Effects of encapsulated thymol and carvacrol mixture on growth performance, antioxidant capacity, immune function and intestinal health of broilers

Abstract This study evaluated the effect of encapsulated thymol and carvacrol mixture (ETCM) on the growth performance, antioxidant capacity, immune function and intestinal health of broilers. In total, 400 one-day-old male Arbour Acres broilers were randomly allocated to two groups with eight replicates of 25 birds each and fed a basal diet (control) or a basal diet supplemented with 150 mg/kg ETCM. The trial lasted 42 days. ETCM supplementation significantly increased (p < .05) average daily gain at d 22–42 and the organ indices of the spleen, pancreas and bursa of Fabricius at d 42. Regarding the serum antioxidant indices, ETCM supplementation significantly increased (p < .05) glutathione peroxidase (GSH-Px) and superoxide dismutase activities and decreased (p < .05) the malondialdehyde content at d 21 and 42. Furthermore, it significantly increased the villus height and villus height-to-crypt depth ratio in the jejunum at d 21 and the ileum at d 42. ETCM supplementation also up-regulated (p < .05) the intestinal mRNA levels of nuclear factor erythroid-2 related factor 2 (Nrf2), GSH-Px, occludin and zonula occludens-1 in the jejunal mucosa, while down-regulated (p < .05) the mRNA levels of nuclear factor kappa B (NF-κB), interleukin 1β, and tumour necrosis factor-alpha. Thus, ETCM positively impacts the growth performance, antioxidant capacity and immune function of broilers. ETCM can also improve intestinal health which may be partially related to the activation of the Nrf2 signalling pathway and the suppression of the NF-κB signalling pathway. HIGHLIGHTS ETCM improves the growth performance, antioxidant capacity and immune function of broilers. ETCM promotes the gut health of broilers, which may be by activating the Nrf2 signalling pathway and suppressing the NF-κB signalling pathway.


Introduction
Over the past few decades, using antibiotics as growth promoters has effectively enhanced the growth performance of animals and maintained their health status (Lillehoj et al. 2018;Mehdi et al. 2018). However, antibiotic overuse has been a long-standing problem worldwide, particularly in the poultry industry. It leads to the major problems of drug residue contamination and bacterial resistance, threatening human health (Mehdi et al. 2018). Therefore, many countries have banned the use of antibiotics as growth promoters . To overcome these challenges, it is imperative to investigate a safe alternative. Among the potential alternatives, the use of essential oils (EO) is preferred because of their non-toxic, residue-free and pollution-free characteristics (Dima and Dima 2015). EO are plant-derived mixtures of volatile compounds and exhibit various biological properties, including antibacterial (Teixeira et al. 2013), anti-oxidation (Hashemipour et al. 2013), and anti-inflammatory properties (Omonijo et al. 2019). Therefore, the development of EO as feed additives is receiving considerable interest and enthusiasm.
The effects of EO have been extensively recorded in poultry production. The addition of EO to the diet of broilers could alleviate the deterioration of intestinal morphology and enhance digestive enzyme activities in the gut and pancreas of birds, thereby improving their growth performance (Hashemipour et al. 2013;Peng et al. 2016). In addition, EO from oregano was found to positively impact the carcase traits (Peng et al. 2016), intestinal immune and antioxidant capacity and caecal microflora of broilers . Furthermore, a recent study in yellow-feather broilers revealed that EO can improve growth performance and intestinal health by improving intestinal antioxidant enzymes activities, barrier function and microbial community (Ding et al. 2022). However, few studies have reported the molecular mechanisms of EO. Moreover, the results of studies have been inconsistent because the actual effects of EO can be influenced by various factors, such as their structure, composition and coating. Among these factors, the functional group of the EO component plays a key role in the function of the EO (Vergis et al. 2015). For instance, the hydroxyl group in the structure of EO component takes up the key position to enhance the antimicrobial properties of the EO. Consequently, the antibacterial properties of phenolic EO are superior to those of other EO (Di Pasqua et al. 2007). Our laboratory confirmed this finding by testing the antibacterial activity of EO. Our unpublished results revealed that the bacteriostatic effects of phenolic EO, particularly thymol and carvacrol, were superior to those of other types of EO. Other studies have also reported significant antimicrobial effects of thymol and carvacrol against various pathogens, including Escherichia coli, Shigella Castellani, Streptococcus mutans and Listeria innocua (Mead 2000;Burt et al. 2007;Guarda et al. 2011). Moreover, previous studies have reported that dietary supplementation with thymol or carvacrol alone could improve the growth performance and intestinal morphology of broilers (Du et al. 2016;Ibrahim et al. 2021). In addition, the mixture of thymol and carvacrol was demonstrated to directly inhibit the growth of pathogenic bacteria in the gut and modulate the intestinal microbial composition of broilers (Yin et al. 2017). However, information about the effect of the thymol and carvacrol blend on broilers is still rarely enough. Therefore, this study evaluated the effect of encapsulated thymol and carvacrol mixture (ETCM) on the growth performance, antioxidant capacity, immune function and intestinal health of broilers.

