Recent meta?analyses and systematic reviews indicate that well?characterised phytogenic feed additives can improve growth and feed efficiency in broilers by 2-5% on average, with additional benefits for gut morphology and antioxidant status [1,2]. However, responses are highly formulation?specific and often depend on the botanical source, extraction method, and inclusion level. The widespread use of Antibiotic Growth Promoters (AGPs) in livestock has been directly linked to the emergence of multi-drug-resistant pathogens and the presence of harmful chemical residues in the human food chain [3,4]. Consequently, regulatory bodies worldwide have imposed stringent bans on sub-therapeutic antibiotic use, catalyzing a search for sustainable, natural alternatives known as Phytogenic Feed Additives (PFAs) [5].

Phytogenic Feed Additives, or botanicals, are plant-derived bioactive compounds-including herbs, spices, and essential oils-that exert multifaceted biological effects. [6]. While their efficacy is well-recognized, the primary challenge in their commercial adoption remains the inherent phytochemical variability caused by genetic factors, soil composition, and post-harvest handling [7]. To mitigate this inconsistency, contemporary research focuses on "synergistic PFA blends," which combine multiple plant species to create standardized, multi-target formulations often protected by registered trademarks [5,7].

Among these emerging formulations, Enancine B^® and Immunocine^® represent a novel class of Cameroonian phytobiotics developed by the Monks at the Monastery Natural Medicine Center in Mbengwi. According to manufacturer information, both formulations are derived from mixed medicinal plants traditionally used for viral and bacterial infections, including malaria, typhoid, hepatitis, HIV/AIDS and COVID?19, and are reported to contain beta?carotene, azulene, alkaloids, tannins, and 1,8?cineole as key secondary metabolites. In the present study, these phytochemical profiles were considered qualitative background information; batch?specific quantitative analysis was not conducted and remains a priority for future work aimed at standardisation and regulatory approval [6,7]. The biochemical basis for using these compounds in broiler nutrition is compelling. Beta-carotene serves as a potent antioxidant and immune modulator; [8] alkaloids and tannins possess broad-spectrum antimicrobial properties by disrupting bacterial cell wall integrity [9]; and 1,8-cineole has been shown to enhance gut morphology and stimulate the secretion of endogenous digestive enzymes [10]. Despite sharing similar bioactive profiles, Enancine B^® and Immunocine^® are positioned in the market at different price points and recommended for distinct therapeutic applications, with Enancine B^® targeting antibacterial and antimalarial pathways, and Immunocine^® focused on high-level antiviral and immune support.

The discrepancy between their commercial valuation and recommended use raises a critical scientific question: Does the increased cost of a PFA formulation translate to a proportional increase in biological potency and growth performance in a livestock model?

This study therefore aims to evaluate the efficacy of Enancine B^® and Immunocine^® as natural alternatives to conventional prophylactic protocols and synthetic antibiotics (Oxytetracycline 80).

We hypothesised that specific inclusion levels of these phytogenic blends would maintain or modestly improve broiler growth performance, feed conversion efficiency, and selected health indicators relative to a conventional prophylactic programme and an oxytetracycline?based growth promoter. A secondary objective was to assess whether differences in commercial pricing are reflected in differential carcass quality and economic returns. By comparing these two formulations across two different inclusion levels, this research seeks to validate their role as sustainable components of "precision nutrition" in antibiotic-free poultry systems.

Ethical Statement and Study Site

The study protocol was approved by the University of Buea Institutional Animal Care Committee (UB-IACUC; Approval No. 27/2023). Animal welfare and handling procedures were conducted in strict accordance with the National Ethical Committee Guidelines (No. FWA-IRB0000194) and the European Committee Council Directive (86/609/EEC). The trial was conducted at the Faculty of Agriculture and Veterinary Medicine Teaching and Research Farm.

Experimental Design and Husbandry

A total of 216-day-old unsexed Cobb 500 broiler chicks, with uniform initial body weights, were sourced from a commercial hatchery (FECAM Sarl, Bafoussam). Random allocation was performed using a computer?generated randomisation list, and personnel recording performance and carcass traits were blinded to treatment codes to minimise allocation and observer bias [11]. The birds were randomly assigned to six dietary treatments (T0-T5) in a Completely Randomized Design (CRD). Each treatment consisted of three replicates (n=12 birds per replicate).

