Comparative Effects of Azadirachta indica‐Derived Silver Nanoparticles and Silver Nitrate on the Productive and Reproductive Performance of Japanese Quails

ABSTRACT

Background

Silver compounds, particularly silver nitrate (AgNO₃), have been widely studied for their effects on poultry health and reproduction. However, limited information is available regarding the impact of green synthesized silver nanoparticles (Ag‐NPs).

Objective

This study aimed to compare the effects of Ag‐NPs and AgNO₃ on productivity, egg quality, reproductive performance, and male sexual behaviour in Japanese quails.

Methods

A total of 480 Japanese quails (8 weeks old) were randomly divided into five experimental groups (96 birds each) with six replicates and a male‐to‐female sex ratio of 1:3. The control group received a basal diet, while four treatment groups were supplemented with Ag‐NPs (10 and 20 mg/kg) or AgNO₃ (10 and 20 mg/kg). The experiment lasted 9 weeks. Productive performance, egg quality traits, fertility, hatchability, and male sexual behaviours were evaluated.

Results

Quails supplemented with 20 mg/kg Ag‐NPs showed significantly improved productive performance and egg quality parameters (p < 0.05). This group also exhibited the highest fertility and hatchability rates. Additionally, male quails in the 20 mg/kg Ag‐NPs group demonstrated enhanced sexual behaviours, including wing flapping, waltzing, mounting, tidbitting, rear approach, and treading, compared to other groups.

Conclusion

Dietary supplementation with Ag‐NPs, particularly at 20 mg/kg, significantly improved productive and reproductive performance, egg quality, and male sexual behaviour compared to AgNO₃ in Japanese quails.

Supplementation with 20 mg/kg Ag‐NPs significantly improved productive performance, egg quality, fertility, hatchability, and male sexual behaviour in Japanese quails compared to AgNO₃, indicating superior efficacy of Ag‐NPs in enhancing reproductive efficiency and overall performance.

1. Introduction

Japanese Quails (Coturnix coturnix japonica) have become a common choice for game bird production due to their domestication. The lucrative nature of commercial quail farming has led to a surge in breeding programmes in recent times. The meat and eggs of quail are in high demand worldwide, with Europe and Japan primarily producing them for meat and eggs, respectively (Bagh et al. 2016). The typical range for the average live weight of a wild Japanese quail falls within the 85–110 g. Early sexual maturity can be influenced by a variety of external and internal factors. Male wild Japanese quail typically reach sexual maturity by the age of 52 days, while females typically reach it by the age of 63 days (Lukanov and Pavlova 2020). Under optimal conditions and suitable light schedules, domestic Japanese quail typically reach sexual maturity around the age of 4–5 weeks, with females beginning regular egg‐laying at the age of approximately 6 weeks (Lukanov and Pavlova 2020). Because of their rapid reproductive cycle and sensitivity to nutritional interventions, Japanese quails are frequently used as an experimental model for evaluating dietary strategies aimed at improving reproductive physiology, fertility, and egg production (Huang et al. 2024).

Recent advances in poultry nutrition increasingly focus on the use of functional additives that can enhance reproductive performance through improved nutrient utilization, oxidative balance, and endocrine regulation (Dablool et al. 2024). Trace minerals and their nano‐forms have received considerable attention because of their ability to influence metabolic pathways associated with follicular development, spermatogenesis, and embryonic development (Tharwat et al. 2025). In particular, nanotechnology has been proposed as a promising approach for improving the bioavailability and targeted delivery of micronutrients in poultry diets (Malik et al. 2023).

Silver has attracted increasing interest in animal nutrition and health due to its antimicrobial, antioxidant, and physiological properties (Rasool et al. 2024). Although silver is not traditionally considered an essential trace element in poultry diets, recent studies suggest that controlled supplementation may influence reproductive performance, egg quality, and microbial balance in the gastrointestinal tract (Satti et al. 2024; Almudayni et al. 2025; Sadan et al. 2025). Furthermore, silver has been investigated as a potential alternative to antibiotic growth promoters because of its strong antimicrobial activity and lower likelihood of inducing microbial resistance (Usman et al. 2025). Studies have indicated that appropriate silver supplementation may improve egg production, fertility, and hatchability in poultry, possibly through improved reproductive health and reduced microbial load in the reproductive tract (Al‐Khalaifah, Fatima, et al. 2025; Al‐Khalaifah, Naz, et al. 2025).

Nanotechnology has further enhanced the functional potential of trace elements by converting them into nanoparticles (NPs) with improved physicochemical properties (S. R. Khan et al. 2024). NPs possess a high surface area‐to‐volume ratio, increased reactivity, and enhanced cellular uptake, which may improve nutrient bioavailability and biological efficiency compared with conventional mineral sources (Almuzaini et al. 2024). In poultry nutrition, NPs of trace minerals have been shown to influence growth performance, egg production, immune responses, and antioxidant status (Haseeb et al. 2025; R. U. Khan et al. 2025). Importantly, these nano‐scale materials may also interact with reproductive physiology by modulating oxidative stress, hormone regulation, and gamete quality, thereby potentially improving fertility and hatchability.

Silver (Ag) is commonly added to poultry feed in two forms: organic and inorganic. The organic form improves Ag deposition in tissues and eggs while decreasing its excretion into the environment through faeces (Bashir et al. 2024). However, extreme supplementation can be harmful to animals. For the synthesis of Ag‐NPs, many approaches, such as biological or synthetic methods, have been documented (Khurana et al. 2019). Ag‐NPs have therefore emerged as a promising alternative due to their enhanced bioavailability and biological activity.

