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. 2023 Sep 17;102(12):103119. doi: 10.1016/j.psj.2023.103119

The physicochemical features of eggshell, thick albumen, amniotic fluid, and yolk during chicken embryogenesis

Jakub Biesek 1,1
PMCID: PMC10585332  PMID: 37832191

Abstract

The study aimed to analyze the hatching egg and physiochemical features of eggshells, thick albumen, amniotic fluid, and yolk during the incubation of Ross 308 chicken eggs. Eggs (n = 755) were incubated for 21 d. Quality analysis of fresh eggs was performed. Eggshells, albumen, and yolk were collected from fresh eggs and incubation d 1, 7, and 14. Eggshell thickness and strength, pH, vitelline membrane strength, fatty acid (FA) in the yolk, pH, viscosity, lysozyme activity, and crude protein content in thick albumen and amniotic fluid were analyzed. Hatching parameters were calculated. Egg weight loss was constant (8.04% overall). Lower egg surface temperature was found on d 7 compared to d 4, 14, and 18. A lower thickness of posthatch eggshells was found. The strength of the vitelline membrane significantly decreased within 24 h (by over 58%). During incubation, there was a decrease in thick albumen/amniotic fluid pH; an opposite trend was found in yolk pH. The vitelline membrane strength was negatively correlated with the albumen pH. Lysozyme activity was higher in fresh thick albumen and up to 2 wk of incubation. On d 7, the lowest activity was found in the amniotic fluid. On d 14, lysozyme activity increased in amniotic fluid. The higher viscosity of the thick albumen was demonstrated on d 7 and 14 of incubation. The lowest viscosity in amniotic fluid was found on the same days. Crude protein content was higher in thick albumen (d 7 and 14) and lowest in amniotic fluid on d 7. The FA content changed between d 0 and 14. The results indicate different use of FA, where PUFA decreased. Eggshell is used in the last week of incubation. The thick albumen is reduced, while the biological value of amniotic fluid is increasing. Lysozyme activity, viscosity, and crude protein content may be interdependent. It may indicate the flow of substances and the transfer of functions from the thick albumen to the amniotic fluid during chicken embryogenesis.

Key words: albumen and amniotic fluid viscosity, crude protein, eggshell thickness, lysozyme activity, yolk’ fatty acid

INTRODUCTION

Hamburger and Hamilton (1951) presented a series of normal chick embryo stages. The authors already claimed that it was crucial for widely used descriptive and experimental embryology and in research, including medical research. During embryonic development, the structure of the egg and its morphological components change. It is an essential element with environmental, defensive, and nutritional functions (Hegab and Hanafy, 2019). At the oviposition stage, its protective structures (biomineralized eggshell, protein antimicrobial peptides, vitelline membrane of egg yolk) undergo dynamic changes. The fetal membranes are formed (chorion-allantoic, amniotic, yolk sac). As a result, primary protective barriers are degraded during embryo development (Hincke et al., 2019).

The eggshell is the egg's outer protective barrier and forms the embryo's environment. Its quality is expressed in cracking strength and thickness. The thickness of the eggshell affects gas exchange and water loss and influences the embryo's development and chick' hatching (Lavelin et al., 2000). The eggshell is thinning during embryogenesis, which is related to the embryo's calcium uptake, resulting in less protection and possible vulnerability toward the end of development (Castilla et al., 2009). Temperature and humidity regulate the processes mentioned (Veldsman et al., 2019). According to the authors, thinner eggshells may result in faster water loss, affecting hatchability. A water loss of 12 to 14%, up to the 18th day of incubation of chicken eggs, is considered optimal. In comparison, an increase in embryo mortality has been demonstrated with a water loss below 9.1% or above 18.5% (Van der Pol et al., 2013). Nakage et al. (2003) showed a relationship between incubation temperature and water loss from partridge-hatching eggs. Weight loss by d 16 of incubation has been shown to range from 8% (35.5°C–36.5°C) to 9.94% (37.5°C).

The role of the albumen is to form embryonic fluid. Albumen contains approx. 10.5% proteins. The proteins in the albumen are transported to the amniotic cavity, the yolk sac, and then to the embryo, more specifically, to the digestive tract. The embryo develops tissues properly (Willems et al., 2014). Egg albumen provides the environment for the embryo, and its components, including bioactive proteins, have an antimicrobial function, protecting the egg yolk from microbial infections. In addition, some proteins (ovotransferrin, lysozyme, ovomucoid) move to the embryo and may perform specific functions in embryonic organs and the circulatory system (Takashi et al., 2019). Lysozyme (muramidase) is an enzymatic protein with antibacterial properties. Together with ovomucin, it forms a complex related to the albumen's pH and viscosity (Caner and Yüceer, 2015). The authors reported that the decrease in Haugh units and viscosity during storage was associated with the weakening of the albumen. It was due to the deterioration of the lysozyme-ovomucin complex, decreased carbohydrate content in ovomucin, and increased pH and water loss. In the hatching egg, the embryo affects albumen liquefaction (Benton et al., 2001). The authors suggested that the ammonia produced by the embryo affects the dynamics of the process. In mid–late-stage incubation, the albumen is reduced in favor of the amniotic fluid, which takes over the embryo's environment role (Tona et al., 2005; Willems et al., 2014).

