Phocharapon Pasri

Sitthipong Rakngam

Nadine Gérard

Pascal Mermillod

Sutisa Khempaka
email: khampaka@sut.ac.th

DIETARY SYNTHETIC AND PHYTOGENIC ANTIOXIDANTS IN HEAT-STRESSED BREEDER HENS

Keywords: dietary antioxidant, breeder hen, heat stress, antioxidant activity, hatchability

This study aimed to investigate the efficacy of a synthetic source (a combination of vitamin E, vitamin C, Selenium, and L-carnitine) and phytogenic sources (a combination of clove, green tea pomace, and Vietnamese coriander) in overcoming heat stress (HS) damage in female breeder hens on production, sperm survival in the oviduct, antioxidant properties, gene expression, and quality of offspring. One hundred SUT female breeder hens were housed in individual cages and divided into four treatment groups: T1) basal diets under thermoneutral temperature (TN); T2) basal diets under HS; 3) basal diets with synthetic antioxidants under HS; and T4) basal diets with phytochemical antioxidants under HS. The result revealed that HS condition had a negative effect on reducing final body weight, FCR, egg weight, and 1-day old chick weight while increasing water intake and altered blood chemicals in breeder hens compared to TN breeder hens (P ˂ 0.01). However, both synthetic and phytogenic antioxidants resulted in increased egg production and hatchability, while decreasing the number of late stages of embryo death during the incubation (P < 0.05). Furthermore, the synthetic antioxidants also improved the uniformity of chicks and the late stage of embryo death compared with the phytogenic antioxidants (P < 0.05). HS breeder hens fed with both antioxidant sources exhibited higher antioxidant capacity in terms of DPPH and ABTS radical scavenging (in yolk, liver, and breast meat) and FRAP radical scavenging (in yolk and liver) and lower liver malondialdehyde than HS breeder hens fed with the control diet (P < 0.05).

Additionally, the gene expression of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) in liver was significantly upregulated, whereas the expression of proinflammatory cytokines (nuclear factor-κB) and heat shock proteins (HSP70 and HSP90) was downregulated in breeder hens that received both antioxidant sources (P < 0.05). This indicates the potential use of various bioactive antioxidant compounds as a combination of synthetic and phytogenic antioxidants against the deleterious effects of HS in breeder hens.

INTRODUCTION

In the context of global climate change, poultry production faces increasingly challenging conditions, particularly during heat stress (HS) episodes. High environmental temperature of 32 to 38°C coupled with high humidity could induce large amounts of reactive oxygen species (ROS) that lead to oxidative stress (OS) which negatively affects the integrity of sperm membrane and DNA in uterovaginal sperm storage tubules, consequently, influencing egg production, maturity, fertility, embryo development, and causing economic losses [START_REF] Fouad | Impact of heat stress on meat, egg quality, immunity and fertility in poultry and nutritional factors that overcome these effects: a review[END_REF]. Female breeder hens are sensitive to HS because their physiology is covered with feathers and there are no sweat glands that are difficult to thermoregulate. Previous studies have focused more on HS mitigation in housing management and genetic improvement against HS than on the body's capacity for ROS scavenging [START_REF] Gharaghani | Effect of fennel (Foeniculum Vulgare Mill.) used as a feed additive on the egg quality of laying hens under heat stress[END_REF]. HS has been reported to significantly reduce various key parameters in female breeder hens, including live weight, feed efficiency, egg production, egg quality, eggshell quality, fertility, hatchability, and the overall livability of hatchlings [START_REF] Ajakaiye | Impact of heat stress on egg quality in layer hens supplemented with L-ascorbic acid and DL-tocopherol acetate[END_REF]. Methods to overcome heat stress damage include supplementation with antioxidant substances, such as vitamin C, vitamin E, selenium (Se), manganese (Mn), zinc (Zn), or phytogenic substances [START_REF] Hu | Polyphenols as potential attenuators of heat stress in poultry production[END_REF]. In addition, the resolution of HS using a mixture of antioxidant substances from synthetic or natural sources that function at all levels of the antioxidant defense network can potentially alleviate the negative impact of HS. Unfortunately, little information is available regarding the use of mixtures from synthetic or phytogenic sources in breeder hens.

The high potential of dietary antioxidants are expected to cover three major levels of antioxidant defense networks: organelles, subcellular compartments, and the extracellular space [START_REF] Horváth | Impact of selected antioxidant vitamins (Vitamin A, E and C) and micro minerals (Zn, Se) on the antioxidant status and performance under high environmental temperature in poultry. a review[END_REF]. Antioxidant substances play a key role in activating antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), during the early stages of ROS formation, necessitating optimal levels of Mn, Cu, Zn, and Se for effective detoxification. However, some ROS, particularly transition metal ions, may remain active, causing lipid peroxidation and damage to DNA and proteins.

To address this issue, second-level antioxidants are required to break the chain by scavenging peroxyl radical intermediates to prevent the propagation of lipid peroxidation. These functions are performed by substances such as vitamins A, E, and C; carotenoids; coenzyme Q10; and L-carnitine. The third level of the antioxidant defense network works continuously from the second level, aiming to repair and remove damaged molecules (lipids, proteins, and DNA) affected by ROS. This process involves the action of heat shock proteins (HSPs), methionine sulfoxide reductases, DNA repair enzymes, and phospholipases (Surai and Kochish, 2019;Surai et al., 2019). Based on the antioxidant functions mentioned above, the challenge for nutritionists and feed formulators is to find suitable antioxidants that can effectively support hens in coping with HS conditions [START_REF] Surai | Antioxidant systems in chick embryo development. part 1. vitamin E, carotenoids and selenium[END_REF].

Previous studies have reported a synergistic effect of dietary vitamins E and C in poultry during HS on antioxidant status (reduced lipid peroxidation and enhanced SOD and CAT activities) [START_REF] Jena | Supplementation of vitamin E and C reduces oxidative stress in broiler breeder hens during summer[END_REF] and productive performance (improved fertile eggs, hatchability, and embryo mortality) [START_REF] Ipek | The effects of vitamin E and vitamin C on sexual maturity body weight and hatching characteristics of Japanese quails (Coturnix coturnix japonica) reared under heat stress[END_REF]. The combination of vitamin E and Se in diets provides highly effective protection against OS and improves the production and reproduction of poultry in comparison to the use of individual antioxidants [START_REF] Harsini | Effects of dietary selenium, vitamin E, and their combination on growth, serum metabolites, and antioxidant defense system in skeletal muscle of broilers under heat stress[END_REF][START_REF] Horváth | Impact of selected antioxidant vitamins (Vitamin A, E and C) and micro minerals (Zn, Se) on the antioxidant status and performance under high environmental temperature in poultry. a review[END_REF]. In addition, vitamin E and Se combinations in laying hen diets have been shown to transfer to the tissue and egg yolk, leading to decreased lipid peroxidation, increased GSH-Px enzyme levels, and enhanced hatchability and chick quality [START_REF] Urso | Vitamin E and selenium in broiler breeder diets: effect on live performance, hatching process, and chick quality[END_REF][START_REF] Celebi | Effect of dietary vitamin E, selenium and their combination on concentration of selenium, MDA, and antioxidant enzyme activities in some tissues of laying hens[END_REF]. The interaction between vitamins C and Se has also been reported to improve egg production and feed efficiency compared with individual supplementation [START_REF] Attia | Laying performance, digestibility and plasma hormones in laying hens exposed to chronic heat stress as affected by betaine, vitamin C, and/or vitamin E supplementation[END_REF]. The combination of vitamins E, C, and Se in the diet can act synergistically as antioxidants to reduce HS and lipid peroxidation in poultry meat [START_REF] Leskovec | Antioxidative effects of supplementing linseed oil-enriched diets with αtocopherol, ascorbic acid, selenium, or their combination on carcass and meat quality in broilers[END_REF]. Furthermore, the use of L-carnitine has gained significant attention due to its crucial role as a novel antioxidant via preventing DNA damage induced by ROS and stimulating antioxidant enzyme activities, transferring long-chain fatty acids to maintain βoxidation in the matrix compartment of mitochondria and energy production by stabilization acetyl Co-A/Co-A ratio for adequate acetyl storage for laying hens [START_REF] Surai | Silymarin as a natural antioxidant: an overview of the current evidence and perspectives[END_REF][START_REF] Agarwal | Role of L-carnitine in female infertility[END_REF]. Dietary L-carnitine supplementation in poultry decreases malondialdehyde (MDA) levels and increases SOD, CAT, and GSH-Px activities under high stocking density stress [START_REF] Çetin | Effect of dietary L-carnitine supplementation and energy level on oxidant/antioxidant balance in laying hens subjected to high stocking density[END_REF]. In addition, some studies have reported improved egg production, hatchability, antioxidant activity, and offspring quality in laying hens and duck breeders fed Lcarnitine [START_REF] Salmanzadeh | The effect of dietary L-carnitine supplementation on egg production, egg weight, and hatching traits of broiler breeder hen from 32 to 36 weeks of age[END_REF][START_REF] Wang | Effects of dietary L-carnitine supplementation on growth performance, organ weight, biochemical parameters and ascites susceptibility in broilers reared under low-temperature environment[END_REF][START_REF] Awad | Effect of sex and Lcarnitine addition on growth performance and carcass quality of Sudani ducklings[END_REF].

