The use of phytogenic feed additives (PFAs) in animal feeding has gained significant attention in recent years, particularly after the ban on antibiotics as growth promoters. PFAs are plant-derived substances and are utilized to improve livestock and poultry productivity. These additives encompass a wide range of herbs, spices, and their derivatives (Windisch et al., 2008). The bioactive compounds found in PFAs, such as polyphenols, organic acids, essential oils, terpenoids, and aldehydes, are known to possess antioxidant and antibacterial properties that can have a positive impact on animal productivity (Shirani et al., 2019). In addition, PFAs can stimulate blood circulation and promote the production of digestive secretions, including mucin, amylase, lipase, trypsin protease, and bile acid (Mohebodini et al., 2021). Previous studies have shown that PFAs can impact the gut microbiome (Barbarestani et al., 2020), improve lipid metabolism (Mohebodini et al., 2019), enhance intestinal mucosal morphology, strengthen the intestinal barrier function (Oso et al., 2019), and boost the immune system of broiler chickens (Pirgozliev et al., 2019). However, our knowledge about their applications in poultry nutrition is still somewhat limited.

Turmeric (Curcuma longa) is a spice that belongs to the Zingiberaceae family and is widely distributed in tropical and subtropical regions of the world, particularly in Asian countries (Amalraj et al., 2017). As a traditional herbal medicine, turmeric has been used for centuries to treat various ailments, including urinary tract infections, liver disorders, inflammation, menstrual disorders, and digestive issues (Salehi et al., 2019). Both in vitro and in vivo studies have demonstrated that turmeric possesses numerous biological and pharmacological properties, such as antioxidant, antibacterial, antifungal, anti-inflammatory, and immunomodulatory effects (Strimpakos and Sharma, 2008; Mirzaei et al., 2017). The pharmacological activity of turmeric is primarily attributed to the presence of various curcuminoid compounds, including curcumin, demethoxycurcumin, and bisdemethoxycurcumin (Salehi et al., 2019).

Black pepper (Piper Nigrum) is another significant, widely-used spice with therapeutic benefits. In traditional medicine, it is utilized to alleviate symptoms of dizziness, asthma, chronic indigestion, flu, fever, chills, and migraine (Gorgani et al., 2017). Piperine, the primary active compound found in black pepper, has been extensively researched in recent years due to its relatively low side effects and a broad range of physiological activities, including antioxidant, antimicrobial, anti-inflammatory, anticarcinogenic, and cholesterol-lowering effects (Vijayakumar and Nalini, 2006; Shityakov et al., 2019). Other benefits of piperine include promoting blood circulation, stimulating appetite, and producing saliva and digestive secretions in poultry (Ogbuewu et al., 2020). Although the pharmaceutical activities of turmeric and black pepper in humans and various experimental animals have been well-documented, there have been limited studies on the effects of these additives on poultry as natural growth promoters.

Given the pharmacological properties of turmeric and black pepper documented in previous in vitro studies, the current research postulated that these phytogenic products would work synergistically to enhance each other’s beneficial effects. Therefore, the present study aimed to investigate the impact of turmeric and black pepper powder, either separately or in combination, on the growth performance, gastrointestinal microbial population, intestinal morphology, blood metabolites, meat quality, and fatty acid profile of Japanese quails. Additionally, the study sought to assess the potential of these additives as alternatives to antibiotics.