The synthesis of Ag‐NPs can be achieved through physical, chemical, and biological methods. Conventional physicochemical methods often involve the use of high energy inputs and potentially hazardous chemicals. In contrast, green synthesis approaches using plant extracts, microorganisms, or biological metabolites have gained considerable attention because they are environmentally friendly, cost‐effective, and capable of producing stable NPs with diverse structural properties (Mathur et al. 2018; Chand et al. 2018). Plant‐mediated synthesis is particularly attractive because plant extracts contain polyphenols, flavonoids, and other bioactive compounds that can act as both reducing and stabilizing agents during NP formation (Satti et al. 2025).

Plants are capable of accumulating metal ions in different tissues, enabling their extracts to facilitate the synthesis and stabilization of metallic NPs in a single step. Consequently, numerous studies have utilized plant leaf extracts for the eco‐friendly synthesis of metal and metal‐oxide NPs with applications in medicine, agriculture, and animal production. Green‐synthesized NPs are increasingly investigated in poultry nutrition due to their potential to enhance biological activity while minimizing environmental and toxicological risks.

Ag‐NPs can be synthesized using both physicochemical and biological processes depending on the reduction agents involved. In physicochemical synthesis, energy sources such as light, chemicals, ultrasound, electricity, laser irradiation, or microwave energy are applied to reduce silver ions. In contrast, biological synthesis relies on enzymes, microbial metabolites, and natural phytochemicals. Biological approaches provide both economic and ecological advantages while enabling the production of NPs with diverse morphologies and crystal structures (Dawadi et al. 2021). The biological properties of Ag‐NPs are strongly influenced by their particle size, shape, surface charge, and aggregation behaviour, which determine their stability, cellular uptake, and biological activity in living systems. Despite the growing interest in NP applications in poultry production, limited information is available regarding the specific effects of Ag‐NPs on reproductive physiology and productive performance in Japanese quails. Most previous studies have focused on growth performance or antimicrobial applications, while fewer investigations have examined reproductive traits such as egg production, fertility, hatchability, and mating behaviour. Moreover, the mechanistic links between NP supplementation and reproductive responses in quail remain insufficiently explored.

Therefore, the present study was designed to evaluate the effects of dietary Ag‐NPs on productive and reproductive performance in Japanese quails. It was hypothesized that Ag‐NP supplementation could enhance egg production, egg quality, fertility, and hatchability through improved physiological and reproductive efficiency. In addition, Ag‐NPs were expected to influence reproductive behaviour in male quails, including courtship displays and mating frequency, thereby contributing to improved overall reproductive performance.

2. Materials and Methods

2.1. Trial Birds

This experiment was conducted in accordance with the guidelines for the care and handling of laboratory animals established by Government College University Faisalabad. A total of 480 eight‐week‐old quails, with uniform body weight (185 ± 10 g) and production rate (70 ± 2%), were randomly assigned to five experimental groups, each consisted of 96 birds, further subdivided into six replicates, with 16 birds per replicate (4 males and 12 females, sex ratio 1:3). The first group served as the control and received the basal diet (Table 1). Groups 2 and 3 were supplemented with Ag‐NPs at concentrations of (10 mg/kg) and (20 mg/kg) of body weight, respectively. Groups 4 and 5 were fed with AgNO₃ at the same concentrations (10  and 20 mg/kg). All treatments were administered orally once daily via gavage to ensure accurate dosing based on weekly measured body weights. The feeding regimen consisted of a commercial basal diet formulated to meet the nutritional requirements for quails (Table 2) as recommended by the National Research Council (1994). Birds were provided feed and water ad libitum and maintained under controlled environmental conditions, with temperature ranging from 20 to 25°C and relative humidity around 70%. A 16‐h light and 8‐h dark photoperiod was maintained to simulate natural daylight and optimize reproductive cycle.

TABLE 1.

Experimental groups and dietary treatments.

Group	Treatment description	Diet supplement	Ag source	Ag dose (mg/kg)
1	Control	Basal diet only	—	0
2	Ag‐NPs 10 (mg/kg)	Basal diet + Ag‐NPs	Ag‐NPs	10
3	Ag‐NPs 20 (mg/kg)	Basal diet + Ag‐NPs	Ag‐NPs	20
4	AgNO3 10 (mg/kg)	Basal diet + AgNO3	AgNO3	10
5	AgNO3 20 (mg/kg)	Basal diet + AgNO3	AgNO3	20

TABLE 2.

Major components and nutritional contents of Japanese quail diets.

Ingredients	Contents (%)
Yellow corn	49.25
Soya bean meal	32.18
Starch	10.15
Limestone	6.50
Di‐calcium phosphate	1.16
Salt (NaCl)	0.30
Alfalfa leaf powder	0.16
Vitamin and mineral premixture a	0.30
Analyzed composition
ME (kcal/kg)	2830
Crude protein	23.63
Crude fibre	2.21
Ether extract	2.19
Calcium	2.82
Phosphorous	0.33
Methionine + cysteine	0.72
Methionine	0.44
Lysine	1.01

^a Each kilogram of vitamin and mineral mixture included the following: 10,000 IU of retinol, 3500 IU of cholecalciferol, 35 IU of tocopherol, 1.67 mg of phylloquinone, 1.67 mg of thiamine, 2 mg of riboflavin, 3.67 mg of pyridoxine, 0.012 mg of cyanocobalamin, 6.67 mg of pantothenic acid, 16.7 mg of nicotinic acid, 1.67 mg of folic acid, 0.07 mg of biotin, 400 mg of choline chloride, 0.03 mg Selenium, 133.4 g of Mg, 90 mg of Mn, 80 mg of Zn, 25 mg of Fe, 1.67 mg of Cu, and 0.8 mg of I.

