Evaluation of Vernonia hymenolepis wash water as phytogenic prophylaxis for broiler chicken production

Abstract

Risk management in commercial broiler production requires prophylaxis to mitigate unforeseen metabolic and health risks. This study evaluated the efficacy of Vernonia hymenolepis Wash Water (VhWW), an agro-processing byproduct, as a natural, non-conventional prophylactic agent. A 42-day feeding trial with 264 Cobb 500 broiler chicks compared ad-libitum VhWW (from four varieties processed with or without salt) against conventional and control groups. Nutrient analysis confirmed green varieties were superior in protein and minerals, while salt processing significantly enhanced the antioxidant activity of purple varieties (P < 0.05). Crucially, the Negative Control (T11) exhibited severe metabolic instability, showing hypertriglyceridemia (Total Triglycerides 193.19 mg/dL), exceeding the normal range) and unacceptable performance uncertainty (worst starter FCR, best finisher FCR). This risk profile was present despite (T11) recording the best Low-Density Lipoprotein (LDL) levels. Conversely, most VhWW treatments successfully maintained Total Triglycerides and Total Cholesterol within the healthy physiological range, demonstrating a critical hepatoprotective and metabolic stabilizing effect. The optimal VhWW protocols (Green Sweet + Salt) achieved superior growth and FCR statistically comparable to controls, but without the high metabolic risk. Economically, VhWW treatments were statistically similar to the lowest-cost T11, offering a superior risk-adjusted return. In conclusion, VhWW is an efficacious, sustainable, and economically competitive prophylactic agent. The study recommends processing the Green Sweet variety with salt (T8) to maximize growth, economic, and metabolic benefits.

1. Introduction

In the context of modern poultry farming, a robust and effective prophylaxis protocol is the most critical and economically viable strategy for sustaining flock health and optimizing productivity (Bilal et al., 2025; Ziebe et al., 2025). The high stocking densities inherent to intensive broiler production render flocks highly susceptible to infectious diseases, resulting in significant morbidity, mortality, and economic losses. Consequently, a proactive, multi-faceted disease prevention approach-encompassing strict biosecurity measures, vaccination programs, and proper nutrition-is essential for success (Abdou et al., 2020; Acheampong, 2024; Bilal et al., 2025).

Historically, antimicrobials have been routinely incorporated into broiler diets as Antibiotic Growth Promoters (AGPs) and as a cornerstone of prophylactic management (Abou-Jaoudeh et al., 2024; Enshaie et al., 2025). However, the extensive and often indiscriminate use of AGPs has globally accelerated the emergence of Antimicrobial-Resistant (AMR) bacterial strains (Enshaie et al., 2025). This global public health threat not only jeopardizes therapeutic options for humans and animals but also intensifies economic losses for farmers and shifts consumer preference toward antibiotic-free products (Igene et al., 2018).

In direct response to the AMR crisis, there is a global push to investigate non-conventional feed resources and herbal plants as sustainable, natural alternatives (Amata, 2014; Oyebade et al., 2023). This momentum has fueled significant interest in phytogenic additives-plant-derived compounds known to enhance animal performance and health (Ivanova et al., 2024; Wang et al., 2024)-and, specifically, their application in phytogenic prophylaxis: the preventative management of disease and sub-optimal performance using these natural compounds (Ewane et al., 2025a).

Among these promising natural resources are plants belonging to the genus Vernonia, which includes four commonly consumed species in Cameroon. This study centers on Vernonia hymenolepis, a species deemed more economically important than the widely studied V. amygdalina (Ucheck Fomum, 2004a). Vernonia. hymenolepis is notably less bitter than its related edible Vernonia species (V. amygdalina Delile, V. colorata, and V. thomsoniana), which theoretically offers greater potential for acceptability and profitability (Ucheck Fomum, 2004a). Despite its economic advantage, V. hymenolepis remains underexplored in poultry production, with most research focusing on V. amygdalina.

Studies on V. amygdalina have established its therapeutic potential due to its rich composition of bioactive compounds, including sesquiterpene lactones, phenolic compounds, flavonoids, and saponins. These phytochemicals confer diverse properties such as antibacterial, antiviral, anti-fungal, anti-inflammatory, and anti-lipidaemic effects (Degu et al., 2024; Tura et al., 2024). Specifically, the high concentration of phenolic compounds and flavonoids provides substantial antioxidant capacity, which is crucial for mitigating oxidative stress common in fast-growing broiler strains (Malila, 2023; Oke et al., 2024). This protection reduces cellular damage and promotes overall health. Furthermore, sesquiterpene lactones and saponins have been linked to enhanced gut health by exhibiting antimicrobial activity against pathogenic bacteria and positively modulating the gut microbiota. Analogous to findings in other species (Serrano, 2013), saponins found in Vernonia species are known to interact with dietary cholesterol and bile acids, suggesting a mechanism that could influence lipid metabolism to achieve a more favorable blood lipid profile (Cao et al., 2024; Owen et al., 2011). By reducing pathogen load and oxidative burden, these compounds collectively enhance the efficiency of nutrient absorption and utilization, thereby supporting metabolic efficiency and protein deposition (Tokofai et al., 2023). Given that V. amygdalina and V. hymenolepis share comparable nutritional and chemical profiles (Ucheck Fomum, 2004a), the latter holds high potential as a potent, natural prophylactic agent.

Vernonia. hymenolepis grows wild in mountainous and high plateau regions across West, Central, East, and Southern Africa, with cultivation primarily confined to Nigeria and Cameroon (Ucheck Fomum, 2004a). In Cameroon, it is locally known as “Bayangi bitterleaf.” The species exhibits four distinct selections based on stem color (green or purple) and taste (sweet or bitter) (Mih et al., 2007). All the four varieties are produced from seeds of the same parent due to cross pollination via insects and wind (Ucheck Fomum, 2004a). However selection pressure is applied constantly by farmers who prefer the less bitter selections. It is only the sweet varieties (Purple Sweet and Green sweet) that are utilized directly for human consumption. The bitter varieties (Purple bitter and Green bitter) are usually destroyed by farmers or used to adulterate other harvested species of Vernonia such as Vernonia amygdalina (Ucheck Fomum, 2004a, 2004b). In traditional practice, while all varieties possess medicinal properties (Ucheck Fomum, 2004a), they require washing before human consumption, and the resulting wash water (VhWW) is typically discarded (Gockowski et al., 2003).

This study intentionally focuses on VhWW-a zero-cost, readily available kitchen by-product-to valorize a currently discarded waste stream. This approach offers a highly accessible, low-cost, and sustainable prophylactic agent, circumventing the resource-intensive extraction processes and higher input costs associated with conventional leaf extracts. Moreover, unlike solvent-based extraction methods (Gabriel et al., 2015; Sulaiman et al., 2024), utilizing VhWW avoids rendering the leaf material unpalatable for human consumption in traditional dishes, thereby mitigating competition between humans and livestock for valuable food resources (Ewane et al., 2025b). Although VhWW is expected to have a lower bioactive content than purified extracts, its high volume availability and suitability for bulk usage as a water additive make it a compelling practical alternative.

To our knowledge, this is the first study to comprehensively evaluate Vernonia hymenolepis wash water (VhWW) as a phytogenic prophylactic agent in commercial broiler production. Specifically, this research assesses the effects of the four plant selections and two processing methods (with or without salt) on various performance and health metrics. The inclusion of salt in the processing of some VhWW treatments was based on traditional local preparation methods, requiring its potential influence on phytochemical properties and efficacy to be systematically evaluated (Jones & Etchells, 1944; Sinha & Khare, 2014).

We hypothesize that VhWW supplementation, when used in conjunction with a conventional vaccination protocol, will effectively serve as a natural prophylactic agent, resulting in comparable or superior growth performance, a favorable blood lipid profile, and improved economic returns compared to conventional antimicrobial control groups.

Therefore, this study was conducted to determine the effects of VhWW from four distinct selections on:

• 1.Growth Performance: Assessing final body weight, body weight gain, and Feed Conversion Ratio (FCR).
• 2.Health Biomarkers: Evaluating changes in key blood lipid profiles (total cholesterol, triglycerides, High-Density Lipoprotein (HDL), and Low-Density Lipoprotein (LDL)) as proxies for physiological health and stress.
• 3.Economic Efficiency: Analyzing benefit-cost ratios (BCR) and feed costs to establish the financial viability of VhWW compared to Conventional, Positive (antimicrobial), and Negative Control protocols.

