All experimental procedures using animals were conducted following the European Guidelines for Care and Use of Animals for Research Purpose, and they were approved by the Local Ethics Committee for Animal Testing of the Aydın Adnan Menderes University (64583101/2019/048).

A total of two hundred and forty, one-day-old mixed-sex Japanese quail (Coturnix coturnix japonica) chicks with an average body weight of 9.38 ± 0.02 g were divided into three groups, each consisting of four replicates with 20 chicks each cage. CS were ground during the preparation of the diets and mixed into the diets with other feeds. Three isocaloric and iso-nitrogenous experimental diets containing control, 25 g/kg chasteberry seeds (CS25), and 50 g/kg chasteberry seeds (CS50) were formulated according to the NRC (1994) (Table 1). The chemical analysis of the diets was carried out by standard methods described by AOAC (2005). The ambient temperature was set at 32 °C for the first three days. After that, the temperature was decreased by 3 °C each week until it reached 21 °C and it remained until the end of study. The lighting schedule was set to 24 hours a day for the first three days of the study. After that, it was reduced to 23 hours a day until the trial was over. During the 35-day experiment period, the water and feed requirements of the animals were provided ad libitum.

The body weight (BW), and feed intake (FI) of quail were recorded weekly throughout the experiment. Body weight gain (BWG) and feed conversion ratio (FCR) were calculated at 0-21, 21-35 and 0-35 days.

At the end of the experiment, 12 male and 12 female quails were randomly selected from each group for carcass traits and analysis. A total of 72 quails were slaughtered. Before slaughtering, the body weight of quails was determined. After weighing, quails were slaughtered, de-feathered, processed (removal of head and feet), and eviscerated (removal of the gastrointestinal tract) one by one. The weight of the hot carcass, liver, heart, gizzard, and proventriculus was separately determined. The relative weight of the liver, heart, gizzard, and proventriculus was calculated as percentages of body weight (g/100 g body weight).

Right breast samples were taken from the carcasses of 72 birds to determine the water-holding capacity (WHC), cooking loss (CL), drip loss (DL), shear force (SF), and color. The samples were frozen and stored in a freezer at –20 °C until further analyses.

A 3-gram sample of meat was put on Whatman No. 1 filter paper, which had been dried and weighed, together with two thin plastic films. The filter paper and plastic films containing the meat sample were then placed between Plexiglas plates. For five minutes, a 2.5 kg load was applied. Wet filter paper and plastic films were promptly weighed following the exact removal of the compressed meat. The WHC percentage was calculated as follows (Joo, 2018):

WHC % = damp filter paper and plastic film weights – filter paper and plastic film weights/meat sample weight × 100.

To evaluate CL, each breast meat sample was weighed, placed into a sealed polyethylene bag, and cooked in a water bath at 80 °C until the sample core temperature reached 75^oC. After allowing the cooked meat samples to cool down to room temperature in tap water, they were reweighed (Honikel, 1998). Using the following formula, CL was determined:

CL % = raw weight – cooked weight/ raw weight ×100

To calculate the DL, the method described by Honikel (1998) was used. In order to avoid any contact between the sample and the bag, each sample was first weighed and then suspended in an inflated plastic container at 4 °C for 24 hours. Subsequently, the samples were re-weighed. Then the DL was calculated as the percentage of weight loss:

DL % = [(initial weight–final weight)/initial weight] × 100

Cooked meat samples were tested for SF. Two strips of meat, each measuring 2.5 × 1.0 × 1.0 cm³, were sliced parallel to the muscle fibers. The Zwick Testing Machine Model Z2.5/TN1S (Zwick GmbH and Co, Germany) was used to measure the maximum SF of the meat samples, using a Warner-Bratzler shear. The meat samples were cut perpendicular to the fiber direction at a test speed of 2 mm/s. The maximum SF was measured in Newton force (N) (dos Santos et al., 2020).

