Hydroalcoholic extract of Withania somnifera leaf and α-tocopherol acetate in diets containing oxidised oil: effects on growth performance, immune response, and oxidative status in broiler chickens

Abstract

This study was conducted to evaluate the effects of hydroalcoholic leaf extract of Withania somnifera (WS) and α-tocopherol acetate (α-Toc) in the diets containing oxidised oil on the growth performance, immune response, and oxidative status in the broiler chickens. A 3-way factorial design (2 × 3 × 2) was applied consisting of the oxidised oil (0 and 2%), WS (0, 100 and 200 mg/kg diet), and α-Toc (0 and 200 mg/kg). In total, 600 one-day-old male broilers were randomly subjected to 12 treatments with 5 replicates (10 birds/replicate). Broilers were provided with feed and water ad libitum during the 42-day experimental period. On day 42, one bird per each replicate was killed to measure the lymphoid organ weights, intestinal morphometric parameters, and MDA content in the thigh meat. Dietary oxidised oil reduced the FI, and BWG (p < .05). In contrast, dietary WS at a concentration of 200 mg/kg alleviated the negative effects of the oxidised oil on the BWG. Birds fed with the dietary oxidised oil revealed lower titres of antibody against the SRBC (p < .05). Antibody titres were significantly increased by the supplementary WS (p < .05), except for IgG, and IgM during primary and secondary responses, respectively. Dietary supplementation of WS at the concentrations of 100 or 200 mg/kg resulted in a higher CBH response after 12 h (p < .01). Birds fed with the diet supplemented with WS exhibited significantly higher spleen weight than those fed with the non-WS-supplemented diets (p < .01). Dietary oxidised oil significantly increased the MDA content in thigh meat, and inversely reduced the activity of serum antioxidant enzymes (p < .05). Birds fed with the WS-supplemented diets showed significantly lower levels of MDA content in thigh meat, and conversely higher values of serum GPx and SOD activities (p < .05). The GPx activity values were significantly higher for the birds fed with the dietary α-Toc compared to those fed with the non- α-Toc-supplemented diets (p < .05). Our findings showed that the presence of WS in the broilers diets as an alternative to α-Toc could be a good approach to improve the performance, immune response, and meat oxidative stability under oxidative stress conditions.

Highlights

• Dietary oxidised oil reduced the FI, and BWG, and the diets supplemented with WS leaf extract alleviated the deleterious effects of the oxidised oil on the BWG.

• Dietary WS revealed the immunostimulatory effects. The birds fed with the WS-supplemented diets showed a higher antibody titre against the SRBC.

• Dietary inclusion of WS in the broilers diets negated the negative effects of the oxidised oil by reducing the MDA content in thigh meat and increasing the activity of the antioxidant enzymes.

Keywords:

• Withania somnifera
• broiler chicken
• oxidised oil
• immune response
• oxidative status

Introduction

Animal and vegetable-derived lipids account for a significant part of the dietary fat, which are regularly used in the poultry feed industry for feed formulation and energy supply (Li et al. 2012). This is mainly the case for the broilers nutrition so that, the diets with higher amount of fat are needed in order to meet the energy demands for fast-growing (Wang et al. 2016). Vegetable oils are mainly consisted of the polyunsaturated fatty acids (PUFA), which have been found to have high amounts of pro-oxidants and cause subsequent oxidative damage in the broilers (Gao et al. 2010). The diets containing the oxidised oils can act as the pro-oxidants during the oxidation of PUFA, making easier the production of reactive oxygen species (ROS) and spreading of oxidation (Lu et al. 2014). The thermally oxidised oils undergo the chemical modification producing various amounts of oxidation products including hydroperoxide, 4-hydroxynonenal, 2,4-heptadienal, and malondialdehyde (MDA) (Choe and Min 2007; Smyk 2015). Degree of oxidation in the meat lipids can be monitored by measurement of the MDA values (Voljc et al. 2013). Antioxidants are capable of delaying or inhibiting the oxidation of lipids and are commonly used in the food industry. Supplementation of the dietary vitamin E is one of the strategies to reduce the development of lipid oxidation in the broiler's meat (Bou et al. 2001). The α-tocopherol (α-Toc) is the most biologically active antioxidant form of vitamin E, playing an essential role in the free radical scavenging and inhibiting the propagation of the lipid peroxidation. It has been previously reported that the dietary inclusion of α-Toc in the broilers produced the meat with higher oxidation stability and α-Toc content (Jensen et al. 1997). Therefore, supplementation with α-Toc reduces the lipid oxidation and improves the meat nutritional value (Bou et al. 2006). Recently, synthetic antioxidants have been replaced by the natural ones, particularly those derived from the medicinal plants, vegetables, fruits, etc., containing the high amounts of active secondary metabolites. (Falowo et al. 2014). It has been reported that the application of natural antioxidants can be helpful not only through inhibition of the oxidation but also through prevention of forming the cytotoxic compounds in the meat muscles (Falowo et al. 2014; Jiang and Xiong 2016). Withania somnifera L Dunal (WS) (family Solanaceae), as an annual medicinal plant is well known for its medicinal properties. Previous findings have revealed some beneficial effects for this plant, including antistress, antioxidant, and immunomodulatory, antitumor, as well as antiinflammatory, and haematopoietic activities (Mishra et al. 2000). It has been previously reported that the plant displayed antioxidant and radical scavenging activities attributing to some natural chemical compounds, such as the anthocyanin, acids, phenolic compounds, and many other essential phytoconstituents (Pietta 2000). Also, the leaves and roots of the plant contain the withanolides, a group of steroidal lactones (Jayaprakasam et al. 2003). Udayakumar et al. (2010) reported that daily administration of the ethanolic root and leaf extracts of WS in alloxan-induced diabetic rats restored the levels of the catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) to the normal. Elberry et al. (2010) reported that orally administration of the methanol extract of aerial parts of WS for 4 weeks in the rats with carbon tetrachloride-induced hepatotoxicity significantly increased the hepatic GPx, glutathione reductase (GR), and glutathione S-transferase (GST) activities and decreased the MDA content. Therefore, the present study was conducted with a factorial design to evaluate the effect of the hydroalcoholic leaf extract of WS compared to α-Toc and their interactions in the diets containing the oxidised oil on the growth performance, immune response, and oxidative status of the broiler chickens.

