Heat stress relief for broiler chickens: organic selenium and a vitamin C and E blend can enhance growth, nutrient digestibility, and blood parameters

Abstract

This study aimed to evaluate the effects of dietary supplementation with organic selenium (OR-Se) and/or vitamins C and E on the rectal temperature (Rectal-T), growth performance, carcase yield, intestinal morphology, plasma thyroid hormone levels, and antioxidant status of broilers reared in hot environmental conditions. Under heat stress conditions (29.4 to 33.6 °C), 360 Arbour Acres chicks were fed in a (2 × 3) factorial design with two dietary levels of organic selenium (0 and 0.6 mg/kg) and three dietary levels of a mix of vitamins C and E (0, 200 and 250 mg/kg). Chicks were randomly assigned to six treatments (T) with three replicates of 20 birds each: the 1st group (T1): basal diet with no supplementation (control group); the 2nd group (T2): basal diet supplemented with 0.6 mg OR-Se/kg; the 3rd group (T3): basal diet with a mixture of vitamins C and E, 200 mg of each/kg diet (Vita-Mix A); the 4th group (T4): basal diet with a mixture of vitamins C and E, 250 mg of each/kg diet (Vita-Mix B); the 5th group (T5): basal diet with (Vita-Mix A) + 0.6 mg OR-Se/kg diet; the 6th group (T6): basal diet with Vita-Mix B and 0.6 mg OR-Se/kg diet for a five-week feeding period. Results showed that chicks fed diets supplemented with OR-Se and/or vitamins (C, E) had lower (p < 0.01) rectal-T, growth performance indicators (except feed intake). Also, compared to un-supplemented birds, the carcase yield, digestibility of ether extract, and antioxidant indices were significantly improved. In conclusion, dietary 0.6 mg OR-Se/kg and/or a mixture of vitamins C and E (both at 200 or 250 mg/kg) improved growth performance, carcase yield, nutrient digestibility, and antioxidant capacity under heat-stress conditions. In addition to the previously mentioned results, it would be more reliable when applied to a large number of chicks to ensure data robustness and reliability for productive performance indices.

Highlights

• Adding 0.06 mg of organic selenium plus a mixture of vitamins C and E to the broiler’s diet (per 1 kg diet) improved growth performance.

• Including 0.6 mg of organic selenium plus a mixture of vitamins C and E (per 1 kg diet) in the broiler led to a better average feed conversion ratio throughout the rearing period and boosted the FCR of broiler chicks.

• Vita-Mix plus organic selenium supplementation led to better antioxidant status (GSH-px, SOD, and MDA) of broiler chicks.

Keywords:

• Broiler
• organic selenium
• digestion
• blood biochemical
• haematology

Introduction

Recently, climate change has attracted the attention of many countries worldwide (Cheng et al. 2022; Dwivedi et al. 2022). While the poultry industry contributes to boosting the economies of several countries, poultry farming is a vital source of revenue for small farmers in developing countries, where chicks are commonly raised in open-sided poultry houses (Mottet and Tempio 2017; Karcher and Mench 2018). As a result of continuous selection for higher production of meat chickens, in addition, birds lack sweat glands, thereby they are more susceptible to high-temperature environments (Soleimani et al. 2011; Fathi et al. 2022).

Heat stress (HS) is a serious problem in poultry production, particularly in poultry reared in open-sided housing systems (Kadim et al. 2008). HS impairs growth performance by reducing feed consumption, feed efficiency, body weight gain, meat quality, and physiological and immunological responses (He et al. 2018; Abdel-Moneim et al. 2021). Increased production of reactive oxygen species (ROS) depletes antioxidant reserves, leading to free radical production, which affects the overall performance, including meat quality (Mishra and Jha 2019), damages cells, disrupts intestinal function, and negatively affects biological molecules such as carbohydrates, lipids, proteins, and DNA (Akbarian et al. 2016).

HS causes a reduction in feed intake and an increase in trace mineral excretion; therefore, HS increases the dietary requirements for trace minerals (Marai et al. 2008). Selenium (Se) is a well-known essential trace element that plays an active role in orchestrating multiple metabolic processes in the body. It improves the antioxidant defence system by regulating the expression of several enzymes such as thioredoxin reductases, deiodinases, and glutathione peroxidases, and acts as a gene regulator, which normalises the expression of these enzymes, improves mitochondrial biogenesis to prevent cell death, and protects the body from oxidative stress (Mehta et al. 2012; Elgendey et al. 2022). The supplementation of Se to stressed birds prevents oxidative damage to the pancreatic mucosa and small intestine (Placha et al. 2014). Se supplementation improved the growth performance, antioxidant capacity, and immunity of chicks exposed to high temperatures (Safiullah et al. 2019). Recent research has shown that the organic forms of Se (OR-Se), selenium-yeast or selenomethionine, may be more accessible to birds than inorganic forms (Ibrahim et al. 2019; Gul et al. 2021).

Vitamins C and E are the primary antioxidants that protect lipids and proteins against oxidation (Pisoschi and Pop 2015; Niki 2021). Vitamin C (ascorbic acid) acts as a co-antioxidant to protect cells from oxidative damage and boosts the immune system (Santos-Sánchez et al. 2019). Supplementing stressed broilers with vitamin C mitigates stress metabolic signs, improves growth performance, carcase yield, immunological and antioxidant status, and decreases mortality (Erfani et al. 2021; Gouda et al. 2022). Vitamin E regulates cellular enzymatic activity, protects macrophages, lymphocytes, and plasma cells from oxidative damage, and promotes their survival, proliferation, and function. Consequently, the supplementation of broiler diets with vitamin E under stress conditions enhances immune responses (Calik et al. 2022b).

To the best of our knowledge, many studies have been conducted on the use of Se and vitamins in stressed broilers; however, little is known about the effect when they are combined and their effects on performance, carcase and intestinal characteristics, nutrient digestibility, thyroid hormones, and antioxidant status. Thus, this study aimed to determine the optimal levels of vitamin mixtures (C and E) and/or OR-Se that can improve the growth performance, nutrient digestibility, carcase and intestinal characteristics, and antioxidant status of broiler chicks reared in open-sided broiler houses in high-temperature environments.

