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Timing matters: laying sequence and location effects on egg quality and embryonic growth

Published online by Cambridge University Press:  16 October 2026

İmge Duru Önol Open the ORCID record for İmge Duru Önol [Opens in a new window] ,
Florence Jenkins ,
Mustafa Önol ,
Nigel Kendall  and
Cormac O’Shea Open the ORCID record for Cormac O’Shea [Opens in a new window]
İmge Duru Önol*
Affiliation:
School of Biosciences, University of Nottingham, UK
Florence Jenkins
Affiliation:
School of Biosciences, University of Nottingham, UK
Mustafa Önol
Affiliation:
School of Biosciences, University of Nottingham, UK
Nigel Kendall
Affiliation:
School of Veterinary Medicine and Science, University of Nottingham, UK
Cormac O’Shea
Affiliation:
School of Biosciences, University of Nottingham, UK Bioveterinary and Microbial Sciences, Technological University of the Shannon Midlands Midwest - Athlone Campus, Ireland
*
Corresponding author: İmge Duru Önol; Email: imge.duruonol@nottingham.ac.uk
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Abstract

In broiler breeders, while floor eggs are associated with a negative impact on hatching performance, the role of laying sequence position remains poorly understood and may contribute a further source of variation to egg quality and embryo development. Two experiments were conducted to evaluate the effects of laying environment and sequence position. In Experiment 1, eggs were classified as first collection floor eggs (FCFE) or second collection floor eggs (SCFE) and incubated to embryonic day 12 (ED12). In Experiment 2, eggs were classified as first collection nest eggs (FCNE), FCFE and SCFE to assess internal and external quality, compositional traits and eggshell mineral composition. In Experiment 1, embryos from FCFE were significantly lighter at ED12 compared with those from SCFE, despite greater initial egg size. In Experiment 2, albumen parameters differed, with first collection eggs, FCNE and FCFE, showing greater albumen weight and proportion than second collection eggs, SCFE. Shell thickness was greater in FCNE and FCFE. Mineral analysis revealed higher sodium concentrations in first collection eggs, lower zinc and molybdenum concentrations in floor eggs, reduced manganese in FCNE and higher arsenic in FCFE. Together, these findings demonstrate that laying sequence and environment alter egg composition, shell quality and mineral deposition, with egg-laying sequence demonstrating potential downstream effects on embryonic development. Improved understanding of the underlying mechanistic contribution of egg sequence to egg quality and embryo development will contribute to our understanding of achieving high and consistent hatch success in broiler breeders.

Keywords

broiler breederegg layingincubationoviposition

Information

Type
Animal Research Paper
Information
The Journal of Agricultural Science , Volume 164 , 2026 , e51
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Copyright
© The Author(s), 2026. Published by Cambridge University Press

Introduction

The quality of the egg is a key determinant of success in poultry production. The composition of nutrients within a fertile egg significantly impacts nutrient uptake by the embryo during incubation (Uni et al., Reference Uni, Yadgary and Yair2012). Given the relatively short lifespan of commercial meat strains, the incubation period constitutes approximately one-third of their overall growth period and is therefore a critical phase governing embryonic nutrition, growth and development (Speier et al., Reference Speier, Yadgary, Uni and Wong2012).

Eggs laid outside designated nesting areas, known as floor eggs, are particularly problematic in the poultry industry. The environment in which an egg is laid significantly influences its microbial load, with floor eggs presenting a greater risk of contamination due to increased contact with surface dirt, higher levels of bacterial presence on the shell and a greater incidence of shell cracks and reduced hatchability (Berrang et al., Reference Berrang, Frank, Buhr, Bailey, Cox and Mauldin1997; De Reu et al., Reference De Reu, Messens, Heyndrickx, Rodenburg, Uyttendaele and Herman2008; Van den Brand et al., Reference Van den Brand, Sosef, Lourens and Van Harn2016; Costa et al., Reference Costa, Lopes, Sousa, Barbosa, Gonçalves, Franco, Lara and Araújo2022). In line with this, Van den Brand et al. (Reference Van den Brand, Sosef, Lourens and Van Harn2016) reported that the hatchability of fertile eggs was substantially lower for floor eggs (approximately 74 %) compared to nest eggs (approximately 93 %), highlighting the impact of egg-laying location on hatchability. Additionally, studies have shown that the average body weight of chicks hatching from floor eggs is lower than that of chicks hatching from nest eggs (Van den Brand et al., Reference Van den Brand, Sosef, Lourens and Van Harn2016; Perić et al., Reference Perić, Mitraković, Tomić, Orehovački and Meijerhof2022).