Materials and methods

Preparation of hydroalcoholic extract of WS leaf

The leaves of WS were freshly collected from the two-year-old plants during September months from Saravan, Baluchestan, Iran. The samples were authenticated by the Herbarium of the Plant Production Department in Higher Educational Complex of Saravan. The leaves were shade dried, were finely ground to a powder, and were mixed with 50% ethanol at room temperature, and were frequently shaken. After 72 h, the liquid soluble substances were separated from the solid material using the vacuum filtration and were concentrated by a rotary evaporator (Laborota 4000, Heidolph, Germany). The dried powder was kept at −20 °C for further use.

Birds and dietary treatments

A total of 600 one-day-old male broiler chickens of the Ross 308 strain were acquired from a local hatchery and were raised from one to 42 days of age in 60 pens and 10 birds per each pen. Feed and water were offered on an ad libitum basis during the experimental period. The air standard temperature, photoperiod, and ventilation were managed according to the operating procedures related to the breeder guidelines of Ross broiler (Ross 2007). The 2 × 3 × 2 factorial arrangement was used in a completely randomised design. Factors were consisted of the oxidised soy oil (0 and 2% diet, Peroxide value 75 mEqO₂/kg), WS leaf extract (0, 100, and 200 mg/kg diet), and α-Toc (0 and 200 mg/kg diet) (Razak, Tehran, Iran). The corn soybean meal based experimental diets were formulated according to the nutrient requirement recommendation manual for breeding the Ross 308 broiler (Ross 2007) (Table 1). In each growing phase, the experimental diets were isocaloric and isonitrogenous. Growth performance was calculated per replicate on 11, 24, and 42 days of age as average body weight gain (BWG), average feed intake (FI), and FI/BWG. Also, all the data were adjusted for mortality. All the animal protocols used in the current experiment were approved by the Animal Ethics Committee of Saravan Higher Educational Complex.

Table 1. Composition of the basal diets (g/kg as fed).

Ingredient	Starter (1–10 days)	Grower (11–23 days)	Finisher (24–42 days)
Corn	522.50	523.7	570.8
Soybean meal	340	378.6	324.50
Corn gluten	48.2	–	–
Non oxidised soybean oil^a	12.2	35	43
Oxidised soybean oil^b	20	20	20
Dicalcium phosphate	17.8	15.6	15.6
Limestone	12.5	11.8	11.5
Salt	4	4.5	4.3
DL-Methionin	3.4	3.2	2.9
L-Lysine	4.6	1.8	1.8
Vitamin premix^c	2.5	2.5	2.5
Mineral premix^d	2.5	2.5	2.5
L-Threonine	1.4	0.8	0.6
Sand	8.4	–	–
Calculated nutrients and energy
AME, (kcal/kg)	3000	3100	3210
Crude protein (g/kg )	230.2	215.2	195.1
L-Lysine (g/kg)	14.4	12.9	11.6
DL-Methionin (g/kg)	7	6.4	5.9
TSAA (g/kg)	10.7	9.8	9.1
Calcium (g/kg)	9.6	9	8.8
Nonphytate P (g/kg)	4.8	4.5	4.4
Total P (g/kg)	7.3	6.9	6.6

^a Peroxide value <1 mEqO₂/kg.

^b Peroxide value 75 mEqO₂/kg.

^c Vitamin premix provided per kilogram of diet: retinyl acetate, 11,000 U; cholecalciferol, 1800 U; DL-α-tocopheryl acetate, 11 mg; menadione sodium bisulphate, 2 mg; riboflavin, 5.7 mg; pyridoxine hydrochloride, 2 mg; cyanocobalamin, 0.024 mg; nicotinic acid, 28 mg; folic acid, 0.5 mg; pantothenic acid, 12 mg; choline chloride, 250 mg.

^d Mineral premix provided per kilogram of diet: Mn, 100 mg; Zn, 65 mg; Cu, 5 mg; Se, 0.22 mg; I, 0.5 mg; and Co, 0.5 mg.

AME: apparent metabolizable energy.

Evaluation of traits and measurements

Immune response

Humoral immunity

Sheep red blood cells (SRBC) were used as an antigen to evaluate the specific antibody responses. On day 25, two birds per each replicate were randomly selected and bled via the brachial vein. Before performing the SRBC challenge, 3 ml of blood per each bird was collected to check the antibody titres. The same birds were immunised with the challenge dose of 7% SRBC. They were bled via the brachial vein within one week after the primary injection, and the blood samples (3 mL per each bird) were collected to assess the primary antibody titres, and the inoculation was repeated. The blood samples were kept at ambient temperature for 2 h until clotting. After centrifugation for 10 min at 580 × g, the sera were collected and were kept at −20 °C for further analyses. Fourteen days after the primary immunisation, the blood samples were taken for analysis of secondary antibody.