Material and methods

The design of this study was directed according to our previous studies that led to the best levels of organic selenium (0.6 or 0.9 mg/kg diet) (Mohamed and Toson 2022) and the positive effect of adding vitamins C and E (Mohamed et al. 2019a,2019b) for broiler chickens, which were reared under heat stress conditions. All studies were approved by the Faculty of Agriculture, Minia University ethics committee under approval No. MU/FA/015/12/22.

Chicks and experimental design

The experiment was conducted on 360 post-hatch Arbour Acres mixed-sex chicks (42.74 ± 1.45 g). All chicks were reared in 18-floor pens (20 chicks/pen), and the floor was littered with fresh wood shavings in an open-sided house for 35 days (the experimental period). Chicks were weighed and randomly allocated to floor pens (2.5 m2 floor area), and the birds were provided with feed and water ad libitum for the five weeks of the experiment. All the chicks were reared under similar conditions. Birds selected for measurements in this study were randomly chosen.

In a factorial design (2 × 3), there were two levels of organic selenium (0 and 0.6 mg/kg of diet), and three levels of vitamins C, E (0 + 0, 0 + 200 + 200, and 0 + 250 + 250 mg/kg of diet, respectively). The experimental treatments (groups) were as follows: The 1st group (T1) had a basal diet with no addition (the control group); the 2^nd group (T2) had a basal diet supplemented with 0.6 mg organic selenium (OR-Se)/kg diet, while OR-Se was derived from yeast Saccharomyces cerevisiae (ALKOSEL® R397, Lallemand Inc., London, United Kingdom); the 3^rd group (T3) had a basal diet with a mixture of 200 mg vitamin C + 200 mg vitamin E/kg diet (Vita-Mix A); the 4th group (T4) had a basal diet with a mixture of 250 mg vitamin C + 250 mg vitamin E/kg diet (Vita-Mix B); the 5th group (T5) had a basal diet with Vita-Mix A + 0.6 mg OR-Se/kg diet; and the 6th group (T6) had a basal diet with Vita-Mix B + 0.6 mg OR-Se/kg diet for a five-week feeding period. Vitamins C (L-ascorbic acid, 96%) and E (tocopherol acetate, 50%) were provided by Megamix (Moscow, Russia).

Uninterrupted light was ensured during the first three days, and then the chicks were exposed to 23 h of light and 1 h of darkness per day. Throughout the trial period, the ambient temperature and relative humidity were recorded daily every 4 h, but ambient temperatures from 12:00 to 17:00 were manually recorded hourly (Figure 1). Chicks were fed common starter (1 to 21 days old) and finisher (22 to 35 days old) diets that met the Arbour Acres guidelines, according to the nutrient specifications of Arbour Acres (Aviagen 2019). The diet compositions of the starter and finisher groups are listed in Table 1. The chemical composition was determined as described by the Association of Official Analytical Chemists (AOAC 2005).

Figure 1. (A And B). Ambient temperature (C°) and relative humidity (%) during the experimental period (1–35 days of age).

Table 1. Ingredient composition and nutrient analysis of the basal diet.

Ingredient, %	Starter (1–21 days)	Finisher (22–35 days)
Yellow corn	52.58	57.98
Soybean meal, 44%	34.50	31.05
Corn gluten meal	5.00	3.00
Sunflower oil	4.00	4.70
Dicalcium phosphate	2.00	1.30
Dl-methionine	0.12	0.12
L-Lysine	0.00	0.31
Limestone	1.20	0.94
Salt	0.30	0.30
Vitamin-mineral premix^a	0.30	0.30
Analytical composition
Dry matter (DM)	92.51	92.88
Crude protein (CP)	22.70	20.89
Crude fibre (CF)	3.65	3.52
Ether extract (EE)	6.25	6.95
Calculated composition
Metabolizable energy (kcal per kg)	3069	3174
Calcium	1.01	0.75
Available phosphorus	0.52	0.38
Methionine + Cystine, digestible	0.88	0.80
Lysine, digestible	1.33	1.12

^a Provided per kilogram of diet: vitamin A, 12,500 IU; vitamin E, 30 IU; vitamin B12, 3 mg; vitamin K3, 2.3 mg; vitamin D3, 4000 IU; B1, 2.2 mg; B3, 65 mg; B2, 8 mg; B6, 4 mg; B5 acid, 24.3 mg; B7, 0.25 mg; folic acid, 1.2 mg; 125.1 mg; choline, 600 mg; Fe, 60 mg; Se, 0.35 mg; Cu, 7.5 mg; Mn, 100 mg; Zn, 110 mg; I, 1.8 mg.

Rectal temperature

The rectal temperature (Rectal-T) of broilers during the last three days of the experiment (33, 34, and 35 days of age) at 12:00, 14:00, and 16:00 was measured daily (three chicks per pen) using a Rectal Temperature Probe (CP100S).

Growth parameters

The survival birds were recorded daily. Live BW and feed intake (FI) were weekly recorded per pen and adjusted for mortality, body weight gain (BWG), and feed conversion ratio (FCR) calculation. The FCR was calculated by dividing FI by BWG.

Carcase and intestinal characteristics

At the end of the experiment (35 days), birds were fasted for 12 h, and three chickens from each pen (nine chicks per treatment) were slaughtered to determine carcase characteristics. The dressed carcase, internal organs (gizzard, heart, liver, and spleen), and abdominal fat weights were determined and presented as a percentage of BW. In addition, the caecal and small intestine (duodenum, jejunum, and ileum) lengths were measured and calculated as grams per 1 kg of broiler BW. Moreover, the relative weights (g per 1 kg of live BW) and pH of digesta (pH metre IT-1101) of these organs were determined.

Nutrient digestibility

At the end of the experiment (35 days), nine chicks from each treatment (three replicates) were housed in separate metabolic cages. Birds were fed experimental diets for four days, and daily measurements of feed intake and excreta were recorded. The excreta were sprayed with 1% boric acid to prevent nitrogen loss due to ammonia release. Proximate analyses of feed and dried excreta were performed according to AOAC (2005). The nitrogen content was determined by separating the faecal protein fraction from the excreta samples (Jakobsen et al. 1960).

