The effect of oregano essential oil on Feed Passage Syndrome in broilers: 1. Assessment under field conditions

Introduction

Over the years, broilers have become more efficient, with lower feed conversion ratios (FCR; Martinez et al., 2020), more efficient energy partitioning (Martinez et al., 2022d), higher lean body mass contents (Maharjan et al., 2020), higher meat production, with relatively more meat in the breast than the leg quarters (Martinez et al., 2022e). For this reason, they are more sensitive to adverse conditions, such as those affecting gut health and, consequently, the ability of the birds to uptake the dietary nutrients efficiently (Martinez et al., 2019) and to use them to gain lean mass. Due to the relatively higher turnover of the gut tissue compared to the rest of the body, minor damages to the gut mucosa induce high expenses of nutrients to support maintenance metabolism (Maharjan et al., 2021).

The Feed Passage Syndrome (FPS) in broilers, whose characteristics were described elsewhere (Martinez et al., 2023), is a subclinical condition that includes undigested feed in the feces, impairs digestive processes, and exacerbates opportunistic pathogens. Among others, Clostridium perfringens (Anadon et al., 2019), coccidiosis, and the intake of trypsin inhibitors (Ruiz et al., 2020) have been considered major factors. However, it has been reported not associated with coccidiosis lesions or dietary factors under commercial conditions (Martinez et al., 2023).

Usually, antimicrobials or acidifiers are used to control the FPS in commercial settings (Anadon et al., 2019). However, antibioticfree alternatives are needed to reduce the risk of bacterial resistance (Martinez, 2021). Plant derivatives, including oregano (Origanum vulgare) essential oil (OEO), are under study due to the antimicrobial action they develop, which has been well documented (Nazzaro et al., 2013). Indeed, a companion paper assessed the effect of the administration of OEO in drinking water to birds naturally challenged with FPS under field conditions, showing positive results on the excreta characteristics and the BW (Martinez et al., 2023). This study assessed OEO fed to broilers challenged with the FPS on performance, excreta characteristics, gut health, carcass yield, antioxidant status, and growth curve.

Material and methods

Birds, experimental treatments, and diets

The experiment was conducted at the Poultry Nutrition and Feeding Research Lab (LINAA), La Molina National Agrarian University (Universidad Nacional Agraria La Molina), Lima, Peru. One hundred ninety-two male day-old Cobb 500 broiler chicks with BW = 48.13 g (pooled SEM = 0.70 g) were tagged, randomly distributed into 24 cages (21.4 birds/m²) containing rice husk-based reused litter and assigned to one of the following treatments: T1: negative control; basal diets. T2: positive control; basal diets with added 75 g neomycin sulfate (neomycin; used to treat the FPS in commercial settings; Eltazi, 2014) per each 1 000 kg of diet (Landoni and Albarellos, 2015). T3: OEO; basal diets with added OEO (Orevitol-M; containing 48 g carvacrol per kg of the product; CKM S.A.C.; Lima, Peru) at a rate of 500 g of product per each 1 000 kg of diet. All birds were subjected to a challenge model to induce the FPS. OEO and neomycin were included in the corresponding treatment diets at the expense of corn and were fed continuously from 1 to 28 days.

The basal mash corn-fish-soybean meal diets were formulated exceeding or following the genetic line recommendations (CobbVantress, 2018). Starter diet provided excess CP (from fish meal) to predispose the growth of C. perfringens and the associated dysbacteriosis (Teirlynck et al., 2011). All birds were fed the same basal diets: Starter, 26.7% CP, 1.45% digestible lysine, and 3 028 kcal metabolizable energy/kg from 1 to 14 days. Finisher, 20.0% CP, 1.04% digestible lysine, and 3 008 kcal metabolizable energy/kg from 15 to 28 days (Supplementary Table S1). Feed and water were provided ad libitum. One dark hour and 23 hours of light were provided continuously from 1 to 28 days of age, and the temperature was controlled with electric brooders.

