Research Article

Breed-Specific Responses to Peppermint Oil Supplementation: Effects on Growth, Antioxidant Status, and Carcass Traits in Growing Rabbits

Amin O. Hendawy1, Abdalrahem A. Amrabit2, Hossam A. Shahba3, Saber S. Hassan1, Mohamed I. Hassan4*, Walid S. Habashy1

1Animal and Poultry Production Department, Faculty of Agriculture, Damanhour University, Damanhour, Egypt; 2Animal Production Department, Faculty of Agriculture, Omar Al-Mukhtar University, Al-Bayda, Libya; 3Animal Production Research Institute (APRI), Agricultural Research Center (ARC), Ministry of Agriculture, Giza, 12611, Egypt; 4Livestock Research Department, Arid Lands Cultivation Research Institute, City of Scientific Research and Technological Applications (SRTA-City), New Borg El-Arab, Alexandria, Egypt.

Received | August 11, 2025; Accepted | October 15, 2025; Published | November 19, 2025

*Correspondence | Mohamed I. Hassan, Livestock Research Department, Arid Lands Cultivation Research Institute, City of Scientific Research and Technological Applications (SRTA- City), New Borg El-Arab, Alexandria, Egypt; Email: [email protected]

Citation | Hendawy AO, Amrabit AA, Shahba HA, Hassan SS, Hassan MI, Habashy WS (2025). Breed-specific responses to peppermint oil supplementation: effects on growth, antioxidant status, and carcass traits in growing rabbits.

Adv. Anim. Vet. Sci., 13(11):2424-2434.

DOI | https://dx.doi.org/10.17582/journal.aavs/2025/13.11.2424.2434

ISSN (Online) | 2307-8316

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Oxidative status of animals is a multifaceted physiological parameter which is influenced by diet, age, production levels, management practices, and environmental conditions (Bernabucci et al., 2002). Oxidative stress results when there is an imbalance between the generation of reactive oxygen species and the antioxidant defense mechanisms disrupt cellular integrity and physiological functions (Pizzino et al., 2017; Hendawy et al., 2020). Adverse effects of this phenomenon include reduced animal health, depressed performance, diminished production efficiency, and thus negative economic consequences on livestock systems (Lee et al., 2017; Hendawy et al., 2022). Therefore, it is important to identify the determinants of oxidative stress and to implement effective mitigation strategies in order to improve animal welfare and production outcomes.

In recent years, natural agents with strong antioxidant properties have gained much attention as alternatives to antibiotics in animal nutrition fields (Hendawy et al., 2019; Hassan et al., 2023a, b). Herbal plants and their extracts are one of the available sources of potential antioxidants. Herbs possess diverse bioactive properties that can beneficially influence contemporary animal nutrition (Jachimowicz et al., 2022). Among these, flavonoids and other plant-derived phenolic compounds function as potent free radical scavengers and inhibitors of lipid peroxidation (Formica and Regelson, 1995). Peppermint (Mentha piperita L.), a perennial aromatic medicinal herb belonging to the Lamiaceae family, contains approximately 1–3% essential oil. The primary constituents of this oil are menthol (37.69%) and menthone (21.79%) (Khodambashi et al., 2012).

Peppermint essential oil has shown antioxidant, antimicrobial, antiviral, anticarcinogenic, antiparasitic, anti-inflammatory, and immunomodulatory actions on animal physiology with particular positive effects on the digestive system (Attia et al., 2017; Hesabi Nameghi et al., 2019). Elspeiy et al. (2020) observed that rabbits receiving peppermint oil exhibited higher average daily gain (ADG) and improved feed conversion ratio (FCR) compared with those on a basal diet. Their findings also indicated that peppermint oil supplementation lowered serum levels of cholesterol, low-density lipoprotein (LDL), triglycerides, and malondialdehyde (MDA), while elevating high-density lipoprotein (HDL), total antioxidant capacity (TAC), and immunoglobulin G concentrations. Ciliberti et al. (2024) reported that peripheral blood mononuclear cells (PBMCs) treated with 10% peppermint oil and stimulated with concanavalin A and lipopolysaccharide exhibited higher proliferation rates than the positive control (PBMCs stimulated with concanavalin A alone). Additionally, peppermint oil treatment increased the concentration of anti-inflammatory cytokines and reduced pro-inflammatory cytokine levels compared with the positive control. Based on these findings, the present study was conducted to evaluate the impact of peppermint oil supplementation on antioxidant status, growth performance, and carcass characteristics in two strains of growing rabbits.

