Combinatorial Effect of Dietary Oregano Extracts and 3,4,5-Trihydroxy Benzoic Acid on Growth Performance and Elimination of Coccidiosis in Broiler Chickens.

We aimed to compare the combinatorial effect of 3,4,5-trihydroxybenzoic acid (THB) and oregano extracts (OE) with THB alone on the growth performance and elimination of deleterious effects in coccidiosis-infected broilers. A total of 210 one-day-old broilers were randomly assigned to one of five dietary treatments, with six replicates each, for 35 days. Dietary treatments were: 1) non-challenged, non-treated (NC); 2) challenged, non-treated (PC); 3) PC+ Salinomycin (0.05 g/kg; AB); 4) PC+THB (0.1 g/kg; THB); and 5) PC+THB+OE (0.1 g/kg; COM). On day 14, all groups except for NC were challenged with a 10-fold dose of Livacox® T anticoccidial vaccine to induce mild coccidiosis. All treatments significantly improved (P<0.05) body weight, average daily gain, and average daily feed intake, compared to PC, on days 21, 28, and 35. However, all treatments significantly reduced (P<0.05) the feed conversion ratio of PC by more than 14.60% on day 35, 11.76% during growing period, and 10.36% through the entire period. Broilers receiving anticoccidial treatments had 54.23% and 51.86% lower lesion scores (P<0.05) at 4 and 7 days post-infection, respectively, compared to PC. Additionally, the villus height of COM was significantly longer (P < 0.05) than that of THB. Although the molecular action of COM remains unclear, OE addition to THB reduced the shedding of oocysts better than THB alone (P<0.05, 9-11 days post-infection). Most importantly, COM effectively minimized the mortality of challenged birds from as high as 11.90% (PC) to 0%, a level similar to NC and AB, while THB maintained a mortality of 2.38%. In conclusion, the anticoccidial effect of THB can be enhanced by the addition of OE for better animal performance and the elimination of deleterious effects from coccidiosis-infected broilers for 35 days. 2022, Japan Poultry Science Association.

Introduction

Poultry coccidiosis is considered a major parasitic disease caused by coccidia, which comprise a wide variety of single-celled protozoa, particularly those of the genus Eimeria (Conway and McKenzie, 2007; Ali ). Coccidiosis leads to high mortality rates and reduced performance, including impaired feed intake, feed efficiency, and the need for excessive medication (Tanweer ; Chand ; Abudabos ), resulting in considerable economic losses (Amerah and Ravindran, 2014; Tanweer ). Eimeria parasites colonize and reproduce in the intestinal regions and infect the digestive tract, causing tissue destruction of the epithelial cells and, eventually, lesion formation (Gilbert ; Soutter ). This phenomenon may lead to bloody diarrhea and osmotic stress in birds. Consequently, infected birds exhibit reduced digestibility and malabsorption of nutrients, eventually resulting in higher morbidity and mortality rates (Persia ; Perez-Carbajal ; Arczewska-Wlosek and Swiatkiewicz, 2012).

Immunity boosting through vaccination and anticoccidial medication is commonly practiced to prevent coccidiosis in poultry (Chand ). Broad-spectrum anticoccidials are supplemented in feed on a prophylactic basis to strengthen the immunity of birds against multiple Eimeria species (Chapman, 1998; Allen and Fetterer, 2002; Conway and McKenzie, 2007). Anticoccidial products can be classified into three major groups based on their origin: chemically synthesized compounds, ionophores/polyether antibiotics (produced by fermentation of Streptomyces spp. or Actinomadura spp.), and a mixture of synthetic compounds and ionophores (Allen and Fetterer, 2002; Peek and Landman, 2011). Polyether antibiotics are effective against Eimeria, as they destroy the sodium/potassium concentration gradient across the parasitic cell membrane. This alters the intracellular pH, increases osmotic pressure (followed by osmotic swelling), induces cell vacuolization, and impedes mitochondrial function (Lavine and Arrizabalaga, 2011; Kala ; Antoszczak ). Moreover, a popular global strategy has been the addition of antimicrobial compounds to broiler feed at subtherapeutic levels to improve the growth performance of birds in terms of body weight (BW) and feed efficiency (Lin ). Nonetheless, haphazard use of antimicrobial compounds has led to the development and spread of antimicrobial resistance against bacterial diseases, which adversely affects consumer perception and the quality of the final product (Stanley ; Samuel ). To prevent the transmission of antimicrobial resistance genes and antibiotic residue accumulation in the food chain, the European Union, South Korea, and the United States banned the use of antimicrobial growth promoters in animal feed in 2006, 2010, and 2017, respectively (Maron ; Alloui ; Giannenas ; Salim ; Roth ). Consequently, there is a tremendous demand for alternatives to antimicrobial growth promoters, such as phytogenics, to reduce the incidence of poultry coccidiosis (Tanweer ; Mohiti and Ghanaatparast, 2015; Ali ).

Phytogenic compounds are a broad range of plant-derived natural bioactive composites that can be supplemented in broiler diets, either as essential oils or herbal extracts, to enhance animal growth performance (Hashemi and Davoodi, 2010; Puvača ). Due to their unique biological and chemical properties, including antimicrobial, antioxidative, and antiparasitic properties, phytogenic compounds are emerging as naturally sourced ingredients used to combat poultry coccidiosis. The plant-derived phenolic compound 3,4,5-trihydroxybenzoic acid (THB), or gallic acid, can mitigate the negative impact of coccidiosis in broilers and improve gut health, BW, and feed efficiency, while reducing the number of oocysts in excreta (Samuel ; Tinh et al., 2019). The antioxidative nature of THB and its ability to scavenge reactive oxygen species (ROS) helps mitigate coccidiosis conditions and serves as a natural antimicrobial agent (Kroes ; Naidoo ; Özçelik ).

