Growth performance and physiological responses of growing japanese quail to methanolic lettuce (Lactuca sativa L.) leaf extract in drinking water

Abstract

The current study assessed the impact of lettuce leaf extract (LLE) supplementation in drinking water as a natural substitute for antibiotics on growth performance, antioxidant status, immune response, gut microbiota, and intestinal morphology in growing Japanese quail. A total of 375 nine-day-old Japanese quail chicks were randomly allocated into five experimental groups for the experiment: a negative control group, a group that received colistin, and three groups that received LLE at 1, 2, or 3 mL/L of drinking water. The supplementation continued until the chicks were 39 days old. The methanolic LLE had a lot of phenolics and flavonoids and was a strong antioxidant. In comparison to the control and colistin groups, birds that got 2- and 3-mL LLE had much higher body weight and body weight gain, as well as a better feed conversion ratio (P<0.05). LLE supplementation positively influenced serum biochemical parameters by elevating albumin levels and decreasing urea concentration (P<0.05), while preserving normal liver and kidney function. LLE also made lipid metabolism much better by lowering concentrations of cholesterol, triglycerides, and low-density lipoprotein and rising concentrations of high-density lipoprotein. The antioxidant status was significantly improved (P<0.05), as shown by higher levels of glutathione peroxidase, glutathione-S-transferase, and glutathione and lower levels of malondialdehyde. Higher levels of immunoglobulin G made the immune response better. The total bacterial count in the cecum was significantly decreased, especially at 2 mL LLE, with no negative effects on the histology of the intestines or liver. Furthermore, LLE supplementation increased the surface area of the intestines (P<0.05), which suggests that the gut was working better. Generally, addition 2–3 mL/L of LLE to drinking water enhanced quail growth, metabolic health, antioxidant resistance, immunity, and gut health.

Introduction

The continuous growth of the global population has intensified the demand for safe and high-quality poultry products. However, the extensive use of conventional antibiotics for disease prevention in poultry production has contributed to the emergence of antimicrobial resistance (AMR), a major global public health concern (Alhujaili et al., 2025). The expansion of intensive production systems has further accelerated the dissemination of antibiotic-resistant bacteria, posing significant risks to both animal and human health (Youssef et al., 2024). Consequently, considerable attention has shifted toward identifying natural alternatives to antibiotic growth promoters.

Phytogenic feed additives have emerged as promising candidates due to their capacity to modulate gut microbiota, enhance immune responses, and improve productive performance (Dosoky et al., 2024; Cufadar et al., 2024; Youssef et al., 2023). Plant-derived bioactive compounds, particularly polyphenols and flavonoids, have demonstrated antioxidant, antimicrobial, and immunomodulatory activities that support animal health (Abd El-Hack et al., 2025; Reda et al., 2026). Essential oils (EOs) have also been widely studied for their biological activities in poultry nutrition (Negm et al., 2025; Mohamed et al., 2025; Kokalj Ladan et al., 2026). Despite these advances, research remains limited regarding the potential of certain edible leafy plants as functional additives, particularly when administered through drinking water.

Lettuce (Lactuca sativa L.), a member of the Asteraceae family native to the Mediterranean region, is widely cultivated and consumed worldwide (Salem et al., 2023). Beyond its nutritional value, lettuce contains considerable amounts of bioactive compounds, including phenolic acids, flavonoids, carotenoids, and chlorophyll derivatives, which exhibit antioxidant and health-promoting properties (Lal et al., 2024). The concentration and profile of these phytochemicals vary among cultivars, with red varieties generally showing higher phenolic content than green types (Shi et al., 2022). These bioactive constituents may contribute to improved oxidative balance, enhanced immune function, and modulation of gut microbial communities.

Despite increasing interest in phytogenic additives, limited data are available on the biological effects of lettuce leaf extract (LLE), particularly when supplied via drinking water, on quail productivity and physiological responses. The primary hypothesis of the present study was that supplementation of drinking water with graded levels of LLE would improve growth performance parameters (body weight gain and feed conversion ratio) in growing quail compared with an unsupplemented control. Secondary hypotheses were that LLE supplementation would (i) enhance systemic antioxidant status, as reflected by GPx, GST, GSH, and MDA levels; (ii) modulate selected cecal bacterial populations; and (iii) improve intestinal morphometric indices, without inducing adverse effects on carcass characteristics or liver and kidney function indicators. Therefore, the present study aimed to evaluate the effects of LLE supplementation in drinking water on growth performance as the primary endpoint, while assessing carcass traits, serum biochemical indices, antioxidant capacity, immune-related parameters, cecal microbiota counts, and intestinal morphology as secondary outcome measures. The study also aimed to identify a biologically effective supplementation level within the tested range, rather than to establish LLE definitively as a replacement for antibiotic growth promoters.

Materials and methods

The experiment was performed at Zagazig University's Poultry Research Farm, part of Egypt's Faculty of Agriculture. The Ethics Committee of the Poultry Department, Agriculture Faculty, Zagazig University approved the experiments, which were conducted in accordance with the Institutional Animal Care and Use Committee's (IACUC) guidelines.