Haematological and biochemical measurements

On day 35, three birds per replicate (pen) were randomly selected, and blood samples were collected from the wing vein into two sterile tubes with heparin. Red blood cells (RBC) and white blood cells (WBC) were counted using a haemocytometer in the first tube (Campbell 1995). Blood haemoglobin (Hb) and packed cell volume (PCV) were estimated using cyanomethemoglobin and micro-hematocrit centrifuges, respectively (Eilers 1967; Daice and Lewis 1991). In the second tube, blood samples were centrifuged at 3,000 xg for 15 min, and the plasma was separated and stored at −20 °C until further analysis. For the plasma biochemical index assay, the plasma within the replicates was pooled and analysed in duplicates.

Plasma triiodothyronine (T3) and thyroxine (T4) concentrations were determined via radioimmunoassay procedures described by Chopra et al. (1971) and kits purchased from Diagnostic Products Corporation (Los Angeles, CA, USA).

Plasma glutathione peroxidase (GSH-px), superoxide dismutase (SOD), and malondialdehyde (MDA) levels were measured using commercial kits from Diagnostic, Egypt.

Statistical analysis

Statistical analysis of the results obtained was performed using SAS software version 9.1.3, (SAS Institute, 2003), and the data were analysed using a two-way factorial design. The results are presented as the mean ± SE. The significance of the mean differences was calculated using Duncan’s test (Duncan 1955). Statistical significance was set at p < 0.05.

The statistical model was as the following equation: $${\mathrm{Y}}_{\text{ij}}\text{k}=\mu +T_{\mathrm{i}}+{\mathrm{E}}_{\mathrm{j}}+T_{\mathrm{i}}{\mathrm{E}}_{\mathrm{j}}+{\mathrm{e}}_{\text{ijk}}$$ with i = 0, 0.6 and j = 0 + 0, 200 + 200, 250 + 250.

Where

Yij = mean of the experimental unit factor i and factor j.

μ = constant inherent to all experimental units.

T_i=  effect of level OR-Se of factor T.

E_j= effect of the levels of a mixture of vitamin C and E.

T_iE_j = effect of interaction between OR-Se and mixture of vitamins

e_ijk = error experimental unit factor i and factor j.

Results

Rectal temperature (rectal-T)

The individual and combined effects of OR-Se and/or a mixture of vitamins C and E on rectal-T in broiler chicks are shown in Table 2. Dietary supplementation with OR-Se and a mixture of vitamins C and E significantly decreased (p < 0.01) the rectal-T of stressed birds, but the interaction effect (at 14:00) had no effect (p > 0.05), compared to those in the control group.

Table 2. Effect of dietary supplementation with organic selenium (or-Se), vitamin C and E mixture on rectal temperature (C°) of stressed broiler chicks.

Treatments	Time/day
	12:00 pm	14:00 pm	16:00 pm
A) Effect of Organic selenium (OR-Se)
–	41.31^a	42.24^a	42.06^a
+	41.20^b	42.04^b	41.79^b
SEM	0.020	0.029	0.024
p-value	0.001	<.0001	<.0001
B) Effect of Vita-Mix
-	41.43^a	42.32^a	42.03^a
+ Vita-Mix A	41.22^b	42.06^b	41.87^b
+ Vita-Mix B	41.12^c	42.05^b	41.88^b
SEM	0.025	0.035	0.030
p-value	<.0001	<.0001	0.001
A*B) Effect of interaction between OR-Se and Vita-Mix
T1	41.45^a	42.43	42.10^a
T2	41.42^a	42.20	41.95^b
T3	41.38^a	42.15	42.07^ab
T4	41.10^b	42.15	42.00^ab
T5	41.05^b	41.97	41.67^c
T6	41.13^b	41.95	41.75^c
SEM	0.035	0.050	0.042
p-value	<.0001	0.879	0.020

T1= basal diet, T2= basal diet + 0.6 mg OR-Se, T3= basal diet + Vita-Mix A, T4= basal diet + Vita-Mix B, T5= basal diet plus 0.6 mg OR-Se + Vita-Mix A, T6= basal diet plus 0.6 mg OR-Se + Vita-Mix B. Vita-Mix A = mixture of 200 mg vitamin C + 200 mg vitamin E/kg diet, Vita-Mix B = mixture of 250 mg vitamin C + 250 mg vitamin E/kg diet, ^{a, b, c} means with different letters in the same column are significantly different at (p < 0.05). SEM, standard error of the mean.

Survival rate

There were no dead chicks during the first three weeks of the age (1–21 days) between the experimental treatments. However, adding OR-Se and vitamins C, E (individually or in combination) had no significant effect on the survival rate of stressed broilers (Table 3). Also, the survival rate was found to be higher (p > 0.05) in these supplemented birds during d 22 to 35.

Table 3. Effect of dietary supplementation with organic selenium (or-Se, vitamin C and E mixture on the survival rate of stressed broiler chicks.

Treatments	Period/day
	22–28	29–35	1–35
A) Effect of Organic selenium (OR-Se)
–	98.89	93.83	92.78
+	99.44	93.86	93.33
SEM	0.68	0.78	0.96
p-value	0.4983	0.2403	0.0614
B) Effect of Vita-Mix
-	99.17	93.29	92.50
+ Vita-Mix A	100.00	93.33	93.33
+ Vita-Mix B	98.33	94.91	93.33
SEM	0.83	0.96	1.18
p-value	0.6405	0.4481	0.6241
A*B) Effect of interaction between OR-Se and Vita-Mix
T1	98.33	93.25	91.67
T2	100.00	93.33	93.33
T3	100.00	93.33	93.33
T4	98.33	94.91	93.33
T5	100.00	93.33	93.33
T6	98.33	94.91	93.33
SEM	1.18	1.35	1.67
p-value	0.7013	0.8569	0.9701

T1: basal diet, T2: basal diet + 0.6 mg OR-Se, T3: basal diet + Vita-Mix A, T4: basal diet + Vita-Mix B, T5: basal diet plus 0.6 mg OR-Se + Vita-Mix A, T6: basal diet plus 0.6 mg OR-Se + Vita-Mix B. Vita-Mix A: mixture of 200 mg vitamin C + 200 mg vitamin E/kg diet, Vita-Mix B: mixture of 250 mg vitamin C + 250 mg vitamin E/kg diet, statistical significance was set at p < 0.05. SEM, standard error of the mean.