2.2. Synthesis of Silver Nanoparticles

The green synthesis of Ag‐NPs was carried out using the aqueous leaf extract of Azadirachta indica (neem), renowned for its bioactive compounds and eco‐friendly reducing agents. Fresh neem leaves were collected, thoroughly washed, and shade‐dried at ambient temperature to preserve phytochemicals. The dried leaves were then ground into a fine powder. Two hundred grams of this powder was soaked in 1000 mL of 95% ethanol and left to macerate for 3 days with intermittent stirring to facilitate the extraction of the active compounds. The ethanolic extract was filtered through Whatman No. 1 filter paper, followed by concentration under reduced pressure using a rotary evaporator to obtain a viscous crude extract. This extract was then added drop wise in a 1:5 ratio to an aqueous solution of 3 mM AgNO₃, stirring continuously at room temperature. The reaction mixture was incubated in the dark for 24 h to prevent photo‐degradation and encourage the formation of Ag‐NPs, indicated by a characteristic colour change to brownish‐yellow (R. U. Khan et al. 2022).

The synthesized Ag‐NPs were characterized for their physicochemical properties using standard analytical techniques. X‐ray diffraction analysis confirmed the nature of Ag‐NPs, and Fourier‐transform infrared spectroscopy was used to identify the functional groups and biomolecules neem extract responsible for reductions in capping ions. UV‐Vis spectroscopy (300–700 nm) showed a characteristic peak around 430–450 nm, matching literature reports for neem‐based Ag‐NPs (peak at ∼429 nm in extract).

2.3. Estimation of Productive Performance

During weeks 9, 11, and 13, eggs were collected and weighed daily using a scale with a precision of 0.01 g. The total number of eggs laid each day was recorded, and all eggs were marked with a marker pen for identification (Khan et al. 2018; Abbasi et al. 2022). Egg mass was calculated using the formula:

$$\text{Egg mass}=(\text{Egg production}\times \text{Egg weight})/100$$

Hen day egg production (HDEP) was calculated as follows (Abbasi et al. 2022):

$$\text{HDEP}=(\text{Number of eggs produced}/\text{Number of live hens})\times 100$$

2.4. Estimation of Egg Quality Parameters

At the end of the ninth, 11th, and 13th weeks, 60 eggs (10 eggs/replicate) from each group were gathered to calculate the egg quality index. The sample size was selected based on standard practices in poultry nutrition studies evaluating egg quality parameters, where 8–12 eggs per replicate are commonly considered sufficient to detect biologically meaningful differences among treatments. The following parameters were studied and calculated:

Shell weight (g): After cracking the eggs, the shells were washed and dried, and the shell membrane was removed before weighing. Shell weight was recorded using a digital scale.

Shell thickness (mm): Shell thickness was measured at three different points (broad end, narrow end, and equator) using a digital micrometre, and the average value was used as shell thickness.

Albumen weight (g): Albumen weight was calculated as follows:

$$\text{Albumen Weight}=\text{Egg Weight}-(\text{Yolk Weight}+\text{Shell Weight})$$

Yolk weight (g): Each egg was carefully cracked, and the yolk was separated from the albumen without breaking the yolk sac. The yolk was weighed individually using a tared digital scale.

Haugh units: To calculate Haugh units, the height of the thick albumen was measured after cracking the egg on a flat surface. The formula used was as follows (El‐Kazaz et al. 2020):

$$\text{Haugh units}=100\times {\log }_{10}(H-1.7\times \mathrm{W}0.37+7.57)$$

Shape index: Egg length and width were measured using a calliper, and the shape index was calculated using the formula:

$$\text{Shape index}=\text{Egg width (mm)}\times 100/\text{Egg length (mm)}$$

Yolk index (%): This was calculated using the formula:

$$\text{Yolk index}(\%)=\text{Yolk height [mm]}\times 100/\text{Yolk Diameter [mm]}$$

2.5. Estimation of Reproductive Performance

During the experimental period, egg collection was carried out in the ninth, 11th, and 13th weeks of age to ensure consistent and reliable egg production. A total of 360 eggs per treatment group (60 eggs per replicate) were collected over 1 week, followed by collection in alternate weeks. Eggs were collected twice daily, preferably in the morning and late afternoon, to minimize contamination. Throughout the setter and hatcher periods, temperatures were maintained consistently at 38.5 and 37°C, respectively. Relative humidity was maintained at 65% during the setter period and 85% during the hatcher period. The total number of eggs placed in the incubator was used to determine fertility and hatchability percentages (M. M. Khan et al. 2018)

$$\text{Fertility}(\%)=\text{Number of fertile eggs}/\text{Total egg set}\times 100$$

$$\text{Hatchability}(\%)=\text{Number of hatched chicks}/\text{Total egg set}\times 100$$

2.6. Sexual Behaviour

For three consecutive weeks, sexual behaviours were continuously observed and documented, starting on the sixth week of life when signs of sexual maturity began to appear and ending on the ninth week. Sexual behaviour of quails was observed between the sixth and ninth weeks because this period typically coincides with the onset of sexual maturity. Observations of mating activity were conducted through continuous visual scanning during that period. Birds were observed for a total of 2 h each day, occurring on 3 specific days of the week, and this observation continued for a period of 3 weeks. Each day of observation was divided into two phases: morning, which spanned from 6 am to 12 pm, and afternoon, which extended from 12 pm to 6 pm. During each of these phases, the observation was carried out for a duration of 1 h, with a rotation between morning and afternoon sessions. Specifically, observations took place from 6:00 am to 12:00 pm on the first day, 8:00 am to 2:00 pm on the second day, and so on. This pattern continued for the remaining weeks. During each hour of observation, the time was divided into 5‐min intervals. Within each of these 5‐min intervals, all birds were monitored, and observations were recorded. This process continued in 5‐min intervals throughout the entire observation period for that day. The frequency of male behaviour was recorded as follows:

• Wing flapping: Wings of male raised above the level of back and flapped.