A colorimeter (Color Flex EZ, Hunterlab, USA) was used to determine the meat color equipped with a standard D65 illuminant and a “2” position of the standard observer, along with a pulse xenon lamp and an 8-mm reading surface area. Before taking the measurements, the device was calibrated using a black-and-white standard plate. Three measurements per sample were taken of the surface area of breast meat, and L*, a*, and b* values were recorded. The hue angle and chroma (saturation index) values were calculated using the a* and b* values using the following formulas (AMSA, 2012):

Chroma = (a*² +b*²)^1/2

Hue angle (º) = arctan (b*/a*)

To determine the oxidant and antioxidant parameters breast samples were taken from the left breast of 12 male quails. Tissue samples were weighed and placed in 50 mM potassium phosphate buffer (pH 7.4). Samples were cooled in an ice bath and homogenized for 20 s. The homogenate was cooled in an ice bath for 30 seconds and subjected to sonication. It was then centrifuged at 10,000 × g at 4°C for 15 min. Superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and malondialdehyde (MDA) experiments were carried out in supernatants.

The content of SOD was determined by method of Sun et al. (1988). CAT activity was measured according to Aebi (1984) method. GSH content was determined using the method developed by Sedlak and Lindsay (1968). MDA levels were determined by the double heating method of Draper & Hadley (1990).

The economic effectiveness of CS supplementation was calculated using the following formulae as described by Shehata et al. (2021).

Total feed cost (TFC) = total feed intake per bird per gram × cost of one gram diet

Feed cost/kg BWG (average cost of each kg BWG from the feed) = feed conversion × cost of one kg diet.

Economic efficiency index (EEI) = (lowest cost of kg BWG from feed/ average cost of kg BWG from the feed) × 100

Total return (TR) = live body weight × price of each gram

Net profit = TR-TFC.

The experiment was conducted as a completely randomized design with three treatments of four replicates each. All data were analyzed by one-way ANOVA using the GLM procedure with SAS software (SAS, 1999). The differences among the means were tested using Duncan multiple-range tests. All statements of significance were based on a probability of p < 0.05.

The growing performances of quails fed diets containing different levels of ground CS are given in Table 2.

On the 21st day of the study, it was determined that the BW and BWG of the quails decreased as the level of CS in the diets increased (p<0.001). As well as, on the 35th day of the experiment, the BW of the quails in the CS50 group were lower than in the other groups (p<0.05). Although the BWG of quails on days 21-35 were not statistically affected by increasing levels of CS, there was a tendency for an increase in the BWG of quails compared to the control group. During the overall period of the study (0-35 days of age), it was observed that BWG of quails in the CS50 group was lower than that of the other groups (p<0.05).

Feed intake of quails on days 21-35 was not affected by the addition of CS. However, during days 0-21 and 0-35, it was observed that the FI of the CS50 group was lower than that of the other groups. Diets containing different levels of CS did not statistically affect the FCR.

The effects of diets containing different levels of CS on carcass and some internal organ weights of male and female quails are given in Table 3.

It was observed that adding CS had no effect on the slaughter weight, warm carcass weight, warm carcass yield, and relative organ weights (liver, gizzard, and heart) in male and female quails.

When the effects of different levels of CS addition on meat quality were analyzed (Table 4), no effect of CS addition was observed in breast meat of male animals (p>0.05).

In female animals, a statistically significant difference was found in WHC, L*, a*, and hue angle values in breast meat (p<0.05). The highest WHC and a* values were observed in the CS50 group, while the lowest L* and hue angle values were also found in the CS50 group. However, the difference between the Control and CS25 groups was not significant, especially in terms of color parameters (p>0.05).

The effects of diets containing different levels of CS on breast meat oxidant and antioxidant parameters of male quails are given in Table 5. It was observed that the addition of different levels of chasteberry seed increased the antioxidant parameters (SOD, CAT, GSH), and decreased the oxidant parameter (MDA) compared to the control group in the current study (p<0.05).

