Effects of black cumin, fenugreek, and sesame seeds as a mixture on performance, intestinal morphology, and blood traits of broilers under chronic heat stress conditions

Abstract

This study aimed to investigate the impact of a seed mixture (SM) (black cumin, fenugreek, and sesame) on broiler chickens’ performance, intestinal morphology, and blood traits under chronic heat stress conditions. 330-day-old male Ross 308 chicks were divided into five treatments (6 replicates and 11 birds per each) consisting of a corn-soybean supplemented with 0, 1.5, 3, 4.5, and 6% SM for 45 days in a completely randomised design. 6% SM supplementation increased body weight (BW) at 45 days of age, average weight gain (AWG) at 26-45 days, and total rearing period under heat stress conditions (p < 0.05). Adding 3% SM to broiler diets increased average feed intake (AFI) at 26-45 and 1-45 days, feed conversion ratio (FCR) at 12-25, and the mortality rate decreased from 28.8% (0% SM) to 12.1% (6% SM) (p < 0.05). Adding SM had no significant effects on the relative weight of carcase segments and internal organs (p > 0.05) except for the duodenum, which increased weight and length at the level of 6% (p < 0.05). The red blood cell (RBC) count and haemoglobin were elevated in chicks inoculated with 3% and 6% SM, respectively (p < 0.05). The mean corpuscular volume (MCV) value increased following the supplementation of SM, while differential white blood cell (WBC) counts did not influence. Aspartate aminotransferase (AST) and Alanine transaminase (ALT) activity declined in chicks that received 6% SM at 45 days of age (p < 0.05). SM supplementation decreased liver steatosis, uric acid, glucose, total protein, and lipid profile concentrations of serum (p < 0.05). Therefore, we conclude that adding SM to the broiler diets at 6% level significantly improves broiler performance, blood traits, and liver health under chronic heat-stress conditions.

HIGHLIGHTS

• Adding a 6% seed mixture to the broiler’s diet during the finisher phase improved weight, reduced mortality rate, better morphometric parameters, increased haemoglobin levels, and healthier liver function.

• Including a 3% seed mixture in the diet increased average feed intake throughout the rearing period and boosted FCR between 12 and 25 days and red blood cell count.

• Seed mixture supplementation led to an increase in mean corpuscular volume levels and a decrease in uric acid, glucose, total protein, and serum lipid profile concentration.

Keywords:

• Fenugreek
• sesame
• black cumin
• heat stress
• broiler
• performance

Introduction

It is clear that the global temperature is rising due to climate change (Chen et al. 2011). Modern broiler chickens cannot tolerate high ambient temperatures because their central body temperature is high (40 °C) due to covering their body with feathers, high metabolic activity, and lacking sweat glands (Caulfield et al. 2014; Nawab et al. 2018; Abo Ghanima et al. 2019; Abdel-Moneim et al. 2021; Greene et al. 2021). These conditions can lead to heat stress, which has substantial adverse effects on poultry production by inducing thirst (Belay et al. 1993), depressing feed intake (Flees et al. 2017; Rajaei-Sharifabadi et al. 2017), immunosuppression (Ghazi et al. 2012; Monson et al. 2018), poor performance (Quinteiro-Filho et al. 2010; Lara and Rostagno 2013), reducing nutrient digestibility (Habashy et al. 2017), and in extreme cases increasing mortality (Furlan et al. 1998).

Many studies have observed that heat stress induces oxidative stress in muscles (Mujahid et al. 2009), which is reflected by an increase in protein oxidation (increased protein carbonyls), lipid peroxidation (increased malondialdehyde), and an increase in leptin blood metabolites such as glucose, cholesterol, and triglycerides (Al-Azraqi 2008). During heat stress, birds reduce heat production by suppressing catabolic hormones {triiodothyronine (T3) and thyroxine (T4)} (Kusnadi and Rahim 2009) and reducing feed intake (Bartlett and Smith 2003), which results in decreasing weight gain (Soleimani et al. 2008).

The mentioned items threaten poultry sustainability and have led to significant economic losses, encouraging researchers to manipulate diets to adjust these conditions. Recently, herbal additives have been studied to promote appetite and overcome the adverse effects of heat stress at the molecular and cellular levels.

Black cumin (Nigella sativa L.) belonging to the family Ranunculaceae is famous for its functional properties, which have been attributed to the bioactive components such as p-cymene, carvone, limonene, thymoquinone, α-thujene, trans-anethole, and longifolene (Nickavar et al. 2003; Singh et al. 2005; Azeem et al. 2014). These components can exert antitoxic and antimicrobial properties by increasing the defense mechanisms against infectious diseases (Abd El-Hack et al. 2020). The seeds of Nigella sativa are composed of protein (20-27%), fat (34.5-38.7%), carbohydrates (23.5-33.2%), crude fibre (8.4%), and ash (4.8%) (Babayan et al. 1978). The seeds also contain a good amount of vitamins and minerals, as well as carotene, which the liver converts to vitamin A (Vaz et al. 2018). Some previous studies have mentioned the positive effects of black seed on broiler performance (Abu-Dieyeh and Abu-Darwish 2008; Erener et al. 2010; Khalaji et al. 2011; Islam et al. 2016).

Fenugreek (Trigonella foenum-graecum L.) is a popular medicinal plant in nature have multiple therapeutic impacts, such as hypoglycaemic, antibacterial, anti-inflammatory, and antimicrobial effects (Xue et al. 2007; Adil et al. 2015; Ali et al. 2021). The seeds include neurin, biotin, and trimethylamine, and their effect on the nervous system promotes appetite (Chevassus et al. 2009). It contains dietary proteins, carbohydrates, minerals, and vitamins, making it a healthy source for humans and livestock (Michael and Kumawat 2003; Ali et al. 2021). Fenugreek seeds can increase feed intake (Mamoun et al. 2014) and improve broiler body weight (Qureshi et al. 2015). Some researchers reported positive effects of Fenugreek seed (Alloui et al. 2011; Yang et al. 2022) on broiler performance.

Sesame (Sesamum indicum L.) seeds belong to the Pedaliaceae family and are widely cultivated in many countries (Kanu et al. 2010). These seeds have a high concentration of oil (44–58%), protein (18–25%), carbohydrate (13.5%), and ash (5%) (Shyu and Hwang 2002; Kahyaoglu and Kaya 2006). The oil fraction is known for its remarkable stability to oxidation and contains endogenous antioxidants such as lignans (sesamin, sesaminol, sesamol, sesamolinol, or sesamolin) (Katsuzaki et al. 1994; Yoshida et al. 1995; Abou-Gharbia et al. 2000) and arachidonic acid levels (Shimizu et al. 1991). Sesame seeds have various health benefits and can help reduce blood lipids (Hirata et al. 1996), enhance antioxidant ability (Hemalatha 2004), and provide anti-inflammatory function (Hsu et al. 2005).These three seeds, besides stimulating the digestive system and growth, can be used as an anti-stressor because they are potentially a great source of antioxidants, have the ability to neutralise free radicals, and can protect the body during stress (Wenk 2003; Kumar and Patra 2017; Asad et al. 2019). We hypothesised that combining black cumin, fenugreek, and sesame seeds in broiler diets could help mitigate the adverse effects of heat stress. Therefore, this study aims to investigate the impacts of the seed mixture of (sesame, fenugreek, and black seed) seeds on broiler performance, intestinal morphology, serum, and blood traits under chronic heat-stress conditions.