• Waltzing: Cock moving around the hen, dropping the wings farthest from her.

• Mounting: Cock steps on the hens back, grips her wing feather with his feet.

• Tidbitting: Male attracts the females by simulating the discovery of food in the litter.

• Rear approach: Cock grasping the hen's neck feathers with his beak.

• Treading: Cock makes small treading movements with his feet followed by mounting and cloacal contact (El‐Kazaz et al. 2020).

2.7. Bioaccumulation in Ovaries and Testes

Five quails each replication were humanely euthanized by cervical dislocation at the conclusion of the trial. The organs of the birds, including the testes and ovaries, were then removed through dissection. After being cleaned with deionized water to get rid of any blood, the organs were chopped into smaller pieces and kept on ice until they could be processed further for study.

2.8. Digestion of Ovaries and Testes Samples

The sample digestion process followed the standard procedure outlined by (Sarmiento‐García et al. 2022). A dry sample weighing 0.5 g was carefully measured and transferred into Teflon tubes. Following this, a concentrated mixture of HClO₄ and HNO₃ in a 1:1 ratio, totalling 6 mL, was added to the sample. The samples were vigorously shaken for a period ranging from 12 to 16 h to guarantee the full completion of the initial digestion process. Following this, the samples were positioned in a water bath maintained at 70°C for a 30‐min duration, after which they were moved to a hot plate to sustain the digestion process until the mixture achieved clarity. The solution was permitted to cool down to room temperature and subsequently poured into a volumetric flask, which was then filled with deionized distilled water (Hussein et al. 2023). Silver concentration was determined using an atomic absorption spectrometer (Aurora Al 1200) at a wavelength of 196.0 nm (Yasmeen et al. 2020).

2.9. Calculation of Silver Concentration

The concentration of silver in ovaries and testes was calculated by using the following formula:

$$\text{Metal concentration}=\text{Reading}\times \text{Dilution factor}/\text{Weight of sample}$$

In this study, the dilution factor was 25 mL for 1 g of sample.

2.10. Statistical Analysis

The effects of Ag‐NPs and AgNO₃ supplementation on productive performance, reproductive outcomes, sexual behaviour, and silver bioaccumulation in the ovaries and testes of Japanese quails were analyzed using a two‐way analysis of variance with treatment (T) and sampling time (t) as fixed factors using SPSS software (Version 21). When significant main effects were detected, means were separated using Tukey's multiple comparison test. In cases where a significant interaction between treatment and time (T × t) was observed, Tukey's post‐hoc test was applied to compare treatment means within each sampling time. Differences among means were considered statistically significant at p < 0.05.

3. Results

3.1. Effects of Ag‐NPs and AgNO₃ Supplementation on Productive Performance of Japanese Quails

Table 3 presents the effects of different silver sources on egg weight, egg mass, and HDEP in Japanese quails over time. As the experiment progressed, egg weight, egg mass, and HDEP increased across all groups. The group receiving the highest dose of Ag‐NPs (20 mg/kg) showed the greatest egg weight (12 g; p < 0.001). Similarly, this group exhibited the highest HDEP (71.33%) compared to other treatments (p < 0.001), while egg mass was non‐significant. A significant dietary treatment × time interaction was observed for all three productive performance parameters. Notably, all productivity parameters peaked in the 11th week in the high‐dose Ag‐NP group (20 mg/kg) relative to the other treatment groups (p < 0.001).

TABLE 3.

Productive performance (mean ± SEM) of Japanese quails given with low and high doses of Ag‐NPs and AgNO_3.

Performance traits	Weeks	Control	Ag‐NPs 10 (mg/kg)	Ag‐NPs 20 (mg/kg)	AgNO3 10 (mg/kg)	AgNO3 20 (mg/kg)	Total mean	SEM	p value
									T	T	T × t
Egg weight (g)	9th	10.00	10.00	12.00	9.00	8.00	10.00c	0.47	0.000	0.000	0.730
	11th	12.00	12.00	12.00	11.00	9.00	12.60a	0.47
	13th	10.00	11.00	12.00	10.00	8.00	11.40b	0.47
	Total mean	10.00c	11.00b	12.00a	10.00c	8.33d	11.33	0.47
Egg mass (g)	9th	6.96	6.97	7.24	6.81	6.87	6.97c	0.46	0.950	0.872	1.000
	11th	7.04	7.31	7.28	7.24	7.00	7.17a	0.46
	13th	6.98	7.30	7.24	6.90	6.90	7.06b	0.46
	Total mean	7.00a	7.19a	7.24a	6.98a	6.92a	7.07	0.46
HDEP %	9th	64.00	67.00	71.00	63.66	57.00	64.53c	0.89	0.000	0.002	0.109
	11th	66.00	70.00	71.66	68.00	63.33	67.79a	0.89
	13th	65.00	69.00	71.33	65.66	62.66	66.31b	0.89
	Total mean	65.33b	69.00a	71.33a	65.00b	61.22c	66.53	0.89

Note: Means within the same row or column bearing different letters (a–e) differ significantly according to Tukey's multiple comparison test (p < 0.05). When a significant interaction (T × t) occurred, Tukey's post‐hoc test was used to compare treatment means within each sampling week.

Abbreviations: Ag‐NPs, silver nanoparticles; AgNO₃, silver nitrate; SEM, standard error of the mean; T, treatment effect; t, time (week) effect; T × t, interaction between treatment and time.