Furthermore, feeding a diet supplemented with essential oils derived from Origanum vulgare improves growth performance and intestinal integrity of broiler chickens when coccidiosis occurs (Giannenas ; Tsinas ; Alp ). Oregano extracts (OEs) comprising terpenoids (isoprenoids), such as carvacrol and thymol, have demonstrated antiprotozoal and antimicrobial properties against Eimeria. These extracts increased cell membrane permeability, resulting in cytoplasmic coagulation, proton motive force reduction, and protein molecule denaturation in parasitic cells (Nazzaro ; Gavaric ; Felici ).

To the best of our knowledge, few studies have examined the efficacy of OE and THB combinations in the treatment of coccidiosis in broilers. Therefore, this study was designed to investigate and compare the effects of the combination of THB+OE and THB alone on the growth performance and elimination of coccidia in infected broilers. The hypothesis is that synergistic anticoccidial effects and improved growth performance can be achieved by supplementing a specific combination of natural phytochemicals (i.e., THB and OE) with divergent anticoccidial mechanisms in infected broilers.

Materials and Methods

The rearing of all birds complied with the guidelines of the Ross® broiler management handbook (Aviagen, 2014a). All animal handling procedures adhered to the guidelines of the Animal Care and Use Committee (Protocol No. 202006A-CNU-092), Chungnam National University, Republic of Korea.

Birds and Management

The experiment was conducted using 210 one-day-old male “Ross® 308” (a trademark of Aviagen Group, Huntsville, AL, USA) broiler chickens for 35 days. Birds were randomly allocated to five dietary treatments as: non-challenged, non-treated(NC), challenged, non-treated (PC), PC+Salinomycin (AB), PC+THB (THB), and PC+THB+OE (COM) (Table 1). Each dietary treatment consisted of six replicate pens. Seven birds with similar initial BWs (44.61±0.07 g) were housed in each raised battery cage (76×61×46 cm). The initial temperature was maintained at 30±1°C for the first 3 days of the experiment and then gradually decreased to 25±1°C until day 14. Thereafter, a temperature of 25±1°C was maintained throughout the experimental period. Each pen was equipped with two nipple drinkers and a metal feeding trough. Birds were offered the experimental diets on an ad libitum basis via a metal trough of the pen and had free access to fresh clean drinking water throughout the experimental period.

Table 1.

Dietary treatments

Treatment	Identity	Details
1	NC	Control diet with no coccidiosis challenge or anti-coccidiosis treatment / non-challenged, non-treated
2	PC	Control diet with coccidiosis challenge and no anti-coccidiosis treatment / challenged, non-treated
3	AB	PC+Salinomycin (0.05 g/kg)
4	THB	PC+THB (0.1 g/kg)
5	COM	PC+THB+OE (0.1 g/kg)

Induction of Coccidial Infection

All the birds were challenged (except those in the NC group) by an overdose (10× recommended dose) of the Livacox® T anticoccidial vaccine (Biopharm, Research Institute of Biopharmacy and Veterinary Drugs, Jílové u Prahy, Czech Republic) on day 14. One milliliter of inoculum (i.e., 0.1 mL of vaccine with 0.9 mL of sterile distilled water) was orally gavaged directly into the crop of each bird in the challenged groups, while those in the NC group were administered the same amount of sterile distilled water. The Livacox® T vaccine contains Eimeria acervulina, E. maxima, and E. tenella strains, which were responsible for coccidiosis in the respective broilers.

Experimental Design, Diets, and Treatments

The experiment was conducted according to a completely randomized design. Five dietary treatments were used (Table 1). Both THB and THB+OE additives were supplied with the product name, “VANTIPEARLTM 201” and “ORSENTIALTM Extend” from Kemin Industries Asia Pte Ltd., Singapore, respectively. All the diets were formulated based on corn and soybean meal to meet the Ross 308 nutrition specification (Aviagen, 2014b; Table 2). All additives were top-dressed to the basal diet. Chromium oxide powder (Cr2O3, > 99.9% purity; Sigma-Aldrich, St. Louis, MO, USA) was added at a concentration of 0.3% for all experimental diets as an internal indigestible marker for digestibility analysis.

Table 2.

Composition (g/kg, as-fed basis) of the experimental diets

Item	Starting period (Days 1–21)	Growing period (Days 22–35)
Corn	48.51	60.96
Wheat, Hard Red Winter	8.40	4.12
Wheat bran	4.10	—
Soybean meal 48%	31.15	27.62
Vegetable Oil	3.30	3.30
Limestone	1.20	0.95
Mono-calcium Phosphate	1.65	1.39
Salt	0.30	0.35
Vitamin-Mineral Premix1	0.30	0.30
Lysine-HCl	0.34	0.30
DL-Methionine	0.20	0.19
L-Threonine	0.13	0.11
L-Cystine	0.12	0.11
Cr2O3	0.30	0.30
Calculated values2
Crude protein, %	22.0	20.0
Metabolizable energy, kcal/kg	3050	3200
Lysine, %	1.40	1.20
Methionine, %	0.62	0.60

Provided per kilogram of diet: vitamin A, 12,000 IU; vitamin D3, 2,500 IU; vitamin E, 30 IU; vitamin K3, 3 mg; D-pantothenic acid, 15 mg; nicotinic acid, 40 mg; choline, 400 mg; and vitamin B12, 12 µg; Fe, 90 mg from iron sulfate; Cu, 8.8 mg from copper sulfate; Zn, 100 mg from zinc oxide; Mn, 54 mg from manganese oxide; I, 0.35 mg from potassium iodide; Se, 0.30 mg from sodium selenite.

