Effects of Dietary Phytobiotic Mixtures on Growth Performance, Nutrient Digestibility, Intestinal Histomorphology, Cecal Microbiota, and Antioxidant Status in Fattening Ducks

Abstract

This study aims to evaluate the effects of two phytobiotic mixtures on performance, nutrient digestibility, histomorphology, microbiota, and antioxidant status in fattening ducks. A total of 180 day-old male mixed-type ducks were randomly assigned to three dietary groups: a control group receiving a basal diet, and two treatment groups (PM1: commercial phytobiotic formulation containing menthol, eucalyptus oil and turmeric leaf oil as key ingredients and PM2: commercial phytobiotic formulation containing garlic oil as a key ingredient) supplemented at 250 g/ton of feed. Ducks were reared for 49 days with six replicates of ten ducks. Performance parameters, including body weight (BW), body weight gain (BWG), and average feed intake (AFI), were significantly improved in phytobiotic groups (p ≤ 0.05). Apparent digestibility of dry matter, crude protein, ether extract, and starch remained unaffected (p > 0.05). Histological analysis showed no significant differences in villus height (VH) or crypt depth (CD). However, cecal microbiota culture-based analysis revealed increased total anerobic bacteria and Lactobacillus counts in PM1 and PM2 (p ≤ 0.05). Antioxidant status demonstrated reduced MDA levels and elevated total phenolic content (TPC) and total antioxidant capacity (TAC) in breast and thigh tissues of treated ducks. Overall, phytobiotic supplementation improved performance and microbiota balance, supporting the potential application of these phytobiotic formulations at the inclusion level of 250 g/ton in fattening ducks’ nutrition.

1. Introduction

Indian herbs, which are deeply rooted in Ayurveda (traditional Indian system of medicine), have gained attention in recent years due to their multiple medicinal properties and cultural significance [1]. Traditionally used for centuries to promote human health and treat various ailments, these herbs are now being explored for applications in animal nutrition and health [2]. There is a growing global interest in natural feed additives that support animal health, immunity, and productivity without compromising food safety or environmental sustainability as a result of increasing restrictions on antibiotic use in livestock and the rise in antimicrobial resistance [3,4].

Recent studies have underlined the beneficial effects of several essential oils, such as eucalyptus, menthol, and turmeric oil, in poultry production [5]. Eucalyptus oil, rich in 1,8-cineole, has shown strong antimicrobial and anti-inflammatory results, contributing to improved respiratory health, enhanced feed conversion ratio, and reduced pathogenic bacterial load in broilers [6,7]. Additionally, menthol has been shown to exert cooling, antioxidant, and appetite-stimulating effects, leading to improved feed intake and growth performance under heat stress conditions [8,9]. Moreover, the rhizome of turmeric contains a range of bioactive secondary metabolites, mainly curcuminoids (such as curcumin) and volatile turmerones (like ar-turmerone). These compounds act as potent antioxidants by scavenging free radicals and reducing lipid peroxidation. Previous studies have found that these turmeric-derived components may serve as natural alternatives to antibiotics, providing strong antioxidant and immune-protective effects while supporting improved growth performance in poultry [10,11]. Also, recent studies have shown that garlic oil supplementation in poultry feed enhances growth performance, improves intestinal health, and modulates immune responses [12].

Although these herbs have been extensively studied in broilers and laying hens, there is still very little research on their effects in ducks. Ducks have different physiological and immune responses compared to broilers and layer hens, which may lead to differential responses to dietary supplements [13]. Moreover, the optimal dosages, synergistic combinations, and long-term effects of these herbs on gut health, nutrient utilization, and overall productivity in ducks remain unexplored [14]. Overcoming these knowledge gaps is important for establishing evidence-based guidelines for the dietary use of these herbs in duck nutrition.