The successful validation of VhWW will provide poultry farmers and policymakers with a sustainable, low-cost, and natural alternative to reduce reliance on conventional antimicrobial prophylactics.

2. Materials and methods

2.1. Study design summary

The study design for this research is summarized in Fig. 1 as a flowchart.

Fig. 1.

Study design flowchart.

2.2. Ethical issues related to the study animal

Unsexed Cobb 500 broiler chickens were utilized as experimental animals. All procedures strictly adhered to the National Ethical Committee Guidelines (No. FWA-IRB00001954) and International standards, including the European Committee Council Directive of November 24, 1986 (86/69/EEC) and the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). Formal ethical approval was obtained from the University of Buea Institutional Animal Care and Use Committee (UB-IACUC) under Permit No. UB-IACUC No.48/2023. Every effort was made to minimize animal suffering and stress throughout the trial.

2.3. Study area and housing

The research was conducted at the Faculty of Agriculture and Veterinary Medicine Teaching and Research Farm (FAVM TRF), University of Buea, Cameroon (latitude 4.1560°N, longitude 9.2632°E). The climate is characterized by high annual precipitation (≈2500 mm) and an average temperature of 23°C.

The Vernonia plants were cultivated on a dedicated field at FAVM TRF. The broiler chickens were housed in a naturally ventilated, open-sided poultry house. Thirty-three identical pens were used, each measuring 1 m × 1.5 m (1.5 m²), resulting in a stocking density of 5.33 birds/m² (0.1875 m²/bird). Birds received continuous lighting for the first week, reduced to 18 h thereafter. The chicks were brooded at slightly decreasing temperatures (at 34°C, 32°C, and 30°C in the first, second, and third weeks, respectively) with the temperature monitored using a thermo-hydrometer (model 288-ATH, SL Technologies).

2.4. Vernonia hymenolepis sample collection and wash water preparation

2.4.1. Plant collection and cultivation

Vernonia hymenolepis seeds were collected from local farmers in Bova and Likombe. The plant was botanically authenticated at the Limbe Botanical Garden Herbarium, Limbe, Cameroon, and a voucher specimen (Accession No. SCA2539) was deposited. Seedlings were selected based on leaf taste and stem color: purple-bitter (PB), purple-sweet (PS), green-bitter (GB), and green-sweet (GS).They were transplanted into 100 randomized field plots (3 m × 2 m) with 20 cm × 20 cm spacing (25 plots per variety).Stems with leaves were harvested rotationally at 15 cm above the ground (Afui Mih & Ndam, 2007; Mih et al., 2007).

2.4.2. Wash water (VhWW) preparation and nutrient assessment

Leaves used in preparation of the VhWW for the feeding trial were harvested daily between 07:00 and 09:00. The VhWW was prepared using modified methods for processing Vernonia amygdalina (Yakubu et al., 2012; Abioye et al., 2014) as follows:

• 1.Abrasion: 1000 g of fresh leaves were manually abraded (squeezed and rubbed) with or without salt (5 g) but no water until foaming was observed.
• 2.Rinsing: Tap water was added stepwise in reduced quantities to rinse out the foam, as the leaves were continuously squeezed and rinsed (initially starting with 3000 ml, then 2000 ml, then 1000 ml, then 500 ml).
• 3.Sweet Varieties: For the sweet varieties (GS, PS), the process was stopped immediately after the foam was completely rinsed out.
• 4.Bitter Varieties: For the bitter varieties (GB, PB), the squeezing and rinsing process continued until the bitterness in the washed leaves (ndole) was barely perceptible to the human palate.
• 5.Volume Measurement: The total volume of VhWW collected from the rinsing and squeezing process was measured and used immediately as the sole source of drinking water for the birds.

The leaves and resulting VhWW were analyzed for Antioxidant Activity (Rate of Inhibition with 2–2-DIPHENYL-1-PICRYHYDRAZYL (DPPH) (Baliyan et al., 2022), Vitamin C (Stan et al., 2014), Crude Protein (AOAC, 2019), and Minerals (Fe, Zn, Cu, Mn) via atomic absorption spectrometry (Magalhaes et al., 2009).

2.5. The feeding trial

2.5.1. Experimental design and treatments

A total of 264 day-old unsexed Cobb 500 chicks were randomly assigned to 11 treatment groups (Table 1) in a Completely Randomized Design (CRD), with 3 replicates of 8 birds each. The 11 treatments comprised 8 VhWW groups (T1-T8) and 3 control groups (T9-T11). The VhWW or control drinking water was provided ad libitum as the birds' sole source of drinking water.

Table 1.

Experimental treatments and administration.

Treatment Code	V. hymenolepis selection and processing method	Prophylactic Protocol	Description of Drinking Water
T1: (PBN)	Purple-Bitter (PB) & No Salt (N)	Standard Vaccinations	VhWW (PB, No Salt) ad libitum
T2:(PBS)	Purple-Bitter (PB) & Salt (S)	Standard Vaccinations	VhWW (PB, Salt) ad libitum
T3:(PSN)	Purple-Sweet (PS) & No Salt (N)	Standard Vaccinations	VhWW (PS, No Salt) ad libitum
T4: (PSS)	Purple-Sweet (PS) & Salt (S)	Standard Vaccinations	VhWW (PS, Salt) ad libitum
T5: (GBN)	Green-Bitter (GB) & No Salt (N)	Standard Vaccinations	VhWW (GB, No Salt) ad libitum
T6: (GBS)	Green-Bitter (GB) & Salt (S)	Standard Vaccinations	VhWW (GB, Salt) ad libitum
T7: (GSN)	Green-Sweet (GS) & No Salt (N)	Standard Vaccinations	VhWW (GS, No Salt) ad libitum
T8: (GSS)	Green-Sweet (GS) & Salt (S)	Standard Vaccinations	VhWW (GS, Salt) ad libitum
T9: C1	Conventional Control	Conventional prophylactic protocol (Full Calendar- Table 2)	antibiotics, vitamins, anti-coccidian, anthelminthic, liver protector in Tap Water ad libitum
T10: C2	Positive Control (antibiotic-prophylaxis)	Conventional prophylactic protocol (Full Calendar- Table 2) Reinforced with Oxytetracycline 80 (g5g/10 L water)	antibiotics, vitamins, anti-coccidian, anthelminthic, liver protector Plus Oxytetracycline 80 (g5g/10 L water) in Tap Water ad libitum
T11: C3	Negative Control	Standard Vaccinations	Tap Water ad libitum

2.5.2. Diet and health protocol

All birds received iso-calorific and iso-nitrogenous basal diets: Starter (Days 1–21) and Finisher (Days 22–42) (Table 3). Feed was provided ad libitum. Daily water consumption was measured per replicate by recording the difference between the water/VhWW provided and the amount remaining after 24 h.

Table 3.

Basal diet composition and calculated analysis.

Ingredients	Starter(%,w/w) (Days 0–21)	Finisher(%,w/w) (Days 21 −42)
Maize	54.8	60.76
Soya bean meal	38.05	32.18
Vitamin/mineral premix*	5.15	5.06
Dicalcium phosphate	2	2
Total	100	100
Calculated nutrient analysis
Dry matter	80.65	80.6
Organic matter	87.93	88.16
Ash (% dry matter)	3.13	2.81
Lipid (% dry matter)	2.61	2.73
Crude protein (% dry matter)	22.19	20.23
Metabliosable Energy (Kcal/ kg DM)	2973.84	3057.35

^⁎Premix Composition (Vitamins per kg); Vit A 3000,000 Ul; Vit D3 600,000 Ul; Vit E 4000 mg; Vit K3 500 mg; Vit B1 320 mg; Vit B2 1000 mg; Vit B3 2400 mg; Vit B6 400 mg; Vit B12 7 mg; Vit PP/Ac nicot/niacin 4800 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 8000 mg, Sulfate 2000 mg.

Throughout the 42-day trial, the health status of all birds was continuously monitored via daily clinical observation. This included observing changes in appetite, water consumption, droppings consistency, posture, and general demeanor. Any mortality was recorded immediately.

All birds received a standard vaccination schedule against New castle disease (NCD), Infectious bronchitis (IB), and Infectious Bursa Disease (IBD). The VhWW treatment groups (T1–T8) received NO conventional antibiotics, anti-coccidials, or other medications (listed in Table 2) during the entire feeding trial, ensuring that the observed effects were solely due to VhWW and standard vaccinations. Conversely, T9 (conventional control) followed the full conventional prophylactic protocol (Table 2), and T10 (positive control) received the full protocol reinforced with Oxytetracycline 80 at the rate of 5 g/1000 ml of water per day. Personnel followed standard biosecurity protocols including the use of clean boots, gloves, and designated clothing during all animal care procedures (Abd El-Hack et al., 2022).