The values were calculated according to the values of feedstuffs in NRC (1994) and “Ross 308” Broiler Nutrition Specification (2013).

Growth Performance Evaluation

The BWs of the birds were recorded individually on day 1 and weekly thereafter. Feed disappearance on a pen basis was measured weekly. The mortality-corrected average daily feed intake was calculated. The feed conversion ratio was calculated based on the feed intake and weight gain every week.

Slaughtering of Birds, Sample Collection, and Sample Preparation for Laboratory Analysis

Six birds per treatment (one bird per cage) that were closer to the mean BW were selected and euthanized by cervical dislocation for sample collection on days 18 and 21 (4- and 7-days post-infection). The birds were necropsied and ileum samples for morphological analysis were excised and prepared as described by Wickramasuriya and Pelicano . A 3 cm fragment of the ileum (i.e., identified as the intestinal segment from the Meckel's diverticulum to the ileocecal junction) was collected from each sacrificed bird, rinsed with phosphate buffered saline, stored in 10% formaldehyde, and dehydrated with increasing concentrations of ethanol. Thereafter, the intestinal segments were embedded into paraffin wax blocks to obtain ring-shaped dehydrated ileal tissues. Finally, six diagonal histological sections (4-6 µm) were sliced, stained with hematoxylin and eosin, and placed on glass slides. The height of ten well-aligned villi and their associated crypts were observed using an Eclipse TE2000 inverted microscope (Eclipse TE2000, Nikon Instruments Inc., Melville, NY, USA). The height of the villi and the depth of the crypts were measured by analyzing images of histological sections made from the computerized image-capture software (NIS-Elements Viewer software, Version: 4.20; NIS-Elements, Nikon Instruments Inc.). The height of the villi was defined as the distance from the tip to the base. The depth of the crypt was defined as the distance from the top of the crypt to the muscularis mucosa (Seyyedin and Nazem, 2017).

Ileal digesta samples from each sacrificed broiler were collected and stored at −20°C until further analysis for ileal nutrient digestibility. Before analysis, the samples were oven-dried at 55°C for 24 h, followed by fine grinding and strained through a sieve of < 0.75 mm (Wickramasuriya ). AOAC (2005) standard analytical methodologies were used to determine the dry matter, crude protein, and energy content in digesta samples, and the concentration of Cr2O3 in the feed and digesta was analyzed as described by Huang and Wickramasuriya . The coefficient of digestibility was calculated as 1-[(ID×AF)/(IF×AD)], where ID is the concentration of the indigestible marker in the diet, IF is the concentration of the indigestible marker in the digesta, AD is the particular nutrient concentration in the diet, and AF is the particular nutrient concentration in the digesta.

The dressing percentage was determined from the properly eviscerated hot carcass of broilers (Jahejo ). Subsequently, breast meat and drumstick samples were obtained to evaluate the relative weights corresponding to the carcass weights of broilers.

Lesion Scoring

Lesion scores in the experimental broilers were determined by separately observing the lesions in the digestive tract, including the jejunum (from the insertion of duodenal mesentery to the Meckel's diverticulum), ileum, and ceca. Generally, lesions in these regions are caused by E. acervulina, E. maxima, and E. tenella, respectively (Conway and McKenzie, 2007). The scoring was performed on a scale of 0 to 4, according to the assessment method of Johnson and Reid (1970), where 0=no lesion, 1=mild lesion, 2=moderate lesion, 3=severe lesion, and 4=extremely severe lesion. The total lesion score for the intestine was calculated using the separate lesion scores from the three different intestinal segments.

Fecal Analysis of the Number of Oocysts Per Gram (OPG Count)

Clean excreta (free from feathers and feed) were collected separately on days 7, 8, 9, 10, and 11 post-infection to enumerate oocysts from each cage. The collected samples were refrigerated until analysis. The OPG count was measured using the McMaster method of Soulsby (1982) and according to the procedures of Mwale and Masika (2011) with slight modifications. Briefly, 4 g of fecal sample was suspended in a 56 mL saturated salt solution (flotation solution) and the mixture was filtered carefully using a sterilized cheesecloth. The filtrate was loaded into both chambers of the McMaster counting slide using a micropipette and kept aside for 5 min before counting. The number of total oocysts in the chambers was counted separately by observation at 10× magnification using an Eclipse TE 2000 compound microscope. The OPG count in each replicate was calculated by multiplying the number of oocysts counted in both chambers by a factor of 50. The final results were expressed as log10 oocysts/g feces for each treatment.

Statistical Analyses

Data were analyzed according to a completely randomized design using a general linear model procedure of one-way ANOVA using SPSS software (Version 26; IBM, Armonk, NY, USA). A single pen was used as the experimental unit for all growth performance measurements and OPG counting. Selected individual birds were considered the experimental unit for the mean lesion score, gut morphology, digestibility, and carcass trait analyses. When treatment effects were significant (P<0.05), means were separated using Tukey's multiple range test in SPSS software.

