Thyme Essential Oil and Hyaluronic Acid as Natural Disinfectant Alternatives for Broiler Breeder Fertile Eggs: Effects on Eggshell Microbial Load, Embryonic Viability and Hatchability

ABSTRACT

Background

Eggshell microbial contamination of fertile hatching eggs represents a major biosecurity concern in commercial poultry hatcheries because it can compromise embryonic viability, hatchability and chick quality. In commercial hatchery practice, broiler breeder fertile eggs are often stored before incubation to synchronize production schedules. However, prolonged storage may increase microbial contamination of the eggshell, deteriorate internal egg quality and reduce embryonic viability and hatchability.

Objectives

This study evaluated thyme essential oil and hyaluronic acid (HA) as natural alternatives to chemical disinfectants for sanitation of broiler breeder fertile eggs.

Methods

A total of 1800 Ross 308 hatching eggs were randomly assigned to four main treatments: untreated control, thyme essential oil (1%) emulsified with polysorbate‐20 (PS20), HA (0.1%) and thyme essential oil + HA. Eggs were stored for 0 or 14 days in a 4 × 2 factorial design. A PS20‐only vehicle control was included for selected comparisons.

Results

All sanitization treatments significantly reduced eggshell total aerobic bacterial counts compared with the untreated control (p < 0.001). Storage for 14 days impaired internal egg quality, reducing albumen ratio, albumen index, yolk index and Haugh unit values, while increasing yolk ratio and albumen pH (p < 0.001). Storage also reduced blastoderm viable cell number and increased dead cell number (p < 0.001). Thyme essential oil produced the most favourable outcomes after storage, with higher hatchability of set eggs and hatchability of fertile eggs and lower embryonic mortality than HA‐treated eggs (p < 0.05). Yolk malondialdehyde (MDA) levels were lower in the thyme group than in the control and HA groups after storage (p < 0.01). Although HA reduced microbial load, it was associated with increased blastoderm dead cell counts and higher embryonic mortality after storage.

Conclusions

Thyme essential oil appears to be a promising residue‐free and biologically safe alternative to conventional chemical disinfectants for hatchery sanitation of broiler breeder fertile eggs.

Natural eggshell sanitation using thyme essential oil reduced microbial contamination and lipid peroxidation while improving embryo viability and hatchability in stored broiler breeder fertile eggs. These findings support thyme essential oil as a promising residue‐free alternative for hatchery egg sanitation.

1. Introduction

Egg storage is a common practice in the hatchery industry to synchronize setting schedules and optimize incubator utilization. Nevertheless, extended storage negatively affects internal egg quality and reduces embryo viability, leading to lower hatchability and poorer chick quality (Tona et al. 2003). These adverse effects are mediated by multiple mechanisms, including water loss and albumen quality deterioration, pH changes and increased vulnerability of the embryo at the blastoderm stage. In parallel, microbial contamination of the eggshell may intensify during handling and storage and constitutes a major biosecurity concern because surface microorganisms can penetrate the shell and membranes, contributing to embryonic mortality and post‐hatch performance losses. Consequently, eggshell sanitation prior to incubation is considered an essential hatchery practice (Yu et al. 2025; Yuan et al. 2022). Eggshell contamination can originate from trans‐ovarian and trans‐oviductal routes, but post‐oviposition environmental exposure is often the dominant source, including contact with litter, feathers, equipment and suboptimal hygiene during egg collection and storage (Board and Tranter 1995; Berrang et al. 1999).

Chemical disinfectants, particularly formaldehyde‐based sanitation practices, have traditionally been used to control eggshell microbial contamination in hatching eggs. However, chemical fumigants may pose occupational and environmental concerns and, more importantly for fertile eggs, may adversely affect embryonic cells when exposure conditions are not properly controlled. Therefore, alternative sanitation approaches should not only reduce eggshell microbial load but also preserve eggshell function and embryonic viability.

Plant‐derived essential oils have attracted increasing attention as natural disinfectants due to their antimicrobial and antioxidant properties. Thyme (Origanum onites L.) essential oil is a promising candidate because of its broad antimicrobial activity, largely attributed to phenolic constituents such as carvacrol and thymol (Fachini‐Queiroz et al. 2012; Hosseinzadeh et al. 2015; Shahein and Sedeek 2014). Recent reports indicate that thyme‐derived products can suppress eggshell‐associated microorganisms and poultry‐relevant pathogens, supporting their potential use in hatchery sanitation (Giovagnoni et al. 2023; Haraz et al. 2022).

Another potential strategy involves the application of barrier‐type biopolymers that may reduce contamination by forming a protective film on the eggshell surface. Hyaluronic acid (HA) is a naturally occurring polysaccharide composed of repeating D‐glucuronic acid and N‐acetylglucosamine units and has been explored as a surface coating material due to its biocompatibility and film‐forming properties. However, although such coatings may limit microbial adhesion, their possible influence on eggshell gas exchange and embryonic development during prolonged storage remains insufficiently understood.

Although thyme extracts have been investigated on table eggs or as dietary supplements, comprehensive evaluations of direct eggshell application of thyme essential oil in long‐term stored fertile hatching eggs—particularly including blastoderm viability, oxidative status and chick quality—are limited. Therefore, the present study aimed to compare the effects of eggshell application of emulsified thyme essential oil and HA, alone or in combination, on eggshell microbial load, egg quality, blastoderm viability, hatchability traits, chick quality and yolk oxidative status after 0 or 14 days of storage. We hypothesized that thyme essential oil would better preserve embryonic viability and hatchability during prolonged storage due to its antimicrobial and antioxidant properties, whereas HA might exert divergent effects by altering eggshell permeability.