Ethical statement
The experimental treatments for the animals in this study were approved by the Animal Ethics Committee of Anhui Agricultural University.

Animals, diets, and experimental design
In total, 400 one-day-old male Arbour Acres (AA) chicks were randomly assigned to two treatment groups with eight replicates containing 25 birds each and fed a basal diet (control group) or a basal diet supplemented with 150 mg/kg ETCM (ETCM group). The ETCM used in this study primarily consisted of thymol and carvacrol, which were mixed and microencapsulated in a triglyceride matrix of hydrogenated vegetable oils in our laboratory. The addition level of ETCM used in this study was based on an unpublished gradient addition trial which found that ETCM supplementation at 150 mg/kg could maximise the growth performance of broilers. The broilers were raised from d 1 to d 42 with free intake of mash feed and fresh water. The temperature in the broiler room was gradually lowered by 2 C or 3 C per week, eventually dropping from 34 C to 23 C. The temperature was then maintained until the end of the experiment. Moreover, the lighting program was set to produce 23-h light and 1-h dark. The relative humidity was controlled at 60%-70% during the entire experimental period. The composition and nutrients of the basal diets are displayed in Table S1 and the diets were formulated according to the requirements for broilers specified by the National Research Council (1994). Body weight (BW) at d 21 and d 42 and daily feed consumption were documented on a replicate basis to calculate the average daily BW gain (ADG), average daily feed intake (ADFI) and feed to gain ratio (F/G).

Sample collection
On d 21 and d 42, one bird from each replicate close to the average BW was selected, weighed and euthanized by electrical stunning and exsanguination. Venous blood from wing was collected into polypropylene tubes and centrifuged at 1500 Â g for 10 min at 4 C to obtain serum. The serum was then preserved at À20 C until the analysis of antioxidant related indicators. After dissection was performed, the thymus, spleen, pancreas, and bursa of Fabricius were removed and individually weighed. The organ index was calculated as the ratio of organ weight (OW) to BW using the following formula: organ index (g/kg) ¼ OW (g)/BW (kg). To assess the digestive enzyme activities, the duodenal, jejunal, and ileal digesta were collected in sterile centrifuge tubes, speedily placed in liquid nitrogen and kept at À80 C until analysis. Subsequently, 2-cm segments were cut from the middle of the duodenum, jejunum and ileum, and fixed in 10% buffered formalin for morphological assessment. Mucosal samples were gathered by gently scraping the jejunal wall using sterile slides, rapidly frozen in liquid nitrogen and stored at À80 C until the analysis of related genes expression levels.

Metabolite contents and enzyme activities
The intestinal contents were homogenised in precooled phosphate buffer saline by using a homogeniser, and the homogenate was centrifuged at 2500 Â g for 8 min at 4 C to obtain the supernatant. The supernatant's protein concentration was quantitated using a commercially available kit (Nanjing Jiancheng Biochemistry, Nanjing, China). The content of malondialdehyde (MDA) and the activities of catalase (CAT), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) in serum as well as intestinal amylase, lipase, and trypsin activities were measured using commercially available kits (Nanjing Jiancheng Biochemistry, Nanjing, China) and specific detection methods, as per the manufacturer's protocols.

Intestinal morphology
The intestinal samples were dehydrated, embedded and stained with haematoxylin and eosin for histological analysis. Morphological indices, including villus height (VH) and crypt depth (CD), were measured using an image processing and analysis system (Leica Imaging Systems Ltd., Cambridge, UK). The ratio of VH to CD (VH/CD) was also calculated.