The results of economic efficiency metrics are displayed in Table 6. There was a statistically significant difference between the groups in terms of feed cost, and the lowest feed cost was obtained in the CS50 group (p<0.05). CS50 group was followed by CS25 and control group, respectively. Feed cost/BW, feed cost/BWG and EEI values did not differ between the groups (p>0.05). Although the lowest TR was observed in the CS50 group (p<0.05), there was no significant difference between the groups in net profit values (p>0.05).

In the current study, although the addition of 50 g/kg chasteberry seeds had negative effects on the BW of the animals, it was observed that the BWG of the CS25 and CS50 groups tended to increase at 21-35 days. In addition, it was observed that chasteberry seed supplementation had positive effects on FCR by decreasing FI. It was thought that the decrease in body weight with the addition of chaste seed may be due to the decrease in protein and nutrient efficiency due to the interaction of hydroxyl groups of phenolic compounds such as polyphenols and oxalates, which are considered as anti-nutritional factors, with protein carbonyl groups, as well as having negative metabolic effects related to the inhibition of digestive enzymes (Vargas-Sánchez et al., 2019; Samtiya et al., 2020; Popescu et al., 2021).

While previous studies on diets containing CS and their impact on the growth performance of poultry are not readily available, various studies have examined the effects of CS supplementation on the performance of laying hens. El-Saadany et al. (2022) reported that the addition of 2.5 and 5 g/kg of CS to the diets of laying hens at 58 weeks of age improved feed conversion ratio, egg production, egg weight, egg mass, eggshell thickness, and yolk color and weight. However, in another study conducted by Nazari et al. (2023), it was reported that the addition of 1% and 2% CS to the diets of laying hens at 72 weeks of age had no effect on the performance and egg quality of the animals. Similarly, it was reported that supplementation of flaxseed, which is a phytoestrogen source, such as chasteberry, in the diets of older laying hens increased egg production and egg quality (Salary et al., 2016; Saleh et al., 2019). However, Popescu et al. (2021) reported that broiler chickens fed a flaxseed diet had negative effects on the performance parameters due to anti-nutritional factors compared to laying hens. As a result of various studies conducted, it has been reported that there is a decrease in the performance parameters of broiler chickens fed with different levels of flaxseed ranging from 2-20% (Mridula et al., 2015; Beheshti Moghadam et al., 2017; Zhaleh et al., 2019; El-Bahr et al., 2021; Kumar et al., 2021). Tamasgen et al. (2021) reported that different processing methods can reduce the anti-nutritional factor content of the seed, thus reducing its negative effects. Research has shown that the negative effects of flaxseed can be reduced by irradiation and seed extrusion (Beheshti Moghadam et al., 2017; Gheorghe et al., 2020; Zhaleh et al., 2020).

In the current study, CS supplementation had no effect on the carcass and internal organ weights of male and female animals. Carcass and internal organ results of the current study are consistent with the results of Zadeh et al. (2020) who reported that there was no significant effect on the relative weight of the carcass of broilers fed the finisher diet for one week with different levels of flaxseed compared to the control diet. Similarly, Gheorghe et al. (2020) also reported that the addition of flaxseed in the broiler diet up to %12 had no effect on carcass yield and internal organ weights. However, in contrast to the findings of the current study, Mridula et al. (2015) reported a significant decrease in carcass yield with the addition of flaxseed. Additionally, Tamasgen et al. (2021) did not determine any difference in carcass weights of broilers as a result of replacing 25% and 50% of soybean meal with flaxseed, but they observed an increase in small intestine weight and length. It is reported that this reduction in carcass characteristics and edible portions may be due to the development of the digestive tract and increased length and weight of the small intestine, together with the interference of phytogenic with phenolic compounds and phenolic groups on digestion and availability of dietary protein and essential amino acids (Ghazaghi et al., 2014; Mehri et al., 2015).

