1. Introduction
Coccidiosis is an economically important parasitic disease caused by protozoa, characterized by the presence of lesions in the intestine [
1]. It is one of the most expensive parasitic diseases affecting poultry production as a result of various species of
Eimeria. Typically,
Eimeria species multiply and induce severe alternations in the epithelial tissue of different parts of the intestine through multiplication [
2]. The economic impact of coccidiosis is substantial due to its widespread global occurrence in countries with significant poultry production [
3]. Economic losses associated with this disease encompass weight reduction, diminished feed efficiency, increased costs of anti-coccidial therapy, and high mortality and morbidity [
2,
4]. Moreover,
Eimeria infections can disrupt digestion and absorption of important nutrients, resulting in impaired performance and immunosuppression [
5].
In the present scenario, the primary approach against coccidiosis in poultry farms involves the application of chemical drugs, vaccination, or natural compounds [
2,
6]. Although using preventive chemical agents has proven highly effective in controlling parasitic infections, concerns have arisen due to the development of drug resistance in poultry products [
7,
8]. As a result, researchers have been actively seeking natural alternative health-conscious solutions [
9,
10]. However, in developed countries, the use of anti-coccidial drugs for preventing coccidiosis has been severely restricted for more than one decade and is anticipated to be completely withdrawn in the upcoming years [
11].
A number of research studies have investigated the positive impacts of plant-based compounds on broiler performance, gastrointestinal function, and mortality. In coccidiosis control programs, phytogenic compounds present themselves as a viable alternative to chemical counterparts. These plant-based anti-coccidial compounds not only exhibit inhibitory effects against
Eimeria species but also tend to have minimal adverse effects on the health of birds [
12]. Recent reports have highlighted the anti-coccidial properties of phytogenic compounds, including plant extracts, spices, and essential oils [
6]. While the use of phytogenic products represents an important option for preventing coccidiosis in antibiotic-free poultry production systems [
3], their wider adoption across poultry farms may be encouraged if these compounds originate from agricultural by-products, providing a cost-effective and more potent alternative to chemical options [
6].
The positive impact of citrus peel supplementation in poultry diets has been documented in various aspects including enhanced performance and nutrient digestibility and improved antioxidant enzyme profiles, as well as favorable alterations in blood metabolites [
13,
14,
15,
16,
17]. The pulp of lemons (
Citrus limon) is a common by-product in the food and juice extraction industry, and lemons are the most widely consumed citrus fruit globally [
18,
19]. Citrus fruit pulp accounts for about one-fourth of the total fruit mass and is obtained after extracting the juice and mechanically removing the remaining pulp [
20]. During the extraction process of citrus juice, a significant amount of waste or by-products is generated [
21]. Ramadan et al. [
22] found that lemon peel extracts exhibit antimicrobial activity, suggesting their potential use as sanitizers to mitigate microbial contamination from foodborne bacteria. While a substantial quantity of citrus pulp is utilized as feed for animals (particularly ruminants), the majority of the processing residue is discarded, resulting in environmental pollution [
23]. As a result, citrus-processing industries have been actively seeking alternative applications for these by-products [
24]. A number of studies have reported beneficial effects of citrus such as improved growth performance, carcass quality, antioxidant activity, intestinal histomorphology, and other serum metabolites in broilers [
24,
25,
26]. However, to the best of our knowledge, no study has reported the anticoccidial effect of lemon peel powder in broilers. Therefore, this study was conducted to evaluate different levels of lemon peel powder dietary supplementation on the growth performance, intestinal lesion score, mortality rate, oocysts shedding, and intestinal histomorphology in broilers experimentally exposed to
Eimeria tenella challenge.