Materials and methods

Seeds mixture preparation and analysis

Black cumin, fenugreek, and sesame seeds were purchased from a local supplier in Qaladiza, Iraq. The SM was prepared by combining all three seeds in equal proportions. The SM was ground into a fine powder using a laboratory mill. The ground powder’s crude protein (CP), ether extract (EE), crude fibre (CF), and ash contents were determined using laboratory methods (AOAC 1995). The nitrogen-free extract (NFE) value was calculated by subtracting the total protein, ether extract, ash, and fibre content from 100.

Ethanolic extraction of seed mixture

Fifty grams of the SM powder were extracted (by a maceration method) by adding 500 mL of ethanol (99.8%) shaken at 85 rpm at 25 °C for six hours. The extract was filtered through Whatman No.1 filter paper. The filtrate was evaporated to condense under reduced pressure in a rotatory vacuum evaporator (Heidolph, North America, Wood Dale, IL) by a method described by Nassiri‐Asl and Hosseinzadeh (2016). The condensed extracts were kept in the dark at four-degree Celsius until analysis by gas chromatography mass spectrometry (GC-MS).

GC-MS analysis was carried out with an Agilent 7890 A Gas Chromatograph equipped with an electron impact quadrupole MD 800 mass spectrometer detector and a 19095-400 fused silica column (30 m length and 0.25 mm inner diameter). The stationary phase had a thickness of 0.25 μm. Set the temperature of the initial injection column at 35 °C for 2.50 min, then gradually increase it to 280 °C for 20 min in 7 °C increments. The identity of volatile oil components was established from their GC Kovats retention indices and mass spectra by computer matching with the mass spectra library (Adams, NIST, and Wiley).

Birds, housing and feeding

This experiment was conducted at the Department of Animal Science, University of Raparin, Ranya, Sulaymaneyah, Iraq. All animal care and use procedures were evaluated and approved by the University of Raparin Animal Care and Ethical Committee, which complies with international guidelines (FASS 1999). The treatments were arranged into a completely randomised design trial for 45 days. On arrival, 330-one-day Ross 308 broiler chicks were randomly distributed into 30 cages (five treatments, six replicates, each consisting of 11 chicks) equipped with a manual feeder and a bell drinker per cage. The cages were numbered, and records were taken for three rearing phases, including; 1-11 days, 12-25 days, and 26-45 days of ages. The animals had unlimited access to feed and water. The feed was provided in pellet form. The lighting was set to a 24-h lightness on the first days, followed by a 23:1 lightness-to-darkness ratio to avoid additional stress during electrical outages. As presented in Tables 1–3, the experimental diets were formulated to provide the chicks’ nutritional requirements (Ross Broiler Nutrition Specifications Handbook 2019). The dietary treatments included: a corn-soybean-based diet was supplemented with 0, 1.5, 3, 4.5, and 6% SM for 45 days. A 34 °C was applied continuously to induce heat stress during the entire period, electrical heaters to reach this temperature, and thermostats to control it.

Table 1. Ingredients and nutrients composition of experimental diets (1-11 days).

Ingredient (fed basis %)	Control	Seed mixture (%)
		1.5	3	4.5	6
Corn grain	50.72	49.15	48.71	48.26	47.81
Soy bean meal	43.33	42.79	42.08	41.36	40.65
Soybean oil	1.05	1.62	1.26	0.90	0.55
Dicalcium phosphate	0.73	0.75	0.76	0.78	0.79
CaCO3	1.15	1.15	1.14	1.14	1.14
Common Salt	0.23	0.23	0.23	0.23	0.23
Premix concentrate^1	2.50	2.50	2.50	2.50	2.50
DL-Methionine	0.29	0.31	0.32	0.33	0.34
Seed mixture	0.00	1.50	3.00	4.50	6.00
Calculated analysis
Crude protein, %	23.00	23.00	23.00	23.00	23.00
ME, kcal/kg	3000	3000	3000	3000	3000
Calcium %	0.97	0.97	0.97	0.97	0.97
Na %	0.16	0.16	0.16	0.16	0.16
K %	1.02	1.00	0.99	0.97	0.96
Cl %	0.23	0.23	0.23	0.23	0.23
Total Phos %	0.74	0.73	0.73	0.72	0.72
Avail Phos %	0.48	0.48	0.48	0.48	0.48
Cysteine, %	0.36	0.36	0.35	0.35	0.34
Lysine, %	1.43	1.41	1.39	1.37	0.71
Methionine, %	0.69	0.69	0.69	0.70	1.35
TSAA, %	1.00	1.00	1.00	1.00	1.00
Threonine %	0.95	0.93	0.92	0.90	0.89
Crude Fibre %	3.30	3.42	3.56	3.70	3.84
Linoleic acid, %	1.82	2.08	1.88	1.69	1.49

Abbreviations: ME, Metabolisable energy; Phos, Phosphate; TSAA, total sulphur amino acid.

¹Provided per kg of diet: Protein, 18%; CaCO₃, 19%; Monocalcium phosphate, 7%; NaCl, 4.5%; Methionine, 2%; Lysine, 4%; Threonine, 1%; Choline chloride, 1%; Iron (sulphate), 2800 mg; Zinc (oxide), 2480 mg; Manganese (oxide), 3600 mg; Copper (sulphate), 240 mg; Iodine (IK), 60 mg; Selenium (SeNa), 14 mg; Cobalt (sulphate), 14 mg; Magnesium (oxide), 800 mg; Antioxidant (BHT), 32 mg; Vitamin A, 420000 IU; Vitamin D₃, 80000 IU; Vitamin E, 800 mg; Vitamin C, 40 mg; Vitamin K₃, 80 mg; Vitamin B₁, 80 mg; Vitamin B₂, 200 mg; Vitamin B₆, 80 mg; Vitamin B₁₂, 0.6 mcg; Biotine, 10.4 mg; Folic acid, 20 mg; Nicotinic acid, 400 mg; Pantothenic acid, 160 mg.

Table 2. Ingredients and nutrients composition of experimental diets (12-25 days).

Ingredient (fed basis %)	control	Seed mixture (%)
		1.5	3	4.5	6
Corn grain	53.32	51.703	51.255	50.807	50.486
Soy bean meal	39.57	39.044	38.329	37.614	36.687
Soybean oil	2.54	3.162	2.803	2.443	2.069
Dicalcium phosphate	0.56	0.577	0.590	0.602	0.617
CaCO3	1.07	1.063	1.061	1.059	1.058
Common Salt	0.23	0.228	0.228	0.229	0.229
Premix concentrate^1	2.50	2.5	2.5	2.5	2.5
DL-Methionine	0.211	0.224	0.234	0.245	0.247
Seed mixture	0.00	1.50	3.00	4.50	6.00
Calculated analysis
Crude protein %	21.50	21.50	21.50	21.50	21.50
ME, kcal/kg	3100	3100	3100	3100	3100
Calcium %	0.89	0.89	0.89	0.893	0.89
Na %	0.16	0.16	0.16	0.16	0.16
K %	0.95	0.94	0.92	0.91	0.89
Cl %	0.23	0.23	0.23	0.23	0.23
Total Phos %	0.69	0.68	0.68	0.67	0.67
Avail Phos %	0.44	0.44	0.44	0.44	0.44
Cysteine, %	0.34	0.34	0.33	0.33	0.32
Lysine, %	1.33	1.32	1.29	1.27	1.30
Methionine, %	0.59	0.59	0.60	0.60	0.60
TSAA, %	0.88	0.88	0.88	0.88	0.87
Threonine %	0.89	0.87	0.86	0.85	0.88
Crude Fibre %	3.19	3.31	3.45	3.59	3.72
Linoleic acid, %	2.63	2.91	2.71	2.51	2.31

Abbreviations: ME, Metabolisable energy; Phos, Phosphate; TSAA, total sulphur amino acid.

