Abstract
The purpose of this experiment was to use a single-factor design to investigate the effects of different supplementation levels of plant essential oil/palygorskite composite (EPO-Pal) on growth performance, blood parameters, and intestinal morphology of broiler chicken. A total of 960 one-day-old chicks were randomly assigned into 4 treatments with 6 replicates of 40 chicks each. The broilers were fed the basal diet supplemented with 0, 500, 750, and 1000 mg/kg EPO-Pal for 42 days, respectively. The results showed that the 500 or 750 mg/kg groups significantly increased the body weight, average daily feed intake and average daily gain of the broilers at 28 and 42 days (p < 0.05). Additionally, as compared to the control group, the 1000 mg/kg EPO-Pal group exhibited higher levels of immunoglobulins-A, immunoglobulins-G, immunoglobulins-M, and high-density lipoprotein cholesterol while having lower levels of tumour necrosis factor-α (p < 0.05). The content of interleukin-2 and the villus height-to-crypt depth ratio were significantly increased in the 750 mg/kg EPO-Pal group (p < 0.05). Moreover, the activity of total antioxidant capacity and superoxide dismutase were significantly enhanced in the 500 mg/kg EPO-Pal group (p < 0.05). The results are very promising, and EPO-Pal can be considered as an additive to promote the growth by enhancing immunity, antioxidant activity, and improving the intestinal morphology of broiler chickens.
HIGHLIGHTS
Dietary supplementation of EPO-Pal improved the growth performance by increased average daily feed intake of broiler chickens.
Dietary supplementation of EPO-Pal enhanced plasma antioxidation and immune response of broiler chickens.
Dietary supplementation of EPO-Pal can postively affect the intestinal morphology of broiler chickens.
Introduction
Antibiotic growth promoters or in-feed antibiotics have been widely used in animal production since 1946 when they were discovered to promote animal growth (Moore et al. Citation1946). However, issues such as antibiotic residues and drug resistance have garnered increased attention from consumers. In the interest of food safety, the European Union (EU) banned the use of antibiotics as early as 2006, and China also implemented a ban on antibiotics since 2020 (http://www.moa.gov.cn/gk/, accessed on January 6th, 2020). Consequently, there has been a growing demand for safe, efficient, and residue-free growth-promoting feed additives in animal husbandry (Landy et al. Citation2020; Landy and Kheiri Citation2023).
Plant essential oil (PEO) refers to the extraction of plant secondary metabolites from plant tissues and organs, which contain a large number of aldehydes, ketones, esters, terpenes, phenols and phenylpropanoids (Dhifi et al. Citation2016; Perumal et al. Citation2022). PEO have been widely used because of its effects promoting growth (Tiihonen et al. Citation2010), anti-inflammatory (Luna et al. Citation2019), antioxidant activity (Dhifi et al. Citation2016), and antibacterial properties (Bora et al. Citation2020). As green and natural additives, numerous studies have proven the safety and efficiency of PEO in livestock production, including pigs (Long et al. Citation2021), poultry (Ding et al. Citation2022), ruminants (Palhares et al. Citation2021), among others. However, PEO cannot be used as a feed additive for production because of its volatility, instability, and difficulty preservation (Sharma et al. Citation2019; Shlosman et al. Citation2022). Therefore, there is a need for effective protective materials to facilitate its application.
Palygorskite (Pal) is a hydrous magnesium-rich aluminium silicate clay mineral, which has been approved by the EU as a carrier of feed additive since 2003 due to its characteristics of good thermal stability, acid resistance, and adsorptive capacity (Galán Citation1996; Murray Citation2000). By modifying its charge, altering surfactant groups, and employing other processes to load PEO onto Pal, PEO can be effectively preserved and utilised (Lei et al. Citation2017; Ghrab et al. Citation2018). Our previous research has shown that supplementing 750 mg/kg PEO-Pal improved egg production by enhancing antioxidative and immune functions and altering the intestinal morphology of laying hens (Cheng et al. Citation2022). We speculate that similar effects may be observered in broiler chickens, such as improving the growth performance thereby enhancing economic benefits of broilers.
‘Yunwu No.1’ is a high-quality broiler chicken breed produced by crossbreeding Xuefeng black-bone chickens with fast-growing broilers. It combines the flavour characteristics of traditional Chinese chicken breeds with the rapid growth rates commonly seen in commercial broilers. This makes it of significant economic and research importance. Consequently, this study was designed to investigate the effects of different levels of PEO-Pal on the growth performance, blood parameters, and intestinal morphology of broiler chickens.
Materials and methods
The PEO-Pal
Plant Essential oil/palygorskite composite (PEO-Pal) was supplied by Jiangsu Sinitic Biological Technology Co., Ltd (Jiangsu, China). The preparation method for PEO-Pal was consistent with the procedure previously described in our study (Cheng et al. Citation2022). The main components of PEO-Pal consistent of 11% PEO (including carvacrol and thymol) and 89% palygorskite.