2. Materials and Methods

2.1. Experimental Design and Treatments

The experiment was conducted using 1800 broiler breeder hatching eggs obtained from a commercial Ross 308 breeder flock (74 weeks of age) maintained at a commercial poultry facility (XXX Tavukçuluk A.Ş.). Eggs were collected on the same day and randomly allocated to experimental groups to ensure homogeneous distribution. The study was designed to evaluate natural disinfectant alternatives for hatchery sanitation applied directly to the eggshell surface.

Eggs were assigned to four treatment groups:

1. untreated control,

2. eggs treated with 1% thyme essential oil (Origanum onites L.) emulsified with polysorbate‐20 (PS20),

3. eggs treated with 0.1% HA (molecular weight 1.5–1.8 MDa) and

4. eggs treated with a combination of thyme essential oil and HA.

All treatment solutions were prepared using sterile distilled water.

PS20 (0.1% v/v) was used as a non‐ionic surfactant to disperse thyme essential oil. A PS20‐only vehicle control (0.1% PS20 in sterile distilled water) was included to evaluate potential emulsifier effects. This vehicle control was analysed separately and was not included in the 4 × 2 factorial ANOVA model.

The chemical composition of the thyme essential oil was determined by gas chromatography–mass spectrometry (GC–MS). The oil was characterized by carvacrol (80.43%) as the major component, followed by linalool (5.54%), p‐cymene (2.25%), β‐bisabolene (1.88%) and γ‐terpinene (1.62%).

Each treatment group was further divided into two storage durations (0 and 14 days), resulting in a 4 × 2 factorial experimental design with three replicates per treatment (75 eggs per replicate). Eggs assigned to the storage treatment were maintained at 15°C and 75% relative humidity for 14 days, with eggs turned twice daily at approximately 45°, simulating standard commercial hatchery storage conditions.

Treatment solutions were applied to the eggshell surface by spraying. Eggs were placed on clean trays, and each treatment solution was sprayed uniformly onto the entire eggshell surface using a hand‐held sprayer. The eggs were allowed to air‐dry at room temperature before being transferred to the relevant storage or incubation procedure. The control group received no sanitization treatment, whereas the PS20 group received 0.1% PS20 solution as the vehicle control.

2.2. Egg Quality Measurements

Prior to incubation, eggs were individually weighed using a precision balance (±0.01 g). Egg length and width were measured using a digital caliper to calculate the shape index. For internal quality assessment, seven eggs per group were randomly selected and broken onto a flat glass surface. Albumen height, albumen width, yolk diameter and yolk height were measured using standard procedures (USDA 2020). Albumen, yolk and shell weights and their respective ratios were calculated. Haugh unit values were determined according to Özbaşer Bulut et al. (2025). Albumen and yolk indices were calculated using established formulas. Albumen pH was measured with a calibrated digital pH metre.

Egg weight was recorded at the beginning of storage and again at the end of the 14‐day storage period before incubation for each treatment group. Group‐level egg weight reduction during storage was estimated using the following formula: [(initial mean egg weight − mean egg weight after storage) / initial mean egg weight] × 100. Because egg weight was not recorded during incubation, egg weight loss up to Day 18 of incubation could not be calculated.

Egg quality indices were calculated using the following formulas:

$$\text{Shape}\text{index}(\%)=[\text{egg}\text{width}(\text{mm})/\text{egg}\text{length}(\text{mm})]\times 100$$

$$\begin{array}{ccc}& & \text{Albumen}\text{index}(\%)\hfill \\ & & =[\text{albumen}\text{height}(\text{mm})/\text{albumen}\text{width}(\text{mm})]\times 100\hfill \end{array}$$

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

$$\text{Albumen}\text{ratio}(\%)=[\text{albumen}\text{weight}(\mathrm{g})/\text{egg}\text{weight}(\mathrm{g})]\times 100$$

$$\text{Yolk}\text{ratio}(\%)=[\text{yolk}\text{weight}(\mathrm{g})/\text{egg}\text{weight}(\mathrm{g})]\times 100$$

$$\text{Shell}\text{ratio}(\%)=[\text{shell}\text{weight}(\mathrm{g})/\text{egg}\text{weight}(\mathrm{g})]\times 100$$

$$\text{Haugh}\text{unit}=100\times {\log }_{10}(H-1.7W^{0.37}+7.6)$$

where H is albumen height (mm) and W is egg weight (g).

2.3. Lipid Peroxidation and Total Protein Analysis

Lipid peroxidation in egg yolk was determined by measuring malondialdehyde (MDA) concentrations using a commercial colorimetric assay kit (Elabscience, E‐BC‐K025‐M) according to the manufacturer's instructions. Briefly, 0.5 g of yolk was homogenized in cold phosphate‐buffered saline (PBS) and centrifuged, and the supernatant was analysed spectrophotometrically at 532 nm. Results were expressed as nmol MDA per mg protein. Total protein concentration was determined using the Bradford method.