¹Provided per kg of diet: Protein, 18%; CaCO3, 19%; Monocalcium phosphate, 7%; NaCl, 4.5%; Methionine, 2%; Lysine, 4%; Threonine, 1%; Choline chloride, 1%; Iron (sulphate), 2800 mg; Zinc (oxide), 2480 mg; Manganese (oxide), 3600 mg; Copper (sulphate), 240 mg; Iodine (IK), 60 mg; Selenium (SeNa), 14 mg; Cobalt (sulphate), 14 mg; Magnesium (oxide), 800 mg; Antioxidant (BHT), 32 mg; Vitamin A, 420000 IU; Vitamin D3, 80000 IU; Vitamin E, 800 mg; Vitamin C, 40 mg; Vitamin K3, 80 mg; Vitamin B1, 80 mg; Vitamin B2, 200 mg; Vitamin B6, 80 mg; Vitamin B12, 0.6 mcg; Biotine, 10.4 mg; Folic acid, 20 mg; Nicotinic acid, 400 mg; Pantothenic acid, 160 mg.

Table 3. Ingredients and nutrients composition of experimental diets (26-45 days).

Ingredient (fed basis %)	control	Seed mixture (%)
		1.5	3	4.5	6
Corn grain	56.26	55.82	55.37	54.92	54.47
Soy bean meal	34.54	33.82	33.11	32.39	31.68
Soybean oil	4.97	4.61	4.25	3.89	3.53
Dicalcium phosphate	0.35	0.36	0.38	0.39	0.40
CaCO3	0.96	0.96	0.96	0.96	0.96
Common Salt	0.23	0.23	0.23	0.23	0.23
Premix concentrate^1	2.50	2.50	2.50	2.50	2.50
DL-Methionine	0.18	0.19	0.20	0.22	0.23
Seed mixture	0.00	1.50	3.00	4.50	6.00
Calculated analysis
Crude protein %	19.50	19.50	19.50	19.50	19.50
ME, kcal/kg	3200	3200	3200	3200	3200
Calcium %	0.79	0.79	0.79	0.79	0.79
Na %	0.16	0.16	0.16	0.16	0.16
K %	0.86	0.84	0.83	0.81	0.80
Cl %	0.23	0.23	0.23	0.23	0.23
Total Phos %	0.62	0.62	0.61	0.61	0.61
Avail Phos %	0.39	0.39	0.39	0.39	0.39
Cysteine, %	0.32	0.31	0.31	0.30	0.29
Lysine, %	1.20	1.18	1.16	1.14	1.11
Methionine, %	0.53	0.54	0.54	0.55	0.55
TSAA, %	0.80	0.80	0.80	0.80	0.80
Threonine %	0.81	0.79	0.78	0.76	0.75
Crude Fibre %	3.03	3.17	3.30	3.44	3.58
Linoleic acid	3.91	3.71	3.52	3.32	3.13

Abbreviations: ME, Metabolisable energy; Phos, Phosphate; TSAA, total sulphur amino acid.

¹Provided per kg of diet: Protein, 18%; CaCO3, 19%; Monocalcium phosphate, 7%; NaCl, 4.5%; Methionine, 2%; Lysine, 4%; Threonine, 1%; Choline chloride, 1%; Iron (sulphate), 2800 mg; Zinc (oxide), 2480 mg; Manganese (oxide), 3600 mg; Copper (sulphate), 240 mg; Iodine (IK), 60 mg; Selenium (SeNa), 14 mg; Cobalt (sulphate), 14 mg; Magnesium (oxide), 800 mg; Antioxidant (BHT), 32 mg; Vitamin A, 420000 IU; Vitamin D3, 80000 IU; Vitamin E, 800 mg; Vitamin C, 40 mg; Vitamin K3, 80 mg; Vitamin B1, 80 mg; Vitamin B2, 200 mg; Vitamin B6, 80 mg; Vitamin B12, 0.6 mcg; Biotine, 10.4 mg; Folic acid, 20 mg; Nicotinic acid, 400 mg; Pantothenic acid, 160 mg.

Performance measurements

On a cage basis, the bird’s BW was recorded on arrival at 11, 25, and 45 days of age. AFI, AWG, and FCR were measured during 1-11, 12-25, 26-45 days, and the entire rearing period (1-45 days) while they were adjusted according to the weight of dead broiler chickens.

Blood and serum collection

At 25 and 45 days of age, one bird per replicate was selected randomly, and a blood sample was taken from the brachial vein before slaughter. Then the blood samples were centrifuged at 4000 rpm x g for 10 min to separate sera and kept at −20 °C until required for analysis. Serum cholesterol, triglycerides, very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), high-density lipoprotein (HDL), glucose, total protein (TP), and uric acid (UA) levels were measured by using diagnostic colourimetric kits (Biolabo S.A.S company, France). Another blood sample was collected in heparinised tubes to determine haematological parameters, including RBC count, haematocrit (HCT), haemoglobin (Hb), MCV, mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC). Also, the differential WBC count was evaluated, which included lymphocytes, mid (monocytes, eosinophils, and basophils) cells, granulocytes, and heterophiles. A veterinary auto haematology analyser (model SmartVet) detected the aforementioned haematological parameters. In determining the lymphocyte-to-heterophile ratio (L/H), the number of lymphocytes was divided by the heterophile, according to Lentfer et al. (2015).

Carcase segments and internal organs

At 46 days of age, the chicks previously chosen for blood sample collection were weighed individually and killed by cervical dislocation. The weights of carcase parts (breast, thigh, and drumstick muscle), internal organs (crop, proventriculus, gizzard, pancreas, liver, spleen, heart, bursa of fabricius, and abdominal fat), and the weight and length of intestinal segments (duodenum, jejunum, ileum, and caeca) were recorded using a digital balance with an accuracy of 0.01 g. All weight and length data are expressed as a percentage of live BW (Wagner et al. 1983).

Intestinal morphology

After slaughtering at 46 days of age, about 4 cm were obtained from the central part of the duodenum jejunum and ileum, washed with physiological saline, and fixed in a 10% formalin solution. Tissues were dehydrated through a graded ethanol series, cleared with xylene, impregnated with an automatic tissue processor, and embedded in paraffin wax. Tissue sections were cut (6 μm) from the waxed tissue using an SRM 200 model microtome (Sakura Finetek Europe B.V., Alphen Aan Den Rijn, Netherlands). The sections floated on prewarmed water (50 °C) before mounting on slides to open wrinkles and then fixed to the slides. The slides were stained with haematoxylin and eosin stains. Morphometric indices such as villus height, width, and crypt depth were determined at a magnification of 40x using a light microscope. The values for villus surface areas were calculated using previous data. The mean values of 10 villi per sample were recorded as the average for further analysis (Abdollahi et al. 2021).

Hepatic histopathology

For pathological assessment of the liver, a 1 cm³ sample was removed from the left lobe of the liver with a sharp scalpel at 46 days of age and then fixed in a 10% buffered formalin solution. The rest of the processing steps, including (staining, moulding with paraffin, and preparation of tissue sections), were the same as the intestinal tissues. Steatosis of liver slides was assessed in five fields of view, and the severity of hepatic steatosis was determined using the following scale: 0 (0–19%), 1 (20–39%), 2 (40–59%), 3 (60–79%), and 4 (80–100%) by 40X magnification (Bancroft and Gamble 2008). The level of leukocyte infiltration was assessed and assigned as the following score: 0 indicates no damage, 1 indicates mild damage, 2 indicates moderate damage, and 3 indicates severe damage (Brunt et al. 1999).