The aim of this study was to investigate the effects of two phytobiotic formulations supplemented at 250 g/ton on growth performance, nutrient digestibility, culture-based cecal microbiota composition, intestinal histomorphometry and antioxidant status in fattening ducks. Based on previous studies regarding the activity of phytobiotic compounds, we hypothesized that the dietary supplementation with two phytobiotic formulations may improve growth performance, antioxidant profile and the cecal microbiome in fattening ducks. The findings of the present study may contribute to understanding how medicinal herbs can be incorporated in poultry management as sustainable and effective alternatives to conventional growth promoters in poultry production.

2. Materials and Methods

2.1. Ethics Statement

Breeding, euthanasia, experimentation and biosafety procedures were carried out according to the Greek legislation for the use of experimental animals, the Veterinary and Research Committee of Aristotle University under research project number 73622.

2.2. Experimental Design, Animals, and Dietary Treatments

A 49-day experiment was conducted to evaluate the effects of dietary phytobiotics on growth performance and gut health in ducks. A total of 180 one-day-old, male fattening ducks (mixed type ducks (local breed × Pekin) were obtained from a hatchery (Revis Vasileios Poultry farm, Velestino, Greece) and randomly allocated to three dietary treatments. Each treatment consisted of 6 replicate pens with 10 ducks per pen (n = 60 birds per treatment).

Ducks were housed in raised wire-mesh cages to improve hygiene by allowing droppings to fall through the floor. Temperature, ventilation and lighting schedule were monitored consistently across all treatment groups according to standard recommendations for fattening ducks. Feed and water were provided ad libitum throughout the experimental period. The feeding program included a starter diet from day 1 to 28 and a grower diet from day 29 to 49.

The control group (C) received a basal diet without any additional supplementation. The first treatment group (PM1) was supplemented with 250 g/ton of feed with commercial phytobiotic formulation (AV/EGM/19, Zenex Animal Health India Private Ltd., Village Katha, P.O. and Tehsil Baddi, Solan 173205, Himachal Pradesh, India), containing menthol, eucalyptus oil, and turmeric leaf oil. The second treatment group (PM2) received a different phytobiotic formulation (AV/EGF/20, Zenex Animal Health India Private Ltd., Village Katha, P.O. and Tehsil Baddi, Solan 173205, Himachal Pradesh, India), also at 250 g/ton, which contains garlic oil. Both phytobiotic products were supplied in proprietary commercial form by the manufacturer and incorporated into the feed according to the recommended inclusion rate. Moreover, the level of supplementation was selected according to the manufacturer’s recommended dosage and previous evaluations in poultry nutrition. Detailed composition of active herbal compounds was not available due to proprietary formulation restrictions.

Diets were formulated to meet or exceed the nutrient requirements of fattening ducks, and the ingredient composition and calculated nutrient values are provided in

Table 1. Broiler duck ration.

Ingredient Composition	Starter (1–28 Days)	Grower/Finisher (29–49 Days)
Maize	45	46.6
Wheat (soft)	10	10
Wheat bran	6.59	7.2
Soybean meal (46%)	24	16.4
Corn gluten meal (60%)	3	1
Alfalfa meal	5	5
Soya oil	1	3.7
DDGS	1	6.43
l-Lysine HCl (78%)	0.24	0.19
dl-Methionine (99%)	0.3	0.2
l-Threonine (98%)	0.1	0.24
Vitamin–mineral premix 1	0.25	0.25
Salt (NaCl)	0.4	0.3
MgO	0.3	0.3
NaHCO3	0.1	0.1
Limestone	2.2	1.59
Dicalcium phosphate	0.52	0.5
Nutrient Content
Moisture (%)	11.6	11
Metabolizable energy (kcal kg−1)	2767	3000
Crude protein (%)	20.5	18
Crude fat (%)	3.8	6.79
Crude fiber (%)	5	5.05
Ash (%)	6.28	5.27
Starch (%)	36.61	37.32
Calcium (%)	1.1	0.9
Phosphorus (%)	0.55	0.55
Sodium (%)	0.25	0.28
Lysine (%)	1.15	1
Methionine + Cysteine (%)	0.99	0.85
Threonine (%)	0.83	0.8