The addition of different amounts of CS did not affect the breast meat quality of males. With the addition of 50 g/kg chasteberry seed, the L* and hue angle values of the breast meat of females decreased, while the WHC and a* values increased. However, Mapiye et al. (2011) reported that phytogenic plants may promote lighter meat color. Although studies specifically focusing on CS were not found, when examining research on various phytogenic plants, there are studies reporting that plants such as mint, thyme, cumin, flaxseed, hemp, black cumin, and fever tea have an effect on meat quality (Mnisi et al., 2017; Yalçın et al., 2017; Singh et al., 2018; Tashla et al., 2019; Vargas-Sánchez et al., 2019; El-Bahr et al. 2021). Mnisi et al. (2017) reported an increase in L* and a* values of breast meat and no difference in CL and SF with the addition of 25g/kg fever tea to quail diets. Similarly, it was reported that the addition of hemp seeds to quail diets increased L* and a* values of breast meat and decreased CL, while there was no difference in thawing loss (Yalcin et al., 2017). In another study, it was reported that diets containing 0, 1 or 2% black cumin seeds did not affect the CL and WHC of quail breast meat (Karadağoğlu et al., 2019). Vargas-Sánchez et al. (2019), reported that adding natural ingredients like medicinal plants to poultry diets can help improve the quality of their carcasses and meat by reducing oxidative stress, but this effect depends on the concentration of the ingredients and the type and/or structure of the compounds present. Although it was also reported that high concentrations of some natural ingredients in the diet may have negative effects on poultry carcasses and meat.

Chasteberry plant has been among the medicinal plants utilized by humans throughout history (Zahid et al., 2016; Niroumand et al., 2018; Kamal et al., 2022). In vitro studies on the biological effects of chasteberry have indicated that this plant has antioxidant properties. It has been reported that these biological effects are mostly due to ketosteroids, diterpenoids, flavonoids and iridoids in the composition of chasteberry (Ahangarpour et al., 2016; Souto et al., 2020; Özderin, 2021; Adamov et al., 2022; Zhelev et al., 2022; Boujbiha et al., 2023). In this study, an increase in SOD, CAT, GSH values was observed in parallel with the increasing amount of chasteberry seeds, while a decrease in MDA value was observed. These results show that CS25 and CS50 diets increase antioxidant capacity and decrease oxidant capacity in quails. No study was found to investigate the antioxidant properties of CS on poultry. However, as a result of studies conducted with flaxseed, another plant with phytoestrogenic properties, it was reported that the addition of flaxseed increased antioxidant capacity similar to this study (Anjum et al., 2013; Saleh et al., 2019; Kumar et al., 2021).

In the current study, it was found that the inclusion of CS in the feeds led to a decrease in feed costs. The lowest feed cost was observed in the CS50 group. As the amount of chasteberry seed in the feed increased, the reduction in feed intake was correlated with a decrease in feed costs. The results of this study were in agreement with Simol et al. (2012), who reported that supplementing the diet with mulberry leaves decreased the cost per kilogram of feed. In a similar study, Al-Khalaifah et al. (2020) reported that the addition of microbial fermented or enzymatically treated dried brewer’s grains to broiler diets reduced feed costs. Although there was no difference between the groups in feed cost/BWG values, the lowest value was observed in the CS50 group, which seems to be related to the fact that the best feed conversion was observed in this group (Al-Khalaifah et al., 2020). The highest TR values were found in the control and CS25 groups. These results are related to the high live weight. Although the lowest value in terms of values was found in the CS50 group, there was no difference between the groups in terms of net profit values. Similarly, Shehata et al. (2021) also reported that the addition of 4% and 8% mulberry leaves to Japanese quails had no negative effect on net profit. Therefore, from an economic point of view, it is understood that especially the addition of 25 g/kg chasteberry seed can be used easily.

This study revealed that the supplementation of 25 g/kg chasteberry seeds did not adversely affect the performance, slaughter, carcass parameters, or economic indices compared to the control group; however, it positively changed the antioxidant parameters. These results suggest that chasteberry seed can be safely added up to 25 g/kg in quail diets. Nevertheless, for a better understanding of the mode of action of chasteberry seeds, further studies investigating their effects on both the growth performance and antioxidant parameters of poultry are needed.