Table 2.

Conventional prophylactic protocol (used for T9 and for vaccination schedule across all other treatments).

Day/Age	Type of Medication	Mode of Administration	Dosage	Function
1	Avinew(A),Bioral(B) and Galivac(G)	Beak dipping or Intra ocular	1000D in 10L	Prevention of NCD, IB and IBD (Gumboro)
1–5	Anti-stress and vitamin	Drinking water	5 g in 5L	Against stress
6–8	Antibiotic(oxy)	Drinking water	5 g in 2.5L	Disease prevention
8	Vaccine; A, B, G	Drinking water	1000D in 10L	Booster against viral infection
8–10	Vitamin (Amin total)	Drinking water	5 g in 10L	Growth promoter
11–13	Anti-coccidiosis	Drinking water	5 g in 10L	Prevention of coccidiosis
14–16	Vitamin (Amin total)	Drinking water	5 g in 10L	Growth promoter
17–19	Antibiotic(oxy)	Drinking water	5 g in 10L	Anti-infectious
20–22	Vitamin	Drinking water	5 g in 10L	Growth promoter
21	Vaccine; A, B, G	Drinking water	1000D in 10L	Booster against viral infection
23–25	Anti-coccidiosis	Drinking water	5 g in 10L	Prevention of coccidiosis
26–29	Vitamin	Drinking water	5 g in 10L	Growth promoter
30	Dewormer (anthelmintic	Drinking water	5 g in 2.5L	Against worms
35–37	Liver protector	Drinking water	1 ml in 1L	Diuretic
38–42	Vitamin	Drinking water	5 g in 10L	Growth promoter

Vaccine: A: Avinew, B: Bioral, G: Galivac, NCD; New castle disease, IB; Infectious bronchitis.

2.6. Blood lipid profiles and carcass analysis

2.6.1. Blood sample collection and analysis

At the end of the 42-day trial, blood samples were collected from 4 randomly selected, non-fasted birds per replicate (N = 132 total) between 08:00 and 11:00 on Day 42 to minimize diurnal variation. Approximately 2 mL of blood was aseptically drawn from the brachial (wing) vein using sterile syringes (Owen et al., 2011) and immediately transferred to sterile, plain (non-anticoagulant) tubes. Samples were allowed to clot at room temperature for 30 min. Serum was separated by centrifugation at 2000 × g for 15 min. The resulting serum was then aliquoted and stored at 20 °C until biochemical analysis.

Lipid concentrations, including Total Cholesterol (TC), Total Triglycerides (TG), High-Density Lipoprotein (HDL), and Low-Density Lipoprotein (LDL), were quantitatively determined using a Unico-2400 Spectrophotometer (Unico, Japan). All determinations utilized commercial enzymatic, colorimetric assay kits (Fortress Diagnostics Kits, Antrim, UK) according to the manufacturer’s instructions.

2.6.2. Carcass characteristics

The same 4 birds per replicate were used for carcass analysis after 12-hour overnight fasting. Final live body weight was recorded before humane slaughter, bleeding, and evisceration. Dressing percentage was calculated as:

$$\text{Dressing}\text{Percentage}(\%)=\frac{CarcassWeight}{LiveWeight}\times 100$$

2.7. Economic analysis

An economic analysis was conducted to assess the financial viability using local prevailing market prices (Cost of Chick: USD1.08; Market Price Chicken: USD3.60/ Kg Live weight; V. hymenolepis bitter varieties (PB, GB) USD0.09/bundle and USD0.36/bundle for sweet varieties (PS and GS), Salt USD 0.09/ Kg, Vaccines USD0.009/bird, Starter feed =0.71USD/ Kg, Finisher feed = 0.69USD/ Kg).

The following parameters were measured or calculated:

• 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.

• Cost of VhWW and Medications: This parameter represents the total cost per bird for all veterinary products, medications (including antibiotics and vaccines), and the estimated cost associated with the Vernonia hymenolepis wash water (VhWW).

• Total Production Cost: This was calculated as the sum of the cost of day-old chicks, the total feed cost, VhWW, and the cost of medications per 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 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 chicken meat per kilogram.

• 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:

  $$\text{BCR}=\frac{TotalRevenue}{TotalProductionCost}$$

These parameters were used to compare the economic performance of the Vernonia hymenolepis wash water treatments with the conventional and antimicrobial control groups.

2.8. Statistical analysis

Data were analyzed using IBM SPSS Statistics, version 22 (IBM Corp., Armonk, NY, USA). Prior to conducting the analysis, Shapiro-Wilk's test confirmed the assumption of data normality, and Levene's test verified the homogeneity of variances (for both assumptions, P > 0.05). Differences among the treatment group means were determined using a One-Way Analysis of Variance (ANOVA), with significance established at P < 0.05. Where the ANOVA indicated significant overall differences, Duncan's Multiple Range Test (DMRT) was applied as a post-hoc comparison to identify specific pairwise differences. Results are reported as Mean ± SEM. To enhance the rigor and practical significance of the findings, all P-values are reported, and Effect Sizes (Partial η2) and 95% Confidence Intervals (95% CI) are included for key growth performance and lipid profile parameters

3. Results

The results are presented in four main sections, beginning with the characterization of the Vernonia hymenolepis varieties and the wash water (VhWW) and concluding with the effects on broiler performance, health, and economic returns.

3.1. Vernonia hymenolepis variety characteristics and VhWW production

3.1.1. Yield of Vernonia hymenolepis plant material

The four V. hymenolepis varieties showed significant differences in biomass yield (Table 4). The purple selections (Purple Bitter (PB) and Purple Sweet (PS)) yielded significantly heavier plant bundles (P < 0.05) than the green selections (Green Bitter (GB) and Green Sweet (GS). The PB selection, with an average weight of 1.98±0.29 kg, was the highest yielding variety. The most crucial component for the study, the leaf weight, was highest in the PB variety (0.74±0.14 kg), which was significantly greater (P < 0.05) than all other varieties. No significant differences (P > 0.05) were found among PS, GB, and GS varieties.

Table 4.

Average weight of Vernonia hymenolepis bundle, stems and leaves.

Variety	Av. Bundle Weight (kg)	Stem weight (kg)	Leaves weight(kg)
Purple Bitter	1.98±0.29c	1.24±0.29b	0.74±0.14b
Purple Sweet	1.61±0.03b	1.28±0.07b	0.47±0.16a
Green Bitter	1.13±0.13a	0.65±0.13a	0.48±0.22a
Green Sweet	1.00±0.30a	0.68±0.30a	0.34±0.15a
SEM	0.1618	0.1113	0.0603

^abc Means in the same column with different superscripts were significantly different (p˂0.05).

Values are means (n) ± standard deviation SEM = Standard error of mean.

3.1.2. VhWW volume production

The volume of VhWW produced from 1000 g of leaves largely mirrored the amount of water used for processing, though without a consistent pattern based on the selection (Table 5). The purple bitter varieties (T1 and T2) produced the largest volumes of VhWW (6.74 L and 6.37 L, respectively), and the green sweet varieties (T7 and T8) produced the lowest (4.81 L and 4.43 L respectively). The T1 produced a significantly greater volume (P < 0.05) than T8. No significant differences (P > 0.05) were observed in VhWW volume among the other treatments. The weight of the de-bittered leaves (Ndole) and the processing time did not vary significantly among the selections (P > 0.05).

Table 5.

Vernonia hymenolepis wash water (VhWW) and de-bittered product (Ndole) from processing 1000 g of Vernonia hymenolepis leaves.

Treatment	Input		Output
	Water used (litres)	Time used (minutes)	VhWW produced (litres)	De-bittered product (ndole)(g)
T1:Purple bitter without salt	7.08±0.5738b	25.00±5.000	6.74±0.5654c	849.25±28.8772
T2: Purple bitter with salt	6.51±0.4943ab	23.33±2.887 ```	6.37±0.5981bc	878.96±21.7943
T3: Purple sweet without salt	6.65±2.0165ab	25.00±5.000	5.55±1.9254abc	870.82±22.5425
T4: Purple sweet with salt	5.81±1.9776ab	23.33±2.887	5.52±1.7378abc	877.42±21.6777
T5: Green bitter without salt	6.27±0.1528ab	26.67±2.887	5.53±0.0577abc	851.75±33.6919
T6: Green bitter with salt	6.31±0.4531ab	21.27±2.887	5.82±0.4423abc	870.61±3.4286
T7: Green sweet without salt	5.18±0.6369ab	26.67±2.887	4.81±0.3755ab	850.11±35.1157
T8: Green sweet with salt	4.65±0.3074a	21.27±2.887	4.43±0.1539a	810.19±60.5164
SEM	0.2414	0.716	0.2239	10.4928

^abc Means in the same column with different superscripts were significantly different (p˂0.05).