Formulation of lettuce leaf solution

Lettuce leaf extract (LLE) was obtained from 2 M Group (10^th of Ramadan City, Sharkia Governorate, Egypt). Fresh green lettuce leaves were washed with distilled water to remove debris, air-dried at room temperature (25 ± 2°C), and homogenized prior to extraction. To prepare the aqueous–ethanolic extract used for drinking water supplementation, 500 g of homogenized lettuce leaves were macerated in 1 L of 70% (v/v) ethanol–distilled water solution (analytical grade) at a plant-to-solvent ratio of 1:2 (w/v). The mixture was kept at ambient temperature (25 ± 2°C) for 24 h under continuous agitation (150 rpm) to ensure optimal extraction. The extract was then filtered using Whatman No. 42 filter paper (Hawach Scientific Co., Ltd., Xi’an, China) and stored at 4°C in amber glass bottles until use (not exceeding 72 h).

The prepared extract was freshly diluted in drinking water at the designated concentrations (1, 2, and 3 mL/L) immediately before administration to ensure stability of bioactive compounds. Supplementation commenced at 9 days of age and continued until 39 days of age.

Representative samples from each batch were analyzed for total phenolic content (TPC), total flavonoid content (TFC), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity to confirm batch-to-batch consistency and standardize the extract based on bioactive compound concentration.

Preparation of lettuce samples

For phytochemical analysis, 10 g of air-dried lettuce leaf powder were extracted with 200 mL of 70% methanol (v/v) at a ratio of 1:20 (w/v). The mixture was agitated for 3 h at room temperature (25°C) and filtered using Whatman No. 2 filter paper. Methanol was removed under reduced pressure using a BUCHI Rotavapor B-480 (BÜCHI Labortechnik AG, Switzerland) at 45°C. The concentrated extract was subsequently freeze-dried using a Thermoelectron-Heto Power Dry LL300 freeze dryer (Thermo Fisher Scientific, USA). The dried extract was stored at −20°C in airtight containers protected from light until further analysis (Ashour et al., 2020).

Evaluation of total phenolic compounds (TPCs)

The total phenolic content of the methanolic extract (1 g/mL) from all samples was measured using the Folin-Ciocalteu technique. Gallic acid (Ga), a standard phenolic complex, was applied at different levels (10-1000 mg/mL) to construct the standard curve [y = 0.001x + 0.0563 (R² = 0.9792)], where y denotes Ga absorbance and x indicate the concentration in mg/mL. Each sample (1 mL), 7.5% Na2CO3 (2 mL), and diluted Folin-Ciocalteu (3 mL) were combined and incubated in the dark at 25 °C for 30 min. Lastly, at 760 nm, the absorbance of the combination was quantified employing a spectrophotometer (JENWAY 6405 UV/Vis, U.K.) (Abdel-Shafi et al., 2019).

Estimation of total flavonoids (TF)

TF was measured using the aluminum chloride colorimetric method. One milliliter of extract was mixed with 1 mL of 2% (w/v) AlCl₃ solution in ethanol. After incubation for 15 min at room temperature, absorbance was recorded at 420 nm. Quercetin (10–1000 mg/L) was used to generate the standard curve (R² = 0.944). Results were expressed as mg quercetin equivalents (QE)/g extract (Abdel-Shafi et al., 2019).

Assessment of antioxidant activity

Antioxidant activity was evaluated using the DPPH radical scavenging assay. One milliliter of extract at various concentrations was mixed with 3 mL of freshly prepared 0.1 mM DPPH methanolic solution. After incubation in the dark for 30 min at room temperature, absorbance was measured at 520 nm. Radical scavenging activity (%) was calculated relative to a control, and SC₅₀ values were determined (Abdel-Hamid et al., 2017).

Birds, diets, and design

A completely random method was used to allocate 375 nine-day-old Japanese quail chicks, weighing equally, into five treatment groups. Each group contained five replicates (15 chicks/replicate). The negative control group (T1) received drinking water without additives, whereas the positive control group (T2) received drinking water treated with the antibiotic colistin (1 g/L). The other groups received LLE in drinking water at 1.0 mL/L (T3), 2.0 mL/L (T4), and 3.0 mL/L (T5). Colistin was included as a reference antibiotic control to compare the biological efficacy of LLE with a conventional antimicrobial agent historically used in poultry production in the region. The experiment was conducted in accordance with local veterinary regulatory guidelines. This supplementation was performed three times a week until the chicks were 39 days old. Intermittent supplementation (three times weekly) was adopted to simulate practical farm application conditions and to avoid potential overstimulation or palatability reduction associated with continuous phytogenic administration. Dose selection was guided by preliminary pilot observations and previous studies demonstrating biological efficacy of lettuce-derived phytochemicals in poultry at comparable inclusion levels (Shi et al., 2022; Soliman et al., 2024).

The chicks were subjected to a constant lighting program of 23 h of light and 1 h of darkness. The basal diets were designed according to the nutritional standards recommended by National Research Council (1994), as shown in Table 1. The basal diet had only one phase, which lasted from day 9 to 39. The chicks were grown in conventional cages measuring 1.0 × 0.75 × 0.5 m², which had continuous access to food and water.

Table 1.

Chemical and compositional evaluation of experimental diets.