Cell-mediated immunity

The Cutaneous Basophil Hypersensitivity (CBH) response was used, as explained previously by Kean and Lamont (1994) to determine the cell-mediated immune response. At 37 days of age, 2 birds per each replicate were chosen randomly, and 0.1 mg of phytohemagglutinin-P (PHA-P) dissolved in 0.1 ml of saline was injected into the toe web of the right foot between the second and third digits. The corresponding toe web of the left foot was injected with 0.1 ml of saline, representing as a control. The toe web thickness was measured at 12 and 24 h following the challenge using a digital micrometer (series 500, Mitutoyo, Tokyo, Japan). The toe web swelling response to the PHA-P was measured by subtracting the pre-PHA injection thickness from the post-PHA injection thickness.

Lymphoid organ weights

On day 42, after 12 h of feed and water withdrawal, one bird per each replicate was chosen at random, and was weighed, and killed by cervical dislocation. The lymphoid organs (thymus, spleen, and bursa of Fabricius) were carefully dissected and weighed. Their relative weights were determined as percentages of the live weight.

Meat and blood antioxidant indices

After slaughter, the thighs were removed from the carcase, and muscles were dissected from the bone. The deboned thigh meat samples were minced twice using an electric meat grinder (MG510, 1600 W, Kenwood, UK) through a 4 mm plate and then, were stored at −20 °C in the polypropylene bags for later analysis. After 30 days of the frozen storage, the MDA content was measured. At 42 days of age, blood samples were taken from the brachial vein of the birds (one bird per each replicate) to determine the SOD and GPx activities. The blood samples were allowed to clot at room temperature, and then, centrifuged at 1500 × g for 10 min to obtain the serum. The serum samples were stored at −20 °C until further analysis.

Morphometric characteristics of the intestine

On day 42, the gut of the killed birds was, dissected and relative length of the duodenum, jejunum, and ileum was measured, which was expressed relative to the body weight (cm per 100 g of live BW). Duodenum was defined as the segment encompassing the duodenal loop, and ileum was defined as the segment before the ileocecal junction equalling the length of the caeca. The jejunum was considered as the segment between the duodenum and ileum.

Laboratory analyses

Antibody assay

The microhemagglutination assay was used in 96-well plates according to LePage et al. (1996), to determine the immunoglobin G (IgG), immunoglobin M (IgM), and total antibody titre in response to SRBC. Therefore, the serum samples were heated in a water bath (56 °C for 30 min). Fifty µL of PBS was placed in the first wells of 96-well microtitration plate to determine the total antibody titre. Then, 50 µL of serum was added to the same wells, and the plates were covered by the tape and were incubated (37 °C for 30 min). Plates were removed from the incubator, and 50 µL of PBS was added to 11 subsequent wells in each row to make a two-fold serial dilution for each sample on sequential rows. A two-fold serial dilution of the samples was made on sequential rows. After that, 50 µL of SRBC suspension (2.5%) in PBS was added to each well. The plates were covered again, and were shaken, and incubated for 30 min. After incubation, agglutination of the wells was carefully observed through holding the plates over a mirror, and titres were noted. The titres of IgG mercaptoethanol resistant (MER) and IgM mercaptoethanol sensitive (MES) antibody were assessed using the same procedure applied for the total titres excepting that 50 μL of 0.02 M 2-mercaptoethanol in PBS was added to the first row of the wells instead of PBS alone. The difference between the total and MER titres was considered as MES titre. The antibody titres were expressed as log2 of the reciprocal in the last dilution presenting visible agglutination.

Thiobarbituric acid reactive substance analysis

The thiobarbituric acid reactive substances (TBARS) analysis was used to assess the stability of the lipids against oxidation or MDA concentration in thigh muscles (mg/kg) on day 30 post frozen storage. The analysis was based on a method revised by Kumari Ramiah et al. (2014). Briefly, one g of meat sample was homogenised in 4 mL of potassium chloride (0.15 M) and 0.1 mM butylated hydroxytoluene (BHT). Then, 200 μL of the homogenised sample was extracted and mixed vigorously with 2 mL of thiobarbituric acid solution. Then, the mixed samples were placed in a boiling water bath (95 °C for 60 min) until the pale red pigment was developed. Then, the assay mixtures were cooled at room temperature, 3 mL of n-butanol was added and vortexed for 60 s. Then, the mixture was centrifuged at 4000 xg for 15 min, and the supernatant was removed. MDA levels were colorimetrically determined at the wavelength of 532 nm using the UV-visible spectrophotometer (model PharmaSpec 1700, Shimadzu, Japan).

Assay of serum antioxidant enzyme activities

The activities of GPx and SOD were determined by the commercially available kits of RANSEL and RANSOD (Randox Laboratories Ltd., Crumlin, Country Antrim, UK), respectively (Arthur and Boyne 1985). The assays were conducted on a UV-visible spectrophotometer (model PharmaSpec 1700, Shimadzu, Japan).

Statistical analysis

Data were statistically analysed using the GLM procedure in SAS software (SAS Institute 2003) as a three factorial arrangement, including dietary oxidised oil (0 and 2% diet), WS leaf extract (0, 100, and 200 mg/kg diet), and α-Toc (0 and 200 mg/kg diet). Duncan’s test was used for comparisons between the treatments. Means were considered statistically significant at p ≤ .05. The statistical model of experiment is shown below. $$Y_{\text{ijkl}}=\mu +O{O}_{\mathrm{i}}+W{S}_{\mathrm{j}}+\alpha -{\text{Toc}}_{\mathrm{k}}+{\left(O{O}_{\mathrm{i}}*WS\right)}_{\text{ij}}+{\left(O{O}_{\mathrm{i}}*\mathrm{ }\alpha -\text{Toc}\right)}_{\text{ik}}+{\left(WS*\alpha -\text{Toc}\right)}_{\text{jk}}+{\left(O{O}_{\mathrm{i}}*W{S}_{\mathrm{j}}*\mathrm{ }\alpha -\text{Toc}\right)}_{\text{ijk}}+e_{\text{ijkl}}$$

Where Y_ijkl is the observation expected independent variables, μ: overall mean, OO_i: fixed effect of oxidised oil, WS_j: fixed effect of WS, α-Toc _k: fixed effect of α-Toc, (OO_i * WS) _ij: interaction between oxidised oil and WS, (OO_i * α-Toc) _ik: interaction between oxidised oil and α-Toc, (WS * α-Toc) _jk: interaction between WS and α-Toc, (OO_i * WS_j * α-Toc) _ijk: is the 3-way interaction and, e_ijkl is the random error.