Phytogenic compounds consist of a variety of polyphenols derived from plant materials that can activate the expression of stress response proteins, such as HSPs and antioxidant enzymes, which can repress ROS and interfere with negative inducers in the HS response [START_REF] Hu | Polyphenols as potential attenuators of heat stress in poultry production[END_REF][START_REF] Saracila | Heat stress in broiler chickens and the effect of dietary polyphenols, with special reference to Willow (Salix spp.) Bark supplements-a review[END_REF]. The most bioactive compound in cloves (Syzygium aromaticum) is eugenol, which is a potent natural antioxidant [START_REF] Hemalatha | Phytochemical composition, GC-MS analysis, in vitro antioxidant and antibacterial potential of clove flower bud (Eugenia caryophyllus) methanolic extract[END_REF]. Supplementation of poultry diets with clove oil or powder can improve feed efficiency, egg production, immunity, and antioxidant activity [START_REF] Sehitoglu | The Effect of clove oil supplementation in laying hen diets on performance, egg quality, some blood parameters, and yolk TBARS[END_REF][START_REF] Mahrous | Effects of different levels of clove bud (Syzygium aromaticum) dietary supplementation on immunity, antioxidant status, and performance in broiler chickens[END_REF]. Green tea (Camellia sinensis) is rich in polyphenols, particularly catechins, that exhibit antioxidant, antimicrobial, antifungal, and anticarcinogenic properties [START_REF] Pinto | Quantification of polyphenols and metals in Chinese tea infusions by mass spectrometry[END_REF]. It has been shown that supplementation of green tea in breeder poultry diets (powder or extraction form) can reduce MDA egg yolk concentrations, while increasing fertility, hatchability, and sperm quality [START_REF] Kara | Influence of catechin (flavan-3-ol) addition to breeder quail (Coturnix coturnixjaponica) diets on productivity, reproductive performance, egg quality and yolk oxidative stability[END_REF][START_REF] Chen | Dietary green tea powder supplementation enriched egg nutrients and physicochemical property in an indigenous chicken breed[END_REF][START_REF] Wang | The mixed application of organic and inorganic selenium shows better effects on incubation and progeny parameters[END_REF]. Vietnamese coriander (Persicaria odorata) has a high phytochemical composition, especially gallic acid, quercetin, ferulic acid, apigenin, and essential oils, which contribute to its antioxidant and biological activities [START_REF] Pawłowska | Changes in the phenolic contents and composition of Persicaria odorata fresh and dried leaves[END_REF]. Supplementing broiler or laying hen diets with Vietnamese coriander leaf meal has shown beneficial effects on growth performance, digestibility, egg production, egg weight, and lipid peroxidation in meat [START_REF] Basit | Effects of graded dose dietary supplementation of Piper betle leaf meal and Persicaria odorata leaf meal on growth performance, apparent ileal digestibility, and gut morphology in broilers[END_REF][START_REF] Ooi | Effect of local medicinal herbs as feed additives on production performance and faecal parameters in laying hens[END_REF][START_REF] Glinubon | Effect of dietary supplementation with Vietnamese coriander (Persicaria odorata) extract on growth performance, carcass characteristics and meat quality of broilers[END_REF]. In our previous in vitro study, we examined 17 edible plant materials; clove, green tea pomace, and Vietnamese coriander showed notably high levels of phenolic and total flavonoid content as well as strong antioxidant activity in terms of DPPH, ABTS, and FRAP. The combination of these plants in equal parts (1:1:1 ratio, v:v:v) produced synergistic antioxidant properties and improved cell safety, making them suitable candidates for use as phytogenic antioxidant feed additives [START_REF] Pasri | Antioxidant properties and cytotoxic effects of selected edible plants in Southeast Asia for further use as phytogenic antioxidant additives[END_REF].

Unfortunately, no information is available on the combined effects of vitamins E, C, Se, L-carnitine, and phytogenic on the productive performance and antioxidant activities of breeder hens under HS that would be applicable to the development of a potential group of feed additives to alleviate the adverse effects of HS. Therefore, the aim of this study was to investigate the efficacy of two sources of antioxidant substances, synthetic (combination of vitamin E, vitamin C, Se, and L-carnitine) and phytogenic (combination of clove, green tea pomace, and Vietnamese coriander), to overcome HS damage in breeder hens and prolong sperm survival in the oviduct, antioxidant properties, gene expression, and quality of offspring.

MATERIALS AND METHODS

Ethics Statement

All animal experiments were approved by the Animal Care and Use Committee of Suranaree University of Technology (SUT-IACUC-012/2020).

Housing, Birds and Experimental diets

This study maintained controlled temperature (°C) and humidity (%) levels according to the temperature and humidity stress indices for laying hens [START_REF]Understanding heat stress in layers: Management tips to improve hot weather flock performance[END_REF]. Thermoneutral (TN) and chronic HS were implemented according to the methodology described by [START_REF] Duangjinda | Hsp70 genotypes and heat tolerance of commercial and native chickens reared in hot and humid conditions[END_REF]. The TN was set up at 23±1°C with the humidity of 4070% by using an air conditioner. The HS room was kept at a temperature of 36±1°C for 4 h daily (from 1 pm to 5 pm) using a gas heater with thermostat-controlled equipment, while during the rest of time, the temperature was maintained at the same conditions in the thermoneutral zone.

A total of 100 female SUT breeder hens (33 weeks old) were housed in individual cages with dimensions of 40 × 45 × 40 cm 3 (length × width × height) and divided into four treatment groups, each consisting of 25 females, using a Completely Randomized Design. Group 1 was raised in a TN room, whereas groups 2, 3, and 4 were subjected to HS for 4 h daily.

In this study, two sources of antioxidants (synthetic and phytogenic) were evaluated in female SUT breeder hens under HS conditions. The experimental diets consisted of four treatments as follows: T1) basal diets under thermoneutral temperature, T2) basal diets under HS; 3) basal diets with combined synthetic antioxidants (200 mg of vitamin C/kg, 150 mg of vitamin E/kg, 0.30 mg of Se yeast/kg, and 150 mg of carnitine/kg) under HS, and T4) basal diets with 1% phytochemical antioxidants (a mixture of clove, green tea pomace, and Vietnamese coriander powders, 1:1:1 ratio/w:w:w) under HS. Diets were formulated to meet the nutrient requirements according to the NRC (1994) and Ross 308 parent stock standard recommendations (Aviagen. 2021) (15% CP, 2800 kcal ME/kg), as shown in Table 1. All breeder hens were provided 16 h of light per day, received 140 g of feed daily, and had unrestricted access to water throughout the experimental period. Each experimental diet was fed to breeder hens for approximately one month prior to starting the trial.

Blood Chemical Analysis

After the hens were subjected to HS for 4 h, 12 breeder hens from each treatment group were randomly selected for blood chemical analysis using an Abbott i-STAT 1 handheld blood gas analyzer (Abbott Point of Care Inc. IL, USA) equipped with a CG8+ cartridge (Abaxis item number 600-9001). This CG8+ cartridge performed various parameters such as partial pressure of carbon dioxide (PCO2) and oxygen (PO2), pH, saturation of oxygen (sO2), concentration of bicarbonate ions (HCO3 -), total concentration carbon dioxide (TCO2), concentration ionized calcium (iCa), sodium (Na), potassium (K), glucose (Glu), hematocrit (Hct), hemoglobin (Hgb), and base excess (BE). Blood from each breeder hen was collected and placed in a lithium heparin tube quickly, which should be used for analysis within 3 min. One hundred μL of blood was dropped into the CG8+ cartridge, which was subsequently inserted into the Abbott i-STAT 1 handheld blood gas analyzer [START_REF] Barrett | Effects of acute and chronic heat stress on the performance, egg quality, body temperature, and blood gas parameters of laying hens[END_REF].

Growth Performance and Productive Performance Measurements

Productive performance parameters were measured over a 7-week period from 38 to 44 weeks of age. Body weight was recorded at the beginning and end of the experiment. Daily records were made for the number of eggs, egg weight, feed intake, and water intake in each treatment throughout the experimental period and were used to calculate egg production, feed conversion ratio, average egg weight, average daily feed, and water intake.

The fertile period length of sperm was determined from eggs collected over 21 days (38-41 weeks of age). Semen samples were collected from 60 Lueng Hang Khao breeder males by pooling and diluting with Beltsville poultry semen extender II (1:1/v:v) prior to artificial insemination. The breeder hens were artificial inseminated two days continually (0.1 mL of pooled semen/time) and then the hens in groups 2, 3, and 4 were exposed to HS at 36ºC for 4 h/day. On day 3, eggs from all treatments were collected over a 21-day period and stored in a cool room at 15ºC. Every 7 days, these eggs were then placed in automatic incubator (Model 192,Petersime Incubation Equipment Co.,Ltd.,Zulte,Belgium) with optimal conditions at 37.67±0.20°C and 62-65% relative humidity at the hatchery of university farm. The fertile period of the sperm was candled on day 7 of incubation. When the infertile eggs broke, the germinal disc region was monitored for embryonic development. The number of days for the fertile period of sperm was counted from the last day of the fertile egg prior to a sequence of three consecutive days with detected infertile eggs [START_REF] Biswas | Effect of vitamin E on production performance and egg quality traits in Indian Native Kadaknath hen[END_REF][START_REF] Ahammad | Effects of fluid secreted from the uterus on duration of retile egg production in hen, and survivability and penetrability of fowl sperm in vitro[END_REF].