Values are means (n) ± standard deviation SEM = Standard error of mean.

````

3.2. Nutrient and antioxidant content of V. hymenolepis

3.2.1. Nutrient content of V. hymenolepis leaves

The chemical analysis of the intact leaves showed significant variation in nutrients across the four selections (Table 6).

Table 6.

Nutrient content of four selections of Vernonia hymenolepis leaves.

Selection	Vit. C (g/100g)	Protein (g/100g)	Fe (mg/100g)	Zn (mg/100g)	Cu (mg/100g)	Mn (mg/100g)
Purple Bitter	0.051±0.07a	29.52±0.47b	25.08±0.007c	9.284±0.50c	0.010±0.01a	0.155±0.005c
Purple Sweet	0.066±0.06b	26.57±0.47a	12.56±0.005b	6.571±0.49b	0.010±0.01a	0.001±0.001a
Green Bitter	0.073±0.03b	29.96±0.47c	52.09±2.004d	12.15±0.15d	0.251±0.03b	0.181±0.003d
Green Sweet	0.100±0.03c	33.08±0.47d	0.016±0.007a	0.029±0.01a	0.192±0.08b	0.014±0.006b
SEM	0.0054	0.6957	5.8177	1.35292	0.034	0.02442

Values are means (n) ± standard deviation SEM = Standard error of mean.

<sup>abc</sup> Means in the same column with different supercripts were significantly different (p˂0.05).

The Green Sweet (GS) variety was the most protein and Vitamin C endowed, with 33.08±0.47 g/100 g protein and 0.100±0.03 g/100 g Vitamin C, significantly higher (P < 0.05) than all other varieties. The Green Bitter (GB) variety was significantly richer (P < 0.05) in most minerals (Fe, Zn, Mn) compared to all other varieties, particularly Fe (52.09±2.004 mg/100 g) and Zn (12.15±0.15 mg/100 g). Generally, the Green varieties were better endowed with Protein and Vitamin C, while the Purple varieties were richer in Fe, Zn, and Mn.

3.2.2. Nutrient and antioxidant content of VhWW

The nutrients extracted into the VhWW were generally low compared to the leaves, but the extraction efficiency varied by mineral and processing method (Table 7, Table 8). Protein and Vitamin C were highly diluted and did not differ significantly (P > 0.05) across the VhWW treatments. The Green Sweet wash waters (T7, T8) had the significantly highest Zn content, consistent with the leaf analysis. Non-salt treated VhWW consistently had a significantly higher Cu content (P < 0.05) than their salt-treated counterparts, indicating salt may hinder Cu extraction. Salt addition increased Mn extraction in the Purple varieties (T4 was significantly highest), but decreased Mn extraction in the Green varieties.

Table 7.

Analyzed nutrient content of VhWW produced from de-bittering Vernonia hymenolepis leaves using two processing methods.

Treatment	Vit. C (g/100g water)	Protein (g/100g)	Fe (mg/100g)	Zn (mg/100g)	Cu (mg/100g)	Mn (mg/100g)
T1: Purple Bitter No Salt	0.011±0.009	1.00±0.00	0.200±0.045b	1.200±0.057b	0.300±0.00d	0.100±0.10ab
T2: Purple Bitter with salt	0.011±0.010	1.00±0.00	0.100±0.0046a	0.100±0.057a	0.100±0.00a	0.200±0.00b
T3: Purple Sweet No Salt	0.015±0.012	1.00±0.00	0.133±0.057a	1.067±0.462b	0.267±0.12cd	0.133±0.06a
T4: Purple Sweet with Salt	0.013±0.017	1.00±0.00	0.200±0.000b	1.600±0.004b	0.100±0.00ab	0.400±0.00c
T5: Green Bitter No Salt	0.008±0.007	1.00±0.00	0.207±0.0023b	1.585±0.029b	0.207±0.00bc	0.107±0.00a
T6: Green Bitter with Salt	0.015±0.013	2.00±0.00	0.099±0.0005a	1.199±0.005b	0.099±0.00a	0.099±0.00a
T7: Green Sweet No Salt	0.009±0.007	1.00±0.00	0.300±0.0001c	2.500±0.007c	0.200±0.00bc	0.200±0.00b
T8: Green Sweet with Salt	0.012±0.010	1.00±0.00	0.133±0.577a	2.800±1.21c	0.133±0.06a	0.133±0.06a
Standard Error of Mean	0.008	0.069	0.014	0.183	0.017	0.02

Values are means (n) ± standard deviation.

<sup>abc</sup> Means in the same column with different supercripts were significantly different (p˂0.05).

Table 8.

DPPH radical scavenging activity of VhWW and the standard antioxidant Butylated hydroxytoluene (BHT).

Treatment	*15µg/ML	*30µg/ML	*45µg/ML	*60µg/ML	*75µg/ML	Absorbance
T1: Purple Bitter No Salt	29.33±0.58a	37.00±0.00ab	55.00±0.00a	65.67±3.51ab	67.58±3.50a	0.44±0.002b
T2: Purple Bitter with salt	42.00±0.00e	49.00±0.00f	67.00±0.00e	77.67±3.51ce	79.95±3.00c	0.99±0.00h
T3: Purple Sweet No Salt	29.00±0.00a	36.00±0.00a	55.00±0.00a	65.33±3.51a	67.47±3.50a	0.43±0.001a
T4: Purple Sweet with Salt	38.00±0.00d	45.00±0.00e	63.00±0.00d	73.67±3.51cde	75.96±3.00c	0.82±0.004g
T5: Green Bitter No Salt	36.00±0.00c	43.00±0.00d	61.00±0.00c	72.00±0.0bcde	73.99±3.00bc	0.73±0.001e
T6: Green Bitter with Salt	31.67±0.58b	39.00±0.00c	57.33±0.57b	67.33±3.51abcd	69.55±3.50ab	0.53±.005d
T7: Green Sweet No Salt	30.33±0.58ab	38.00±0.00bc	56.00±0.00a	66.67±3.51abc	68.58±3.50ab	0.48±0.003c
T8: Green Sweet with Salt	36.00±0.00c	43.00±0.00d	62.00±0.00cd	72.33±3.51bcde	74.49±3.50bc	0.75±0.002f
Standard: Butylated Hydroxytoluene	56.33±2.57f	63.67±2.57g	82.00±2.00f	94.67±9.29f	96.58±3.50d
Standard Error of Mean	1.602	1.591	1.582	1.79	1.78	0.04

Values are means (n) ± standard deviation.

*Percentage inhibition at.

<sup>abcdef</sup> Means in the same column with different supercripts were significantly different (p˂0.05).

All VhWW samples had significantly lower antioxidant activity than the BHT standard (P < 0.05).Salt addition significantly increased (P < 0.05) antioxidant activity in the Purple varieties (T2>T1; T4>T3). Salt had no significant effect on the Green varieties. T2 (PB with salt) had the highest activity among all VhWW samples.

3.3. Broiler growth performance and feed utilization

Performance parameters were analyzed across two phases (Starter and Finisher) and the overall trial (Table 9). The effect sizes and confidence intervals for these parameters are also presented (Table 10).

Table 9.

Growth perfromance of broiler chicken supplemented with Vernonia hymenolepis wash water.

Treatment	Daily Feed Intake (g)	Water Intake (ml)	Body Weight Gain (g)	Feed Conversion Ratio	Mortality (%)
Starter
T1: (Purple bitter wiithout salt)	36.68±3.85	78.61±4.36ab	504.83±37.00ab	1.53±0.14ab	0.00
T2: (Purple bitter with salt)	37.49±2.18	88.04±4.77b	477.33±21.31ab	1.65±0.06ab	0.00
T3: (Purple sweet without salt)	37.87±1.13	76.90±3.52ab	454.00±58.96ab	1.77±0.26ab	0.00
T4: (Purple sweet with salt)	38.58±2.31	82.70±1.73ab	481.17±75.09ab	1.71±0.27ab	0.00
T5: (Green bitter without salt)	36.16±1.78	78.40±11.87ab	533.67±110.39ab	1.47±0.31ab	0.00
T6: (Green bitter with salt)	38.53±0.21	83.65±0.55ab	486.50±87.50ab	1.70±0.3ab	0.00
T7: (Green sweet without salt)	32.76±7.02	76.39±8.68a	398.17±94.62a	1.73±0.06ab	4.16±7.22
T8: (Green sweet with salt, GSS)	35.32±2.51	83.42±3.83ab	464.00±41.26ab	1.60±0.03ab	4.16±7.22
T9: (Conventional prophylactic protocol))	37.92±1.53	74.79±7.47a	471.67±113.91ab	1.74±0.31ab	0.00
T10: (Positive control -antibiotic-prophylaxis)	37.49±2.00	80.36±5.77ab	552.50±24.88b	1.43±0.05a	4.16±7.22
T11: (Negative control, spring water)	35.60±3.34	72.35±3.12a	401.50±46.80a	1.86±0.10b	0.00
Standard Error of the Mean	0.53	1.15	13.11	0.04	0.64
Finisher
T1: (Purple bitter wiithout salt)	100.40±6.13abc	222.34±23.69ab	1779.83±144.99ab	1.67±0.20b	0.00
T2: (Purple bitter with salt)	105.45±4.16bc	242.38±4.89b	1823.17±177.06ab	1.60±0.11b	0.00
T3: (Purple sweet without salt)	104.28 ± 7.46bc	235.27±10.93b	1946.50±117.46ab	1.63±0.03b	0.00
T4: (Purple sweet with salt)	102.91±7.46bc	235.38±4.92b	1864.50±106.66ab	1.54±0.23b	4.76±8.25
T5: (Green bitter without salt)	104.32±7.92bc	241.59±20.57b	2001.17±138.75b	1.57±0.22b	0.00
T6: (Green bitter with salt)	103.08±4.38bc	240.91±10.80b	1834.83±278.68ab	1.45±0.14ab	0.00
T7: (Green sweet without salt)	88.58±3.96a	203.60±18.04a	1666.50±257.61ab	1.58±0.04b	0.00
T8: (Green sweet with salt)	104.27±3.62bc	250.48±11.05b	2083.17±22.83b	1.41±0.09ab	4.76±8.24
T9: (Conventional prophylactic program)	106.98±12.60c	230.05±22.01a	1870.00±117.04ab	1.61±0.19b	0.00
T10: (Positive control -antibiotic-prophylaxis)	109.67±3.09c	235.46±10.71b	1967.50±45.92ab	1.63±0.76b	4.76±8.24
T11: (Negative control, spring water)	92.97±11.28ab	204.01±22.36a	1952.33±135.79ab	1.25 ± 0.04a	0.00
Standard error of the mean	0.53	1.15	13.11	0.04	0.64

Values are means (n) ± standard deviation <sup>abc</sup> Means in the same column with different supercripts were significantly different (p˂0.05).

Table 10.