Animals, experimental design, and management
All the birds and the experimental protocols were approved by the Institutional Animal Care and Use Committee of Hunan Agricultural University, Hunan, China. The ‘Yunwu No.1’ broiler chicken were sourced from Hunan Yunfeifeng Agricultural Commercial Company (Hunan, China). This experiment utilised a single-factor experimental design. A total of 960 one-day-old healthy female broiler chicks, each with a similar weight (33.80 ± 0.21), were randomly and evenly divided into 4 treatment groups, with 6 replicates consistenting of 40 chicks in each. The dietary treatments were as follows: (1) basal diet; (2) basal diet + 500 mg/kg PEO-Pal; (3) basal diet + 750 mg/kg PEO-Pal; (4) basal diet + 1000 mg/kg PEO-Pal. The PEO-Pal was incorporated into the feed through gradual mixing, and the composition and ingredients of the diets are detailed in Table . The experimental diets were formulated to meet the nutrient requirements of the birds according to the China Agricultural Standard (NY/T 33-2004).
Table 1. Composition and nutrient levels of the experimental diets (dry matter basis).
Each replicate consisted of 8 cages, with 5 chicks raised in each cage (50 × 50 × 36 cm; length × width × height). All the chicks were fed their respective experimental diets for a duration of 42 days. The chicks were fed twice a day (06:30 am and 2:30 pm) and given ad libitum access to water throughout the experiment. The temperature in the chicken coop was 33 °C for the first three days, and then gradually decreased 1 °C every three days until the final temperature reaches 20 °C. The lighting regimen used was 24 h light throughout the entire experimental period. Immunisation procedures are carried out according to company standards.
Sample collection
At the 22 d and 43 d of the experiment, 12 chicks (2 chicks per replicate) were randomly selected from each treatment after fasting for 12 h. Blood samples (3 mL) were obtained via the jugular vein using the vacutainer tube. Blood samples were placed at room temperature for 1 h, subsequently centrifuged at 3000 × g for 10 min to separate plasma, and plasma were stored in 1.5 mL Eppendorf tube at －20 °C for further analysis. After collected blood samples, the chicks were exsanguinated and dissected to obtain immune organ (include spleen and bursa of Fabricius). And immune organ index is obtained by the weight of the immune organ divided by live weight of the bird. And the duodenum, jejunum and ileum were taken 2 cm, washed with normal saline and fixed in 4% paraformaldehyde for intestinal morphology measurements.
Growth performance
The chicks were weighed on 1 d, 15 d, 29 d, and 43 d respectively, and recorded the feed intake at the same time. To ensure data credibility, mortality and health status were visually recorded daily. The growth performance index including body weight (BW), average daily feed intake (ADFI), average daily gain (ADG), and feed/gain ratio (F/G ratio).
Blood parameters
The plasma biochemical indexes include glucose (GLU), total protein (TP), albumin (ALB), globulin (GLB), triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and glutamic oxalacetic transaminase (AST) were measured by Mindray automatic analyser (BS-300, Shenzhen Mindray Bio-Medical Electronics Co., Ltd, Shenzhen, Guangdong, China) according to the commercial kits (Shenzhen Mindray BioMedical Electronics Co., Ltd, Shenzhen, China). And the plasma antioxidant index including total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activity were determined with commercial radioimmunoassay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the instructions of the manufacturer. Additionally, the plasma immune indexes include immunoglobulins A (IgA), immunoglobulins G (IgG), immunoglobulins M (IgM), tumour necrosis factor-α (TNF-α) and interleukin-2 (IL-2) were assayed with enzyme-linked immunosorbent assay (ELISA) kit (ZCIBIO Technology Co., Ltd, Shanghai, China) according to the instructions of the manufacturer.
Intestinal morphology
The intestinal tissue fixed samples were embedded in paraffin, sectioned (4 mm), followed by hematoxylin-eosin staining. Morphological measurements were performed on 10 villi chosen from each segment by CaseViewer Image analysis software, and measurements of the villus height (VH) and crypt depth(CD) were taken and the villus height-to-crypt depth ratio (VH/CD) were calculated subsequently.
Statistical analysis
All data were analysed by one-way analysis of variance (ANOVA) using SPSS 22.0 statistical software (SPSS Institute Inc., Chicago, Illinois), and the statistical differences among the groups were determined by Duncan’s multiple range test, and the replicate was used as the experimental unit. The results are expressed as arithmetic mean ± standard deviation, p < 0.05 was considered statistically significant.
Results
Growth performance
As presented in Table , the BW of 500 and 750 mg/kg PEO-Pal groups showed a significant increase compared with the control group at 28 and 42 days, respectively (p < 0.05). The ADFI of the PEO-Pal groups and the ADG of 500 mg/kg PEO-Pal group were significantly improved compared to the control group during the day15–28 (p < 0.05). At 29–42 days, the ADFI of 750 mg/kg PEO-Pal group and the ADG of 500 and 750 mg/kg PEO-Pal groups were significantly increase compared with the control group (p < 0.05). And throughout the entire experimental period, the ADFI of the PEO-Pal groups and the ADG of 500 and 750 mg/kg PEO-Pal groups were significantly increase compared with the control group (p < 0.05).
Table 2. Effects of Plant Essential oil/palygorskite composite (PEO-Pal) on growth performance of broiler chickenTable Footnote1.
Immune organ index
As indicated in Table , there was no significant differences in immune organ indices among the four groups at both 21 days or 42 days (p > 0.05).
Table 3. Effects of Plant Essential oil/palygorskite composite (PEO-Pal) on immune organ index of broiler chickenTable Footnote1.