3. Results
Table 2 shows that supplementation with lemon peel powder had a significant effect on FI in broilers. At week 1, FI was significantly (
p < 0.05) higher in the NC group and PC group than the LPP6 group, with no significant (
p > 0.05) differences between the AT group and the LPP3 group. At week 2, FI was significantly (
p < 0.05) higher in the NC group, PC group, and the AT group followed by the LPP3 group and the LPP6 group. FI at week 3 was significantly (
p < 0.05) higher in the NC group, which had no significant difference with the PC group and the AT group, followed by the LPP3 group and the LPP6 group. In the starter phase, FI was significantly (
p < 0.05) higher in the NC group, the PC group, and the AT group followed by the LPP3 group and the LPP6 group. At week 4, the highest FI was observed in the NC group in comparison with the AT group followed by the LPP6 group, the LPP3 group, and the PC group. At week 5, FI in the NC group was significantly (
p < 0.05) higher than the AT group followed by the LPP6 group, the LPP3 group, and the PC group. FI in the finisher phase was significantly (
p < 0.05) higher in the NC group than in the AT group followed by the LPP6 group, the LPP3 group, and the PC group. For the overall period, FI was significantly (
p < 0.05) higher in the NC group in comparison with the AT group, followed by the LPP6 group, the LPP3 group, and the PC group. There was no significant (
p > 0.05) difference between the lemon peel-supplemented groups in overall FI.
Table 3 illustrates the results of weight gain, which indicates that there was no significant difference in week 1, week 2, and the starter phase. At week 3, weight gain in the LPP6 group had no significant (
p < 0.05) difference compared to the LPP3 group, the NC group, and the PC group but was significantly (
p > 0.05) higher than the AT group. At week 4, weight gain in the NC group was significantly (
p > 0.05) higher than the AT group followed by the LPP6 group, the LP3 group, and the PC group. At week 5, WG was significantly (
p > 0.05) higher in the NC group as compared with the AT group, followed by the LPP6 group, the LPP3 group, and the PC group. In the finisher phase, weight gain in the NC group was significantly (
p > 0.05) higher than the AT group followed by the LPP6 group, the LPP3 group, and the PC group. For the overall period, WG was significantly (
p > 0.05) higher in the NC group than the AT group followed by the LPP6 group, the LPP3 group, and the PC group. There was no significant (
p < 0.05) difference between the LPP3 and LPP6 groups in overall weight gain.
Table 4 illustrates the effect of adding lemon peel powder to the broiler diets on the FCR. At week 1, there was no significant (
p > 0.05) difference in the FCR among the groups. At week 2, the FCR was significantly (
p < 0.05) lower in the LPP6 group and LPP3 group compared with the AT, NC, and PC groups. At week 3, the FCR in the LPP6 group was similar to the LPP3 group followed by the NC group, PC group, and the AT group. In the starter phase, the LPP6 group and LPP3 group had a significantly (
p < 0.05) lower FCR than the NC group and the PC group followed by the AT group. At week 4, the FCR of the NC group and the AT group was significantly (
p < 0.05) lower than the LPP6 group and the LPP3 group followed by the PC group. At week 5, the FCR in the NC group was significantly (
p < 0.05) lower in comparison with the AT group followed by the LPP6 group and the LPP3 group. Moreover, the FCR of the PC group was the highest (
p < 0.05) in week 5. In the finisher phase, the FCR in the NC group was significantly (
p < 0.05) lower than the AT group followed by the LPP6 group, the LPP3 group, and the PC group. For the overall period, the FCR of the NC group was significantly (
p < 0.05) lower compared with the AT group and the LPP6 group followed by the LPP3 group and the PC group.
The effect of supplementation with lemon peel powder on the lesion score and mortality in the broiler diets challenged with coccidiosis is shown in
Table 5. The result shows that the NC group, which was not infected, displayed a normal cecal epithelium. Cecal lesions were significantly (
p < 0.05) higher in the PC group as compared with the LPP3 group, the LPP6 group, and the AT group. Cecal lesions were absent from the NC group, while the lowest lesions were observed in the AT-treated group. The mortality rate was higher in the PC group compared with the LPP3 and LPP6 groups. The lowest mortality was recorded in the NC and AT groups.