The birds were housed in standard deep-litter pens. Environmental parameters were monitored using a digital thermo-hygrometer (Model 288-ATH, SL Technologies). Brooding temperatures were maintained at 33⁰C during the first week and gradually reduced to 31⁰C thereafter. Stocking density was adjusted from 20 birds/m² (starter phase) to 10 birds/m² (finisher phase). Feed and water were provided ad libitum.

Dietary Treatments and Additives

The basal diets (Starter and Finisher) were formulated to meet or exceed the nutritional requirements for broilers (Table 1). The phytogenic additives, Enancine B^® and Immunocine^®, were obtained from the Monastery Medical Center (Mbengwi, Cameroon).

Table 1: Basal Diet of Experimental Birds during Starter and Finisher Phases.

^*Premix Composition (Vitamins per kg); Vit A 3,000,000 Ul; Vit D3 600,000 Ul; Vit E 4,000 mg; Vit K3 500 mg; Vit B1 320 mg; Vit B2 1,000 mg; Vit B3 2400 mg; Vit B6 400 mg; Vit B12 7 mg; Vit PP/Ac nicot/niacin 4,800 mg; Biotin 10 mg; Choline chloride 100,000 mg; Folic acid 160 mg; Cupper II sulphate 200 mg; zinc oxide 10,000 mg; manganese oxide 14,000 mg; Calcium iodate 200 mg; Lysine 7800 mg; Meth 200,000 mg; Iron sulphate 8,000 mg, Sulfate 2,000 mg.

The treatments were provided as additives in drinking water and defined as follows:

• T0 (conventional Control): Conventional prophylactic calendar (anti stress agents. vitamins, antibiotics, anti-coccidia, anthelminthic. diuretics etc.; see Table 2). It should be noted that T0 represents a standard multi?component prophylactic programme rather than a true negative control, and thus comparisons with T2-T5 evaluate the ability of EB and IM to replace a complex chemical calendar rather than a medication?free baseline.
• T1 (Positive Control): Oxytetracycline 80% (0.5 g/L in drinking water).
• T2 & T3: Enancine B^® aqueous infusion at 0.5 g/L and 2.0 g/L, respectively.
• T4 & T5: Immunocine^® aqueous infusion at 0.5 g/L and 2.0 g/L, respectively.

Birds in all the treatments received the various vaccinations as indicated in the prophylaxes calendar (Table, 2).

Table 2: Conventional Prophylactic Calendar for Disease Prevention for Broilers.

Data Collection and Performance Metrics

Growth Performance

Feed intake (FI) and water consumption were measured daily. Body weight (BW) was recorded weekly per replicate. The Feed Conversion Ratio (FCR) was calculated as the ratio of average daily feed intake to average daily weight gain.

Hematological and Serum Biochemical Analysis

At the end of the starter and finisher phases hematological and serum biochemical profiles were analyzed. Blood lipid profile was additionally analysed at the end of the finisher phase. Three birds were randomly selected for each replicate and from which 2mL blood samples were collected (using a syringe) from each birds’ wing vein, and kept in sets of tubes.

Hematology: Blood collected in EDTA tubes was analyzed for Red Blood Cell (RBC) count (Neubauer hemocytometer), Hemoglobin (Hb; Tallquist method), Packed Cell Volume (PCV), and White Blood Cell (WBC) count (Giemsa stain).

Serum Biochemistry: Serum was harvested via centrifugation (1500 rpm for 15 min). Alanine transaminase (ALT) and Alkaline phosphatase (ALP) were quantified using commercial diagnostic kits (Chronolab and Sanymed) according to manufacturer protocols.

Carcass and Morphometric Evaluation

On Day 42, three birds per replicate were sacrificed. After bleeding and manual defeathering, carcass weight and dressing percentage were determined. Visceral organs (liver, heart, gizzard, lungs) and cut-up parts (breast, drumstick, thighs, wings) were weighed using a high-precision electronic scale, (WANT WT-GF 0.1 g from WANT Balance Instrument Co Ltd- China). Intestinal length was measured with a calibrated tape, and breast muscle pH was recorded using a digital pH meter (MODENA, Apluste) inserted 2 minutes post-slaughter.