Beyond the laying location, the sequential pattern in which eggs are laid (referred to as the egg sequence or clutch) also influences egg quality. Eggs are laid in sequences with intervals of approximately 24 to 26 h between ovipositions, resulting in each egg within a sequence being laid progressively later in the day. Once a sequence concludes, a non-laying period, or ‘pause’, follows before the next sequence commences, which can extend up to 40 h (Robinson et al., Reference Robinson, Hardin, Robinson and Williams1991). These processes are regulated by the coordinated activity of reproductive hormones and physiological factors such as energy metabolism, with the hypothalamo-pituitary-gonadal axis playing a central role in follicle development and ovulation. As poultry are long day breeders, light plays a crucial role by activating the pineal and pituitary glands, which regulate reproductive hormones, including follicle-stimulating hormone (FSH), luteinizing hormone (LH) and 17β-estradiol, all of which are essential for the growth and number of ovarian follicles (Onagbesan et al., Reference Onagbesan, Metayer, Tona, Williams, Decuypere and Bruggeman2006). The pineal gland functions as a circadian oscillator, responding to photic signals to produce melatonin, thereby signalling darkness and influencing circadian regulation (Okano and Fukada, Reference Okano and Fukada2003). This circadian mechanism is essential for coordinating the timing of ovulation and subsequent egg-laying. This cycle, spanning approximately 25 to 26 h, includes the progressive development of the yolk, albumen, membranes and shell (Zhang et al., Reference Zhang, Du, Lai, Shu, Zhu, Tian, Li, Wang, Yang, Zhang and Zhao2022). Consequently, the first egg in a sequence is typically laid early in the morning, approximately 3.5 h after the onset of light during the photoperiod (Gumułka et al., Reference Gumułka, Kapkowska and Maj2010). This timing reflects a tightly regulated physiological response, ensuring synchronization with the internal circadian rhythms of the bird, which are profoundly influenced by light exposure (Lewis et al., Reference Lewis, Perry and Morris1995). Thus, light emerges as a critical external factor, modulating hormonal activity and the timing of egg-laying through its interaction with the dark phase (Wilson et al., Reference Wilson, Jennings and Cunningham1985).

Egg sequences vary between the two main types of commercial birds, with laying hens typically having longer sequences, while broiler breeders tend to lay three to four eggs in a sequence with pauses in between (Zuidhof et al., Reference Zuidhof, Renema and Robinson2007; Gumułka et al., Reference Gumułka, Kapkowska and Maj2010) . The order in which eggs are laid in a sequence affects both the weight and composition of the eggs. Previous research has highlighted the temporal dynamics of egg weights, indicating that eggs laid early in the morning, and thus early in the sequence order, tend to be heavier than those laid later in the day (Robinson et al., Reference Robinson, Hardin, Robinson and Williams1991; Novo et al., Reference Novo, Gama and Chaveiro Soares1997; Zakaria et al., Reference Zakaria, Plumstead, Romero-Sanchez, Leksrisompong, Osborne and Brake2005; Tůmová et al., Reference Tůmová, Zita, Hubený, Skřivan and Ledvinka2008).

The quality of the eggshell is crucial not only for protecting the egg but also for ensuring optimal conditions for embryo development in fertilized eggs (Narushin and Romanov, Reference Narushin and Romanov2002). The timing of oviposition within a sequence also influences eggshell quality. Eggs laid later in the day tend to have thicker shells and exhibit fewer deformities compared to those laid earlier (Arafa et al., Reference Arafa, Harms, Miles, Christmas and Choi1982; Yannakopoulos et al., Reference Yannakopoulos, Tserveni-Gousi and Nikokyris1994; Tůmová and Ebeid, Reference Tůmová and Ebeid2005; Tůmová et al., Reference Tůmová, Zita, Hubený, Skřivan and Ledvinka2007). The mineralized eggshell provides structural protection and also serves as a major calcium reservoir for embryonic skeletal development (Chien et al., Reference Chien, Hincke and McKee2009). While mechanistic modelling suggests that the timing of oviposition within the light cycle alters calcium (Ca) absorption, bone mobilization and phosphorus excretion during shell formation (Kebreab et al., Reference Kebreab, France, Kwakkel, Leeson, Kuhi and Dijkstra2009), recent studies have shown that Ca and phosphorus (P) metabolism also varies across the oviposition cycle, with shifts in serum concentrations, excretion and transporter expression that reflect the changing demands of eggshell formation (Ren et al., Reference Ren, Sun, Liu, Li, Han, Cheng, Yan and Yang2019; Yan et al.,Reference Yan, Wang, Chen, Shi, Liao, Pan, Liu, Yang and Ren2023; Sinclair-Black et al., Reference Sinclair-Black, Garcia-Mejia, Blair, Angel, Arbe, Cavero and Ellestad2024). These temporal dynamics are mirrored in the composition of the eggshell itself, with Ca levels typically higher in eggs laid earlier in the day, while P and magnesium (Mg) concentrations increase in eggs laid later (Tůmová et al., Reference Tůmová, Gous and Tyler2014).

Therefore, in addition to the well-established detrimental effect of floor eggs on hatching performance, the egg sequence may also be having an important impact and could account for some of the variation observed between floor and nest eggs. The concept of egg sequence is key to understanding variations in egg quality and production, which are influenced by a range of physiological and environmental factors. Despite the importance of egg quality, research on egg sequence, particularly in broiler breeders, remains limited. This study aimed to explore how the laying environment and the egg sequence affect egg composition, shell mineralization and embryonic growth. Gaining insights into these dynamics is essential for improving incubation performance and optimizing production outcomes in commercial poultry systems.

Materials and methods

Egg collection and sequence classification

The procedure involving animals was reviewed and approved by the University of Nottingham institutional animal ethics committee (approval no. 244). For both experiments, eggs were collected from Ross-308 broiler breeder flocks and classified based on their approximate position in the laying sequence, following a time-specific collection pattern. This classification was based on the established relationship between oviposition time and sequence progression in broiler breeder hens, whereby the first egg of a sequence is typically laid earlier in the day than subsequent eggs. In both experiments, the first collection of floor eggs (first collection floor eggs, FCFE) was assumed to predominantly represent the first egg in a new egg sequence, laid after a pause. The second collection of floor eggs (second collection floor eggs, SCFE) was assumed to represent eggs laid second in the sequence. In the second experiment, an additional experimental group, the first collection nest eggs (FCNE) was included, was assumed, like FCFE, to represent the first egg in a new sequence after a pause, differing only in laying location.