Ingredients	(%)
Yellow Corn (8.8%)	57.10
Soybean meal (44%)	31.00
Gluten meal (62%)	8.00
Soybean oil	0.70
Di Calcium phosphate	1.45
Limestone	1.05
Vit-min Premix*	0.30
NaCl	0.3
DL Methionine (58%)	0.1
Total	100
Calculated analysis⁎⁎
CP%	23.51
ME Kcal/kg	2965
Ca%	0.85
P (Available)%	0.36
Lysine %	1.13
Meth + Cys. %	0.94
CF%	3.53

^⁎Growth vitamins and Mineral premix each 2 kg consists of: Vit A 12000, 000 IU; Vit D3, 2000, 000 IU; Vit. E. 10g; Vit k3 2 g; Vit B1, 1000 mg; Vit B2, 49g; Vit B6, 105 g; Vit B12, 10 mg; Pantothenic acid, 10 g; Niacin, 20 g , Folic acid , 1000 mg ; Biotin, 50 g; Choline Chloride, 500 mg, Fe, 30 g; Mn, 40 g; Cu, 3 g; Co, 200 mg; Si, 100 mg and Zn , 45 g.

^⁎⁎Calculated according to National Research Council (1994).

Data collection

Water consumption (WC) was measured daily several times for all groups in the experimental study. The water/feed ratio (W/F ratio) was measured throughout the study. To determine live body weight (LBW), individual chicks were weighed on days 9, 19, 30, and 39. Additionally, we calculated body weight gain (BWG) for the periods of 9-20, 21-30, 31-39, and 9-39 days. The mean values for feed intake (FI) and feed conversion ratio (FCR) were estimated.

Carcass traits

At the conclusion of the study, we randomly selected five chicks from each treatment group (one bird per replication) for carcass inspections at 39 days of age. The birds were starved of sustenance during the night, weighed, and slaughtered in line with Islamic rites, which entail severing the jugular vein with a sharp knife and letting it bleed for 2-4 minutes to evaluate the quality of the carcass. Furthermore, the weights of the carcass components and edible meat were recorded.

Blood biochemical parameters

Blood samples were collected during the slaughter process in non-heparinized tubes and centrifuged at 5,000 rpm for 15 min at 4°C. The acquired serum was split and stored at −20°C until biochemical assessment. Commercial kits supplied by Bio Diagnostic Co. (Giza, Egypt) were used to determine the immunological properties of the serum, including immunoglobulin G (IgG), immunoglobulin M (IgM), total protein (TP), albumin (ALBU), globulin (GLOB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, creatinine, total cholesterol (TC), triglycerides (TG), low-density lipoproteins (LDL), and high-density lipoproteins (HDL) levels. According to Abd El-Hack et al. (2017) method oxidative markers such as malondialdehyde (MDA), glutathione peroxidase (GPx), glutathione-S transporter (GST), and reduced glutathione (GSH) were identified.

Microbial analysis in cecal samples

The cecal contents were collected from quails that had been slaughtered in a sterile manner. To determine the number of bacteria in the quail cecum, 10 g of cecal contents were mixed. The contents were placed in sterile glass containers, carbon dioxide was pumped into them, and then they were placed in a deep freezer at -80°C prior to any microbial analysis. To count the bacteria, we mixed 10 g of homogenizer with 90 ml of peptone water and shook for 30 min to obtain a 10⁻¹ dilution. The samples were then diluted to 10⁻⁷. To count the total number of bacteria (TBC) and Clostridium spp. we used selective culture media according to Oxoid protocols (Oxoid Ltd., 2006).

Histopathological technique

Samples of quail intestines and livers were preserved in a 10% diluted neutral formalin solution for 24 h. With an increased amount of alcohol, it acted as a drying agent, followed by xylene I and xylene II, which are the cleaning agents. Next, we filled the members with paraffin wax to prepare paraffin molds. A microtome (Leica RM 2155, London, UK) was used to cut the paraffin wafers to a thickness of 5 µm. The tissue sections were stained with hematoxylin and eosin (H&E) for histological examination (Al-Aziz et al., 2025).

The length (VL), width (VW), and absorption surface area (ASA) of the intestinal villi were assessed. The parameters were measured from 50 well-oriented and intact villi per intestinal section, and the mean value for each parameter was calculated per bird. Villus height was measured from the apex to the base at the villus–crypt junction, and villus width was measured at the midpoint of the villus height. Tissue sections were examined using a light microscope equipped with a high-resolution digital camera and image analysis software (Leica Microsystems, Wetzlar, Germany). ASA was calculated using the formula: ASA (mm²) = 2π × (VW/2) × VL (Rehman et al., 2016).

Statistical Analysis

The data were analyzed using one-way ANOVA with a completely randomized scheme, using SPSS software (SPSS, 2019). We then used Duncan’s post-hoc test (Duncan, 1955) to detect differences between the means, and the significance of the results was assessed using a significance level of P<0.05. Using orthogonal polynomial contrasts, the linear (L) and quadratic (Q) consequences of increasing LLE levels were computed. Individual birds were utilized for blood and carcass analyses, and the cage was used as the growth performance experimental component. We employed the following statistical model:

$$\text{Yij}=\mu +\text{Ti}+\text{eij}$$

Where Yij = Detected value for the affected treatment, μ = Detected mean for the affected treatment, Ti = Treatment effect, and eij = Error associated with separate observation.

Results

Phytochemical content and antioxidant capacity of lettuce leaf extract

Table 2 demonstrations that the methanolic lettuce leaf extract (LLE) had a high total phenolic content (233.66 mg GAE g⁻¹ extract) and a high TF content (38.33 mg QE g⁻¹ extract). The extract exhibited significant antioxidant activity, evidenced by a concentration-dependent enhancement in DPPH radical scavenging activity, achieving 79.33% inhibition at 1000 mg/ml.