Table 6 presents the effect of supplementation with lemon peel powder on oocyst OPG of feces in broilers challenged with coccidiosis. The results showed that OPG were significantly (
p < 0.05) higher in the PC group compared with the NC and AT groups on 7, 10, and 14 DPI. No significant change was observed in OPG on 7 DPI between the PC, LPP3, and LPP6 groups. However, on day 10 and day 14 post-infection, OPG decreased significantly (
p < 0.05) in the LPP3 and LPP6 groups compared with the PC group.
Table 7 shows the effect of supplementation of lemon peel powder on villus height, crypt depth, the villus height to crypt depth ratio, and the width of the caecum of broilers challenged with coccidiosis. Villus height and width and VH:CH decreased significantly (
p < 0.05) in the PC group compared with the NC and AT groups. Interestingly, villus height and width and the ratio of VH and CD were significantly (
p < 0.05) higher in the LPP3 and LPP6 groups compared with the PC group. Similarly, crypt depth decreased significantly (
p < 0.05) in the LPP3 and LPP6 groups compared with the PC group. An examination of the cecal epithelium (
Figure 1) revealed that in the PC group, prevalent signs observed in challenged and untreated chickens included the presence of blood, hemorrhages, and sloughing of the epithelium. Various types of inflammatory cells and
Eimeria oocysts were also detected in the intestinal epithelium of these chickens. Conversely, both the infected groups supplemented with lemon peel powder (at 3 g/kg and 6 g/kg) exhibited fewer lesions and mild cecal sloughing. The group treated with amprolium after infection showed only a few lesions with minor damage to the intestinal epithelium.
4. Discussion
The processing of lemons results in a significant amount of peel waste, which contains numerous high-value substances with substantial potential for industrial applications. Lemon peels are particularly abundant in valuable nutritional components, notably vitamin C, pectin, fibers, and various phytochemical compounds. These include phenolic constituents and essential oils, which contribute to the impressive nutraceutical potential of lemon peels [
21]. Studies are scarce on the effect of lemon peel powder as a feed additive in the broiler diet. This is the first study to investigate the effect of LPP in broilers challenged with
E. tenella. In this study, the impact of LPP on growth indicators,
Eimeria oocyst excretion, and intestinal health in broiler chickens subjected to experimental
Eimeria infection was examined. The findings demonstrate that LPP mitigates intestinal lesions following challenges with
Eimeria oocysts and also leads to a reduction in the excretion of
Eimeria oocysts from the gastrointestinal tract and partially restored cecal histopathological microscopic structures compared with the challenged and non-treated group. In the present study, the impact of LPP6 was similar to LPP3. In the present study, broilers challenged with
Eimeria exhibited enhanced growth performance when supplemented with LPP3 and LPP6 compared with the challenged and non-treated group. Under no coccidial challenge, several studies have investigated the impact of lemon peel in different forms (extract, juice) on growth indices in chickens. Akbarian et al. [
24] reported no significant effect on FI, weight gain, and the FCR in broilers fed diets supplemented with lemon peel extract (LPE) at 200 and 400 mg/kg under high ambient temperature. According to Ishaq et al. [
26], a lower FCR was observed in groups treated with
C. aurantium extract may be attributed to the extract’s ability to reduce intestinal inflammation and injuries in broilers. Similarly, Salehifar et al. [
25] also reported no significant effect of 0.2%, 0.6%, and 1.0% lemon pulp powder on the growth performance of broilers under high ambient temperatures. Dudko et al. [
29] found a significant effect on weight gain in Polish sheep supplemented with a blend of essential oil from
Origanum vulgare (lamiaceae) and Citrus spp. (citraceae) under coccidiosis challenge. Ishaq et al. [
26] reported that ethanolic leaf extract of
Citrus aurantium at the levels of 125, 250, and 500 mg/kg resulted in higher daily FI but no change in weight gain in broiler chickens. The notable reduction in body weight gain observed in the PC group in the current study is similar to the outcome of previous reports. This phenomenon may be attributed to the negative implications for digest stability, absorption capacity, and nutritional assimilation.