Results

Growth Performance

No mortality was observed in the NC, AB, and COM groups during the 35-day trial. All deaths in the other groups were reported following the coccidial challenge, with a record of 2.38% and 11.90% for THB and PC, respectively.

There was no difference (P>0.05) in the BW of broilers among the dietary treatments within 14 days after hatching (Table 3). The BWs of NC and PC birds were the highest (P<0.05) and lowest (P<0.05), respectively, compared to the birds in the other groups, from days 21 to 35. Supplementation with antibiotics significantly improved (P<0.05) the BW of broilers, compared to those fed THB and COM, from days 21 to 35. The COM group displayed numerically improved (P<0.1) BW of coccidia-challenged broiler chickens compared to the THB group by 2.03% on day 28 and 4.24% on day 35.

Table 3.

Effect of dietary treatments on growth performance of coccidiosis challenged broiler chickens.1

Period	Experimental Diets					SEM2	P-value
	NC	PC	AB	THB	COM
Body weight, g
Day 1	44.67	44.67	44.57	44.50	44.62	0.106	0.989
Day 7	158.19	145.02	148.17	146.88	143.40	2.241	0.258
Day 14	333.31	310.98	326.43	328.17	310.29	5.580	0.611
Day 21	788.11c	537.53a	725.83bc	695.21b	689.32b	17.217	<0.001
Day 28	1376.d	881.50a	1253.23c	1142.23b	1165.37bc	32.855	<0.001
Day 35	2003.39d	1253.37a	1840.81c	1692.12b	1763.94bc	48.721	<0.001
Average daily gain, g/d
Day 7	16.22	14.34	14.80	14.63	14.11	0.318	0.254
Day 14	25.02	23.71	25.47	25.90	23.84	0.704	0.841
Day 21	64.97c	32.36a	57.06bc	52.43b	54.15b	2.237	<0.001
Day 28	84.04d	49.14a	77.72cd	63.86b	68.01bc	2.567	<0.001
Day 35	89.57c	53.12a	83.94bc	78.55b	85.51bc	2.677	<0.001
Day 1–21	35.40c	23.47a	32.44bc	30.99b	30.70b	0.820	<0.001
Day 22–35	86.81d	51.13a	80.83cd	71.21b	76.76bc	2.382	<0.001
Day 1–35	55.96d	34.53a	51.80c	47.08b	49.12bc	1.397	<0.001
Average daily feed intake, g/d
Day 7	23.40	22.67	23.00	22.57	22.89	0.305	0.936
Day 14	39.16	38.32	38.00	38.46	35.87	0.601	0.513
Day 21	92.59c	58.90a	86.18bc	81.86b	81.39b	2.365	<0.001
Day 28	121.91d	80.27a	114.12cd	96.60ab	101.08bc	3.180	<0.001
Day 35	131.23c	91.37a	123.77bc	117.13b	125.33bc	2.932	<0.001
Day 1–21	51.71c	39.96a	49.06bc	47.63b	46.72b	0.821	<0.001
Day 22–35	126.57d	85.82a	118.94cd	106.86b	113.21bc	2.797	<0.001
Day 1–35	81.66c	58.31a	77.01bc	71.32b	73.31b	1.567	<0.001
Feed conversion ratio, g/g
Day 7	1.45	1.60	1.58	1.55	1.65	0.039	0.582
Day 14	1.57	1.64	1.50	1.54	1.56	0.043	0.903
Day 21	1.43a	1.83b	1.51a	1.58ab	1.53a	0.039	0.006
Day 28	1.46	1.64	1.48	1.51	1.49	0.024	0.142
Day 35	1.47a	1.73b	1.48a	1.49a	1.47a	0.024	<0.001
Day 1–21	1.48a	1.69b	1.53a	1.56ab	1.58ab	0.024	0.047
Day 22–35	1.47a	1.68b	1.48a	1.50a	1.48a	0.019	<0.001
Day 1–35	1.48a	1.69b	1.51a	1.53a	1.54a	0.018	<0.001

Values are the mean of six replicates per treatment; a–d Values in a row with different superscripts differ significantly (P<0.05). NC, non-challenged, non-treated; PC, challenged, non-treated; AB, PC+Salinomycin; THB, PC+3,4,5-trihydroxybenzoic acid; COM, PC+3,4,5-trihydroxybenzoic acid+oregano extracts

Pooled standard error of the mean.

NC broilers had the highest ADG and ADFI (both P<0.05), followed by the AB-treated birds during the starting, growing, and whole phases of the experimental periods. In contrast, PC broilers had the lowest ADG and ADFI (both P<0.05) at the same time points. The COM group displayed improved ADG during the growing period and the overall experimental period (both P<0.01), compared to the THB group. In addition, the COM group displayed numerically enhanced (P>0.05) ADFI during the growing period of infected broilers in favor of the THB diet.

Feed efficiency was significantly different among treatments during the starting, growing, and overall phases of the experimental periods (all P<0.05). NC broilers had better FCR (P<0.05) than PC broilers during these phases. However, no significant difference was found (P>0.05) between challenged birds fed a diet supplemented with antibiotics, THB, COM, and NC birds. Feed efficiency was markedly improved (P<0.05) by supplementing infected birds with COM for the growing period compared to those fed THB. Interestingly, broilers fed COM exhibited a similar feed efficiency to AB broilers during the growing period (P>0.05).