2.4. Microbiological Analysis

Eggshell microbial contamination was evaluated using a surface swab method. Briefly, 10 eggs per group were randomly selected, and the entire eggshell surface was swabbed using sterile swabs pre‐moistened with PBS. Swabs were transferred into sterile tubes containing diluent, vortexed thoroughly and serially diluted. Aliquots were plated onto plate count agar (PCA) and incubated at 37°C for 24–48 h to determine total aerobic bacterial counts. Results were expressed as log10 CFU/egg. A vehicle control group containing 0.1% PS20 without thyme essential oil was included to account for potential emulsifier‐related effects and was processed under the same conditions.

2.5. Blastoderm Cell Viability and Count

Blastoderm cell number and viability were determined prior to incubation using the filter ring technique described by Petitte et al. (1990). Blastoderms from seven eggs per group were isolated, enzymatically dissociated with trypsin, stained with trypan blue and counted using a haemocytometer under a light microscope (Zeiss, Germany). Viable and non‐viable cells were recorded separately.

2.6. Incubation Procedure and Hatchability

Eggs were incubated in a VGS‐490 incubator at 37.6°C and 58% relative humidity for the first 18 days, followed by 36.5°C and 75% relative humidity during the final 3 days (hatcher phase). Eggs were automatically turned every 2 h during the first 18 days. Fertility, hatchability of total eggs, hatchability of fertile eggs and embryonic mortality were calculated using standard formulas (Onbaşilar et al. 2011). Embryonic mortality was classified as early (0–6 days), mid (7–18 days) or late (19–21 days).

2.7. Chorioallantoic Membrane (CAM) Analysis

For eggs stored for 14 days, CAM vascularization was evaluated on embryonic Day 8 after incubation. Representative CAM images were analysed using ImageJ (FIJI) software (RRID: SCR_003070). Images were converted to 8‐bit format, thresholded and vascular area was quantified as a percentage of total area. Data were normalized to the control group (control = 100). Due to limited sample size, CAM results were interpreted cautiously.

3. Statistical Analysis

Sample size was determined using G*Power software based on a medium effect size (f = 0.25), α = 0.05 and statistical power of 0.80. Data normality and homogeneity of variance were assessed using the Shapiro–Wilk and Levene tests, respectively. Continuous variables, including egg quality traits, total aerobic bacterial counts, blastoderm cell numbers, yolk MDA and yolk total protein concentrations, were analysed using two‐way ANOVA with storage duration, sanitization method and their interaction as fixed effects, where applicable. When significant effects were detected, means were compared using Tukey's post hoc test. Hatchability‐related outcomes were analysed as proportional/binomial data using Pearson's chi‐square test for group‐wise comparisons and a binomial generalized linear model to evaluate the main effects of storage duration, sanitization method and their interaction. Egg weight reduction during storage was calculated from group mean values and presented descriptively. Statistical analyses were performed using SPSS v25.0 and GraphPad Prism software. The main factorial analyses were performed using the four principal treatment groups: control, thyme, HA and HA + thyme. The PS20‐only vehicle control was included for descriptive and selected reference comparisons but was not included in the 4 × 2 factorial models used to calculate main effects and interactions. Differences were considered significant at p < 0.05.

4. Results

Eggshell sanitation treatments significantly influenced microbial contamination levels on the eggshell surface (Table 1). Application of thyme essential oil, HA and their combination markedly reduced total aerobic bacterial counts compared with the untreated control at both storage durations. In non‐stored eggs, microbial counts were highest in the control group (4.24 ± 1.35 log10 CFU/egg) and decreased substantially in the thyme, HA and thyme + HA groups (1.12 ± 0.36, 1.48 ± 0.43 and 1.29 ± 0.47 log10 CFU/egg, respectively).

TABLE 1.

Effects of storage duration and eggshell sanitization methods on total aerobic bacterial counts.

Storage duration	Sanitization method	Total aerobic bacterial counts (log10 CFU/egg)
Non‐stored	Control	4.24 ± 1.35aA
	PS20	3.16 ± 0.84aA
	Thyme	1.12 ± 0.36bA
	HA	1.48 ± 0.43bA
	Thyme + HA	1.29 ± 0.47bA
Stored (14 days)	Control	6.14 ± 1.54aB
	PS20	4.49 ± 1.27bB
	Thyme	1.32 ± 0.38cB
	HA	1.86 ± 0.42cA
	Thyme + HA	1.78 ± 0.23cB
Storage duration	Non‐stored	2.25 ± 0.04a
	Stored (14 days)	3.11 ± 0.05b
Sanitization method	Control	5.19 ± 0.07a
	PS20	3.82 ± 0.05b
	Thyme	1.66 ± 0.02c
	HA	1.22 ± 0.01d
	Thyme + HA	1.53 ± 0.02c
Source of variation	p‐value
Storage duration	0.001
Sanitization method	0.001
Storage duration × sanitization method	0.001

Note: Values are presented as mean ± SEM. Different lowercase superscript letters within the same storage duration indicate significant differences among sanitization methods (p < 0.05). Different uppercase superscript letters within the same sanitization method indicate significant differences between storage durations (p < 0.05). Main effects and the storage duration × sanitization method interaction were evaluated using the 4 × 2 factorial model including control, thyme, hyaluronic acid and hyaluronic acid + thyme groups. PS20 was included as a vehicle control and is shown for reference only; it was not included in the factorial model used to calculate main effects and interaction.

Abbreviations: HA, hyaluronic acid; PS20, polysorbate 20.

After 14 days of storage, the control group showed the highest bacterial count, whereas thyme, HA and thyme + HA groups showed lower bacterial counts.