Results

Growth performance

As shown in Table 2, the significant main effects of oxidised oil on FI were observed during the whole experiment (p < .05). The birds fed with dietary oxidised oil showed significantly lower FI compared to those received no dietary oxidised oil (3850.09 vs. 3981.33 g; p < .05). The values of BWG were significantly reduced by addition of the oxidised oil (2274.43 vs. 2350.69 g; p < .05). The BWG was significantly influenced by the interaction between oxidised oil and WS. The interaction showed that the addition of dietary oxidised oil in birds received no dietary WS resulted in significantly lower BWG (2193 vs. 2449.65 g; p < .01). Also, this interaction indicated that the supplementary WC (200 mg/kg) exerted a stimulatory effect on BWG when added to diets containing 2% oxidised oil (2398.98 vs. 2193.06 g; p < .01). The feed conversion efficiency (FCE) was not significantly influenced by the dietary treatments (p > .05). Addition of α-Toc did not influence the broiler’s performance (p > .05).

Table 2. Effect of Oxidised oil, Withania somnifera (WS), and α-tocopherol (α-Toc) on body weight gain (BWG), feed intake (FI), and feed conversion efficiency (FCE) at 42 days of age.

Treatment			FI (g)	BWG (g)	FCE (g/g)
Oxidised oil (%)	WS (mg/kg)	α-Toc (mg/kg)
0	0	0	3933.0	2380.75^abc	1.65
0	0	200	4105.0	2449.65^a	1.67
0	100	0	4048.5	2411.70^a	1.67
0	100	200	4055.3	2366.01^abc	1.71
0	200	0	3882.1	2284.33^abc	1.70
0	200	200	3864.9	2211.72^bc	1.75
2	0	0	3831.2	2193.06^c	1.74
2	0	200	3818.2	2197.00^c	1.73
2	100	0	3822.2	2272.81^abc	1.68
2	100	200	3864.9	2323.62^abc	1.66
2	200	0	3825.2	2261.12^abc	1.69
2	200	200	3938.8	2398.98^ab	1.64
SEM			100.406	57.124	0.036
Main effect
Oxidised oil		0	3981.33^a	2350.69^a	1.69
		2	3850.09^b	2274.43^b	1.69
WS		0	3921.86	2305.11	1.70
		100	3947.70	2343.54	1.68
		200	3877.57	2289.04	1.69
α-Toc		0	3890.37	2300.63	1.69
		200	3941.05	2324.50	1.69
				P
Oxidised oil			0.028	0.025	0.937
WS			0.610	0.389	0.780
α-Toc			0.386	0.472	0.740
Oxidised oil × WS			0.241	0.002	0.250
Oxidised oil × α-Toc			0.960	0.227	0.138
WS × α-Toc			0.928	0.900	0.961
Oxidised oil × WS × α-Toc			0.525	0.240	0.859

^a–cMeans within each column with no common superscript differ significantly (p < .05).

Immune response

Humoral immunity

As shown in Table 3, birds receiving oxidised oil showed significantly lower titres for IgG, IgM, and total antibody titres against the SRBC in both primary and secondary challenges than those receiving no supplementary oxidised oil (p < .05). Supplementary WS significantly influenced the antibody titres during the primary and secondary challenges, except for IgG, and IgM during primary and secondary responses, respectively. A marginal trend towards significance was found for IgG (p = .059), and a favourable trend was noted for IgM (p = .091). For the total antibody titres in the secondary challenge, the birds received both levels of supplementary WS had significantly higher titre values (9.25 and 9 vs. 8.25; p < .01). However, for the primary challenge, the difference was more pronounced at the level of 100 (8 vs. 7.40; p < .05). There was a significant interaction in the total antibody response during secondary challenge between the dietary oxidised oil and WS (p < .05). The interaction demonstrated that supplementation of WS at 200 mg/kg in birds receiving neither oxidised oil nor WS significantly increased the total antibody titre, regardless of dietary α-Toc level (10 vs. 7.80; p < .05). The anti-SRBC antibody responses during both challenges were not significantly influenced by the dietary α-Toc (p > .05).

Table 3. Effect of Oxidised oil, Withania somnifera (WS), and α-tocopherol (α-Toc) on primary and secondary antibody responses.

