The growing demand for animal protein remains a major challenge, particularly in developing countries such as Egypt, where population growth continues to increase pressure on conventional livestock systems (Abdel-Rahman & Ashour, 2023). Rabbit production is an efficient component of sustainable livestock systems (Šufliarský et al., 2024). However, rabbit productivity is particularly vulnerable during the weaning and post-weaning periods, when intestinal instability can impair growth and increase mortality (El-Ashram et al., 2020). The transition to solid feed, maternal separation, and immature digestive and immune systems induce nutritional and microbial challenges (Blas et al., 2012), leading to intestinal dysbiosis, oxidative stress, impaired barrier function, and reduced productive performance (Migdał et al., 2025). Increased oxidative stress during this stage disrupts intestinal barrier integrity by elevating reactive oxygen species (ROS) production, thereby altering tight junction function and increasing epithelial permeability (Hendawy et al., 2020). Simultaneously, dysbiosis within the cecal microenvironment, characterized by the proliferation of pathogenic bacteria, further exacerbates intestinal mucosal injury and triggers localized inflammatory responses (Liang et al., 2022). Therefore, maintaining intestinal homeostasis during weaning is a major objective in modern rabbit nutrition.

Historically, antibiotic growth promoters were widely used to improve productive efficiency and control enteric disorders; however, concerns regarding antimicrobial resistance and drug residues have led to increasing restrictions on their use in animal production (Cervantes, 2015). Consequently, interest has shifted toward natural alternatives, particularly phytogenic feed additives such as essential oils (EOs), which exhibit antimicrobial, antioxidant, anti-inflammatory, and immunomodulatory activities (Movahedi et al., 2024); (Ebeid et al., 2025). The bioactive phenolic compounds in EOs contribute to free radical scavenging, redox regulation, microbial modulation, and improved digestive processes, supporting their increasing application in monogastric animal nutrition (Zeng et al., 2015).

Among phytogenic additives, oregano essential oil (OEO) has emerged as one of the most biologically active and extensively studied (Cui et al., 2024). Its major phenolic constituents, carvacrol and thymol, account for most of its antimicrobial and antioxidant activities, while minor compounds such as γ-terpinene and p-cymene further contribute to its bioactivity (Ghazi et al., 2015). In rabbits, OEO supplementation has been associated with improved feed efficiency, intestinal morphology, modulation of the gut microbiota, antioxidant status, and immune responses (Ayoub et al., 2021); (Areda et al., 2026). These responses have been linked in previous studies to the biological activities of oregano phenolics, including disruption of bacterial membranes, modulation of intestinal fermentation, and improved nutrient utilization, as well as antimicrobial activity against major enteric pathogens such as Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus(Li et al., 2021); (Cui et al., 2024). However, the physiological mechanisms underlying OEO action in rabbits remain insufficiently characterized, as previous studies have primarily evaluated individual responses rather than the integrated effects of OEO on digestive function, cecal microbiota, systemic metabolism, and immune homeostasis (Abd El-Aziz et al., 2024); (Ahmed et al., 2024).

Importantly, previous investigations have mainly focused on dietary OEO supplementation without considering the potential role of genetic background in phytogenic responsiveness (Jinling et al., 2022); (He et al., 2023). Moreover, research on OEO administration through drinking water in rabbits remains limited despite its practical advantages under commercial production conditions (Abd El-Aziz et al., 2024). To date, the dose-dependent effects of water-supplemented OEO on growth performance, nutrient digestibility, cecal microbiota, serum biochemical traits, and humoral immunity, particularly in relation to genotype × phytogenic interactions, have not been comprehensively investigated. This knowledge gap is important because host genotype may influence microbial colonization, digestive efficiency, immune responses, and metabolic adaptation to phytogenic compounds (Abdelsalam et al., 2025); (Hendawy et al., 2025).

Therefore, the present study was conducted to evaluate the effects of two levels of OEO supplementation in drinking water on productive performance, nutrient digestibility, caecal microbial populations, serum biochemical parameters, and humoral immunity in V-line and Alexandria-line growing rabbits. The study further aimed to clarify the optimal OEO inclusion level and to determine whether rabbit genetic background modulates the physiological efficacy of oregano phytochemicals under post-weaning conditions.

Weaning is a critical stage in rabbit production because the immature digestive and immune systems predispose young rabbits to intestinal dysbiosis, impaired growth, and increased mortality (Benlemlih et al., 2020). OEO, rich in the bioactive phenolics thymol and carvacrol, has attracted considerable attention as a phytogenic additive owing to its antimicrobial, antioxidant, anti-inflammatory, and digestive-stimulatory properties(Li et al., 2021); (Cui et al., 2024).In the present study, OEO supplementation through drinking water improved growth performance, nutrient digestibility, cecal microbial populations, metabolic profile, and immune responses; however, the magnitude of these effects varied between the V-line and Alexandria rabbit lines. Because information on OEO supplementation in rabbits remains limited, the present findings were interpreted in the context of previous studies involving oregano essential oil and other phytogenic additives in rabbits and monogastric species.