Plasma immune response
The effects of dietary PEO-Pal on plasma immune response results are reported in Table . At day 21, the concentration of IgA and IgM in 1000 mg/kg PEO-Pal group was significantly higher than that in the control group (p < 0.05), while the concentration of TNF-α was significantly lower than that in the control group (p < 0.05). At day 42, compared with the control group, the concentration of IgA and IgG in 1000 mg/kg PEO-Pal group was significantly increased (p < 0.05) and the concentration of TNF-α in 1000 mg/kg PEO-Pal group was significantly decreased (p < 0.05), additionally, the content of IL-2 in 750 mg/kg PEO-Pal group was significantly improved (p < 0.05).
Table 4. Effects of Plant Essential oil/palygorskite composite (PEO-Pal) on plasma immune indexes of broiler chickenTable Footnote1.
Plasma antioxidant capacity
The results of plasma antioxidant capacity are presented in Table . There was no significant difference in plasma antioxidant capacity among the 4 groups at 21 days (p > 0.05). However, during day 42, the content of T-AOC and SOD in 500 mg/kg PEO-Pal group was significantly higher than the control group (p < 0.05).
Table 5. Effects of Plant Essential oil/palygorskite composite (PEO-Pal) on plasma antioxidant capacity of broiler chickenTable Footnote1.
Plasma biochemical indexes
The results of plasma biochemical indexes in broiler chick are presented in Table . Compared with the control group, the content of HDL-C was significantly higher in the 1000 mg/kg PEO-Pal group at 42 days (p < 0.05).
Table 6. Effects of Plant Essential oil/palygorskite composite (PEO-Pal) on plasma biochemical indexes of broiler chicken at 42 dayTable Footnote1.
Intestinal morphology
The results of broiler chick intestinal morphology are presented in Table . The VH/CD of the jejunum in 750 mg/kg PEO-Pal group was significantly higher than that in the other groups (p < 0.05). There were no significant differences in VH and CD of all intestinal segments among all the groups (p > 0.05). moreover, the VH/CD of the duodenum and ileum did not show significant difference among the 4 groups (p > 0.05).
Table 7. Effects of Plant Essential oil/palygorskite composite (PEO-Pal) on intestinal morphology of broiler chicken at 42 dayTable Footnote1.
Discussion
While numerous studies have demonstrated the growth-promoting properties and safety of plant essential oils (PEO), research results often vary due to differences in the type and dosage of PEO, as well as the breed and age of animals. China, in particular, boasts a rich variety of resources, and there may be different effects when supplementing the diet with PEO in different breeds. Our previous research has also indicated that PEO is suitable for Chinese local chicken breeds (Liu et al. Citation2020), but there are no report in ‘Yunwu No.1’ broiler chicken. Based on this, we hypothesise that PEO-Pal could effectively improve broiler chicken production performance, but the specific mechanism is not very clear. So we conducted further investigations to understand the functional mechanisms of PEO-Pal by evaluating its effects on immune response, antioxidant capacity, and intestinal morphology.
Previous studies have demonstrated that plant essential oils primarily enhance the growth performance of animals through several mechanisms. Firstly, they can improve the palatability of feed, thereby increasing daily feed intake in animals (Clouard and Val-Laillet Citation2014). Secondly, plant essential oils can enhance nutrient utilisation by modulating the intestinal microflora and, consequently, improve digestive capabilities (Windisch et al. Citation2008; Bravo et al. Citation2014). Lastly, plant essential oils can regulate the secretion of growth-related hormones, thereby promoting animal growth (Ariza-Nieto et al. Citation2011; Li et al. Citation2012). In our study, we observed significant improvements in body weight, average daily gain, and average daily feed intake due to dietary supplementation with PEO-Pal. However, it’s important to note that the results of our study differ from some previous research. For instance, Xue et al. (Citation2020) reported that dietary supplementation with 100 mg/kg of mixed plant essential oil had no influence on the growth performance of broilers. On the other hand, Ding et al. (Citation2022) found that supplementing the diet with 400 mg/kg of essential oils improved average daily gain and body weight while reducing the feed conversion ratio in chickens. Park and Kim (Citation2018) also observed increased body weight gain and reduced feed conversion ratio in broiler chicks fed a diet containing 0.03% essential oils. These variations in results may be attributed to differences in dosage and the specific varieties of essential oils used in the studies
The spleen and bursa of Fabricius are important immune organs in poultry, and they serve as indicators of the body’s immune capability (Rivas and Fabricant Citation1988). The bursa of Fabricius plays a unique role in avian immune function, primarily contributing to humoral immunity. The spleen, being the largest peripheral immune organ in poultry, also plays a crucial role in immune responses. The weight of these immune organs is closely linked to their cell proliferation rates and their overall contribution to the body’s immune function (Hassan et al. Citation2010). Several previous studies have reported that dietary supplementation with plant essential oils has no significant impact on the immune organs of animals (Kim et al. Citation2016; Wang et al. Citation2019; Oladokun et al. Citation2021). Our findings in this study align with these previous research results. Specifically, we observed no significant differences in immune organ indices among all experimental groups at both 21 and 42 days of age.