Eggshell sanitation treatments did not significantly affect egg quality traits (p > 0.05), whereas storage duration exerted a pronounced effect on several internal egg quality parameters (Table 2). Storage significantly influenced albumen ratio, yolk ratio, albumen index, yolk index, albumen pH and Haugh unit values (p < 0.001). Overall, albumen ratio decreased from 57.33% ± 0.06% at Day 0 to 54.31% ± 0.06% after 14 days, while yolk ratio increased from 31.88% ± 0.04% to 34.81% ± 0.05%. In parallel, albumen pH increased from 8.37 ± 0.01 to 9.39 ± 0.002, and Haugh unit values declined markedly from 89.95 ± 0.16 to 63.91 ± 0.22 following 14 days of storage. No significant treatment × storage interaction was observed for egg quality parameters (p > 0.05).

TABLE 2.

Effects of storage duration and eggshell sanitization methods on egg quality traits.

Storage duration (day)	Sanitization method	Egg weight (g)	Shape index (%)	Albumen ratio (%)	Yolk ratio (%)	Albumen index (%)	Yolk index (%)	Shell ratio (%)	Albumen pH	Haugh unit (HU)
	Control	71.53 ± 0.48	78.89 ± 0.54	57.68 ± 0.25	31.46 ± 0.19	11.19 ± 0.36	42.34 ± 0.50	10.84 ± 0.11	8.33 ± 0.04	90.04 ± 0.86
	PS20	70.81 ± 0.43	78.95 ± 0.51	57.30 ± 0.32	31.71 ± 0.23	11.24 ± 0.27	42.60 ± 0.65	10.98 ± 0.11	8.29 ± 0.03	90.23 ± 0.92
0	Thyme	69.67 ± 0.45	78.34 ± 0.54	57.30 ± 0.33	32.02 ± 0.18	11.49 ± 0.29	43.09 ± 0.50	10.67 ± 0.16	8.46 ± 0.03	90.60 ± 0.89
	HA	70.10 ± 0.35	78.95 ± 0.52	57.35 ± 0.31	31.98 ± 0.21	11.45 ± 0.35	42.03 ± 0.55	10.67 ± 0.10	8.34 ± 0.03	89.56 ± 0.85
	Thyme + HA	70.03 ± 0.44	78.89 ± 0.54	57.03 ± 0.35	32.24 ± 0.27	11.52 ± 0.35	41.79 ± 0.48	10.71 ± 0.12	8.42 ± 0.03	89.32 ± 0.78
	Control	69.14 ± 0.17	78.80 ± 0.42	54.50 ± 0.27	34.93 ± 0.29	5.67 ± 0.09	32.84 ± 0.39	10.55 ± 0.05	9.40 ± 0.01	62.86 ± 1.56
	PS20	69.08 ± 0.15	78.95 ± 0.44	54.08 ± 0.30	35.35 ± 0.32	5.70 ± 0.17	32.89 ± 0.37	10.77 ± 0.03	9.40 ± 0.01	63.07 ± 1.52
14	Thyme	69.50 ± 0.45	79.25 ± 0.44	54.47 ± 0.37	34.58 ± 0.30	5.44 ± 0.19	34.69 ± 0.32	10.94 ± 0.12	9.38 ± 0.01	64.75 ± 1.25
	HA	67.69 ± 0.82	78.28 ± 0.80	55.33 ± 0.12	33.64 ± 0.11	5.05 ± 0.09	33.29 ± 0.32	11.02 ± 0.06	9.38 ± 0.01	64.85 ± 0.40
	Thyme + HA	69.37 ± 0.55	78.60 ± 0.35	53.80 ± 0.27	34.90 ± 0.24	5.33 ± 0.12	32.58 ± 0.35	10.95 ± 0.05	9.43 ± 0.002	64.02 ± 0.87
Total
0		70.43 ± 0.08	78.71 ± 0.10	57.33 ± 0.06	31.88 ± 0.04	11.13 ± 0.04	42.34 ± 0.10	10.77 ± 0.02	8.37 ± 0.01	89.95 ± 0.16
14		68.96 ± 0.09	78.85 ± 0.07	54.31 ± 0.06	34.81 ± 0.05	5.44 ± 0.03	33.26 ± 0.07	10.82 ± 0.02	9.39 ± 0.002	63.91 ± 0.22
	Control	70.33 ± 0.19	78.85 ± 0.23	56.09 ± 0.18	33.20 ± 0.18	8.43 ± 0.23	37.59 ± 0.41	10.70 ± 0.04	8.86 ± 0.04	76.45 ± 1.17
	PS20	69.94 ± 0.16	79.33 ± 0.22	55.69 ± 0.19	33.53 ± 0.19	8.47 ± 0.22	37.67 ± 0.43	10.77 ± 0.04	8.84 ± 0.04	76.65 ± 1.17
	Thyme	69.59 ± 0.21	78.80 ± 0.24	55.89 ± 0.20	33.30 ± 0.15	8.37 ± 0.25	38.89 ± 0.37	10.80 ± 0.07	8.92 ± 0.04	77.67 ± 1.09
	HA	68.90 ± 0.31	78.61 ± 0.18	56.34 ± 0.14	32.81 ± 0.10	7.98 ± 0.28	37.66 ± 0.39	10.84 ± 0.04	8.86 ± 0.04	77.21 ± 0.97
	Thyme + HA	69.70 ± 0.24	78.74 ± 0.22	55.42 ± 0.19	33.57 ± 0.16	8.15 ± 0.26	37.18 ± 0.39	11.00 ± 0.05	8.92 ± 0.003	76.67 ± 1.01
p (value)
Storage		0.065	0.854	< 0.001	< 0.001	< 0.001	< 0.001	0.570	< 0.001	< 0.001
Sanitization method		0.834	0.989	0.925	0.948	0.882	0.689	0.844	0.655	0.992
Storage × sanitization method		0.853	0.929	0.995	0.812	0.995	0.987	0.298	0.979	0.988

Note: PS20 is a vehicle control (shown for reference only) and was analysed separately (not included in the 4 × 2 factorial model).

