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Article

Oregano (Origanum vulgare L.) Essential Oil Feed Supplement Protected Broilers Chickens against Clostridium perfringens Induced Necrotic Enteritis

by
Xiaolu Jin
1,
Guanggen Huang
1,
Zheng Luo
2,
Yongfei Hu
1 and
Dan Liu
1,*
1
State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, 2 Yuanmingyuan West Road, Beijing 100193, China
2
Kemin (China) Technologies Co., Ltd. 25 Qinshi Road, Sanzao, Zhuhai 519040, China
*
Author to whom correspondence should be addressed.
Agriculture 2022, 12(1), 18; https://doi.org/10.3390/agriculture12010018
Submission received: 30 November 2021 / Revised: 16 December 2021 / Accepted: 17 December 2021 / Published: 24 December 2021
(This article belongs to the Section Farm Animal Production)
Browse Figures
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Abstract

:
Oregano (Origanum vulgare L.) is a well-known traditional medicine and a cooking spice. Recent practice has also applied the essential oil from oregano (OEO) in poultry due to its great potential for an antibiotic alternative. Our objective was to evaluate the potential effects of OEO (with carvacrol and thymol as the main active ingredient) on preventing necrotic enteritis (NE) caused by Clostridium perfringens (Cp) in chickens. In the feeding trial, a total of 450 one-day-old commercial Arbor Acres broilers were randomly assigned in 5 experimental groups during a 26-day production period (d19 to d 26 was the Cp challenge stage), and each group consisted of 6 replicate pens (15 birds each pen). All treatments were: basal diet (control group); basal diet and Cp challenge (model group); Cp challenge and 10 mg/kg enramycin (positive control group); Cp challenge and 200 mg/kg OEO product (OEO low dosage group, OEOL); Cp challenge and 300 mg/kg OEO product (OEO high dosage group, OEOH). OEO feed supplement at both dosages had significant effects on increasing the body weight gain (BWG) and reversing the dropped feed intake (FI) induced by Cp challenge. Histopathological changes in the ileums of broiler chickens with NE induced by Cp were alleviated by OEO, which was mutually confirmed by the intestinal lesion scores. Dosage did not influence the protective effect of OEO on intestinal lesion scores. Furthermore, OEO was found to have limited effects on tight junction-related gene expressions (Occludin and ZO-1). The broilers of the OEOL and OEOH groups significantly decreased the expression of TNF-α mRNA in the ileum and only the OEOH group was found to inhibit the IFN-γ expression of IFN- induced by Cp challenge. Finally, despite the fact that in vitro antibacterial effects by OEO were observed, considering its high minimum inhibitory concentration (MIC) value, we inferred that the protective effects by OEO against Cp challenge were not attributable to its direct antibacterial effects. We proposed OEO as a promising substitute for antibiotics against NE induced by Cp during poultry production.