Challenge model for Feed Passage Syndrome

The challenged model combined the following factors: 1. Excess of CP in the diet (fish meal) from 1 to 14 days (Supplementary Table S1). 2. Birds had access to reused litter since the first day, and the remaining litter in the cages was removed on day 18. 3. A gavage with a mixed inoculum of Eimeria spp., including E. acervulina, E. maxima, E. tenella, E. necatrix, and E. brunetti, with at least 24 x 10⁵ live non-attenuated oocysts per bird (10 doses of Immucox for Chicken II vaccine; Vetech Laboratories, Inc. –CEVA Animal Health Inc.–, Ontario, Canada) administered at 14 days. 4. Gavages were administered at 18, 19, and 20 days with an inoculum of 10⁸ CFU of C. perfringens per dose. The administration of the coccidia inoculum prior to the C. perfringens inoculum was intended to cause mild damage to the mucosal lining, promoting the proliferation of C. perfringens and the associated dysbacteriosis (Pedersen et al., 2008). The two-week period between the coccidia inoculum administration and the assessment of results on day 28 were expected to allow the gut lining to heal after the coccidia damage, as reported previously with birds in cages (Martinez, 2022). This model has been shown to induce the FPS (Martinez and Vilchez, 2015; Martinez, 2022).

Clostridium perfringens used

The C. perfringens inoculum was prepared at the Clinical Pathology and Molecular Biology Laboratory, Faculty of Veterinary Medicine, University of San Marcos, Lima, Peru, as follows: An isolate was obtained from a subclinical enteritis field outbreak from broilers of three weeks of age showing stunted growth, ruffled feathers, with no apparent diarrhea, and showing mild congestion of the ileum mucosa at necropsy. Several C. perfringens strains were identified, and the type-A one that showed possessing the enterotoxinproducing gene was cultured. The following phenotypic characteristics were established: (i) Presence of alpha (total) and beta (partial) hemolytic activity when grown in TSA agar with 5% defibrinated sheep blood, (ii) presence of lecithinase activity when grown in TSA agar with 5% egg yolk at 37 ºC for 24 h under anaerobiosis, and (iii) reduction of sulfite when grown in TSC agar at 37 ºC for 24 h under anaerobiosis. The presence of the alpha toxin- and the enterotoxin-codifying genes (both by PCR) was established as the genotypic characteristic. The number of CFU was deducted from the 17-h growth of inoculum of 10 ml into 200 ml of sterile brain–heart infusion broth (BHI). Finally, 200 ml of BHI broth containing 1 x 10⁸ CFU/ml was diluted with 400 ml of sterile BHI broth to obtain a final concentration of 1 x 10⁸ CFU/3 ml.

Reused litter material

The litter was used in one previous flock. It was obtained from a commercial broiler farm (Huacho, Lima, Peru) with no infectious respiratory background attributable to virus and with satisfactory performance results. After harvesting, the litter was flamed to reduce feathers, stacked in piles, sprayed with water, and let rest for seven days, reaching a temperature 60 ºC. Then, the litter was spread out and sanitized (quaternary ammonium and glutaraldehyde combination). The microbial profile of the litter was determined to be as follows: C. perfringens, 100 CFU/g; cecal coliform, < 3 MPN/g; mesophilic aerobic bacteria, 57 x 10⁴ CFU/g; oocyst count, 1441 u/g.

Performance and excreta evaluation

All birds were weighed at 1, 14, and 28 days of age, and the individual BW gain (BWG; g/bird) was determined (Supplementary Table S2). If a bird was found dead, its weight and leftover feed were recorded. The feed intake (FI; g/bird) was calculated by recording the amount of feed provided and leftovers and considering the actual daily population per cage (Supplementary Table S3). The FCR was calculated with the BWG. These variables were determined for 1–14, 15–28, and 1–28 days. Additionally, the final BW at 28 days was also reported.

Throughout the third and fourth weeks, the excreta was inspected twice weekly and three times per day. The total number of droppings and those showing the following characteristics (Martinez and Vilchez, 2016) were recorded: watery dropping, containing undigested feed, containing desquamated mucosa, bloody, and normal ones (if well-formed and none of the previous characteristics were observed). Then, the prevalence (%) of each finding was calculated per experimental unit (Supplementary Table S4). Pictures of the main findings were obtained with a common digital camera. They were described, the findings quantified, and the pictures reported complying with image standards.