The progressive improvements in final BW, ADG, FCR, and PI with increasing OEO supplementation indicate enhanced nutrient utilization rather than a simple increase in feed intake. Similar improvements in BW, ADG, and feed intake at moderate OEO supplementation levels have been reported in broilers (Yang et al., 2025). The linear improvement in productive performance up to 0.0250% OEO suggests that its antimicrobial, digestive-enhancing, and immunomodulatory properties collectively improved nutrient availability and utilization (Abdel-Wareth et al., 2022); (He et al., 2023). These responses are particularly relevant in rabbits, where hindgut fermentation plays a pivotal role in nutrient recovery and energy supply (Gidenne, 2015). Consistent with the present findings, dietary OEO improved growth performance, reduced diarrhea incidence, and enhanced intestinal barrier integrity in growing rabbits (Li et al., 2021), while OEO combined with antimicrobial peptides further improved growth performance and intestinal health in meat rabbits (Jinling et al., 2022).

The improvement in productive performance was closely associated with enhanced digestive efficiency and gastrointestinal stability. Oregano phenolics improve digestive function, intestinal absorptive capacity, and microbial balance while reducing inflammatory stress, thereby enhancing nutrient utilization (Abdel-Wareth et al., 2022); (He et al., 2023). This interpretation is supported by the reduced mortality rate, particularly the absence of mortality in Alexandria-line rabbits receiving 0.0250% OEO, indicating improved intestinal health and physiological resilience. Similar improvements in feed efficiency and growth performance following OEO supplementation have been reported in poultry, attributed to enhanced nutrient digestibility, modulation of the intestinal microbiota, and reduced oxidative stress (Feng et al., 2021); (Yang et al., 2025). The differential responses between rabbit lines further suggest genotype-dependent adaptation to phytogenic supplementation (Hendawy et al., 2025). Consistent with this observation, (Abdelsalam et al., 2025) reported greater feed intake in V-line than Alexandria-line rabbits following probiotic supplementation, indicating line-dependent responses to functional feed additives.

The improved nutrient digestibility observed in OEO-supplemented rabbits provides a physiological basis for the enhanced productive performance. The increased digestibility of DM, OM, CP, EE, CF, NDF, and ADF indicates that OEO enhanced digestive efficiency and microbial fermentation (Abdel-Wareth et al., 2022). Similar improvements in nutrient utilization have been reported in rabbits supplemented with oregano-based additives, including enhanced digestibility of DM, EE, CF, and CP (Omer et al., 2013); (Ahmed et al., 2024). Likewise, supplementation of drinking water with OEO improved fiber digestibility, particularly CF and NDF digestibility, in V-line rabbits (Sayedahmed et al., 2026). Because the cecum is the primary site of microbial fermentation of structural carbohydrates in rabbits (Saleh et al., 2025), the improved NDF and ADF digestibility observed in the present study suggests enhanced hindgut fermentation and more efficient utilization of fibrous substrates (Gidenne, 2015), thereby contributing to improved growth performance and feed efficiency. Digestibility responses also differed between rabbit lines, with the V-line exhibiting greater NDF and ADF digestibility, indicating that the physiological response to OEO was influenced by genetic background (Abdelsalam et al., 2025). In contrast, the Alexandria line exhibited a stronger immunological response, particularly higher IgM concentrations, suggesting that OEO may differentially modulate digestive and immune functions according to genotype. Similar genotype-dependent responses to phytogenic supplementation have previously been reported in rabbits(Abd El-Aziz et al., 2022).

Modulation of cecal microbial populations appears to be a key mechanism underlying the beneficial effects of OEO on nutrient digestibility, intestinal health, and physiological status (Ahmed et al., 2024). The marked reduction in Clostridium perfringens demonstrates the selective antimicrobial activity of oregano phenolics, particularly carvacrol, which disrupts bacterial membrane integrity and impairs bacterial survival (Li et al., 2021); (Visan & Negut, 2024). Consequently, reduced C. perfringens colonization likely mitigated toxin-mediated epithelial damage and intestinal inflammation, thereby preserving barrier integrity, improving nutrient absorption, and reducing the metabolic cost of immune activation (Feng et al., 2021); (Sluis et al., 2024). This interpretation is further supported by the progressive reduction in WC, indicating improved intestinal barrier function. Concurrently, the increased Lactobacillus population at moderate OEO supplementation suggests the establishment of a more favorable cecal microbial ecosystem (Yang et al., 2025). Beneficial bacteria promote short-chain fatty acid production, which supports mucosal integrity and intestinal homeostasis (Li et al., 2021). Likewise, the lower cecal pH observed in supplemented rabbits is consistent with enhanced microbial fermentation and suppression of opportunistic pathogens (Cui et al., 2024). Collectively, these microbial changes likely enhanced hindgut fermentation and fiber degradation, thereby improving nutrient digestibility and productive performance.