Fertility, hatchability, and embryonic mortality rate were performed over a period of 41-44 weeks of age. Each breeder hen was artificially inseminated twice per week with pooled semen in the afternoon, and induced daily with heat stress at 36°C for 4 h. Then eggs in each treatment were collected daily and stored in a cool room at 15°C. Each week, the eggs were incubated in an automatic incubator for 21 days. Fertility and early and final embryonic mortality rates were detected by candling on days 10 and 18, whereas hatchability was measured on day 21 of incubation by counting the number of 1-day old chicks. Productive performances were calculated using formulas: fertility (%) = [(number of fertilized eggs/total eggs set) × 100]; early embryonic mortality rate (%) = [(number of fertilized eggs before day 10/total eggs set) × 100]; late embryonic mortality rate (%) = [(number of fertilized eggs after day 10/total eggs set) × 100]; hatchability (%) = [(number of day old chicks/ fertilized eggs) × 100], and total hatchability (%) = [(number of 1-day old chicks/total eggs set) × 100] [START_REF] Salmanzadeh | The effect of dietary L-carnitine supplementation on egg production, egg weight, and hatching traits of broiler breeder hen from 32 to 36 weeks of age[END_REF][START_REF] Urso | Vitamin E and selenium in broiler breeder diets: effect on live performance, hatching process, and chick quality[END_REF].

Sample Collection and Sample Extraction for Antioxidant Activity

At the end of the experiments, 25 breeder hens were randomly selected and slaughtered after heating at 36 ºC for 4 h. Liver and breast tissues were collected, immediately frozen in liquid nitrogen, and stored at -80°C until further gene expression and antioxidant activity analyses. Two grams each of egg yolk, liver, and breast tissue were extracted with 2 mL of 99% ethanol in a centrifugal tube. The samples were grinded for 20 s by ultra-homogenizer after that centrifuged at 12,000 x g at 4°C for 10 min. The supernatants were used to estimate the antioxidant activity.

2,2-Diphenyl-1-picrylhydrazyl (DPPH • ) Scavenging Activity Assay

DPPH • scavenging activity was determined according to the method described by [START_REF] Nuengchamnong | Rapid screening and identification of antioxidants in aqueous extracts of Houttuynia cordata using LC-ESI-MS coupled with DPPH assay[END_REF]. For each sample extraction (100 µL), 100 µL of 0.6 mM DPPH in ethanol was added to a 96 well microplate. The mixture was gently shaken and incubated in dark for 30 min. The absorbance of the reaction mixture was measured at 517 nm using a microplate spectrophotometer (Thermo Scientific TM , Multidkan TM GO, Japan). Ethanol was used as a reagent blank instead of the sample. The DPPH • -scavenging activity was calculated as follows: inhibition (%) = [(absorbance of blank-absorbance of sample)/(absorbance of blank)] × 100. All measurements were performed in triplicate.

Scavenging Activity Assay of 2, 2ˈ-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS •+ ) Radical

The ABTS •+ cation radical assay was conducted as described by [START_REF] Re | Antioxidant activity applying an improved ABTS radical cation decolorization assay[END_REF]. The ABTS •+ stock solution was prepared by mixing 7.4 mM ABTS with 2.6 mM of potassium persulfate in a 10 mM phosphate buffer solution at pH 7.4. The mixture was then left to react overnight (12-16 h) in the dark at 4°C. Before starting the reaction, the freshly prepared ABTS •+ stock solution was adjusted to an absorbance value of 0.70±0.02 at 734 nm by dilution with a 10 mM cooled phosphate buffer. For the assay, 180 µL of ABTS •+ working solution was added to 20 µL of the extracted sample solvents in a 96 well microplate.

After 6 min of inoculation at room temperature, absorbance at 734 nm was measured using a microplate spectrophotometer. The absorbance of the blank was measured using ethanol used in the reaction. The ABTS •+ cation radical was calculated using the following equation: inhibition (%) = [(absorbance of blank-absorbance of sample)/(absorbance of blank)] × 100. The results were compared to the standard curve and reported in mM equivalent trolox/g sample weight (mM TE/g sample).

The Ferric Reducing Antioxidant Power (FRAP) Assay

The FRAP assay was conducted according to the method described by [START_REF] Benzie | The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": the FRAP assay[END_REF]. The working FRAP reagent required fresh preparation before use, consisting of 0.3 M acetate buffer (pH 3.6), 10 mM 2,4,6-Tris (2-pyridyl)-s-triazine in 40 mM hydrochloric acid, and 20 mM iron chloride, mixed at a ratio of 10:1:1/v:v:v. The reagent was incubated at 37°C for 15 min. The 200 uL of the FRAP working reagent was added to 20 µL of sample extraction solvents in a 96 well microplate and incubated for 30 min, and the absorbance was then measured using a microplate spectrophotometer at 593 nm. The FRAP value was calculated using a calibration curve of trolox (25100 mM/mL) and the results were reported as mM Trolox equivalents per gram of sample weight (mM TE/g sample).

Thiobarbituric Acid Reactive Substances (TBARs)

Egg yolk, liver, and breast tissue (2 g) were homogenized with 6 mL of DI water and 34 µL of 7.2% butylated hydroxytoluene (BHT) in ethanol using ultra-homogenizer for 40 s.

Subsequently, 2 mL of the homogenized sample was mixed with 4 mL of TBA-TCA solution (20 mM TBA in 15% TCA) in a 15 mL tube and boiled at 95°C for 20 min in an ultrasonic bath (Ultrasonic cleaner 3200 EP S3, Soltec, Italy, 40 KHz and 180 W). After cooling, the mixture was centrifuged at 5,000 x g for 10 min at room temperature. The supernatant (200 µL) was then transferred to a 96 well microplate, and absorbance readings were taken at 532 nm. To quantify lipid peroxidation, MDA was used as a standard at concentrations ranging 5-

Hepatic Gene Expression

Total RNA was extracted from liver tissue using QIAamp ® DNA Stool Mini kits (Qiagen, Hilden, Germany) and purified using a QIAamp spin column (Qiagen, Hilden, Germany). RNA purity and quantification were assessed using a Nanodrop spectrophotometer at 260 nm/280 nm. Subsequently, 1 μg of high quality RNA sample was applied for complementary DNA (cDNA) synthesis using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). For real time PCR, each reaction's master mix (8 µL) contained 5 µL of SYBR Green, 0.2 µL of forward primer, 0.2 µL reverse primer, and 2 µL of cDNA samples in a 96 well microplate. The real time PCR was performed using the QuantiNova™ SYBR Green PCR kit (Qiagen, Hilden, Germany) and analyzed in triplicate as described by [START_REF] Humam | Effects of feeding different postbiotics produced by Lactobacillus plantarum on growth performance, carcass yield, intestinal morphology, gut microbiota composition, immune status, and growth gene expression in broilers under heat stress[END_REF]. The primer sequences for SOD, CAT, GSH-Px, nuclear factor-κB (NF-κB), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), and β-actin are presented in Table 2. Reverse transcription-quantitative real-time PCR (RT-qPCR) was accomplished using the CFX96 real-time PCR system (BioRad, Hercules, California, USA). The RT-qPCR reactions were conducted as follows: initial heat activation at 94°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing for 30 s, and final extension at 72°C for 30 s. Relative quantification of the target gene expressions was normalized using β-actin as the reference gene and calculated using the 2 -∆∆CT method [START_REF] Livak | Analysis of relative gene expression data using realtime quantitative PCR and the 2 -∆∆c T method[END_REF].

Statistical Analysis

Statistical analysis of the data was performed using ANOVA in a Completely Randomized Design (CRD) with SPSS software (version 16.0). Tukey's test was used to assess significant differences among treatments. Additionally, orthogonal contrasts were used to compare the following conditions: 1, thermoneutral vs. heat stress; 2, supplementation vs. nonsupplementation; and 3, synthetic vs. phytogenic conditions. A significance level at P ˂ 0.05 was used (SPSS Inc., Chicago, IL).

RESULTS

Productive Performances

The effects of antioxidant supplementation in the form of synthetic (vitamins C, E, Se, and L-carnitine) and phytogenic (clove, green tea pomace, and Vietnamese coriander) substances on production performance are shown in Table 3. In the orthogonal contrast test, HS breeder hens had lower final body weight (FBW), FCR, egg weight (EW), and day-old chick weight, but higher hatchability and water intake (WI) than TN hens (P ˂ 0.01). Both the synthetic and phytogenic antioxidant supplementation groups showed increased egg production (EP) and hatchability as well as reduced feed intake (FI), WI, EW, early dead and last dead embryos, and old chick weight compared to the non-supplementation group (P < 0.05). However, no significant differences were observed in any of the measured parameters between the synthetic and phytogenic antioxidants (P > 0.05). Interestingly, based on the Tukey analysis, it was found that the supplementation of antioxidants in the HS group could increase EP, similar to that in the TN group (P > 0.05). In addition, both forms of antioxidant supplementation resulted in significantly higher hatchability compared than in the TN group (P = 0.0001). However, a reduction in FBW, FI, EW, and day-old chicks was observed in HS breeder hens that received phytogenic antioxidants compared to TN hens, whereas no such significant differences were observed for synthetic antioxidants (P > 0.05). Additionally, supplementation with synthetic antioxidants demonstrated an improvement in the last stage of embryo development during incubation and chick uniformity compared with phytogenic antioxidants (P ˂ 0.05).