Economic Analysis

An economic evaluation was performed using the prevailing local market prices (FCFA) and converted to US dollars at the rate of USD1 = 559.42 FCFA. The primary indices included:

Cost of Prophylaxis: This cost was calculated in two in two main categories then combined for all treatments. The first category was the Cost of veterinary prophylaxis, which was calculated by adding the total cost per bird of all conventional veterinary products and medications (including antibiotics, anticoccidials, anthelminthics, vitamins, and vaccines) in each treatment. The second category was the Cost of phytogenic additives which was evaluated as total cost per bird of Enancine B^® or Immunocine^® used in T2-T5, calculated from product price and inclusion rate.

Feed Cost: The total cost of feed consumed per bird was calculated based on the price of the starter and finisher diets and the total feed intake recorded for each bird.

Feed Cost per kg Weight Gain: This metric was calculated by multiplying the cost of feed per kilogram by the Feed Conversion Ratio (FCR) for each treatment.

Total Expenses: This was calculated as the sum of the cost of day-old chicks, the total feed cost, Cost of additives consumed per bird.

Total Revenue: The total value of the chickens produced was calculated by multiplying the average final body weight of the birds by the prevailing market price of finished broiler chicken per kilogram of live weight.

Gross margin = (Total Revenue) - (Total cost of production)

Benefit-cost ratio (BCR): This metric was calculated as a profitability index to assess the return on investment. It was defined as the ratio of the total revenue to the total production cost, using the formula:

Statistical Analysis

Data were subjected to a one-way Analysis of Variance (ANOVA) using SPSS (Version 22). Assumptions of normality (Shapiro-Wilk) and homogeneity of variances (Levene's test) were verified. Differences between treatments means were separated using Duncan’s Multiple Range Test. Results are reported as Means, with significance set at P < 0.05. For key performance, carcass, and economic variables, effect sizes (partial η² values) were calculated and reported to aid interpretation of biological relevance.

Growth Performance

The effects of varying inclusion levels of Enancine B^® (EB) and Immunocine^® (IM) on the growth performance of broiler chickens during the starter and finisher phases are summarized in Table 3. In the starter phase (Day 0-21), daily weight gain (DWG) exhibited significant variation (P=0.035), with the positive control (T1) and conventional control (T0) recording the highest gains (26.3 g and 25.33 g, respectively). Conversely, birds receiving 0.5g/L EB (T2) showed the lowest DWG (22.3 g). No significant differences were observed in daily feed intake (DFI), daily water intake (DWI), or feed conversion ratio (FCR) during this period (P > 0.05).

In the finisher phase (Day 21-42), DWG remained significantly influenced by the treatments (P = 0.019), where T1 (69.0 g) significantly outperformed the other groups except for T3 (63.3 g). DFI showed a marginal trend toward significance (P = 0.061), with the highest intake observed in T1 (147.7 g) and the lowest in T5 (127.0 g). FCR and DWI did not differ significantly among the groups (P > 0.05). Overall, although the oxytetracycline group (T1) achieved the highest daily weight gain in both phases, EB and IM treatments-maintained growth performance and FCR within the same statistical range as the conventional prophylactic control (T0), indicating that these phytogenic strategies can sustain productive performance under antibiotic?reduced protocols.

Table 3: Growth Performance of Broiler Chicken on Different Inclusion Levels of Enancine B^® and Immunocine^® During the Stater Phase (Day 0 to Day 21 and 21 To 42 Days).

a,b= means followed by the same letters in a row, were not significantly different (P>0.05), SEM= Standard error of the mean.

Carcass Characteristics and Visceral Organ Proportions

Carcass metrics and internal organ weights are presented in Table 4. Market weight, slaughter weight, and carcass weight were significantly affected by treatment (P < 0.05). The positive control (T1) achieved the highest market weight (2036.7 g) and carcass weight (1825.5 g) compared to the conventional control and phytogenic groups. Notably, the dressing percentage showed significant differences (P = 0.025), with T4 (0.5g/L IM) yielding the highest value (98.7%) compared to T0 (97.6%).