Interestingly, Lactobacillus abundance was highest at 0.0125% OEO but declined at 0.0250%, suggesting a concentration-dependent microbial response to oregano phenolic compounds. The present pattern suggests that moderate OEO supplementation may differentially affect bacterial populations, whereas higher concentrations may also suppress beneficial commensal populations (Yang et al., 2025). Despite the lower Lactobacillus count at 0.0250% OEO, growth performance continued to improve, suggesting that the observed productive responses were not necessarily dependent on changes in the abundance of a single bacterial genus and may have been influenced by broader effects of OEO on intestinal function and nutrient utilization. These findings are consistent with (Ruan et al., 2021), who reported increased Lactobacillus abundance and reduced pathogenic bacteria following OEO supplementation, and with (Kirkpinar et al., 2011), who observed decreased Clostridium counts in oregano oil-supplemented animals. Nevertheless, the decline in Lactobacillus abundance at the higher OEO inclusion level highlights the importance of considering potential effects on beneficial commensal bacteria when evaluating higher OEO concentrations.

The improvements in serum biochemical and immunological parameters further support the modulation of the gut–metabolic–immune axis by OEO supplementation (Visan & Negut, 2024). Enhanced CP digestibility likely increased amino acid availability for hepatic protein synthesis, thereby contributing to the higher serum total protein and albumin concentrations observed in the supplemented groups (Ahmed et al., 2024). Because albumin synthesis depends on adequate amino acid supply and hepatic metabolic activity, the increased albumin concentration also reflects improved liver synthetic function and metabolic efficiency (Farag et al., 2023). Likewise, the increases in globulin, IgG, and IgM indicate enhanced humoral immunity, which may be attributed to reduced pathogenic bacteria and improved intestinal integrity following OEO supplementation (Feng et al., 2021); (Li et al., 2021). Under these conditions, the lower inflammatory burden likely reduced the metabolic demand associated with immune activation, allowing greater nutrient allocation toward immunoglobulin synthesis(Hassan et al., 2023). In addition, the antioxidant properties of thymol and carvacrol protect immune cells from oxidative damage, thereby maintaining lymphocyte function and supporting antibody production (Beghelli et al., 2016). These mechanisms are supported by previous findings showing that OEO supplementation reduced oxidative stress and inflammatory biomarkers and improved the physiological status of rabbits (Hassan et al., 2023); (Sayedahmed et al., 2026). The greater increase in IgM concentration observed in Alexandria-line rabbits, particularly at 0.0250% OEO, together with similar findings reported in rabbits supplemented with OEO or probiotics, indicates that the immunological response to phytogenic supplementation may vary among rabbit genotypes (Abdelsalam et al., 2025); (Sayedahmed et al., 2026).

The improvement in serum lipid profile further confirms the integrated metabolic effects of OEO supplementation. The reductions in total lipids, cholesterol, triglycerides, and LDL, together with increased HDL concentrations, indicate that oregano phytochemicals modulated lipid metabolism by inhibiting hepatic lipogenesis, reducing intestinal cholesterol absorption, and protecting against lipid peroxidation (Abdel-Wareth et al., 2022); (Sayedahmed et al., 2026). Enhanced hindgut fermentation may also have contributed to these responses, as propionate produced by microbial fermentation of fiber can inhibit hepatic HMG-CoA reductase activity, thereby suppressing cholesterol synthesis (Gidenne, 2015). Additionally, carvacrol and thymol may reduce micellar cholesterol solubility and enhance bile acid excretion, collectively decreasing circulating lipid concentrations (Omer et al., 2013); (Abd El-Aziz et al., 2024). Similar hypolipidemic effects have been reported in rabbits receiving oregano-derived phytogenics and other essential oil formulations (Areda et al., 2026).

OEO supplementation improved growth performance, nutrient digestibility, cecal microbial balance, metabolic profile, and humoral immunity in growing rabbits. These responses were associated with reduced pathogenic bacterial load, enhanced digestive efficiency, and improved intestinal physiological status. The magnitude of these effects varied among rabbit lines, with the V-line showing greater digestive and fermentative responsiveness, whereas the Alexandria line exhibited stronger immunological resilience. Collectively, the present findings demonstrate that OEO can serve as an effective nutritional strategy to support intestinal health, metabolic efficiency, and productive performance in rabbit production systems.

The authors declare no conflicts of interest.

During the preparation of this manuscript, the authors used Google Gemini v2.5 and Grammarly v1.2 in some sentences to improve the readability and language quality. After using these tools, the authors carefully reviewed and edited the content as needed and take full responsibility for the publication.