Treatment			Primary antibody response				Secondary antibody response
Oxidised oil (%)	WS (mg/kg)	α-Toc (mg/kg)	IgG	IgM	Total		IgG	IgM	Total
0	0	0	2.00	5.60	7.60		2.40	5.40	7.80^e
0	0	200	2.20	5.60	7.80		2.20	6.40	8.60^bcde
0	100	0	2.20	6.00	8.40		3.00	6.80	9.80^ab
0	100	200	2.00	6.20	8.20		2.80	6.80	9.60^abc
0	200	0	2.80	6.00	8.80		3.00	7.00	10.00^a
0	200	200	2.60	5.60	8.20		3.00	6.80	9.80^ab
2	0	0	2.00	5.00	7.00		2.20	6.00	8.20^de
2	0	200	2.00	5.20	7.20		2.20	6.20	8.40^cde
2	100	0	2.00	6.20	8.20		260	6.00	8.60^bcde
2	100	200	2.00	5.20	7.20		2.40	5.60	8.00^de
2	200	0	2.00	5.40	7.40		2.20	5.80	8.00^de
2	200	200	2.00	5.00	7.00		2.40	6.80	9.20^abcd
SEM			0.200	0.316	0.336		0.230	0.378	0.380
Main effect
Oxidised oil		0	2.30^a	5.83^a	8.16^a		2.73^a	6.53^a	9.26^a
		2	2.00^b	5.33^b	7.33^b		2.33^b	6.06^b	8.40^b
WS		0	2.05	5.35^b	7.40^b		2.25^b	6.00^b	8.25^b
		100	2.05	5.90^a	8.00^a		2.70^a	6.30^ab	9.00^a
		200	2.35	5.50^ab	7.85^ab		2.65^a	6.60^a	9.25^a
α-Toc		0	2.16	5.70	7.90		2.56^a	6.16	8.73
		200	2.13	5.46	7.60		2.50^a	6.43	8.93
						P
Oxidised oil			0.012	0.008	0.0001		0.004	0.037	<0.001
WS			0.059	0.048	0.040		0.015	0.091	0.001
α-Toc			0.774	0.207	0.129		0.619	0.228	0.367
Oxidised oil × WS			0.059	0.905	0.246		0.195	0.084	0.012
Oxidised oil × α-Toc			0.774	0.365	0.609		0.619	1.00	0.763
WS × α-Toc			0.718	0.440	0.197		0.648	0.307	0.166
Oxidised oil × WS × α-Toc			0.718	0.248	0.543		0.939	0.152	0.134

^a-eMeans within each column with no common superscript differ significantly (p < .05).

Cell-mediated immunity

The cell-mediated immune response determined by the inoculation of PHA-P, was significantly influenced by the WS-supplemented experimental diets after 12 h (p < .01) (Table 4). Significant differences were noted in the toe web thickness between the birds fed with the WS-supplemented diets and those received no dietary WS (0.311 and 0.317 vs. 0.302 mm; p < .01). Dietary oxidised oil and α-Toc did not influence the toe web swelling response to the PHA-P (p > .05).

Table 4. Effect of Oxidised oil, Withania somnifera (WS), and α-tocopherol (α-Toc) on swelling response to phytohematoglutinin.

Treatment			Swelling response (mm)
Oxidised oil (%)	WS (mg/kg)	α-Toc (mg/kg)	12 h	24 h
0	0	0	0.296	0.302
0	0	200	0.306	0.306
0	100	0	0.316	0.312
0	100	200	0.310	0.314
0	200	0	0.312	0.330
0	200	200	0.314	0.304
2	0	0	0.302	0.317
2	0	200	0.306	0.314
2	100	0	0.310	0.316
2	100	200	0.308	0.318
2	200	0	0.314	0.318
2	200	200	0.328	0.316
SEM			0.005	0.005
Main effect
Oxidised oil		0	0.309	0.311
		2	0.311	0.316
WS		0	0.302^b	0.309
		100	0.311^a	0.315
		200	0.317^a	0.317
α-Toc		0	0.308	0.315
		200	0.312	0.312
			P
Oxidised oil			0.459	0.122
WS			0.003	0.215
α-Toc			0.273	0.247
Oxidised oil × WS			0.314	0.358
Oxidised oil × α-Toc			0.627	0.414
WS × α-Toc			0.255	0.108
Oxidised oil × WS × α-Toc			0.513	0.143

^a–bMeans within each column with no common superscript differ significantly (p < .05).

Lymphoid organ weights

As presented in Table 5, birds fed with the WS-supplemented diets exhibited significantly higher spleen relative weight compared to those fed with non-WS-supplemented diets (0.154 and 0.153 vs. 0.135 g; p < .05) (Table 5). No significant effects of WS were observed for thymus, and bursa of Fabricius (p > .05). The lymphoid organ weights were not influenced by the dietary oxidised oil, and α-Toc (p > .05).

Table 5. Effect of Oxidised oil, Withania somnifera (WS), and α-tocopherol (α-Toc) on relative weight of the thymus, spleen, and bursa of Fabricius of broilers at 42 days of age.

Treatment			Thymus (g)	Spleen (g)	Bursa (g)
Oxidised oil (%)	WS (mg/kg)	α-Toc (mg/kg)
0	0	0	0.658	0.152	0.113
0	0	200	0.607	0.140	0.195
0	100	0	0.514	0.152	0.120
0	100	200	0.642	0.160	0.100
0	200	0	0.663	0.151	0.138
0	200	200	0.665	0.153	0.098
2	0	0	0.490	0.129	0.119
2	0	200	0.639	0.122	0.119
2	100	0	0.692	0.154	0.111
2	100	200	0.584	0.151	0.146
2	200	0	0.529	0.150	0.104
2	200	200	0.566	0.158	0.083
SEM			0.071	0.010	0.034
Main effect
Oxidised oil		0	0.625	0.151	0.128
		2	0.583	0.144	0.113
WS		0	0.599	0.135^b	0.136
		100	0.608	0.154^a	0.119
		200	0.606	0.153^a	0.106
α-Toc		0	0.591	0.148	0.117
		200	0.617	0.147	0.124
				P
Oxidised oil			0.321	0.266	0.496
WS			0.981	0.049	0.464
α-Toc			0.533	0.915	0.772
Oxidised oil × WS			0.211	0.354	0.517
Oxidised oil × α-Toc			0.993	0.962	0.947
WS × α-Toc			0.920	0.638	0.355
Oxidised oil × WS × α-Toc			0.105	0.835	0.354

^a–bMeans within each column with no common superscript differ significantly (p < .05).