Table 2.

Total phenolic (TP), and total flavonoid contents (TF), and 2,2-diphenyl1-picryl-hydrazyl-hydrate (DPPH) activity of the methanolic extract acquired from lettuce leaf extract.

Items	Lettuce leaf extract content
TP (mg GAE g-1 extract)	233.66
TF (mg QE g-1 extract)	38.33
DPPH activity (SC50; mg mL-1) at different concentrations
100	15.66
250	25.33
500	55.66
1000	79.33

100, 250, 500, 1000 = concentrations of extract (mg/mL).

Growth, water consumption and water/feed ratio

Table 3 displays how adding LLE to drinking water influences growth, feed intake, WC, and the W/F ratio. At 9 days of age, there was no variation linearly or quadratically in LBW between treatments (P>0.05). Nevertheless, there was a considerable change starting on day 19 (P=0.000). On 19, 30, and 39 days, birds that got 2 or 3 mL of LLE/L water had much higher linearly and quadratically LBW than those in the control and colistin groups. The 3 mL LLE group had the highest final body weight.

Table 3.

Influences of lettuce leaf extract (LLE) supplementation in drinking water on growth performance, water intake, and water-to-feed ratio of growing quail.

Items	Treatments (ml / L water)					SEM	P value*
	Control	Colistin	1 ml LLE	2 ml LLE	3 ml LLE		T	L	Q
Live body weight (g)
9 days	43.01	42.97	42.92	42.94	43.02	0.07	0.997	0.976	0.731
19 days	119.81b	114.38d	117.99c	122.15a	123.02a	0.66	0.000	0.000	0.000
30 days	187.56c	192.55ab	186.27c	190.60b	193.97a	0.71	0.000	0.003	0.000
39 days	237.08c	244.09b	237.75c	238.79c	248.93a	1.09	0.000	0.000	0.011
Body weight gain (g/bird/day)
9-19 days	6.98b	6.49d	6.82c	7.20a	7.27a	0.06	0.000	0.000	0.000
20-30 days	6.16b	7.11a	6.21b	6.22b	6.45b	0.08	0.000	0.000	0.000
31-39 days	5.50b	5.73ab	5.72ab	5.35b	6.11a	0.08	0.018	0.021	0.025
9-39 days	6.26c	6.49b	6.28c	6.32c	6.64a	0.03	0.000	0.000	0.011
Feed intake (g/bird/day)
9-19 days	18.84a	17.78b	17.75b	18.85a	18.92a	0.14	0.001	0.001	0.018
20-30 days	23.91	25.55	24.35	23.33	24.39	0.29	0.178	0.539	0.499
31-39 days	35.28a	34.70a	31.94b	32.44b	34.24a	0.31	0.000	0.004	0.001
9-39 days	26.01a	26.01a	24.68b	24.87b	25.85a	0.15	0.003	0.042	0.022
Feed conversion ratio (g feed/g gain)
9-19 days	2.70	2.74	2.60	2.62	2.60	0.01	0.078	0.323	0.184
20-30 days	3.89	3.60	3.92	3.75	3.78	0.05	0.214	0.869	0.817
31-39 days	6.45a	6.07ab	5.61b	6.08ab	5.61b	0.10	0.041	0.010	0.016
9-39 days	4.16a	4.01ab	3.93b	3.94b	3.89b	0.02	0.011	0.001	0.007
Water consumption (ml/bird/day)
9-19 days	48.97a	46.89b	45.45c	49.98a	50.10a	0.41	0.000	0.002	0.000
20-30 days	70.03a	66.20ab	63.27b	69.05a	68.71a	0.70	0.015	0.007	0.049
31-39 days	104.30a	103.18ab	98.16c	98.73bc	102.82ab	0.78	0.021	0.007	0.009
9-39 days	74.44a	72.09a	68.96b	72.59a	73.87a	0.49	0.001	0.000	0.044
Water / feed ratio
9-19 days	2.60	2.64	2.56	2.65	2.65	0.02	0.482	0.430	0.598
20-30 days	2.93a	2.60b	2.61b	2.96a	2.82ab	0.04	0.006	0.003	0.021
31-39 days	2.96	2.98	3.08	3.05	3.00	0.02	0.717	0.377	0.372
9-39 days	2.86	2.77	2.80	2.92	2.86	0.01	0.070	0.272	0.217

^a,b,cMeans within the same row with different superscripts are significantly different (P≤0. 05).

^⁎T, overall impacts of treatments; L, linear impacts of increasing LLE levels of broiler; Q, quadratic impacts of increasing LLE levels of broiler.

LLE supplementation significantly linearly and quadratically boosted BWG during many growth periods (P<0.05). The 3 mL LLE group had the most overall weight gain during the whole experiment (9–39 days), followed by the 2 mL LLE group. Throughout the starter (9–19 days) and finisher (31–39 days) phases (P<0.01), FI was greatly affected. But there were no differences during the grower phase (20–30 days). The overall feed intake (9–39 days) was markedly reduced in birds administered 1- and 2-mL LLE in comparison to the control group.

FCR didn't change much during the early growth phases, but it did get a lot better during the finisher phase and throughout the entire experiment (P<0.05). Birds that got 1–3 mL of LLE had a superior overall FCR than the control, and the 3 mL LLE groups had the lowest value.