The use of PFAs has become increasingly popular in the post-antibiotic era due to their various biological properties, including antibacterial, antioxidant, and anti-inflammatory effects. Phenolic and flavonoid compounds, which are secondary metabolites of plants, are primarily responsible for their antioxidant and antimicrobial activities. In the current study, turmeric extract’s phenolic and flavonoid contents were higher than black pepper extract’s, resulting in higher DPPH scavenging activity for turmeric. The feeding trial results indicated that quail chicks fed diets supplemented with AGP and TUP had higher body weight gain and lower FCR than the other groups. However, adding BPP to the experimental diets did not enhance growth performance at any trial stage. Likewise, Khan and Ahmad (2022) reported that TUP at a 0.1% inclusion rate increased BWG and reduced FCR in broiler chickens. Yarru et al. (2009) also suggested adding TUP at a 0.5% inclusion rate to diets to improve BWG in aflatoxin-exposed broiler chickens. Furthermore, prior research has demonstrated that adding different PFAs to broiler diets, such as Pulicaria gnaphalodes powder and Achyrabthes japonica extract, can improve BWG, FCR, nutrient digestibility, gut microbiota, and intestinal morphology (Shirani et al., 2019; Park and Kim, 2020). Plant extracts, spices, and herbs can improve nutrient digestion in poultry by stimulating various digestive functions, such as appetite, saliva secretion, bile acid secretion, and digestive enzyme activity, including lipase, amylase, and protease (Jang et al., 2007; Oso et al., 2019). These outcomes can ultimately contribute to improved growth performance. The improved growth performance in birds fed the TUP diet in the present study was associated with the higher concentrations of phenolic and flavonoid compounds in turmeric extract. Nevertheless, some studies on PFAs in poultry diets have yielded inconsistent results regarding growth performance, such as the report by Jang et al. (2007). The lack of impact of diets containing BPP on growth performance in the current study is likely due to factors like inclusion dosage, BPP composition, active ingredient concentrations, and their biological activity.

In terms of carcass characteristics, breast yield was found to be higher in birds fed AGP, TUP, and MIX than in the other groups, indicating the growth-promoting effect of these feed additives. Furthermore, the present study’s findings demonstrate that using herbal additives (TUP, BPP, and MIX) can reduce abdominal fat in Japanese quails. Similarly, previous studies have demonstrated that dietary supplementation with 100–300 mg/kg turmeric rhizome extract reduces abdominal fat in broiler chickens (Wang et al., 2015). Xie et al. (2019) investigated the effect of curcumin (the primary constituent of turmeric) on the lipid metabolism of broiler chickens and found that curcumin could reduce abdominal fat by decreasing hepatic and plasma lipid concentration and regulating the expression of genes associated with lipogenesis and lipolysis, such as acetyl-CoA carboxylase. According to other studies, active substances present in medicinal plants can also reduce abdominal fat by inhibiting the synthesis of adipose tissue through the modulation of fatty acid transportation (Shirani et al., 2019).

Meat pH is a significant factor that affects meat quality attributes such as color and WHC. A decrease in postmortem pH can result in protein denaturation, leading to paleness and decreased WHC of the meat. The data from this study indicated that the MIX treatment increased the meat sample pH value after 7 days of refrigerated storage compared to the CON and AGP treatments. The WHC of meat is another critical parameter that indicates the muscle tissue’s ability to retain moisture and directly affects meat taste and tenderness. A lower WHC reflects losses in the meat’s nutritional value through released exudates, resulting in tougher and less flavorful meat (Cao et al., 2012). The simultaneous inclusion of TUP and BPP in the diet and their synergistic effect can control oxidation reactions in meat to maintain water storage space between myofibrils and increase WHC in meat (Huff-Lonergan and Lonergan, 2005). Rajput et al. (2014) also reported that natural antioxidants, such as carotenoids in curcumin, can improve meat’s WHC by modulating the redox state and enhancing the antioxidant capacity in muscle. Meat color is a crucial criterion related to meat freshness and quality, affecting consumer acceptance of meat. It is evaluated by determining the lightness, redness, and yellowness (Niu et al., 2017). According to Jiang et al. (2014), consumers prefer redness; a lower yellowness index indicates less pale meat. In the present study, diets supplemented with TUP and MIX decreased the lightness index in meat samples after 7 days of refrigerated storage compared to the other diets. Niu et al. (2017) suggested that the decrease in the lightness index might be related to the increased antioxidant activity of PFAs, which protects cells from damage and prevents cell juice extravasation, ultimately decreasing light reflection.