Experiment 1: incubation

A total of 200 floor eggs were collected from a Ross-308 broiler breeder flock. Eggs were collected at two flock ages: 33 wk (Run 1) and 44 wk (Run 2). For each run, 50 eggs were collected at 07:00 h (FCFE) and 50 eggs were collected at 08:30 h (SCFE).

Eggs were washed for 2 min using a Rota-maid washer and ROTOSAN® solution (15 mL/L). After air drying, eggs were fumigated with Virkon™ TDX solution. The external surface of each egg was evaluated and profiled using the Alltech ‘20 Common Eggshell Quality Problems’ chart. Eggshell quality was assessed, and abnormalities were categorized, translucent and wrinkled eggs were categorized by the affected surface area as minimal (<25 %), moderate (25–75 %) or extensive (>75 %). Each egg was weighed using a digital balance (Ohaus SPU6001), and its length and width were measured using a digital calliper (±0.1 mm). The shape index (SI) was calculated as: SI = (width/length) × 100. Eggs were incubated using a Sanyo MIR-154 (Sanyo, Tokyo, Japan), with temperature and relative humidity maintained according to the breeder’s management recommendations. Egg weight was tracked on Embryonic Day 4, 8 and 12 (ED4, ED8 and ED12, respectively) using a digital balance (Ohaus SPU6001). On ED12, eggs were candled and classified as infertile, dead or alive. Embryos were euthanised by storing eggs at −20°C for 1 h. Embryo length was measured using a digital calliper, and embryo weight was recorded a digital balance (Ohaus SPU6001). Eggshell thickness was measured at the blunt end, pointed end and equator using a digital calliper (±0.1 mm).

Experiment 2: egg quality and mineral composition

In Experiment 2, 150 eggs were collected from a 27-week-old Ross-308 breeder flock. Eggs were categorized into three groups as FCFE and FCNE collected at 07:00 h and SCFE collected at 10:00 h (n = 50/group). An additional group, FCNE, was included in Experiment 2 to determine whether the differences observed in floor eggs were influenced by laying location in addition to collection time.

Each egg was weighed using an Ohaus SPU6001 balance (OHAUS Corporation, Parsippany, New Jersey, USA), and its length and width were measured using a digital calliper. The SI was calculated as: SI = (width/length) × 100. Eggshell quality was assessed, and abnormalities were categorized. The albumen and yolk widths were measured using a digital Vernier calliper. Albumen height was measured with an albumen height gauge equipped with an electronic display (Technical Services and Supplies, York, UK). Yolk colour was assessed using a DSM Yolk Colour Fan (DSM, Switzerland, 2005), with a scale ranging from 1 to 16 units (Ortiz et al., Reference Ortiz, Lawson, Jarrett, Ring, Scoles, Hoverman, Rocheford, Karcher and Rocheford2021). The Haugh unit was calculated using the formula: 100 × log10 (h − 1.7 × w0.37 + 7.6), where h represents the albumen height (mm) and w represents the egg weight (g) (Şekeroglu and Altuntaş, Reference Şekeroğlu and Altuntaş2009). Albumen and yolk were separated, and individual weights were recorded (Ohaus SPU6001). The albumen and yolk ratios were determined relative to whole-egg weight. The yolk index was calculated as: (yolk height/yolk width) × 100, and the albumen index as: (albumen height/albumen width) × 100. Eggshells were cleaned, air-dried and measured for thickness at the blunt end, pointed end and equator using a digital calliper to the nearest 0.001 mm. Albumen and yolk samples were stored at −80°C, while eggshell samples were stored at −20°C for further analysis. Eggshell mineral composition was assessed on a subset of 15 eggs/group. Eggshell samples were freeze-dried using a Christ Gamma 1–16 LSCplus freeze dryer (Martin Christ, Osterode, Germany). Approximately 0.2 g was digested in nitric acid, Milli-Q water and hydrogen peroxide and analysed via ICP-MS (iCAP-Q, Thermo Fisher Scientific, Bremen, Germany).

For nutrient composition profiling, yolk and albumen samples were dried using a freeze dryer (Christ Gamma 1–16 LSCplus with LyoCube). Yolk fat content was determined by Soxhlet extraction (Gerhardt Analytical Systems, Königswinter, Germany) with petroleum ether (AOAC, 1998). Albumen protein content was quantified by using nitrogen analysis (EA 1112 elemental analyser, Thermo Scientific, UK) using aspartic acid standards (Elemental Lab, Okehampton, UK), applying a conversion factor of 6.25. Three technical replicate measurements were taken per sample.

Statistical analysis

In both experiments, the individual egg was considered the statistical unit. Statistical analyses were performed using SAS University Edition (SAS Institute Inc., Cary, NC, USA). Effects were considered to be significant at P < 0.05. For continuous response variables analysed by ANOVA or t-test, normality of model residuals was evaluated primarily by inspection of Q–Q plots. Homogeneity of variance was assessed using Levene’s test and residual versus fitted plots. For comparisons between two groups, Student’s t-test was used when the assumption of equal variances was satisfied; otherwise, Welch’s t-test was applied.