Regarding cut-up parts, percentage breast weight was significantly higher in T3 (30.08%) and T5 (31.21%) compared to the conventional control (24.97%; P = 0.035). Visceral organ proportions remained largely uniform, although the percentage lung weight was significantly higher in T3 (1.17 %) and T5 (1.27 %) relative to the other treatments (P = 0.019). No significant differences were recorded for the percentages of back muscle, drumstick, wing, head and neck, heart, gizzard, or liver (P > 0.05). Breast muscle pH was also statistically similar across all groups (P = 0.102).

Blood Lipid Profile, Hematology, and Serum Biochemistry

As shown in Table 4, the blood lipid profile - including total cholesterol, triglycerides, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) - did not vary significantly across treatments (P > 0.05).Hematological parameters at Day 21 and Day 42 (Table 5) revealed no significant differences in Red Blood Cell (RBC) count, Packed Cell Volume (PCV), Hemoglobin (Hb) concentration, or White Blood Cell (WBC) count (P > 0.05). Similarly, liver enzyme activity at Day 21 (ALP and ALT) remained unaffected by the treatment protocols (P > 0.05). However, at Day 42, while ALP remained stable, ALT levels showed significant variation (P = 0.035). Birds in the T2 (48.50 U/l) and T3 (45.50 U/l) groups exhibited significantly higher ALT activity compared to the conventional control (T0: 26.50 U/l).

Table 4: Effects of Enancine B^® and Immunocine^® Supplementation in Drinking Water on Carcass Characteristics of Broiler Chicken.

a,b= means followed by the same letters in a row, were not significantly different (P>0.05), SEM= Standard error of the mean

Table 5: Hematological Parameters of Broilers Fed Different Levels of Enancine B^® and Immunocine^® in Drinking Water at Days 21and 42.

a,b= means followed by the same letters in a row, were not significantly different (P>0.05), SEM= Standard error of the mean

Table 6: Cost Analysis of Enancine B^®, Immunocine^® of Broiler Chicken (in USD).

a, b= means followed by the same letters in a row, were not significantly different (P>0.05), SEM= Standard error of the mean

Economic Analysis

The economic implications of substituting conventional prophylaxis with EB and IM are detailed in Table 6. The cost of prophylaxis varied significantly (P < 0.001), with the lowest costs observed in T1 (1.19) and T2 (1.22), and the highest in T0 (3.05). While total feed costs and total expenses did not reach statistical significance (P > 0.05), gross margin and the benefit-cost ratio (BCR) were significantly impacted.

The positive control (T1) and T4 (0.5g/L IM) achieved the highest BCR (0.736 and 0.718, respectively), which were significantly higher than the BCR for the conventional control (T0: 0.669) and high-level Immunocine^® (T5: 0.663; P = 0.006). Gross margin followed a similar trend (P = 0.017), with T1 recording the maximum profit (62.37), while T5 recorded the minimum (44.29).Thus, under the prevailing Cameroonian market prices used in this study, 0.5 g/L Immunocine^® (T4) achieved a benefit-cost ratio statistically comparable to that of the oxytetracycline?based protocol (T1), indicating that phytogenic strategies can be economically competitive alternatives to AGPs in commercial settings.

The global shift toward antibiotic-free poultry systems has prioritized the search for standardized phytogenic blends that offer reproducible results. This study evaluated two such blends - Enancine B^® (EB) and Immunocine^® (IM) - which, despite sharing a common biochemical backbone of secondary metabolites, demonstrated divergent effects on broiler physiology and production economics.

As noted in the introduction, both formulations contain beta-carotene, azulene, alkaloids, tannins, and 1,8-cineole. However, our results show that Immunocine^® (T4) yielded a superior dressing percentage (98.7%) compared to Enancine B^®. Dressing percentage was calculated as eviscerated carcass weight (including giblets) divided by live body weight×100; the high values observed (up to 98.7%) reflect this definition and should not be directly compared with studies using cold carcass or retail cut yields (e.g., Aviagen [12]). Furthermore, EB showed a greater propensity to elevate serum ALT levels at higher doses. Although ALT activity was significantly elevated in EB?treated birds, the observed values remained within or only slightly above the upper range reported for clinically healthy broilers in intensive systems [13], suggesting a dosage?related increase in hepatic metabolic load rather than overt liver damage. Nevertheless, these patterns warrant caution when considering higher EB inclusion rates. In phytomedicine, biological activity is often dictated not by the presence of compounds, but by their quantitative ratios and synergistic concentrations.