MATERIALS AND METHODS

The experiment was conducted at El-Bostan Poultry Farm, Faculty of Agriculture, Damanhour University. All experimental procedures were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee of Damanhour University (Approval No. DUFA-2024-10). Peppermint oil (Mentha piperita) was purchased from a local commercial supplier in Egypt. The oil was food-grade, with no additives, and was used as received for preparing the experimental doses.

 

Animals and experimental design

As shown in Figure 1, a total of 96 growing rabbits (48 Alexandria and 48 V-line) were included in the study. At 35 days of age, their average initial body weights were 1112 g and 1023 ± 29.77 g, respectively. Galvanized wire cages with a single tier (55 × 45 × 35 cm) were used for housing the experimental rabbits within a naturally ventilated building. Each breed was divided into three groups: a control group receiving a placebo (2 ml of water), a second group receiving 0.25 ml peppermint oil/kg of BW, and a third group receiving 0.5 ml peppermint oil/kg of BW. Each group was composed of 16 rabbits per breed divided into four replicates (4 rabbits per cage). The supplementation was administered orally twice a week (Sunday and Wednesday). The experimental period extended for five weeks. Ventilation and temperature were naturally regulated. The temperature, relative humidity, and photoperiod varied from 23 to 27°C, 60 to 65%, and 14 hours, respectively, during the experimental period.

All rabbits across the experimental treatments were provided with identical standard diets formulated according to NRC (NRC, 1977). The diets were provided in pelleted form and offered ad libitum, and fresh drinking water was also available ad libitum throughout the trial. The feed composition and proximate chemical analysis were consistent with those reported in the previous study by Shahba and Mansour (2022). The commercial pelleted diet had 2464 kcal/kg of metabolizable energy, 17.24% crude protein, 13.46% crude fiber, and 2.80% fat.

Feed intake and body weight

Feed intake was recorded per cage weekly throughout the research period. Rabbits were weighed individually at the beginning of the experiment as an initial weight and weekly. Initial and final weight of individual rabbits were used to determine ADG as follows: ADG = (Final weight − Initial weight)/Experimental period (d). The FCR was calculated as: FCR = average dry matter (DM) intake per rabbit/ADG. Performance index = (final live BW (kg)/feed conversion ratio) x 100.

Blood biochemical constituents

Six rabbits were chosen at random from each treatment at the conclusion of the trial, and blood samples were taken through the inner ear vein in sterile tubes devoid of anticoagulants. Samples were stored in an ice tank right away. Serum was separated from blood samples by centrifuging them for 15 minutes at 4000 rpm. The serum was then kept at -20°C until analysis. As directed by the manufacturer, assay kits were used to measure the levels of total cholesterol, LDL, HDL, VLDL, triglycerides, total protein, albumin, TAC, superoxide dismutase (SOD), and MDA in serum (Biodiagnostic Company, Dokki, Giza, Egypt). The variation between total protein and albumin was used to determine the serum globulin level (g/dl).

Carcass characteristics

To assess carcass characteristics, including the weights of the carcass, head, full and empty stomachs, abdominal fat, intestine, cecum, pancreas, liver, kidney, heart, spleen, and lung, six rabbits were chosen at random from each treatment at the conclusion of the experiment. The weight of the liver, kidney, lungs, spleen, heart, pancreas, and abdominal fat was reported as a percentage of live body weight.

Gut microbial count

The total cecal bacterial count was determined following the method described by the American Public Health Association (Richardson and Association, 1985) Specific bacterial groups, including Clostridium spp., Escherichia coli, and Proteus spp., were identified according to the procedures outlined by Mackie and McCartney (1953). The colony-forming unit (CFU) technique was employed, with incubation conducted at 30 °C for 2 to 7 days.