Blood Chemistry Parameters

Based on orthogonal contrasts, breeder hens subjected to HS had lower blood values of PCO2, PO2, BE, HCO3 -, TCO2, and iCa, while pH and sO2 were higher than the TN breeder hens (P < 0.01) (Table 4). In addition, HS led to decreased blood Na, K, Hct, and Hb and increased blood Glu compared to TN (P < 0.01). However, under HS conditions, supplementation with both synthetic and phytogenic antioxidant substances did not significantly alter any of the blood parameters compared to the non-supplementation group (P > 0.05). In particular, Tukey's test revealed that HS breeder hens supplemented with phytogenic antioxidants had lower blood Na and K levels than the TN group (P < 0.05).

Antioxidant Activities in Liver, Breast, and Yolk

The effects of antioxidant supplementation in the form of synthetic and phytogenic substances in breeder hen diets on antioxidant activity are shown in Table 5. According to the orthogonal contrast test, the HS breeder hen groups had significantly higher antioxidant activities in terms of DPPH radical scavenging in egg yolk and liver, and FRAP radical scavenging in the liver, but had lower levels of MAD in egg yolk than breeder hens in the TN group (P < 0.05). Both antioxidant supplementation groups exhibited significantly highest antioxidant activities in DPPH, FRAP, and ABTS radical scavenging in all samples (except FRAP in breast tissue), as well as a decrease in MDA in egg yolk compared to the nonsupplementation group during HS (P ˂ 0.05). The breeder hens fed with synthetic antioxidants exhibited the highest DPPH and FRAP radical scavenging activity and the lowest MDA levels in egg yolk compared to those fed with phytogenic antioxidants (P ˂ 0.01). Furthermore, supplementation with synthetic antioxidants in breeder hen diets also improved the values of DPPH, FRAP, and MDA in the liver and yolk compared to non-supplementation under both HS and TN conditions (P ˂ 0.05).

Moreover, DPPH (in egg yolk and breast tissue), FRAP (in egg yolk and liver), and ABTS radical scavenging (in egg yolk, liver, and breast tissue) were elevated, whereas MDA levels (in egg yolk) were reduced in breeder hens fed phytogenic dietary supplementation compared to breeder hens without supplementation under HS (P > 0.05).

Gene Expression in Liver

The gene expression levels related to antioxidant enzymes, pro-inflammatory cytokines, and heat shock proteins are presented in Table 6. In orthogonal contrasts, upregulation of SOD and GHS-Px genes and downregulation of HSP70 and HSP90 genes were observed in HS breeder hens compared to those in TN breeder hens (P ˂ 0.05). However, no significant differences in the expression of the CAT and NF-κB genes were found between the HS and TN groups (P > 0.05). During HS, it was found that both synthetic and phytogenic antioxidants can alter gene expression in the liver by upregulating SOD, CAT, and GSH-Px and down regulating NF-κB, HSP70, and HSP90 compared to non-supplementation (P ˂ 0.01).

It is interesting to note that phytogenic antioxidants were found to induce the expression of the GSH-Px gene in a similar manner to synthetic antioxidants (P ˂ 0.05). In addition, both synthetic and phytogenic antioxidants showed similar effects on the expression of SOD, CAT, NF-kB, HSP70, and HSP90 under HS conditions (P > 0.05).

DISCUSSION

Exogenous dietary antioxidants are widely accepted as effective substances for mitigating the adverse effects of HS on breeder hens in terms of favorable productive and reproductive performance, immunity, embryonic development, and antioxidant activity [START_REF] Ibtisham | The effect of ginger powder and Chinese herbal medicine on production performance, serum metabolites and antioxidant status of laying hens under heat-stress condition[END_REF][START_REF] Amevor | Combination of quercetin and vitamin E supplementation promotes yolk precursor synthesis and follicle development in aging breeder hens via liver-blood-ovary signal axis[END_REF][START_REF] Darmawan | Dietary phytogenic extracts favorably influence productivity, egg quality, blood constituents, antioxidant and immunological parameters of laying hens: a metaanalysis[END_REF]. Our results revealed the beneficial effects of synthetic (vitamins C, E, Se, and L-carnitine) and phytogenic (clove, green tea pomace, and Vietnamese coriander) antioxidants on egg production, hatchability, embryo development, and antioxidant activity of breeder hens under HS.

In this study, both synthetic and phytogenic antioxidant sources were found to improve egg production and hatchability in breeder hens subjected to HS. However, in the orthogonal contrast test, the FI in the HS hen groups supplemented with antioxidants were lower than that in the HS hen group without supplementation or the TN hen group. This led to insufficient nutrient uptake. However, these groups of hens were still able to maintain egg production. This is probably because birds anabolize fat storage in the body to conserve the nutrients needed for maintenance and production, which can be confirmed from the reduced BW of HS hens. In general, HS poultry require higher maintenance energy than usual for thermoregulation.

However, the decrease in FI during HS results in sufficient nutrient intake, as chickens compensate by breaking down glycogen or fat stored through gluconeogenesis [START_REF] Nawaz | Poultry response to heat stress: its physiological, metabolic, and genetic implications on meat production and quality including strategies to improve broiler production in a warming world[END_REF]Jastreski et al., 2017). In addition, [START_REF] Xie | Effects of acute and chronic heat stress on plasma metabolites, hormones and oxidant status in restrictedly fed broiler breeders[END_REF] reported that elevated plasma glucose levels in HS broiler breeders indicate changes in carbohydrate and lipid metabolism for maintaining the metabolic rate, which is consistent with our results, which revealed increased blood glucose in HS breeder hens. In addition, this study found reductions in PCO2, PO2, BE, HCO3 -, TCO2, and iCa, along with an increase in pH and sO2 in the blood of HS breeder hens.

The current study indicates that both antioxidant sources play interconnected roles in metabolic pathways, which effectively assists breeder hens in combating the negative effects of HS. During HS, vitamin C serves as a co-factor for dopamine beta-hydroxylase, converting dopamine into norepinephrine in neural tissues during the HS, promoting gluconeogenesis, which indirectly causes an increase in heart rate, blood pressure, blood glucose, and skeletal muscle blood flow [START_REF] Shakeri | Strategies to combat heat stress in broiler chickens: Unveiling the roles of selenium, vitamin E and vitamin C[END_REF]. Vitamin E prevents liver damage and maintains vitellogenin synthesis, which is important for yolk formation and consequences of egg production [START_REF] Khan | Effect of vitamin E in heat-stressed poultry[END_REF]. L-carnitine is crucial for transferring long-chain fatty acids from the cytoplasm to the mitochondrial matrix for βoxidation and energy production, facilitating the anabolism of stored fat in bird bodies, which ultimately provides energy for follicle development and egg production [START_REF] Zhai | The effect of male and female supplementation of L-carnitine on reproductive traits of White Leghorns[END_REF][START_REF] Awad | Effect of sex and Lcarnitine addition on growth performance and carcass quality of Sudani ducklings[END_REF]. In addition, Se is essential for optimal poultry performance during HS, indirectly regulating triiodothyronine (T3) and thyroxine (T4) hormones, which affect the metabolic rate, protein synthesis, and nutrient metabolism [START_REF] Shakeri | Strategies to combat heat stress in broiler chickens: Unveiling the roles of selenium, vitamin E and vitamin C[END_REF].

The improved hatchability and antioxidant capacity observed in breeder hens that received dietary phytogenic antioxidants in this study can be attributed to the deposition of bioactive antioxidant compounds. In phytogenic sources (clove, green tea pomace, and Vietnamese coriander), several bioactive compounds are associated with antioxidant properties, such as eugenol (from clove), catechin (from green tea pomace), and catechin, quercetin, kaempferol, and ellagic acid (from Vietnamese coriander). These polyphenols are potent antioxidants because their chemical structure contains more than two hydroxyl groups (PhenOH), which allow them to break bonds and release hydrogen and electrons. This effectively eliminates excess ROS (such as O2, H2O2, OH • , RO • , and RO2 • ) and transforms them into a phenoxyl radical (PhenO), which is more stable and less likely to initiate chain reactions than the initial radicals. Polyphenols can also transfer electrons to bind to metal-ion free radicals (such as Fe 2+ , Cu 2+ , or Cu + ) [START_REF] Saracila | Heat stress in broiler chickens and the effect of dietary polyphenols, with special reference to Willow (Salix spp.) Bark supplements-a review[END_REF]. Dietary catechins or tea polyphenols have been shown to be transferred from the blood to the ovaries, magnum, and other organs, providing antioxidant capacity and stability for polyunsaturated fatty acids (PUFA) in eggs [START_REF] Ariana | Effects of powder and extract form of green tea and marigold, and α-tocopheryl acetate on performance, egg quality and egg yolk cholesterol levels of laying hens in late phase of production[END_REF]. [START_REF] Chen | Dietary green tea powder supplementation enriched egg nutrients and physicochemical property in an indigenous chicken breed[END_REF] observed catechin deposition in chickens fed with green tea, which led to increased DPPH, ABST, and OH radical scavenging activities. Additionally, clove oil or buds with high eugenol levels showed the potential to decrease MDA in eggs and increase the enzyme activity of reduced glutathione (GSH), SOD, and glutathione S-transferase (GST) in the breast muscle [START_REF] Mahrous | Effects of different levels of clove bud (Syzygium aromaticum) dietary supplementation on immunity, antioxidant status, and performance in broiler chickens[END_REF][START_REF] Sehitoglu | The Effect of clove oil supplementation in laying hen diets on performance, egg quality, some blood parameters, and yolk TBARS[END_REF]. Feeding broilers with a mixture of herbal extracts (mulberry leaf, Japanese honeysuckle, and Goldthread) resulted in the accumulation of phenolic compounds in breast tissue and demonstrated the potential to increase DPPH and ABST and reduce TBARs [START_REF] Jang | Antioxidative potential of raw breast meat from broiler chicks fed a dietary medicinal herb extract mix[END_REF]. In this study, various bioactive polyphenol compounds, such as eugenol, catechin, quercetin, kaempferol, and ellagic acid, were detected, all of which could complement the mechanisms of action to eliminate ROS. However, there are limitations to the metabolism and bioavailability of polyphenols, as some are poorly absorbed in the small intestine and require enzymatic hydrolysis by gut microbes [START_REF] El-Hack | Pharmacological, nutritional and antimicrobial uses of Moringa oleifera Lam. leaves in poultry nutrition: an updated knowledge[END_REF]. The rapid absorption and elimination of polyphenols is a major factor leading to their low accumulation in tissues compared to that of synthetic antioxidants; thus, frequent supplementation could potentially improve their biological activity [START_REF] Hidalgo | Metabolism of anthocyanins by human gut microflora and their influence on gut bacterial growth[END_REF].