Growth performance

Table 4 presents the growth parameters (BW, BWG, FI, and FCR) as affected by supplementation with OR-Se and/or a mixture of vitamins (C and E). Regarding, the individual and the interaction effects of adding OR-Se and different levels of a mixture of vitamins to stressed broiler diets, there was an improvement (p < 0.05) in the growth parameters, except for FI. In particular, the effect of the interaction between OR-SE and different levels of vitamins (low and high) showed the highest BW, BWG, and lowest FCR compared to the control group throughout the entire period.

Table 4. Effect of dietary supplementation with organic selenium (or-Se), vitamin C and E mixture on growth performance of heat stressed broilers.

Treatments	Parameters
	Body weight, (g)		Body weight gain, (g)/day of age/Period			Feed intake, (g)/day of age Period			Feed conversion ratio (g)/day of age/period
	21	35	1–21	22–35	1–35	1–21	22–35	1–35	1–21	22–35	1–35
A) Effect of organic selenium (OR-Se)
–	840.58^b	1817.05^b	797.50^b	976.47^b	1773.97^b	1086.57	1900.68	2987.25	1.37	1.95^a	1.69^a
+	870.37^a	1906.73^a	827.96^a	1036.36^a	1864.33^a	1094.86	1916.55	3011.41	1.32	1.85^b	1.62^b
SEM	8.18	10.34	8.14	3.36	10.33	16.31	28.68	40.90	0.018	0.027	0.022
p-value	0.024	<.0001	0.021	<.0001	<.0001	0.725	0.702	0.684	0.104	0.025	0.043
B) Effect of Vita-Mix
–	830.86^b	1782.58^b	788.46^b	951.72^c	1740.18^b	1099.23	1875.04	2974.27	1.40^a	1.97	1.71^a
+ Vita-Mix A	863.62^a	1907.59^a	820.85^a	1043.97^a	1864.82^a	1096.05	1946.83	3042.88	1.34^ab	1.87	1.64^ab
+ Vita-Mix B	871.94^a	1895.51^a	828.89^a	1023.57^b	1852.46^a	1076.87	1903.99	2980.86	1.30^b	1.86	1.61^b
SEM	10.02	12.67	9.97	4.11	12.66	19.97	35.12	50.09	0.022	0.033	0.027
p-value	0.031	<.0001	0.033	<.0001	<.0001	0.700	0.378	0.580	0.029	0.061	0.052
A*B) Effect of interaction between OR-Se and Vita-Mix
T1	809.51^b	1719.08^c	766.61^b	909.58^d	1676.19^c	1102.82	1871.83	2974.65	1.44^a	2.06^a	1.77^a
T2	852.22^ab	1846.08^b	810.31^ab	993.86^c	1804.17^b	1095.65	1878.25	2973.89	1.35^ab	1.89^b	1.65^b
T3	845.53^ab	1869.77^b	802.59^ab	1024.24^b	1826.83^b	1095.94	1966.72	3062.65	1.37^ab	1.92^ab	1.68^ab
T4	866.70^a	1862.30^b	823.30^a	995.60^c	1818.90^b	1060.95	1863.50	2924.46	1.29^b	1.87^b	1.61^b
T5	881.70^a	1945.40^a	839.10^a	1063.70^a	1902.80^a	1096.16	1926.94	3023.10	1.30^b	1.81^b	1.59^b
T6	877.18^a	1928.72^a	834.47^a	1051.54^a	1886.01^a	1092.79	1944.47	3037.26	1.31^b	1.85^b	1.61^b
SEM	14.17	17.91	14.10	5.82	17.90	28.25	49.67	70.84	0.031	0.047	0.038
p-value	0.035	<.0001	0.034	<.0001	<.0001	0.769	0.492	0.552	0.047	0.046	0.048

T1: basal diet, T2: basal diet + 0.6 mg OR-Se, T3: basal diet + Vita-Mix A, T4: basal diet + Vita-Mix B, T5: basal diet plus 0.6 mg OR-Se + Vita-Mix A, T6: basal diet plus 0.6 mg OR-Se + Vita-Mix B, Vita-Mix A: mixture of 200 mg vitamin C + 200 mg vitamin E/kg diet, Vita-Mix B: mixture of 250 mg vitamin C + 250 mg vitamin E/kg die ^{a, b, c} means with different letters in the same column are significantly different at (p < 0.05). SEM, standard error of the mean.

Carcase traits

The effects of dietary supplementation with feed additives (OR-Se and vitamins), either individually or in combination, on the carcase yield and organ percentage of broilers under summer conditions are shown in Table 5. Adding OR-Se with different levels of vitamins (C, E) significantly improved carcase yield, but did not differ (p > 0.05) in the other carcase characteristics (gizzard, liver, heart, abdominal fat, and spleen percentages). Broiler chickens fed diets supplemented with OR-Se and vitamins (T5, T6) had higher values in the carcase yield (%) than other treatments.

Table 5. Effect of dietary supplementation with organic selenium (or-Se), vitamin C and E mixture on carcase traits of heat-stressed broilers.

Treatments	LBW, (g)	%
		Carcase yield	Gizzard	Liver	Heart	Abdominal fat	Spleen
A) Effect of Organic selenium (OR-Se)
–	1827.23^b	71.99^b	1.88	1.831	0.5011	1.325^a	0.152^b
+	1908.55^a	72.99^a	1.93	1.839	0.5263	1.174^b	0.165^a
SEM	7.343	0.069	0.024	0.0172	0.009	0.029	0.003
p-value	<.0001	<.0001	0.166	0.773	0.056	0.001	0.012
B) Effect of Vita-Mix
-	1789.87^b	71.66^b	1.901	1.826	0.506	1.392^a	0.152
+ Vita-Mix A	1908.19^a	72.93^a	1.924	1.859	0.520	1.125^b	0.162
+ Vita-Mix B	1905.61^a	72.89^a	1.899	1.819	0.515	1.233^c	0.162
SEM	8.994	0.085	0.029	0.021	0.011	0.036	0.004
p-value	<.0001	<.0001	0.804	0.362	0.673	<.0001	0.145
A*B) Effect of interaction between OR-Se and Vita-Mix
T1	1727.99^c	70.72^c	1.85	1.80	0.49	1.50	0.14
T2	1851.75^b	72.59^b	1.95	1.85	0.52	1.29	0.16
T3	1874.23^b	72.59^b	1.91	1.88	0.50	1.22	0.15
T4	1879.45^b	72.67^b	1.89	1.81	0.52	1.26	0.16
T5	1942.14^a	73.26^a	1.94	1.84	0.54	1.03	0.17
T6	1931.76^a	73.11^a	1.91	1.82	0.51	1.20	0.16
SEM	12.72	0.12	0.04	0.03	0.02	0.05	0.01
p-value	0.018	<.0001	0.536	0.372	0.154	0.294	0.465

T1: basal diet, T2: basal diet + 0.6 mg OR-Se, T3: basal diet + Vita-Mix A, T4: basal diet + Vita-Mix B, T5: basal diet plus 0.6 mg OR-Se + Vita-Mix A, T6: basal diet plus 0.6 mg OR-Se + Vita-Mix B, Vita-Mix A: mixture of 200 mg vitamin C + 200 mg vitamin E/kg diet, Vita-Mix B: mixture of 250 mg vitamin C + 250 mg vitamin E/kg die, ^{a, b, c} means with different letters in the same column are significantly different at (p < 0.05). SEM, standard error of the mean.