Growth Performance and Statistical Parameters of Broiler Chickens Supplemented with Vernonia hymenolepis Wash Water.

This table presents the means ± SEM for growth performance indicators alongside the main statistical results (Partial η2 and its 95% CI) from the ANOVA for treatment effects across the starter and finisher phases.

Parameter	Period	F-value	P-value	Partial η2	95% CI for η2
Daily Feed Intake (g)	Starter (1–21 d)	F(10,33)=1.84	0.092	0.358	[0.000,0.484]
	Finisher (22–42 d)	F(10,33)=5.73	<0.001	0.635	[0.370,0.718]
Water Intake (ml)	Starter (1–21 d)	F(10,33)=3.45	0.003	0.511	[0.206,0.634]
	Finisher (22–42 d)	F(10,33)=7.92	<0.001	0.706	[0.497,0.763]
Body Weight Gain (g)	Starter (1–21 d)	F(10,33)=1.72	0.118	0.343	[0.000,0.473]
	Finisher (22–42 d)	F(10,33)=2.58	0.018	0.439	[0.091,0.566]
Feed Conversion Ratio	Starter (1–21 d)	F(10,33)=1.98	0.067	0.375	[0.000,0.510]
	Finisher (22–42 d)	F(10,33)=4.25	<0.001	0.563	[0.280,0.668]
Mortality (%)	Starter (1–21 d)	F(10,33)=1.00	0.462	0.232	[0.000,0.392]
	Finisher (22–42 d)	F(10,33)=1.00	0.462	0.232	[0.000,0.392]

Individual Means (± SEM) for all 11 treatments are presented in Table 9.

3.3.1. Water consumption and feed intake

Water intake was highest in the salt-treated sweet varieties (T2 and T8) and significantly higher (P < 0.05) than the controls (T9, T11) and the least consumed VhWW (T7). This suggests that the addition of salt, and the sweet variety profile enhanced palatability and drove water intake.

The T7 group (GS without salt) consumed significantly less feed in the Finisher phase (P < 0.05) than the Conventional (T9) and Positive (T10) controls.

3.3.2. Body weight gain and feed conversion ratio (FCR)

Performance in weight gain and feed conversion ratio are compared in Table 11.

Table 11.

Comparative performance of weight gain and feed conversion ratio.

Phase/Parameter	VhWW Treatment Groups with Best Performance	Control Group Comparison
Starter body weight gain	T10 (Positive Control) was highest. T5 (GB-N) and T1 (PB-N) were statistically similar to T10.	T10 was significantly higher than T7 and T11 (P < 0.05).
Finisher body weight gain	T8 (GS with salt) recorded the highest BWG (2083.17±22.83 g). T5 was also high.	T8 was numerically and statistically superior to T7. No VhWW group was significantly different from T10.
Starter Feed conversion ratio	T10 (1.43±0.05) was significantly better than T11 (1.86±0.10) (P < 0.05).	Most VhWW groups were statistically similar to T10. T8 was the only VhWW group with an FCR numerically close to T10.
Finisher Feed conversion ratio	T11 (Negative Control, 1.25±0.04) had a significantly better FCR (P < 0.05) than T10 (1.63±0.76).	T8 (1.41±0.09) showed the best FCR among VhWW treatments and was statistically comparable to T11.

3.3.3. Mortality

Mortality was low across all groups (ranging 0% to 4.76%) and did not differ significantly (P > 0.05).

3.4. Carcass characteristics and blood lipid profile

3.4.1. Carcass characteristics

Most carcass parameters, including Average Live Weight, Slaughter Weight, Dressed Weight, and Dressing Percentage, were statistically similar (P > 0.05) across the control and VhWW treatments (Table 12). The exception was that T8 (GS with salt) yielded significantly higher (P < 0.05) average Live Weight (2.13±0.02 kg), Slaughter Weight, and Dressed Weight than its T7 (GS without salt) counterpart. Also, the T3 (PS without salt) recorded the highest dressing percentage (93.03±2.22%), which was significantly different (P < 0.05) from its T7 (GS without salt (83.75±7.22%)) counterpart.

Table 12.

Carcass characteristics of broiler chicken fed Vernonia hymnolepis wash water.

Treatment	Average Live Weight	Average Slaughter Weight	Avergage DressedWeight	Dressing Percentage%
T1: (Purple bitter wiithout salt)	1.82±0.14a	1.71±0.18ab	1.70±0.18abc	90.60±3.30ab
T2: (Purple bitter with salt)	1.86±0.17ab	1.76±0.17abc	1.62±0.08ab	87.08±3.65ab
T3: (Purple sweet without salt)	1.99±0.13ab	1.89±0.12abc	1.85±0.78bc	93.03±2.22b
T4: (Purple sweet with salt)	1.90±0.10ab	1.80±0.12abc	1.74±0.13bc	91.21±3.60ab
T5: (Green bitter without salt)	2.01±0.14b	1.95±0.13bc	1.84±0.05bc	88.52±6.09ab
T6::(Green bitter with salt)	1.86±0.27ab	1.78±0.28abc	1.64±0.22ab	87.48±4.63ab
T7: (Green sweet without salt)	1.71±0.26a	1.63±0.26a	1.42±0.18a	83.75±7.22a
T8: (Green sweet with salt)	2.13±0.02b	2.06±0.05c	1.94±0.02c	89.65±0.76ab
T9: (Conventional prophylactic program)	1.91±0.12ab	1.81±0.10abc	1.74±0.13bc	91.15±0.91ab
T10: (Positive Control -antibiotic-prophylaxis)	2.01±0.04ab	1.92±0.05abc	1.77±0.06bc	88.27±4.29ab
T11: (Negative control, spring water)	1.97±0.13ab	1.88±0.07abc	1.71±0.16bc	85.59±1.44ab
Standard Error of Mean	0.029	0.04	0.08	0.74

Values are means (n) ± standard deviation abc= Means in the same column with different superscripts were significantly different (p˂0.05).

3.4.2. Blood lipid profile

The VhWW had significant effects on the blood lipid profile, an indicator of metabolic health (Table 13). The effect sizes and confidence intervals for the blood lipid profile are also presented (Table 14).

Table 13.

Blood lipid profile of broiler chicken fed Vernonia hymnolepis wash water.

Treatments	Total cholesterol	Total Triglycerides	High Density Lipoprotein (HDL)	Low Density Lipoprotein (LDL)
T1: (Purple bitter without salt)	153.89±20.95b	102.15±37.75ab	50.78±9.07ab	59.54±17.76abcd
T2: (Purple bitter with salt)	165.37±22.51b	180.50±12.39cde	56.72±15.19ab	57.97±29.80abcd
T3: (Purple sweet without salt)	170.10±39.35b	82.84±12.08a	48.25±10.92a	47.33±31.26ab
T4: (Purple sweet with salt)	143.49±35.77ab	143.83±28.4dbc	44.14±9.71a	93.00±7.25d
T5: (Green bitter without salt)	160.46±31.06b	143.44±4.04dbc	54.83±9.42ab	92.54±14.65d
T6: (Green bitter with salt)	144.67±25.16ab	184.09±8.86de	72.22±4.38b	52.96±25.18abc
T7: (Green sweet without salt)	85.810±16.43a	93.713±33.67a	41.82±1.61a	86.01±12.84cd
T8: (Green sweet with salt)	166.04±25.77b	139.60±9.10bc	53.53±5.91ab	71.91±8.00abcd
T9: (Conventional prophylactic program)	135.52±46.98ab	89.35±26.79a	61.67±20.68ab	67.06±24.23abcd
T10: Positive Control (Antibiotic-prophylaxis)	149.86±44.71b	120.35±35.32ab	58.80±12.73ab	82.14±17.89bcd
T11: (Negative control, spring water)	115.77±34.52ab	193.19±12.85e	48.82±15.92a	43.23±5.54a
Normal range (mg/dL)	125 −200	60 – 165	26 −65	≤ 100
Standard Error of Mean	6.32	7.5	2.23	4.18

Values are means (n) ± standard deviation abc= Means in the same column with different superscripts were significantly different (p˂0.05).

Table 14.

Blood Lipid Profile and Statistical Parameters of Broiler Chickens Supplemented with Vernonia hymenolepis Wash Water.

This table presents the means ± SEM for blood lipid parameters alongside the main statistical results (Partial η2 and its 95% CI) from the ANOVA for treatment effects.

Parameter	Normal Range (mg/dL)	F-value	P-value	Partial η2	95% CI for η2
Total Cholesterol	125−200	F(10,55)=4.12	<0.001	0.428	[0.198,0.554]
Total Triglycerides	60−165	F(10,55)=31.47	<0.001	0.851	[0.768,0.892]
HDL Cholesterol	26−65	F(10,55)=3.25	0.002	0.371	[0.143,0.508]
LDL Cholesterol	≤100	F(10,55)=4.89	<0.001	0.471	[0.245,0.585]

Individual Means (± SEM) for all 11 treatments are presented in Table 13.

The T7 (GS without salt) had the lowest Total Cholesterol (TC) (85.81±16.43 mg/dL), significantly lower (P < 0.05) than seven other groups. All groups, however, fell within the normal range (125 – 200 mg/dL), except T7 and T11.

The T3 (PS without salt) and T7 had the lowest Triglycerides (TG) levels, statistically comparable to the Conventional Control (T9). Notably, the Negative Control (T11) recorded an elevated TG level (193.19±12.85 mg/dL), exceeding the normal range (60–165 mg/dL).

The T6 (GB with salt) had the highest High-Density Lipoprotein (HDL (72.22±4.38 mg/dL)), significantly higher (P < 0.05) than five other treatments.

The T4 (PS with salt) and T5 (GB without salt) recorded the highest Low-Density Lipoprotein (LDL) levels, significantly higher (P < 0.05) than T3, T6, and T11. The Negative Control (T11) had the lowest LDL (43.23±5.54 mg/dL).