Statistical analysis

The following formula was used to statistically analyze the data using the General Linear Model (GLM) process of the SAS Institute’s statistical analysis system (SAS, 2002) employing factorial analysis of variance: Yijk is equal to µ + Bi + Tj + Eijk. Where Yijk is the statistical measure’s observation, µ is the overall mean, Bi is the breed effect, Tj is the treatment effect, and Eijk is the experimental random error. Arcsine transformation was used to standardize the data distribution prior to analysis. Duncan (1955) was used to determine the least square mean (LSM) + standard errors and test for significance at (p≤0.05).

RESULTS

Body weight and body weight gain

Peppermint oil supplementation did not significantly affect BW from week 5 to week 10 or overall BWG (Table 1). However, during weeks 8–9 and 9–10, the 0.25 m/kg group exhibited higher BWG compared with both the control and 0.5 ml/kg groups. Breed differences were also observed: Alexandria rabbits had higher BW at weeks 5, 6, 8, and 9, and greater BWG between weeks 9–10, whereas V-line rabbits gained more during weeks 6–7. A tendency for treatment × strain interaction was detected for BWG during weeks 9–10.

Feed intake, feed conversion ratio, and performance index

Peppermint oil significantly influenced FI at weeks 6, 9, and 10, as well as total FI (Table 2). The 0.25 ml/kg group consumed more feed at week 9 and overall compared with the control and 0.5 ml/kg groups, while the 0.5 ml/kg group had the lowest intake at week 10. The FCR was affected at week 10, where the control group showed the highest value and the 0.5 ml/kg group the lowest. Strain effect was significant for FCR at week 8 and week 10, with V-line rabbits showing higher FCR at week 8 and lower FCR at week 10. The PI and live body weight were not significantly affected by peppermint oil or strain. A significant treatment × strain interaction was observed for total FI and FCR at week 10.

Blood parameters

Peppermint oil supplementation significantly reduced cholesterol, LDL, and MDA concentrations (Table 3), with the 0.25 and 0.5 mL/kg groups showing lower values

 

Table 1: Effect of peppermint oil (PO) level on body weight and body weight gain in Alexandria and V-line rabbits during the growing period.

Table 2: Effect of peppermint oil (PO) level on feed intake, feed conversion ratio and the production index in Alexandria and V-line rabbits during the growing period.

 

Table 3: Effect of peppermint oil (PO) level on blood parameter in Alexandria and V-line rabbits during the growing period.

 

than the control. Meanwhile, the peppermint oil groups had higher levels of HDL, total protein, albumin, globulin, TAC, and SOD compared to the control group. No effect was observed on triglycerides or VLDL. Strain differences were noted for triglycerides, VLDL, globulin, and MDA. Several significant treatments × strain interactions were found, particularly for cholesterol, LDL, albumin, SOD, and MDA.

Cecal microbial populations

Peppermint oil supplementation significantly decreased total bacterial count, Escherichia coli, and Clostridium spp. (Table 4). Strain effect was also significant; the V-line rabbits had higher microbial counts than Alexandria rabbits in all microbial groups. A significant treatment × strain interaction was detected for Clostridium spp.

 

Table 4: Effect of peppermint oil (PO) level on the cecum microbial populations (log10cfu/g) in Alexandria and V-line rabbits during the growing period.

Item		Total bacteria count	Escherichia coli	Clostridium
PO level
0		12.90a	11.08a	3.12a
0.25		11.60b	8.84b	2.30b
0.5		9.05c	7.29c	2.03b
SEM		0.277	0.300	0.0933
Breed
Alx		7.88b	5.87b	0.884b
V-line		14.48a	12.28a	4.085a
SEM		0.226	0.245	0.076
PO level and breed interaction
0	Alx	9.49	8.09	0.888d
	V-line	16.31	14.07	5.355a
0.25	Alx	8.39	5.25	0.923d
	V-line	14.81	12.44	3.678b
0.5	Alx	5.78	4.27	0.841d
	V-line	12.32	10.32	3.22c
SEM		0.393	0.426	0.132
p- value
Treatment		<.0001	<.0001	<.0001
Strain		<.0001	<.0001	<.0001
Interaction		0.8760	0.2874	<.0001
Linear		0.0024	<.0001	<.0001
Quadratic		<.0001	<.0001	<.0001

 

Different superscripts in each column indicate significant differences (p <0.05). 0: 0 ml peppermint oil /kg of body weight; 0.25: 0.25 ml peppermint oil /kg of body weight; 0.5: 0.5 ml peppermint oil /kg of body weight; SEM: standard error of the mean; Alx: Alexandria rabbits; V-line: V-line rabbits.