Liver enzyme activity

The serum AST, ALT, and Alkaline phosphatase (ALP) levels were measured to determine liver health at 25 and 45 days of age. Professional kits from Biolabo S.A.S., Les Hautes Rives, 02160 Maizy, France, were used to measure these enzymes colourimetrically (Sahoo et al. 2019).

Apparent ileal digestibility

The titanium dioxide (TiO2 from Merck Company, Germany) was added as a marker at 4 g per 1000 g diet, provided four days before slaughtering at 46 days of age. The total ileum contents of slaughtered chicks were squeezed gently into plastic cans and frozen at −20 °C until further analysis to measure apparent ileal digestibility. The dried samples’ dry matter, organic matter, and ash were determined (AOAC 1995), and ileal digestibility was calculated by the Short et al. (1996) method.

Statistical analysis

All collected data were subjected to analysis of variance as a completely randomised design trial using the General Linear Model procedures of the SAS Institute (2001). Tukey’s test compared differences between means when the model was declared significant (p < 0.05).

Results

Seed mixture analysis

The chemical composition of SM samples used in the present study, including CP, CF, EE, ash, and NFE, is shown in Table 4 and is presented on a dry matter basis.

Table 4. Chemical composition of seed mixture on a DM basis (%).

Parameters	DM	Ash	CP	CF	EE	NFE
Seed Mixture	93.31	3.86	22.51	12.32	33.76	33.19

Abbreviation: CF, Crude fibre; CP, Crude protein; DM, Dry matter; EE, Ether extract; NFE, Nitrogen-free extract.

Bioactive components of herbal mixture extract

GC-MS analysis identified the bioactive compounds of the seeds mixture. The ethanolic extract chromatogram of the seed mixture showed several peaks in Figure 1 and reported eight higher-pick compounds in Table 5. The most abundant compounds are anethole (48.46%), α-phellandrene (9.60%), D-limonene (9.59%), α-pinene (8.27%), 1.9-octadecenoic acid (6.67%), linoleic acid (5.27%), oleic acid (4.08%) and 1.2-Propanone (3.77%). According to the studies, these eight compounds are all monoterpenes or phenylpropene, the most potent antibacterial and antioxidant compounds found in medicinal herbs (Koeduka et al. 2009; Lin et al. 2014; Salehi et al. 2019; Anandakumar et al. 2021).

Figure 1. Chromatographic profile (GC-MS) of the of seed mixture ethanolic extract.

Table 5. Pharmacological activities of the eight most expressed bioactive compounds in the seed mixture (SM) ethanolic extract.

R. time (minute)	Compound name	Com. %	Pharmacological activity	References
13.567	Anethole	48.458%	Antioxidant and antimicrobial activities	(Senatore et al. 2013; Contant et al. 2021)
9.774	α-Phellandrene	9.597%	Antioxidant activity	(De Christo Scherer et al. 2019)
9.774	D-Limonene	9.597%	Antioxidant activity	(Anandakumar et al. 2021; Murali et al. 2013)
7.794	α-Pinene	8.277%	Antioxidant activity	(Xanthis et al. 2021; Shahriari et al. 2018)
22.979	9,12-Octadecadienoic acid	6.671	Antioxidant activity	(Ahmed et al. 2022; Zitouni et al. 2022)
23.115	Linoleic acid	5.269%	Antioxidant activity	(Beeharry et al. 2003)
22.868	oleic acid	4.079%	Antioxidant activity	(Cho et al. 2010)
15.311	1.2-Propanone	3.767%	Antioxidant activity	(Tumosienė et al. 2020)

R. time: Retention time; Com.: Compound.

Performance

The effects of different levels of SM (0, 1.5, 3, 4.5, and 6%) on BW, AWG, and the mortality rate of broiler chicks under heat stress from 1 to 45 days of age are presented in Table 6. Experimental diets had no significant effects on broiler’s BW at 11 and 25 days of age and average weight gain at 1-11 and 12-25 days of age, but they were numerically different. Supplementation of diets with different levels of SM increased body weight regularly at 45 days and weight gain at the finisher phase and total rearing period (p < 0.05). Notably, birds fed a diet containing 6% of the SM showed maximum live body weights and average weight gain compared to the control (p < 0.05). The mortality rate gradually declined with an increase in SM level in the broiler chicken diet during the total rearing period (p < 0.05). Such that birds fed a diet containing 6% of the SM had the lowest mortality rate compared with the control, which recorded a higher level of mortality under the same heat stress.

Table 6. Effects of seed mixture (SM) on body weight, average weight gain, and mortality (%) in broiler chicken under heat stress condition.

Treatments	Body weight (gram)			Weight gain (gram)				Mortality (%)
	11 d	25 d	45 d	1-11 d	12-25 d	26-45 d	1-45 d	1-45 d
Control	262	1091	2426^b	222	778	1336^c	2386^c	28.8^a
1.5 % SM	264	1055	2494^ab	223	791	1465^ab	2480^abc	22.7^ab
3.0 % SM	269	1064	2503^ab	228	795	1409^bc	2463^bc	21.8^ab
4.5 % SM	268	1087	2540^ab	233	824	1487^ab	2500^ab	16.7^bc
6.0 % SM	269	1100	2622^a	236	839	1517^a	2528^a	12.1^c
	---------------------------Probability--------------------------
Probability	0.9491	0.7813	0.0428	0.7150	0.5028	0.0124	0.0098	0.0081
SEM	3.163	12.26	19.25	3.125	11.24	18.32	23.68	1.245

^a-c Means in the same column with different letters are different significantly (p < 0.05).

The effects of experimental diets on the AFI and FCR of broilers under the same heat stress conditions from 1 to 45 days of age are presented in Table 7. There were no differences between the experimental group regarding AFI during 1-11 and 12-25 days of age (p > 0.05). However, chicks fed diets containing 3% SM increased AFI compared to the control and 1.5% SM during 26-45 and 1-45 days of age (p < 0.05). FCR was not affected significantly by SM administration during 1-11 days of age. FCR for birds fed a diet containing 1.5% of the SM was the lowest among all treatments numerically during the 12-25 days of age, with a significant difference compared to birds that received 3% of the SM. There were no differences in FCR among all treatments between 26-45 and 1-45 days of age (p > 0.05).

Table 7. Effects of seed mixture (SM) on average feed intake and feed conversion ratio in broiler chicken under heat stress condition.

Treatments	Average feed intake (gram)				Feed conversion ratio (gram/gram)
	1-11 d	12-25 d	26-45 d	1-45 d	1-11 d	12-25 d	26-45 d	1-45 d
Control	266	1140	2622^b	4034^c	1.203	1.38^ab	1.95	1.76
1.5 % SM	265	1113	2654^b	4175^bc	1.188	1.34^b	1.94	1.68
3.0 % SM	280	1158	2871^a	4352^a	1.227	1.50^a	2.04	1.78
4.5 % SM	278	1151	2782^ab	4262^ab	1.257	1.40^ab	1.98	1.71
6.0 % SM	272	1126	2781^ab	4179^b	1.275	1.41^ab	1.85	1.66
	Probability
Probability	0.3808	0.8018	0.0395	0.0065	0.2422	0.0161	0.7816	0.7713
SEM	2.934	12.12	32.45	45.52	0.014	0.016	0.046	0.033

^a-c Means in the same column with different letters are different significantly (p < 0.05).

Carcase Segments

As shown in Table 8, the relative weight of carcase, breast, thigh, and drumstick muscle to live body weight (g/100g) was not influenced by experimental diets under heat stress conditions (p > 0.05).