3.2. Effects of Ag‐NPs and AgNO₃ Supplementation on Egg Quality of Japanese Quails

The effects of different silver sources over time on various egg‐quality parameters in Japanese quails are presented in Table 4. Birds receiving a high dose of Ag‐NPs (20 mg/kg) showed significant increases in shell weight (1.00 g), shell thickness (0.52 mm), yolk weight (4.91), Haugh unit (73.66), and Shape index (75) compared to the control and other treatments (p < 0.001), while albumin weight and yolk index were non‐significant. All parameters reached their peak values in week 11 in the high‐dose Ag‐NP group (p < 0.001). These results indicate that Ag‐NP supplementation positively influenced egg quality, with the highest effects observed at the 20 mg/kg dose.

TABLE 4.

Egg quality parameters (mean ± SEM) of Japanese quails supplemented with low and high doses of Ag‐NPs and AgNO₃.

Egg quality traits	Weeks	Control	Ag‐NPs 10 (mg/kg)	Ag‐NPs 20 (mg/kg)	AgNO3 10 (mg/kg)	AgNO3 20 (mg/kg)	Total mean	SEM	p value
									T	t	T × t
Shell weight (g)	9th	0.92	0.95	0.99	0.93	0.95	0.95c	0.10	0.824	0.390	1.000
	11th	0.94	0.97	1.02	0.95	0.98	0.97a	0.10
	13th	0.93	0.96	1.01	0.94	0.96	0.96b	0.10
	Total mean	0.93c	0.96b	1.00a	0.94bc	0.96bc	0.96	0.10
Shell thickness (mm)	9th	0.09	0.08	0.42	0.21	0.21	0.20c	2.98	0.000	0.006	0.055
	11th	0.29	0.28	0.62	0.26	0.27	0.30a	2.98
	13th	0.19	0.18	0.52	0.22	0.22	0.27b	2.98
	Total mean	0.19bc	0.18bc	0.52a	0.24b	0.25b	0.25	2.98
Albumin weight (g)	9th	3.89	3.29	4.00	3.53	4.11	3.76c	0.18	0.140	0.000	0.840
	11th	4.40	4.36	4.85	4.26	4.43	4.46a	0.18
	13th	3.96	4.29	4.45	4.15	4.40	4.25b	0.18
	Total mean	4.08	4.17	4.43	3.98	4.31	4.19	0.18
Yolk weight (g)	9th	3.40	3.05	4.80	3.00	2.75	3.40c	0.21	0.000	0.000	0.000
	11th	3.70	4.05	5.10	3.12	2.85	3.76a	0.21
	13th	3.60	4.02	4.85	3.02	2.80	3.66b	0.21
	Total mean	3.56c	3.71b	4.91a	3.05d	2.80e	3.60	0.21
Haugh unit	9th	43.84	70.46	70.55	62.93	66.25	62.81c	5.67	0.040	0.330	0.770
	11th	65.81	73.24	75.64	64.03	67.78	69.30a	5.67
	13th	43.95	71.97	74.79	63.95	65.36	64.00b	5.67
	Total mean	51.20c	71.89b	73.66a	63.63ab	66.46ab	65.37	5.67
Shape index	9th	58.00	58.00	65.00	56.00	66.00	60.06c	4.71	0.314	0.000	0.529
	11th	78.00	78.00	85.00	76.00	76.00	78.06a	4.71
	13th	68.00	68.00	75.00	66.00	75.00	70.04b	4.71
	Total mean	68.00	68.00	75.00	66.00	66.00	68.60	4.71
Yolk index	9th	37.00	39.91	49.00	31.13	26.25	36.66c	4.91	0.010	0.000	0.819
	11th	57.00	53.50	69.00	51.13	46.26	55.38a	4.91
	13th	47.00	43.25	59.00	41.13	36.25	45.33b	4.91
	Total mean	47.00ab	45.55ab	59.00a	41.13b	36.25b	45.78	4.91

Note: Means within the same row or column bearing different letters (a–e) differ significantly according to Tukey's multiple comparison test (p < 0.05). When a significant interaction (T × t) occurred, Tukey's post‐hoc test was used to compare treatment means within each sampling week.

Abbreviations: Ag‐NPs, silver nanoparticles; AgNO₃, silver nitrate; SEM, standard error of the mean; T, treatment effect; t, time (week) effect; T × t, interaction between treatment and time.

3.3. Effects of Ag‐NPs and AgNO₃ Supplementation on Reproductive Performance of Japanese Quails

The effects of different silver treatments (Ag‐NPs and AgNO₃) over time on fertility (%) and hatchability (%) in Japanese quails are summarized in Table 5. Birds receiving Ag‐NPs at 20 mg/kg showed significantly higher fertility (83.00%) and hatchability (85.00%) compared to the control and AgNO₃ groups (p < 0.001). These results indicate that Ag‐NP supplementation, particularly at 20 mg/kg, significantly improves fertility and hatchability in quails, whereas AgNO₃ and the effect of time have minimal impact.

TABLE 5.

Reproductive performance (mean ± SEM) of Japanese quails given with low and high doses of Ag‐NPs and AgNO₃.

Variables (%)	Weeks	Control	Ag‐NPs 10 (mg/kg)	Ag‐NPs 20 (mg/kg)	AgNO3 10 (mg/kg)	AgNO3 20 (mg/kg)	Total mean	SEM	p value
									T	t	T × t
Fertility	9th	75.00	77.00	80.00	72.00	69.00	73.20c	1.97	0.000	0.001	0.096
	11th	79.00	88.00	86.00	74.00	71.00	79.60a	1.97
	13th	77.00	79.00	83.00	73.00	70.00	76.40b	1.97
	Total mean	77.00bc	79.00ab	83.00a	73.00cd	70.00e	76.40	1.97
Hatchability	9th	76.00	82.00	84.00	72.00	69.00	76.06c	0.57	0.000	0.000	0.980
	11th	78.00	84.00	86.00	75.00	72.00	79.00a	0.57
	13th	77.00	83.00	85.00	74.00	71.00	78.00b	0.57
	Total mean	77.00c	83.00b	85.00a	74.00d	70.00e	77.73	0.57

Note: Means values with distinct letters (a–e) in a row exhibit a substantial variation at p < 0.05.