However, the results of the plasma immune response tests indicated that PEO-Pal can enhance the immune competence of chickens. Immunoglobulins play a crucial role in the immune system as they regulate the inflammatory response, and their levels serve as important indicators of immune function (Magnadóttir Citation2006; Elghandour et al. Citation2020). Among the key immunoglobulins, IgA primarily contributes to mucosal immunity, IgG plays a central defensive role throughout the inflammatory response due to its longer half-life, and IgM exhibits the fastest response and plays a pivotal role in early inflammation (Elghandour et al. Citation2020; Kim et al. Citation2022).
Our study indicated that dietary supplementation with 1000 mg/kg of PEO-Pal had a positive impact on the concentrations of IgA, IgG, and IgM. Furthermore, in comparison to the control group, the 1000 mg/kg PEO-Pal group showed a significant reduction in TNF-α levels, while the 750 mg/kg PEO-Pal group exhibited a remarkable increase in IL-2 levels. Cytokines encompass both pro-inflammatory factors, such as TNF-α, and anti-inflammatory factors, like IL-2. As intercellular signalling molecules, the primary role of cytokines in the immune system is to mediate and regulate immune responses (Hou et al. Citation2015). Previous studies have demonstrated that PEO can enhance an animal’s immune response by increasing immunoglobulin levels and the presence of anti-inflammatory factors, while inhibiting the activity of pro-inflammatory factors (Liu et al. Citation2019; Sivandzade et al. Citation2019; Su et al. Citation2021). Our research also proves this point.
Oxidative stress refers to an increase in the generation of free radicals in the body or a decrease in its ability to scavenge them, which caused accumulation of free radicals and then result in oxidative damage (Sohal and Allen Citation1990). Oxidative stress is also a significant factor contributing to the reduction in animal growth performance and the quality of animal products. In this paper, we observed that the group supplemented with 500 mg/kg PEO-Pal showed an increase in the activity of T-AOC and SOD in the plasma of broiler chickens compared to the control group. T-AOC represents the total antioxidant capacity of the body when exposed to external stimuli (Siegel and Latimer Citation1984). SOD and GSH-Px are the primary antioxidant enzymes. SOD activity reflects the body’s ability to scavenge free radicals, and its primary function is to catalyse the disproportionation reaction of superoxide anions and free radicals, thereby protecting cells and tissues from damage (Giardino et al. Citation2002). GSH-Px, an enzyme that catalyses the decomposition of hydrogen peroxide, helps maintain the integrity of cell membrane structure and function (Surai et al. Citation2018). Previous studies have shown that dietary supplementation with essential oils can significantly increase T-AOC, SOD, and GSH-Px activities in the plasma, jejuna, and ileal mucosa of chickens (Ruan et al. Citation2021). Other studies by Zhang et al. (Citation2021) and Su et al. (Citation2018) have also demonstrated that dietary essential oils can enhance the activity of T-AOC, SOD, and GSH-Px in chicken plasma. PEO enhance the antioxidant function of plasma through two aspects, on the one hand, the phenolic hydroxyl group of PEO can bind to peroxy radicals, thereby alleviating oxidative stress (Brenes and Roura Citation2010; Asghari et al. Citation2021), on the other hand, PEO can influence the activity of antioxidant enzymes by regulating nuclear factor erythroid-2 related factor2 (Meeran and Prince Citation2012; Kang et al. Citation2015).
Plasma biochemical indices are important markers for assessing the health status and physiological metabolism of an organism, and they can be influenced by a variety of factors (Vicari et al. Citation2008). Hesabi et al. (Citation2019) found that a blend of essential oils significantly altered the levels of cholesterol, aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase, while not affecting glucose, triglycerides, HDL, and LDL. Ibrahim et al. (Citation2019) demonstrated that essential oils improved dyslipidemia, leading to significant reductions in total cholesterol, triacylglycerol, and low-density lipoprotein-cholesterol, along with an increase in high-density lipoprotein-cholesterol. And Maral et al. (Citation2022) also demonstrated that essential oils significantly increased the content of HDL-C and decreased the content of LDL-C in athlete plasma. In our paper, the addition of 1000 mg/kg PEO-Pal to the diet significantly increased the plasma concentration of HDL-C in broiler chickens.The differences in our results compared to previous studies may be attributed to factors such as the chicken breed, nutritional composition of the diet, dosage of the additive, or other variables. Previous research has suggested that thymol and carvacrol, two components of essential oils, can promote adipocyte metabolism, reduce sterol regulatory element-binding protein-1c levels, and inhibit lipid accumulation (Saravanan and Pari Citation2016; Choi et al. Citation2017). HDL-C is considered a beneficial form of cholesterol due to its ability to reduce overall cholesterol levels and its anti-inflammatory, antioxidant, and anti-apoptotic properties (Rosenson et al. Citation2016).