Hatching performance differed among treatments, particularly after prolonged storage (Table 3) (χ ², p = 0.001). After 14 days of storage, hatchability of set eggs was higher in the thyme group (68.44%) than in the stored control (57.78%) and stored HA group (54.22%). Hatchability of fertile eggs was 93.33% in stored thyme eggs versus 86.67% in stored control and 74.39% in stored HA eggs. Embryonic mortality was lowest in stored thyme eggs (6.67%) and highest in stored HA eggs (25.61%) (Table 3).

TABLE 3.

Effects of storage duration and eggshell sanitization methods on hatching performance.

Storage duration (day)	Sanitization method	Hatchability of set eggs (%)	Fertility (%)	Hatchability of fertile eggs (%)	Embryonic mortality (%)
0 days	Control	68.89ab	73.33ab	93.94	6.06
	Thyme	75.11a	80.00a	93.89	6.11
	HA	56.89c	60.00c	94.81	5.19
	Thyme + HA	64.00bc	68.89b	92.90	7.10
	PS20	69.33ab	74.22ab	95.12	4.88
	χ 2	18.875	24.389	0.500	0.857
	p‐value	0.001	0.001	0.973	0.931
14 days	Control	57.78b	66.67	86.67b	13.33b
	Thyme	68.44a	73.33	93.33a	6.67c
	HA	54.22b	72.89	74.39c	25.61a
	Thyme + HA	53.33b	63.11	84.51b	15.49b
	PS20	58.22b	68.44	85.07b	14.94b
	χ 2	13.377	7.797	23.499	23.499
	p‐value	0.010	0.099	0.001	0.001
Storage duration	0 days	66.84	72.62	92.04	5.88
	14 days	58.40	68.89	84.77	15.23
	χ 2	17.137	2.092	20.661	37.233
	p‐value	0.001	0.148	0.001	0.001
Sanitization method	Control	63.33b	70.00b	90.48ab	9.52b
	Thyme	71.78a	76.67a	93.62a	6.67b
	HA	55.56c	69.78b	79.62c	15.61a
	Thyme + HA	58.67cb	66.00b	88.89b	11.11ab
	PS20	63.78b	71.33ab	89.41ab	9.66b
	χ 2	29.078	12.922	34.764	14.865
	p‐value	0.001	0.012	0.001	0.005
Storage duration × sanitization method	p‐value	0.138	0.021	0.020	0.001

Note: Values are presented as percentages. Different lowercase superscript letters within the same section and column indicate significant differences among groups (p < 0.05). Group‐wise comparisons of proportional outcomes were performed using Pearson's chi‐square test. Main effects and the storage duration × sanitization method interaction were evaluated using the 4 × 2 factorial/binomial model including control, thyme, hyaluronic acid and hyaluronic acid + thyme groups. PS20 was included as a vehicle control and is shown for reference only; it was not included in the factorial model used to calculate main effects and interaction.

Abbreviations: HA, hyaluronic acid; PS20, polysorbate 20.

Blastoderm cell viability was significantly affected by storage duration (Table 4). Mean viable blastoderm cell number decreased from 31,708.33 ± 61.72 cells/egg at Day 0 to 24,633.33 ± 82.21 cells/egg after 14 days (p < 0.001), whereas dead cell counts increased from 2844.16 ± 41.73 to 10,133.33 ± 87.56 cells/egg (p < 0.001). A significant storage duration × sanitization method interaction was observed for blastoderm dead cell number (p = 0.003), with the HA group stored for 14 days showing the highest dead cell count. Total blastoderm cell number did not differ with storage (p > 0.05).

TABLE 4.

Effects of storage duration and eggshell sanitization methods on blastoderm cell number and viability.

Storage duration (day)	Sanitization method	Blastoderm viable cells	Blastoderm dead cells	Blastoderm total cells
0 days	Control	32,083.33 ± 127.29	2610.00 ± 32.14	34,633.33 ± 134.71
	Thyme	31,733.33 ± 83.88	2850.00 ± 275.37	34,583.33 ± 220.05
	HA	31,500.00 ± 440.95	3133.33 ± 200.92	33,830.00 ± 429.90
	Thyme + HA	31,516.66 ± 283.98	2783.33 ± 110.97	34,300.00 ± 218.58
14 days	Control	24,866.66 ± 302.45	10,066.66 ± 236.48	34,933.33 ± 107.15
	Thyme	25,633.33 ± 183.58	8733.33 ± 50.90	34,366.66 ± 203.67
	HA	23,733.33 ± 350.13	11,433.33 ± 38.38	35,133.33 ± 403.76
	Thyme + HA	24,300.00 ± 133.33	10,300.00 ± 33.33	34,566.66 ± 150.30
Storage duration	0 days	31,708.33 ± 61.72	2844.16 ± 41.73	34,537.50 ± 59.55
	14 days	24,633.33 ± 82.21	10,133.33 ± 87.56	34,750.00 ± 60.66
Sanitization method	Control	28,475.00 ± 666.91	6338.33 ± 684.86ab	34,783.33 ± 60.93
	Thyme	28,683.33 ± 560.49	5791.66 ± 544.32a	34,475.66 ± 90.86
	HA	27,616.66 ± 731.01	7283.33 ± 760.43b	34,883.33 ± 198.44
	Thyme + HA	27,908.33 ± 618.82	6541.66 ± 687.15ab	34,433.33 ± 87.34
Source of variation	p‐value
Storage duration		< 0.001	< 0.001	0.522
Sanitization method		0.116	< 0.001	0.706
Storage duration × sanitization method		0.356	0.003	0.882