3.5. Economic evaluation

The economic analysis (Table 15) highlighted the financial viability of using VhWW as a prophylactic agent.

Table 15.

Economic performance of broiler chicken fed VhWW.

Treatment	Feed cost (USD/ bird)	Cost of VhWW /Medications (USD/bird)	Total Production Cost (USD/bird)	Total Revenue (USD/bird)	Feed Cost per kg weight gain (USD)	Benefit Cost Ratio (BCR)
T1: (Purple bitter without salt)	1.719±0.097bc	0.016±0.001bc	2.649±0.098ab	6.053±0.467ab	0.943±.0111b	2.29±0.19abc
T2: (Purple bitter with salt)	1.793±0.066bc	0.018±0.000bc	2.725±0.066abc	6.191±0.577ab	0.906±0.061b	2.28±0.26abc
T3: (Purple sweet without salt)	1.784±0.094bc	0.0623±0.003d	2.759±0.096bc	6.606±0.383ab	0.918±0.096b	2.39±0.08bc
T4: (Purple sweet with salt)	1.775±0.074bc	0.0635±0.000d	2.752±0.074bc	6.329±0.346ab	0.868±0.127b	2.30±0.01abc
T5: (Green bitter without salt)	1.763±0.122bc	0.026±0.002c	2.702±0.124abc	6.783±0.448b	0.889±0.125b	2.52±0.25bc
T6: (Green bitter with salt)	1.777±0.057bc	0.023±0.000bc	2.713±0.058abc	6.235±0.292ab	0.822±0.079ab	2.30±0.31abc
T7: (Green sweet without salt)	1.522±0.137a	0.085±0.007d	2.521±0.048a	5.671±0.863a	0.893±0.021b	2.27±0.47abc
T8: (Green sweet with salt)	1.751±0.043bc	0.107±0.004e	2.771±0.048bc	7.059±0.077b	0.795±0.052ab	2.55±0.25bc
T9: (Conventional prophylactic program)	1.818±0.177bc	0.188±0.018f	2.919±0.195c	6.345±0.398ab	0.907±0.105b	2.17±0.01ab
T10: (Positive Control (Antibiotic-prophylaxis)	1.846±0.064c	0.771±0.038g	3.529±0.098d	6.672±0.145ab	0.921±0.067b	1.89±0.09a
T11: (Negative control, spring water)	1.613±0.183ab	0.002±0.00a	2.526±0.183a	6.628±0.452ab	0.709±0.042a	2.62±0.30c
SEM	0.023	0.037	0.049	0.119	0.017	0.05

The Positive Control (T10) had the highest Total Production Cost (3.529 USD/bird), driven by the high cost of the antibiotic prophylaxis (0.771USD/bird). The Negative Control (T11) and T7 had the lowest production costs.

The T8 (GS with salt) yielded the highest Total Revenue (7.059 USD), reflecting its superior final body weight.

The Negative Control (T11) recorded the highest Benefit Cost Ratio (BCR) (2.62±0.30), followed by T8 (2.55±0.25) andT5 (2.52±0.25).

Crucially, the BCR for the top VhWW treatments (T8, T5) was statistically comparable (P > 0.05) to the best-performing control (T11) and significantly higher (P < 0.05) than the antibiotic-based T10 (1.89±0.09).

5. Discussion

The core findings of this study establish the novel utility of Vernonia hymenolepis Wash Water (VhWW), an agro-processing byproduct, as an effective, natural, and economically superior phytogenic prophylactic agent in broiler production. The results demonstrate that optimal efficacy is highly dependent on both plant genotype and the processing methodology.

The nutritional assessment confirms V. hymenolepis leaves as a rich source of essential nutrients, validating its traditional use. The Green Sweet (GS) variety exhibited high concentrations of crude protein and Vitamin C, which are pivotal for rapid growth and immune function (NRC, 1994). Critically, the VhWW effectively captured these water-soluble components, making the processing byproduct a valuable nutritional supplement. The differential nutrient profiles among the four tested varieties (purple bitter, purple sweet, green bitter, green sweet) highlight that genetic selection is the foundational step for maximizing the prophylactic benefit.

The effect of salt (NaCl) on mineral content in the wash water showed a complex, non-linear, and non-uniform trend. For three varieties (PB, GS, GB), salt addition resulted in a lower concentration of macro- and trace minerals in the (VhWW) compared to non-salt treatments. This counterintuitive result challenges the simple expectation of enhanced osmotic leaching. A plausible explanation is the "salting-out effect” or mineral-protein complexation. In a high-ionic-strength environment created by NaCl, the solubility of certain minerals or the proteins/phenolic compounds to which they are chelated may decrease (Sinha & Khare, 2014), causing them to precipitate or remain bound within the cellular matrix of the leaf residue, thereby reducing their release into the wash water (Oboh & Madojemu, 2010). The purple sweet variety's deviation from this trend for Fe and Mn suggests unique structural or protein differences that require further investigation. This variation in response, despite all four being derived from the same local stock, underscores the power of local selection and micro-genotypic differences in influencing nutrient binding properties.

A pivotal finding was the variety-dependent influence of salt processing on antioxidant activity (AA). Salt treatment significantly increased AA in the purple varieties but had no significant effect on the green varieties. This difference strongly points to the role of anthocyanins, the water-soluble pigments responsible for purple coloration and potent AA (Alam et al., 2021). The fact that these pronounced phytochemical differences exist across varieties originating from the same stock provides a strong argument that minor genetic divergence, likely driven by agronomic selection, leads to substantial metabolic pathway shifts (e.g., the activation of anthocyanin synthesis). It is hypothesized that salt stress either enhanced the biosynthesis of anthocyanins as a protective mechanism or significantly improved the solubility and subsequent release of these specific compounds from the vacuole into the VhWW. Conversely, the primary antioxidants in the green varieties (e.g., chlorophylls, other phenolics) were less affected by salt, indicating that stem color may serve as a reliable marker for predicting the optimal processing method.

The inclusion of VhWW as the sole source of drinking water proved highly effective, particularly in the later stage of production. Crucially, the treatments did not negatively affect palatability, with the salt-treated sweet varieties (T2, T8) even showing a significantly higher water intake-an important factor for ensuring adequate intake of bioactive compounds.

The Green Sweet with Salt (T8) and Green Bitter without Salt (T5) protocols achieved the highest body weight gain in the finisher phase and maintained excellent Feed Conversion Ratios that were statistically indistinguishable from the Negative Control (T11). This demonstrates that these specific (VhWW) combinations provide a level of performance efficiency comparable to an un-supplemented diet, but with the added metabolic benefits discussed below.

The blood lipid profile results yield the most compelling evidence for VhWW’s prophylactic role, particularly when considering the full context of the negative control (T11).

The T11 group exhibited a severe metabolic paradox: it demonstrated the best level of LDL cholesterol, which remained within the healthy range for poultry, yet simultaneously showed a markedly elevated level of total Triglycerides (193.19 mg/dL). This high triglyceride concentration significantly exceeded the normal physiological range for broilers (Regar et al., 2019;) indicating a severe, non-sustainable state of dyslipidemia and associated excessive fat deposition-a recognized quality defect in intensive poultry farming (Fan et al., 2021; Malila, 2023).

This metabolic inconsistency in T11 underscores a critical risk: while the broilers successfully maintained healthy LDL levels (likely reflecting efficient cholesterol transport and utilization), their ability to process and clear fat from circulation was severely impaired, leading to hypertriglyceridemia. The high triglycerides represent a profound failure in hepatic lipid metabolism under the stress of rapid growth, which is a greater and more immediate metabolic liability than the LDL concentration (Lu et al., 2019)