In Experiment 1, initial egg weight, egg length, egg width, SI, ED12 embryo weight, ED12 embryo length and mean eggshell thickness were initially analysed by two-way ANOVA using the PROC GLM procedure. Mean eggshell thickness for each egg was calculated as the average of measurements obtained at the blunt end, equator and pointed end. Collection group (FCFE or SCFE), flock age/run (33 or 44 wk; Runs 1 and 2, respectively) and the collection group × flock age/run interaction were included as fixed effects. As neither flock age/run nor the collection group × flock age/run interaction significantly affected these response variables (P ≥ 0.05), data from the two runs were pooled, and FCFE and SCFE were subsequently compared using two-tailed t-tests. Egg-weight loss at ED4, ED8 and ED12 was analysed using a repeated-measures linear mixed model with the PROC MIXED procedure. Collection group, flock age/run, embryonic day and their interactions were included as fixed effects, with embryonic day specified as the repeated factor and individual egg as the subject. Eggshell abnormality frequencies were compared among treatment groups using Pearson’s chi-square test. Fertility and embryonic survival to ED12 were analysed separately as binary outcomes. Fertility was classified as fertile or infertile using all eggs, whereas embryonic survival was classified as alive or dead among fertile eggs only. Binary logistic regression was used to evaluate the effects of collection group, flock age/run and their interaction.

In Experiment 2, data were obtained from a single 27 wk old flock. Egg weight, egg length, egg width, SI, albumen and yolk measurements, Haugh unit, albumen and yolk weights and ratios, albumen and yolk indices, mean eggshell thickness, nutrient composition and individual eggshell mineral concentrations were analysed by one-way ANOVA using the PROC GLM procedure, with experimental group (FCNE, FCFE or SCFE) included as the fixed effect. Statistical differences among treatments were assessed using Tukey’s test. Two a priori contrasts were specified: FCNE versus FCFE, and FCFE versus SCFE. For nutrient composition analyses conducted in technical triplicate, replicate measurements from each sample were averaged to obtain a single egg-level value before statistical analysis.

Results

Experiment 1

The effect of grouping eggs based on collection time into FCFE and SCFE are presented in Figure 1. Throughout the experiment, significant differences were observed between eggs from FCFE and SCFE in several parameters. Initial egg weight at embryonic day 0 (ED0) was significantly higher in FCFE compared to SCFE (P = 0.034), and the final egg weight after 12 day of incubation (ED12) also remained greater in FCFE (P = 0.006). Additionally, a significant difference in absolute egg weight loss was observed between groups (P = 0.021), as was percentage weight loss (P = 0.008) between ED0 to ED12. Egg length was significantly longer in FCFE (P = 0.017), but there were no differences in egg width or SI between collections.

Figure 1.

Experiment 1: egg and embryo traits for first and second collection floor eggs. (a) Egg weight on embryonic day 0 (ED0) for first collection floor eggs (FCFE) and second collection floor eggs (SCFE). (b) Egg weight on embryonic day 12 (ED12). (c) Embryo weight on ED12. (d) Embryo length on ED12. (e) Egg weight loss between ED0 and ED12 expressed as a percentage of initial egg weight. (f) ED12 embryo weight expressed as a proportion of ED12 egg weight. Each point represents a single egg or embryo, and horizontal lines indicate group means. Exact P values for differences between FCFE and SCFE are shown on the panels. FCFE, first collection floor eggs; SCFE, second collection floor eggs; ED, embryonic day.

Multiple scatter plots compare egg and embryo traits for first and second collection floor eggs.
Figure 1. Long description

Panel A: A scatter plot shows egg weight on embryonic day 0 (ED0) for first collection floor eggs (FCFE) and second collection floor eggs (SCFE). The y-axis represents egg weight in grams, ranging from 50 to 70 grams. The x-axis labels are FCFE and SCFE. The plot indicates a slight difference in egg weight between the two groups, with a P value of 0.034. Panel B: Another scatter plot displays egg weight on embryonic day 12 (ED12) for FCFE and SCFE. The y-axis represents egg weight in grams, ranging from 30 to 80 grams. The x-axis labels are FCFE and SCFE. The plot shows a more pronounced difference in egg weight between the two groups, with a P value of 0.006. Panel C: A scatter plot illustrates embryo weight on ED12 for FCFE and SCFE. The y-axis represents embryo weight in grams, ranging from 2 to 12 grams. The x-axis labels are FCFE and SCFE. The plot reveals a significant difference in embryo weight between the two groups, with a P value of less than 0.001. Panel D: A scatter plot presents embryo length on ED12 for FCFE and SCFE. The y-axis represents embryo length in millimeters, ranging from 40 to 90 millimeters. The x-axis labels are FCFE and SCFE. The plot shows a notable difference in embryo length between the two groups, with a P value of less than 0.001. Panel E: A scatter plot depicts egg weight loss between ED0 and ED12 expressed as a percentage of initial egg weight for FCFE and SCFE. The y-axis represents the percentage of egg weight loss, ranging from 0 to 20 percent. The x-axis labels are FCFE and SCFE. The plot indicates a difference in egg weight loss between the two groups, with a P value of 0.004. Panel F: A scatter plot shows ED12 embryo weight expressed as a proportion of ED12 egg weight for FCFE and SCFE. The y-axis represents the proportion of embryo weight to egg weight, ranging from 0 to 20 percent. The x-axis labels are FCFE and SCFE. The plot reveals a significant difference in the proportion of embryo weight to egg weight between the two groups, with a P value of less than 0.001.

Embryo weight was significantly lower in FCFE compared to SCFE on ED12 of incubation (P < 0.001) as was embryo weight as a percentage of ED12 egg weight (P < 0.001). Finally, embryo length was significantly shorter in FCFE compared to SCFE (P = 0.001). Figure 2 presents the distribution of shell abnormalities. SCFE exhibited a significantly higher proportion of calcium-coated eggs (P = 0.044) as well as a greater incidence of minimal wrinkled eggs (P = 0.042). FCFE had a significantly higher proportion of soiled eggs (P < 0.001). However, there were no overall significant differences observed in the total number of shell abnormalities or shell characteristics between collections (P ≥ 0.05). There were no significant differences between collections in the percentages of eggs classified as alive, infertile, early dead, mid dead or rotten after 12 day of incubation. These results highlight that while weight-related parameters and specific shell abnormalities differed by collection time, other factors such as egg width and SI were unaffected.