RNA isolation and real-time PCR analysis
Total RNA was extracted from the jejunal mucosa using TRIzol Reagent (Yeasen, Shanghai, China) based on manufacturer's protocols. The concentration and purity of RNA were determined by Nanodrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA) by considering the ideal absorbance ratio (A260/280) of 1.8-2.0. The integrity of RNA was verified by electrophoretic analysis on a 1.5% agarose gel. The RNA samples were reverse transcribed into cDNA for next analysis using HifairV R II 1st Strand cDNA Synthesis Kit (Yeasen, Shanghai, China) and gene expression levels were quantified using Real-Time PCR with a Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) and Hieff V R qPCR SYBR V R Green Master Mix (Yeasen, Shanghai, China). The operation was according to the protocols of manufacture. The primers (Table S2) for b-actin (housekeeping gene), nuclear factor erythroid-2 related factor 2 (Nrf2), CAT, GSH-Px, SOD1, nuclear factor kappa B (NF-jB), tumour necrosis factor-alpha (TNF-a), interleukin (IL)-1b, IL-6, IL-10, occludin, and zonula occludens-1 (ZO-1) were commercially synthesised by General Biol Corporation (Anhui, China). The reaction conditions and parameter settings were according to the settings in our previous research (F. ). The relative mRNA expression levels of the target genes were determined using the 2 ÀDDCt method.

Statistical analysis
Data were analysed using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA) and expressed as the mean-± standard error. Student's t-test was performed to analyse and compare the difference between the control and ETCM groups. Differences were considered significant at p < .05.

Growth performance
As shown in Table 1, the ADG in the ETCM group was significantly increased (p < .05) at d 22-42. Moreover, ETCM supplementation tended to increase the BW at d 42 (p ¼ .059) and ADG at d 1-42 (p ¼ .061), and decrease the F/G at d 1-42 (p ¼ .064). However, ETCM had no marked effect (p > .05) on the ADFI in all phases.

Organ index
As shown in Table 2, broilers in the ETCM group had higher (p < .05) organ indices of the spleen, pancreas and bursa of Fabricius than those in the control group at d 42. ETCM tended to increase the spleen index at d 21 (p ¼ .059). The thymus index was similar in both the groups (p > .05).

Serum antioxidant status
As presented in Table 3, broilers in the ETCM group recorded the lower (p < .05) content of MDA and the higher (p < .05) activities of GSH-Px and SOD at d 21 and d 42 than those in the control group. Moreover, ETCM treatment tended to increase the T-AOC activity at d 42 (p ¼ .060). However, the indexes of CAT did not markedly affect by ETCM treatment (p > .05).

Digestive enzyme activities
As shown in Table 4, the activities of amylase and lipase were significantly increased (p < .05) with ETCM treatment in duodenum and ileum at d 21. Broilers in the ETCM group were observed a higher (p < .05) ileal amylase activity at d 42. However, the trypsin activity was not affected by ETCM addition throughout the study (p > .05).

Intestinal morphology
As shown in Table 5, ETCM treatment increased (p < .05) the duodenal VH and jejunal VH/CD at d 21.   Moreover, it increased the ileal VH and VH/CD at d 42 (p < .05). Furthermore, ETCM tended to increase the jejunal VH at d 21 (p ¼ .067). However, the CD was statistically similar between the two groups (p > .05).

Expression of genes in jejunal mucosa
As shown in Table 6, ETCM up-regulated (p < .05) the relative mRNA levels of Nrf2, GSH-Px, ZO-1 and Occludin, whereas it down-regulated (p < .05) the mRNA expression levels of NF-jB, IL-1b, and TNF-a. Moreover, ETCM tended to increase the mRNA expression level of CAT (p ¼ .070). However, no significant influence was observed on the relative mRNA levels of SOD1, IL-6 and IL-10 (p > .05).