 

Carcass traits

Peppermint oil significantly affected head weight, full and empty stomach weights, and pancreas weight, with the 0.25 ml/kg group showing higher head weight, while the 0.5 ml/kg group had greater full and empty stomach weights and lower pancreas weight (Table 5). Strain effect was significant for live body weight, with V-line rabbits being heavier than Alexandria rabbits. Significant treatment × strain interactions were observed for full and empty stomachs, pancreas, spleen, abdominal fat, intestine, and cecum.

Carcass percentage traits

Peppermint oil significantly affected full stomach percentage, pancreas percentage, spleen percentage, and abdominal fat percentage (Table 6). Strain effect was significant for head, pancreas, kidney, abdominal fat, intestine, and cecum percentages, with Alexandria rabbits generally showing higher relative weights. Significant treatment × strain interactions were detected for carcass, full stomach, pancreas, liver, spleen, kidney, abdominal fat, intestine, and cecum percentages.

DISCUSSION

In the present investigation, we examined the peppermint oil supplementation impact on rabbits’ health and performance, studying its potential as a natural feed additive to improve feed efficiency, mitigate oxidative stress, and support the overall health and welfare of livestock. By examining several parameters, we aimed to clarify the mechanisms by which peppermint oil can contribute to the development of more sustainable and health-conscious animal husbandry practices. The findings of this study showed a complex interaction of how peppermint oil supplementation interacts with breed differences to influence several characteristics of rabbit physiology and performance. The enhancements in lipid profile, antioxidant status, and bacterial counts with peppermint oil supplementation suggest a multifaceted beneficial effect on rabbit health and metabolism. These effects likely contribute to the observed improvements in feed efficiency and body weight gain in late stages of growth.

With respect to growth performance, peppermint oil supplementation did not significantly affect overall BW, BWG, or FCR. However, rabbits receiving 0.25 ml/kg BW peppermint oil showed higher total FI compared with the other groups, and BWG was significantly increased during weeks 8–9 and 9–10. This pattern may reflect a biphasic (hormetic) response of peppermint oil, where a lower dose (0.25 ml/kg BW) stimulates appetite and digestive efficiency, while a higher dose (0.5 ml/kg BW) exerts stronger antimicrobial and antioxidant effects that might slightly influence palatability or fiber fermentation. The FCR at week 10 was improved in rabbits supplemented with 0.5 ml/kg BW compared with the control group. In accordance, Zeweil et al. (2017) reported that peppermint essential oil supplementation (400 mg of peppermint essential oil/ kg diet) to V-line rabbits had no significant effect on final BW, ADG, and FCR. In contrast, Elspeiy et al. (2020) showed that weaned male California rabbits

 

Table 5: Effect of peppermint oil (PO) level on carcass traits in Alexandria and V-line rabbits during the growing period.

 

Table 6: Effect of peppermint oil (PO) level on carcass percentage (%) traits in Alexandria and V-line rabbits during the growing period.

 

received peppermint oil had lower feed intake but exhibited enhanced final BW, ADG, and FCR. This discrepancy could be explained by the fact that Mentha piperita L. varies based on environmental parameters as sunshine exposure, soil and water quality, pH, humidity, pesticide use, and genetics (Waterman and Mole, 2019). Over all, the observed breed-specific responses may relate to physiological and genetic variations in metabolism and energy partitioning. V-line rabbits, being genetically selected for lean growth, might utilize nutrients more efficiently, whereas Alexandria rabbits with larger digestive organs may rely more on fiber fermentation and lipid deposition, leading to different responses to peppermint oil supplementation.

The increased total FI observed in the 0.25 g/kg peppermint oil group is noteworthy, particularly as this group also demonstrated enhanced FCR during specific growth stages. This indicates that peppermint oil could potentially boost appetite while also enhancing feed efficiency, likely due to its influence on gut health and metabolic processes. The increased weight gain noted in the groups supplemented with peppermint oil, especially the second group, supports the enhancements observed in feed efficiency. The impact was particularly evident during the later stages of growth, indicating that the advantages of peppermint oil may build up over time or become increasingly noticeable as the rabbits develop. The improvement in FCR at 0.5 ml/kg BW could also be attributed to reduced oxidative stress and metabolic energy loss, as evidenced by the lower MDA and improved antioxidant status.