Figure 2.

Experiment 1: eggshell abnormalities in first- and second collection floor eggs. The proportion of eggs exhibiting each abnormality type (translucent, wrinkled, soiled, cracked, pimple, broken and mended, misshapen, brown fleck, calcium spot and calcium coated) in first collection floor eggs (FCFE) and second collection floor eggs (SCFE). FCFE, first collection floor eggs; SCFE, second collection floor eggs.

A bar graph comparing eggshell abnormalities in two types of eggs.

Experiment 2

The internal and external egg quality characteristics for the three groups FCNE, FCFE, SCFE are summarized in Table 1. The egg weight did not differ significantly among the groups (P = 0.080); however, pairwise comparison revealed that FCFE (55.4 g) was significantly heavier than SCFE (53.8 g; P = 0.026). There were no significant differences in egg height or width across the groups. Internal egg quality varied across groups. Albumen width was significantly greater in FCFE (69.4 mm) and FCNE (68.3 mm) compared to SCFE (65.9 mm) (P = 0.004). Albumen weight and percentage were significantly higher in FCNE and FCFE compared to SCFE (P = 0.020 and P = 0.046, respectively). However, no significant differences were found in albumen height or Haugh units. Yolk characteristics showed that yolk width was significantly larger in FCNE (41.1 mm) compared to FCFE (40 mm) and SCFE (40 mm) (P = 0.002), while yolk weight and percentage did not differ significantly between groups. Yolk colour differed significantly among the groups (P < 0.001), SCFE had significantly higher yolk colour than both FCFE and FCNE. There were no significant differences in yolk fat content or albumen protein content across the samples. Significant differences were observed in shell characteristics, whereby FCNE and FCFE had thicker shells compared to SCFE (P < 0.001).

Table 1.

Experiment 2; internal and external egg quality characteristics of eggs designated as first or second collections and from different egg-laying locations

A table comparing internal and external egg quality characteristics across three groups.
Table 1. Long description

The table presents internal and external egg quality characteristics for three groups: FCNE, FCFE, and SCFE. It has 21 rows and 6 columns. The columns are labeled FCNE, FCFE, SCFE, SEM, and P value. The rows include various egg quality metrics such as egg weight, egg height, egg width, shape index, albumen height, albumen width, albumen weight, albumen weight percentage, Haugh unit, yolk height, yolk width, yolk weight, yolk weight percentage, yolk color score, shell weight, shell weight percentage, shell thickness, albumen dry matter percentage, albumen crude protein percentage, yolk dry matter percentage, and yolk crude fat percentage. Each row provides specific values for each group and includes statistical measures such as SEM and P value to indicate significance.

SEM: standard error of the mean; FCNE: first collection nest eggs; FCFE: first collection.

floor eggs; SCFE: second collection floor eggs (FCNE n = 50, FCFE n = 50, SCFE n = 50.

means ± SEM); FE %: fresh egg percentage

a,b means with different superscripts differed significantly according to Tukey’s test (p < 0.05).

The eggshell mineral concentrations are summarized in Table 2. Sodium (Na) concentration was significantly lower in SCFE (0.79 mg/g) compared to FCNE (0.85 mg/g) and FCFE (0.83 mg/g) (P = 0.007). Magnesium (Mg) concentration also differed among groups, with FCNE having a higher concentration than SCFE, while FCFE was intermediate (P = 0.026). For trace minerals, molybdenum (Mo) and zinc (Zn) were significantly higher in FCNE than in FCFE (P = 0.002, P = 0.040). For manganese (Mn) and arsenic (As), FCFE eggs had significantly higher concentrations than FCNE eggs (P = 0.007 and P = 0.040, respectively). No significant differences were found in the concentrations of calcium (Ca), phosphorus (P), potassium (K), sulphur (S) or other trace elements (P ≥ 0.05).

Table 2.

Experiment 2; mineral composition of eggshells as eggs designated as from first and second collection times and different egg-laying locations

A table comparing mineral concentrations in eggshells from different collection times and locations.
Table 2. Long description

The table presents mineral concentrations in eggshells, comparing early and later collection times and different egg-laying locations. It has 16 rows and 6 columns. The columns are labeled Mineral concentration, FCNE, FCFE, SCFE, SEM, and P value. The rows are grouped into Macro minerals (mg/g) and Trace minerals (mg/kg). Macro minerals include Ca, P, Na, K, S, and Mg. Trace minerals include Mo, Zn, Fe, Cu, Mn, Pb, Co, Ni, Se, Cd, and As. Each row lists the mineral concentration values for FCNE, FCFE, and SCFE, along with SEM and P value. Notable trends include significant differences in Na and Mg concentrations among the groups, with specific P values indicating statistical significance.

SEM: standard error of the mean; FCNE: first collection nest egg; FCFE: first collection.

floor eggs; SCFE: second collection floor eggs

a,b means with different superscripts differed significantly according to Tukey’s test (P < 0.05). Macro minerals are expressed as mg/g and trace minerals as mg/kg.