¹ Supplying per kg feed: 12,000 IU vitamin A, 5000 IU vitamin D3, 30 mg vitamin E, 3 mg vitamin K, 5 mg thiamin, 6 mg riboflavin, 6 mg pyridoxine, 0.02 mg vitamin B12, 60 mg niacin, 15 mg pantothenic acid, 1.5 mg folic acid, 0.25 biotin, 10 mg vitamin C, 500 mg choline chloride, 100 mg Zn, 120 mg Mn, 20 mg Fe, 15 mg Cu, 0.2 mg Co, 1 mg I, 0.3 mg Se, and phytase in optional amounts per kg of feed.

.

2.3. Feed Evaluation

Additional information regarding the composition of the phytobiotic formulations is presented in

Table 2. Chemical composition of phytobiotic formulations.

Chemical Composition %	PM1	PM2
Moisture	4.76	4.83
Total protein	13.21	13.32
Fat	6.74	6.89
Fiber	0.50	0.50
Ash	20.12	20.56

Values are extracted on a fed basis. Active phytobiotic ingredients were estimated by the manufacturer.

[15]. To assess the antioxidant potential of the phytobiotic additives used in the study, the total phenolic content (TPC) of the raw phytobiotic mixtures PM1 and PM2 was determined prior to their inclusion in the diets. The basal diet itself was also evaluated separately. The TPC was measured using the Folin–Ciocalteu colorimetric method and results are expressed as micrograms of gallic acid equivalents per gram of dry weight (μg GAE/g DW) in

Table 3. Antioxidant activity expressed as total phenolic content (μg GAE/g DW) of control diet and raw phytobiotic mixtures.

(μg GAE/g DW)	Groups			p-Value
	C	PM1	PM2
TPC	92.95 a	352.68 b	285.57 c	<0.05

C group (Control) received a basal diet; PM1 group was supplemented with 250 g/ton of a phytobiotic formulation AV/EGM/19 containing menthol, eucalyptus oil, turmeric leaf oil; PM2 group received AV/EGF/20 at 250 g/ton, which contains garlic oil. ^a,b,c Different letters denote significant (p ≤ 0.05) differences between treatments.

.

2.4. Determination of Growth Performance

Growth performance parameters, including body weight (BW), average feed intake (AFI), body weight gain (BWG), and feed conversion ratio (FCR), were assessed at three key time points: on day 1, day 28, and at the end of the experiment on day 49. BW was measured individually for each duck using a digital scale with a precision of ±1 g, and results were recorded in grams. AFI was determined per pen by recording the weight of feed offered and subtracting the weight of feed remaining at each time point, accounting for any visible feed spillage. FCR was calculated per pen as the ratio of total FI (g) to total BWG (g) [15].

2.5. Sampling, Histology, and Histomorphometric Analysis

At the end of the trial, one duck per replicate (six per group) was randomly selected and euthanized by exposure to a rising concentration of carbon dioxide in an airtight chamber. After euthanasia, necropsy was performed. The gastrointestinal tract was removed, and 1 cm segments of the jejunum and ileum were collected from the mid-portion of each segment and immediately fixed in 10% buffered formalin for at least 24 h for histomorphometric assays under light microscopy. Tissues were dehydrated, cleared in xylene, and embedded in paraffin wax. Formalin-fixed intestinal tissues were processed, sectioned at 4 μm, and stained with haematoxylin and eosin [16]. Histomorphometric measurements were performed on well-oriented villi and crypts using the imageJ software (NIH, Bethesda, MD, USA, v.1.54m). Villus height (VH) was measured as the distance from the villous tip to the villous–crypt junction, and crypt depth (CD) as the distance from the base of the crypt to the villous–crypt junction, following the criteria of Gava et al. [17]. For each intestinal segment, 20 villi and 20 corresponding crypts were measured per section, and mean values were calculated for each duck as a single experimental unit. Measurements were performed blind to treatment group to avoid observer bias.