Table 8. Effects of seed mixture (SM) on relative weight of the carcase segments to live body weight (g/100g) in broiler chicken under heat stress condition at 46 day of age.

Treatments	Carcase	Breast	Thigh	Drumstick
Control	64.4	23.77	4.92	4.32
1.5 % SM	65.0	25.85	5.18	4.36
3.0 % SM	67.1	27.17	5.23	4.42
4.5 % SM	66.0	25.64	5.32	4.47
6.0 % SM	65.4	24.89	5.28	4.52
	----------Probability-----------
Probability	0.1348	0.1297	0.4089	0.7071
SEM	0.354	0.426	0.070	0.047

^a-c Means in the same column with different letters are different significantly (p < 0.05).

Internal organs

Effects of the experimental diets on the relative weight of internal organs to live body weight (g/100g) at 46 days of age are presented in Table 9. There was no significant difference in the crop, Proventriculus, gizzard, pancreas, liver, spleen, heart, bursa of Fabricius, and abdominal fat (p > 0.05) in broilers reared under heat stress conditions.

Table 9. Effects of seed mixture (SM) on relative weight of internal organs to live body weight (g/100g) in broiler chicken under heat stress condition at 46 day of age.

Treatments	Crop	Proventriculus	Gizzard	Pancreas	Liver	Spleen	Heart	Bursa of fabriciuos	Abdominal fat
Control	1.047	0.373	0.802	0.205	2.16	0.108	0.445	0.043	1.515
1.5 % SM	0.644	0.382	0.985	0.210	2.59	0.098	0.398	0.042	1.430
3.0 % SM	0.455	0.458	1.024	0.198	2.36	0.123	0.378	0.053	1.247
4.5 % SM	0.752	0.442	0.918	0.228	2.27	0.100	0.404	0.047	1.390
6.0 % SM	0.447	0.464	0.935	0.218	2.35	0.093	0.393	0.043	0.972
	--------------------probability---------------------------------
Probability	0.0681	0.3317	0.0896	0.8063	0.1836	0.6125	0.3833	0.0555	0.1734
SEM	0.078	0.017	0.027	0.008	0.057	0.006	0.011	0.001	0.078

^a-c Means in the same column with different letters are different significantly (p < 0.05).

Intestinal weight and length

The effects of experimental diets on the relative weight and length of the duodenum, jejunum, ileum, and caecum are shown in Table 10. There were no significant differences among experimental groups regarding the relative weight and relative length of the jejunum, ileum, and caecum (p > 0.05) after feeding on a diet supplemented with SM under heat stress conditions. However, the relative weight and length of the duodenum were highest in the birds that received 6% of the SM compared to the control (p < 0.05), but there was no significant difference with other treatments.

Table 10. Effects of seed mixture (SM) on intestinal relative weight (g/100g) and length (cm/100g) to live body weight in broiler chicken under heat stress condition at 46 day of age.

Treatments	Weight				Length
	Duodenum	Jejunum	Ileum	Caecum	Duodenum	Jejunum	Ileum	Caecum
Control	0.743^b	2.76	2.10	0.650	1.237^b	3.55	3.62	0.913
1.5 % SM	0.767^ab	2.24	1.92	0.713	1.278^ab	3.38	3.27	0.752
3.0 % SM	0.782^ab	1.69	1.38	0.583	1.295^ab	2.95	2.86	0.766
4.5 % SM	0.815^ab	2.31	1.47	0.638	1.387^ab	3.25	2.96	0.747
6.0 % SM	0.958^a	2.34	2.14	0.572	1.528^a	3.45	3.45	0.752
	----------------------probability-----------------------------
Probability	0.0113	0.1212	0.0517	0.5064	0.0490	0.6168	0.2008	0.1725
SEM	0.036	0.128	0.107	0.027	0.034	0.123	0.117	0.026

^a-c Means in the same column with different letters are different significantly (p < 0.05).

Haematological parameters

All the estimated haematological parameters, such as RBC, Hb, HCT, MCV, MCH, and MCHC of broiler chickens under the same heat stress, are summarised in Table 11. No significant change was observed between experimental groups about RBC, Hb, HCT, MCV, MCH, and MCHC at day 25 of age (p > 0.05). Significantly feeding diets containing 3% of the SM increased RBC counts (p < 0.05) compared to other groups except for the control at 45 days. Also, birds that consumed 6% of the SM showed a significant elevation in Hb compared to 4.5% of the SM, but there was no significant difference compared to the control group at 45 days of age. Different levels of SM significantly increased MCV compared to the control (p < 0.05). There were no significant changes in the HCT, MCH, and MCHC between groups at 45 days of age.

Table 11. Effects of seed mixture (SM) on hematological parameters at 25 and 45 days in broiler chicken under heat stress condition.

Treatments	25 days						45 days
	RBC (×10^6 cell/µL)	Hb (g/dL)	HCT (%)	MCV (fL)	MCH (pg/cell)	MCHC (%)	RBC (×10^6 cell/µL)	Hb (g/dL)	HCT (%)	MCV (fL)	MCH (pg/cell)	MCHC (%)
Control	2.09	13.36	22.25	104	61.4	58.97	2.41^ab	13.30^ab	25.98	105^b	55.08	52.3
1.5 % HM	1.85	12.77	21.72	111	62.7	57.20	2.34^b	13.02^ab	27.05	109^a	55.60	50.9
3.0 % HM	2.12	13.80	23.33	114	62.6	59.37	2.65^a	13.45^ab	27.62	109^a	56.65	52.0
4.5 % HM	2.15	13.28	22.48	106	61.7	60.18	2.42^b	12.82^b	26.28	111^a	57.08	52.5
6.0 % HM	2.02	12.20	25.65	104	60.7	62.37	2.50^b	14.10^a	27.15	109^a	56.16	51.6
	-----------------------Probability--------------------------------
P. value	0.4019	0.4940	0.2273	0.0622	0.7707	0.5298	0.0281	0.0485	0.5612	0.0038	0.3874	0.3724
SEM	0.051	0.271	0.564	1.27	0.507	8.81	0.034	0.149	0.351	0.568	0.351	2.73

^a-c Means in the same column with different letters are different significantly (p < 0.05).

Abbreviation: RBC, Red blood cell; Hb, haemoglobin; HCT, hematocrite; MCV, Mean corpuscular volume; MCH, Mean corpuscular haemoglobin; MCHC, Mean corpuscular haemoglobin concentration.

White blood cells

Table 12 shows the effects of SM supplementation on broilers’ differential white blood cell count, including lymphocyte, Mid cells, granulocyte, heterophile, and lymphocyte to a heterophile ratio (L/H). Results indicated that inoculation of SM in the broiler diet under heat stress could not affect the differential leukocyte counts at 25 days or the total rearing period (p > 0.05).

Table 12. Effects of seed mixture (SM) on differential white blood cell counts at 25 and 45 days in broiler chicken under heat stress condition.

Treatments	25 days					45 days
	Gran (%)	Hetero (%)	Lymph (%)	L/H	Mid (%)	Gran (%)	Hetero (%)	Lymph (%)	L/H	Mid (%)
Control	38.52	34.42	60.25	1.75	5.32	38.62	33.10	61.38	1.86	5.52
1.5 % SM	40.74	36.00	58.67	1.64	4.66	38.47	32.53	61.53	1.91	5.93
3.0 % SM	42.70	36.80	57.80	1.57	5.40	37.47	31.02	62.53	2.05	6.45
4.5 % SM	40.20	35.05	59.60	1.70	5.35	39.12	35.75	59.05	1.66	5.20
6.0 % SM	40.74	37.10	57.62	1.56	5.27	37.36	31.56	62.24	2.00	6.20
	------------Probability----------------------------------------
Probability	0.5026	0.3015	0.3159	0.3083	0.6866	0.8378	0.2368	0.2841	0.2740	0.3949
SEM	0.67	0.45	0.45	0.03	0.19	0.53	0.64	0.49	0.05	0.21

^a-c Means in the same column with different letters are different significantly (p < 0.05).