The functions and changes in the egg yolk are also related to the above. The vitelline membrane separates the yolk from the albumen and is its integral part. It protects against pathogens and supports the proper development of the embryo. Its strength after 48 h of incubation decreases by 73% (Marzec et al., 2016). The authors showed a significantly positive correlation between the strength of the vitelline membrane and the yolk index. The vitelline membrane's characteristics may depend on the properties of the albumen and yolk that create a microenvironment for the embryo (Reijrink et al., 2008). The development of the embryo depends on the availability of nutrients, and the yolk is an essential source of nutrients. Components pass from the yolk to the embryo via the vascularized membrane of the yolk sac or directly into the intestine via the yolk stalk, depending on the stage of development (Ding et al., 2022). The yolk is the primary energy source, therefore lipids and fatty acids, the concentration of which changes during embryogenesis. However, fatty acid interconversions can differ and result from the activity of different enzymatic processes (Şahan et al., 2014).

In a previous pilot study (Biesek et al., 2023), it was found that the protective barriers of the embryo are changed, and fractions are liquefied during incubation. Due to its activity, it was concluded that lysozyme passes from the albumen into the amniotic fluid. Thus, further research was undertaken on the changes in morphological components and their relationships during embryogenesis regarding the physiochemical features of the eggshell, thick albumen, amniotic fluid, and yolk.

The presented study aspires to confirm the assumptions and complement current knowledge about the changes occurring in the structures of the hatching egg, which may be a critical element in the modern line of broiler chicken hatching technology in embryonic development and embryo protection. Thus, the study aimed to analyze the relationship between the characteristics of hatching eggs and changes in the physicochemical characteristics of the eggshell (thickness, strength), thick albumen and amniotic fluid (pH, viscosity, lysozyme activity, and crude protein content), and yolk (pH, strength of the vitelline membrane, fatty acid profile) during the incubation of Ross 308 broiler chicken hatching eggs in nutritional and protective aspect.

MATERIALS AND METHODS

The research obtained permission No. 2/2022 of the Committee for the Care of Animals (Ethics Committee) of the Bydgoszcz University of Science and Technology, Poland. The research was carried out following the applicable regulations.

Egg Incubation and Sample Collection

Hatching eggs were obtained from a breeding flock of Ross 308 meat-type hens. The flock was 32-wk old (Drobex-Agro Ltd., Makowiska, Poland). Eight hundred eggs were purchased. The eggs were selected in terms of structure. Malformed, broken eggs were eliminated. Seven hundred seventy-three hatching eggs were chosen for further stages. Before incubation, 18 fresh eggs were collected for quality analysis. Then, on d 1, 7, and 14, 18 eggs each (54 eggs) were selected. Thus, the initial number of incubated eggs was 701.

Incubation was performed in a single-stage incubator (Jarson, Gostyń, Poland) and hatcher (Jarson, Gostyń, Poland). Incubation lasted 21 d. Before, the eggs were stored for 1 d, according to the hatchery recommendation. All stages of egg handling, including storage, transport, and disinfection, are described by Biesek et al. (2023). The incubation was divided into 2 stages. Stage 1—the incubator—lasted from 1 to 18 d. The following parameters were set: temperature—37.7°C, humidity—55/60%, ventilation—50/60%. In the incubator, eggs placed in trays (vertical) were rotated 45° every hour. On the seventh day of incubation, the eggs were candled to remove unfertilized eggs or eggs with dead embryos. During routine work, the incubator was opened for approx. 15 min.

On the 18th day, the eggs were candled again to eliminate dead embryos and then transferred to the hatcher (stage 2). At this stage, the eggs were placed in not rotated trays (eggs placed horizontally). The following parameters were set: temperature—37.5°C, humidity—70/75%, ventilation—80%. Hatching lasted until the 21st day. The chicks (incubated individually) were weighed on the day of hatching. Hatching data were recorded, and the fertilization rate, chick share in egg, hatch rate from set eggs, hatch rate from fertilized eggs, and early and mid-late embryo mortality rate were calculated (Biesek et al., 2023).