2.6. Microbiota Analysis

Samples of cecal content (approximately 1–5 g) were collected aseptically from ducks and homogenized in sterile buffered peptone water at a 1:10 (w/v) ratio using a stomacher. Serial tenfold dilutions were prepared, and appropriate dilutions were plated for bacterial enumeration. Total aerobic bacteria were enumerated on Plate Count Agar and incubated aerobically at 30 °C for 48 h. Total anerobic bacteria were cultured on Reinforced Clostridial Agar under anerobic conditions at 37 °C for 48 h. Enterobacteriaceae were determined on Violet Red Bile Glucose Agar incubated at 37 °C for 24 h [18]. Enterococci were counted on Slanetz and Bartley agar incubated at 37 °C for 48 h [19]. Lactobacilli were assessed on de Man, Rogosa and Sharpe (MRS) agar incubated anerobically at 37 °C for 48–72 h [18]. All results were expressed as log10 colony-forming units per gram of sample (log10 CFU/g).

2.7. Digestibility Analysis

Titanium dioxide (TiO₂; E171 titanium dioxide, IMCD Benelux N.V., Mechelen, Belgium) was incorporated as a digestibility marker in the experimental diets of ducks at a concentration of 0.3%. Diets were given to ducks from day 29 for 5 days, while excreta collection on plastic mats was performed on days 33 and 34. Feed and excreta content of moisture, nitrogen and fat was determined according to the Association of Official Analytical Chemists (AOAC) [20]. Gross energy was measured using an adiabatic bomb calorimeter (IKA^® Calorimeter System C 5000 Control, IKA^®-Werke GmbH & Co. KG, Staufen, Germany). Measurement of titanium dioxide was performed by inductively coupled plasma optical emission spectrometry following the procedure of van Bussel et al. [21]. Apparent digestibility coefficients (%) were calculated using titanium dioxide as an indigestible marker according to the following equation:

Apparent digestibility (%) = 100 − [100 × (TiO₂ concentration in feed/TiO₂ concentration in excreta) × (nutrient concentration in excreta/nutrient concentration in feed)].

Approximately 100–200 mg of finely ground sample was weighed into a digestion vessel, followed by the addition of concentrated HNO₃ and concentrated H₂SO₄ under a fume hood. The mixture was heated until complete digestion was accomplished, then diluted to volume with deionized water. The final solutions were analyzed by ICP-AES against acidified aqueous and matrix-matched standards. All glassware and digestion vessels were soaked in freshly prepared 10% v/v HNO₃ overnight and, finally, washed three times with Milli-Q quality water. An axial viewing plasma spectrometer model Perkin Elmer Optima 3100 XL (Perkin Elmer Inc., Waltham, MA, USA) was used.

2.8. Antioxidant Parameters

To evaluate TPC levels in thigh and breast, tissue samples were collected from two ducks per replicate (12/group) and evaluated following the protocol described by Jang et al. [22]. Particularly, a total of 2 g of tissue was homogenized with 6 mL of distilled water. Subsequently, 3.6 mL of dichloromethane was added to the homogenate and mixed using a vortex. The sample was then centrifuged, and the resulting supernatant was collected and measured. To prepare the diluted sample, 1 mL of the supernatant was mixed with 4 mL of distilled water. Subsequently, 1 mL of the diluted sample was combined with 500 μL of Folin–Ciocalteu reagent and 1 mL of 7% Na₂CO₃ solution. The mixture was thoroughly mixed using a vortex and incubated at room temperature for 60 min. After the incubation period, the total phenolic content was determined using a spectrophotometer (PharmaSpec, Shimadzu Corporation, Kyoto, Japan) set to measure absorbance at 700 nm.