Abbreviations: Gran, Granulocyte; Hetero, Heterophil; Lymph, Lymphocyte; L/H, Lymphocyte to heterophil ratio; Mid, sum of basophil, monocyte, and eosinophil.

Lipid profile of serum

Table 13 shows the effect of SM supplementation in broiler diets on serum lipid profiles under heat stress conditions. The results showed no significant changes among treatments about serum cholesterol, triglyceride, and HDL concentrations in broilers reared under heat stress at 25 days of age (p > 0.05). There was a significant difference in serum LDL and VLDL at 25 days of age (p < 0.05). Birds fed 1.5 and 4.5% of the SM showed maximum LDL concentration compared to the control and 6% of the SM groups (p < 0.05). Also, supplementation of broilers diets by 1.5 and 3% SM significantly increased VLDL concentration compared to control and 4.5% SM supplemented groups (p < 0.05). At 45 days of age, cholesterol level decreased due to 4.5% SM supplementation compared to other groups (p < 0.05). Triglyceride and LDL concentrations significantly declined in the 3 and 4.5% SM-supplemented group (p < 0.05). Adding 3% SM to broiler’s diets increased HDL concentration under heat stress conditions (p < 0.05). However, VLDL concentration has not change among experimental groups at 45 days of age (p > 0.05).

Table 13. Effects of seed mixture (SM) on serum lipid profile at 25 and 45 days in broiler chicken under heat stress condition.

Treatments	25 days					45 days
	Cholesterol (mg/dL)	Trigyceride (mg/dL)	HDL (mg/dL)	LDL (mg/dL)	VLDL (mg/dL)	Cholesterol (mg/dL)	Trigyceride (mg/dL)	HDL (mg/dL)	LDL (mg/dL)	VLDL (mg/dL)
Control	101	70.9	48.4	38.5^b	14.1^b	87.89^a	51.15^a	32.04^b	45.61^a	10.23
1.5 % SM	115	95.3	30.0	65.5^a	19.1^a	82.37^a	38.51^ab	34.22^b	42.80^ab	7.70
3.0 % SM	104	93.4	30.7	59.9^ab	18.7^a	80.82^a	25.67^b	57.43^a	15.21^c	5.13
4.5 % SM	104	61.2	31.3	63.7^a	12.2^b	48.09^b	23.18^b	27.89^b	18.48^bc	5.28
6.0 % SM	106	87.8	36.0	38.6^b	17.6^ab	78.11^a	30.65 ^ab	40.00^b	31.98^abc	6.13
	-------------------------Probability------------------------------
Probability	0.4491	0.2672	0.2758	0.0158	0.0369	0.0025	0.0098	0.0011	0.0024	0.0629
SEM	2.66	5.64	3.13	3.07	0.94	3.63	3.22	2.72	3.10	0.51

^a-c Means in the same column with different letters are different significantly (p < 0.05).

Abbreviations: HDL, High density lipoprotein; LDL, Low density lipoprotein; VLDL, Very low density lipoprotein.

Serum biochemical traits and enzymes activity

The effects of experimental diets on the serum concentration of glucose, UA, TP, and enzyme activity (AST, ALT, and ALP) at 25 and 45 days of age are shown in Table 14. The AST, ALT, and ALP were not affected by SM supplementation in broiler diets under heat stress at 25 days of age (p > 0.05). However, at 45 days of age, AST numerically decreased with the elevation of SM per cent, while 1.5% SM supplementation significantly decreased it compared to the control (p < 0.05). Also, diets containing 1.5, 4.5, and 6% SM significantly declined the ALT compared to the control (p < 0.05). SM supplementation in the broiler diets did not influence ALP and UA under heat stress conditions at 45 days of age. At 25 days of age, UA concentration decreased as a result with supplementation of 3 and 6% SM compared to control (p < 0.05), while dietary treatments did not influence UA at 45 days of age (p > 0.05). Adding 3, 4.5, and 6% SM to broiler diets effectively decreases blood glucose levels at 25 and 45 days of age (p < 0.05). Total protein levels had no change among experimental groups at 25 days of age (p > 0.05), while 6% SM significantly decreased the TP concentration at 45 days of age (p < 0.05).

Table 14. Effects of seed mixture (SM) on liver enzymes, srum uric acid, glucose and total protein at 25 and 45 days in broiler chicken under heat stress condition.

Treatments	25 days						45 days
	AST (U/L)	ALT (U/L)	ALP (U/L)	UA (mg/dL)	Glucose (mg/dL)	TP (mg/dL)	AST (U/L)	ALT (U/L)	ALP (U/L)	UA (mg/dL)	Glucose (mg/dL)	TP (mg/dL)
Control	34.90	20.94	803	73.75 ^a	218 ^a	2.59	87.25^a	9.89^a	463	31.6	181 ^a	2.97^a
1.5 % SM	21.23	20.36	826	45.00^ab	214 ^a	3.40	66.60^ab	4.56^b	637	32.2	171 ^a	3.51^a
3.0 % SM	43.62	13.95	828	36.87^b	164^b	2.83	60.49^ab	7.20 ^ab	777	21.2	140^b	3.01^a
4.5 % SM	54.09	15.70	744	46.67^ab	175^b	2.85	52.93^ab	3.49^b	616	25.0	150^ab	3.02^a
6.0 % SM	57.58	15.70	824	40.00^b	99^b	3.06	40.43 ^b	4.36^b	486	35.2	141^b	2.92^b
	----------------------Probability---------------------------------
Probability	0.0866	0.5875	0.5353	0.0457	0.0030	0.2841	0.0331	0.0051	0.3150	0.0657	0.0379	0.0035
SEM	4.89	1.54	16.99	3.85	13.41	0.17	5.28	0.70	51.24	0.18	5.71	0.06

^a-c Means in the same column with different letters are different significantly (p < 0.05).

Abbreviations: AST, Aspartate transaminase; ALT, Alanine transaminase; ALP, Alkaline phosphatase, UA, Uric acid; TP, Total protein.

Hepatic histopathology

Table 15 shows the effect of experimental diets on liver steatosis (liver fat vacuole score) and leukocyte infiltration. Liver steatosis decreased significantly in birds fed 3% SM compared to control and other levels of SM under heat stress conditions (p < 0.05). In contrast, the leukocyte infiltration showed no statistical difference and recorded a mild rate in all treatments under the same heat stress conditions (p > 0.05).

Table 15. Effects of seed mixture (SM) on liver histopathology in broiler chicken under heat stress condition at 46 day of age.

Treatments	Steatosis	Leukocyte infilteration score
Control	3.83^a	Mild
1.5 % SM	3.50^a	Mild
3.0 % SM	2.00^b	Mild
4.5 % SM	3.83^a	Mild
6.0 % SM	3.67^a	Mild
	-------Probability-------
Probability	0.0001	0.7368
SEM	0.198	0.107

^a-c Means in the same column with different letters are different significantly (p < 0.05).