Antioxidant indices

As demonstrated in Table 6, the dietary oxidised oil significantly increased the MDA content in thigh meat (0.261 vs. 0.225 mg/kg; p < .05), and inversely reduced the activity of GPX, and SOD antioxidant enzymes (138.50 vs. 122.10 and 139.66 vs. 130.76 U/ml, respectively; p < .05). The birds fed with the WS-supplemented diets showed lower levels of MDA content in thigh (0.208 and 0.223 vs. 0.297 mg/kg; p < .001) and conversely showed higher values of GPx and SOD enzyme activities (138.60 and 136.26 vs. 116.75, and 139.78 and 140 vs. 126.33 U/ml, respectively; p < .05). The GPx activity values were significantly higher for the birds fed with the dietary α-Toc compared to those fed with non- α-Toc-supplemented diets (138.34 vs. 122.80 U/ml; p < .05). However, the effects of α-Toc on MDA content in thigh were at the margin of significance (p = .057). A significant two-way interaction was noted between the oxidised oil and WS for MDA content (p < .01). The interaction showed that the addition of WS at the levels of 100 or 200 mg/kg to diets containing oxidised oil resulted in significantly lower values of MDA compared with groups not receiving WS, irrespective of dietary α-Toc level (0.167 and 0.262 vs. 0.387; p < .01).

Table 6. Effect of Oxidised oil, Withania somnifera (WS), and α-tocopherol (α-Toc) on antioxidant enzymes activity, and thigh meat malondialdehyde (MDA) content at 42 days of age.

Treatment			MDA content (mg/kg)	GPx activity (U/mL)	SOD activity (U/mL)
Oxidised oil (%)	WS (mg/kg)	α-Toc (mg/kg)
0	0	0	0.235^c	106.80	126.80
0	0	200	0.232^c	133.20	122.80
0	100	0	0.220^c	141.20	145.80
0	100	200	0.222^c	160.60	146.00
0	200	0	0.235^c	136.80	148.80
0	200	200	0.205^c	152.40	147.80
2	0	0	0.387^a	108.00	130.50
2	0	200	0.335^ab	119.00	126.00
2	100	0	0.222^c	121.80	132.40
2	100	200	0.167^c	130.80	133.75
2	200	0	0.262^bc	122.20	130.00
2	200	200	0.192^c	133.00	131.75
SEM			0.030	12..841	6.343
Main effect
Oxidised oil		0	0.225^b	138.50^a	139.66^a
		2	0.261^a	122.10^b	130.76^b
WS		0	0.297^a	116.75^b	126.33^b
		100	0.208^b	138.60^a	139.78^a
		200	0.223^b	136.26^a	140.00^a
α-Toc		0	0.260	122.80^b	135.89
		200	0.225	138.34^a	135.14
				P
Oxidised oil			0.047	0.037	0.023
WS			<0.001	0.040	0.010
α-Toc			0.057	0.045	0.787
Oxidised oil × WS			0.002	0.609	0.077
Oxidised oil × α-Toc			0.172	0.499	0.882
WS × α-Toc			0.826	0.950	0.839
Oxidised oil × WS × α-Toc			0.979	0.959	0.985

^a–cMeans within each column with no common superscript differ significantly (p < .05).

GPx: glutathione Peroxidase; SOD: superoxide dismutase.

Morphometric characteristics of the small intestine

As depicted in Table 7, no significant differences were observed between the experimental treatments for the relative length of small intestine segments (p > .05).

Table 7. Effect of Oxidised oil, Withania somnifera (WS), and α-tocopherol (α-Toc) on relative length of small intestine.

Treatment			Relative length (cm/100g of BW)
Oxidised oil (%)	WS (mg/kg)	α-Toc (mg/kg)	Duodenum	Jejunum	Ileum
0	0	0	0.726	1.63	1.57
0	0	200	0.826	1.60	1.71
0	100	0	0.732	1.66	1.64
0	100	200	0.788	1.64	1.60
0	200	0	0.733	1.64	1.51
0	200	200	0.708	1.67	1.62
2	0	0	0.731	1.68	1.61
2	0	200	0.733	1.72	1.59
2	100	0	0.719	1.60	1.66
2	100	200	0.729	1.53	1.47
2	200	0	0.672	1.51	1.51
2	200	200	0.707	1.66	1.62
SEM			0.044	0.059	0.073
Main effect
Oxidised oil		0	0.752	1.64	1.61
		2	0.715	1.62	1.58
WS		0	0.754	1.66	1.62
		100	0.742	1.61	1.59
		200	0.705	1.62	1.56
α-Toc		0	0.719	1.62	1.58
		200	0.748	1.64	1.60
				P
Oxidised oil			0.161	0.469	0.493
WS			0.282	0.451	0.541
α-Toc			0.254	0.619	0.719
Oxidised oil × WS			0.979	0.091	0.865
Oxidised oil × α-Toc			0.589	0.516	0.209
WS × α-Toc			0.766	0.282	0.085
Oxidised oil × WS × α-Toc			0.447	0.551	0.689