Although both the antioxidant sources maintained egg production, a reduction in egg weight was observed. HS is known to reduce FI in poultry, resulting in insufficient nutrient intake, particularly protein, which leads to a decrease in egg weight. [START_REF] Khatibi | Effect of diet nutrients density on performance and egg quality of laying hens during the post-peak production phase of the first laying cycle under subtropical climate[END_REF] revealed that laying hen diets with protein levels of 15.0-15.5% CP under a subtropical climate can improve production, egg weight, and egg mass compared to diets with 14.2-14.7% CP.

Although both antioxidants in this study reduced egg weight compared to the control and HS without supplementation, the weight was still in the range of 50-70 g, which was suitable for hatching following the Cobb 500 or Ross 308 breeder guidelines. In general, egg weight is associated with chick weight, indicating that both antioxidant sources can help maintain egg weight, resulting in a normal chick weight, which benefits the poultry industry.

Neither the HS conditions nor antioxidant supplementation affected the fertility rate or fertile period length of the sperm. However, this phenomenon remains unclear, and related research on this subject is limited. HS can adversely affect the reproductive performance of both male and female poultry; however, its effects on male fertility, specifically spermatogenesis, have been extensively studied [START_REF] Fouad | Impact of heat stress on meat, egg quality, immunity and fertility in poultry and nutritional factors that overcome these effects: a review[END_REF]. In this study, we attempted to minimize the sperm factor error by using artificial insemination techniques and pooling sperm. Based on our results, the fertility and fertile period length of sperm in breeder hens aged 4750 weeks were 9899% and 15 d, respectively. Notably, this fertility rate was higher than that of the Cobb 500 breeder hens (95%) (Cobb-Vantress Inc. 2018). This is consistent with previous reports in which the combination of vitamins C, E, and Se in ISA brown laying hen diets did not affect the fertility rate (7780%) or fertile period length of sperm (17 days), possibly because the hens reached their maximum productivity aligned with their genetic potential [START_REF] Pasri | Effects of dietary energy, vitamin C, vitamin E and selenium in maternal diets on productive performance, fertile period length of sperm and antioxidant activity in uterine fluid[END_REF].

Notably, the hatchability rate in HS hens supplemented with both antioxidant sources was higher than that in TN and HS hens without supplementation. This is consistent with the results of increased antioxidant activities (DPPH, ABTS, and FRAP radical scavenging) and decreased lipid peroxidation (MDA) in the egg yolk, liver, and breast tissues of breeder hens.

Both sources of dietary antioxidants were observed to enhance hatchability, probably because of the function of antioxidants that are deposited in the egg yolk and act on the elimination of ROS that occurs during embryonic metabolism. Embryonic tissues typically contain a high proportion of PUFA, rendering them vulnerable to lipid peroxidation [START_REF] Zhai | The effect of male and female supplementation of L-carnitine on reproductive traits of White Leghorns[END_REF]. ROS can also destroy other biological molecules, such as DNA, proteins, and carbohydrates, which are the leading causes of infertility and embryonic mortality [START_REF] Surai | Antioxidant systems in chick embryo development. part 1. vitamin E, carotenoids and selenium[END_REF]. Various types of antioxidants have been observed in the egg yolk and tissues of chick embryos, which contribute to the achievement of high-quality and viable chicks, including antioxidant enzymes such as SOD, GSH-Px, and CAT, water-soluble antioxidants such as vitamin C, taurine, Lcarnitine, and glutathione, and fat-soluble antioxidants such as vitamin E, carotenoids, coenzyme Q, and Se [START_REF] Urso | Vitamin E and selenium in broiler breeder diets: effect on live performance, hatching process, and chick quality[END_REF]Surai et al., 2019).

The synergistic effects of a combination of various antioxidant substances, especially vitamin C, vitamin E, Se, and L-carnitine, have been reported [START_REF] Abdel-Azeem | The role of nutritive solutions during embryogenesis in improving hatchability and posthatch growth performance[END_REF][START_REF] Leskovec | Antioxidative effects of supplementing linseed oil-enriched diets with αtocopherol, ascorbic acid, selenium, or their combination on carcass and meat quality in broilers[END_REF][START_REF] Shakeri | Strategies to combat heat stress in broiler chickens: Unveiling the roles of selenium, vitamin E and vitamin C[END_REF]. This revealed that a combination of these substances could function more effectively in the antioxidant defense network. Vitamin E plays an antioxidant role in cells and prevents the oxidation of low-density lipoproteins in cell membranes by donating electrons to lipid peroxyl radicals to stop chain-breaking antioxidant reactions before interacting with other lipids [START_REF] Ebeid | Vitamin E and organic selenium enhances the antioxidative status and quality of chicken semen under high ambient temperature[END_REF]. Vitamin C acts as a potent reducing agent in ROS scavenging by interacting with the tocopheroxyl radical and regenerating reduced tocopherol [START_REF] Akbari | An overview of the characteristics and function of vitamin C in various tissues: relying on its antioxidant function[END_REF]. Se plays a crucial role in antioxidant activity as an essential coenzyme of GSH-Px, which facilitates the disposal of hydrogen peroxide generated after the superoxide is catalyzed by SOD in the cellular antioxidant defense network, contributing to the detoxification of lipid peroxides [START_REF] Lykkesfeldt | Oxidants and antioxidants in disease: oxidative stress in farm animals[END_REF][START_REF] Mishra | Oxidative stress in the poultry gut: potential challenges and interventions[END_REF].

Vitamins C and E supplementation in broiler breeder hen diets during the summer has been shown to improve FRAP activity and reduce MDA levels in erythrocytes compared with the control [START_REF] Jena | Supplementation of vitamin E and C reduces oxidative stress in broiler breeder hens during summer[END_REF]. Generally, MDA serves as a marker of oxidative stress and represents cell/tissue damage caused by lipid peroxidation, and its reduction is linked to the antioxidant defense system through antioxidant supplementation [START_REF] Shakeri | Strategies to combat heat stress in broiler chickens: Unveiling the roles of selenium, vitamin E and vitamin C[END_REF][START_REF] Tang | Heat stress in broilers of liver injury effects of heat stress on oxidative stress and autophagy in liver of broilers[END_REF]. It has also been reported that L-carnitine can prevent the formation of ROS in the mitochondria, such as xanthine oxidase, cytochrome p450, cyclooxygenase, lipoxygenase, nitric oxide synthase (NOS), and NADPH oxidases (NOXs), to maintain a normal electron transport chain during the elevated metabolic rates of rapid embryo development or HS exposure [START_REF] Surai | Antioxidant systems in chick embryo development. part 1. vitamin E, carotenoids and selenium[END_REF]. The use of L-carnitine in female breeder hen diets has been shown to increase carnitine deposition in egg yolks and promotes the utilization of yolk lipids for energy production during embryogenesis [START_REF] Zhai | The effect of male and female supplementation of L-carnitine on reproductive traits of White Leghorns[END_REF][START_REF] Awad | Effect of sex and Lcarnitine addition on growth performance and carcass quality of Sudani ducklings[END_REF].