The MDA concentration in tissue indirectly mirrors lipid peroxidation, which is the outcome of attenuated antioxidant protection (Jazi et al., 2020). Long-term storage is one of the reasons for the increased lipid peroxidation in meat after slaughter (Aziza et al., 2010). In the current study, lower MDA content in breast muscle during 7 days of storage at 4°C was observed when quails were fed the MIX diet. Thus, the simultaneous inclusion of TUP and BPP may provide a more efficient free radical scavenging activity in birds. Similarly, improvements in the lipid peroxidation of meat have been reported in several studies when herbal additives rich in phenolic compounds or flavonoids were added to the diet (Aziza et al., 2010; Cao et al., 2012).

The current study found that the tested feed additives reduced pH and coliforms in the crop and ileum while increasing the LAB population in the ileum of birds. The growth inhibition of coliforms by TUP and BPP was similar to that of AGP. Curcumin possesses high antibacterial activity against Clostridium difficile, as shown by an in vitro study by Mody et al. (2020). Additionally, the administration of curcumin in humans has been reported to markedly shift the ratio of beneficial and pathogenic bacteria by enhancing the population of lactobacilli, bifidobacteria, and butyrate-producing bacteria and decreasing the number of enterococci and enterobacteria, as reported by Zam (2018). Moreover, several in vivo studies have demonstrated the potent antibacterial activity of black pepper essential oil against pathogenic Escherichia coli and Staphylococcus. (Zhang et al., 2017; Abdallah and Abdolla, 2018). Some studies with Japanese quails (Mehri et al., 2015) and broiler chickens (Chowdhury et al., 2018) indicate in vivo antibacterial activity of different PFAs (peppermint and cinnamon) against intestinal pathogenic microbes such as coliform and E. coli. One of the critical features of PFAs is their hydrophobicity, allowing them to easily enter the bacterial cell membrane, leading to cell membrane disintegration, leakage of intracellular material, reduction of proton force, and, eventually, death of the bacterial cell (Zeng et al., 2015; Barbarestani et al., 2020). Therefore, reducing the population of pathogenic bacteria, such as coliforms within the gastrointestinal tract, may increase the number of beneficial bacteria, such as LAB. Another possible explanation for the positive effect of PFAs on the growth of beneficial bacteria is that LAB can metabolize phenolic compounds as nutritional substrates, suggesting the prebiotic-like impact of phenolic compounds (Iqbal et al., 2020).

Intestinal epithelial integrity and structure play an important role in nutrient digestion, absorption, and overall gut health (Soumeh et al., 2019). Intestinal villi are the leading site for absorption of nutrients; therefore, longer villi may indicate a greater surface area, which in turn can enhance nutrient absorption capacity, while deeper crypt may suggest fast cellular turnover and regeneration processes of tissue due to damage that is induced by pathogens (Jazi et al., 2018b). Therefore, VH: CD can reflect digestive and absorptive capacities. The present study showed that the MIX treatment group had a higher VH: CD ratio in the duodenum, while the AGP, TUP, and MIX groups had higher VH and VH: CD ratio in the jejunum. The tested feed additives increased the ileum’s VH compared to the control group. Improving the intestinal mucosal structure in response to dietary PFAs may be linked to an increased population of beneficial bacteria, which can produce antibacterial compounds and compete with harmful pathogens like coliforms. This can prevent their colonization and minimize their negative impact on the intestinal structure (Jazi et al., 2017). Feeding broiler chickens with PFAs may also stimulate mucus secretion into the intestine, which can act as a dynamic protective surface and prevent the adherence of pathogens to intestinal epithelial cells (Cornick et al., 2015; Zeng et al., 2015). Furthermore, the antioxidant activity of PFAs may protect villi from oxidative stress caused by digestive processes (Zeng et al., 2015). The present study found that combining TUP and BPP in the diet improved the population of gastrointestinal microbiota and intestinal morphology more effectively, indicating synergistic effects between TUP and BPP.