Furthermore, the performance data introduces a high degree of risk and uncertainty associated with the T11 protocol. While T11 achieved the best Feed Conversion Ratio (FCR) in the finisher phase, its FCR in the starter phase was the worst among all groups. This dramatic phase-dependent fluctuation suggests that the T11 diet lacked the necessary metabolic support to ensure consistent performance. This instability-manifested as poor early growth efficiency and a severe late-stage metabolic overload (hypertriglyceridemia) - presents an unacceptable risk profile for commercial producers (Vasconcelos et al., 2024).

In stark contrast, most VhWW treatments (along with the positive controls) successfully maintained both Total Triglycerides and Total Cholesterol within the healthy range, eliminating the metabolic instability observed in T11. This suggests that the bioactive compounds-specifically the polyphenols, steroidal saponins, and sesquiterpene lactones (e.g., vernolides) known to be present in Vernonia species (especially the closely related V. amygdalina and vernolepin specifically present in V. hymenolepis) (Ucheck Fomum, 2004a; Degu et al., 2024) in the VhWW improved hepatic lipid metabolism, acting as a crucial metabolic stabilizer and hepatoprotective agent (Tokofai et al., 2021). The anti-hyperlipidemic and growth-promoting effects are hypothesized to involve multiple, synergistic pathways.

Polyphenols (flavonoids and anthocyanins) and steroidal saponins are the primary agents of anti-hyperlipidemic mechanism via hepatic and enteric regulation. Saponins, known to exhibit cholesterol-binding and emulsifying properties (Cao et al., 2024) likely interfere with micelle formation and cholesterol absorption in the gut. This reduction in circulatory cholesterol induces compensatory metabolic changes. Furthermore, the polyphenols are known to directly modulate key enzymes in hepatic lipid synthesis, such as HMG-CoA reductase and fatty acid synthase, in monogastrics, thereby reducing endogenous cholesterol and fatty acid production (Wang et al., 2025). This dual action (reduced absorption and reduced synthesis) provides a robust explanation for the successful mitigation of dyslipidemia.

The observed variability in VhWW efficacy-where some protocols are optimal (T8 and T5), while others (T2, and T6) resulted in significantly higher triglyceride levels, statistically comparable to the hypertriglyceridemic Negative Control (T11)- can be explained by a complex "Genotype-Process-Compound Matrix" influencing extraction and biological antagonism.

The role of salt in mitigating dyslipidemia in V. hymenolepis is consistent with findings in its congener, V. amygdalina (Ewane et al., 2025a). This dynamic indicates that salt does not merely increase osmotic leaching; rather, it influences the extraction ratio of specific, critical metabolites.

Salt-assisted washing in the optimal genotype -process-compound matrix T8 (Green Sweet + Salt) is hypothesized to create an environment that specifically enhances the solubility and transfer of key hypolipidemic saponin molecules while maintaining beneficial antioxidant levels.

For the T5 (Green Bitter No Salt) genotype-process-compound matrix, simple water abrasion may be sufficient to extract a "cleaner" profile of bioactive compounds that successfully regulates lipids, suggesting its inherent structure facilitates the release of essential saponins without needing salt assistance.

The protocols T2 (Purple Bitter + Salt) and T6 (Green Bitter + Salt) all showed significantly higher triglyceride levels, nearing the metabolic risk of the Negative Control (T11). This strongly suggests that in these specific genotype/processing combinations, the wash water contained a sub-optimal ratio of active ingredients, potentially due to:

• Antagonistic Compound Leaching where salt, in the context of the Purple (T2) and Green Bitter (T6) genotypes, may have inadvertently enhanced the extraction of non-polar or semi-polar bitter compounds (like specific sesquiterpene lactones) that, in the concentrations achieved, interfere with optimal hepatic function or the metabolic action of the primary saponins. This interference leads to a failure to process the rapid-growth-induced lipid load, mimicking the dyslipidemia observed in the un-supplemented T11 group.

• Conversely, the high ionic strength may have caused the salting-out' or precipitation of the most potent, specific hypolipidemic saponin fractions, resulting in a wash water that is deficient in the exact compounds needed to control plasma triglycerides.

• It is also Plausible that the altered mineral profile due to ion exchange caused by salt may itself contribute to metabolic strain, indirectly increasing the triglyceride levels, especially in genotypes (like the Purple varieties) where structural differences may prevent protective compound co-extraction.

• The wide disparity across treatments underscores that VhWW cannot be considered a monolithic entity. Effective prophylaxis is only achieved when the processing method is precisely calibrated to the specific genotype, creating a final extract that maximizes the concentration of synergistic hypolipidemic agents while minimizing the concentration of compounds that exert a metabolic load.

• Selective antimicrobial properties of sesquiterpene lactones (e.g., vernolepin, vernolide and vernodalol) against specific gut pathogens (Erasto et al., 2006) contribute to a healthier gut microbiota. This modulation of the intestinal environment, coupled with the known effects of saponins to enhance the activity of digestive enzymes and improve gut histomorphology (Serrano, 2013), leads to better nutrient utilization and thus the superior FCR and growth performance observed in T8 and T5.

• The water-soluble antioxidants (e.g., Vitamin C and phenolics) are vital for scavenging Reactive Oxygen Species (ROS), which are increasingly generated under the metabolic stress of intense, rapid growth in broilers (Oke et al., 2024). By reducing systemic oxidative stress, the VhWW preserves cellular integrity and function, particularly in the metabolically active liver, allowing for optimal lipid processing and detoxification, which further supports the observed hepatoprotective effect. This regulatory effect resulted in a healthier carcass composition, as evidenced by the superior live and dressed weights in T8 and T5.

The efficacy of VhWW protocols aligns well with studies supporting the use of water-soluble plant extracts in broiler production, particularly concerning the dual benefits of growth promotion and lipid regulation. The results are comparable to those reported for the aqueous leaf extract of Moringa oleifera, which has also demonstrated significant improvements in weight gain, FCR, and a desirable reduction in total cholesterol and triglycerides in broilers (Sukria et al., 2025) Similarly, Azadirachta indica (Neem) leaf supplementation has shown positive effects on growth and economic returns by modulating gut microflora (Paul et al., 2020; Sutradhar et al., 2025) However, VhWW offers a decisive set of advantages that distinguish it from these other phytogenic sources:

• As an agro-processing byproduct, VhWW fundamentally alters the economic equation, offering a zero-cost source of bioactive compounds. This provides a clear financial superiority over the use of primary ingredients like raw Moringa leaf meal or commercial extracts of Neem, which require dedicated cultivation, harvesting, and processing costs(Mandey & Sompie, 2021; Aminullah et al., 2025)

• This study uniquely demonstrates that efficacy is maximized by matching the VhWW genotype to the extraction method (e.g., salt for purple, no-salt for green), a level of optimization not commonly reported in single-species phytogenic studies. And one that ensures a consistently potent prophylactic product.

• Administering VhWW as drinking water ensures immediate and consistent daily uptake of water-soluble compounds, allowing for rapid intervention against metabolic stressors, especially in the critical finisher phase. This method bypasses potential degradation or inconsistent mixing issues often associated with feed-based inclusion of labile phytochemicals.