Intestinal morphology plays a crucial role in the process of digestion and absorption, and indicators such as villus height (VH), crypt depth (CD), and the VH/CD ratio are commonly used to assess intestinal structure. The VH/CD ratio, in particular, is positively correlated with the digestive and absorptive functions of the intestine (Dubrovsky and Dunn Citation2018; Mazzoni et al. Citation2022). Du et al. (Citation2016) reported that dietary supplementation with thymol and carvacrol improved the villus height and VH/CD ratio in the ileum of broilers. Hesabi et al. (Citation2019) demonstrated that diets containing 150 or 200 mg/kg of essential oils could increase the ileal villus height and VH/CD ratio in broilers. Furthermore, our previous study showed that the addition of 750 mg/kg PEO-Pal to the diet improved the VH/CD ratio in the jejunum of laying hens (Cheng et al. Citation2022). In this study, we observed that the VH/CD ratio in the jejunum of the 750 mg/kg PEO-Pal group was significantly higher than in the other groups. Numerous studies have provided evidence that PEO can improve the intestinal health of animals. PEO helps maintain a balanced intestinal microbiota, preventing the destructive effects of pathogenic bacteria on the intestine, and preserving the architecture of intestinal epithelial cells. Meanwhile, its antioxidant function can promote the growth of the intestinal villi (Castillo et al. Citation2006; Jang et al. Citation2007; Yang et al. Citation2014).
In conclusion, dietary supplementation with PEO-Pal can improve growth performance by boosting immunity and antioxidant activity while also positively affecting intestinal morphology in broiler chicken. Our findings suggest that dietary supplement with 500 mg/kg PEO-Pal during days 1–28 and increase the dosage of PEO-Pal to 750 mg/kg on days 29–42, which may obtain optimal growth performance in broiler chickens. This study provides a theoretical basis for the scientific use of PEO-Pal.
Authors’ contributions
XYQ conceptualised and designed the experiment; CQH, YL, JX and YBW performed the experiment; YLY and MCL provided resource and supervision; YYD and YD writing original draft, review and editing. All authors have read and agreed to the published version of the manuscript.
Acknowledgements
This work was financially supported by Hunan Provincial Science and Technology Plan Project - Innovation Platform and Talent Project (2022NK4156) and Black-Bone Chicken Forest And Antibiotic-Free Breeding Technology Development And Demonstration Project (2021KJC-JS073).
Data availability statement
Raw data were generated at the Hunan Agricultural University. Data availability statement: The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Disclosure statement
The authors declare that there are no conflicts of interest.
Correction Statement
This article has been corrected with minor changes. These changes do not impact the academic content of the article.
References
- Ariza-Nieto C, Bandrick M, Baidoo SK, Anil L, Molitor TW, Hathaway MR. 2011. Effect of dietary supplementation of oregano essential oils to sows on colostrum and milk composition, growth pattern and immune status of suckling pigs. J Anim Sci. 89(4):1079–1089. doi: 10.2527/jas.2010-3514.
- Asghari M, Abdi-Benemar H, Maheri-Sis N, Salamatdoust-Nobar R, Salem AZ, Zamanloo M, Anele UY. 2021. Effects of emulsified essential oils blend on performance, blood metabolites, oxidative status and intestinal microflora of suckling calves. Anim Feed Sci Tech. 277:114954. doi: 10.1016/j.anifeedsci.2021.114954.
- Brenes A, Roura E. 2010. Essential oils in poultry nutrition: main effects and modes of action. Anim Feed Sci Technol. 158(1-2):1–14. doi: 10.1016/j.anifeedsci.2010.03.007.
- Bravo D, Pirgozliev V, Rose SP. 2014. A mixture of carvacrol, cinnamaldehyde, and capsicum oleoresin improves energy utilization and growth performance of broiler chickens fed maize-based diet. J Anim Sci. 92(4):1531–1536. doi: 10.2527/jas.2013-6244.
- Bora H, Kamle M, Mahato DK, Tiwari P, Kumar P. 2020. Citrus essential oils (CEOs) and their applications in food: an overview. Plants. 9(3):357. doi: 10.3390/plants9030357.
- Castillo M, Martín-Orúe SM, Roca M, Manzanilla EG, Badiola I, Perez JF, Gasa J. 2006. The response of gastrointestinal microbiota to avilamycin, butyrate, and plant extracts in early-weaned pigs. J Anim Sci. 84(10):2725–2734. doi: 10.2527/jas.2004-556.
- Clouard C, Val-Laillet D. 2014. Impact of sensory feed additives on feed intake, feed preferences, and growth of female piglets during the early postweaning period. J Anim Sci. 92(5):2133–2140. doi: 10.2527/jas.2013-6809.
- Choi JH, Kim SW, Yu R, Yun JW. 2017. Monoterpene phenolic compound thymol promotes browning of 3T3-L1 adipocytes. Eur J Nutr. 56(7):2329–2341. doi: 10.1007/s00394-016-1273-2.
- Cheng H, Chen JF, Tang SG, Guo SC, He CQ, Qu XY. 2022. Effects of essential oil/palygorskite composite on performance, egg quality, plasma biochemistry, oxidation status, immune response and intestinal morphology of laying hens. Poult Sci. 101(4):101632. doi: 10.1016/j.psj.2021.101632.
- Dhifi W, Bellili S, Jazi S, Bahloul N, Mnif W. 2016. Essential oils’ chemical characterization and investigation of some biological activities: a critical review. Medicines. 3(4):25. doi: 10.3390/medicines3040025.
- Du E, Wang W, Gan L, Li Z, Guo S, Guo Y. 2016. Effects of thymol and carvacrol supplementation on intestinal integrity and immune responses of broiler chickens challenged with Clostridium perfringens. J Anim Sci Biotechnol. 7:1–10.