Abbreviations: Ag‐NPs, silver nanoparticles; AgNO₃, silver nitrate; SEM, standard error of the mean; T, treatment; t, time; T × t, treatment × time interaction.

3.4. Effects of Ag‐NPs and AgNO₃ Supplementation on the Sexual Behaviour Frequencies of Male Japanese Quails

The effects of different silver sources over time on male sexual behaviour in Japanese quails are presented in Table 6. Males supplemented with Ag‐NPs at 20 mg/kg exhibited the highest frequencies of wing flapping (84.26), waltzing (15.24), mounting (65.52), debiting (3.67), rear approach (76.38), and treading (85.38) compared to other groups (p < 0.001). A significant dietary treatment × time interaction was observed for wing flapping, tidbitting, and rear approach (p < 0.05) but not for waltzing, mounting, or treading (p > 0.05). All behavioural frequencies peaked in week 11 in the high‐dose Ag‐NP group (p < 0.001).

TABLE 6.

Sexual behaviour (mean ± SEM) of Japanese quails supplemented with low and high doses of Ag‐NPs and AgNO₃.

Sexual behaviour	Weeks	Control	Ag‐NPs 10 (mg/kg)	Ag‐NPs 20 (mg/kg)	AgNO3 10 (mg/kg)	AgNO3 20 (mg/kg)	Total mean	SEM	p value
									T	t	T × t
Wing flapping	9th	62.64	76.03	83.48	61.53	61.70	69.08c	0.072	0.000	0.251	0.000
	11th	63.92	80.91	84.82	84.82	62.80	75.45a	0.072
	13th	63.88	80.15	84.48	61.75	62.14	70.48b	0.072
	Total mean	63.48c	79.03b	84.26a	62.72c	62.21c	70.34	0.072
Waltzing	9th	9.12	12.50	16.20	11.05	10.00	11.77c	0.025	0.000	0.286	0.023
	11th	9.18	12.52	16.28	11.73	10.68	12.08a	0.025
	13th	9.16	12.50	16.25	11.35	10.26	11.90b	0.025
	Total mean	9.15e	12.50b	15.24a	11.71c	10.31d	11.58	0.025
Mounting	9th	50.15	63.25	64.82	51.39	52.25	56.37c	0.004	0.000	0.000	0.004
	11th	50.80	63.92	65.94	51.82	52.92	57.08a	0.004
	13th	50.50	63.70	65.82	52.06	52.82	56.98b	0.004
	Total mean	50.48e	63.62b	65.52a	51.75d	52.66c	56.81	0.004
Tidbitting	9th	0.00	2.00	2.00	1.00	1.00	1.20c	0.070	0.001	0.001	0.001
	11th	2.00	4.00	5.00	2.00	3.00	3.20a	0.070
	13th	1.00	3.00	4.00	1.00	2.00	2.20b	0.070
	Total mean	1.00d	3.32b	3.67a	1.32c	1.36c	2.13	0.040
Rear approach	9th	59.20	74.02	76.12	60.50	58.53	56.87c	0.176	0.000	0.000	0.059
	11th	59.81	74.50	76.76	61.98	59.78	66.57a	0.176
	13th	59.25	74.16	76.28	61.76	59.31	66.15b	0.176
	Total mean	59.42d	74.22b	76.38a	61.41c	59.20de	66.13	0.176
Treading	9th	62.55	80.52	85.05	71.00	70.02	73.83c	0.058	0.001	0.001	0.001
	11th	63.98	81.91	86.02	71.35	70.28	74.51a	0.058
	13th	63.92	81.05	85.07	71.25	70.12	74.28b	0.058
	Total mean	63.48d	81.16b	85.38a	71.20c	70.14cd	74.27	0.058

Note: Means values with distinct letters (a–e) in a row exhibit a substantial variation at p < 0.05.

Abbreviations: Ag‐NPs, silver nanoparticles; AgNO₃, silver nitrate; SEM, standard error of the mean; T, treatment; t, time; T × t, treatment × time interaction.

3.5. Effects of Ag‐NPs and AgNO₃ Supplementation on Bioaccumulation in Ovaries and Testes of Japanese Quails

The data on bioaccumulation, presented in Table 7, highlight a notable advantage of green Ag‐NPs over AgNO. The mean bioaccumulation values in ovaries and testes differed significantly among the quail groups (p < 0.05). The highest bioaccumulation was observed in the Ag‐NPs (20 mg/kg) group, with values of 0.80 in ovaries and 3.87 (µg/g) in testes, compared to the other treatments.

TABLE 7.

Bioaccumulation in ovaries and testes (mean ± SEM, µg/g) of Japanese quails supplemented with low and high doses of Ag‐NPs and AgNO₃.