In the same trend, the individual effect of OR-Se and vitamins (high and low levels) led to an increase in carcase yield (%) and a reduction (p < 0.05) in abdominal fat deposition (%). Nevertheless, there were no significant changes in the gizzard, liver, heart, and spleen percentages of supplemented birds, compared to the control.

Intestinal characteristics

As presented in Table 6, supplemental OR-Se or different levels of the vitamin mixture (Vita-Mix A and Vita-Mix B) significantly altered the intestinal characteristics (weight, length, and pH) of stressed birds; however, the interaction was not significant. When stressed chicks were fed diets containing OR-Se or a vitamin C and E mixture, the weights of the duodenum and jejunum increased (p < 0.05), but the weights of the ileum and caeca did not change (p > 0.05) when compared to those of the un-supplemented chicks (control).

Table 6. Effect of dietary supplementation with organic selenium (or-Se), vitamin C and E mixture on intestinal characteristics of heat-stressed broilers.

Treatments	Weight, (g)				Length, (cm)				pH
	Duodenum	Jejunum	Ileum	Caeca	Duodenum	Jejunum	Ileum	Caeca	Duodenum	Jejunum	Ileum	Caeca
A) Effect of organic selenium (OR-Se)
–	6.12^b	11.40^b	8.82	2.07	17.70	38.48	29.87	3.81	5.73^a	5.99^a	5.88^a	6.07^a
+	6.42^a	11.60^a	8.98	2.15	17.80	38.89	30.59	3.88	5.60^b	5.80^b	5.70^b	5.91^b
SEM	0.068	0.056	0.110	0.035	0.191	0.220	0.478	0.063	0.047	0.041	0.041	0.037
p-value	0.003	0.018	0.333	0.083	0.713	0.201	0.291	0.460	0.049	0.003	0.004	0.004
B) Effect of Vita-Mix
-	6.08^b	11.29^b	8.72	2.08	17.71	38.43	29.48	3.70^b	5.61	5.90	5.81	6.07
+ Vita-Mix A	6.37^a	11.67^a	9.04	2.14	17.96	38.71	30.63	3.86^ab	5.69	5.89	5.73	5.93
+ Vita-Mix B	6.37^a	11.54^a	8.94	2.11	17.59	38.91	30.58	3.97^a	5.69	5.90	5.83	5.97
SEM	0.084	0.069	0.135	0.061	0.234	0.270	0.586	0.078	0.057	0.050	0.051	0.046
p-value	0.023	0.001	0.256	0.659	0.529	0.460	0.298	0.053	0.543	0.984	0.335	0.118
A*B) Effect of Interaction between OR-Se and Vita-Mix
T1	5.88	11.15	8.73	2.06	17.61	38.02	29.07	3.64	5.69	6.02	5.97	6.20
T2	6.27	11.43	8.72	2.11	17.80	38.84	29.89	3.76	5.53	5.78	5.66	5.93
T3	6.27	11.61	8.89	2.07	17.82	38.57	30.47	3.86	5.73	5.99	5.81	5.99
T4	6.20	11.46	8.85	2.07	17.68	38.86	30.08	3.93	5.77	5.96	5.86	6.02
T5	6.47	11.74	9.18	2.20	18.10	38.84	30.80	3.87	5.64	5.79	5.64	5.88
T6	6.53	11.62	9.02	2.15	17.51	38.97	31.09	4.01	5.61	5.84	5.80	5.92
SEM	0.119	0.097	0.191	0.061	0.331	0.381	0.829	0.110	0.081	0.071	0.072	0.065
p-value	0.725	0.724	0.734	0.818	0.893	0.625	0.915	0.898	0.778	0.635	0.212	0.364

T1: basal diet, T2: basal diet + 0.6 mg OR-Se, T3: basal diet + Vita-Mix A, T4: basal diet + Vita-Mix B, T5: basal diet plus 0.6 mg OR-Se + Vita-Mix A, T6: basal diet plus 0.6 mg OR-Se + Vita-Mix B, Vita-Mix A: mixture of 200 mg vitamin C + 200 mg vitamin E/kg diet, Vita-Mix B: mixture of 250 mg vitamin C + 250 mg vitamin E/kg die, ^{a, b, c} means with different letters in the same column are significantly different at (p < 0.05). SEM, standard error of the mean.

There were no significant differences in the lengths of the intestinal parts (duodenum, jejunum, ileum, and caeca) as influenced by the individual effects of OR-Se or a mixture of vitamins, with one exception: supplemental Vita-Mix B significantly increased (p < 0.05) the caeca length. Supplementation with OR-Se alone significantly decreased the pH of the intestinal parts; but adding a vitamin mixture did not change (p > 0.05) the pH of these parts compared with those of the control.

Nutrient digestibility

Nutrient digestibility (DM, CP, EE, CF, and NFE) affected by dietary supplementation (OR-Se and/or vitamins) in stressed broilers is shown in Table 7. The combined effects of OR-Se and/or vitamins C and E (Vita-Mix A, Vita-Mix B) improved (p < 0.05) EE digestibility. Furthermore, chicks fed diets with OR-Se and lower levels of vitamins C, E (Vita-Mix A) had higher EE digestibility (p < 0.05) than the chicks in other groups. However, the interaction effect had no changes (p > 0.05) in CP, DM, CF, and NFE compared with the un-supplemented group.

Table 7. Effect of dietary supplementation with organic selenium (or-Se), vitamin C and E mixture on nutrient digestibility of heat-stressed broilers.