We also found that synthetic and phytogenic sources upregulated the expression of gene-related antioxidant enzymes, such as SOD, CAT, and GSH-Px, in the liver. This result is consistent with the finding that vitamin E, vitamin C, and L-carnitine have the ability to activate the transcriptional factor activity of activator protein-1 (AP-1), nuclear factor erythroid 2 related factor 2 (Nrf2), and NF-κB DNA binding site; this activation helps in regulating the expression of adhesive molecules, cytokines, and antioxidant enzyme genes, ultimately providing additional protection in HS condition [START_REF] Surai | Silymarin as a natural antioxidant: an overview of the current evidence and perspectives[END_REF][START_REF] Min | Vitamin E and vitamin C supplementation improves antioxidant status and immune function in oxidative-stressed breeder roosters by up-regulating expression of GSH-Px gene[END_REF]. Se mainly regulates GSH-Px mRNA through the biological processes of selenoproteins, requiring a selenocysteine insertion sequence that incorporates a Se-specific elongation factor, selenocysteinyl-tRNA, and a selenocysteine insertion sequence mRNA stem-loop structure into the Se-insertion complex during translation. This complex subsequently modulates a unique endonucleolytic cleavage site, resulting in increased GSH-Px mRNA expression [START_REF] Weiss | Selenium regulation of classical glutathione peroxidase expression requires the 3' untranslated region in Chinese hamster ovary cells[END_REF][START_REF] Puangmalee | Effect of organic trace mineral on gene expression of antioxidant enzyme and meat quality responsible enzyme in young chick[END_REF]. Studies have reported that feeding chickens vitamins C, E, Se, and L-carnitine leads to increased expression of SOD, CAT, and GSH-Px mRNA in the liver, which is considered the initial step in the antioxidant defense against free radicals and superoxide [START_REF] Elgendey | Selenium and/or vitamin E upregulate the antioxidant gene expression and parameters in broilers[END_REF]. Polyphenols exert their influence on gene expression in the liver of breeder hens through an indirect mechanism involving the synthesis of ROS-removing enzymes, which can stimulate the Keap1-Nrf2 complex by modifying cysteine residues in Kelch-like ECH-associated protein 1, leading to the translocation of Nrf2 into the nucleus. After that, Nrf2 binds to the antioxidant electrophile/antioxidant response element (EpRE/ARE) sequence [START_REF] Lee | Antioxidant capacity of phytochemicals and their potential effects on oxidative status in animals-a review[END_REF][START_REF] Saracila | Heat stress in broiler chickens and the effect of dietary polyphenols, with special reference to Willow (Salix spp.) Bark supplements-a review[END_REF], resulting in the upregulation of cellular antioxidant enzymes, such as SOD, CAT, GSH-Px, and GST [START_REF] Hosseini-Vashan | Growth, immune, antioxidant, and bone responses of heat stress-exposed broilers fed diets supplemented with tomato pomace[END_REF]Bernetoniene and Kopustinskinen 2018). Interestingly, our study revealed that phytogenic antioxidants have a greater capacity to upregulate GSH-Px gene expression than synthetic antioxidants do. This difference is likely attributable to the fact that synthetic antioxidants contain only one type of Se, which is the main precursor for GSH-Px synthesis, whereas the phytogenic antioxidants found in cloves, green tea pomace, and Vietnamese coriander consist of a variety of polyphenols with antioxidant properties, including eugenol, gallic acid, catechin, ellagic acid, quercetin, and kaempferol. The characteristic chemical structures of certain polyphenols can also activate antioxidant enzymes via modification of the transcription pathway [START_REF] Saracila | Heat stress in broiler chickens and the effect of dietary polyphenols, with special reference to Willow (Salix spp.) Bark supplements-a review[END_REF]. Epigallocatechin gallate from green tea exhibits outstanding antioxidant activity in poultry, surpassing that of vitamin E by 25 times and vitamin C by 100 times [START_REF] El-Hack | Impact of green tea (Camellia sinensis) and epigallocatechin gallate on poultry[END_REF]. In vitro tests have demonstrated that clove oil exhibits pronounced positive effects on antioxidant properties. including radical scavenging of DPPH, ABTS, H2O2, superoxide anion radical, and chelating activities, compared to Butylated hydroxyanisole (BHA), BHT, tocopherol, and trolox [START_REF] Gulcin | Antioxidant activity of clove oil -a powerful antioxidant source[END_REF]. This can be attributed to the presence of natural antioxidants, essential fatty acids and lipid-soluble bioactive molecules typically found in clove oil [START_REF] Sehitoglu | The Effect of clove oil supplementation in laying hen diets on performance, egg quality, some blood parameters, and yolk TBARS[END_REF].

While the bioactive compounds present in Polygonum odoratum L such as flavonoids, alkaloids, phenolic compound, and tannin showed moderately potent antioxidant activity (50.25 ± 0.61 mg/mL) compared to vitamin E (14.79 ± 0.78) and BHT (19.71 ± 0.79 mg/mL) (Somparn et al.,20.14).

We found that NF-κB, HSP70, and HSP90 mRNA expression was down-regulated in HS hens receiving synthetic and phytogenic antioxidants compared to HS hens without supplementation. In general, when animals are exposed to HS, two possible mechanisms are involved in homeostasis that make the body tolerant to HS. First, in response to HSPs, cells promote the expression of HSPs genes to protect against cell damage, particularly HSP70, which plays an important role in the HS response in chickens [START_REF] Soleimani | Physiological responses of 3 chicken breeds to acute heat stress[END_REF]. HSP70 in cells also helps maintain protein refolding, promotes the degradation of misfolded proteins, and reduces cell inflammation [START_REF] Varasteh | Differences in susceptibility to heat stress along the chicken intestine and the protective effects of galacto-oligosaccharides[END_REF][START_REF] Xu | Effect of chronic heat stress on some physiological and immunological parameters in different breed of broilers[END_REF]. Second, the oxidative stress response is a defense system against HS, and its gene plays a key role in the regulation is Nrf2 [START_REF] Lian | Beyond heat stress: intestinal integrity disruption and mechanism-based intervention strategies[END_REF][START_REF] Surai | Redox homeostasis in poultry: regulatory roles of NF-κB[END_REF]. Both response systems have been reported to improve the antioxidant capacity, reduce lipid oxidation, and increase digestive enzyme activity in the gastrointestinal tract [START_REF] Shehata | Modulation of heatshock proteins mediates chicken cell survival against thermal stress[END_REF]. Hence, dietary antioxidants (e.g., vitamins C, E, Se, catechin from green tea, quercetin from Vietnamese coriander, and other phytochemicals) may inhibit NF-κB binding to inflammation-related genes. This in turn, can lower the expression of pro-inflammatory cytokines and decrease the levels of HSPs (HSP60, HSP70, and HSP90), which serve as mediators for inducing NF-κB expression [START_REF] Jang | Effects of vitamin C or E on the pro-inflammatory cytokines, heat shock protein 70 and antioxidant status in broiler chicks under summer conditions[END_REF][START_REF] Akbarian | Association between heat stress and oxidative stress in poultry; mitochondrial dysfunction and dietary interventions with phytochemicals[END_REF]Bernatoniene and Kopustinskien, 2018;[START_REF] Kumbhar | Impacts of selenium and vitamin E supplementation on mRNA of heat shock proteins, selenoproteins and antioxidants in broilers exposed to high temperature[END_REF][START_REF] Chansiw | Anti-inflammatory and antioxidant activities of the extracts from leaves and stems of Polygonum odoratum Lour. Anti-Inflamm[END_REF][START_REF] Manaig | Heat stress management strategies using plant extracts in poultry[END_REF].

This study revealed the benefits of a combination of synthetic antioxidants (vitamins C and E, Se, and L-carnitine) and phytogenic antioxidants (clove, green tea pomace, and Vietnamese coriander) on breeder hens exposed to HS. Both sources of antioxidants demonstrated significant improvements in egg production and hatchability, and reduced embryo mortality. In addition, both antioxidant sources also alleviate the adverse effects of HS through antioxidant defenses and inflammation by reducing lipid peroxidation in yolk and tissues, up-regulating the relative expression of SOD, CAT, GSH-Px mRNA, and downregulating of NF-κB, HSP70, and HSP90 mRNA expressions in liver. This finding suggests the promising application of a combination of synthetic and phytogenic antioxidant bioactive compounds to combat the deleterious effects of HS in breeder hens. This knowledge may lead to the development of innovative strategies for HS management by integrating dietary supplements with antioxidants. 2 Orthogonal contrasts: 1= thermoneutral (T1) vs. heat stress condition (T2, T3, T4); 2, nonsupplement (T2) vs. supplement (T3, T4); 3, synthetic antioxidants (T3) vs. phytogenic (T4).

SOD F-5'-CACTGCATCATTGGCCGTACCA-3' NM_001031215.1 R-5'-GCTTGCACACGGAAGAGCAAGT-3' CAT F-5'-TGGCGGTAGGAGTCTGGTCT-3' NM_205064.1 R-5'-GTCCCGTCCGTCAGCCATTT-3' GSH-Px F-5'-GCTGTTGCCTTCCTGAGAG-3' NM_001277853.1 R-5'-GTTCCAGGAGACGTCGTTGC-3' HSP70 F-5'-GATCTGGGCACCACGTATTCT-3' FJ217667.1 R-5'-GGTTCATTGCCACTTGGTTCTT-3' HSP90 F-5'-ACACATGCCAACCGCATTTA-3' NM_001109785.1 R-5'-CCTCCTCAGCAGCAGTATCA-3' NF-κB F-5'-GAAGGAATCGTACCGGGAACA-3' NM_205134 R-5'-CTCAGAGGGCCTTGTGACAGTAA-3' β-actin F-5'-TTGGTTTGTCAAGCAAGCGG-3' NM 205518.1 R-5'-CCCCCACATACTGGCACTTT-3'
Abbreviations: PCO2, Partial pressure of carbon dioxide; PO2, Partial pressure of oxygen; BE, Base excess; HCO3 -, concentration of bicarbonate ions; TCO2, total concentration carbon dioxide; sO2, saturation of oxygen; iCa, concentration ionized calcium. Na, sodium; K, potassium; Glu, glucose; Hct, hematocrit; Hgb, hemoglobin. 2 Orthogonal contrasts: 1, thermoneutral (T1) vs. heat stress condition (T2, T3, T4); 2, nonsupplement (T2) vs. supplement (T3, T4); 3, synthetic antioxidants (T3) vs. phytogenic (T4).