Necropsy evaluation, antioxidant status, and processing weights

At 21 and 28 days, one bird per cage was selected, weighed, euthanized by cervical dislocation, and allowed bleeding by severing the neck skin and puncturing both femoral arteries (Martinez et al., 2022c). Necropsies were performed at the Laboratory of Avian Pathology, Faculty of Veterinary Medicine, University of San Marcos, Lima, Peru. The density of lesions (u/cm²) was recorded, and a pooled mean value was determined per cage with observations at both ages. The gut mucosa congestion was registered, and the number of cages affected at any age (21 or 28 days) was determined. The bursa of Fabricius, spleen, and thymus were extracted at 28 days and weighed. The diameter of the bursa was determined with a commercial bursameter (Sellaoui et al., 2012), which is a ruler with eight holes corresponding to values of one to eight with sizes equal to one to eight-eights of an inch. The morphometric indexes of the bursa and spleen were calculated as follows:

where MI was the morphometric index, OW was the organ weight (g), and BW was the BW. The relationship between the weights of the bursa and thymus was calculated and reported (Supplementary Table S2).

Six blood samples from each of the negative control and OEO treatments were collected on day 28 into tubes with heparin lithium (BD Vacutainer Lithium Heparin, Ref. 367884; Becton, Dickinson, and Co.; NJ, USA) to determine superoxide dismutase (SOD) activity. The SOD activity was determined by the colorimetric method developed by McCord and Fridovich in 1969 (Abiaka et al., 2000) using a spectrophotometer (Agilent 8453 UV–visible Spectroscopy System; Agilent Technologies Deutschland GmbH, Waldbronn, Germany) and a commercial kit (Ransod; reference SD125; Randox Laboratories; Crumlin, United Kingdom). The method used xanthine and xanthine oxidase to generate superoxide radicals, which reacted with 2-(4-iodophenyl)-3-(4-nitrophe nyl)-5-phenyl tetrazolium chloride (INT) to produce red formazan color and was read at 505 nm. The SOD activity was measured by the degree of inhibition of the mentioned reaction, and one SOD activity unit was considered the one producing a 50% inhibition of the reduction value of INT (inhibition of the INT reduction occurs proportional to the presence of SOD). The SOD enzymatic activity was expressed in SOD units per ml of entire blood (U/ml). In a previous study assessing the antioxidant effect of OEO in broilers, including antimicrobials as the positive control group, Giannenas et al. (2005) did not determine the antioxidant status in the positive control group as the antimicrobials used were not expected to influence it. Following these researchers, and considering that neomycin has been shown not to produce an increase in the blood SOD content or activity (Adeyemi et al., 2021; Wang et al., 2022), SOD activity was not determined in the positive control group in the present study, but only in the negative control and OEO treatments.

After necropsies at 28 d, the selected birds were processed as described elsewhere (Martinez et al., 2022b). The carcass was obtained after defeathering, eviscerating, and removing the feet and head (Martinez et al., 2022a). The weights of both the carcass and breast were recorded. The breast included the whole breast meat (Pectoralis major and P. minor) without the skin. The carcass and breast yields (%) were calculated with the weights of the part and the BW of the sampled bird. However, to control the influence of sampling and avoid biases, the treatment carcass and breast weight values were determined with the mean BW of the birds in the cage at 28 days and the yield (%) value determined on the sampled bird (Supplementary Table S2). One bird per cage was also selected at days 7 and 14, euthanized as indicated above, and saved frozen to preserve a backup sample for potential future body composition determinations (Supplementary Table S3). However, these birds were ultimately not used.