Treatments	%
	Dry matter (DM)	Crude protein (CP)	Ether extract (EE)	Crude Fibre (CF)	Nitrogen-free extract (NFE)
A) Effect of organic selenium (OR-Se)
–	78.01	87.86^b	81.54^b	30.23	79.09
+	78.21	88.91^a	83.47^a	31.69	79.20
SEM	0.391	0.192	0.338	1.21	0.458
p-value	0.723	0.002	0.002	0.450	0.869
B) Effect of Vita-Mix
–	77.76	87.87^b	81.73^b	30.04	78.87
+ Vita-Mix A	79.06	88.99^a	83.57^a	32.84	80.23
+ Vita-Mix B	77.52	88.30^ab	82.22^b	30.01	78.33
SEM	0.479	0.235	0.414	1.480	0.560
p-value	0.090	0.018	0.023	0.331	0.086
A*B) Effect of interaction between OR-Se and Vita-Mix
T1	77.33	87.00	80.12^c	29.14	78.40
T2	78.20	88.73	83.34^ab	30.93	79.35
T3	78.76	88.39	82.13^b	31.73	79.79
T4	77.95	88.19	82.38^b	29.83	79.08
T5	79.35	89.58	85.01^a	33.97	80.66
T6	77.09	88.41	82.06^b	30.18	77.58
SEM	0.678	0.333	0.585	2.093	0.792
p-value	0.420	0.112	0.020	0.896	0.254

T1: basal diet, T2: basal diet + 0.6 mg OR-Se, T3: basal diet + Vita-Mix A, T4: basal diet + Vita-Mix B, T5: basal diet plus 0.6 mg OR-Se + Vita-Mix A, T6: basal diet plus 0.6 mg OR-Se + Vita-Mix B, Vita-Mix A: mixture of 200 mg vitamin C + 200 mg vitamin E/kg diet, Vita-Mix B: mixture of 250 mg vitamin C + 250 mg vitamin E/kg die, ^{a, b, c} means with different letters in the same column are significantly different at (p < 0.05). SEM, standard error of the mean.

Regarding the individual effect, supplementation with OR-Se or a vitamin mixture improved (p < 0.01) CP and EE digestibility. The supplementation of OR-Se or lower levels of vitamins C and E (Vita-Mix A) individually to stressed broiler diets improved (p < 0.05) CP and EE digestibility, but higher levels of vitamins C and E (Vita-Mix B) had no significant effect on CP digestibility.

Haematological blood parameters

Red blood cells, WBC, Hb, and PCV parameters of broilers fed organic Se and/or a mixture of vitamins C and E in a high-temperature environment are shown in Table 8. The interaction effects of OR-Se and vitamins significantly increased PCV but had no effect (p > 0.05) on RBCs, Hb, and WBC values.

Table 8. Effect of dietary supplementation with organic selenium (or-Se), vitamin C and E mixture on haematological parameters of heat-stressed broilers.

Treatments	RBCs,10^6/mm^3	WBCs,10^3/mm^3	PCV, %	Hb, g/dL
A) Effect of organic selenium (OR-Se)
–	2.89^b	20.22	30.37^b	9.05^b
+	3.02^a	20.31	30.92^a	9.36^a
SEM	0.033	0.060	0.178	0.090
p-value	0.007	0.334	0.035	0.017
B) Effect of Vita-Mix
–	2.86^b	20.27	30.04^b	8.80^b
+ Vita-Mix A	2.99^a	20.26	31.11^a	9.40^a
+ Vita-Mix B	3.01^a	20.26	30.78^a	9.42^a
SEM	0.040	0.074	0.219	0.110
p-value	0.028	0.989	0.004	0.0002
A*B) Effect of interaction between OR-Se and Vita-Mix
T1	2.76	20.16	29.32^b	8.53
T2	2.96	20.38	30.76^a	9.07
T3	2.93	20.24	31.06^a	9.24
T4	2.97	20.27	30.73^a	9.38
T5	3.06	20.28	31.15^a	9.56
T6	3.04	20.26	30.94^a	9.46
SEM	0.057	0.104	0.309	0.155
p-value	0.5279	0.5092	0.0495	0.3464

T1: basal diet, T2: basal diet + 0.6 mg OR-Se, T3: basal diet + Vita-Mix A, T4: basal diet + Vita-Mix B, T5: basal diet plus 0.6 mg OR-Se + Vita-Mix A, T6: basal diet plus 0.6 mg OR-Se + Vita-Mix B, Vita-Mix A: mixture of 200 mg vitamin C + 200 mg vitamin E/kg diet, Vita-Mix B: mixture of 250 mg vitamin C + 250 mg vitamin E/kg die, ^{a, b, c} means with different letters in the same column are significantly different at (p < 0.05). SEM: standard error of the mean, RBCs: Red blood cells, WBCs: White blood cells, PCV: Packed cell volume, Hb: Hemoglobin..

Individual effects of organic Se and vitamins C and E supplementation markedly increased RBCs, PCV, and Hb levels; nevertheless, WBC levels did not change (p > 0.05) compared to that of the control.

Thyroid hormones

As shown in Table 9, the individual effects of feed additives (OR-Se and vitamins) in stressed broiler diets resulted in a significant increase in serum T3; however, they did not change (p > 0.05) serum T4 levels, and their interaction effect was not significant on serum T3 or T4 compared with that of the control group.

Table 9. Effect of dietary supplementation with organic selenium (or-Se), vitamin C and E mixture on thyroid hormones (T3, T4) of heat-stressed broiler chicks.

Treatments	Triiodothyronine ((T3), ng/mL))	Thyroxine (T4), ng/mL)
A) Effect of organic selenium (OR-Se)
–	3.85^b	18.75
+	4.32^a	19.17
SEM	0.066	0.298
p-value	0.0003	0.340
B) Effect of Vita-Mix
–	3.76^b	18.55
+ Vita-Mix A	4.29^a	18.88
+ Vita-Mix B	4.21^a	19.46
SEM	0.080	0.365
p-value	0.001	0.247
A*B) Effect of interaction between OR-Se and Vita-Mix
T1	3.50	18.31
T2	3.99	18.61
T3	4.07	19.35
T4	4.02	18.80
T5	4.59	19.15
T6	4.35	19.57
SEM	0.114	0.517
p-value	0.387	0.946

T1: basal diet, T2: basal diet + 0.6 mg OR-Se, T3: basal diet + Vita-Mix A, T4: basal diet + Vita-Mix B, T5: basal diet plus 0.6 mg OR-Se + Vita-Mix A, T6: basal diet plus 0.6 mg OR-Se + Vita-Mix B, Vita-Mix A: mixture of 200 mg vitamin C + 200 mg vitamin E/kg diet, Vita-Mix B: mixture of 250 mg vitamin C + 250 mg vitamin E/kg die, ^{a, b, c} means with different letters in the same column are significantly different at (p < 0.05). SEM, standard error of the mean.