Organs	Weeks	Control	Ag‐NPs 10 (mg/kg)	Ag‐NPs 20 (mg/kg)	AgNO3 10 (mg/kg)	AgNO3 20 (mg/kg)	Total mean	SEM	p value
									T	t	T × t
Ovaries	9th	0.10	0.40	0.75	0.15	0.60	0.40c	0.024	0.000	0.000	0.704
	11th	0.10	0.45	0.81	0.25	0.72	0.47b	0.024
	13th	0.11	0.51	0.86	0.31	0.79	0.66a	0.024
	Total mean	0.10e	0.45c	0.80a	0.23d	0.71b	0.46	0.024
Testis	9th	0.15	2.08	3.74	0.05	0.40	1.28c	0.048	0.000	0.000	0.000
	11th	0.15	2.10	3.93	0.27	0.52	1.39b	0.048
	13th	0.16	2.56	3.94	0.35	0.82	1.57a	0.048
	Total mean	0.15de	2.24b	3.87a	0.22d	0.58c	1.44	0.048

Note: Means values with distinct letters (a–e) in a row exhibit a substantial variation at p < 0.05.

Abbreviations: Ag‐NPs, silver nanoparticles; AgNO₃, silver nitrate; SEM, standard error of the mean; T, treatment; t, time; T × t, treatment × time interaction.

4. Discussion

In this study, the productive performance of Japanese quails was primarily analyzed, including egg weight, egg mass, and HDEP throughout the experimental period. Among all treatments, the productive performance was the highest in the Ag‐NPs (20 mg/kg) group. The improved performance observed in the Ag‐NP groups may be associated with the enhanced physicochemical properties of NPs, including increased surface area and improved bioavailability compared with conventional mineral forms (Satti et al. 2024; Al‐Khalaifah, Fatima, et al. 2025; Al‐Khalaifah, Naz, et al. 2025). However, it should be noted that antioxidant enzyme activity or endocrine parameters were not directly measured in the present study, and therefore the underlying physiological mechanisms remain speculative. The observed improvement in egg weight and egg mass may reflect improved nutrient utilization and metabolic efficiency rather than direct antioxidant stimulation. NPs have previously been reported to enhance mineral absorption and cellular uptake, which could indirectly influence ovarian follicular development and egg formation (Al Tikriti and Al‐Nassery 2023). Poultry allocates a considerable proportion of their metabolic resources toward egg formation, which directly influences egg size at oviposition. Consequently, improved nutrient utilization resulting from NP supplementation may partly explain the higher productive performance observed in the Ag‐NP treatments.

The potential role of silver in endocrine regulation has also been discussed in previous studies. Silver has been suggested to influence thyroid hormone metabolism through interactions with enzymes involved in iodothyronine regulation (Al Tikriti and Al‐Nassery 2023). However, circulating thyroid hormones (T3 or T4) were not measured in the current experiment; therefore, the involvement of thyroid‐mediated pathways cannot be confirmed and should be interpreted cautiously. From a broader physiological perspective, these findings suggest that NP‐based mineral supplementation may influence metabolic pathways involved in growth and reproduction, although additional biochemical and endocrine analyses are required to verify these mechanisms.

According to this study, egg quality parameters—shell weight, shell thickness, albumin weight, yolk weight, Haugh unit, shape index, and yolk index—showed the highest values at the 20 mg/kg dose of Ag‐NPs. These improvements may be linked to enhanced mineral utilization and improved metabolic efficiency rather than specific hormonal regulation. Supplementation with nano‐minerals has previously been associated with improved nutrient absorption and gut microbial balance, which can indirectly influence egg formation and shell mineralization (Barakat et al. 2023). Nevertheless, gut microbiota composition and digestive enzyme activity were not evaluated in the present study; therefore, such explanations remain hypothetical.

Eggshell strength was also enhanced in Ag‐NP supplemented groups. Improved shell characteristics may be related to enhanced mineral availability for eggshell formation; however, calcium metabolism and endocrine regulators of shell formation were not measured in this experiment. Key indicators of egg quality, as reported by Franco et al. (2020), include Haugh unit, albumen‐to‐yolk ratio, albumen weight and index, and egg shape index. In this study, the Haugh unit increased significantly in quails fed higher concentrations of Ag‐NPs, consistent with previous reports (Al‐Khalaifah, Fatima, et al. 2025; Al‐Khalaifah, Naz, et al. 2025). Eggs from the control group exhibited lower Haugh unit values compared to those from Ag‐NP‐supplemented groups. Improved albumen quality may reflect better protein stability or reduced degradation during egg formation; however, antioxidant enzymes such as glutathione peroxidase were not measured in this study, therefore their contribution cannot be confirmed. Compared to AgNO₃, Ag‐NPs demonstrated a greater positive impact on quail performance and egg quality (Bano et al. 2022). The superior performance of Ag‐NPs compared with AgNO₃ may be attributed to differences in bioavailability and cellular uptake between NP and ionic forms of silver.

This study revealed that fertility and hatchability rates increased progressively during the experimental period. Quails receiving the higher Ag‐NP dose (20 mg/kg) showed improved fertility and hatchability compared with other groups. Fertility rates were higher in quails receiving Ag‐NPs than in those supplemented with AgNO₃. The observed improvement in reproductive performance may be associated with improved overall physiological condition and egg quality in the NP‐treated groups. However, reproductive hormones, antioxidant enzyme activity, and ovarian histology were not measured; therefore, direct mechanisms such as GPx activation or endocrine modulation cannot be confirmed. Previous studies have suggested that trace mineral supplementation may influence reproductive performance by improving gamete quality and embryo viability (M. M. Khan et al. 2018). The hatchability percentage and fertility rate were significantly higher (p ≤ 0.05) in previous studies (Ahmed and Al‐Barzinji 2020). Fertility is one of the most important factors in Japanese quail breeding. The fertility rate of Japanese quail eggs increased at all levels of Ag supplementation in the study conducted by Pratheebha and Revathi (2018). Moreover, fertility percentage increased even midway through the trial in quails receiving Nano‐Ag. The enhanced egg fertility observed in this study is consistent with previous findings (Abbasi et al. 2022; Al‐Khalaifah, Fatima, et al. 2025; Al‐Khalaifah, Naz, et al. 2025). Throughout the trial, the control group's fertility was consistently surpassed by the Nano‐Ag treatment group. The effects were more pronounced at the higher Nano‐Ag dose (20 mg/kg) compared to the lower concentration (10 mg/kg). The study by Azam et al. (2023) also emphasized that higher Ag concentrations may produce stronger biological responses; however, further studies are required to determine the optimal dose that maximizes reproductive performance without causing potential toxicity.