Nutritional efficiency

The assessment of nutritional efficiency (Martinez and Uculmana, 2016) included the protein efficiency ratio (PER), which was determined with the following equation:

where PER was the protein efficiency ratio (g of BWG per g of protein intake), ADBWG was the average daily BWG (g/bird/day) in the period, ADFI was the average daily FI (g/bird/day) in the period, and CP was the dietary value in the period (%). The energy efficiency ratio (EER) was calculated as follows:

where EER was the energy efficiency ratio (g of BWG per each 100 kcal of metabolizable energy consumed), ADBWG was the average daily BWG (g/bird/day) in the period, ADFI was the average daily FI (g/bird/day) in the period, and ME was the dietary metabolizable energy (kcal/g). The feed-to-carcass and feed-to-breast conversion ratios were calculated as follows:

where FXCR was the feed-to-carcass or feed-to-breast conversion ratio (g feed per g of carcass or breast), FI was the feed intake in the period (g/bird), and XW was the carcass or breast weight (g), respectively. The protein-to-carcass and protein-to-breast efficiency ratios were also calculated as follows:

where PXER was the protein-to-carcass or protein-to-breast conversion ratio (g protein intake per g carcass or breast), FI was the feed intake in the period (g/bird), CPC was the concentration (%) of CP in the diet, and XW was the carcass or breast weight (g). The four variables above were assessed from 0 to 28 days, and protein and energy efficiency ratios also from 15 to 28 days (Supplementary Table S2).

Growth curve

A procedure previously reported (Martinez et al., 2023) was applied to determine the influence of the treatments on the growth pattern. Individual growth curves were fitted per cage considering the challenge produced a shift in the growth curve, not affecting the asymptotic BW (Chasser et al., 2019). For this reason, the Gompertz 3P type IIa model was used (Tjorve and Tjorve, 2017). The adult asymptotic BW was considered common for all experimental units and was obtained by fitting a Gompertz 3P model (script embedded in Supplementary File 1; report in Fig. S1) to the standard BW data of the genetic line (Cobb-Vantress, 2018; Supplementary Table S5). Considering this common asymptote BW value and the data from each experimental unit (Supplementary Table S6), independent Gompertz 3P models were fitted to each of them (scripts embedded in Supplementary Files 2–25; reports in Figs. S2–S25). Predicted BW values were interpolated by applying the fitted Gompertz models to the corresponding cages (Supplementary Table S2).

Using both measured data from 1 to 28 days and predicted data beyond 28 days (Supplementary Table S7), the ages at which birds would attain specific BW benchmarks were calculated, including the Peruvian market BW of 2.7 kg (Minagri, 2022) and the US market’s average BW of four weight categories: 1.6 kg, 2.3 kg, 3.2 kg, and 4.2 kg (USDA, 2022). To achieve this, two Gompertz 3P models were fitted independently per treatment, and the mentioned ages were determined by inverse prediction (script embedded in Supplementary File 26; report in Fig. S26).

Data processing and statistical procedures

Gompertz 3P models were fitted as reported by (Martinez et al., 2023). Treatments were compared under a Completely Randomized Design with three treatments, except for SOD activity, which considered two treatments as indicated above. Data of performance, processing weights, dietary efficiencies, excreta characteristics, and growth curve parameters were analyzed considering the cage as the replication, while necropsy evaluations (lesions and lymphoid indexes) were analyzed considering the selected bird per cage as the replication. Data on mucosa congestion and morphometry of lymphoid organs were analyzed with the KruskalWallis test, and treatments were compared with the Wilcoxon test (script embedded in Supplementary File 27; report in Fig. S27). The ANOVA of all other variables was performed, and treatments were compared with Tukey-Kramer HSD multiple comparison test (script embedded in Supplementary File 28, reports in Figs. S28a–S28c). The pooled SEM was calculated with the equation provided by Martinez et al. (2023) (Supplementary Table S8). P-values were considered statistically significant if < 0.05. Data processing, modeling, and all statistical procedures were performed in JMP (JMP Pro 16 software; SAS Institute Inc., Cary, NC) at the Center of Excellence for Poultry Science, University of Arkansas.

Results

The negative control birds, subjected to the challenge model but receiving no treatment, showed evidence of a process compatible with the FPS (Table 1), as summarized by Martinez et al. (2023). The induced FPS was characterized by wet droppings (76%; Fig. 1), containing undigested feed (65%; Fig. 2), and desquamated mucosa (3%; Fig. 3) with no bloody droppings observed.

During both the third and fourth weeks, feeding OEO produced more normal droppings (P < 0.0001), reducing the frequency of all abnormalities in the excreta (Table 1). During the fourth week, the percentage of droppings with desquamated mucosa was reduced (P = 0.0002) in both the neomycin- and OEO-fed birds. No bloody droppings were observed. In both weeks, the excreta of neomycin-fed birds showed better characteristics than the control birds, but the OEO-fed birds still showed more normal droppings.