The serum concentrations of cholesterol and triglycerides can reflect lipid metabolism (Sharifi et al., 2023). The current study revealed that the tested feed additives reduced the concentration of cholesterol. Kim and Kim (2010) reported a significant reduction in serum cholesterol levels in rats fed an excessive fat diet supplemented with curcumin, possibly due to increased expression of cholesterol 7α-hydroxylase, a rate-limiting enzyme in bile acid formation from cholesterol. Xie et al. (2019) suggested that curcumin reduces cholesterol by inhibiting ATP-citrate lyase, a key enzyme in fatty acid synthesis that converts citrate to acetyl CoA. Piperine, the active compound in black pepper, is known to reduce plasma and liver cholesterol by changing cholesterol transporter proteins and reducing lipogenic gene expression (Tunsophon and Chootip, 2016). Shirani et al. (2019) suggested that the hypocholesterolemic effect of PFAs may result from the active ingredient’s inhibitory effects on the 3-hydroxy-3-methyl glutaryl-CoA enzyme, a key enzyme for cholesterol biosynthesis. Serum levels of ALT, ALP, and AST serve as indicators of liver health and functionality, as their concentrations increase when liver cells are destroyed (Mohebodini et al., 2019). However, in the present study, the experimental treatments did not affect the concentrations of these enzymes and therefore had no negative impact on the liver.

Researchers have recently focused on modifying fatty acid compounds in poultry meat, as the level of SFA are correlated to hypercholesterolemia and coronary heart disease (Ahmed et al., 2015; Shirani et al., 2019). Our results demonstrated that dietary supplementation with TUP, BPP, and MIX increased the concentrations of total MUFA, PUFA, and PUFA/SFA ratio while decreasing total SFA content. However, the MIX diet had a more pronounced effect on the meat fatty acid profile, possibly due to the synergistic effects. Similar observations were made in broiler chickens fed phytogenic products containing TUP by Daneshyar et al. (2011). It has been suggested that palmitic acid (C16:0) often raises blood cholesterol levels, while oleic acid (C18:1) has the opposite effect on blood cholesterol. Additionally, PUFA may decrease abdominal fat deposition by enhancing the expression of acyl-CoA oxidase (the main enzyme of the beta-oxidation process) (Crespo and Esteve-Garcia, 2001; 2002). The decrease in SFA in the meat of birds may be related to an increase in the amount of oleic acid (C18:1) and MUFA, given that stearic acid (C18:0) is converted to oleic acid (C18:1) more rapidly (Ahmed et al., 2015). In addition, the enhanced PUFA concentration may be due to the improvement of the antioxidant defense system, which could protect PUFA oxidation and cause the production of stable products (Mohebodini et al., 2021). The PUFA: SFA and n-6: n-3 ratios are typically used to assess the nutritional value of meat. Ahmed et al. (2015) suggested that meat with a PUFA: SFA ratio less than 0.4 and n-6: n-3 ratio greater than 5 is considered unfavorable because they may increase cholesterolemia. In the current study, although the n-6: n-3 ratio was not affected by the experimental treatments, the PUFA: SFA ratio in birds fed the additives was higher than the minimum recommended values, indicating a positive effect of the tested PFAs on meat quality. In addition to the above indices, a more comprehensive approach to the nutritional evaluation of meat could involve using indices based on the functional effects of fatty acids, such as the HH ratio (Santos-Silva et al., 2002). The highest HH index values were recorded in birds fed the MIX diet, indicating a better nutritional value of meat in this group. Therefore, these findings suggest that consuming these meats may lower humans’ risk of coronary disease.

The present study indicates that the simultaneous application of TUP and BPP to improve growth performance is ineffective. However, TUP alone can result in improvements in BWG and FCR. Additionally, because the simultaneous inclusion of TUP and BPP can positively affect the population of gastrointestinal microbiota and intestinal morphology, their combination may be more effective under stressful conditions. Combining TUP and BPP in the diet also improves breast meat quality and shelf life, indicating the synergistic effects between TUP and BPP.