Antioxidant activity

As shown in Table 10, the interaction effect of OR-Se and vitamins (C and E) supplementation in broiler diets significantly enhanced (p < 0.05) SOD and MDA values; however, had no effect (p > 0.05) on serum GSH-px. Additionally, chicks fed diets with 0.6 mg OR-Se and a mixture of 200 mg vitamin C + 200 mg vitamin E/kg diet (Vita-Mix A) had higher levels of GSH-px and SOD and lower levels (p < 0.05) of MDA, when compared with the other treatments and the control.

Table 10. Effect of dietary supplementation with organic selenium (or-Se), vitamin C and E mixture on serum antioxidant activity of heat-stressed broiler chicks.

Treatments	Glutathione peroxidase (U/ml)	Superoxide dismutase (U/ml)	Malondialdehyde (nmol/mL)
A) Effect of organic selenium (OR-Se)
–	23.50^b	135.40^b	2.43^a
+	25.51^a	148.45^a	2.02^b
SEM	0.297	1.300	0.033
p-value	0.001	<.0001	<.0001
B) Effect of Vita-Mix
–	23.72^b	134.70^b	2.50^a
+ Vita-Mix A	25.15^a	146.36^a	2.06^b
+ Vita-Mix B	24.65^ab	144.72^a	2.10^b
SEM	0.364	1.593	0.040
p-value	0.047	0.001	<.0001
A*B) Effect of interaction between OR-Se and Vita-Mix
T1	22.37	126.22^d	2.68^a
T2	25.06	143.17^bc	2.33^b
T3	24.02	137.33^c	2.38^b
T4	24.11	142.66^bc	2.23^b
T5	26.28	155.40^a	1.75^d
T6	25.18	146.78^b	1.97^c
SEM	0.514	2.252	0.056
p-value	0.301	0.016	0.019

T1: basal diet, T2: basal diet + 0.6 mg OR-Se, T3: basal diet + Vita-Mix A, T4: basal diet + Vita-Mix B, T5: basal diet plus 0.6 mg OR-Se + Vita-Mix A, T6: basal diet plus 0.6 mg OR-Se + Vita-Mix B, Vita-Mix A: mixture of 200 mg vitamin C + 200 mg vitamin E/kg diet, Vita-Mix B: mixture of 250 mg vitamin C + 250 mg vitamin E/kg die, ^{a, b, c} means with different letters in the same column are significantly different at (p < 0.05). SEM, standard error of the mean.

Similarly, the individual effect of these feed additives significantly increased (p < 0.05) the levels of GSH-px and SOD and linearly decreased the MDA values, compared to that of the un-supplemented treatment (control).

Discussion

Dietary supplementation with OR-Se and vitamins (C, E) had no significant effect on the survival rate. A similar trend was documented by Pardechi et al. (2020) who indicated that adding sodium selenite, selenised yeast, and nanoelemental selenium (at three levels 0.1, 0.2, and 0.5 mg/kg) to broiler diets did not change clearly (p < 0.05) survival rates of these birds compared with un-supplemented birds. In contrast, Calik et al. (2022a) documented that, chicks fed diets supplemented with 250 mg of vitamin E and 1 mg OR-Se had a lower mortality rate (1.92% vs. 7.01%), compared with others in the un-supplemented group. In the present study, chicks exposed to a high temperature (up to 33.1 °C) during the afternoon (higher environmental temperature than optimum ambient temperatures) had higher rectal temperatures (up to 42.15 °C) compared with that of chicks reared in normal cases, while Zampiga et al. (2021) demonstrated that chicks raised in thermoneutral conditions (< 22 °C, after 21 days of age) recorded up to 41.3 °C. However, the addition of OR-Se and/or a mixture of vitamins (C and E) significantly decreased rectal-T and mortality rate compared to that of the control (T1). These findings are in agreement with those of Shakeri et al. (2018), who indicated that the supplementation of vitamin E (250 mg of dl-α-tocopheryl acetate kg/diet) with OR-Se (0.3 to 0.8 mg of selenised yeast/kg) significantly decreased rectal-T of chicks exposed to HS. These results show that the supplemented chicks could adequately thermoregulate and dissipate the extra heat. Rectal-T under HS conditions is a good indicator of heat resistance in broiler chickens (Chen et al. 2013).

Adding OR-Se with or without graded levels of vitamins (C, E) to stressed broiler diets improved growth performance indicators, the percentages of carcase and spleen, and reduced (p < 0.05) abdominal fat; however, FI and the percentages of gizzard, liver, and heart did not differ compared with those of the un-supplemented group. Similarly, chicks subjected to HS and fed diets with 250 mg/kg Vit E and 1 mg/kg OR-Se had significantly greater BW, BWG, FCR, and carcase yield throughout the experiment (1 to 35 days of age) compared to those of the control (Calik et al. 2022a). In contrast, Habibian et al. (2016) indicated that supplementation with varying doses of vitamin E (α-tocopherol acetate, 125 and 250 mg/kg) and/or OR-Se (selenomethionine, 0.5 and 1 mg/kg) had no effect (p > 0.05) on growth parameters of broilers under HS. Furthermore, dietary supplementation with vitamins C and E (200 and 100 mg/kg, respectively) did not affect the carcase characteristics (carcase yield, abdominal fat, liver, gizzard, and heart) of chicks exposed to chronic HS at 25 to 42 days of age (Attia et al. 2017). The current findings suggest that the positive effects of OR-Se and a mixture of vitamins (C and E) on stressed broilers may be because of their effects on stimulating the thyroid gland, which improves feed efficiency, reflecting BW and BWG, which may be attributable to the antioxidant role of Se and vitamins against HS effects, as described in more recent studies (Elgendey et al. 2022; Gouda et al. 2022; Calik et al. 2022b).