At the necropsy, all cages of the negative control treatment showed birds sampled with congested mucosa, the same as the neomycin-fed birds (Table 2). The OEO-fed birds showed a lower percentage of cages with birds affected (P = 0.0101). The OEOand neomycin-fed birds showed a mean density of lesions 14 and 21% lower than the negative control birds; however, it was not possible to determine if those differences were attributable to the treatments (P > 0.05). Lymphoid variables of neomycin-fed birds did not show differences compared to the negative control birds. However, the OEO-fed birds showed smaller bursas (diameter and morphometric index) (P < 0.03) and a lower ratio when compared to the weight of the thymus (P < 0.05). The OEO-fed birds showed a higher SOD activity than the negative control birds (P < 0.05; Fig. 4).

Table 1 Excreta characteristics of broilers challenged with a model for Feed Passage Syndrome and fed oregano essential oil (OEO) or neomycin sulfate.¹

Fig. 1. The challenge induced watery droppings in broilers. The dropping shown in the picture was scored as wet and corresponded to droppings also scored wet under a natural field challenge (Martinez et al., 2023). It also shows high gas production associated with the dysbacteriosis induced by the challenge.

Fig. 2. The challenge induced the presence of droppings with desquamated mucosa in broilers. The dropping shown was scored positive for desquamated mucosa and corresponded to droppings also scored accordingly under a natural field challenge (Martinez et al., 2023). The color of the dropping was not related to the challenge or the treatments.

Fig. 3. The challenge induced the presence of droppings with undigested feed in broilers. The dropping shown was scored positive for undigested feed and corresponded to droppings with the same score under a natural field challenge (Martinez et al., 2023). The color of the dropping was not related to the challenge or the treatments.

After the challenge (15–28 days), feeding the OEO showed no effect (P > 0.05) on the feed intake (FI); however, the BWG was higher (P = 0.0124), and the FCR more efficient (P = 0.0161) than the negative control birds (Table 3). Before the microbial inoculums were administered (0–14 days), the OEO-fed birds showed higher BWG (P = 0.0004) and lower FCR (P = 0.0022) compared with the negative control ones. Similar effects were observed in the overall period due to the OEO (P < 0.01). In all the cases, the BWG and FCR of neomycin-fed birds were approximately intermediate compared to the negative control and OEO treatments but were not different from either (P > 0.05). Only from 0 to 14 days, the BWG in neomycin-fed birds was improved compared to the negative control ones (P < 0.05).

The OEO treatment influenced the growth curve compared to the negative control treatment, as evidenced by differences in the growth rate and inflection point (P < 0.02; Table 3; Fig. 5). The effect of the neomycin treatment was located between the OEO and the negative control birds (P > 0.05). Consequently, the ages to reach specific target weights were lower (P < 0.05) for the neomycin treatment but even lower (P < 0.05) for the OEO birds.

No treatment effect was observed in the carcass or breast yield (P > 0.05; Table 3). However, the weights of the breast and carcass of OEO-fed birds were heavier than the negative control ones (P < 0.04).

The OEO-fed birds showed improved PER, EER, feed-to-carcass and feed-to-breast conversion ratios, and better protein-tocarcass and protein-to-breast efficiency ratios than the negative control ones (P < 0.05). However, no effect (P > 0.05) on these variables was detected in the neomycin treatment compared to the negative control one. Better responses in these variables in the OEO-fed birds compared to the neomycin-fed birds were detected (P < 0.05) in the feed-to-carcass conversion ratio and the proteinto-carcass efficiency ratio.

Table 2 Necropsy evaluation of broilers challenged with a model for Feed Passage Syndrome and fed oregano essential oil (OEO) or neomycin sulfate.¹

Fig. 4. Antioxidant status (superoxide dismutase activity) of broilers challenged with a model for Feed Passage Syndrome and fed oregano essential oil (OEO). Bars indicate the SE (n = 6 per treatment). a,bMeans with a different letter are significantly different (P = 0.0417).