Discussion

In the present study, oxidative stress was induced via inclusion of the thermally oxidised soy oil. On the contrary, the hydroalcoholic leaf extract of WS was used as a potent and enriched source of the antioxidant agents to alleviate the detrimental effects of the peroxidized oils. Therefore, the use of this medicinal plant as an alternative to vitamin E was evaluated in the broiler's nutrition. The reducing effects of the oxidised oil on FI observed in the present experiment are in agreement with Liang et al. (2015), who reported that moderately increasing the soybean oil peroxide value reduced the broiler’s average daily FI. Our results are consistent with the previous findings (Anjum et al. 2004; McGill et al. 2011; Tavarez et al. 2011). The negative effects of the oxidised oils on FI may result from several factors. Thermal oxidation of the oils may lead to rancidity of the feed, production of the toxic lipid peroxides, such as ketones, aldehydes, polymerised oils, and esters which, in turn, results in the development of disagreeable odours, flavours, and reduction of the feed palatability (Kumagai et al. 2004; Liu et al. 2014). The findings of the current experiment indicated the suppressive effects of the oxidised oil on the BWG. In agreement with the results of the current experiment, Anjum et al. (2004) reported that feeding diets containing the oxidised soy oil reduced the average daily gain by 4.2% compared to diets with fresh soy oil. The present data are also consistent with those of Hung et al. (2017), who reported that feeding the broilers with the diets containing peroxidized oil reduced the average daily gain by 11.1% in comparison with the birds fed on the diets with unperoxidized oil. In the oxidative stress, the detrimental effects caused by the generation of reactive oxygen species (ROS) are exerted through the action of oxidant agents (Lin et al. 2004). Previous reports have suggested that the lower values in the BWG in the broilers suffering from oxidative stress are attributed to the lower endocrine and metabolic responses, and downregulation of the genes interfering with the transport of peptides, amino acids, and sugars (Lin et al. 2004; Ebrahimi et al. 2015). Chae et al. (2002) reported that feeding the animals with the oxidised oils resulted in lower retention of the energy and fat, which consequently depressed the BWG. It has been found that the, dietary WS alleviates the adverse effects of the oxidised oil on the BWG. Different parts of a plant, including leaves, fruits, and roots contain the glycosides, withanolides, flavonoids, tannins, alkaloids, phenolics, and saponin (Dhuley et al. 1993). The effects observed for this plant may be mainly attributed to the phytochemical diversity. It has been shown that the phenolic compounds have redox properties making them efficient hydrogen donors (Liang et al. 2010). Also, it has been found that the flavonoids have anti-microbial activity against the pathogenic intestine bacteria, prevent the physical adhesion of the pathogens to the intestinal surface of epithelium, and thereby improving the intestinal health, digestion, and nutrient absorption (Parkar et al. 2008). In confirmation of the results of the present study, Takahashi and Akiba (1999) reported that feeding the broilers with the diets containing the oxidised oil decreased the production of antibodies during the primary challenge against a bacterial pathogen. Also, the present findings are consistent with the previous studies reporting that the oxidised fat-supplemented diets suppressed the antibody production in diverse poultry species (Dibner et al. 1996; Laika and Jahanian 2015). Immunoglobulins are vital components of the humoral immune response, and their lower titres are related to the humoral immune deficiencies (Moise et al. 2010). In the oxidative stress, the products of lipid peroxidation may influence the animal's immune response via activation of stress pathways and, subsequently resulting in overproduction of the inflammatory mediators from the macrophages (Yun et al. 2009). Besides, it has been previously shown that the hydroperoxides impaired the synthesis of RNA, DNA, and proteins in the splenocytes, and thymocytes, and increased the amount of TBARS in lymphocytes, which in turn influenced on the immune responses (Oarada and Terao 1992). Regarding the immunostimulatory effects of WS, the results of the current study are in line with the study by Davis and Kuttan (2000), who reported that intraperitoneal injection of the hydroalcoholic root extract of WS ((20 mg/dose/animal) daily for 5 days increased the titre of circulating antibody against SRBC antigen in the mice. Similarly, Malik et al. (2007), reported that oral administration of the aqueous alcoholic root extract of WS at 30 mg/kg BW in SRBC for the mice immunised for 15 days increased the IgG, and IgM titres. The immunostimulatory effects of the WS leaf extract might be attributed to the biologically active phytochemicals, including steroidal lactones (withanolides, and withaferins), alkaloids, saponins, polyphenols, and flavonoids (Mirjalili et al. 2009). It has been reported that the leaves of WS contain higher amounts of flavonoids and polyphenols compared to its fruits and roots (Alam et al. 2011). The plant components may directly contribute to activation of the inherent defense mechanisms against the pathogens affecting the cellular receptors and stimulate the expression of the genes involving in the immune responses (Bricknell and Dalmo 2005). Moreover, the withanolides present in the leaves and roots induce the expression of nitric oxide synthase gene exerting the immunostimulatory effects (Iuvone et al. 2003).