Discussion

In both experiments, eggs were collected and sampled early and late in the morning, to capture, at a population level, eggs broadly representative of early and later positions in the laying sequence respectively, as supported by the consistent egg weight gradient observed between collection groups. In Experiment 1, there was no significant effect of collection time on the proportion of eggs classified as alive, infertile or dead at various stages of incubation. This indicates that both SCFEs and FCFEs contributed similarly to reductions in fertility and hatchability. However, clear differences in embryonic growth emerged between groups. Embryos from FCFE were significantly lighter at ED12, even though these eggs were initially heavier and lost less weight during incubation. The initial egg size and nutrient content determine substrate availability and necessitate corresponding adjustments in metabolic pathways during embryonic development (Hu, Reference Hu2013). However, these differences were not explained by the egg weight on ED12 alone, suggesting that additional factors – such as water vapour conductance or differences in shell functional properties – may have influenced the trajectory of embryonic growth. Comparable trends have been reported by Zakaria et al. (Reference Zakaria, Plumstead, Romero-Sanchez, Leksrisompong and Brake2009) indicating that mid-sequence eggs experienced greater weight loss during storage than those laid earlier or later, a pattern they associated with increased shell porosity and variation in albumen quality, although oviposition time did not affect weight loss during incubation. Such mechanisms may help explain the elevated weight loss observed in SCFEs in the present study. Notably, embryo length mirrored embryo weight, further reinforcing the interpretation of slower developmental progression in FCFEs. This contrasts with findings by Ruiz and Lunam (Reference Ruiz and Lunam2002), who reported that chicks from morning-laid eggs were consistently heavier than those from afternoon-laid eggs, supporting the assumption that egg weight is positively associated with chick or embryo weight. To our knowledge, the magnitude of differences observed in the current study have not been previously reported, making it difficult to speculate on the specific mechanisms contributing to these variations. The current results suggest that this relationship may not hold under all conditions. Variation in pre-incubation thermal exposure is a potential contributing factor to the observed differences in growth. Earlier studies (Fasenko et al., Reference Fasenko, Robinson, Armstrong, Church, Hardin and Petitte1991; Akil and Zakaria, Reference Akil and Zakaria2015) proposed that prolonged pre-incubation at ambient temperature can lead to partial embryonic development prior to storage-induced diapause. While this may initially explain the advanced embryonic growth observed in FCFEs, studies have shown that embryos progressing beyond stage XIII of the Eyal-Giladi and Kochav (Reference Eyal-Giladi and Kochav1976) classification – toward primitive streak formation – may be more vulnerable to developmental arrest during storage due to ongoing cellular migration and differentiation (Bellairs, Reference Bellairs1986; Fasenko et al., Reference Fasenko, Robinson, Whelan, Kremeniuk and Walker2001). In contrast, embryos that remain at stage XIII, a more quiescent stage, are better suited to resume development following storage. Taken together, these observations suggest that SCFEs, which likely experienced shorter exposure to ambient conditions prior to collection, may have been developmentally less advanced at the onset of storage and therefore more stable during the early stages of incubation. It is worth noting that any difference in pre-incubation thermal exposure between collection groups is itself downstream of sequence-dependent differences in oviposition timing and therefore cannot be considered entirely independent of laying sequence. However, the underlying mechanisms remain unclear. Findings from Experiment 2 provide some support for this, as differences in egg characteristics across collection times may help to explain the variation in embryonic growth outcomes observed in Experiment 1.

Shell quality also showed variation between collection times in Experiment 1. While the overall incidence of abnormal shells was not affected, the nature and distribution of defects differed between collection times. FCFEs were more likely to exhibit structural flaws such as misshapen, cracked, translucent and calcium-spotted shells, whereas SCFEs were more often affected by shell surface irregularities such as pimples, wrinkles, soiling or signs of mending. The presence of calcium-coated shells exclusively among SCFEs may indicate transient oviposition delays, during which additional calcium is deposited on the shell surface (Reynard and Savory, Reference Reynard and Savory1999). The greater frequency of soiled eggs in FCFEs, despite post-collection washing, may reflect extended contact with the floor or residual surface contamination. These traits, commonly used as proxies for shell quality, may reflect underlying variation in shell gland function, calcium metabolism or the timing of shell calcification (Solomon, Reference Solomon1997; Roberts, Reference Roberts2004; Kebreab et al., Reference Kebreab, France, Kwakkel, Leeson, Kuhi and Dijkstra2009). Although the precise mechanisms remain unclear, these patterns suggest that the processes underlying shell formation are not static throughout the laying period and may shift over the course of shell formation. These findings indicate temporal variation in shell quality that warrants further investigation.