Egg Quality Analyzes

An analysis of morphological and qualitative features of 18 fresh hatching eggs was performed. The width and length were considered, and the egg-shape index was calculated. Eggs (Radwag, Radom, Poland) were weighed, and egg surface area was calculated using a formula: 4.835×W0.662 (W—egg weight, g). Yolk, albumen, and dry eggshell were weighed. The eggshells were dried for 3 h at 105°C in a SUP 100M oven. Yolk, albumen, and eggshell share in the egg (%) were calculated. In addition, thick albumen height was measured using a TA.XT plus C texture analyzer (Stable Micro Systems, Godalming, UK). Haugh units were calculated using HU = 100 logs (H + 7.57 − 1.7 W0.37), where H—thick albumen height; W—egg weight.

Eggshell Surface Temperature and Egg Weight Loss

Throughout the incubation period (d 1, 4, 7, 10, 14, 18), the temperature from the incubator system was noted. Also, the eggshell surface temperature (EST) of selected 18 hatching eggs was measured. The eggs were numbered to measure the temperature of the eggs in the same spot. Eggs were also weighed to control water loss (egg weight loss). The EST was measured using a laser thermometer (Microlife, Rzeszów, Poland).

Considering the average weight, the other hatching eggs were randomly selected for further analysis on d 1, 7, and 14.

Eggshell Strength and Thickness

Eggshell strength was analyzed using an Egg Force Reader (Orka Food Technology, Utah, USA; Cereus Wena, Toruń, Poland). Eggshells were dried for 3 h at 105°C (SUP 100M heater). Eggshell thickness was measured with an electronic micrometer screw (QTC, TSS, York, UK). The membranes were removed with a scalpel. Posthatching, the thickness of the eggshell without the eggshell membrane was measured.

Yolk’ Vitelline Membrane Strength

The strength of the egg yolk vitelline membrane was determined on d 0 and 1 (TA.XT plus C texture analyzer, Stable Micro Systems, Godalming, UK). The Exponent Connect software was used. Eighteen replicates were analyzed at each time.

Albumen and Amniotic Fluid and Yolk pH

Using a pipette, the thick albumen and amniotic fluid were sampled in the cavities. The pH value was measured using a SevenEasy pH meter (Mettler-Toledo AG, Schwerzenbach, Switzerland). The glass-tipped electrode was dipped into the egg yolk, albumen, and amniotic fluid. Eighteen repetitions were performed at each time. pH for thick albumen was determined on d 0, 1, 7, and 14, and for the amniotic fluid on d 7 and 14. Thick albumen and amniotic fluid were frozen (−18°C).

Lysozyme Enzymatic Activity in Thick Albumen and Amniotic Fluid

Thick albumen and amniotic fluid samples were thawed (4°C for 12 h). Phosphate buffer was prepared within the Enzymatic Assay of Lysozyme EC 3.2.1.17. (Merck, KGaA, Darmstadt, Germany) (Merck, 2022). The buffer was also used for a Micrococcus lysodeikticus (luteus) bacteria suspension. The absorbance was in the range of 0.6 to 0.7 (λ = 450 nm). From each sample, 1 mL was taken and refilled with buffer to a volume of 10 mL.

For the enzymatic activity of lysozyme analysis, 2.4 mL of the bacterial suspension and 0.1 mL of the thick albumen/amniotic fluid sample were added to the spectrophotometric cuvette and placed in the spectrophotometer (SP830 Plus, Metertech, Taipei, Taiwan). The enzymatic activity of lysozyme (U/ mL) was calculated using the formula: (ΔA450/minTest−ΔA450/minBlank)(df)(0.001)(0.1) (df, dilution factor; 0.001, change in absorbance (ΔA450) as per the Unit Definition; 0.1, volume (mm) of added solution).

Thick Albumen and Amniotic Fluid Viscosity

A Brookfield Ametek, DVNext, LV viscometer (Labo Plus, Warsaw, Poland) was used for the viscosity of the thick albumen and amniotic fluid. A cone/plate measuring system was used to measure the viscosity of small-volume samples. A total of 0.5 mL of supernatant per plate was taken from the centrifuged sample. Based on the pilot study, the shear rate (20 rpm = 150,000 1s) was chosen. The values were the average of the 30-s analysis.

The above methods of incubation, egg quality, and physical and chemical characteristics were performed following the methodology of Biesek et al. (2023).

Crude Protein Content in Thick Albumen and Amniotic Fluid

The crude protein content analyses in thick albumen and amniotic fluid were carried out (Polish Committee for Standardization, 2015; Congjiao et al., 2019). The Kjeldahl method was used. The crude protein content in thick albumen was determined on d 0, 1, 7, and 14 and for the amniotic fluid on d 7 and 14.