The results obtained regarding the CBH response are in accordance with the findings reported by Malik et al. (2007) who showed that oral administration of the aqueous-alcoholic extract of WS at 30 mg/kg BW for 15 days in the mice challenged with SRBC significantly increased the delayed-type hypersensitivity in terms of the footpad thickness. The CBH response to the PHA-P is a thymus-dependent response mediated by the diverse subpopulations of T lymphocytes (Corrier and DeLoach 1990). The WS plant is a rich source of the withanolides, especially withanolide A, making it considerably efficient in the immune challenges against the antigens. It has been found that WS extract stimulates the B and T cell proliferation, promotes the secretion of immunoglobulins, and macrophage activation, and subsequently, enhances the humoral and cell-mediated immune responses (Malik et al. 2007). Our results suggested that the WS leaf extract exerts potentiating effects on both humoral and cellular immune responses. The present results regarding the lymphoid organ weights are consistent with those the study by Ansari et al. (2013), who reported that the dietary supplementation of WS root Powder in the broilers diets significantly increased the spleen weight compared to those, fed on non-WS-supplemented diets. Similarly, Davis and Kuttan (2000) reported that the intraperitoneal injection of WS root extract (20 mg/animal/day) for 5 days significantly enhanced the size, and weight of the spleen compared to the control group. The spleen, thymus, and bursa of Fabricius are the main lymphoid organs in the poultry, and their development status influences the function of immune system (Fan et al. 2013). The spleen plays a key role in the modulation of immune responses, and antibody generation. The increase in the spleen weight may be related to improvement of the proliferative ability of the spleen cells (Tarantino et al. 2013). Supplementary WS has been suggested to increase the proliferative ability of the spleen cells. It has been found that the ROS actively damage the spleen lymphocytes and influence its weight and size (Koo et al. 2013). Dietary antioxidant phytochemicals, including the polyphenols, and flavonoids provides the protection against the free radicals, and prevent the adverse effects on the spleen cell proliferation and differentiation (Li et al. 2016).

The higher concentrations of MDA in thigh meat and lower activities of GPx, and SOD between the groups fed with the oxidised oil in the present experiment are consistent with the previous findings in the study by Hu et al. (2020) who reported that significant enhancement of MDA content in thigh meat, and lower SOD, and GPx activities during oxidative stress were induced by the heat exposure (34 °C, 8 h/day). These results are consistent with the study by Leskovec et al. (2018), who found that the broilers exposed to the oxidative stress were induced by a high n − 3 dietary polyunsaturated fatty acids showed the higher MDA contents in breast muscle and lower whole blood GPx activity. Also, similar results have been found in other studies (Tavarez et al. 2011; Delles et al. 2014; Liang et al. 2015; Tan et al. 2018; Lindblom et al. 2019). Our results regarding the effects of dietary WS on the antioxidant indices are in line with the previous findings of Khalil et al. (2015), who showed that oral pre-treatment of the rats with WS leaf extract (100 mg/kg BW) for 4 weeks alleviated the oxidative stress induced by the isoproterenol via reducing the MDA levels and elevating the SOD and GPx activities in the heart tissues. The results are consistent with those of the study by Rajasankar et al. (2009) who reported that treatment of the Parkinson's disease with WS leaf extract (100 mg/kg BW for 7 days) in a mouse model closely related to the oxidative stress and generation of ROS, decreased the elevated levels of the MDA and increased the GPx, and SOD activities in the brain tissue. The present findings showed that the WS leaf extract had the antioxidant activities, and exerted great inhibitory effects on the lipid peroxidation attributing to the high flavonoid and phenolic contents (Alam et al. 2011). Flavonoids are a group of polyphenolic secondary metabolites with known attributes, including free radical scavenging activity, inhibitory effects on the oxidative and hydrolytic enzymes, and anti-inflammatory properties (Panche et al. 2016). In our study, the dietary supplementation of α-Toc led to the higher activity of the serum GPx. In agreement with the present results, Cinar et al. (2014) reported that the dietary inclusion of vitamin E alleviated the oxidative stress induced by toxic levels of Cu and increased the total blood GPx activity in the broiler chickens. Similarly, Cinar et al. (2010) reported that vitamin E attenuated the deleterious effects of the Cadmium-induced oxidative stress and improved the erythrocytes GPx activity. The α-Toc is one of the most essential antioxidants in the poultry nutrition and protects the cells from ROS-mediated oxidative damage (Surai et al. 2019). It has been reported that the oxidative stress reduces the ratio of reduced glutathione (GSH)/oxidised glutathione (GSSG), indicating higher oxidant production. On the other hand, dietary supplementation of α-Toc has been shown to enhance the GSH/GSSG ratio (Ryan et al. 2010).

The structural alterations in the digestive tract, such as the length of small intestine segments, can be associated with the changes in its nutrient absorption. It has been shown that the oxidative stress could damage the intestinal mucosal barrier, and decreases the nutrient digestibility, as a result of the excessive level of ROS that oxidising and destroying a wide variety of biological molecules and finally leading to the impairment of intestinal tissues (Payne and Southern 2005). These results are consistent with a previous study of Tan et al. (2019), who reported that the dietary supplementation of the oxidised fish oil did not influence the morphology of the small intestine. It has also been demonstrated that the dietary oxidised soybean oil did not influence the morphological characteristics of the jejunum and ileum (Tan et al. 2018).

Conclusions

The results of the present study suggested that the dietary oxidised oil reduced the FI, and BWG, and supplementing the diets with WS leaf extract alleviate the deleterious effects of oxidised oil on BWG. Dietary WS revealed immunostimulatory effects. The birds fed with the WS-supplemented diets showed a higher antibody titre against the SRBC. Dietary oxidised oil exerted negative effects on the antioxidant indices through increasing the MDA content in thigh meat and reducing the activity of antioxidant enzymes. On the contrary, dietary inclusion of the WS in the broilers diets negated the effects by reducing the MDA content in thigh meat and increasing the activity of the antioxidant enzymes. Our results illustrate that the dietary supplementation of WS leaf extract at the concentrations of 100 or 200 mg/kg to the broilers diets as an alternative to the α-Toc could be an excellent approach to improve the performance, immune response, and meat oxidative stability under oxidative stress conditions. However, further research is required to elucidate the exact mechanisms through which the active secondary metabolites of the WS leaf extract influence the oxidative stress markers in the broiler chickens.

Ethical approval

The Animal Ethics Committee of Saravan Higher Educational Complex approved all the animal protocols used in the current experiment.

Disclosure statement

The authors declare that they have no conflicts of interest associated with this manuscript.