Experiment 2 extended this by assessing whether time of collection was also associated with variation in other structural and compositional egg traits. It confirmed the pattern already observed in Experiment 1: eggs collected earlier were heavier than eggs collected later. These findings align with prior studies which have consistently demonstrated that oviposition time significantly impacts egg weight, with eggs laid earlier in the day tending to be heavier than those laid later (Robinson et al., Reference Robinson, Hardin, Robinson and Williams1991; Patterson, Reference Patterson1997; Zakaria et al., Reference Zakaria, Plumstead, Romero-Sanchez, Leksrisompong, Osborne and Brake2005; Tůmová et al., Reference Tůmová, Englmaierová and Zita2009). There are two possible underlying biological processes to consider here. Firstly, differences in egg weight may partly reflect variation in maternal investment during egg formation, as resources are deposited maternally into avian eggs and the amount of major resources is generally related to egg size, while maternal diet and nutritional condition may also influence the composition of resources allocated to eggs (Williams, Reference Williams1994; Nager et al., Reference Nager, Monaghan, Houston, Arnold, Blount and Verboven2006; Rao et al., Reference Rao, Xie, Yang, Chen, Grossmann and Zhao2009; Rubolini et al., Reference Rubolini, Romano, Navara, Karadas, Ambrosini, Caprioli and Saino2011). In wild birds, mothers influence embryonic development by allocating nutrients, with the amounts varying across the laying sequence and with maternal condition or social environment (Reed and Vleck, Reference Reed and Vleck2001; Reed and Clark, Reference Reed and Clark2011). Maternal mechanisms have been proposed to regulate hatching synchrony (Boonstra et al., Reference Boonstra, Clark and Reed2010). In both synchronously hatching Canada geese and asynchronously hatching American coots, later-laid eggs receive lower parental investment, yet their embryos exhibit developmental strategies that mitigate these disadvantages. While egg components were once thought to unilaterally direct development, growing evidence suggests that embryos are not passive recipients but can actively sense and respond to their environment, even modulating maternally derived factors such as yolk steroids potentially to their own benefit (Reed and Clark, Reference Reed and Clark2011). Secondly, the first egg in a new sequence is often retained in the reproductive tract for an extended period of time, which may account for the greater egg weight observed. This prolonged residence is particularly relevant for the shell gland, where the amount of deposited shell is a linear function of uterine residence time after plumping (Tůmová and Ebeid, Reference Tůmová and Ebeid2005; Ketta and Tůmová, Reference Ketta and Tumová2016). Prolonged transit may also influence albumen deposition in the magnum, as albumen synthesis proceeds continuously in the tubular gland cells while the rate of secretion rises when an egg is present (Muramatsu et al., Reference Muramatsu, Hiramoto and Okumura1991). Although direct evidence for such a time-dependent effect is more limited.

The timing of egg-laying is known to influence various components of the egg. The yolk weight and percentage did not vary with collection time. This is broadly consistent with previous work by Gumułka et al., (Reference Gumułka, Kapkowska and Maj2010), who reported no consistent trend across oviposition times. However, other studies have reported greater yolk mass in eggs laid earlier in the day (Tůmová and Ebeid, Reference Tůmová and Ebeid2005; Tůmová and Gous, Reference Tůmová and Gous2012), suggesting that yolk dynamics may be more context dependent. In the present study, the greater egg weight observed in early-collection eggs was not attributable to yolk content, indicating that albumen allocation is the more responsive component of egg mass under these conditions. Albumen proteins are deposited around the yolk during passage through the magnum, where synthesis proceeds continuously but the rate of secretion rises when an egg is present in the region (Muramatsu et al., Reference Muramatsu, Hiramoto and Okumura1991). Both types of first collection egg had higher absolute weight, proportional weight and albumen width than SCFEs, a consistent reduction that points to a sequence-linked mechanism.

Eggshell thickness was also influenced by the collection time, with thicker shells observed in first collection eggs. This corresponded to an increase of approximately 3% in thickness compared with SCFEs. This difference may be associated with a longer laying interval, as the duration of eggshell formation and the time an egg spends in the shell gland influence shell deposition and eggshell thickness (Nys, Reference Nys1986; Tůmová and Ebeid, Reference Tůmová and Ebeid2005; Ketta and Tumová, Reference Ketta and Tumová2016). Longer laying intervals have also been positively correlated with the duration of eggshell formation, deposition rate and quality (Chen et al., Reference Chen, He, Li, Song, Huang, Shi, Li, Li, Xu and Zheng2021). While previous research has often focused on factors such as genotype, temperature, bird age and nutrition (Michalak et al., Reference Michalak, Chojnacka, Dobrzański, Górecki, Zielińska, Korczyński and Opaliński2011; Gualhanone et al., Reference Gualhanone, Furlan, Fernandez-Alarcon and Macari2012; Abdelqader et al., Reference Abdelqader, Al-Fataftah and Daş2013; Tůmová et al., Reference Tůmová, Gous and Tyler2014; Kibala et al., Reference Kibala, Rozempolska-Rucińska, Kasperek and Zięba2015), the findings of this study highlight the role of laying sequence disruptions in influencing eggshell characteristics. These structural differences may carry functional consequences: thicker shells reduce water vapour conductance and weight loss (Ar et al., Reference Ar, Paganelli, Reeves, Greene and Rahn1974), while also modulating gas exchange critical for embryonic development (Burton and Tullett, Reference Burton and Tullett1983). As highlighted in Experiment 1, variations in shell thickness may influence water vapour loss and gas conductance during incubation. While shell conductance was not measured directly, the observed differences in shell characteristics provide a plausible link between laying pattern and embryonic growth, as shell conductance has been proposed as a potential mechanism underlying laying-sequence differences in embryonic metabolism in Canada geese (Boonstra et al., Reference Boonstra, Clark and Reed2010).

As the timing of shell calcification may differ depending on when the egg is laid, we considered whether variation in calcium availability might influence shell quality (Mongin and Sauveur, Reference Mongin and Sauveur1974). Final stages of shell formation occur overnight, when hens are not feeding, and calcium must be drawn primarily from the medullary bone (Bain et al., Reference Bain, Nys and Dunn2016). Late-laid eggs may follow a different temporal pattern relative to feed intake as calcium absorption is not tightly synchronized with shell deposition, hens rely on reserves from the crop or medullary bone to support shell formation (Mongin and Sauveur, Reference Mongin and Sauveur1974). This may contribute to variation in shell quality depending on laying time.