- Dubrovsky G, Dunn JC. 2018. Mechanisms for intestinal regeneration. Curr Opin Pediatr. 30(3):424–429. doi: 10.1097/MOP.0000000000000630.
- Ding Y, Hu Y, Yao X, He Y, Chen J, Wu J, Wu S, Zhang H, He X, Song Z. 2022. Dietary essential oils improves the growth performance, antioxidant properties and intestinal permeability by inhibiting bacterial proliferation, and altering the gut microbiota of yellow-feather broilers. Poult Sci. 101(11):102087. doi: 10.1016/j.psj.2022.102087.
- Elghandour MMY, Tan ZL, Abu Hafsa SH, Adegbeye MJ, Greiner R, Ugbogu EA, Cedillo Monroy J, Salem AZM. 2020. Saccharomyces cerevisiae as a probiotic feed additive to non and pseudo‐ruminant feeding: a review. J Appl Microbiol. 128(3):658–674. doi: 10.1111/jam.14416.
- Galán E. 1996. Properties and applications of palygorskite-sepiolite clays. Clay Miner. 31:443–453.
- Giardino R, Giavaresi G, Fini M, Torricelli P, Guzzardella GA. 2002. The role of different chemical modifications of superoxide dismutase in preventing a prolonged muscular ischemia/reperfusion injury. Artif Cells Blood Substit Immobil Biotechnol. 30(3):189–198. doi: 10.1081/bio-120004339.
- Ghrab S, Eloussaief M, Lambert S, Bouaziz S, Benzina M. 2018. Adsorption of terpenic compounds onto organo-palygorskite. Environ Sci Pollut Res Int. 25(19):18251–18262. doi: 10.1007/s11356-017-9122-2.
- Hassan HMA, Mohame MA, Youssef AW, Hassan ER. 2010. Effect of using organic acids to substitute antibiotic growth promoters on performance and intestinal microflora of broilers. Asian Australas J Anim Sci. 23(10):1348–1353. doi: 10.5713/ajas.2010.10085.
- Hou YC, Wu JM, Wang MY, Wu MH, Chen KY, Yeh SL, Lin MT. 2015. Modulatory effects of Astragalus polysaccharides on T-cell polarization in mice with polymicrobial sepsis. Mediators Inflamm. 2015:826319–826310. doi: 10.1155/2015/826319.
- Hesabi NA, Edalatian O, Bakhshalinejad R. 2019. Effects of a blend of thyme, peppermint and eucalyptus essential oils on growth performance, serum lipid and hepatic enzyme indices, immune response and ileal morphology and microflora in broilers. J Anim Physiol Anim Nutr (Berl). 103(5):1388–1398. doi: 10.1111/jpn.13122.
- Ibrahim FA, Usman LA, Akolade JO, Idowu OA, Abdulazeez AT, Amuzat AO. 2019. Antidiabetic potentials of Citrus aurantifolia leaf essential oil. Drug Res (Stuttg). 69(4):201–206. doi: 10.1055/a-0662-5607.
- Jang IS, Ko YH, Kang SY, Lee CY. 2007. Effect of a commercial essential oil on growth performance, digestive enzyme activity and intestinal microflora population in broiler chickens. Anim Feed Sci Technol. 134(3-4):304–315. doi: 10.1016/j.anifeedsci.2006.06.009.
- Kang JS, Han MH, Kim G-Y, Kim CM, Chung HY, Hwang HJ, Kim BW, Choi YH. 2015. Schisandrae semen essential oil attenuates oxidative stress-induced cell damage in C2C12 murine skeletal muscle cells through Nrf2-mediated upregulation of HO-1. Int J Mol Med. 35(2):453–459. doi: 10.3892/ijmm.2014.2028.
- Kim SJ, Lee KW, Kang CW, An BK. 2016. Growth performance, relative meat and organ weights, cecal microflora, and blood characteristics in broiler chickens fed diets containing different nutrient density with or without essential oils. Asian-Australas J Anim Sci. 29(4):549–554. doi: 10.5713/ajas.15.0426.
- Kim S, Yoo S, Lee JI, Kim S, Chang HY, Kim D, Jeong SH, Lee KS, Lee HW. 2022. Anti-HBc IgG levels: a predictor of HBsAg seroclearance in chronic hepatitis b patients with nucleos (t) ide analogue-induced HBeAg seroclearance. Dig Dis Sci. 67(1):321–328. doi: 10.1007/s10620-021-06845-2.
- Landy N, Kheiri F, Faghani M. 2020. Evaluation of cottonseed bioactive peptides on growth performance, carcase traits, immunity, total antioxidant activity of serum and intestinal morphology in broiler chickens. Ital J Anim Sci. 19(1):1375–1386. doi: 10.1080/1828051X.2020.1844085.
- Landy N, Kheiri F. 2023. Effects of Hydrolyzed Cottonseed Protein on Growth Performances, Carcass Traits, Immunity, Microbial and Morphological Responses of the Small Intestine and Total Antioxidant Capacity of Serum, and Small Intestine in Broiler Chickens. Iran J Appl Anim Sci. 13:121–132.
