiler when compared with the control (Chen et al., 2013). Our findings were supported by Zhu et al. (2015) who reported increased activity of antioxidant enzymes and decreased MDA level with increasing level of dietary GABA in Hy-Line Brown hens under heat stress. Additionally, Zhang et al. (2012) observed that increasing supplementation of GABA increased the level of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and decreased the level of malondialdehyde (MDA).

Inflammation was induced by the innate immune system in response to tissue damage or invade pathogens (Chen et al., 2018). In response to heat stress, the levels of proinflammatory cytokines increased, which may result in hemorrhage and necrosis in liver and spleen (Jang et al., 2014; Ohtsu et al., 2015). Former studies indicated that the pro-inflammatory cytokines production were associated with the overproduction of ROS under stress (Jang et al., 2014).

Glucocorticoids inhibit many of the initial events in an inflammatory response that impact both the innate and adaptive immune responses (Busillo et al., 2011). Thus, the primary anti-inflammatory action of glucocorticoids is to decrease the high levels of proinflammatory genes encoding cytokine and chemokine, to resolve the inflammatory process and restore homeostasis. Interleukin-10 is known to be an inhibitor of proinflammatory cytokines; it is possible that the beneficial effect of DEX partly results from the upregulation of IL-10–producing cells (Osho and Adeola, 2020). Glucocorticoids can inhibit inflammation by canceling the activity of transcription factors (such as nuclear factor-kB and activator protein-1) that control the production of proinflammatory cytokines by interacting with glucocorticoid-responding elements when bound to glucocorticoid receptors (Dejager et al., 2014). As, bits of cytokines may serve as cell-signaling molecules, which activated the nuclear factor kappa B signal pathway, hence promoting the production of cytokines (Xu et al., 2018). In this study, dietary GABA supplementation decreased serum IL-10 and IL-6 levels as it has anti-inflammatory effect (Duthey et al., 2010). Our results indicated that DEX-induced inflammation was repressed with GABA supplementation. Additionally, GABA has some positive effects on cellular immune function, such as activation or suppression of cytokine secretion, modification of cell proliferation, and even cell migration (Jin et al., 2013). The levels of lipopolysaccharide-induced tumor necrosis factor-α and inflammatory cytokines (IL-1β, IL-6) in serum were significantly decreased by GABA at 80 mg/kg under beak-trimming stress as reported by Xie et al. (2013).

In order to assess chickens exposed to stressors a great array of physiological parameters relating to metabolism, immunity, antioxidant response, and stress response in blood or serum samples is monitored (Puvadolpirod and Thaxton, 2000). For example, H:L ratio is considered as one of the best-recognized stress indicators in poultry. If chickens are exposed to stressors, heterophil numbers increase but lymphocyte numbers decrease leading to an increase of the H: L ratio (Siegel, 1995). This effect is probably due to GABA’s potential role in decreasing the glucocorticoid level, which causes a rapid outflow of heterophils from the bone marrow into the circulatory system (Al Wakeel et al., 2017; Melvin and Reece, 1996; Jeong et al., 2020).

Dietary GABA supplementation increased the serum concentrations of ALT, AST, and ALP. These effects were similar to the results obtained by Hu et al. (2008), Jeong et al. (2020) and El-Naggar et al. (2019) but are inconsistent with those of Hu et al. (2016), who detected reduced concentrations of AST, ALT and LDH and increases in the ALP enzyme activity upon GABA administration. The increases of ALT and AST levels were found in 4-tert-butylphenol-caused liver damage (Cui et al., 2022), liver and kidney (Gao et al., 2014). Our findings were in contrast with an earlier report (Wang et al., 2016) on the hepatoprotective property by GABA. Nonetheless, it should be kept in mind that the observed AST values are within normal physiological ranges from about 140 to 190 IU/L (An et al., 2018).

In general, It was reported that chickens exposed to stress conditions increased concentrations of triglyceride in serum samples (Zhang et al., 2012). In addition, an increase in total cholesterol was detected in heat-stress-raised chickens (Houshmand et al., 2012; Onbasilar et al., 2008). This increased concentration of total cholesterol and HDL is expected as HDL transports cholesterol to the liver from body tissues in excess needs (Tall, 1998).

In this study, GABA increased triglyceride concentration and decreased LDL and HDL concentrations in the serum of birds under stress. Similar increase in triglyceride concentration was shown by Zhigang et al. (2013) in Cherry Valley ducks supplemented with 100 mg GABA/kg diet. The increased triglyceride concentration may have been associated with the increases in abdominal fat content in the broiler (El-Naggar et al., 2019) and increased fat mobilization in ducks (Zhigang et al., 2013) upon dietary GABA supplementation.

Our finding is similar to that of (Dai et al., 2011; Jeong et al., 2020; Zhigang et al., 2013). This effect may be due to increased serum levels of GABA (Hu et al., 2016), which can stimulate the turnover of fat and the release of free fat acids and glucose into serum to be available to all cells as energy sources (Dai et al., 2011). Based on these findings, GABA might have an important role in the metabolism of nutrients especially lipids.

Conclusions

In conclusion, the results of our study indicated that stress mimicked by in-feed DEX treatment could significantly increase BW loss, induce oxidative stress, and suppress immune function. However, dietary GABA improved growth performance and immune function in broiler chickens, especially in the presence of stress, which explains the ability of GABA to decrease catabolism and oxidative injury of tissues. Therefore, GABA supplementation may be a potential agent to relieve oxidative stress and inflammation response in immunosuppressed broiler chickens. The results of the present study suggested that GABA supplementation at the rate of 100 mg/kg feed can better alleviate the deleterious effect of oxidative stress in terms of growth performance, oxidative damage, and inflammation responses in broilers.

Disclosure statement

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Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.

Notes

1 DG⁺, group received 1 mg/kg DEX and 100 mg/kg GABA

2 DG⁺⁺, group received 1 mg/kg DEX and 200 mg/kg GABA