TAC levels were assessed following the method described by Prieto et al. [23] using a phosphomolybdate reagent. Tissue extracts (100 μL) were vortexed with 1 mL of the reagent and incubated in a water bath at 95 degrees Celsius for 90 min. After cooling, the absorbance was measured at 695 nm using a spectrophotometer (PharmaSpec, Shimadzu Corporation, Kyoto, Japan). A standard curve was prepared using ascorbic acid, and a blank was prepared using the phosphomolybdate reagent without the tissue sample. TPC and TAC were estimated only on day 1 to evaluate the immediate antioxidant capacity of fresh tissue after slaughter. However, MDA was additionally calculated at day 5 postmortem to evaluate lipid oxidation during refrigerated storage, as lipid peroxidation develops progressively after storage.

The determination of lipid peroxidation (malondialdehyde—MDA) was employed as a marker, according to Ahn et al. [24]. Tissue samples weighing 1 g were homogenized with a mixture of 8 mL 5% trichloroacetic acid (TCA) and 5 mL 0.8% Butylated Hydroxytoluene (BHT) dissolved in hexane. Subsequently, the samples underwent centrifugation at 3000 g for 5 min, after which 1.5 mL of the underlying layer was collected. To this, 2.5 mL of 0.8% thiobarbituric acid (TBA) was added, and the mixture was incubated in a water bath at 70 °C for 30 min. The resulting sample’s absorbance was measured at 532 nm using a spectrophotometer (PharmaSpec, Shimadzu Corporation, Kyoto, Japan). MDA levels in the samples were determined by creating a reference curve using known quantities of MDA and expressed as nmol/gr tissue.

2.9. Statistical Analysis

The minimum required sample size was estimated before initiation of the experiment using power analysis for one-way ANOVA, based on the guidelines of Charan and Katharia [25] and resources from IDRE. Calculations were based on an alpha level (a = 0.05) and were performed using G*Power version 3.1.9.2 (Universität Kiel, Germany), ensuring a statistical power of ≥0.80.

Experimental data were analyzed using SPSS software (version 20.0; IBM Corp., Armonk, NY, USA). For growth performance parameters, the pen was considered the experimental unit, whereas the individual birds were considered the experimental unit for tissue, microbiological, digestibility and histological analysis. One-way ANOVA was used to compare treatment means, and where significant differences were detected, Tukey’s post hoc test was applied for multiple comparisons. Results were considered statistically significant at p ≤ 0.05.

3. Results

3.1. Performance

On day 28, ducks supplemented with the PM1 phytobiotic mixture exhibited significantly higher BW and BWG compared to the control group (p ≤ 0.05), while the PM2 group showed intermediate values. AFI and FCR during this early phase remained unaffected by dietary treatment (p > 0.05) (

Table 4. Impact of different phytobiotic mixtures on various performance parameters of ducks.

Age	Parameter	Groups			SEM	p-Value
		C	PM1	PM2
Day 1	BW (g)	52.22	52.83	52.47	0.14	0.222
Day 28	BW (g)	1912.25 b	1968.52 a	1932.45 b	7.90	0.005
	BWG (g)	1860.03 b	1915.58 a	1879.98 b	7.84	0.005
	AFI (g)	2595.67	2774.83	2702.83	34.00	0.089
	FCR	1.40	1.45	1.44	0.02	0.351
Day 49	BW (g)	3225.08 b	3383.30 a	3376.7 a	26.46	0.012
	BWG (g)	1313.39 b	1414.78 ab	1444.25 a	23.41	0.046
	AFI (g)	3117.67 b	3310.83 a	3381.50 a	38.86	0.007
	FCR	2.37	2.35	2.35	0.03	0.949
Total experimental period	BWG (g)	3173.42 b	3330.47 a	3324.23 a	26.42	0.012
	AFI (g)	5713.33 b	6085.67 a	6084.33 a	61.24	0.007
	FCR	1.80	1.83	1.83	0.01	0.643

C group (Control) received a basal diet; PM1 group was supplemented with 250 g/ton of a phytobiotic formulation AV/EGM/19 containing menthol, eucalyptus oil, turmeric leaf oil; PM2 group received AV/EGF/20 at 250 g/ton, which contains garlic oil. BW: body weight. BWG: body weight gain. AFI: average feed intake;FCR: feed conversion ratio. SEM: standard error of mean. ^a,b Different letters denote significant (p ≤ 0.05) differences between treatments.