Intestinal morphology

The effects of SM supplementation on the intestinal morphology of broilers are presented in Table 16. No significant differences existed in the duodenum, jejunum, and ileum morphology indices (villus height, crypt depth, villus height to crypt depth ratio, and villus surface area among all treatments (p > 0.05) under the same heat stress conditions. However, supplementation of different levels with SM numerically increased villus height, villus height to crypt depth ratio, and improved intestinal health compared to control.

Table 16. Effects of seed mixture (SM) on villus height (VH), crypt depth (CD), villus height to crypth depth ratio (VH/CD) and villus surface area (SA) in broiler chicken under heat stress condition at 46 day of age.

Treatments	Duodenum				Jejunum				Ileum
	VH (µm)	CD (µm)	VH/CD (µm)	SA (mm^2)	VH (µm)	CD (µm)	VH/CD (µm)	SA (mm^2)	VH (µm)	CD (µm)	VH/CD (µm)	SA (mm^2)
Control	1142	247	4.62	0.185	860	222	3.87	0.076	886	239	3.81	0.116
1.5 % SM	1439	194	7.48	0.189	1177	173	6.77	0.121	800	137	5.89	0.101
3.0 % SM	1222	222	5.59	0.163	1114	209	5.49	0.109	937	192	4.90	0.088
4.5 % SM	1484	252	6.10	0.192	865	217	4.02	0.090	928	187	5.08	0.101
6.0 % SM	1550	205	7.23	0.171	1205	201	5.98	0.113	1005	154	6.65	0.138
	------------------------probability-----------------------------
Probability	0.0658	0.3074	0.2901	0.4034	0.6783	0.9023	0.0759	0.8631	0.7585	0.1557	0.1655	0.4612
SEM	55.57	9.82	0.425	0.005	81.27	13.78	0.395	0.010	35.65	13.51	0.401	0.009

^a-c Means in the same column with different letters are different significantly (p < 0.05).

Apparent ileal digestibility

As mentioned in Table 17, the digestibility of dry matter, organic matter, and ash show no significant difference among treatments (p > 0.05). However, supplementation of 3% SM numerically improved dry matter, organic matter, and ash digestibility under heat-stress conditions.

Table 17. Effects of seed mixture (SM) on apparent ileal digestibility in broiler chicken under heat stress condition at 46 day of age.

Treatments	Dry matter	Organic matter	Ash
Control	95.89	79.98	81.05
1.5 % SM	94.05	79.28	85.12
3.0 % SM	98.05	89.77	94.06
4.5 % SM	96.53	80.97	87.85
6.0 % SM	93.13	74.31	83.90
	--------Probability------------
Probability	0.3177	0.5738	0.4797
SEM	0.796	2.876	2.282

^a-c Means in the same column with different letters are different significantly (p < 0.05).

Discussion

Based on our data, adding 6% of SM to the diet increased the BW of broiler chickens at 45 days of age and the AWG at 26-45 and 1-45 days of age under heat stress conditions. Corticosterones are released to compensate for physiologic disruptions caused by heat stress in the body (Star et al. 2008). Vahdatpour et al. (2008) reported that increased plasma levels of corticosterone reduced the FI and BW of broiler chickens. It seems that SM has been able to reduce the harmful effects of heat stress on performance due to the antioxidant activities of SM compounds. In such a way, Tewari et al. (2020) reported that fenugreek improved hepatic antioxidant defence enzyme activities, including superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx) in the ageing mice. Rastad et al. (2016) investigated that elevation of the SOD, catalase (CAT), and GPx activity could decrease the corticosterone levels in broiler chickens. Black seed could reduce corticosterone levels under heat stress conditions in broiler chickens (EL-Shoukary et al. 2014); fenugreek administration declines corticosterone under stress conditions in Nile tilapia (Basha et al. 2018). Therefore, supplementing SM with broiler diets can significantly improve AFI, BW, and AWG when heat stress increases during the finisher phase. In the same line with our results, EL-Shoukary et al. (2014) reported that introducing 1% black seed to broiler diets increased AWG from 1 to 3 weeks and improved FCR under heat stress conditions (p < 0.05). Khalaji et al. (2011) reported that the addition of 1% black seed to the broiler’s diet significantly increased BW and decreased FCR (p ≤ 0.01 and p ≤ 0.05, respectively). Yang et al. (2022) reported that supplementation of 50 and 100 mg fenugreek seed extract to 1 kilogram of broilers diets significantly increased final BW and average daily gain and improved the feed-to-gain ratio compared to control (p < 0.01). Alloui et al. (2011) observed that adding 3 g of fenugreek seed per 1 kilogram of broilers diets significantly (p < 0.05) increased BW and FI at 21 and 42 days of age. Also, a diet containing fenugreek could significantly improve FCR compared to the control (p < 0.05).

FI increased the following supplementation of SM to broiler diets except for the group that received 1.5% SM. Exposure to high ambient temperatures disrupts the metabolic and endocrine responses and causes several molecular, cellular, and immune dysfunctions (Sun et al. 2015). Heat stress causes a reduction in nutrient intake and alters nutrient digestibility and mRNA expression of nutrient transporters, impairing nutrient availability and growth performance (Habashy et al. 2017). Some researchers demonstrated that heat exposure changes the jejunal glucose and lipid transporters (Sun et al. 2015), and the relative mRNA expression of oligopeptide, sugar, and fatty acid transporters in the ileum and Pectoralis major muscles (Habashy et al. 2017). Habashy et al. (2017) reported that heat stress affects protein and fat retention in the Pectoralis major muscles due to reduced protein synthesis or increased protein breakdown.

Fenugreek is mentioned as an appetiser due to its galactomannan and neurin, which stimulate the brain’s appetite centre (Adil et al. 2015). Also, the aromatic compounds of black seed increase the activity of the digestive system, diet palatability, appetite, and digestion of nutrients in poultry (Gilani et al. 2004). Therefore, adding SM to the broiler diets caused increased feed intake throughout the entire rearing period, particularly at the finisher phase (26-45 days). Our result is in agreement with Yatoo et al. (2012), Weerasingha and Atapattu (2013), Mamoun et al. (2014), and Kumar et al. (2017). Generally, increasing feed consumption reduces the adverse effect of heat stress on weight gain and performance. Also, adding SM to diets improves heat tolerance in broiler chickens and creates a lower level of heat increment due to the considerable amount of oil content (33.76% based on our result).

Increasing FCR due to SM supplementation in broiler diets belongs to an increase in feed consumption, while growth has not changed in the same proportion. The mortality declined due to SM supplementation, especially in the 6% group. It is clear that adding SM to broiler diets prevents free radical production (in particular ROS), therefore protecting the integrity of the cell phospholipid membranes and saving the cells from pathogen attack, apoptosis, and several diseases (Ebeid 2009; Mahmoud et al. 2016).

The increase in the relative weight and length of the duodenum due to 6% SM might belong to the protective effect of SM compounds against cell apoptosis and free radical removal. Therefore, this leads to the reduction of damage to the intestinal cells and the reduction of their need for turnover (Ebeid 2009; Mahmoud et al. 2016).