Compared to COM, THB numerically improved the feed efficiency of infected birds by 1.28% and 0.65% for the starting period and overall experimental period, accordingly (1.58 vs. 1.56 and 1.54 vs. 1.53, respectively).

Lesion Score

The effect of anticoccidial dietary supplementation on the overall mean lesion score in broiler chickens for 35 days was determined (Table 4). No lesions were found in the digestive tract of non-infected birds (NC), whereas PC broilers had the most severe lesions (P<0.05) at either 4 or 7 days post-infection (dpi). AB broilers had the lowest (P<0.05) scores among the infected groups. The differences between THB- and COM-fed birds were not significant. COM numerically improved gut integrity by suppressing lesions at both 4 and 7 dpi in infected birds (1.93 and 2.14, respectively) when compared to THB (2.06 and 2.36, respectively). The data in Table 5 are the separate lesion scores for ceca, jejunum, and ilium in the digestive tract of broilers. COM-fed broilers had lower lesion scores in all intestinal segments compared to those fed THB, with a significant difference detected in the jejunum at 4 dpi (P<0.05).

Table 4.

Effect of dietary treatments on overall mean lesion score of coccidiosis challenged broiler chickens.1

Period	Experimental diets					SEM	P-value
	NC	PC	AB	THB	COM
Lesion Score
4 dpi3	0.00a	3.24d	0.46b	2.06c	1.93c	0.314	<0.001
7 dpi3	0.00a	3.58d	0.67b	2.36c	2.14c	0.425	<0.001

Values are the mean of six replicates per treatment; a–d Values in a row with different superscripts differ significantly (P<0.05). NC, non-challenged, non-treated; PC, challenged, non-treated; AB, PC+Salinomycin; THB, PC+3,4,5-trihydroxybenzoic acid; COM, PC+3,4,5-trihydroxybenzoic acid+oregano extracts

Pooled standard error of the mean

Days post-infection

Table 5.

Effect of dietary treatments on lesion score in caeca, jejunum, and ilium of coccidiosis challenged broiler chickens.1

Period	Experimental diets					SEM	P-value
	NC	PC	AB	THB	COM
Caeca
4 dpi3	0.00a	3.33d	0.78b	2.00c	1.83c	0.309	<0.001
7 dpi3	0.00a	3.58d	0.83b	2.33c	2.25c	0.419	<0.001
Jejunum
4 dpi3	0.00a	2.83e	0.44b	2.28d	1.89c	0.294	<0.001
7 dpi3	0.00a	3.33c	0.50a	2.42bc	2.08b	0.416	<0.001
Ileum
4 dpi3	0.00a	3.56c	0.17a	1.89b	2.06b	0.359	<0.001
7 dpi3	0.00a	3.83d	0.67b	2.33c	2.08c	0.449	<0.001

Values are the mean of six replicates per treatment; a–e Values in a row with different superscripts differ significantly (P<0.05). NC, non-challenged, non-treated; PC, challenged, non-treated; AB, PC+Salinomycin; THB, PC+3,4,5-trihydroxybenzoic acid; COM, PC+3,4,5-trihydroxybenzoic acid+oregano extracts

Pooled standard error of the mean

Days post-infection

OPG Counts

As expected, oocysts were undetected in the excreta obtained from the non-infected group, while PC birds had the highest OPG count from 7 to 11 dpi (P<0.05; Table 6). The oocyst shedding pattern was increased (P<0.05) at 7, 8, and 9 dpi, but was decreased (P<0.05) at 10 and 11 dpi in all treatments. The COM diet reduced (P<0.05) the OPG count in the coccidia-infected birds at 9, 10, and 11 dpi. These values were higher (P<0.05) than those fed AB. The collective findings indicate that supplementation with COM appears to be more efficacious than THB alone in reducing the OPG count in infected broilers.

Table 6.

Effect of dietary treatments on oocysts per gram in feces of coccidiosis challenged broiler chickens.1

Period	Experimental diets					SEM	P-Value
	NC	PC	AB	THB	COM
OPG count (Log10 Oocysts)
7 dpi3	0.00a	4.06d	2.75b	3.64c	3.55c	0.391	<0.001
8 dpi3	0.00a	4.11d	3.01b	3.78c	3.68c	0.401	<0.001
9 dpi3	0.00a	4.16e	3.16b	3.85d	3.76c	0.408	<0.001
10 dpi3	0.00a	4.09e	2.96b	3.75d	3.61c	0.398	<0.001
11 dpi3	0.00a	4.02e	2.62b	3.63d	3.46c	0.387	<0.001

Values are the mean of six replicates per treatment; a–e Values in a row with different superscripts differ significantly (P<0.05). NC, non-challenged, non-treated; PC, challenged, non-treated; AB, PC+Salinomycin; THB, PC+3,4,5-trihydroxybenzoic acid; COM, PC+3,4,5-trihydroxybenzoic acid+oregano extracts

Pooled standard error of the mean

Days post-infection

Ileal Digestibility of Nutrients

There were no differences (P>0.05) in the digestibility coefficients of dry matter and energy of infected broilers at both 4 and 7 dpi. The birds in the NC group had a higher (P<0.05) crude protein digestibility coefficient than those of the other groups at both 4 and 7 dpi (Table 7). Both THB and COM improved protein digestibility in challenged birds, when compared to PC birds on both test dates (P<0.05). However, birds fed AB had similar protein digestibility profiles to those of NC birds at 4 dpi.

Table 7.