Peppermint oil supplementation significantly reduced cholesterol, LDL, and MDA, while increasing HDL, total protein, globulin, TAC, and SOD compared with the control group. In agreement with our findings, Elspeiy et al. (2020) reported that peppermint oil supplementation at a level of 800 mg/kg diet decreased the concentrations of serum cholesterol, LDL, triglyceride, and MDA and increased the concentrations of serum HDL and TAC. In the same line, broiler chicks fed 0.1% peppermint oil exhibited a decrease in the concentrations of serum cholesterol, LDL, triglycerides, and MDA while serum total protein, glutathione activity, and HDL significantly increased (Hussein et al., 2023). Conversely, Morshedy et al. (2019) revealed peppermint oil did not affect most of blood biochemical parameters including total lipids, triglycerides, cholesterol, LDL, VLDL, HDL, and MDA in the growing rabbits. Finally, the more pronounced improvement in HDL and reduction in LDL observed in Alexandria rabbits at 0.25 ml/kg BW may indicate that their hepatic metabolism and bile acid turnover are more responsive to the choleretic and hypolipidemic effects of peppermint oil compared with V-line rabbits.

It has been demonstrated that peppermint volatile oil, which contains menthol and thymol, reduces the activity of enzymes such hepatic reductase and hydroxymethyl glutaryl coenzyme A that are essential for the creation of cholesterol. It suggests that volatile phenolic components like menthol, menthone, mentyl acetate, menthofuran, limonene, polygen, cineole, and azulen may be the cause of a drop in total cholesterol levels in the case of a phenolic compound like peppermint extract (Arab et al., 2016). Furthermore, the present compounds in peppermint stimulate liver cell activity, which increases bile acid concentration. Bile acids play a critical role in the digestion of lipids; therefore, elevated bile acid concentrations in the duodenum facilitate digestion of fats and fat-soluble vitamins (Crossland, 1980; Arab et al., 2016). Mimica-Dukić et al. (2003) indicated that peppermint can stimulate bile secretion from the gall bladder because of its antioxidant and antibacterial activity and is subsequently able to decrease serum total cholesterol level. Thus, peppermint supplementation may help regulate cholesterol metabolism by reducing absorption in the intestine and lowering serum lipid levels, as demonstrated in chickens (Zwain, 2022).

Breed differences were observed in the levels of triglycerides and VLDL, with V-line rabbits showing higher levels. This may suggest genetic variations in lipid metabolism between breeds, possibly related to differences in energy utilization or fat deposition patterns. Several pharmacological studies have demonstrated the potent antioxidant activity of peppermint, evidenced by its high antioxidant capacity, free radical hunting ability, and reducing power (Singh et al., 2015; Stringaro et al., 2018; Wu et al., 2019). Oils and extracts derived from Mentha species are capable of neutralizing free radicals and reactive oxygen species (Singh et al., 2015; Brown et al., 2019). This antioxidant potential is largely attributed to the presence of phytochemicals such as polyphenols, flavonoids, lignans, stilbenes, and monoterpenes within the plant (McKay and Blumberg, 2006; Pavlić et al., 2021). These phytochemical compounds mitigate oxidative stress through several mechanisms that include: The mechanisms by which antioxidants scavenge free radicals are classified primary into hydrogen atom transfer and single electron transfer, although most antioxidant reactions do not happen through a single mechanism but rather through a combination of the two (Prior et al., 2005; Brown et al., 2019). Furthermore, Mentha plants have antioxidant activity through direct radical scavenging, self-oxidation to less reactive species, singlet oxygen quenching, metal ion chelation, and neutralization of secondary oxidation products and the inhibition of pro-oxidative enzymes (Brown et al., 2019; Pavlić et al., 2021).