The proprietary formulations used by the Monks likely utilize different concentrations of these bioactive markers. One plausible explanation is that Immunocine^® contains higher concentrations or more favourable ratios of key constituents such as 1,8?cineole, which has been associated with improved nutrient absorption and gut morphology in poultry [14]. Similarly, its higher market price may reflect differences in extraction or standardisation protocols. However, because batch?specific phytochemical quantification was not performed in this study, these interpretations remain hypothetical and should be tested in future work using marker?based quality control [1].

While the positive control (Oxytetracycline) maintained a lead in absolute market weight (2036.7 g), the parity in growth performance between the phytogenic groups (T2-T5) and the conventional prophylactic control (T0) validates EB and IM as effective Natural Growth Promoters (NGPs). The ability of these plant-based blends to match a complex chemical prophylactic calendar (Table 1) suggests that their bioactive components - alkaloids and tannins - successfully managed the microbial load without the need for synthetic intervention.

A critical observation was the significant elevation of Alanine transaminase (ALT) in EB-treated birds (T2 and T3) during the finisher phase. While all hematological parameters remained within healthy ranges, the rise in ALT suggests a dosage-specific metabolic demand on the liver.

These findings are consistent with the hypothesis that, despite similar ingredient classes, Enancine B^® may contain higher or more bioactive alkaloid fractions than Immunocine^®, resulting in different hepatic clearance demands at higher inclusion levels. Direct pharmacokinetic and dose-response studies would be necessary to substantiate this mechanism.

The study addressed the question: Does increased cost translate to proportional biological potency? The Benefit-Cost Ratio (BCR) results suggest a "ceiling effect." The higher market price of Immunocine^® appears to be partly justified by its superior carcass dressing percentage and competitive benefit-cost ratio at 0.5 g/L, although performance advantages over Enancine B^® were not universal across all traits. The lower-cost Enancine B^® at 0.5 g/L (T2) offered a highly competitive BCR (0.697) for farmers focused on standard production. However, for "precision nutrition" aiming for maximum dressing percentage and high-health status, the premium cost of T4 (0.5 g/L IM) is justified by its biological performance, yielding a BCR (0.718) that closely rivals the antibiotic control (0.736).

This research provides evidence that Enancine B^® and Immunocine^® can serve as effective components of antibiotic?reduced broiler production systems, maintaining growth performance and economic returns comparable to a conventional prophylactic programme under the conditions tested.

Formulation Divergence: Despite sharing a qualitative phytochemical profile, the two products act as distinct biological tools. Immunocine^® at 0.5 g/L is a superior "high-performance" additive, optimizing carcass dressing percentage.

Dosage and Safety: Enancine B^® is an effective low-cost alternative, but its use at high concentrations must be monitored due to increased metabolic load on the liver.

Economic Validation: The higher market price of Immunocine^® does indeed translate into a tangible biological advantage (carcass yield and immune support), though the 0.5 g/L inclusion level remains the most economically viable threshold for both products.

For Farmers and Producers

Precision Selection: Producers seeking the highest carcass yield and dressing percentage should prioritize Immunocine^® at 0.5 g/L. For standard growth performance with a focus on initial cost-saving, Enancine B^® at 0.5 g/L is a suitable substitute for synthetic antibiotics.

Avoid Over-supplementation: Increasing the dosage to 2.0 g/L for either product does not yield significant performance gains and negatively impacts the Benefit - Cost Ratio.

For Policy Makers and the Monastery Natural Medicine Center

Phytochemical Standardization: To transition these products into world-class commercial feed additives, the Monastery should implement standardized "marker-based" quality control to ensure consistent concentrations of 1,8-cineole and alkaloids across batches.

Labeling and Veterinary Extension: Policy makers should support the labeling of "Phytogenic-Raised" poultry. Extension services should emphasize that while these products share ingredients, they are not interchangeable in their metabolic impact, as shown by the ALT variations.