• The robust and successful mitigation of severe dyslipidemia, in a manner statistically equivalent to the antibiotic-based control (T10), positions VhWW as a high-value, natural metabolic insurance strategy, offering a superior risk-adjusted alternative to conventional prophylaxis.

• The economic evaluation is crucial for the practical adoption of VhWW protocols. The Positive Control with antibiotic reinforcement (T10) resulted in the lowest Benefit-Cost Ratio (BCR) due to high input costs. While the Negative Control (T11) delivered the highest BCR due to minimal cost, this financial benefit is fundamentally compromised by the unacceptable high metabolic risk (severe hypertriglyceridemia) and performance instability (worst starter FCR) observed. The risk of sudden mortality, organ damage, and poor carcass quality associated with this dyslipidemia effectively negates any perceived economic advantage.

• The optimal VhWW protocols (T8 and T5) achieved BCR values (2.55 and 2.52) that were statistically non-significant from the high-risk (T11) yet they successfully mitigated the metabolic instability and ensured consistent FCR. This places VhWW protocols as a superior, low-cost "metabolic insurance" and an economically sound alternative to expensive and controversial conventional prophylaxis.

6. Conclusion

This study concludes that Vernonia hymenolepis Wash Water (VhWW) is an efficacious, sustainable, and economically competitive phytogenic prophylactic agent for broiler chickens.

The optimal strategy involves using the Green Sweet variety processed with salt (T8) which delivers enhanced palatability, superior growth performance, and a strong Benefit-Cost Ratio (2.55). Crucially, the use of VhWW eliminates the severe metabolic instability (hypertriglyceridemia and inconsistent FCR across phases) associated with the un-supplemented Negative Control (T11), providing necessary metabolic insurance and resulting in better carcass quality. The VhWW is proven to be a natural, safer, and more economically viable alternative to conventional antibiotic prophylaxis in intensive broiler production systems.

7. Recommendations

7.1. Practical recommendations

• •Processing the green variety of V. hymenolepis with salt to maximize their growth-promoting and economic benefits is recommended for adoption by poultry producers as a cost-effective, natural drinking water supplement for broiler chickens throughout the production cycle (day 1 to 42).
• •Growers should prioritize the Green Sweet variety to maximize protein and vitamin delivery.
• •The Green bitter and purple bitter varieties which are presently destroyed or used to adulterate Vernonia amygdalina commercially, should be co- utilized in livestock prophylaxis
• •The administration of VhWW as the sole source of drinking water is recommended for ensuring consistent and immediate daily uptake of bioactive compounds, particularly throughout the critical finisher phase

7.2. Research recommendations

Phytochemical Quantification and Mechanism: A mandatory next step is to use advanced analytical techniques (e.g., LC-MS/MS or GC–MS) to fully identify, quantify, and compare the specific fractions of saponins, flavonoids, and sesquiterpene lactones in the VhWW across all four varieties and processing methods. This is crucial for definitively linking the observed anti-hyperlipidemic effects to specific compounds.

Organ Safety and Histopathology: Conduct a histopathological analysis of major metabolic and excretory organs (liver, kidney, spleen, and intestines) across all treatment groups. This is required to provide definitive assurance of long-term organ safety and confirm the absence of tissue damage or metabolic stress from continuous VhWW administration.

Industrial Scalability and Stability: Future studies should focus on optimizing methods for large-scale, industrial preparation, concentration, and long-term storage stability of VhWW concentrates without compromising the potency and bioactive integrity of the labile phytochemicals.

8. Study strengths and limitations

8.1. Key study strengths

Novel Circular Economy Approach: This is a pioneering study to validate the prophylactic use of a food processing byproduct VhWW, significantly contributing to waste reduction and offering a highly cost-effective, sustainable solution.

Protocol Optimization: We provided definitive data on the synergistic effects of genotype and processing, yielding an optimized protocol (GS + Salt) that maximizes performance and economic return. This finding is mechanistically supported by comparative data from V. amygdalina, confirming that salt-assisted washing is a vital tool for achieving the optimal Genotype-Process-Compound matrix necessary for effective metabolic control

Metabolic Risk Identification and Mitigation: The study uniquely identified severe hypertriglyceridemia and performance instability as a critical, high-risk consequence of the un-supplemented commercial broiler production (T11). It demonstrated VhWW’s capacity to successfully regulate lipid metabolism and provide consistent FCR, offering a risk-adjusted, high-value alternative to the economically but metabolically unstable Negative Control.

Economic Justification: The economic analysis offers a clear, practical argument for adoption, establishing VhWW as a superior economic and low-risk alternative to conventional, antibiotic-based prophylaxis.

8.2. Acknowledged study limitations

Phytochemical Resolution: While the study provided performance and metabolic benefits, the discussion on the mechanisms (e.g., specific compound antagonism) is limited by the absence of high-resolution phytochemical profiling (e.g., via LC-MS). The precise quantification of the most potent hypolipidemic molecules is currently inferred rather than directly measured.

Lack of Definitive Organ Safety Data: The study lacks histopathological data. Although favorable blood parameters suggest hepatoprotection, definitive, tissue-level assurance regarding the long-term safety and integrity of the liver and kidney under continuous VhWW use requires targeted organ analysis.

Generalizability Constraint: The feeding trial was confined to a single commercial broiler strain (Cobb 500) under one specific set of environmental and management conditions. This limits the immediate generalizability of the findings to different genetic lines or diverse global production climates.

Antioxidant Activity Context: The direct antioxidant potential of VhWW was lower than the synthetic standard BHT, suggesting its in vivo benefits may rely more heavily on complex indirect mechanisms, such as gene expression modulation, enzyme induction, or gut microbiota manipulation, rather than simple, direct radical scavenging.

Ethical statement

Unsexed Cob500 broiler chickens were used as experimental animals for this study. The experiments were carried out following the National Ethical Committee Guidelines (No. FWA-IRB00001954) and International (European Committee Council Directive of November 24, 1986(86/69/EEC); Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) for the care and use of laboratory animals. All efforts were made to minimize the suffering and stress of chickens used at each stage of the study. Ethical approval was given by the University of Buea Institutional Animal Care and Use Committee (UB-IACUC) via permit No.UB-IACUC No.48/2023

CRediT authorship contribution statement

Divine Ewane: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Lawrence Monah Ndam: Writing – original draft, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Conceptualization. Brandon Rahim Fongang Keubiwou: Writing – original draft, Visualization, Validation, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Boris Nghombuoche: Writing – original draft, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Ewane Ekuh Bazil: Writing – review & editing, Validation, Resources, Investigation.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.