).

By day 49, both phytobiotic-supplemented groups (PM1 and PM2) demonstrated significantly greater BW compared to the control (p ≤ 0.05). BWG was highest in the PM2 group, which differed significantly from the control, while PM1 produced intermediate results (p ≤ 0.05). AFI during the finisher phase was also significantly increased in both phytobiotic groups (p ≤ 0.05) and FCR remained statistically similar across all treatments (p > 0.05) (Table 4).

Across the entire trial period, cumulative BWG and AFI were significantly increased in both PM1 and PM2 groups compared to the control (p ≤ 0.05), whereas FCR remained unchanged (p > 0.05) (Table 4).

3.2. Histomorphometric and Histological Analysis

Table 5. Impact of different phytobiotic mixtures on the morphometry in μM of the gut of ducks.

Gut Segment	μM	Groups			SEM	p-Value
		C	PM1	PM2
Jejunum	VH	983.0	997.6	996.7	16.16	0.929
	CD	208.1	200.6	205.8	2.90	0.596
	VH:CD	4.72	4.97	4.84	2.70	0.592
Ileum	VH	544.1	550.8	549.6	4.78	0.852
	CD	168.4	170.4	165.9	2.80	0.832
	VH:CD	3.23	3.23	3.31	2.85	0.852

C group (Control) received a basal diet; PM1 group was supplemented with 250 g/ton of a phytobiotic formulation AV/EGM/19 containing menthol, eucalyptus oil, turmeric leaf oil; PM2 group received AV/EGF/20 at 250 g/ton, which contains garlic oil. VH: villus height; CD: crypt depth. SEM: standard error of mean.

displays the histomorphometrical measurements in the gut of ducks. In the jejunum, VH and CD were not significantly different among the control, PM1, and PM2 groups (p > 0.05) (Figure 1). Similarly, in the ileum, neither VH or CD showed significant variation across treatments (p ≤ 0.05). The same results were observed in VH: CD ratio (Table 5).

3.3. Microbiota

Notably, total anerobic bacterial counts were significantly increased in both phytobiotic-treated groups (PM1 and PM2) compared to the control (p ≤ 0.05). Similarly, the population of beneficial Lactobacilli was significantly elevated in ducks receiving either PM1 or PM2 (p ≤ 0.05). Conversely, no significant differences were observed among treatments for total aerobic bacteria, Enterobacteriaceae, or Enterococci (p > 0.05) (

Table 6. Impact of different phytobiotic mixtures on the microbiota of cecum of ducks.

Intestinal Part	Bacteria Species (log10 CFU/g)	Groups			SEM	p-Value
		C	PM1	PM2
Cecum	Total anerobic	8.64 b	9.14 a	9.04 a	0.08	0.010
	Total aerobic	6.66	6.54	6.50	0.06	0.582
	Enterobacteriaceae	6.45	6.69	6.72	0.08	0.306
	Enterococci	3.38	3.40	3.39	0.05	0.992
	Lactobacilli	4.36 b	4.69 a	4.76 a	0.06	0.012

C group (Control) received a basal diet; PM1 group was supplemented with 250 g/ton of a phytobiotic formulation AV/EGM/19 containing menthol, eucalyptus oil, turmeric leaf oil; PM2 group received AV/EGF/20 at 250 g/ton, which contains garlic oil. SEM: standard error of mean. ^a,b Different letters denote significant (p ≤ 0.05) differences between treatments.

).

3.4. Digestibility

The inclusion of phytobiotic mixtures in the diets of ducks had no statistically significant effect on the apparent digestibility of DM, CP, EE, or starch (p > 0.05 for all parameters). Despite slight numerical differences between groups, digestibility values remained comparable across all the treatments (

Table 7. Impact of different phytobiotic mixtures on digestibility % of ducks.