1. Introduction

Poultry production is of great importance for animal husbandry, which is facing new challenges and needs to find more solutions to remain sustainable. Necrotic enteritis (NE) is a common but serious poultry disease which is a great threat worldwide [1]. Although Clostridium perfringens (C. perfringens, Cp) is a common commensal bacterium of the chicken intestine, the overgrowth of Cp leads to acute clinical symptoms, mainly intestinal disorders such as severe necrosis and inflammation, damaged intestinal mucosa, and significantly decreased digestion and absorption of nutrients which greatly affect chicken growth performance. It is estimated that, worldwide, Cp causes NE that costs the poultry industry ~two billion US dollars annually [2]. Traditionally, antibiotics showed great effectiveness in the control of NE [3], however, the use of antibiotics for feed in EU and many other countries has been banned since 2006. Furthermore, it started to be strictly restricted in many other countries including USA and China [4,5,6]. Prophylactic use of antibiotics and antimicrobial growth promoters (AGPs) in animal nutrition brings many debates, including threats to health and negative environmental impacts, therefore novel approaches to control NE in the replacement of antibiotics are urgently needed [7]. Among the different alternative substitutes for antibiotics, the usage of essential oils (EO) has been shown to have its effectiveness and potential for pre- and post-harvest antimicrobial strategies [8].
EOs are prepared using different fragrance extraction techniques from parts of plants, including leaves, roots, barks, stems, flowers, and seeds [9,10,11]. Strictly, they are not like edible oils, but usually have poor solubility in water, as are oils. EOs are complex mixture of hundreds of volatile compounds which have been reported with many biological benefits, such as anti-microbial, anti-inflammatory, anti-oxidant activities and many others [12]. Moreover, EOs been also have widely applied as effective reagent for the treatment of various diseases both in human medicine and animal husbandry [8]. Several trials have successfully shown the positive impacts of EOs against Cp induced NE during poultry production alone or in combination (blend) forms, such as Rosmarinus officinalis L. (rosemary), Thymus vulgaris L. (thyme) Pimpinella anisum L. (anise) and Origanum vulgare L.(oregano) [13].
Oregano is a well-known traditional medicine and a cooking spice. It also works as a useful poultry feed supplement for boosting the immunity and increasing birds’ growth performance [14]. Due to its rich in aromatic compounds, we hypothesize that EOs from oregano (OEO) might be useful for preventing NE for chickens. The aim of this study was to investigate the potential effects of OEO, which was chemically characterized with its main bioactive ingredient, on preventing NE caused by Cp in broilers chickens.

2. Materials and Methods

2.1. Chemical Analysis on the Oregano Essential Oil (OEO)

Thymol and carvacrol standard were purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China) with compound purities at ~98%. The pure OEO was obtained from by Kemin (Zhuhai, China) Technologies Co., Ltd., Zhuhai, China. Thymol and carvacrol in the pure OEO were analyzed using a HPLC with an Agilent ZORBAX SB-C18 (4.6 × 100 mm, 3.5 μm particle size) analytical column using an isocratic elution with a binary mobile phase (acetonitrile and water, 33:67, v/v) was used. During assay, an aliquot of 10 μL of diluted mixed standard or samples of pure OEO was injected in duplicate into the analytical column at 30 °C at a flow rate of 1 mL/min. Thymol and carvacrol were detected at 276 nm and quantitatively determined using an external calibration method [15].

2.2. Animal Trial Experimental Design

The experiments were ethically approved by the China Agricultural University Laboratory Animal Welfare and Animal Experimental Ethic committee (AW22121202-1-1). A total number of 450 one-day-old commercial Arbor Acres broilers were randomly assigned in 5 experimental groups, and each group consisted of 6 replicate pens (15 birds each pen). All treatments were as follows: basal diet; basal diet + Cp challenge; Cp challenge + 10 mg/kg enramycin; Cp challenge + 200 mg/kg OEO; Cp challenge + 300 mg/kg OEO. The OEO product, also known as OrsentialTM used in the animal trial was a commercial product obtained from by Kemin (China) Technologies Co., Ltd., Zhuhai, China, which contained 5% pure Origanum vulgare L. Essential Oil (OEO) as active components and defatted rice bran and silica as carriers. Broilers were raised in a controlled environment and allowed ad libitum access to water and feed. Repetitions of different treatments were equally distributed among the cages as much as possible to reduce variations at the cage level. The entire experiment was divided into two phases: the non-Cp challenge phase (d0–18) and the Cp challenge phase (d19–26) using one basal diet formulation. All diets were designed following the instructions of NRC (1994) and the Chinese chicken feeding standard (NY/T-33–2004) (Table 1). The Cp challenge was performed on the basis of our previous study [16]. A field strain of Cp type A (CVCC2030) was cultured on tryptone-sulfite-cycloserine agar, and a single colony was then inoculated into cooked meat medium and subsequently cultured in an incubator at 37 °C for 8 h. In the infected groups, chickens were orally inoculated with 1 mL bacterial solution containing 108 CFU/mL Cp once a day from Day 19 to Day 25. Chickens in the basal diet group were subjected to the same gavage procedure described above but with sterilized medium. Body weight of chickens was measured by replicate on day 0, day18, and on the last day (day 26). Feed intake (FI), body weight gain (BWG) and the feed conversion ratio (FCR) were calculated. Mortality was recorded during the whole experiment.