In the present study, both supplements demonstrated equal effectiveness in terms of lesion scores, oocysts per gram (OPG), and histological dimensions. Furthermore, the assessment of LPP3 and LPP6 doses revealed that while they exhibit some anti-coccidial effects at these concentrations, these effects are considerably less pronounced compared with AT in terms of mitigating typical coccidiosis symptoms or enhancing growth performance. Numerous studies have investigated the antimicrobial properties of lemon peels, demonstrating their broad-spectrum effectiveness [
30]. Ishaq et al. [
26] reported a reduction in oocysts shedding and improvement in the lesion score in broilers supplemented with 125, 250, and 750 mg/kg
Citrus aurantium L. ethanolic leaf extract against experimental
E. tenella infection. Ishaq et al. [
26] suggested that the reduction in oocyst shedding observed with lemon peel extract may be attributed to its saponin content. This study indicates that saponins bind with sterol molecules on the parasite’s cell layer, leading to their destruction and subsequently reducing excretion in chicken feces. This study demonstrated a noteworthy reduction in cecal and lesion scores among the treated groups. This damage may be caused by the replication of developmental
Eimeria parasites, potentially leading to inflammatory reactions and secondary bacterial infections, which could be associated with the observed lesions in the bird’s caeca [
31]. Ishaq et al. [
26] suggested that the decrease in oocyst shedding is attributed to the presence of antioxidant and free radical scavenging properties in essential oils from
C. aurantium leaves likely contributed to an observed improvement in cecal lesions. Antioxidant compounds are recognized for their ability to diminish the number of harmful free radical molecules in the body, providing protection against their detrimental effects. In this study, lemon peel powder effectively reduced the mortality rate of broiler chickens compared with the challenged and non-treated group. The findings indicate that the lower oocyst excretion rate, increased survivability, reduced bloody diarrhea, and improved cecal condition in the LPP-treated groups, compared with the infected-untreated group, may be attributed to immune-enhancing compounds like tannins, alkaloids, and flavonoids present in lemon peels [
21,
22]. Phenolic extracts from lemon peels were found to effectively inhibit the growth of Staphylococcus aureus and Bacillus cereus. Recent research on citrus fruits revealed that lemon peel extract exhibited stronger antibacterial effects compared with pomelo peels but was slightly less potent than lime peels [
32]. Ramadan et al. [
22] reported that methanol and ethanol extracts from lemon peels are effective against several foodborne pathogens (
Escherichia coli,
Salmonella Typhimurium,
Listeria monocytogenes) in in vitro tests using disc diffusion and minimum inhibition concentration assays, as well as in situ experiments on chicken skin. Treatment with 5 mg/mL of the extract significantly reduced
L. monocytogenes and
P. fluorescens levels.
The presented study demonstrated that the deterioration in villus dimensions caused by
E. tenella challenge was partially reversed by including LPP3 and LPP6 supplements in the broilers’ diets. The application of LPP treatment led to an improvement in the histological features of broilers infected with
Eimeria challenge. This could be attributed to the antiprotozoal properties of lemon peel, as well as its potent antioxidant and anti-inflammatory activities. These attributes serve to safeguard host tissue from damage caused by
Eimeria oocysts. Akbrarian et al. [
24] reported an improvement in intestinal histological features in broilers supplemented with 200 and 400 mg/kg citrus peel extracts under high ambient temperatures. Similar improvements in histological structures were noted in broilers fed with 0.2%, 0.6%, and 1% lemon peel powder (LPP) in high ambient temperatures [
25]. The effectiveness of lemon peels can be mainly attributed to their key chemical constituents, particularly vitamin C, fibers, pectin, and beneficial phytochemical compounds. These are due to the desirable properties of essential oils and phenolic constituents [
21]. As natural products, lemon peels show great potential as sources for novel anticoccidial agents that target
Eimeria while also providing protective and healing properties for infected host tissues.