Discussion
EO are receiving increasing attention as a potential alternative to antibiotics because of their growthenhancing property (Hashemipour et al. 2013;Reis et al. 2018;Galli et al. 2020). A study reported the increase of ADG and feed efficiency was recorded in Ross 308 broilers offered 200 mg/kg of a phytogenic product containing thymol and carvacrol (Hashemipour et al. 2013). Moreover, Peng et al. (2016) reported that dietary supplementation with oregano EO increased the final BW and ADG of AA broilers. Nevertheless, other studies reported no marked effect of dietary supplementation with thymol or carvacrol on the growth performance of broilers (Lee, Everts, Kapperst, Yeom, et al. 2003;Jang et al. 2007). This study evaluated the effect of ETCM addition on the growth performance of AA broilers and revealed no significant impact on growth performance at d 1-21. However, ETCM supplementation significantly increased the ADG at d 22-42. An increasing trend was also noted in the BW at d 42, and ADG and feed efficiency at d 1-42 following ETCM treatment. These results suggest that dietary addition of ETCM seems to have a cumulative effect with time and has positive impact on broilers' growth performance, which may be that ETCM enhanced the maturity of the digestive tract and perfected gastrointestinal function, thereby increasing the utilisation of feed nutrients. In addition, varying effects were observed from EO supplementation across various studies, which could be attributable to some key elements, including the types and doses of EO, composition of basal diets, daily management and feed environment as well as the genetic characteristics of broilers (Zeng et al. 2015;Jimenez-Moreno et al. 2016;Liu et al. 2018).
It is well known that digestive enzymes are involved in nutrient digestion and their activities play a crucial role in feed utilisation and growth performance of broilers. A study revealed that EO could stimulate digestive juice secretion (Platel and Srinivasan 2004). The pancreas is an important organ for the secretion of digestive juices, including various digestive enzymes. Enlargement of the pancreas can increase the secretion of pancreatic juice, and the digestive enzymes in the juice can positively affect the process of nutrient digestion by improving the chance of feed-enzyme contact. The present results showed  1.00 ± 0.08 1.14 ± 0.03 .209 TNF-a 1.00 ± 0.08 0.56 ± 0.10 .033 ZO-1 1.00 ± 0.08 1.64 ± 0.11 .012 Occlduin 1.00 ± 0.07 1.33 ± 0.04 .022 Differences were considered significant at p < .05. Control, basal diet; ETCM, basal diet þ 150 mg/kg encapsulated thymol and carvacrol mixture. Nrf2: nuclear factor erythroid-2 related factor 2; CAT: catalase; GSH-x: glutathione peroxidase; SOD1: superoxide dismutase 1; NF-jB: nuclear factor kappa B; IL: interleukin; TNF-a: tumour necrosis factor-alpha; ZO-1: zonula occludens-1.
ETCM significantly increased the relative weight of the pancreas in broilers, which was consistent with Kucukyilmaz et al. (2012). Moreover, Hashemipour et al. (2013) found supplementation with thymol and carvacrol increased intestinal trypsin and lipase activities in broilers. A study on female broilers revealed that EO containing thymol improved the amylase activity in the intestinal digesta (Lee, Everts, Kappert, Frehner, et al. 2003). In our work, we found that the activities of amylase and lipase in duodenum and ileum were improved by ETCM treatment, which was in agreement with Yang et al. (2018). These positive results suggest that dietary ETCM supplementation effectively improves the digestive function of broilers. Nevertheless, existing the information is limited, and further investigation the deep-seated mechanism is required. Antioxidant enzyme activities and oxidation product contents are considered effective biomarkers for evaluating the antioxidant status of animals. GSH-Px and SOD are important components of the enzyme system, and they can effectively scavenge free radicals and inhibit peroxidation (Li et al. 2013). MDA is one of the final products of membrane lipids peroxidation, and its content is regarded as an index for detecting the degree of peroxidation (Li et al. 2013). Previous studies have confirmed that EO could remove free radicals, prevent oxidation and improve antioxidant enzyme activities in vivo. It was also reported by Hashemipour et al. (2013) that supplemented EO containing thymol to the diets of broilers could enhance the level of GSH-Px and SOD activities in serum and lowered MDA content. Zhang et al. (2021) reported that oregano EO led to higher GSH-Px and SOD activities in serum of broilers. Furthermore, Ding et al. (2020) reported that oregano EO addition enhanced the serum SOD activity and decreased the serum MDA content in Pekin ducks. In our study, dietary ETCM supplementation dramatically lowered the serum MDA content of broilers at d 21 and d 42, indicating the lower level of lipid peroxidation. Moreover, ETCM addition markedly raised the activities of SOD and GSH-Px at d 21 and d 42, suggesting an improvement of antioxidant capacity. Based on the aforementioned results, we speculate that thymol and carvacrol can enter the blood circulation of broilers as antioxidant components and their hydroxyl group can then supply active hydrogen to remove free radicals through the dehydrogenation reaction, thereby improving antioxidant capacity of broilers.