Effect of dietary treatments on nutrient digestibility coefficient of coccidiosis challenged broiler chickens.1

Period	Experimental Diets					SEM2	P-value
	NC	PC	AB	THB	COM
Dry Matter
4 dpi3	0.70	0.57	0.69	0.64	0.65	0.020	0.228
7 dpi3	0.77	0.57	0.71	0.65	0.67	0.026	0.137
Crude Protein
4 dpi3	0.83b	0.60a	0.80b	0.73ab	0.76ab	0.024	0.012
7 dpi3	0.87b	0.66a	0.81ab	0.75ab	0.78ab	0.024	0.049
Energy
4 dpi3	0.75	0.57	0.76	0.62	0.67	0.027	0.099
7 dpi3	0.80	0.60	0.77	0.66	0.73	0.028	0.121

Values are the mean of six replicates per treatment; a–b Values in a row with different superscripts differ significantly (P<0.05). NC, non-challenged, non-treated; PC, challenged, non-treated; AB, PC+Salinomycin; THB, PC+3,4,5-trihydroxybenzoic acid; COM, PC+3,4,5-trihydroxybenzoic acid+oregano extracts

Pooled standard error of the mean

Days post-infection

Ileal Morphology

The effects on the gut morphology of coccidia-challenged broiler chickens fed diets containing different dietary treatments are summarized in Table 8. There was an effect of diet (P<0.05) on villus height, crypt depth, and the villus height: crypt depth (V:C) ratio of coccidia-infected broilers. NC broilers had the healthiest (P<0.05) gut morphology, followed by AB-, COM-, and THB-fed birds (P<0.05). The PC broilers exhibited the worst (P<0.05) ileal morphology. Broiler-fed COM displayed longer villi (P<0.05) but shorter crypts, and hence a higher V:C ratio, than those fed THB at 4 and 7 dpi.

Table 8.

Effect of dietary treatments on villus height, crypt depth and V: C ratio of coccidiosis challenged broiler chickens.1

Period	Experimental Diets					SEM2	P-value
	NC	PC	AB	THB	COM
Villus height (µm)
4 dpi3	974.24e	605.84a	880.04d	753.73b	821.50c	19.247	<0.001
7 dpi3	1133.91d	693.91a	955.09c	817.11b	898.59c	23.004	<0.001
Crypt depth (µm)
4 dpi3	76.96a	109.54c	90.20b	94.14b	93.44b	1.783	<0.001
7 dpi3	85.34a	118.38c	94.83ab	99.08b	95.70ab	2.003	<0.001
Villus height: Crypt depth ratio (V: C)
4 dpi3	12.71d	5.55a	9.38c	8.39b	8.81bc	0.358	<0.001
7 dpi3	13.28d	5.87a	10.22c	8.64b	9.13bc	0.390	<0.001

Values are the mean of six replicates per treatment; a–e Values in a row with different superscripts differ significantly (P<0.05). NC, non-challenged, non-treated; PC, challenged, non-treated; AB, PC+Salinomycin; THB, PC+3,4,5-trihydroxybenzoic acid; COM, PC+3,4,5-trihydroxybenzoic acid+oregano extracts

Pooled standard error of the mean

Days post-infection

Carcass Traits

There were no differences (P>0.05) in dressing percentage and relative breast meat weight of broilers among dietary treatments at 4 and 7 dpi (Table 9). Nonetheless, the NC broilers had the highest relative drumstick weight, and the PC broilers had the lowest relative weight on both test dates compared to those of the other treatments (all P<0.05). No significant difference was observed between the NC, AB, THB, or COM treatments in terms of relative drumstick weight.

Table 9.

Effect of dietary treatments on dressing percentage, relative breast meat weight, and relative drumstick weight of coccidiosis challenged broiler chickens.1

Period	Experimental diets					SEM2	P-value
	NC	PC	AB	THB	COM
Dressing percentage3, %
4 dpi6	88.96	84.64	87.86	87.11	87.38	0.512	0.078
7 dpi6	89.24	85.84	89.05	87.70	87.85	1.015	0.836
Relative breast meat weight4, %
4 dpi6	25.86	23.65	25.11	24.14	24.91	0.444	0.584
7 dpi6	26.16	25.65	26.05	25.33	25.76	0.361	0.963
Relative drumstick weight5, %
4 dpi6	9.91b	8.93a	9.86b	9.76b	9.81b	0.123	0.048
7 dpi6	10.64b	9.67a	10.39ab	9.97ab	10.14ab	0.131	0.036

Values are the mean of six replicates per treatment; a–b Values in a row with different superscripts differ significantly (P<0.05). NC, non-challenged, non-treated; PC, challenged, non-treated; AB, PC+Salinomycin; THB, PC+3,4,5-trihydroxybenzoic acid; COM, PC+3,4,5-trihydroxybenzoic acid+oregano extracts

Pooled standard error of the mean

Dressing percentage=[Carcass weight/Live body weight]×100%

Relative breast meat weight=[Breast meat weight/Carcass weight]×100%

Relative drumstick weight=[Drumstick weight/Carcass weight]×100%

Days post-infection

Discussion

Oregano-derived phytogenic compounds are effective against coccidiosis by suppressing adverse growth performance and ameliorating the deleterious effects of coccidiosis in broilers (Reisinger ; Remmal ; Alarcon-Rojo ). The main active components of OE, carvacrol and thymol, are potent molecules against Eimeria tenella, E. acervulina, and a combination of Eimeria spp. (Giannenas ; Christaki ). In contrast, THB, an equally important hydrolyzable tannin component, has anticoccidial activity by stimulating immunity against Eimeria spp. in broilers (Choi and Kim, 2020). Nonetheless, until the present study, the effect of diet supplementation using a combination of OE and THB on coccidiosis in broilers remains unknown. It is important to consider OE and THB in broiler diets to optimize growth performance and prevent adverse reactions during treatment of poultry coccidiosis.