2.3. The Histology Analysis

Upper ileum sections were obtained from all sacrificed birds (one bird was chosen randomly from each biological replicate) after removing the digesta and washing using PBS. Then the ileum samples were fixed using 4% paraformaldehyde for hematoxylin and eosin (H&E) staining following standard protocols [17]. Histological images were observed and taken under a light microscope with an attached image capture system (Nikon Eclipse Ci, Tokyo, Japan).

2.4. Intestinal Lesion Score Evaluations

Intestinal scoring was evaluated on the basis provided from previous study [18]. On the last day of the trial, one bird from the pen was chosen randomly and scarified for lesion scoring by cervical dislocation. After cutting opened the intestines, NE lesions were measured based on the description and observations on the intestine. 0 = normal intestinal appearance; 0.5 = severely congested serosa and mesentery engorged with blood; 1 = thin walled and friable intestines with small red petechiae; 2 = focal necrosis, grey appearance and small amounts of gas production; 3 = sizable patches of necrosis, gas-filled intestine and small flecks of blood; and 4 = severe extensive necrosis, marked hemorrhage, large amounts of gas in the intestine [19].

2.5. Total RNA Extraction and Reverse Transcription

Ileal tissues from one randomly chosen 26-day-old bird per replicate were collected for total RNA extraction using Trizol method (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The reverse transcription was conducted using a PrimeScript RT Reagent Kit (Takara Biotechnology Inc., Dalian, China). Real-time PCR reactions were performed using SYBR®Premix Ex TaqTM (Tli RNaseH Plus, TaKaRa, Dalian, China) under the 7500c Real-time PCR Detection System (Applied Biosystems, Carlsbad, CA, USA). The primers were designed to flank introns with the Primer 5 software (Premier Biosoft, Palo Alto, CA, USA). The primer sets are listed in the Supplemental Table S1. All measurements were carried out in triplicate, with calculations on the average values. The data were calculated using 2−ΔΔCT method, normalized to the expression of the housekeeping gene (β-actin), and expressed as a fold change compared to the control group [20].

2.6. In Vitro Anti-Bacterial Effects by the OEO

The minimum inhibitory concentration (MIC) of the OEO against C. perfringens (CVCC2030) was determined via a conventional broth dilution method as described in our previous study [2]. The minimal bactericidal concentration (MBC) was defined as the lowest concentration of EO with which no viable bacteria were detected. All assays were performed in triplicate. For the bacterial morphology observations, Cp cells (~2 × 107 CFU/mL) were incubated with OEO at MIC or MBC or an equal volume of vehicle (methanol) at 37 °C for 6 h. After centrifugation at 1000 × g for 10 min, bacterial cells were prepared for scanning electron microscopy analysis with a Hitachi S-4800 scanning electron microscope following the manufacturer’s instruction.

2.7. Statistical Analyses

Quantitative data are presented as the arithmetic mean ± standard deviation (SD) for each treatment group. The data including growth performance, lesion score, gene expressions of both intestinal tight junction and pro-inflammation cytokine collected for quantitative parameters were analyzed using analysis of variance technique (ANOVA) under a completely randomized design. Significant differences among the treatments were measured by using Duncan’s multiple comparison. Variability in the data was expressed as the standard error of means, and a significant difference was declared when p < 0.05. All statistical tests were performed using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA).