This study also analyzed how exposure to green‐synthesized Ag‐NPs influenced sexual behaviour in Japanese quails. Quails exhibited dose‐dependent increases in male sexual behaviours, with the highest behavioural responses observed in birds receiving 20 mg/kg Ag‐NPs, consistent with the findings of Ahsan et al. (2024). Previous research suggested that silver supplementation may influence reproductive hormones, such as testosterone, which could affect male sexual activity (El‐Kazaz et al. 2020). However, it is important to emphasize that testosterone concentrations were not measured in the present study, and therefore behavioural changes cannot be directly attributed to hormonal alterations. Instead, improved sexual behaviour may be indirectly related to improved physiological condition or reduced microbial stress in the Ag‐NP groups. In contrast, quails receiving AgNO₃—particularly at the higher dose—showed reduced behavioural performance. This negative effect may be associated with the higher reactivity of ionic silver, which can generate oxidative stress or metabolic disturbances when administered in excess. Similar adverse effects of ionic silver compounds on reproductive behaviour have been reported in previous toxicological studies (Alwan and Al‐Saeed 2021; Tiedemann et al. 2014).

The deposition of silver was detected in the ovaries and testes of quails supplemented with Ag‐NPs, particularly at the higher dose (20 mg/kg). The present study showed significant results (p < 0.05) regarding silver deposition in both reproductive organs. The higher deposition observed in Ag‐NP treatments likely reflects enhanced absorption and tissue distribution of NPs compared with ionic silver forms. Previous research indicates that Nano‐Ag can cross biological membranes more efficiently than conventional mineral forms (Ema et al. 2017). Experimental evidence also suggests that NP supplementation results in greater tissue accumulation compared with AgNO₃. However, it is important to interpret these findings cautiously, as tissue deposition does not necessarily indicate beneficial physiological effects and may also reflect accumulation that could pose toxicological risks at higher concentrations.

The current study also revealed a positive association between ovarian silver bioaccumulation, fertility, and hatchability. Increased deposition of Ag‐NPs in the ovaries corresponded with higher fertility and hatchability rates in quails. In particular, the 20 mg/kg dosage group exhibited the highest bioaccumulation of Ag‐NPs, which coincided with improved reproductive performance compared to other groups. These findings are consistent with previous reports suggesting that NP supplementation can influence reproductive performance in poultry (Zhang et al. 2020). Nevertheless, histopathological evaluation of reproductive tissues and long‐term toxicity assessments were not conducted in this study, which limits conclusions regarding the safety of sustained silver accumulation in reproductive organs.

Several limitations of the present study should be acknowledged. First, biochemical indicators such as antioxidant enzymes (e.g., SOD, GPx), thyroid hormones (T3 and T4), and reproductive hormones were not measured, limiting mechanistic interpretation of the observed responses. Second, the experimental duration of 9 weeks may not fully capture long‐term physiological or toxicological effects of NP supplementation. Third, silver bioaccumulation analysis was conducted on a relatively small sample size, and histopathological evaluation of tissues was not performed. Finally, gut microbiota composition and immune responses were not investigated despite the proposed antimicrobial properties of Ag‐NPs.

Future research should therefore focus on integrating physiological, biochemical, and molecular analyses to clarify the mechanisms underlying NP‐mediated improvements in poultry reproduction. Studies evaluating oxidative stress biomarkers, endocrine responses, gut microbiota dynamics, and tissue histopathology are particularly needed to establish the safety and optimal dosage of Ag‐NP supplementation in poultry production systems.

5. Conclusions

The present study successfully demonstrated that Ag‐NPs boosted productive performance, reproductive performance, and sexual behaviour in Japanese quails, outperforming AgNO₃. Supplementation of 20 mg/kg improved egg weight, egg mass, HDEP, egg quality, fertility, hatchability, and mating. These findings emphasize the potential of Ag‐NPs. Silver buildup in ovaries and testes rose with dosage, showing Ag‐NPs accumulate effectively in tissues. Future research should examine the long‐term effects of Ag‐NP supplementation on quail health and productivity. Investigating optimal dosing, cost‐effectiveness, and interactions with other dietary components will be essential.

Author Contributions

Hafeez Abdul Razaq and Shabana Naz: conceptualization. Swaira Ashfaq: methodology. Muhammad Usama: software. Rifat Ullah Khan, Ala Abudabos, and Rasha Alonaizan: validation. Sania Satti: formal analysis. Nudrat Fatima: investigation. Hifza Shehzadi: resources. Swaira Ashfaq and Sania Satti: data curation. Swaira Ashfaq and Shabana Naz: writing – original draft preparation. Rifat Ullah Khan and Hafeez Abdul Razaq: writing – review and editing. Nudrat Fatima: visualization. Shabana Naz: supervision. Shabana Naz: project administration. Ala Abudabos and R.A.: funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the King Saud University, Riyadh, Saudi Arabia, through the Ongoing Research Funding Program (ORF‐2026‐591).

Ethics Statement

The Committee on Animal Rights and Welfare, GC University Faisalabad, Pakistan approved this study (GCUF/ERC/460). ChatGPT was used to improve the English language, and the resulting text was carefully reviewed and revised by the authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

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