Note: Values are presented as mean ± SEM. Different lowercase superscript letters within the same column indicate significant differences among sanitization methods (p < 0.05). Main effects of storage duration and sanitization method, and their interaction, were evaluated by two‐way ANOVA followed by Tukey's post hoc test.

Abbreviation: HA, hyaluronic acid.

Relative CAM vessel density appeared higher in all treated groups compared with the control after 14 days of storage (Figure 1). Relative vessel density values (control = 100) were 116.7 in the thyme group, 105.0 in the HA group and 113.4 in the thyme + HA group.

FIGURE 1.

Chorioallantoic membrane (CAM) vascularization after 14 days of egg storage. Representative CAM images are shown for (a) control, (b) thyme, (c) HA and (d) thyme + HA, with (e–h) the corresponding processed images generated after image processing using ImageJ (FIJI, NIH, USA) following 8‐bit conversion and thresholding. Vessel density was quantified as the percentage of vascular area relative to the total analysed area and normalized to the control group (control = 100). Relative vessel density values were 116.7 (thyme), 105.0 (HA) and 113.4 (thyme + HA). Because of the limited sample size, CAM outcomes were evaluated descriptively and were not subjected to inferential statistical analysis.

Yolk MDA levels did not differ among treatments in non‐stored eggs (p > 0.05). Storage increased MDA levels in all groups (p < 0.01); however, after 14 days the thyme essential oil group exhibited lower MDA concentrations than the control and HA groups (p < 0.01) (Figure 2).

FIGURE 2.

Effects of treatment and storage duration on egg yolk malondialdehyde (MDA) levels. Data are expressed as mean ± SEM (n = 7). Different letters above the bars indicate statistically significant differences among groups (one‐way ANOVA followed by Tukey's multiple comparison test; p < 0.05).

Yolk total protein concentration differed among groups (p < 0.0001) (Figure 3). Storage increased protein concentration in the control group, and among stored eggs protein levels in the thyme group were comparable to the stored control, whereas the HA and thyme + HA groups showed lower values (p < 0.05).

FIGURE 3.

Effects of storage duration and experimental treatments on egg yolk total protein concentrations. Values are expressed as mean ± SEM (n = 7). Different letters indicate significant differences among groups (one‐way ANOVA with Tukey's multiple comparison test; p < 0.05).

Egg weight reduction during 14 days of storage is presented in Table 5. Overall, mean egg weight decreased from 70.43 ± 0.08 g at the beginning of storage to 68.96 ± 0.09 g after 14 days, corresponding to an approximate group‐level reduction of 2.09%. The greatest numerical reductions were observed in the HA group (3.44%) and the control group (3.34%), whereas the thyme group showed the lowest numerical reduction (0.24%).

TABLE 5.

Egg weight reduction during 14 days of storage in hatching eggs treated with different eggshell sanitization methods.

Sanitization method	Initial egg weight (g)	Egg weight after 14 days of storage (g)	Difference (g)	Approximate reduction (%)
Control	71.53 ± 0.48	69.14 ± 0.17	2.39	3.34
PS20	70.81 ± 0.43	69.08 ± 0.15	1.73	2.44
Thyme	69.67 ± 0.45	69.50 ± 0.45	0.17	0.24
HA	70.10 ± 0.35	67.69 ± 0.82	2.41	3.44
Thyme + HA	70.03 ± 0.44	69.37 ± 0.55	0.66	0.94
Overall	70.43 ± 0.08	68.96 ± 0.09	1.47	2.09

Note: Values are presented as mean ± SEM. Egg weight reduction was estimated from group mean values using the formula: [(initial mean egg weight − mean egg weight after storage) / initial mean egg weight] × 100. Therefore, this parameter represents group‐level egg weight reduction during storage. Egg weight loss during incubation was not measured.

Abbreviations: HA, hyaluronic acid; PS20, polysorbate 20.

5. Discussion

Prolonged pre‐incubation storage was the dominant factor impairing internal egg quality and blastoderm viability in the present study. After 14 days of storage, Haugh unit, albumen ratio, albumen index and yolk index decreased, whereas yolk ratio and albumen pH increased. These findings are consistent with previous reports showing that extended storage reduces albumen viscosity and structural integrity, increases albumen pH through CO₂ loss and alters the internal egg environment required for early embryonic maintenance (Jadhav and Siddiqui 2007; Kralik et al. 2014; Lall et al. 2018; Alshaikhi et al. 2020). The deterioration of these internal quality traits is particularly relevant for fertile eggs because the blastoderm remains dependent on a stable physicochemical microenvironment before incubation. Moreover, the present results are consistent with recent studies showing that storage duration adversely affects egg quality, embryonic development, hatchability and chick quality in poultry (Biesek et al. 2024; Tainika et al. 2024).