3. Results & Discussion

3.1. Active Compounds Characterization of Carvacrol and Thymol from OEO

Origanum vulgare L. has been traditionally used for medicinal purposes but also widely applied in as food additive, veterinary medicine, pesticide and feed supplement [21]. The present study used pure oregano essential oil (OEO) was obtained from its mother plant processed using steam distillation after separation of the aqueous phase, which is in strict accordance with the requirement by the International Organization for Standardization. Furthermore, according to European Commission Regulation No 1334/20083, OEO can be applied as a feed additive in all animal feed, without extra evaluations [22,23]. Previous studies reported the major active components of oregano essential oil (OEO) from Origanum vulgare L. were carvacrol and thymol [21]. We therefore determinates these two active compounds in the pure OEO. We separated and quantified carvacrol and thymol in the pure OEO by HPLC by comparison with the retention times of authentic standards (Figure 1). The quantities of carvacrol and thymol in the pure OEO was 445 mg/g and 221 mg/g, respectively. Therefore, carvacrol and thymol concentrations in the experimental diet were 4.45 mg/kg and 2.21 mg/kg (low dosage experimental group) and 6.68 mg/kg and 3.32 mg/kg (high dosage experimental group), respectively. This composition and inclusion levels of OEO provided us a better understanding on the inclusion level of the phytogenic additives we used.

3.2. OEO Imporved the Growth Performance in Cp Challenged Chickens

Despite many trials using the OEO in poultry production, its effects on growth performance were still controversial. Some of previous studies suggested that there were no significant impacts on chickens’ growth performance [24,25]. However, more studies noticed that OEO was found to improve growth performance, such as increasing the average daily gain (ADG), final body weight (BW) [26] and decreasing the feed conversion ratio (FCR) [27]. In this study, dietary supplementation of 200 and 300 mg/kg OEO (in with containing 5% pure OEO and 95% silicon dioxide as caking inhibitor) had no significant influence the growth performance of the broilers during d 0 to 18 (Table 2). During the Cp challenge stage (d19–d26), OEO feed supplement at both low (200 mg/kg) and high dosage (300 mg/kg) groups had significant effects on increasing the body weight gain (BWG) and reversing the dropped feed intake (FI) induced by Cp challenge (Table 2). We chose the dosages of OEO based on colleagues and our previous studies. Botsoglou et al. [28] showed that inclusion of 50 and 100 mg/kg OEO in the feed did not improve either feed conversion ratio or growth performances. A similar effect was reported by Hassanin et al. [29] when 0.005 and 0.01% was added in broilers feed. On the other hand, the study of Vlaicu and colleagues found that 0.01% OEO significantly improved broilers’ performances [30]. Nevertheless, Alagawany et al. concluded a study in which 500 mg/kg diet of OEO is the appropriate level of inclusion in broiler diets for best performance and health; higher doses could have negative effect on performances due to some toxic effects [14]. Based on these inconsistent data relating the growth performance, we hypothesize that potential effects of OEO on growth performance were closely related to the dosage chosen, or by some factors like oil extraction process, different growing geographical regions of the plants, storage conditions and so on. Therefore, supplementation of OEO in our study showed to be effective on improving growth performance in Cp challenged chickens [14,31].