1

  Abbreviations: SOD, superoxide dismutase; CAT, catalase; GSH-Px, glutathione

Table 1 .

 1 Ingredients and calculated nutrient composition of experimental diets (as-fed basis).

		25-50 weeks of age	After 50 weeks of age
	Ingredients (%)
	Corn	64.60	63.50
	Soybean meal, 44 %CP	18.20	16.52
	Full fat soybean meal	6.70	9.00
	Calcium carbonate	8.50	8.90
	Monocalcium phosphate	0.94	1.00
	Salt	0.41	0.44
	DL-methionine	0.135	0.134
	Copper	0.00040	-
	Iodine	0.00022	0.00022
	Vitamin B2	0.00043	0.00043
	Vitamin D3	0.00030	0.00030
	Manganese	0.00490	-
	Biotin	0.00140	0.00140
	Zine	0.01230	0.00390
	Premix 1	0.50	0.50
	Calculated compositions (%)
	Metabolizable energy (kcal/kg)	2,800	2,800
	Dry matter	90.22	90.34
	Crude protein	15.49	15.44
	Crude fiber	3.09	3.07
	Ether extract	3.84	4.19
	Calcium	3.51	3.71
	Total Phosphorus	0.53	0.54
	Available phosphorus	0.31	0.32
	Digestible Lysine	0.70	0.70
	Digestible Methionine	0.35	0.35
	Digestible Methionine + Cystine	0.57	0.57
	Digestible Threonine	0.50	0.50
	1 Premix (0.5%) provided the following (per kg of diet) ; vitamin A, 15,000 IU; vitamin D3,
	3,000 IU; vitamin E, 25 IU; vitamin K3, 5 mg; vitamin B1, 2 mg; vitamin B2, 7 mg; vitamin
	B6, 4 mg; vitamin B12, 25 mg; pantothenic acid, 11.04 mg; nicotinic acid, 35 mg; folic acid, 1

Table 2 .

 2 Primer sequences used in real-time PCR.

	Gene	Primer sequences 1	Accession No.

Table 3 .

 3 Effect of dietary antioxidant supplementation in breeder hen diets under heat stress condition on productive performances.

	Items		Treatments 1 T1 T2	T3	T4	Pooled SEM	Contrasts 2 1 2	3
	IBW (g)		2795.21 2891.37	2747.18	2785.16 26.644 0.836	0.057	0.614
	FBW (g)		3314.48 a 3176.57 ab 3102.67 ab 3048.19 b 34.971 0.013	0.215	0.571
	FI (g/day/hen)	137.20 a 137.22 a	135.92 ab 135.56 b 0.2673 0.103	0.025	0.634
	FCR		2.12 b	2.15 ab	2.16 a	2.15 ab	0.0044 0.007	0.600	0.431
	WI (mL/day/hens) 290.60 b 406.57 a	370.27 a	372.05 a	7.7241 0.001	0.015	0.913
	EP (g)		88.64 a	83.50 b	88.62 a	89.78 a	0.6707 0.372	<0.001 0.524
	EW (g)		64.61 a	63.83 b	62.94 c	62.92 c	0.0943 <0.001 0<.001 0.925
	Fertility (%)		98.86	98.43	99.34	99.14	0.1925 0.798	0.086	0.709
	Hatchability (%)	87.63 b	87.79 b	91.81 a	91.71 a	0.6394 0.046	0.009	0.957
	Fertile	period
	length of sperm	15.04	14.64	14.5	14.33	0.157	0.141	0.556	0.707
	(day)
	Day old chick weight (g)	46.30 a	45.55 a	44.89 ab	43.66 b	0.3195 0.009	0.037	0.074
	Early dead (%)	2.06 b	4.70 a	2.76 ab	2.67 ab	0.3512 0.096	0.022	2.67
	Last dead (%)	5.85 ad	6.16 a	2.83 c	3.27 b	0.5029 0.123	0.011	0.744
	Abnormal chicks (%)	2.55	1.38	1.02	2.21	0.3215 0.172	0.769	0.192
	Chick uniformity (%)	75.85 ab	76.39 ab	82.23 a	70.94 b	1.6265 0.833	0.953	0.007
	a-b Means within each row with different superscripts are significantly different (P < 0 .05).
	1 T1, thermoneutral zone (23±1°C) + basal diet without supplementation; T2, heat stress
	(36±1°C, 4 h/day) + basal diet without supplementation; T3, heat stress condition (36±1°C, 4
	h/day) + basal diet with synthetic antioxidants; T4, heat stress condition (36±1°C, 4 h/day) +
	basal diets with phytogenic.

Table 4 .

 4 Effect of dietary antioxidant supplementation in breeder hen diets under heat stress condition on blood chemistry. Means within each row with different superscripts are significantly different (P < 0 .05).

	Items	Treatments 1 T1 T2	T3	T4	Pooled SEM	Contrasts 2 1 2	3
	pH	7.37 b	7.47 a	7.46 a	7.46 a	0.007	<0.001 0.458	0.859
	PCO2 (mmHg)	50.73 a	36.90 b	36.51 b	36.82 b	0.9762 <0.001 0.886	0.867
	PO2 (mmHg)	40.09 a	37.36 b	36.72 b	37.18 b	0.3859 <0.001 0.627	0.641
	BE (mmol/L)	4.72 a	3.17 ab	2.55 b	2.41 b	0.2842 0.002	0.299	0.85
	HCO3 -(mmol/L) 29.94 a	26.84 b	26.35 b	26.20 b	0.3098 <0.001 0.368	0.842
	TCO2 (mmol/L)	31.33 a	27.94 b	27.44 b	27.35 b	0.3098 <0.001 0.406	0.902
	sO2 (%)	70.17 b	77.29 a	76.11 a	77.05 a	0.7313 <0.001 0.662	0.609
	iCa (mmol/L)	1.78 a	1.53 b	1.51 b	1.58 b	0.0263 <0.001 0.860	0.225
	Na (mmol/L)	148.35 a 146.43 ab 146.37 ab 146.00 b 0.3218 0.004	0.744	0.687
	K (mmol/L)	5.13 a	4.92 ab	5.10 ab	4.86 b	0.0348 0.023	0.448	0.011
	Glu (mg/dL)	229.77 b 241.17 a	246.16 a	246.47 a 1.6465 <0.001 0.159	0.947
	Hct (%PCV)	28.27 a	25.64 b	25.72 b	25.47 b	0.3174 <0.001 0.942	0.781
	Hb (g/dL)	9.62 a	8.71 b	8.75 b	8.66 b	0.1076 <0.001 0.966	0.760
	a-b 1 T1, thermoneutral zone (23±1°C) + basal diet without supplementation; T2, heat stress
	condition (36±1°C, 4 h/day) + basal diet without supplementation; T3, heat stress condition
	(36±1°C, 4 h/day) + basal diet with synthetic antioxidants; T4, heat stress condition (36±1°C,
	4 h/day) + basal diets with phytogenic.

Table 5 .

 5 Effect of dietary antioxidant supplementation in breeder hen diets under heat stress condition on antioxidant activity in egg yolk, liver, and breast. Means within each row with different superscripts are significantly different (P < 0 .05).

	Items	Treatments 1 T1 T2	T3	T4	Pooled SEM	Contrasts 2 1 2	3
	DPPH (%)
	Egg yolk	17.23 c	18.20 c	47.03 a	22.18 b	2.2021	<0.001 <0.001 <0.001
	Liver	81.82 b	86.24 ab	87.48 a	86.43 ab	0.7426	0.003	<0.001 0.567
	Breast	19.49 ab	18.60 b	21.05 a	21.35 a	0.3478	0.212	0.004	0.733
	FRAP (mM TE/g sample)
	Egg yolk	0.42 b	0.36 b	0.71 a	0.40 b	0.0299	0.072	<0.001 <0.001
	Liver	1.87 b	2.14 b	2.49 a	2.53 a	0.0628	<0.001 0.001	0.676
	Breast	0.23	0.29	0.29	0.29	0.0111	0.023	0.962	0.934
	ABTS (mM TE/g sample)
	Egg yolk	5.46 a	5.18 b	5.43 a	5.42 a	0.0336	0.052	0.001	0.921
	Liver	35.56 ab	34.18 b	35.23 ab	37.28 a	0.4011	1.000	0.027	0.051
	Breast	40.28 ab	38.29 b	45.37 a	45.02 a	0.8393	0.103	<0.001 0.855
	MDA (uM/g sample)
	Egg yolk	28.14 ab	32.74 a	10.76 c	23.94 b	1.6502	0.001	<0.001 <0.001
	Liver	10.92 a	10.90 a	8.65 b	11.53 a	0.3397	0.398	0.274	0.002
	Breast	8.26	8.71	7.74	8.68	0.1433	0.716	0.134	0.015
	a-c 1 T1, thermoneutral zone (23±1°C) + basal diet without supplementation; T2, heat stress
	condition (36±1°C, 4 h/day) + basal diet without supplementation; T3, heat stress condition
	(36±1°C, 4 h/day) + basal diet with synthetic antioxidants; T4, heat stress condition (36±1°C,
	4 h/day) + basal diets with phytogenic.

Table 6 .

 6 Effect of dietary antioxidant supplementation in breeder hen diets under heat stress condition on gene related to antioxidant enzyme, pro-inflammatory cytokines, and heat shock proteins expressions. Means within each row with different superscripts are significantly different (P < 0 .05).