The adverse effect of storage was also evident at the cellular level. Storage markedly reduced viable blastoderm cell number and increased dead cell counts, indicating that prolonged pre‐incubation storage compromises embryonic cell survival before incubation begins. This finding agrees with earlier studies showing that the avian blastoderm is sensitive to storage‐related stress and that reduced viable cell numbers before incubation may contribute to impaired embryonic development and hatchability (Hamidu et al. 2010; Pokhrel et al. 2021). Therefore, the decline in hatchability observed after storage in the present study appears to reflect not only deterioration of internal egg quality but also direct loss of blastoderm cellular viability.

Eggshell microbial contamination increased after storage, confirming that prolonged storage can increase the microbial challenge on fertile eggs. This finding is consistent with previous reports indicating that eggshell microorganisms may proliferate during storage and may penetrate the shell pores and membranes, thereby increasing the risk of embryonic mortality and reduced chick quality (Board and Tranter 1995; Berrang et al. 1999; Yu et al. 2025; Yuan et al. 2022). In the present study, all sanitization treatments reduced total aerobic bacterial counts compared with the untreated control, indicating that thyme essential oil, HA and their combination were effective in lowering eggshell microbial load. These findings indicate that microbial control is necessary but not sufficient for successful hatching egg sanitation.

However, the present data also illustrate an important hatchery principle: Reduction of eggshell microbial load does not automatically translate into improved hatchability if other physiological constraints are introduced. While microbial control is necessary to reduce contamination pressure (Yu et al. 2025; Yuan et al. 2022), the egg is a living biological system, and interventions that alter shell function may offset microbial benefits.

Among the tested treatments, thyme essential oil produced the most favourable overall profile after 14 days of storage, as reflected by lower eggshell bacterial counts, reduced yolk MDA levels, lower embryonic mortality and higher hatchability compared with HA‐treated eggs. The thyme group exhibited reduced eggshell microbial counts, improved hatchability relative to other stored groups, lowered embryonic mortality (particularly early mortality), improved chick quality and significantly lowered yolk MDA compared with control and HA. These outcomes are biologically plausible given the known properties of thyme essential oil and its phenolic constituents. Carvacrol and thymol are widely reported to exert antimicrobial activity through membrane‐disrupting effects and interference with microbial metabolism (Christaki et al. 2012; Shahein and Sedeek 2014). Recent studies also support the use of thyme‐derived products as antimicrobial interventions relevant to eggs and poultry‐related microbial control (Haraz et al. 2022; Giovagnoni et al. 2023; El‐Soufi et al. 2025; Ergün et al. 2023).

Beyond antimicrobial effects, the antioxidant capacity of thyme phenolics may be critical in the context of prolonged storage, where oxidative processes intensify. Avian yolk contains high levels of polyunsaturated fatty acids, making it particularly susceptible to lipid peroxidation under oxidative stress (Dunning et al. 2014). MDA, a classical marker of lipid peroxidation, increased after storage in all groups, confirming that storage imposes oxidative challenge to yolk lipids. Similar oxidation dynamics under prolonged storage and processing conditions have been described in earlier biochemical work on lipid peroxidation products and their propagation (Vercellotti et al. 1992). The significantly lower MDA in the thyme group suggests attenuation of oxidative damage, which may indirectly support embryonic cell survival and vascular development, thereby improving hatchability and post‐hatch quality.

The finding that hatchability in the stored thyme group approached the non‐stored control further strengthens the practical significance of thyme essential oil as a hatchery‐relevant sanitation option under long storage scenarios. Plant‐derived disinfectants are increasingly explored as alternatives to conventional chemical sanitation practices due to concerns regarding chemical residues, environmental impact and occupational exposure. Their effectiveness depends on achieving microbial control while preserving eggshell physiology and embryo viability (Copur et al. 2011; Oliveira et al. 2024; Iraqi et al. 2025).

HA application reduced eggshell bacterial counts, supporting a barrier‐mediated mechanism. HA is a biocompatible polysaccharide with viscoelastic and adhesive properties that can form a continuous film on biological surfaces (Zhang et al. 2019; Laffleur et al. 2018; Martin et al. 2019). Previous evidence suggests that HA itself has limited intrinsic antimicrobial activity but can become functionally relevant as a carrier or network‐forming matrix that affects adhesion and surface interactions (Fan et al. 2023). The present microbiological results are consistent with such a physical barrier concept.

Although HA reduced eggshell bacterial counts, this microbial reduction did not translate into improved hatchability. In contrast, HA‐treated eggs stored for 14 days showed increased blastoderm dead cell counts and the highest embryonic mortality. Therefore, the poorer incubation outcome in the HA group appears unlikely to be explained by microbial load alone. A more plausible interpretation is that HA application may have altered the eggshell surface microenvironment, cuticle properties or moisture dynamics during storage. The additional evaluation of storage‐related egg weight reduction showed that the HA group had the greatest numerical reduction during storage. However, because egg weight reduction was estimated from group mean values and egg weight loss during incubation was not measured, this interpretation should be considered hypothesis‐generating rather than confirmatory. Accordingly, HA‐associated impairment in hatchability cannot be conclusively attributed to moisture loss or altered shell conductance based on the present data alone.