The blood system is sensitive to changes in ambient temperature and is an indicator of broilers’ responses to stressors. Changes in blood cells reflect variations in blood traits such as HCT, WBCs, and Hb. Blood parameters such as RBC count, Hb, HCT, MCV, MCH, and MCHC at 25 days of age were not influenced by the inclusion of SM in the broiler diets under heat stress. Salam et al. (2013) and Waheed et al. (2017) showed that supplementation with black seed had no effects on the broiler blood parameters mentioned above. Also, Wahhed et al. (2017) investigated that fenugreek administration to broiler did not influence the RBC count, HCT, MCV, MCH, and MCHC. Alimohamadi et al. (2014) reported that RBC counts, Hb concentration, and HCT percentage were significantly higher in the chicks fed diets containing 8 g of black seed per kilogram of diets compared with those fed the control diet (p < 0.05). Adding sesame to the broiler diet at different levels (2, 4, 6, and 8%) under heat-stress conditions did not change the blood profile significantly (Oganija and Apata 2022). At 45 days of age, the RBC count, Hb, and MCV value were influenced by diets. Elevation of RBC count and Hb following supplementation of 3 and 6% SM, respectively, could be attributed to the lowered lipid peroxide in the erythrocyte membrane, resulting in decreased erythrocyte susceptibility to haemolysis (Alimohamadi et al. 2014). The MCV value increased in chickens fed with different levels of seed mixture. Acute and chronic heat stress affects the size of RBCs in chickens, making them microcytic (Sola-Ojo et al. 2019; Al-Mushhadani and Al-Hayali 2020). Khan et al. (2012) investigated that black seed administration to broilers’ diets significantly increased MCV value. Also, Al-Mushhadani and Al-Hayali (2020) reported that supplementation diets with sesame seeds increased the MCV value in broilers under heat stress.

It is expected that under stressful situations, the release of adrenocorticotropic hormone (ACTH) causes a reduction in the circulating lymphocytes, an elevation in the L/H ratio, and the involution of lymphoid tissue (thymus, spleen, and bursa). These events suppress humoral immunity and make the chicks susceptible to illness (Virden and Kidd 2009; Oganija and Apata 2022). However, according to our results, supplementing the SM to broiler diet under heat stress had no significant effect on differential WBC count, including granulocyte, heterophile, lymphocyte, Mid cells, and L/H ratio. Our finding agreed with Oganija and Apata (2022), which reported that administration of 2, 6 and 8% sesame oil to broiler diets did not change heterophile, lymphocyte, or monocyte, respectively basophile, eosinophile, and L/H ratio under heat stress. Salam et al. (2013) observed that supplementation of different levels of black seed to broiler diets did not change WBC count, monocyte, and eosinophile per cent under high ambient temperatures. Otherwise, some studies disagreed with this result; Shoukary et al. (2015) reported that supplementation of black seed to broiler diets increased lymphocyte, L/H ratio and decreased heterophile, monocyte, and eosinophile per cent under heat stress. Khalaji et al. (2011) reported that monocyte percentages were lower in chicks fed 1% black seed. Al-Mushhadani and Al-Hayali (2020) showed that including sesame seed in broiler diets decreased lymphocyte, monocyte, and granulocyte under heat stress.

The increase in blood lipids under heat stress was explained by Rashidi et al. (2010), that high temperature reduced feed intake, and broilers compensate for their need for energy by lipolysis of body lipids that it causes increasing the blood cholesterol and triglycerides. Stress can elevate total cholesterol levels by increasing LDL and decreasing HDL (Mundim et al. 2017). However, Tawfeek et al. (2014) reported that heat stress did not significantly influence blood plasma total cholesterol, VLDL cholesterol, and triglycerides. The SM components can adjust the lipid profile of the blood. Al-Beitawi et al. (2009) reported that supplementing broiler diets with 3% crushed and non-crushed black seeds reduced plasma cholesterol and triglycerides concentrations. Also, they increased the plasma HDL compared to 1.5, 2, and 2.5% crushed black seeds (Al-Beitawi et al. 2009). The reduction in triglycerides and cholesterol levels may be due to active ingredients such as thymoquinone that lower cholesterol synthesis by inhibiting HMG-CoA reductase activity, an allosteric enzyme necessary for cholesterol synthesis in the liver (Crowell 1999). It has been demonstrated that thymoquinone, one of the components of black cumin, can stimulate bile acid excretion and suppress de novo cholesterol synthesis (Badary et al. 2000). Consequently, our results suggest that SM with hypolipidaemic and hypocholesterolemic properties could improve the serum lipid profile under heat-stress conditions.

ALT and AST are important serological indexes reflecting liver injury (Tang et al. 2022). The reduction of these enzymes following the supplementation of 6% SM indicates that this mixture could improve the health status of the liver, which is reduced by heat stress. This effect is probably due to the antioxidant components of SM. Rama Rao et al. (2008) reported that the alkaline phosphatase activity decreased linearly following the replacement of the soybean meal with 0.33, 0.67, and 1.00 proportions of sesame in broiler diets. Liver steatosis (fat vacuole score) was the least in the experimental group that received 3% SM. This reduction in steatosis indicates the reduction of damage to the liver due to adding SM to diets. Leukocyte infiltration score had no change in our research, while Tang et al. (2022) reported that applying two weeks of heat stress-induced inflammatory infiltration in the liver of broilers.

The stress has a catabolic effect and produces the adrenocorticotropic hormone, yielding glucocorticoids. These events produce more serum glucose, uric acid, triglycerides (Borges et al. 2007), and protein (Whitehead and Keller 2003). Rashidi et al. (2010) reported that high environmental temperatures increased broilers’ serum glucose levels, cholesterol, and triglyceride levels. Kutlu and Forbes (1993) observed a decline in the protein concentration of plasma and an elevation in glucose and cholesterol in plasma as a result of the heat environment in broiler chicks. The decline in the concentration of glucose at 25 and 45 days of age, uric acid at 25 days of age, and total protein at 45 days of age as a result of SM supplementation indicates that SM had the potential to reduce the adverse effect of heat stress on their balance, especially at the level of 6%. Rama Rao et al. (2008) reported a linear decline in serum protein concentration due to the soybean meal replacement by 0.33, 0.67, and 1.00 proportions of sesame. Previous studies have reported that herbal seeds have a hypoglycaemic effect. Supplementing sesame, black cumin, and fenugreek seeds can decrease serum glucose levels, particularly in diabetic individuals (Meral et al. 2004; Roberts 2011; Al-Rikaby et al. 2020; Yargholi et al. 2021; Shabil et al. 2023). As a result, the decrease in serum glucose levels, especially at the 6% level of the SM, can be attributed to the hypoglycaemic effect of these seeds.

Although the addition of different levels of SM numerically increased villus height, villus height to crypt depth ratio, and villus surface area of the duodenum, jejunum, and ileum, these changes were not significant. Heat stress disrupts the intestinal mucosal barrier integrity and increases intestinal barrier permeability, leading to the high absorption of toxic agents into the enterocyte. Therefore, maintaining the intestinal barrier in an integrated form is essential for body homeostasis (Banan et al. 2001). Yang et al. (2022) reported that supplementation of 50 and 100 mg fenugreek seed extract to 1 kilogram of broilers diets significantly increased jejunum villus high compared to control (p < 0.01) and decreased ileum crypt depth compared to the zink bacitracin group (p < 0.05).

Conclusions

According to the results, adding a seed mixture to the broiler diets could compensate for the adverse effects of heat stress on performance and health with a more potent effect at a 6% level. It even had a hypolipidemic effect and decreased liver steatosis under heat-stress conditions. However, supplementation with a seed mixture positively affected broiler performance, increasing feed intake and weight gain and reducing mortality in broilers under heat stress conditions. Therefore, the inclusion of this mixture could be a beneficial way to adjust the negative effect of heat stress on broilers, particularly in hot climate places or in the summer.

Acknowledgements

The authors gratefully acknowledge Gold Fish for providing the premix concentrate.

Disclosure statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

To access all the data, request it through the link provided below: https://docs.google.com/spreadsheets/d/1gI1IKqztaVqU33W9NmjhYrAp37IlmVaa/edit?usp=sharing&ouid=108496959837745938947&rtpof=true&sd=true