Water consumption was considerably affected linearly and quadratically by treatment throughout most experimental periods (P<0.05). Birds receiving 1 mL LLE consumed the least amount of water overall, whereas the control and higher LLE levels appeared higher water intake. The water-to-feed ratio was largely unaffected, except during the grower phase (20–30 days), where considerable differences were observed (P<0.01).

Carcass traits

Carcass traits are exhibited in Table 4. Pre-slaughter weight and carcass proportion were not considerably concerned by therapies (P>0.05). Heart and spleen comparative weights also presented no significant variations among treatments. In contrast, liver comparative weight was considerably raised linearly and quadratically in all LLE-treated groups and the colistin group (P<0.01). Gizzard proportion was significantly influenced by therapy (P<0.05), with the highest value detected in birds receiving 2 mL LLE/L water.

Table 4.

Influences of lettuce leaf extract (LLE) supplementation in drinking water on carcass traits of growing quail.

Items	Treatments (ml / L water)					SEM	P value*
	Control	Colistin	1 ml LLE	2 ml LLE	3 ml LLE		T	L	Q
Pre-slaughtered weight	250.00	254.40	242.40	227.00	269.20	1.80	0.077	0.724	0.074
Carcass %	73.66	69.41	71.98	74.04	68.84	0.82	0.163	0.378	0.744
Heart %	0.96	0.95	0.91	0.88	0.92	0.01	0.576	0.210	0.680
Liver %	1.75b	2.67a	2.80a	2.64a	2.95a	0.12	0.005	0.002	0.047
Gizzard %	1.40bc	1.63ab	1.51abc	1.70a	1.38c	0.03	0.031	0.016	0.008
Spleen %	0.07	0.05	0.07	0.06	0.07	0.01	0.540	0.883	0.464

^a, b, c Means within the same row with different superscripts are significantly different (P≤0. 05).

^⁎T, overall impacts of treatments; L, linear impacts of increasing LLE levels of broiler; Q, quadratic impacts of increasing LLE levels of broiler.

Blood biochemical parameters

Blood biochemical indicators are displayed in Table 5. Treatment did not have a significant effect on TP, GLOB, albumin/globulin ratio (A/G), or creatinine levels (P>0.05). The concentration of albumin, conversely, was considerably greater linearly and quadratically in birds that got 2 or 3 mL of LLE than in the control and colistin groups (P<0.01).

Table 5.

Influences of lettuce leaf extract (LLE) supplementation in drinking water on blood biochemical parameters of growing quail.

Items	Treatments (ml / L water)					SEM	P value*
	Control	Colistin	1 ml LLE	2 ml LLE	3 ml LLE		T	L	Q
Liver and kidney functions
TP (mg/dL)	6.65	6.72	6.76	6.76	6.85	0.02	0.327	0.146	0.830
ALB (mg/dL)	3.10b	3.09b	3.10b	3.15a	3.15a	0.01	0.011	0.002	0.041
GLOB (mg/dL)	3.55	3.63	3.66	3.61	3.70	0.02	0.565	0.187	0.877
A/G (%)	0.88	0.85	0.85	0.87	0.85	0.01	0.641	0.594	0.601
ALT (IU/L)	11.82bc	12.15ab	12.43a	11.72c	12.23a	0.07	0.006	0.004	0.002
AST (IU/L)	145.65ab	147.07a	145.82ab	141.85c	142.54bc	0.61	0.018	0.005	0.029
Urea (mg/dL)	43.78a	40.16b	36.08c	39.06b	37.99bc	0.59	0.000	0.000	0.001
Creatinine (mg/dL)	0.70	0.68	0.63	0.70	0.68	0.01	0.644	0.487	0.447
Lipid profile
TC (mg/dL)	62.15a	61.78a	59.94a	55.80b	54.90b	0.69	0.000	0.000	0.017
TG (mg/dL)	79.37a	78.50a	77.21a	59.09b	52.55c	1.31	0.000	0.000	0.000
HDL (mg/dL)	30.39c	31.34c	33.65b	33.65b	35.68a	0.40	0.000	0.000	0.001
LDL (mg/dL)	16.21a	14.73a	10.62b	11.14b	9.90b	0.50	0.000	0.001	0.000

^a, b, c Means within the same row with different superscripts are significantly different (P≤0. 05).

^⁎T, overall impacts of treatments; L, linear impacts of increasing LLE levels of broiler; Q, quadratic impacts of increasing LLE levels of broiler.

TP: total protein; ALB: albumin; GLOB: globulin; A/G: albumin/globulin ratio; AST: aspartate aminotransferase; ALT: alanine aminotransferase; TC: total cholesterol; TG: triglycerides; HDL: high-density lipoprotein; and LDL: low-density lipoprotein.

LLE supplement had a big effect on liver enzyme activities. The 1- and 3-mL LLE groups had higher ALT activity, but the 2- and 3-mL LLE groups had much lower AST activity than the colistin treatment. The concentration of urea was considerably lower linearly and quadratically (P<0.001) in all groups treated with LLE, especially in 1 mL of LLE group, contrasted to the control group. In terms of lipid profile, birds that got 2- and 3-mL LLE had much reduced concentrations of TC and TG (P<0.001). As the level of LLE went up, the level of HDL went up too. The highest level was in the 3 mL LLE group. Conversely, the concentration of LDL decreased considerably, in wholly the LLE groups compared to the control.