Several studies have reported improved growth performance (i.e., BW, feed efficiency, and feed intake) of coccidiosis-infected broiler chickens receiving diets containing dietary oregano, compared to the infected control group (Tsinas ; Alp ; Yitbarek, 2015; Franciosini ). Despite the anti-coccidiocidal effect on animals, the improvement of growth performance in coccidia-infected broilers depends on other factors, such as gut microflora, gut histomorphology, and mucus production (Bozkurt ). Oregano and its key components of carvacrol and thymol protect the integrity and microbial ecosystems in the gut, while stimulating the secretion of endogenous digestive enzymes, mucus, and saliva, which improve digestion, growth performance, and feed efficiency in challenged broilers (Silva ; Alloui ; Yitbarek, 2015). These effects could be achieved by enhancing the inner surface area of the intestine and prolonging the feed retention time in the gut. OEs increase nutrient absorption and transport through the villi and amplify feed efficiency in broilers (Garcia ; Bozkurt ; Simitzis, 2017). Hernandez and Cross demonstrated that dietary OE containing carvacrol and thymol improved the apparent ileal digestibility of dry matter and crude protein in broilers. Consistent with these findings, in the present study a significantly higher digestibility coefficient of crude protein with COM than with PC and a numerical improvement when compared to THB were observed. Bozkut et al. (2016) reported that pancreatic and intestinal chymotrypsin activities in coccidiosis challenged birds could be improved by feeding OE to increase protein digestibility. Both Jamroz and Yitbarek (2015) reported enhanced intestinal trypsin activity by incorporating phytogenic compounds, such as essential oils, into the broiler diet. The higher feed intake observed in the OE-fed birds (COM) in the present study might be due to the improvement in flavor and odor of the feed contributed by the essential oil components, thus enhancing dietary palatability (Frankič ). Interestingly, THB-fed broilers showed numerically higher feed efficiency than COM-fed broilers, and a non-significant difference was observed with the AB-fed broilers during the entire experimental period. Tinh et al. (2019) reported similar results, in which birds fed a diet with THB (100 g/ton) showed an equally better feed efficiency than those fed lasalocid (75 g/ton). This finding could be attributed to the significantly lower feed intake of THB-supplemented feed, and the higher concentration of the pure form of polyphenols that improved the gut architecture in the infected birds, thus influencing effective nutrient absorption and, ultimately, obtaining better feed efficiency. Mašek reported an improved growth performance of THB-fed broilers. This could be attributed to the increased short-chain fatty acid concentration, which aids nutrient digestion and proper absorption (Liao ; Iqbal ), in turn leading to improved gut morphology in broilers. These findings suggest that OE and THB provided individually can improve the growth performance of either infected or non-infected chickens, with synergistic effects evident with their combined use in broiler diets.

Supplementation of OE can reduce the severity of gut lesions caused by Eimeria spp. (Giannenas ; Tsinas ; Mohiti and Ghanaatparast, 2015; Pop ). This could be due to the antioxidative compounds in oregano, which reduces the cytotoxic effect caused by ROS and helps to mitigate the intestinal damage caused by Eimeria invasion (Idris ). Carvacrol and thymol can scavenge hydroxyl radicals and convert them to stable compounds, thus diminishing oxidative stress and in turn reducing the severity of intestinal lesions in infected broilers (Giannenas ; Alagawany ; Bozkurt ; Upadhaya ). In addition, the antiparasitic property of OE might reduce coccidia oocysts, thereby reducing protozoa-induced lesions (Scheurer ; Mohiti-Asli and Ghanaatparast-Rashti, 2015). Similarly, Tosi et al. (2016) and Qaid reported reductions in gut lesions of Eimeria-infected broilers receiving diets supplemented with dietary plant-based meals and extracts, such as THB, when compared to the infected control group. THB has wound healing abilities in mammals by activating specific healing factors, such as focal adhesion kinases, cytoplasmic protein tyrosine kinase, c-Jun N-terminal kinases, and extracellular signal-regulated kinases (Yang ; Pal, 2018). Moreover, the ferric-reducing antioxidative activity and the ability to scavenge hydroxyl radicals of dietary THB could attenuate intestinal lesions and cytotoxicity caused by Eimeria spp. (Archer ; Qaid ). Hence, enhanced antioxidative activities and altered gut structure due to Eimeria invasion could be better ameliorated by combining OE with THB, compared to THB alone.