Fatty Acid Content in the Yolk and Yolk Sac

The chloroform-methanol extraction method was used (Wang et al., 2000). For fatty acid (FA) profile analyses, 6 eggs from d 0 and 14 were selected. Each yolk was analyzed in duplicate (12 replicates per group). Yolks were lyophilized. A gas chromatograph (Agilent 7890B, Santa Clara, CA) equipped with a flame ionization detector (FID) and a fused silica capillary SP-2380 column, 30 m × 0.25 mm × 0.2 µm (Supelco, Bellefonte, PA), with a constant flow of 1.0 mL/min helium as carrier gas was used for FA profile identification and quantification.

In this research, FA were analyzed: C14:0—myristic acid; C14:1n-5—myristoleic acid; C15:0—pentadecanoic acid; C15:1n-5—10-pentadecenoic acid; C16:0—palmitic acid; C16:1n-7—palmitoleic acid; C17:0—margaric acid; C17:1n-7—heptadecanoic acid; C18:0—stearic acid; C18:1n-9t—elaidic acid; C18:1n-9—oleic acid; C18:2n-6cc—linoleic acid; C18:3n6—ɣ-linolenic acid; C18:3n-3—α-linolenic acid; C21:0—heneicosanoic acid; C20:2—eicosadienoic acid; C20:4n-6—arachidonic acid; C20:5n-3—eicosapentaenoic acids; C24:0—lignoceric acid; C24:1n-9—nervonic acid; C22:6n-3—docosahexaenoic acid. The SFA—saturated fatty acids (C14:0; C15:0; C16:0; C17:0; C18:0; C21:0; C24:0); MUFA—monounsaturated fatty acids (C14:1n-5; C15:1n-5; C16:1n-7; C17:1n-7; C18:1n-9t; C18:1n-9; C24:1n-9); PUFA—polyunsaturated fatty acids (C18:2n-6cc; C18:3n-6; C18:3n-3; C20:2; C20:3n-3; C20:4n-6; C20:5n-3; C22:6n-3), and TFA—trans fatty acids (C18:1n-9t) were analyzed.

Statistical Calculation

The data were calculated in a statistical program (Statistica 13.3, Statsoft, TIBCO, Kraków, Poland). The morphological composition of hatching eggs was presented as descriptive statistics (the mean values, standard deviation (±SD), standard error of the mean (SEM), and minimum and maximum values). Mean values, ±SD and SEM, were calculated for all the features. Levene's test for homogeneity was used. Statistically significant differences were analyzed by Tukey's test. Statistical verification was performed with the P value <0.05. The figures were prepared in Excel (Microsoft, Washington, USA). The correlation between some morphologic and physicochemical features was also calculated, considering the P value <0.05.

RESULTS

Hatching Results

The fertilization rate of hatching eggs of Ross 308 broiler chickens was 98.29%. The hatchability from laid eggs was 93.44%, and from fertilized eggs—95.07%. Healthy hatched chicks accounted for as much as 99.70% of all hatched chicks. On average, the percentage of chicks in an egg was 76.32% (Table 1).

Table 1.

Hatchability of broiler chickens.

Parameter Values
Total number of eggs 701
Fertilized eggs 689
Unfertilized eggs (d 7) 10
Eggs with early dead embryos (d 1–7) 7
Eggs with mid-late dead embryos (d 8–21) 13
Eggs incubated in a hatcher (from d 18) 659
Total of hatched chicks 657
Healthy chicks 655
Unhatched eggs 2
Egg weight (g) 60.62 ± 0.54
Crippled and weak chicks 2
Chicks weight (g) 46.27 ± 0.80
Chick share in egg (%) 76.32 ± 1.19
Fertilization rate (%) 98.29
Hatch rate from set eggs (%) 93.44
Hatch rate from fertilized eggs (%) 95.07
Early embryo mortality rate (%) 1.02
Mid-late embryo mortality rate (%) 2.18
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Quality of Fresh Hatching Eggs

Table 2 presents the eggs' structure, morphological composition, and qualitative features. Hatching eggs were characterized by low variability (±SD). The average weight of the egg was 59.98 g, while the difference between the lowest and the highest value was 2.89 g. The egg's morphological components share was 31.02% of the yolk, 60.08% of the albumen, and 8.90% of the eggshell. A height of 6.34 mm characterized the egg albumen, and the difference between the minimum and maximum values was 3.86 mm. The freshness index determined using Haugh units was at the level of 78.61. The difference between the lowest and highest value was 29.62.