3.3. Feed Supplement with OEO Ileal Histopathological Changes in Broilers Challenged by C. perfringens

NE is a reemerging threaten to poultry production in the modern animal husbandry which requires us to reduce, replace and refine the usage of antibiotics and certain antimicrobial feed supplements [31]. Probiotics, prebiotics, plant extract as well as the active molecules from plant origins have shown great potential in mitigating NE in poultry [32]. For example, several species of genus Bacillus, Lactobacillus, have been shown with anti-Cp activity [33]. They usually supported the reduction of C. perfringens in the intestines positively by altering the intestinal microbial population and have improved the growth performance. Compared with these probiotics, plant extracts offer an alternative approach to put the underutilized some very cheap raw materials for good usages. Plant and plant-derived products, like garlic (Allium sativum), grape (Vitis vinifera), green tea (Camellia sinensis) as well as many others were found to positively affect growth performance and improve intestinal health alone or in combination with other compounds [34,35]. In addition to the growth-promoting effects, OEO may also be related to the decrease ileus inflammation during the Cp infection to the broilers. We further tested the potential impacts of OEO feed supplement on histopathological changes in ileums of broiler chickens challenged by Cp. As shown in Figure 2, chickens from the control group had no intestinal lesions. In contrast, upper ileum tissues were found with typical subclinical necrotic enteritis characteristics and severe intestinal inflammation in the model group. Feed supplement with OEO reduced the negative impacts caused by Cp with ameliorated ileitis, which is consistent with the previous findings in which they applied an essential oil, which contained 25% thymol and 25% carvacrol as active components [36].

3.4. Feed Supplement with OEO Improved the Gut Health of Broilers Challenged by C. perfringens

We next measured the effects of several key genes encoding inflammation responses and intestinal barrier permeability on the broiler jejunum challenged by Cp. As shown in Figure 3A, no intestinal lesions were observed in the unchallenged birds. In the challenged birds, intestinal lesion scores were significantly increased to 1.67 ± 0.52 (p ≤ 0.001), while dietary addition of enramycin (0.25 ± 0.27) also depressed the intestinal lesion (p = 0.004). The intestinal lesion scores in OEO Low dosage Group and OEO High dosage Group were 0.67 ± 0.26 and 0.50 ± 0.32, which were significantly lower than Cp -challenged Model group (p = 0.027 and 0.011). Interestingly, the dosage did not influence the protective effect of OEO on intestinal lesion scores. NE birds infected by Cp are shown with increased gut barrier permeability and losses of intestinal barrier functions [37], which not only greatly affects nutrient absorption, but also increases the possibility to be infected with other pathogens, like Campylobacter jejuni, Salmonella enterica or Eimeria maxima [38]. Regarding the intestinal barrier function-related gene expressions, Cp challenges lead to decreased Occludin expressions, but no significant effects on ZO-1 gene expressions. In addition, OEO fed to the broilers significantly dropped the expression of TNF-α mRNA in the ileum and only high dosage of OEO was found to inhibit the IFN-γ expression. Consistent with the previous findings, we inferred that OEO inhibited the inflammatory signaling pathways [37,38,39], therefore reasonable pathophysiological mechanisms and potential therapeutic strategies could be further developed in vitro for Cp induced chicken intestinal epithelial cell stresses.

3.5. In Vitro Antibacterial Activity of OEO against C. perfringens

We next tested the in vitro antibacterial activity of the pure OEO against Cp. The MIC and MBC of the pure OEO against Cp were 375 μg/mL and 750 μg/mL, respectively, which were measured based on broth dilution method. As shown in Figure 4, surface morphology of Cp following exposure with OEO was visualized by SEM. PBS and methanol (vehicle) treated bacteria showed a regular intact rod shape and with an intact smooth surface, whereas propolis treatment at MIC concentration showed a relative longer, with rougher surface. Meanwhile, OEO treatment at MBC concentration to the Cp showed an extensively rough and collapsed surface. A number of previous studies investigated the antibacterial activity of OEO against different foodborne pathogens, including the Cp [40]. Our previous report obtained MICs of thymol and carvacrol at 187.5 and 375 μg/mL [36]. Nevertheless, the in vitro antibacterial effects of the OEO we applied is stronger than previous studies obtained. The possible interference of thymol and carvacrol on the Cp growth cannot be ruled out. Collectively, these data clearly showed OEO had potent in vitro antibacterial activity against Clostridium perfringens. As we calculated that the carvacrol and thymol concentrations in the experimental diet were 4.45 mg/kg and 2.21 mg/kg (low dosage experimental group) and 6.68 mg/kg and 3.32 mg/kg (high dosage experimental group), such dosages are far less to reach the direct optimal antibacterial concentrations. We therefore inferred that the protective effects by OEO against Cp challenge were not attributable to its direct antibacterial effects.