	Items	Treatments 1 T1 T2	T3	T4	Pooled SEM	Contrasts 2 1	2	3
	SOD	1.00 b 0.71 c	1.56 a	1.45 a	0.0001	<0.001	<0.001	0.121
	CAT	1.00 ab 0.35 b	1.49 a	1.54 a	0.0240	0.672	0.003	0.907
	GHS-Px	1.00 c 0.76 c	2.40 b	5.42 a	0.0001	<0.001	<0.001	<0.001
	NF-κB	1.00 ab 1.25 b	0.64 ab	0.42 a	0.5257	0.285	0.009	0.381
	HSP70	1.00 b 1.11 b	0.16 a	0.15 a	0.2252	<0.001	<0.001	0.799
	HSP90	1.00 b 1.01 b	0.20 a	0.17 a	0.0058	0.011	0.001	0.872
	a-c

1 T1, thermoneutral zone (23±1°C) + basal diet without supplementation; T2, heat stress condition (36±1°C, 4 h/day) + basal diet without supplementation; T3, heat stress condition (36±1°C, 4 h/day) + basal diet with synthetic antioxidants; T4, heat stress condition (36±1°C, 4 h/day) + basal diets with phytogenic.

Orthogonal contrasts: 1) thermoneutral (T1) vs. heat stress conditions (T2, T3, T4); 2) nonsupplement (T2) vs. supplement (T3,

T4); 3) synthetic antioxidants (T3) vs. phytogenic (T4).

Orthogonal contrasts: 1, thermoneutral (T1) vs. heat stress condition (T2, T3, T4); 2, nonsupplement (T2) vs. supplement (T3,

T4); 3, synthetic antioxidants (T3) vs. phytogenic (T4).

ACKNOWLEDGMENTS

The authors would like to thank the financial support provided by the Royal Golden Jubilee Ph.D. Programme (grant number PHD/0165/2560), the National Research Council of Thailand (NRCT5-RSA63009-03), the Thailand Research Fund (TRF), Thailand Science Research and Innovation (TSRI), and Suranaree University of Technology (SUT).

DECLARATION OF COMPETING INTEREST

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.


Julie Bachellerie

Patrick Schmidt

Reconnaissance et quantification du traitement thermique du silex analyses de surface comme nouvel outil analytique

Introduction

Reconnaître macroscopiquement un traitement thermique

Matériel et méthode

Il y a plusieurs problèmes liés à l'identification macroscopique du lustre de chauffe: 1) l'impossibilité de le reconnaître avec certitude en l'absence de plages mates résiduelles associées, 2) sa reconnaissance basée sur une observation à l'oeil nu = pas de quantification objective, 3) son degré d'intensité très variable selon les types de silex

Or ce lustre de chauffe serait relatif aux modifications des propriétés mécaniques de la roche pendant la chauffe (réduction progressive de la porosité intergranulaire; Schmidt et al., 2019), entraînant un changement du microrelief de la surface.

Dans ce cadre, des analyses de rugosité de surface ont été menées au microscope confocal.

Le traitement thermique est un procédé technique intentionnel qui entraîne une modification des propriétés mécaniques du matériau pouvant aboutir à une amélioration des qualités de taille de la roche et du tranchant des bords de l'objet.

Macroscopiquement, il peut provoquer une altération de l'état de surface du matériau de plusieurs ordres:

-La rubéfaction et le blanchiment notamment, pas systématiques et insuffisants pour caractériser l'intentionnalité de la chauffe.

-Le lustre de chauffe, lié à une action délibérée puisqu'il n'est visible que sur les enlèvements réalisés après la chauffe. Le négatif de l'enlèvement présente alors un aspect lustré et graisseux, souvent visible à l'oeil nu. Sa reconnaissance est facilitée lorsqu'il est associé à des plages résiduelles antérieures à la chauffe. Dans ce cas, on parlera de double état de surface (ou double lustre), qui constitue alors le seul critère macroscopique fiable de reconnaissance d'un traitement thermique.

Matériel:

• Blocs de silex géologique de provenances différentes (silex Bergeracois, silex Tertiaire de Dordogne.)

→ Collection de référence d'éclats produits expérimentalement (pour chaque bloc, 5 éclats ont été extraits avant chauffe, puis à chaque palier de chauffe à 200, 250, 300, 350 et 400°C.)

• Microscope confocal (Keyence VK-100), objectif x50

Méthode:

1. Acquisition (sans contact) de modèle 3D de surfaces de faces ventrales d'éclats, 740 x 540 μm 2. Traitement des modèles à partir du logiciel libre Gwyddion:

-Extraction de 4 profils linéaires de manière aléatoire sur chaque modèle (afin d'éviter les défauts inhérents à la matière première et les irrégularités des éclats), -Suppression de la composante ondulatoire (application d'un « cut-off » = 0.15) pour ne garder que celle liée à la rugosité, -Enregistrement des paramètres de rugosité (rugosité arithmétique = Ra)

Résultats et discussion

-Réduction progressive de la rugosité observée à partir de 200 °C et jusqu'à 300 °C. Au-dessus de 300 °C les valeurs de rugosité restent sensiblement les mêmes, avec toutefois, une légère réaugmentation de la rugosité à 400 °C.

-Dispersion des valeurs plus importante pour les éclats non chauffés, cette dispersion tend à diminuer à mesure que la température augmente.

Les résultats obtenus confirment l'évolution de la rugosité de surface des échantillons en fonction:

-des températures de chauffe: baisse progressive de la rugosité dès 200-250°C et réaugmentation à 400°C (surchauffe = présence de microfractures) -du type de matière première, ce qui s'avère en accord avec les observations macroscopiques faites lors des chauffes expérimentales.

A partir de de 200-250°C, dans le cas du silex Bergeracois et Tertiaire, la surface de fracture est plus lisse, entraînant l'apparition d'un lustre de chauffe visible macroscopiquement au cours des expérimentations. Cette évolution est progressive jusqu'à 300-350°C environ, correspondant à l'augmentation de l'intensité du lustre de chauffe pour ces matières premières, ce qui pourrait également coïncider avec l'amélioration des qualités de taille de ces silex (par ex. Schmidt et al. 2012 ;Schmidt et al. 2019).

→

  Mesures de rugosité présentent un fort potentiel analytique, puisqu'elles permettraient de quantifier l'intensité du lustre de chauffe Potentiel de cet outil pour la reconnaissance et l'étude du traitement thermique ? → Contrairement aux observations visuelles, les résultats obtenus via les analyses de surface ne sont pas déformées par d'autres phénomènes influençant la réflectance de la lumière → Caractère non destructif et rapide des mesures + applicable à tout type d'échantillon en silex, y compris pour des pièces épaisses → Montre une bonne complémentarité des analyses IR (Bachellerie et al. 2021) avec des mesures de rugosité plus sensibles aux changements provoqués à basse température → Si les résultats obtenus sur le matériel géologique sont concluants, il est nécessaire de multiplier les tests, tant sur du matériel géologique que sur des artefacts lithiques Bilan et perspectives Feuille de laurier traitée thermiquement, MNP. photo J. Jugie Microscope confocal à balayage laser Keyence VK-100, Eberhard Karls Universität, Allemagne Évolution de la rugosité de surface Ra des silex tertiaire et Bergeracois, en fonction de la température de chauffe. Les modèles dont ont été extraits ces données ont été réalisés avec un objectif x50 Représentations 2D et 3D des modèles de surface obtenus au microscope confocal à balayage laser sur les faces inférieures d'éclats de silex Bergeracois. En a. échantillon non chauffé, en b. échantillon chauffé à 200°C, en c. échantillon chauffé à 300°C, en d. échantillon chauffé à 400°C. Tous les échantillons ont été extraits du même bloc. Fragments de feuilles de laurier traitées thermiquement en silex Bergeracois provenant des gisements de Laugerie-Haute et Pré-Aubert. Les surfaces mates correspondant aux négatifs d'enlèvements antérieurs au traitement thermique sont figurés en orangées, tandis que les surfaces lustrées obtenues après traitement thermique sont en blanc. Négatifs d'enlèvements postérieurs au traitement thermique = lustre de chauffe Négatifs d'enlèvements antérieurs au traitement thermique = surface mate

Nous remercions K.G. Nickel et C. Berthold pour leur aide concernant la réalisation des analyses de surface au sein du département de géosciences de l'université deTübingen. PS a reçu le soutien financier de la Deutsche Forschungsgemeinschaft (DFG) (numéro de subvention SCHM 3275/2-1).En Europe, la chauffe intentionnelle du silex apparaît pour la première fois dans le Paléolithique récent, et plus précisément durant le Solutréen (circa 25,5-23,5/23 ka cal BP). Si les premières mentions de traitement thermique en contexte solutréen remontent aux années 60-70(Bordes, 1969; Inizan et al., 1976), les données demeurent relativement éparses et les interrogations persistent sur l'ampleur de ce phénomène technique, ses modalités de mise en oeuvre, ainsi que sa place au sein du système technique solutréen.Un travail de caractérisation du traitement thermique a donc récemment été mené sur des assemblages lithiques solutréens afin de cerner d'avantage la portée de cette innovation technique (Bachellerie, 2022).La première étape de ce travail fut de réaliser un diagnostic macroscopique des stigmates de chauffe, ce qui pose la question de la reconnaissance et de la quantification de ce procédé.

Remerciements