In the current study, the individual effects of dietary OR-Se or different levels of the vitamin mixture (C, E) in stressed broilers had a significant effect (p < 0.05) on the weight of intestinal organs (duodenum, jejunum, ileum, caeca) in contrast, had no effect on length of these organs (except caeca significantly increased by Vita-Mix B addition). Also, the pH of these intestinal organs markedly decreased by OR-Se addition alone but, the individual effect of a mixture of vitamins had no effect on it, compared with those of the control group. According to Wang et al. (2021), HS (33 ± 1 °C) decreased the jejunum weight (g/kg) and length of the ileum and jejunum (cm/kg) in broiler chickens during the experiment (21 to 42 days of age). In contrast, Rezvani and Shojaee (2021) found that supplementing 1 ml/L of vitamin E-selenium to drinking water increased (p < 0.05) intestinal length but had no significant effect on intestinal weight. HS increases levels of cholecystokinin, which helps increase intestinal motility in the duodenum and jejunum, increasing the digesta transit rate that is reflected in nutrient digestibility (incomplete digestion) and a decrease in the amount of nutrients that may be fermented in the caeca (Mazzoni et al. 2022). In the current study, this may explain the positive effect of adding OR-Se and/or a vitamin mixture to broiler diets under summer conditions by improving the weight of the duodenum and jejunum compared with that seen with the non-supplemented treatment. In addition, supplemented birds had a lower pH in the ileum and caeca, which can improve the environment for some resident microorganisms and reduce pathogen colonisation (Abd El-Hack et al. 2020).

Supplementation of stressed broilers with OR-Se or a vitamin mixture improved (p < 0.01) CP and EE digestibility, but their interaction effect had no effect (p > 0.05) on the nutrient digestibility (except EE). Similar to the current findings, supplementation with vitamins C and E (200 and 150 mg/kg, respectively) linearly improved CP digestibility in laying hens exposed to chronic HS (Attia et al. 2016). However, adding vitamin C (50 mg/L) or a mixture of vitamin E and Se (1 ml/L) to drinking water during HS had no significant effect on DM, CP, and EE digestibility in broilers (Rezvani and Shojaee 2021). The negative effects of HS (32 °C) on nutrient digestibility by decreasing the activities of amylase, chymotrypsin, and trypsin in stressed broilers can lead to decreased CP digestibility (Hai et al. 2000). According to the results of this experiment, the positive effects of supplementation with OR-Se and/or a mixture of vitamins on nutrient digestibility can be seen in an improvement in feed efficiency, which is reflected in growth performance.

Under summer conditions, birds fed supplemented diets exhibited a significant improvement in haematological parameters, except for WBC, when compared to those with un-supplemented diets. In turn, HS causes an imbalance in protein homeostasis within the cell and negatively affects cytokine production, leading to cell death (Greene et al. 2022). Gouda et al. (2015) indicated that broilers fed diets containing Se (0.03 mg/kg) or vitamin E (200 IU/kg diet) had increased RBC, Hb, and PCV values during the summer season. Therefore, based on our results, we suggest that the effects of OR-SE and/or vitamins C and E on stressed broilers could be attributed to their antioxidant properties and ability to protect cells from oxidative stress and improve the function and proliferation of these cells (Abdel-Moneim et al. 2021).

The individual effects of dietary OR-Se or graded levels of vitamins increased serum T3 levels but did not affect serum T4 levels. In addition, the interaction effect of these additives had no noticeable impact on the T3 or T4 levels compared to that of the control. These results are consistent with those noted by Abdel-Moneim et al. (2022), who showed that supplementing Se nanoparticles at levels of 0.1 or 0.2 mg/kg diet for stressed broilers significantly increased T3. HS causes negative changes in the plasma levels of corticosterone and T3, which has negative effects on T3 expression in the pituitary thyrotropin (Beckford et al. 2020). Our findings may be attributable to the positive effects of both OR-Se and vitamins C and E individually and in combination, whereas Lin et al. (2014) reported that selenoproteins play an essential role in the metabolism of thyroid hormones and can affect the conversion of T4 to T3. In addition, supplementation with antioxidant vitamins under HS conditions for 35 days contributed to protection against oxidative damage (Gouda et al. 2020).

GSH-px and SOD levels were significantly increased by OR-Se and/or vitamin C and E supplementation in stressed broiler diets; however, MDA was markedly decreased compared to the control’s. The same trend was observed by Kumbhar et al. (2018), who found that dietary Se and/or vitamin E supplementation caused a linear increase in GSH-px and SOD and a decrease in MDA in broilers exposed to high temperatures. In our study, the improvement in antioxidant indices may be attributed to the synergistic effect of Se and vitamins C and E, which inhibit the production of free radicals and have a greater effect as inhibitors of lipid peroxidation, thereby protecting cell constituents from oxidative damage under HS conditions (Jang et al. 2014; Malyar et al. 2021). Future research should be able to take advantage of some limitations of our study. A larger number of birds is needed to expand the results in particular of productive performance. Nevertheless, the present study also has several strengths, for example, it is not a limitation that e prioritised a wide range of studied parameters rather than large numbers of birds in each treatment.

Conclusions

The supplementation of OR-Se (0.6 mg/kg), Vita-Mix A, Vita-Mix B, or their combinations enhanced rectal-T, growth performance, carcase yield, and blood constituents of broiler chickens under summer conditions. These results revealed that chicks derived greater benefits from supplemental diets when they were provided in combination, possibly because of the synergistic effects of these antioxidants. Furthermore, this study suggests the potential use of OR-Se and a mixture of vitamins C and E to alleviate the adverse effects of HS and improve performance and physiological states.

Ethical Approval

All studies were approved by the Faculty of Agriculture, Minia University ethics committee under approval No. MU/FA/015/12/22.

Acknowledgments

The authors extend their appreciation to the Deputyship for Research and Innovation, “Ministry of Education” in Saudi Arabia for funding this research work through project no. (IFKSUOR3-560-2).

Disclosure statement

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

Data availability statement

The data that support the findings of this study are available on request from the corresponding author.

Additional information

Funding

This work was funded by the Deputyship for Research and Innovation, “Ministry of Education” in Saudi Arabia, project no. [IFKSUOR3-560-2].