Importantly, the combination of thyme essential oil and HA did not outperform thyme essential oil alone. This suggests that the presence of HA may have reduced the net benefit of thyme essential oil, possibly by modifying the shell surface environment, affecting the distribution of the sprayed formulation or altering moisture dynamics during storage. Therefore, for hatchery practice, HA‐based applications may require optimization of concentration, viscosity and application volume before they can be considered suitable for use on stored fertile eggs.

Overall, these findings emphasize that microbial reduction alone is not sufficient for a hatching egg sanitation strategy. A useful intervention must reduce eggshell contamination while preserving the shell surface, internal egg quality and embryonic viability during storage and subsequent incubation.

Storage increased yolk protein concentration in the control group. This observation is often consistent with a concentration effect secondary to moisture loss during storage rather than true enhancement of protein content. However, protein quantity does not necessarily reflect functional integrity; oxidative stress can modify proteins structurally and functionally even when total concentration appears unchanged. In this context, the thyme group showed protein levels comparable to stored control while exhibiting lower MDA, which may indicate that reduced lipid peroxidation occurred without substantial deviation in measured protein concentration. Conversely, the lower protein values in HA and combination groups could reflect altered water dynamics, differential yolk composition changes or broader metabolic shifts due to altered shell function. Although mechanistic inference should be cautious, these patterns reinforce that storage‐related biochemical endpoints should be interpreted alongside internal quality, embryo viability and hatchability outcomes.

The CAM analysis showed increased relative vessel density in all treated groups, with the highest increase in the thyme group. Because this component was descriptive and limited in sample size, it should be interpreted as exploratory. Nonetheless, the directionality is biologically coherent with the broader dataset: Improved oxidative balance and reduced stress can support embryonic developmental trajectories, including vascular organization. Importantly, the HA group also showed increased vessel density yet exhibited poor hatchability, indicating that vessel density alone is insufficient to predict successful hatching and that other physiological constraints (e.g., gas exchange, cellular viability) likely dominate under prolonged storage. Future work with larger sample size, standardized imaging fields and inclusion of inferential statistics would strengthen the CAM component and clarify its relationship to hatchability.

From a hatchery management perspective, the present findings suggest that a spray intervention must satisfy two conditions to be useful under long‐term storage: (i) meaningful reduction of eggshell microbial load and (ii) preservation of physiological shell function required for embryo survival. Thyme essential oil met both requirements within the tested conditions, whereas HA, although effective in reducing microbial load, was associated with poorer incubation outcomes. These data support thyme essential oil as a promising natural alternative for long storage periods, consistent with growing interest in plant‐derived antimicrobials in poultry systems (Copur et al. 2011; Oliveira et al. 2024).

The study has limitations that should be acknowledged. First, eggshell gas conductance and egg weight loss during incubation were not directly measured; therefore, the proposed permeability‐ or moisture‐related interpretation for HA remains inferential. Although storage‐related egg weight reduction was estimated from group mean values, individual egg weight loss and Day‐18 incubation weight loss were not available. Second, CAM quantification was exploratory because of the limited sample size; expanding this component and linking it with embryonic stage‐specific outcomes would improve mechanistic depth. Finally, future studies should optimize HA concentration, viscosity and application volume to determine whether a thinner or modified coating can preserve hatchability while maintaining microbial barrier effects.

6. Conclusion

In conclusion, 14 days of egg storage significantly impaired internal egg quality, increased oxidative stress, reduced blastoderm cell viability and decreased hatchability, confirming the multifactorial nature of storage‐induced embryonic loss. While all tested sprays reduced eggshell microbial contamination, thyme essential oil provided the most consistent biological protection under prolonged storage, improving hatchability and chick quality and lowering yolk lipid peroxidation. In contrast, HA reduced microbial counts but was associated with reduced hatchability, suggesting that barrier‐mediated alterations to shell function may negatively influence embryonic survival under the conditions tested. Collectively, these findings indicate that plant‐derived agents combining antimicrobial and antioxidant activities may offer practical benefits for long‐term storage of hatching eggs as safe and residue‐free alternatives to conventional chemical disinfectants, provided that eggshell permeability and embryo physiology are preserved.

Author Contributions

Conceptualization: Sinan Vicil and Fatma Tülin Özbaşer Bulut. Methodology: Sinan Vicil, Fatma Tülin Özbaşer Bulut and Hakan Bulut. Investigation: Sinan Vicil, Fatma Tülin Özbaşer Bulut and Muazzez Gürgan Eser. Formal analysis: Sinan Vicil, Fatma Tülin Özbaşer Bulut and Hakan Bulut. Software: Sinan Vicil. Visualization: Sinan Vicil. Data curation: Fatma Tülin Özbaşer Bulut. Validation: Fatma Tülin Özbaşer Bulut. Writing – original draft: Sinan Vicil, Fatma Tülin Özbaşer Bulut and Hakan Bulut. Writing – review and editing: Sinan Vicil, Fatma Tülin Özbaşer Bulut, Muazzez Gürgan Eser and Hakan Bulut. Supervision: Sinan Vicil, Fatma Tülin Özbaşer Bulut and Hakan Bulut. Project administration: Sinan Vicil, Fatma Tülin Özbaşer Bulut and Hakan Bulut. Funding acquisition: Hakan Bulut. Resources: Sinan Vicil and Fatma Tülin Özbaşer Bulut.

Funding

The authors have nothing to report.

Ethics Statement

The study was conducted in accordance with institutional guidelines and approved by the Animal Experiments Local Ethics Committee of Tekirdağ Namık Kemal University (approval no: T2025/5; date: 03 July 2025).

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.