Digestibility in %	Groups			SEM	p-Value
	C	PM1	PM2
DM	74.8	74.5	74.7	0.19	0.772
CP	75.1	75.8	75.9	0.31	0.523
EE	92.5	92.4	93.0	0.48	0.877
STARCH	98.5	98.9	98.5	0.16	0.521

C group (Control) received a basal diet; PM1 group was supplemented with 250 g/ton of a phytobiotic formulation AV/EGM/19 containing menthol, eucalyptus oil, turmeric leaf oil; PM2 group received AV/EGF/20 at 250 g/ton, which contains garlic oil. SEM: standard error of mean.

).

3.5. Antioxidant Status

The impact on antioxidant status in ducks of phytobiotic mixtures is shown in

Table 8. Impact of different phytobiotic mixtures on various antioxidant analysis factors of ducks.

Antioxidant Analysis	Anatomical Part	Day	Groups			SEM	p-Value
			C	PM1	PM2
MDA (nmol/gr tissue)	breast	1	2.54 a	1.21 b	0.97 b	0.18	<0.001
		5	9.75 a	2.47 b	2.10 b	0.64	<0.001
	thigh	1	3.46 a	2.01 b	2.05 b	0.20	0.002
		5	14.01 a	3.81 b	4.19 b	0.86	<0.001
TPC (μg GAE/g DW)	breast	1	1298.3 b	1882.9 a	1919.7 a	73.02	<0.001
	thigh	1	1439.6 b	1906.0 a	1856.9 a	61.91	<0.001
TAC (% of ascorbic acid)	breast	1	22.2 b	45.4 a	43.1 a	2.34	<0.001
	thigh	1	15.9 b	29.6 a	29.8 a	1.43	<0.001

C group (Control) received a basal diet; PM1 group was supplemented with 250 g/ton of a phytobiotic formulation AV/EGM/19 containing menthol, eucalyptus oil, turmeric leaf oil; PM2 group received AV/EGF/20 at 250 g/ton, which contains garlic oil. MDA: malondialdehyde; TPC: total phenolic content; GAE: gallic acid equivalents; TAC: total antioxidant capacity. SEM: standard error of mean. ^a,b Different letters denote significant (p ≤ 0.05) differences between treatments.

. MDA levels were significantly reduced in both breast and thigh muscles of ducks fed phytobiotic mixtures (PM1 and PM2) compared to the control group at both sampling points (day 1 and day 5 postmortem). Similarly, TPC in breast and thigh tissues was significantly elevated in the phytobiotic-fed groups compared to controls (p ≤ 0.05). TAC also showed significant improvements in both muscle types for the PM1 and PM2 groups compared to the control (p ≤ 0.05), further supporting the antioxidant-enhancing effect of the phytobiotic formulations.

4. Discussion

The examination of growth and health through dietary interventions is of particular interest in ducks. Previous studies have demonstrated that supplementation with specific phytobiotics can improve BWG and antioxidant status in broilers and laying hens [26,27]. In this study, we expect the findings to reveal whether dietary phytobiotic mixtures can similarly enhance growth performance, nutrient digestibility, intestinal histomorphology, cecal microbiota composition, and antioxidant status in fattening ducks, to provide insights into their potential as natural alternatives to conventional growth promoters.

The evaluation of growth performance in ducks showed significant differences in BW and BWG among the experimental groups. Both phytobiotic-supplemented groups (PM1 and PM2) exhibited higher final BW and BWG compared to the control, indicating the positive impact of dietary phytobiotic additives on duck performance. Khattak et al. [27] reported that a natural blend of essential oils enhanced growth performance in broilers. Similarly, in ducks fed diets supplemented with Moringa leaf powder, researchers found significant improvements in BWG and FCR, while AFI did not differ significantly among treatments [28]. Interestingly, AFI was significantly affected during the second period of the trial, with the PM2 group demonstrating a higher value compared to the control group. A research study by Abouelezz et al. [29] did not find significant changes in AFI in ducks supplemented with oregano. Another study supported that most phytobiotic treatments did not significantly alter feed intake [29]. The results of AFI may suggest that specific combinations or dosages of phytobiotics may exert a more pronounced effect under certain conditions, highlighting the need for further investigation.