- Li P, Piao X, Ru Y, Han X, Xue L, Zhang H. 2012. Effects of adding essential oil to the diet of weaned pigs on performance, nutrient utilization, immune response and intestinal health. Asian-Australas J Anim Sci. 25(11):1617–1626. doi: 10.5713/ajas.2012.12292.
- Lei H, Wei Q, Wang Q, Su A, Xue M, Liu Q, Hu Q. 2017. Characterization of ginger essential oil/palygorskite composite (GEO-PGS) and its anti-bacteria activity. Mater Sci Eng C Mater Biol Appl. 73:381–387. doi: 10.1016/j.msec.2016.12.093.
- Luna EC, Luna IS, Scotti L, Monteiro AFM, Scotti MT, de Moura RO, de Araujo RSA, Monteiro KLC, de Aquino TM, Ribeiro FF, et al. 2019. Active essential oils and their components in use against neglected diseases and arboviruses. Oxid Med Cell Longev. 2019:6587150–6587152. doi: 10.1155/2019/6587150.
- Liu SD, Song MH, Yun W, Lee JH, Kim HB, Cho JH. 2019. Effect of carvacrol essential oils on immune response and inflammation-related genes expression in broilers challenged by lipopolysaccharide. Poult Sci. 98(5):2026–2033. doi: 10.3382/ps/pey575.
- Liu X, Liu W, Deng Y, He C, Xiao B, Guo S, Zhou X, Tang S, Qu X. 2020. Use of encapsulated Bacillus subtilis and essential oils to improve antioxidant and immune status of blood and production and hatching performance of laying hens. Ital J Anim Sci. 19(1):1583–1591. doi: 10.1080/1828051X.2020.1862715.
- Long S, Liu S, Wang J, Mahfuz S, Piao X. 2021. Natural capsicum extract replacing chlortetracycline enhances performance via improving digestive enzyme activities, antioxidant capacity, anti-inflammatory function, and gut health in weaned pigs. Anim Nutr. 7(2):305–314. doi: 10.1016/j.aninu.2020.12.004.
- Moore PR, Evenson A, Luckey TD, McCoy E, Elvehjem C, Hart EB. 1946. Use of sulfasuxidine, streptothricin, and streptomycin in nutritional studies with the chick. J Biol Chem. 165(2):437–441. doi: 10.1016/S0021-9258(17)41154-9.
- Murray HH. 2000. Traditional and new applications for kaolin, smectite, and palygorskite: a general overview. Appl Clay Sci. 17(5-6):207–221. doi: 10.1016/S0169-1317(00)00016-8.
- Magnadóttir B. 2006. Innate immunity of fish (overview). Fish Shellfish Immunol. 20(2):137–151. doi: 10.1016/j.fsi.2004.09.006.
- Meeran MF, Prince PS. 2012. Protective effects of thymol on altered plasma lipid peroxidation and nonenzymic antioxidants in isoproterenol‐induced myocardial infarcted rats. J Biochem Mol Toxicol. 26(9):368–373. doi: 10.1002/jbt.21431.
- Maral H, Ulupınar S, Türk Baydır A, Özbay S, Altınkaynak K, Şebin E, Şiktar E, Kishalı NF, Buzdağlı Y, Gençoğlu C, et al. 2022. Effect of Origanum dubium, Origanum vulgare subsp. hirtum, and Lavandula angustifolia essential oils on lipid profiles and liver biomarkers in athletes. Z Naturforsch C J Biosci. 77(5-6):177–187. doi: 10.1515/znc-2021-0142.
- Mazzoni M, Zampiga M, Clavenzani P, Lattanzio G, Tagliavia C, Sirri F. 2022. Effect of chronic heat stress on gastrointestinal histology and expression of feed intake-regulatory hormones in broiler chickens. Animal. 16(8):100600. doi: 10.1016/j.animal.2022.100600.
- Oladokun S, MacIsaac J, Rathgeber B, Adewole D. 2021. Essential Oil Delivery Route: effect on Broiler Chicken’s Growth Performance, Blood Biochemistry, Intestinal Morphology, Immune, and Antioxidant Status. Animals. 11(12):3386. doi: 10.3390/ani11123386.
- Park JH, Kim IH. 2018. Effects of a protease and essential oils on growth performance, blood cell profiles, nutrient retention, ileal microbiota, excreta gas emission, and breast meat quality in broiler chicks. Poult Sci. 97(8):2854–2860. doi: 10.3382/ps/pey151.
- Palhares CJ, Gesteira CS, Belli AL, Samarini MF, R Pereira LG, R Tomich T, A Carvalho W, S Silva RO, L Voorsluys A, V Jacob D, et al. 2021. Effects of a blend of essential oils in milk replacer on performance, rumen fermentation, blood parameters, and health scores of dairy heifers. PLoS One. 16(3):e0231068. doi: 10.1371/journal.pone.0231068.
- Perumal AB, Huang L, Nambiar RB, He Y, Li X, Sellamuthu PS. 2022. Application of essential oils in packaging films for the preservation of fruits and vegetables: a review. Food Chem. 375:131810. doi: 10.1016/j.foodchem.2021.131810.
- Rivas AL, Fabricant J. 1988. Indications of immunodepression in chickens infected with various strains of Marek’s disease virus. Avian Dis. 32(1):1–8.