4. Conclusions

This study clearly shows that OEO containing carvacrol and thymol as the key ingredient in the feed supplement improved the subclinical NE induced by C. perfringens in broiler chickens by regulating host inflammatory responses and increasing intestinal barrier function. Although OEO was shown with in vitro anti-bacterial effects against C. perfringens, the calculated MIC will not be sufficient for its action in vivo, therefore further in-depth studies are still warranted to evaluate the OEO effects on the its pathophysiological mechanisms. This study also proposed OEO as a promising substitute for antibiotics against NE or other inflammatory-related intestinal diseases during poultry production.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/agriculture12010018/s1, Table S1: Primers sequences used for the real-time PCR.

Author Contributions

Conceptualization, X.J. and D.L.; methodology, D.L.; software, G.H.; validation, Z.L. and D.L.; formal analysis, X.J. and D.L.; investigation, Z.L. and D.L.; resources, X.J. and D.L.; data curation, X.J. and D.L.; writing—original draft preparation, X.J.; writing—review and editing, Y.H. and D.L.; funding acquisition, Y.H. and D.L. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by Shandong Provincial Key R&D Program (2019JZZY020602) and the 2115 Talent Development Program of China Agricultural University.

Institutional Review Board Statement

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the China Agricultural University Laboratory Animal Welfare and Animal Experimental Ethic committee (AW22121202-1-1) on 1 April 2020.

Data Availability Statement

Data supporting reported results can be requested from the corresponding author via email.

Conflicts of Interest

Z.L. was an executive director of Kemin Industries (China) Co., Ltd. and provided the experimental material for this study. The remaining authors have no conflicts of interest to declare. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript.