We are grateful to the Ministry of Higher Education, Cameroon (MINESUP), and the University of Buea, who provide a research and modernization allowance to the corresponding author. We also acknowledge the use of facilities provided by the Faculty of Agriculture and Veterinary Medicine, as well as the Professor Fujii Agroecology Laboratory.

Funding Sources

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Ethical Considerations

The study protocol was approved by the University of Buea Institutional Animal Care Committee (UB-IACUC; Approval No. 27/2023). Animal welfare and handling procedures were conducted in strict accordance with the National Ethical Committee Guidelines (No. FWA-IRB0000194) and the European Committee Council Directive (86/609/EEC).

Competing Interests

The authors declare no competing interests in the research and the publication.

Corresponding Author

Divine EWANE, Faculty of Agriculture and Veterinary Medicine, University of Buea, Cameroon

Data Availability

Data are available from the corresponding author, upon reasonable request.

Author Contribution

ED: Conceptualization; formal analysis; investigation; methodology; project administration; resources, supervision, validation, writing original draft, review and editing.

NLM: Conceptualization; formal analysis, investigation; project administration; resources supervision, validation writing original draft

NMN: Conceptualization, Data curation, formal analysis, investigation, methodology, project administration, resources, validation, writing original draft

AEA: Conceptualization Data curation, formal analysis, investigation, methodology, project administration, resources, validation, writing original draft

KBRF: Data curation, formal analysis; resources, supervision, validation, writing original draft, review and editing.

MOP: Validation, writing original draft, review and editing

Consent to Publish

All Co-authors have given their full consent to publish.

1. Abdelli N, Sola-Oriol D, Perez JF, Manzanilla EG. Phytogenic feed additives in poultry: Achievements, prospective and challenges. Animals. 2021; 11: 347.
2. Wang L, Li Z, Zhang Y, Wang Y, Zhang X. Effects of essential oils and plant extracts on growth performance and health of broiler chickens: A meta-analysis. J Anim Sci Biotechnol. 2022; 13: 82.
3. Wisniewski P, Trymers M, Chajecka-Wierzchowska W, Tkacz K, Zadernowska A, Modzelewska-Kapitula M. Antimicrobial resistance in the context of animal production and meat products in Poland-a critical review and future perspective. Pathogens. 2024; 13: 1123.
4. Enshaie E, Nigam S, Patel S, Rai V. Livestock Antibiotics Use and Antimicrobial Resistance. Antibiotics. 2025; 14: 621.
5. Wang J, Deng L, Chen M, Che Y, Li L, Zhu L, et al. Phytogenic feed additives as natural antibiotic alternatives in animal health and production: A review of the literature of the last decade. Anim Nutr. 2024; 17: 244-264.
6. Abdelli N, Sola-Oriol D, Perez JF. Phytogenic Feed Additives in Poultry: Achievements, Prospective and Challenges. Animals. 2021; 11: 3471.
7. Nantapo CW, Marume U. Strategic technologies to improve phytogenic feed additive efficacy in pigs and poultry. Anim Nutr. 2025; 23: 286-303.
8. Bufka J, Vankova L, Sykora J, Krizkova V. Exploring carotenoids: Metabolism, antioxidants, and impacts on human health. J Funct Foods. 2024; 118: 106284.
9. Farha AK, Yang QQ, Kim G, Li HB, Zhu F, Liu HY, et al. Tannins as an alternative to antibiotics. Food Biosci. 2020; 38: 100751.
10. Di Y, Cao A, Zhang Y, Li J, Sun Y, Geng S, et al. Effects of Dietary 1,8-Cineole Supplementation on Growth Performance, Antioxidant Capacity, Immunity, and Intestine Health of Broilers. Animals. 2022; 12: 2415.
11. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research. PLoS Biol. 2010; 8: e1000412.
12. Broiler management handbook. Huntsville, AL: Aviagen Group; 2019.
13. Hussein EOS, Abudabos AM, Ali MHM, Ahmed ST, Suliman GM. Effects of phytogenic feed additives on growth, carcass traits and blood serum metabolites in broilers: A meta-analysis. Rev Bras Zootec. 2019; 48: e20180298.
14. Di C, Wang X, Ye Y, Huang Y, Zhou G. Dietary essential oils and broiler performance: Effects on intestinal morphology, microbiota, and immunity. Poult Sci. 2022; 101: 101661.