Phytogenic compounds inhibit the growth of Eimeria spp. in different phases of the parasitic life cycle (i.e., sporogony and merogony stages). This in turn reduces the production and release of oocysts in feces (Muthamilselvan ). The hydrophobic nature and high lipid solubility of carvacrol and thymol could reduce the fluidity of the phospholipid bilayer in the cell membrane of microorganisms, including Eimeria spp., thereby decreasing the permeability of vital cations, such as K+ and H+ (Ultee ; Ochoa-Velasco ). Consequently, parasitic cell destruction occurs through energy loss, ion leakage (particularly calcium), and cellular constituents commensurate with a decrease in adenosine triphosphate generation (Ultee ; Jitviriyanon ; Sidiropoulou ). Numerous studies have reported that carvacrol and thymol are responsible for the inhibition of glycoprotein synthesis and the cysteine protease cruzain enzyme in parasites (Dos Santos ). This restricts propagation and diminishes survival in the parasitic life cycle (Stokes ; Monzote ). Several studies (Chand ; and Ali ) reported similar oocyst shedding patterns as those of the present study (see Table 6). The higher biological potential and the short prepatent period of the parasites may decrease the rate of oocysts during the initial infection stage, and the acquisition of immunity during the latter period reduces oocyst excretion through chicken feces (Pattison et al., 2007). A reduction in OPG counts emphasized the protective role of an additive against Eimeria infection (Yim et al., 2011). In addition, several studies have reported that OE containing thymol and carvacrol contributes to a reduction in the number of oocysts shed through the feces of infected broilers (Giannenas ; Küçükyilmaz et al., 2012; Mohiti and Ghanaatparast, 2015). The finding agrees with our results and suggests that OE constituents have a defensive capability against E. tenella, E. acervulina, and a combination of Eimeria spp. (Giannenas ; Christaki ; Küçükyilmaz et al., 2012). In the present study, PC birds had significantly higher oocyst counts than AB-treated birds, whereas COM birds had intermediate-level counts that were significantly lower than the THB-treated birds. Alp obtained a similar observation by providing a diet containing 300 mg/kg of oregano to infected birds. We concur with the authors' recommended use of OE as a phytogenic anticoccidial agent. Although this approach was not as effective as antibiotics, it was still effective in reducing oocyst excretion beyond that of infected non-treated birds. Similarly, a series of recent studies have reported that either plant meals or phytogenic plant extracts that contain THB could reduce the OPG count of coccidiosis-infected birds, compared to infected non-treated groups (Naidoo ; Lee ; Lahlou ). Tannin derivatives (including THB) can improve the resistance and resilience of animals against gastrointestinal parasitic infections by generating complexes with essential parasitic enzymes, inhibiting oxidative phosphorylation or electron transport (thus decreasing parasite metabolism), and chelating metal iron to make them unavailable for vital biological activities (i.e., heme formation, reduction of the ribonucleotide precursor of DNA) for the parasite (Chung ; Min and Hart, 2003). Because THB has a low molecular weight and poor affinity to proteins (and thus is easily hydrolyzed), it can be easily absorbed into the intestinal bloodstream, consequently providing antioxidative and anti-inflammatory benefits to chickens (Manach ; Choi and Kim, 2020).

Ileal morphology is an indicator of both broiler gut health and nutrient absorption capacity (Apperson and Cherian, 2017). Villus height, crypt depth, and V:C ratio are histomorphometric parameters that can be used to assess the development and functional status of the small intestine (Laudadio ; Nabizadeh, 2012; Seyyedin and Nazem, 2017). Coccidiosis in broilers distresses intestinal integrity, causing tissue damage in the mucosa and submucosa, shortening the villi, and impairing digestive enzyme activities (Kettunen ; Williams, 2005; Perez-Carbajal ). Consequently, detrimental effects can occur during digestion and nutrient absorption in the broiler gut (Amerah and Ravindran, 2015; Ali ). Earlier studies reported that broilers fed diets with OE at con centrations of 12 and 24 mg/kg, and with a mixture of carvacrol and thymol at concentrations of 100 and 200 mg/kg, increased villus height and V:C ratio, and reduced crypt depth in an experimental infection of Eimeria spp. in broiler chickens for 42 days (Hashemipour ; Bozkurt ). Moreover, the commensal relationship between intestinal bacteria and THB in mammals improves the diversity of the gut microbiome and enhances short-chain fatty acid synthesis (i.e., butyric acid), which maintains proper gut integrity (Leeson ; Bortoluzzi ). Additionally, carvacrol and thymol reportedly increased the production and secretion of mucin from the villi surface, which improved the morphology and pathogenic activities of the broiler gut (Jamroz ). Samuel reported a significant improvement in the V:C ratio with a decrease in crypt depth by incorporating THB into the broiler diet at a level of 75-100 mg/kg, compared to that of the equivalent control group. A reduction in crypt depth indicates slow cellular turnover in the intestine, which is an indication of good intestinal health and overall growth of the birds (Oso ).

The results of the present study regarding carcass traits agree with those of Eler , who described that the relative breast meat weight was unaffected by dietary oregano supplementation, although carcass yield and relative leg meat weight differed. Hong et al. (2012) did not detect a difference in carcass dressing percentage and hot carcass yield of OE-fed chickens, which was consistent with our results, indicating that birds fed COM did not show any differences in dressing percentage. However, increased carvacrol and thymol levels may reduce the visceral organ weight in broiler chickens (Cázares-Gallegos ), which aids in amplifying the dressing percentage. The present findings do not support this concern. Furthermore, improved breast muscle yields were reported from broilers fed a diet supplemented with ≥100 mg/kg THB (Samuel ) and Rumex nervosus leaves meal (the active compound in the leaves is THB) (Azzam ).

In conclusion, COM outperformed THB alone in achieving better coccidia elimination in broiler chickens in the 35-day study. These results could provide useful data in related fields and support our hypothesis that a synergistic anticoccidial effect can be achieved through a specific combination of natural phytochemicals in challenged broilers.