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Figure 1. High-performance liquid chromatography (HPLC) chromatograms of carvacrol and thymol in the pure oregano essential oil (OEO). Detection wavelength was at 276 nm. Mixed authentic standards (up) and pure OEO sample (down) were shown. Retention times (min) of carvacrol and peaks of OEO were shown.
Figure 1. High-performance liquid chromatography (HPLC) chromatograms of carvacrol and thymol in the pure oregano essential oil (OEO). Detection wavelength was at 276 nm. Mixed authentic standards (up) and pure OEO sample (down) were shown. Retention times (min) of carvacrol and peaks of OEO were shown.
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Figure 2. Effects of OEO feed supplement on histopathological changes in ileums of broiler chickens with necrotic enteritis induced by C. perfringens. Ileum sections from the broiler chickens were proceeded based on H&E staining. Typical ileum images from (A) control group; (B) Cp induced necrotic enteritis group; (C) Cp birds fed with low dosage OEO group (200 mg/kg) and (D) Cp birds fed with high dosage OEO group (300 mg/kg).
Figure 2. Effects of OEO feed supplement on histopathological changes in ileums of broiler chickens with necrotic enteritis induced by C. perfringens. Ileum sections from the broiler chickens were proceeded based on H&E staining. Typical ileum images from (A) control group; (B) Cp induced necrotic enteritis group; (C) Cp birds fed with low dosage OEO group (200 mg/kg) and (D) Cp birds fed with high dosage OEO group (300 mg/kg).
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Figure 3. Effects of OEO feed supplement on gut health parameters in broiler chickens with necrotic enteritis induced by C. perfringens. (A) Heat map showed the lesion score of the chickens. High histological scores are shown in blue and low histological scores in white. (B) Tight junctions related gene expressions in the ileum. (C) Inflammation related gene expressions in the ileum. Means with no common letters differed significantly (p < 0.05). (p ≤ 0.05). Treatment information: Ctrl Group, basal diet; A Cp Group, Cp challenge +10 mg/kg enramycin; Cp Group, basal diet and Cp challenge; OEOL Group, Cp challenge +200 mg/kg of OEO; OEOH Group, Cp challenge +300 mg/kg of OEO.
Figure 3. Effects of OEO feed supplement on gut health parameters in broiler chickens with necrotic enteritis induced by C. perfringens. (A) Heat map showed the lesion score of the chickens. High histological scores are shown in blue and low histological scores in white. (B) Tight junctions related gene expressions in the ileum. (C) Inflammation related gene expressions in the ileum. Means with no common letters differed significantly (p < 0.05). (p ≤ 0.05). Treatment information: Ctrl Group, basal diet; A Cp Group, Cp challenge +10 mg/kg enramycin; Cp Group, basal diet and Cp challenge; OEOL Group, Cp challenge +200 mg/kg of OEO; OEOH Group, Cp challenge +300 mg/kg of OEO.
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Figure 4. Scanning electron microscopy analysis on the C. perfringens following treatment of OEO. C. perfringens (~2 × 107 cfu/mL) were incubated with PBS (A), vehicle (methanol, (B)) or OEO concentrations at MIC (375ug/mL, (C)) and MBC (750 ug/mL, (D)) at 37 °C for 6 h under anaerobic conditions. The bacterial surface morphology was observed with a Hitachi S-4800 SEM.
Figure 4. Scanning electron microscopy analysis on the C. perfringens following treatment of OEO. C. perfringens (~2 × 107 cfu/mL) were incubated with PBS (A), vehicle (methanol, (B)) or OEO concentrations at MIC (375ug/mL, (C)) and MBC (750 ug/mL, (D)) at 37 °C for 6 h under anaerobic conditions. The bacterial surface morphology was observed with a Hitachi S-4800 SEM.
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Table
Table 1. Basal diet composition (as-fed basis).
Table 1. Basal diet composition (as-fed basis).
Ingredient, %.Basal Diet
Corn57.52
Soybean meal (Crude protein >46%)36.20
Soy oil2.14
Limestone1.13
Dicalcium phosphate1.97
Salt0.35
Methionine (99%, DL-Form)0.19
Choline (50%)0.25
Vitamin premix 10.025