Antioxidant status and immunoglobulin levels

Antioxidant parameters and immunoglobulin concentrations are exhibited in Table 6. LLE supplementation considerably improved antioxidant enzyme activities linearly and quadratically. GPx, GST, and GSH concentrations increased considerably with cumulative LLE concentration (P<0.001), while MDA level was considerably decreased in all LLE groups in comparison to the control.

Table 6.

Influences of lettuce leaf extract (LLE) supplementation in drinking water on antioxidant parameters and immunoglobulin levels of growing quail.

Items	Treatments (ml / L water)					SEM	P value*
	Control	Colistin	1 ml LLE	2 ml LLE	3 ml LLE		T	L	Q
Antioxidant parameters
GPx (U/mg protein)	169.22b	162.86c	171.20b	172.20ab	176.40a	1.10	0.000	0.000	0.036
GSTs (U/mg protein)	6.74c	7.68a	7.43b	7.72a	7.93a	0.09	0.000	0.000	0.003
GSH (U/mg protein)	74.50c	74.92c	78.27b	79.96a	80.82a	0.55	0.000	0.000	0.031
MDA (nmol/ml)	5.35a	5.05a	4.31b	4.16b	3.71b	0.15	0.001	0.000	0.000
Immunoglobulin levels
IgG (mg/dL)	220.14b	224.51ab	230.90ab	234.54a	235.73a	1.95	0.043	0.003	0.003
IgM (mg/dL)	18.66	19.53	16.37	20.17	20.45	0.72	0.299	0.417	0.308

^a,b, cMeans within the same row with different superscripts are significantly different (P≤0. 05).

^⁎T, overall impacts of treatments; L, linear impacts of increasing LLE levels of broiler; Q, quadratic impacts of increasing LLE levels of broiler.

GPx: Glutathione Peroxidase; GST: Glutathione S-Transferase; GSH: Reduced Glutathione; MDA: Malondialdehyde; IgG: immunoglobulin G; and IgM: immunoglobulin M.

Immunoglobulin G concentrations were considerably higher in birds receiving 2- and 3-mL LLE (P<0.05), while IgM concentrations were not significantly influenced by treatment.

Cecal microbial counts

The impacts of LLE on cecal microbial populations are revealed in Fig. 1. Total bacterial count (TBC) was considerably decreased linearly and quadratically in the 2 mL LLE group (Fig. 1A) contrasted to the control and 1 mL LLE groups (P<0.05). Clostridium counts were numerically reduced in LLE-treated groups; nevertheless, these differences were not statistically significant, linearly or quadratically (Fig. 1B).

Fig. 1.

Influences of lettuce leaf extract (LLE) supplementation in drinking water on cecal bacterial count of growing quail.

(A) Total bacterial count (TBC; log10 CFU/g cecal content); (B) Clostridium spp. count (log10 CFU/g cecal content). Data are presented as means ± SEM (n = 5 birds per treatment). Treatments included control (no additive), colistin (1 g/L), and LLE at 1, 2, and 3 mL/L of drinking water. Means with different letters differ significantly according to one-way ANOVA followed by Duncan’s multiple range test (P≤0.05). For TBC (A), the overall treatment effect was significant (P<0.05), with significant linear and quadratic responses to increasing LLE levels (P<0.05), showing the lowest bacterial count in the 2 mL LLE group compared with the control and 1 mL LLE groups. For Clostridium spp. (B), numerical reductions were observed in LLE-treated groups; however, neither overall treatment effect nor linear or quadratic contrasts reached statistical significance (P>0.05).

Intestinal morphology

Histological analysis of intestinal sections from all experimental groups (Fig. 2) demonstrated a normal architectural arrangement of the mucosal villi, submucosa, and muscular layers. We did a quantitative histomorphometric analysis of VL, VW, and ASA for all groups (Table 7). The control and colistin groups had the lowest histomorphometric rates (Fig. 2A and B). Conversely, birds receiving 1 mL LLE demonstrated the most considerable enhancement in intestinal morphology, marked by improved villus structure and the largest absorption surface area among all treatments (Fig. 2C). There were no considerable variations in villus length and width among treatments (P>0.05). However, the absorption surface area was significantly greater linearly and quadratically in birds receiving 1- and 2-mL LLE compared to the control group (P<0.01). A progressive reduction in intestinal morphometric parameters was recorded at raised supplementation levels (2- and 3-mL LLE; Fig. 2D and E) in comparison to the 1 mL LLE group.

Fig. 2.

Photomicrograph of H&E-stained sections of intestine of Quails (scale bar 200μm) showing: A-E: normal histological structures of mucosal villi, submucosa and muscular layer at all examined groups. A, B: the least values of histomorphometry at both groups 1 and 2 between all groups. C: The best improved group among all treated groups at group.3. D-E: Gradual decline of values of intestinal parameters at group.4 then group.5 when compared to group.3.

Table 7.

Influences of lettuce leaf extract (LLE) supplementation in drinking water on intestinal morphology of growing quail.