The intriguing difference in the eggshell thickness suggests that there may be differences in eggshell mineral composition. It is emphasized that the mineral composition of eggshells can vary based on dietary supplementation and environmental conditions (Drabik et al., Reference Drabik, Karwowska, Wengerska, Próchniak, Adamczuk and Batkowska2021)., which may change throughout the day. For instance, hens that lay eggs in the morning may have different access to nutrients compared to those that lay later in the day, potentially leading to variations in the mineral content of the eggshells produced during these times. In the present study, the higher eggshell sodium (Na) levels in first collection eggs may reflect temporal changes in Na+ related ion transport in the shell gland during egg formation (Lavelin et al., Reference Lavelin, Meiri, Genina, Alexiev and Pines2001). In terms of trace minerals, the results showed that zinc (Zn) and molybdenum (Mo) levels were significantly lower in FCFE and SCFE compared to FCNE. The observed differences may reflect underlying hen-level factors, such as differences in mineral metabolism, stress or access to feed associated with floor-laying behaviour. Previous research has shown that eggshell mineral content can be influenced by the hen’s physiological status, dietary intake and environmental or management conditions (Ketta and Tůmová, Reference Ketta and Tumová2016). Thus, the reduced Zn and Mo content in FCFE and SCFE eggshells may indicate that floor-laying hens experience physiological or nutritional challenges that impact mineral deposition in the shell, independent of oviposition time. The higher As content observed in first sequence floor eggshells may partly reflect greater contact with floor or litter material, as arsenic can occur in poultry manure and associated surface soils and environmental metal exposure may vary geographically (Liu et al., Reference Liu, Tian, Cao, Li, Dong and Li2021; Drabik et al., Reference Drabik, Skałecki, Spasowska, Melnyk, Kutrzuba and Batkowska2024). Eggs laid on the floor are in direct contact with dust, litter or bedding material, which can serve as sources of arsenic deposition. Levels of lead (Pb), cobalt (Co), nickel (Ni), copper (Cu) and iron (Fe) did not differ significantly among the egg types. Overall, in this study the findings suggest that the mineral composition of eggshells was more influenced by location than sequence.

Conclusion

Oviposition time influenced both egg composition and embryonic growth. Eggs laid early in the sequence were heavier and had greater albumen weight and width, yet embryos from these eggs were lighter and shorter at mid-incubation compared with those from later-laid eggs. By contrast, smaller eggs laid later in the sequence supported more advanced embryonic growth, suggesting that nutrient utilization might differ across the laying sequence. Shell properties also varied, with early-laid eggs exhibiting greater thickness and higher sodium concentrations, while later-laid eggs produced thinner shells with more irregularities. These differences in albumen allocation and shell structure may affect water balance and gas conductance during incubation. Whether such variation in egg composition translates into measurable effects on hatchability, chick viability or post-hatch performance remains an important question for future research.

Acknowledgements

The authors gratefully acknowledge the financial support of the Study Abroad Postgraduate Education Scholarship (YLSY) awarded by the Republic of Türkiye Ministry of National Education. The authors also wish to thank Catherine Williams (NUVetNA, School of Veterinary Medicine and Science, University of Nottingham) for her valuable technical support during this work.

Author contributions

Imge Duru Onol contributed to data curation, formal analysis and investigation, supported project administration and funding acquisition and undertook visualization and writing of the original draft. Florence Jenkins contributed to data curation, formal analysis and investigation and assisted with writing of the original draft. Mustafa Onol contributed to data curation and investigation. Nigel Kendall contributed to methodology, validation and formal analysis. Cormac J. O’Shea was responsible for conceptualization, methodology and supervision, contributed to data curation and formal analysis, provided resources and project administration and supported writing through review and editing.

Funding statement

This research received no specific grant from any funding agency, commercial or not-for-profit sectors.

Competing interests

Cormac O’Shea is an Editor of The Journal of Agricultural Science, but was excluded completely from the review process for this paper. The other authors declare there are no conflicts of interest.

Ethical standards

All procedures involving vertebrate animals were reviewed and approved by the University of Nottingham Institutional Animal Ethics Committee (approval number 244) and adhered to the relevant national and institutional guidelines for the care and use of animals in research.

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Figure 0

Figure 1. Figure 1 long description.Experiment 1: egg and embryo traits for first and second collection floor eggs. (a) Egg weight on embryonic day 0 (ED0) for first collection floor eggs (FCFE) and second collection floor eggs (SCFE). (b) Egg weight on embryonic day 12 (ED12). (c) Embryo weight on ED12. (d) Embryo length on ED12. (e) Egg weight loss between ED0 and ED12 expressed as a percentage of initial egg weight. (f) ED12 embryo weight expressed as a proportion of ED12 egg weight. Each point represents a single egg or embryo, and horizontal lines indicate group means. Exact P values for differences between FCFE and SCFE are shown on the panels. FCFE, first collection floor eggs; SCFE, second collection floor eggs; ED, embryonic day.

Figure 1

Figure 2. Experiment 1: eggshell abnormalities in first- and second collection floor eggs. The proportion of eggs exhibiting each abnormality type (translucent, wrinkled, soiled, cracked, pimple, broken and mended, misshapen, brown fleck, calcium spot and calcium coated) in first collection floor eggs (FCFE) and second collection floor eggs (SCFE). FCFE, first collection floor eggs; SCFE, second collection floor eggs.

Figure 2

Table 1. Experiment 2; internal and external egg quality characteristics of eggs designated as first or second collections and from different egg-laying locationsTable 1 long description.

Figure 3

Table 2. Experiment 2; mineral composition of eggshells as eggs designated as from first and second collection times and different egg-laying locationsTable 2 long description.