The findings of histomorphometric analysis of the jejunum and ileum revealed no statistically significant changes in VH, CD and the ratio VH/CD among the experimental groups. Similar results were reported by Ding et al. [30]. However, Bao et al. [31] have found improved intestinal histomorphometry in ducks fed encapsulated essential oils. These discrepancies may be caused by differences in the type, dosage, formulation or duration of phytobiotic supplementation, as well as variations in duck genetics, diet composition, or environmental conditions [32].

The cecal culture-based analysis revealed that total aerobic bacterial counts were not significantly affected. This result may suggest that these populations are relatively resilient to dietary interventions. In contrast, total anerobic bacterial counts were significantly increased in PM1 and PM2 groups compared to the control. This selective modulation may be attributed to the bioactive compounds in the phytobiotics, such as menthol, eucalyptus oil, turmeric leaf oil, and garlic oil, which can influence microbial metabolism by serving as substrates for fermentation or by altering gut redox conditions [33]. Specifically, the observed increase in Lactobacilli in PM1 and PM2 may indicate a shift toward a more favorable microbial profile. Previous studies have shown that increased populations of beneficial bacteria may be associated with changes in microbial metabolism and the production of metabolites such as short-chain fatty acids (SCFAs) [33,34].

The inclusion of phytobiotic formulations in fattening duck diets did not significantly affect the macronutrient digestibility. Therefore, the improved growth performance (BW and BWG) observed in the phytobiotic-treated groups cannot be solely attributed to enhanced nutrient absorption [33]. The lack of significant changes in nutrient digestibility despite the improved BW and BWG may suggest that the observed productive responses cannot be explained exclusively by enhanced nutrient utilization [35]. Alternative interpretations may include improved metabolic efficiency, modulation of physiological responses, or changes in feed utilization efficiency induced by phytobiotic supplementation.

The observed increase in beneficial Lactobacilli counts together with the increase in antioxidant status may indicate a possible interaction between intestinal microbial populations and host antioxidant responses [36]. Previous studies have suggested that microbial metabolites such as SCFAs may activate antioxidant-related pathways, including Nrf2 signaling, which regulates the expression of antioxidant enzymes [37]. Also, studies have shown that the activation of AMPK-related pathways has been associated with improved growth performance [38]. Although these mechanisms were not directly evaluated in the present study, they may represent potential biological explanations for the observed findings. Such effects could partially explain why ducks fed the phytobiotic mixtures exhibited better BW and BWG, along with improved antioxidant profiles, even though nutrient digestibility and gut structure did not show significant changes. Future studies are required to evaluate whether these proposed mechanisms contribute to the observed responses in fattening ducks.

Consistent with the mechanisms described above, antioxidant status was improved in ducks receiving PM1 and PM2 groups in breast and thigh compared to control. These results may suggest that the bioactive compounds in the phytobiotic mixtures can enhance tissue antioxidant defenses, potentially by scavenging free radicals and modulating endogenous antioxidant pathways [39,40]. Enhanced antioxidant status may protect muscle lipids from peroxidation and improve overall meat quality [26,41,42].

5. Conclusions

In conclusion, the present study demonstrates that supplementation with phytobiotic formulations at 250 g/ton in fattening ducks’ diets could be a promising nutritional strategy. The findings of the present study showed improved growth performance (including BW and BWG), regulation of cecal microbial populations based on culture methods, and increased antioxidant status, and support the application of phytobiotics as sustainable alternatives to conventional growth-promoting feed additives. Future studies should elucidate different dosages and formulations and investigate the underlying molecular mechanisms responsible for the observed responses in fattening ducks.