Mineral premix 20.2
Ethoxyquin (66%)0.03
Total100.00
Calculated composition 3, %
Crude protein21.0
Metabolizable energy (kcal/kg)2950
Calcium1.00
Available phosphorus0.45
Lysine1.11
Methionine0.5
1 Provided per kg of diet: Vitamin premix (1 kg) contained: vitamin A, 50 MIU; vitamin D3, 12 MIU; vitamin K3, 10 g; vitamin B1, 10 g; vitamin B2, 32 g; vitamin B12, 0.1 g; vitamin E, 0.2 MIU; biotin, 0.5 g; folic acid, 5 g; pantothenic acid, 50 g; niacin, 150 g copper, 4 g; zinc, 90 g; iron, 38 g; manganese, 46.48 g; selenium, 0.1 g; iodine, 0.16 g; cobalt, 0.25 g. 2 Provided per kg of diet: 150 g copper, 4 g; zinc, 90 g; iron, 38 g; manganese, 46.48 g; selenium, 0.1 g; iodine, 0.16 g; cobalt, 0.25 g. 3 Calculated value based on the analyzed data for the experimental diets.
Table
Table 2. The effect of supplementation oregano (Origanum vulgare L.) essential oil on growth performance in Cp-challenged broilers 1.
Table 2. The effect of supplementation oregano (Origanum vulgare L.) essential oil on growth performance in Cp-challenged broilers 1.
Treatment 2Control GroupCP-Challenged Model GroupAntibiotics Positive Control GroupOEO Low Dosage GroupOEO- High Dosage Groupp Value 3SEM
d0–d18 Non-challenge phase
BWG, g36.06 ± 0.5235.47 ± 0.6835.44 ± 0.6435.88 ± 0.8434.52 ± 0.810.5930.31
FI, g51.61 ± 1.5750.87 ± 0.9851.65 ± 1.0552.37 ± 0.9551.19 ± 1.280.9220.503
FCR1.43 ± 0.031.44 ± 0.021.46 ± 0.011.46 ± 0.021.48 ± 0.010.350.009
BW,g654.97 ± 8.88644.94 ± 11.5644.55 ± 10.83651.94 ± 14.26628.81 ± 13.820.5935.267
d19–d26 Challenge phase
BWG, g66.12 ± 2.23 a54.95 ± 1.86 b59.21 ± 1.73 ab62.48 ± 2.33 a63.32 ± 3.1 a0.0221.192
FI, g109.55 ± 2.41 a99.9 ± 2.35 b98.34 ± 1.23 b106.03 ± 2.35 ab110.82 ± 5.12 a0.021.551
FCR1.66 ± 0.031.82 ± 0.051.67 ± 0.061.7 ± 0.031.75 ± 0.040.0690.021
Overall
BWG, g46.46 ± 0.66 a42.21 ± 0.65 b43.67 ± 0.59 ab45.09 ± 1.09 ab44.49 ± 1.37 ab0.040.466
FI, g71.66 ± 1.267.84 ± 1.2867.81 ± 0.6470.94 ± 1.0171.83 ± 2.430.130.688
FCR1.54 ± 0.021.61 ± 0.021.55 ± 0.021.58 ± 0.021.61 ± 0.020.1280.011
BW,g1250.04 ± 17.08 a1139.48 ± 16.84 b1177.47 ± 15.3 ab1214.27 ± 28.41 ab1198.7 ± 35.72 ab0.0412.11
1 Treatment information: Control Group, basal diet; Cp -Challenged Model Group, basal diet and Cp challenge; Antibiotics Positive Control Group, Cp challenge +10 mg/kg enramycin; OEO Low dosage Group, Cp challenge +200 mg/kg of OEO; OEO High dosage Group, Cp challenge + 300 mg/kg of OEO. BWG, body weight gain; FI, Feed intake; FCR, feed conversion ratio.2 Mean ± SEM; Mean was calculated from the average of 15 birds each pen, which was considering as one biological replicate and n = 6 of each experimental group. 3 a,b Mean values within a column with unlike superscripts letters are significantly different (p < 0.05). A total number of 450 one-day-old commercial Arbor Acres broilers were randomly assigned in 5 experimental groups, and each group consisted of 6 replicate pens (15 birds each pen).
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Jin, X.; Huang, G.; Luo, Z.; Hu, Y.; Liu, D. Oregano (Origanum vulgare L.) Essential Oil Feed Supplement Protected Broilers Chickens against Clostridium perfringens Induced Necrotic Enteritis. Agriculture 2022, 12, 18. https://doi.org/10.3390/agriculture12010018

AMA Style

Jin X, Huang G, Luo Z, Hu Y, Liu D. Oregano (Origanum vulgare L.) Essential Oil Feed Supplement Protected Broilers Chickens against Clostridium perfringens Induced Necrotic Enteritis. Agriculture. 2022; 12(1):18. https://doi.org/10.3390/agriculture12010018

Chicago/Turabian Style

Jin, Xiaolu, Guanggen Huang, Zheng Luo, Yongfei Hu, and Dan Liu. 2022. "Oregano (Origanum vulgare L.) Essential Oil Feed Supplement Protected Broilers Chickens against Clostridium perfringens Induced Necrotic Enteritis" Agriculture 12, no. 1: 18. https://doi.org/10.3390/agriculture12010018

APA Style

Jin, X., Huang, G., Luo, Z., Hu, Y., & Liu, D. (2022). Oregano (Origanum vulgare L.) Essential Oil Feed Supplement Protected Broilers Chickens against Clostridium perfringens Induced Necrotic Enteritis. Agriculture, 12(1), 18. https://doi.org/10.3390/agriculture12010018

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