Concerning the antimicrobial effect of peppermint oil, the total bacterial count, Escherichia coli, and Clostridium spp., were significantly decreased in the cecum of growing rabbits fed peppermint compared to the control group. The significant reduction in pathogenic bacteria such as E. coli and Clostridium spp. likely created a more favorable gut environment, potentially reducing intestinal inflammation and competition for nutrients, which may have indirectly benefited the growth and activity of fiber-fermenting bacteria essential for rabbit health. This antibacterial effect was dose-dependent, with the highest dose showing the greatest impact. These findings have significant implications for gut health, nutrient absorption, and potentially for reducing the need for antibiotics in rabbits rearing. Similarly, total bacteria count and Escherichia coli were decreased in the jejunum of growing Japanese quail fed peppermint essential oil compared to growing Japanese quail fed basal diet only (Abbas et al., 2021). On the other hand, Ghazaghi et al. (2014) indicated that total count of microbial populations was increased in the ileal of growing Japanese quail fed dried spearmint (Mentha spicata) powder through reduction in Escherichia coli and increasing in Lactobacillus bacteria populations. The antibacterial effect of peppermint essential oil may be due to menthol and menthone which are the main active compounds of peppermint essential oil. Like antibiotics, essential oils particularly those with lipophilic character e.g., peppermint essential oil interact with the cell membranes of gram negative and gram-positive bacteria (Benchaar et al., 2006; Tajkarimi et al., 2010). Because the cyclic hydrocarbons found in several essential oils are hydrophobic, they can build up in the lipidic membrane of bacteria by taking up space between the fatty acid chains. The membrane structure undergoes conformational changes as a result of this contact, which causes it to expand and fluidify. Ions leak through the cell membrane as a result of this membrane instability, which lowers the transmembrane ionic gradient. Ionic pumps are typically used by bacteria to counterbalance these effects, preventing cell death. However, a significant amount of energy is diverted to this function with energy depletion, which slows down bacterial growth (Lv et al., 2011; Chouhan et al., 2017; Elfadadny et al., 2024).

Supplementation of peppermint oil did not affect live BW and carcass weight but altered organ weights. It decreased pancreas weight while increasing the weights of spleen, empty stomach, and intestine at a dose of 0.5 ml peppermint oil/kg of BW. The lower pancreas weight in treated groups may indicate improved digestive efficiency and reduced need for enzyme synthesis due to enhanced gut health. The heavier spleen at 0.5 ml/kg BW aligns with higher globulin levels and reduced pathogenic bacteria, suggesting enhanced humoral immunity. The increased intestinal weight could reflect improved mucosal integrity and gut-associated lymphoid tissue (GALT) stimulation resulting from a more balanced gut microbiota.

CONCLUSION

In conclusion, peppermint oil supplementation represents a viable natural approach to enhance the metabolic health and gut environment of growing rabbits. The present findings clearly demonstrate that its efficacy is breed-dependent. A supplementation level of 0.25 ml/kg BW is recommended for Alexandria rabbits to optimize feed intake and lipid metabolism, while 0.5 ml/kg BW is more suitable for V-line rabbits to improve feed efficiency and antioxidant capacity. This evidence-based, precision-nutrition strategy can contribute to more sustainable rabbit production and reduced dependence on in-feed antibiotics.

ACKNOWLEDGEMENT

The authors gratefully acknowledge the facilities and assistance provided by their institutions during the course of this study.

NOVELTY STATEMENT

This study is the first to compare peppermint oil supplementation in Alexandria and V-line rabbits, revealing breed-specific responses. Peppermint oil improved lipid profile, antioxidant status, and gut microbial balance without adverse effects on growth, indicating its potential as a natural alternative to antibiotics in rabbit production.

AUTHOR’S CONTRIBUTION

All authors contributed to the conceptualization, methodology, resources, and formal analysis.

Amin O. Hendawy carried out the investigation, visualization, and writing of the original draft.

Abdalrahem A. Amrabit contributed to conceptualization, methodology, and resources.

Hossam A. Shahba handled data curation, investigation, and visualization.

Saber S. Hassan provided supervision and manuscript review. Mohamed I. Hassan contributed to conceptualization, drafting, and editing of the manuscript.

Walid S. Habashy performed data curation, investigation, and visualization.

Generative AI and AI-assisted technology statement

All authors confirm that no generative AI tools, such as large language models (e.g., ChatGPT, Copilot) or text-to-image generators, were used in the conception, writing, or editing of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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