Items	Treatments (ml / L water)					SEM	P value*
	Control	Colistin	1 ml LLE	2 ml LLE	3 ml LLE		T	L	Q
Villous length (VL) μm	567.00	711.00	783.50	677.67	814.67	1.47	0.061	0.214	0.143
Villous width (VW) μm	117.00	103.00	130.50	135.00	105.33	1.40	0.222	0.144	0.118
Absorption surface area (ASA) "mm2"	0.06d	0.07c,d	0.10a	0.09a,b	0.08b,c	0.01	0.008	0.008	0.014

^{a, b, c}Means within the same row with different superscripts are significantly different (P≤0. 05).

^⁎T, overall impacts of treatments; L, linear impacts of increasing LLE levels of broiler; Q, quadratic impacts of increasing LLE levels of broiler.

Histological assessment of liver segments (Fig. 3A–E) revealed normal hepatic architecture through all experimental groups, characterized by well-organized hepatic cords, central veins, sinusoids, and Kupffer cells. Hepatocytes demonstrated polygonal morphology with centrally positioned nuclei, and mild intracytoplasmic vacuolations were mainly noticed in the controls, representative of the absence of detrimental hepatic changes associated to LLE supplementation.

Fig. 3.

Photomicrograph of H&E-stained sections of liver of Quails (scale bar 20μm) showing: A-E: normal histology of hepatic cords, kupffur cells, sinusoids, and central veins. The hepatocytes polygonal in shape with centrally located nuclei with intracytoplasmic vacuolations primarily at group.1 (A). Hepatocytes (HP), patent hepatic blood vessels (arrows).

Discussion

The current investigation exhibits that the addition of LLE to drinking water produces various beneficial influences on growth, physiological state, antioxidant defense, immune response, gut microbiota, and intestinal morphology in growing quail. These inspirations are primarily dose-dependent and appear to be boosted by the increased phenolic and flavonoid content, as well as the strong antioxidant properties of LLE (Shi et al., 2022).

The enhancement in growth performance, especially at 2–3 mL/L, may be ascribed to improved nutrient absorption and metabolic efficiency facilitated by plant-derived polyphenols. Not all performance parameters were consistently influenced across growth phases; however, the overall improvement in body weight gain and feed conversion ratio indicates enhanced feed efficiency rather than increased feed intake. This corroborates earlier research demonstrating that phytogenic compounds can augment digestive enzyme activity, promote gut health, and diminish subclinical inflammation (Hashemi and Davoodi, 2011; Adedokun and Olojede, 2019; Soliman et al., 2024). Lettuce supplementation has also been linked to similar improvements in growth. Consequently, the observed responses indicate a moderate yet biologically significant enhancement, characterized by a distinct dose-dependent trend.

The physiological responses documented in this study reinforce the functional significance of LLE. The bioactive components of lettuce, such as β-carotene, lutein, phenolics, and flavonoids, are recognized for their ability to alleviate oxidative stress and enhance cellular function (Yang et al., 2022). Differences in water intake between treatments may indicate slight metabolic or osmotic changes caused by polyphenols (Guerreiro et al., 2022). The small effect on the water-to-feed ratio shows that LLE did not negatively affect hydration balance or feeding behavior.

Carcass characteristics remained largely unaffected, indicating that LLE supplementation did not induce undesirable morphological or metabolic alterations. The small rise in liver weight may be due to increased metabolic and detoxification activity, which is in line with the role of plant extracts high in antioxidants (Negm et al., 2025). The rise in gizzard weight at moderate levels of supplementation may also suggest better digestive function, which could help explain why feed efficiency improved. These results align with prior studies indicating no adverse impacts of phytogenic additives on carcass characteristics (Pourmahmoud et al., 2013).

The biochemical indicators of blood serum confirm the physiological benefits of the LLE. Elevated ALBU content in birds treated with 2 and 3 ml of LLE indicates an improvement in protein synthesis and liver function. ALBU is a sensitive indicator of nutritional and metabolic health, with high concentrations indicating increased anabolic activity (Kosmadakis, 2025). Lower serum urea concentrations in LLE-treated groups led to improved protein uptake and reduced nitrogen waste, along with increased feeding efficiency. Modulation of liver enzyme activity following lettuce leaf extract supplementation suggests a potential influence on hepatic function, possibly related to its antioxidant constituents, which are known to stabilize cellular membranes and mitigate oxidative damage (Ganesan et al., 2018). A study by Long et al. (2018) revealed elevated ALBU levels, along with a tendency toward increased GLOB and TP, when various vegetable oils were used compared to a control group. Lettuce contains numerous antioxidants and bioactive phytochemicals that may support tissue resilience under oxidative stress conditions. Oxidative stress is a recognized contributor to liver injury, as it promotes lipid peroxidation and cellular damage (Allameh et al., 2023). In the present study, LLE significantly enhanced antioxidant defense markers, including GPx, and reduced lipid peroxidation as reflected by lower MDA levels, which may contribute to maintaining hepatic cellular stability (Bestwick et al., 2001). Importantly, histopathological examination of liver tissues did not reveal structural abnormalities in LLE-supplemented groups, supporting the absence of over-hepatic damage at the tested levels. Therefore, the combined biochemical and histological findings suggest that LLE did not induce pathological liver alterations under the experimental conditions; however, the observed ALT elevation in certain groups indicates that further investigation—particularly under long-term or higher-dose exposure—is required before definitively concluding hepatoprotection.