- Rosenson RS, Brewer HJ, Ansell BJ, Barter P, Chapman MJ, Heinecke JW, Kontush A, Tall AR, Webb NR. 2016. Dysfunctional HDL and atherosclerotic cardiovascular disease. Nat Rev Cardiol. 13(1):48–60. doi: 10.1038/nrcardio.2015.124.
- Ruan D, Fan Q, Fouad AM, Sun Y, Huang S, Wu A, Lin C, Kuang Z, Zhang C, Jiang S. 2021. Effects of dietary oregano essential oil supplementation on growth performance, intestinal antioxidative capacity, immunity, and intestinal microbiota in yellow-feathered chickens. J Anim Sci. 99:skab033.
- Siegel HS, Latimer JW. 1984. Interaction of high temperature and Salmonella pullorum antigen concentration on serum agglutinin and corticosteroid responses in white rock chickens. Poult Sci. 63(12):2483–2491. doi: 10.3382/ps.0632483.
- Sohal RS, Allen RG. 1990. Oxidative stress as a causal factor in differentiation and aging: a unifying hypothesis. Exp Gerontol. 25(6):499–522. doi: 10.1016/0531-5565(90)90017-v.
- Saravanan S, Pari L. 2016. Protective effect of thymol on high fat diet induced diabetic nephropathy in C57BL/6J mice. Chem Biol Interact. 245:1–11. doi: 10.1016/j.cbi.2015.11.033.
- Su G, Zhou X, Wang Y, Chen D, Chen G, Li Y, He J. 2018. Effects of plant essential oil supplementation on growth performance, immune function and antioxidant activities in weaned pigs. Lipids Health Dis. 17(1):139. doi: 10.1186/s12944-018-0788-3.
- Surai PF, Kochish II, Fisinin VI. 2018. Glutathione peroxidases in poultry biology: part 1. Classification and mechanisms of action. World’s Poul Sci J. 74(2):185–198. doi: 10.1017/S0043933918000284.
- Sivandzade F, Prasad S, Bhalerao A, Cucullo L. 2019. NRF2 and NF-κB interplay in cerebrovascular and neurodegenerative disorders: molecular mechanisms and possible therapeutic approaches. Redox Biol. 21:101059. doi: 10.1016/j.redox.2018.11.017.
- Sharma R, Rao R, Kumar S, Mahant S, Khatkar S. 2019. Therapeutic potential of citronella essential oil: a review. Curr Drug Discov Technol. 16(4):330–339. doi: 10.2174/1570163815666180718095041.
- Shlosman K, Rein DM, Shemesh R, Koifman N, Caspi A, Cohen Y. 2022. Encapsulation of thymol and eugenol essential oils using unmodified cellulose: preparation and characterization. Polymers (Basel). 15(1):95. doi: 10.3390/polym15010095.
- Su G, Wang L, Zhou X, Wu X, Chen D, Yu B, Huang Z, Luo Y, Mao X, Zheng P, et al. 2021. Effects of essential oil on growth performance, digestibility, immunity, and intestinal health in broilers. Poult Sci. 100(8):101242. doi: 10.1016/j.psj.2021.101242.
- Tiihonen K, Kettunen H, Bento MH, Saarinen M, Lahtinen S, Ouwehand AC, Schulze H, Rautonen N. 2010. The effect of feeding essential oils on broiler performance and gut microbiota. Br Poult Sci. 51(3):381–392. doi: 10.1080/00071668.2010.496446.
- Vicari T, van den Borne JJ, Gerrits WJ, Zbinden Y, Blum JW. 2008. Postprandial blood hormone and metabolite concentrations influenced by feeding frequency and feeding level in veal calves. Domest Anim Endocrinol. 34(1):74–88. doi: 10.1016/j.domaniend.2006.11.002.
- Windisch W, Schedle K, Plitzner C, Kroismayr A. 2008. Use of phytogenic products as feed additives for swine and poultry. J Anim Sci. 86(14 Suppl):E140–8. doi: 10.2527/jas.2007-0459.
- Wang L, Zhang Y, Fan G, Ren JN, Zhang LL, Pan SY. 2019. Effects of orange essential oil on intestinal microflora in mice. J Sci Food Agric. 99(8):4019–4028. doi: 10.1002/jsfa.9629.
- Xue F, Shi L, Li Y, Ni A, Ma H, Sun Y, Chen J. 2020. Effects of replacing dietary Aureomycin with a combination of plant essential oils on production performance and gastrointestinal health of broilers. Poult Sci. 99(9):4521–4529. doi: 10.1016/j.psj.2020.05.030.
- Yang KM, Jiang ZY, Zheng CT, Wang L, Yang XF. 2014. Effect of Lactobacillus plantarum on diarrhea and intestinal barrier function of young piglets challenged with enterotoxigenic Escherichia coli K88. J Anim Sci. 92(4):1496–1503. doi: 10.2527/jas.2013-6619.
- Zhang LY, Peng QY, Liu YR, Ma QG, Zhang JY, Guo YP, Xue Z, Zhao LH. 2021. Effects of oregano essential oil as an antibiotic growth promoter alternative on growth performance, antioxidant status, and intestinal health of broilers. Poult Sci. 100(7):101163. doi: 10.1016/j.psj.2021.101163.