Intestinal morphology is a key factor affecting nutrient absorption in the intestine. VH, CD and VH/CD are usually measured to evaluate intestinal morphology and function. A higher VH and VH/CD indicate a natural increase in epithelial cell turnover and a well-differentiated intestinal mucosa, suggesting improvements in digestive and absorptive capacities (Su et al. 2021). Moreover, a deeper CD is indicative of enterocyte cell renewal and accelerates tissue turnover, which results in greater nutrient utilisation to maintain the normal physiological function of the intestine, eventually leading to poor growth performance (Berrocoso et al. 2017). A previous study reported that oregano EO significantly decreased CD and rose VH/CD of jejunum in broilers at d 42, whereas it had no significant effect on the VH (Peng et al. 2016). Zhang et al. (2021) reported broilers fed with oregano EO supplementation had higher jejunal VH and ileal VH/CD at d 42. Su et al. (2021) reported that EO containing carvacrol and thymol increased VH/CD at d 21 and decreased CD at d 42 in the jejunum. We found that the VH and VH/CD were significantly increased by ECTM supplementation in jejunum at d 21 and ileum at d 42, indicating an improved intestinal morphology. In addition, Windisch et al. (2008) reported that the improvement in intestinal morphology induced by herbal EO was attributed to the enhancement of antioxidant activity. Thus, ETCM most likely promotes antioxidant capacity, as a result, the improvement of gut morphology was observed.
The integrity of the intestinal barrier not only has a major impact on nutrient digestion and absorption but also plays a critical role in protecting the intestine from harmful substances (Du et al. 2016;Pirgozliev et al. 2019). ZO-1, claudin-1 and occludin are essential tight junction proteins of the intestinal barrier that play a vital role in regulating intestinal permeability and integrity (P. Zhao, Yan, et al. 2021). A study in Feng et al. (2021) showed that dietary EO containing thymol and carvacrol supplementation led to a higher expression level of occludin and claudin-1 in broilers' ileum. Du et al. (2016) revealed that the mRNA level of ZO-1 was up-regulated in small intestine of hens by oregano EO addition. The similar results were observed in our work, revealing dietary ETCM supplementation up-regulated the expressions levels of occludin and ZO-1 in jejunum mucosa, suggesting an improvement in intestinal barrier integrity. Besides, intestinal barrier integrity has an important bearing on intestinal antioxidation status and immunity. As a major transcription regulator, Nrf2 can increase or decrease the levels of various genes related to antioxidation and protect the body from oxidative stress damage. The expression levels of antioxidant genes can be increased through the activation of the Nrf2 signalling pathway. A study in Mountzouris et al. (2020) reported that EO containing thymol and carvacrol rose the mRNA expressions of CAT, SOD1, GSH-Px and Nrf2 in broilers' intestine. Similarly, our results presented that ETCM enhanced the expressions of Nrf2 and GSH-Px in jejunal mucosa, indicating antioxidant capacity was improved. This positive finding may be attributed to the fact that ETCM may activate the Nrf2 signalling pathway, thereby protecting the intestinal mucosa from harmful substances . Moreover, NF-jB, a key transcription factor, plays a pivotal role in the response to external stimulus and regulates inflammatory and immune responses (Stefanson and Bakovic 2014). Lower expression levels of genes encoding pro-inflammatory cytokines, such as interferon-c (IFN-c), IL-1b and TNF-a, can retard the activation of the NF-jB signalling pathway. Du et al. (2016) reported that a mixture of thymol and carvacrol inhibited the mRNA levels of TNF-a and toll-like receptors in the ileum. Moreover, dietary phytogenic additive comprising carvacrol supplementation downregulated the intestinal expression levels of IFN-c and IL6 (Pirgozliev et al. 2019). Similarly, the present results showed ETCM addition down-regulated the mRNA levels of NF-jB, IL-1b and TNF-a in the jejunal mucosa, indicating a lower inflammation reaction and an improved immune function may be partially attributed to the inhibitory effect of ETCM on the NF-jB signalling pathway. Indeed, Sahin et al. (2013) reviewed that the activation of Nrf2 and high expression of genes related to antioxidant enzymes (e.g. CAT, SOD and GPH-Px) blocked the activation of NF-jB by decreasing various stimulus, such as H 2 O 2 and pro-inflammatory cytokines, such as IL-1 and TNF-a, which was most likely achieved by depressing the degradation of the NF-jB inhibitor-alpha protein (IjBa). IjBa sequestered NF-jB and blocked NF-jB translocation and the subsequent transcription activation of inflammatory components, thereby effectively inhibiting NF-jB signalling pathway (Mountzouris et al. 2020). Thus, the improvements in intestinal health including intestinal morphology, antioxidant capacity, immune status and intestinal barrier function induced by ETCM addition were found to be closely related to the activation of the Nrf2 signalling pathway and inhibition of the NF-jB signalling pathway.

Conclusions
This study found that ETCM has a positive impact on the growth performance, antioxidant capacity and immune function of broilers. Moreover, ETCM can improve intestinal health which may be partially related to the activation of the Nrf2 signalling pathway and the suppression of the NF-jB signalling pathway in the intestinal mucosa of broilers. However, further study is needed to reveal the exact mechanism of action.

Supplementary material
The following supporting information can be downloaded, Table S1: The composition and nutrients (g/kg diet) of the basal diets; Table S2: Sequences of Real-Time PCR Primers.

Ethical approval
The experimental treatments for the animals in this study were approved by the Animal Ethics Committee of Anhui Agricultural University.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Data availability statement
The data that support the findings of this study are available from the corresponding author, upon reasonable request.