The improvement in lipid metabolism observed with lettuce leaf extract supplementation may be attributed to its rich polyphenolic content. Dietary polyphenols have been reported to influence lipid metabolism and improve serum lipid profiles in poultry and other animal models (Chambers et al., 2019). Similar findings were reported by Suleiman et al., who documented reduced total cholesterol, triglycerides, and low-density lipoprotein levels in broiler chickens supplemented with lettuce oil. Gomosh et al. also demonstrated that dietary oils can lower total cholesterol and LDL concentrations in poultry.

A central finding of this study is the enhancement of antioxidant status, as evidenced by increased GPx, GST, and GSH levels and decreased MDA concentrations. These results confirm that LLE effectively reduces oxidative stress and lipid peroxidation. The mechanism is likely related to both direct free radical scavenging and activation of endogenous antioxidant systems, including Nrf2-related pathways (Adesso et al., 2016; Song et al., 2020; Soliman et al., 2024). Improved oxidative balance may also contribute to better growth performance and immune competence.

Elevated IgG immunoglobulin concentrations in birds receiving high concentrations of LLE indicate an enhanced humoral immune response. It is known that polyphenols affect immune cell activity, cytokine production, and antibody synthesis, thereby enhancing immune surveillance (Yahfoufi et al., 2018). The lack of substantial changes in IgM levels may indicate that lettuce seed extract primarily promotes long-term adaptive immunity, rather than acute immune responses. The biologically active substances found in lettuce or its seed oil can alter the mechanism of action of the immune system. Adding lettuce seed oil to broiler chicken diets has been shown to raise blood levels of immunoglobulins IgG and IgM, along with complement component C3 and lysozyme activity, compared to control groups (Soliman et al., 2024).

The reduction in total bacterial counts following LLE supplementation suggests a modulatory effect on intestinal microbiota, likely driven by its phenolic constituents. Phenolic compounds can disrupt bacterial cell membranes and interfere with pathogen metabolism, thereby suppressing harmful microbes while exerting limited effects on beneficial populations (Ecevit et al., 2022). Although the decrease in Clostridium spp. was not statistically significant, the observed trend supports the potential antibacterial activity of lettuce-derived bioactives. Natural phytogenic additives are increasingly recognized for their role in promoting gut microbial balance in poultry (Abdelli et al., 2021; Rafeeq et al., 2023). Previous studies have demonstrated the antibacterial properties of plant-derived extracts and essential oils against pathogens such as E. coli (Mohammadi et al., 2014; Cetin-Karaca and Newman, 2015; Omer et al., 2018). Plant bioactives may enhance intestinal homeostasis through increased production of lactic acid and short-chain fatty acids, creating an environment less conducive to pathogen proliferation. Similar microbiota-modulating effects of plant extracts in broiler chickens have been reported by Ding et al. (2022).

A limitation of the present study is the reliance on culture-based enumeration methods, which provide quantitative information on selected bacterial groups but do not fully characterize microbial diversity. Future studies should employ molecular approaches such as 16S rRNA gene sequencing to provide comprehensive insights into microbiome modulation.

Enhancing intestinal surface area in birds with 1 and 2 mL of LLE extract improved intestinal function and nutrient absorption. Higher LLE levels led to decreased benefits, indicating that moderate supplementation is preferable. Histological examinations confirmed the safety and intact structure of the intestinal and liver tissues across all treatments (Shi et al., 2022). The histopathological evaluation was descriptive and lacked a blind scoring system, which may limit objectivity. Future studies should incorporate standardized, blinded assessments to improve reliability. Intestinal morphology indicators such as villus height (VH), crypt depth (CD), and VH:CD ratio are important measures of nutrient absorption and gut health (Attia et al., 2017). The antioxidant phytochemicals found in lemon leaf extract protect intestinal epithelial cells from oxidative damage, thus maintaining intestinal health. Bioactive substances may stimulate intestinal cell proliferation and differentiation, promoting the maturation and absorption capacity of the epithelial layer (Santhiravel et al., 2022).

Conclusion

The current findings collectively indicate that LLE functions through antioxidant, hypolipidemic, hepatoprotective, immunomodulatory, and gut health-promoting mechanisms to enhance performance and physiological status in growing quail under the controlled conditions of the present experiment. The most consistent improvements were observed at supplementation levels of 2–3 mL LLE/L of drinking water.

Data availability

Data will be available upon reasonable request from the corresponding authors.

CRediT authorship contribution statement

Mohammed A. Agha: Writing – review & editing, Writing – original draft, Methodology, Investigation, Data curation. Elwy A. Ashour: Writing – review & editing, Methodology, Investigation, Data curation, Conceptualization. Mohamed M. El-Makkawy: Writing – original draft, Investigation, Formal analysis, Data curation, Conceptualization. Islam M. Youssef: Writing – review & editing, Writing – original draft, Visualization, Validation, Formal analysis, Data curation. Ahmed Shabaan: Writing – original draft, Visualization, Validation, Software, Methodology, Investigation. Ali O. Osman: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Hanan M. Alharbi: Writing – review & editing, Writing – original draft, Software, Project administration, Funding acquisition. Khairiah M. Alwutayd: Writing – review & editing, Writing – original draft, Resources, Funding acquisition, Data curation. Mohammad M.H. Khan: Writing – review & editing, Writing – original draft, Visualization, Validation, Data curation, Conceptualization. Mohamed E. Abd El-Hack: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Data curation, Conceptualization.

Disclosures

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.