Author’s point of view

The main outcomes of this study are the following: (1) A model to induce the FPS in broilers, with the main characteristics of the droppings (i.e., wet, undigested feed) registered in pictures and matching a FPS case reported under commercial conditions (Martinez et al., 2023). (2) A new application of a procedure (Martinez et al., 2023) to obtain growth curves per treatment and compare the parameter estimates with an ANOVA. (3) The finding that under the FPS induced, OEO fed to broilers improved the quality of excreta, reduced the hyperplasia of the bursa, improved the antioxidant status (SOD activity) and performance, increased the carcass and breast weights, and favored a better use of the dietary protein and energy to produce BW, carcass, and breast. (4) The finding that in some of these variables, the response was higher to OEO than to neomycin. (5) The finding that the OEO likely influenced the growth curve by reducing the age at the inflection point and increasing the growth rate.
The results of this study confirm the positive effect of the OEO treatment on the characteristics of the excreta and the BW of broilers under a natural field FPS challenge reported elsewhere (Martinez et al., 2023). They are consistent with the antimicrobial action observed for OEO (Lambert et al., 2001). Compared to the characteristics of the FPS challenge reported by Martinez et al. (2023), we included an inoculum of Eimeria spp. Also, considering that the feed intake was not shown to be influenced by the treatments, the improved dietary protein and energy efficiencies and overall performance reported in this study indicate a possible improvement in digestibility, a lower nutrient expense in maintenance (Martinez, 2021), or a combination of both. This mechanism would agree with a lower body temperature (i.e., energy loss as heat production) reported in birds fed OEO (Lee et al., 2020), which would imply an improved dietary energy partitioning (Martinez et al., 2022d). We consider that the reduced hyperplasia of the bursa in our study agrees with this mechanism.
OEO has been shown to down-regulate liver genes involved in fatty acid metabolism, reducing abdominal fat (Sabino et al., 2018). We consider that lowering the abdominal fat (Martinez and Uculmana, 2016) and, therefore, increasing the relative body lean mass (Maharjan et al., 2020) would determine a higher body protein-to-fat content ratio, which has been reported to predict carcasses with higher contents of the breast (higher breast-to-legs ratio) and better market values (Martinez et al., 2022e). We consider this mechanism may partially explain the higher breast weight in OEO-fed birds in our study, as the increase in breast weight in OEO-fed birds (14.9% increase) versus the negative control treatment was 18% higher compared with the increase in BW (12.6%).
In the present study, we assumed the challenge did not influence the maximum BW (asymptote of the Gompertz 3P model) but produced a shift in the growth curve, making the birds reach the commercial market weight later. This assumption may be considered a limitation of our study. However, this effect was reported by Chasser et al. (2019) in birds that also were subclinically affected by C. perfringens. Indeed, as indicated elsewhere (Martinez et al., 2023), fitting independent Gompertz 3P models to treatments may provide biased asymptote estimates. Therefore, we consider this assumption reasonable considering previous findings (Chasser et al., 2019) and a conservative approach to prevent biases when estimating the Gompertz 3P curve parameters (Martinez et al., 2023).

Table 3 Performance, growth curve, processing weights, and dietary efficiencies of broilers challenged with a model for Feed Passage Syndrome and fed oregano essential oil (OEO) or neomycin sulfate.¹

The data presented herein (values and pictures) will support researchers in comparing the findings in birds subjected to different challenge models. Other researchers will also be benefited by reproducing the procedure used in this study to fit growth curves per replication and compare the parameter estimates through an ANOVA, enriching the strength of their research inferences.
The data provided herein may be used to develop new modeling strategies to compare growth curves among experimental treatments. By adopting biologically reasonable assumptions, the Gompertz 3P model may be modified to help stabilize the asymptote when the data used to obtain the growth curves comes from studies run only up to a commercial age.

Fig. 5. Growth curves of broilers challenged with the Feed Passage Syndrome and fed oregano essential oil (OEO) or neomycin sulfate.

Conclusion

The findings in this study indicate that feeding OEO to broilers challenged with the FPS may improve the characteristics of the excreta, reduce the percentage of birds with congestion in the intestine mucosa, revert the hyperplasia of the bursa, and improve the performance.