Table 2.

Descriptive statistics of fresh broiler chicken hatching eggs.

Item Mean value Min. value Max. value ±SD SEM
Width (mm) 44.08 43.02 44.93 0.55 0.13
Length (mm) 55.29 53.82 57.52 1.18 0.28
Egg-shape index (%) 79.78 74.88 82.85 2.50 0.59
Egg weight (g) 59.98 58.44 61.33 0.94 0.22
Egg surface area (cm2) 72.69 71.44 73.76 0.76 0.18
Yolk weight (g) 18.60 16.67 22.83 1.51 0.36
Yolk share (%) 31.02 28.13 39.06 2.74 0.65
Albumen weight (g) 36.05 30.44 38.35 2.03 0.48
Albumen share (%) 60.08 52.09 63.30 2.90 0.68
Thick albumen height (mm) 6.34 3.98 7.84 0.93 0.22
Haugh units 78.61 58.62 88.24 6.91 1.63
Eggshell weight (g) 5.34 4.96 5.92 0.25 0.06
Eggshell share (%) 8.90 8.20 9.85 0.42 0.10
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The results of a correlation between the features of hatching eggs of Ross 308 broiler chickens are shown in Table 3. The egg's width was negatively and significantly correlated with the length of the egg (−0.736), while in the case of the relation between the width and the egg-shape index, a positive, high correlation (0.890) was found. Similarly, egg length and shape index were highly correlated (−0.964; P < 0.05). The weight of the hatching egg was highly, significantly correlated with its surface (0.999). A positive correlation was also shown depending on albumen weight (0.650), thick albumen height (0.613), and Haugh units (0.576).

Table 3.

Correction between the morphologic and quality features of hatching eggs.

Correlation Width (mm) Length (mm) Egg-shape index (%) Egg weight (g) Egg surface area (cm2) Yolk weight (g) Yolk share (%) Albumen weight (g) Albumen share (%) Thick albumen height (mm) Haugh units Eggshell weight (g)
Length (mm) −0.7361
Egg-shape index (%) 0.8901 −0.9641
Egg weight (g) 0.014 −0.124 0.094
Egg surface area (cm2) 0.014 −0.123 0.093 0.9991
Yolk weight (g) 0.132 0.042 0.027 −0.280 −0.281
Yolk share (%) 0.123 0.055 0.014 −0.441 −0.442 0.9851
Albumen weight (g) −0.076 −0.128 0.056 0.6501 0.6511 −0.9031 −0.9601
Albumen share (%) −0.098 −0.103 0.030 0.438 0.438 −0.9761 −0.9911 0.9681
Thick albumen height (mm) 0.245 0.005 0.094 0.6131 0.6131 −0.153 −0.256 0.395 0.268
Haugh units 0.249 0.026 0.082 0.5761 0.5761 −0.140 −0.238 0.372 0.252 0.9951
Eggshell weight (g) −0.122 0.311 −0.264 0.184 0.184 0.218 0.167 −0.203 −0.295 0.022 0.005
Eggshell share (%) −0.127 0.352 −0.294 −0.145 −0.145 0.315 0.317 −0.421 −0.444 −0.182 −0.188 0.9461
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1

Correlation between features is statistically significant, P value <0.05.

Similarly, egg surface area was significantly correlated with these characteristics, as was egg weight (0.651, 0.613, 0.576, consecutively). A significantly positive correlation was found for the weight of the yolk and its percentage share in the egg (0.985), and a significantly negative correlation between the weight of the yolk and the weight and percentage of albumen (−0.903; −0.976, respectively). A highly significant negative correlation was found between the proportion of yolk and the weight and proportion of egg albumen (−0.960 and −0.991, respectively). Albumen weight was significantly correlated with its percentage in the egg (0.968). Analyzing the height of the thick albumen and the Haugh unit, a high, significant positive correlation (0.955) was demonstrated. A similar relationship was found between eggshell weight and percentage (0.946).

Eggshell Surface Temperature and Egg Weight Loss

When analyzing the eggshell surface temperature (EST), a significantly lower value was found on the seventh day of incubation compared to the 4th, 14th, and 18th (P < 0.001). On the other hand, egg weight loss was constant. There was a significant decrease in egg weight at each prescribing date (P < 0.001). Total egg weight loss was 8.04% on d 18, with a daily average of 0.45%, similar to d 1 of incubation, where the egg weight loss was 0.49% (Figure 1).

Figure 1.

Figure 1

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The temperature of the eggshell (EST) and incubator and the egg weight loss during incubation. a,bMeans lacking a common superscript differ (P < 0.05); ±SD, standard deviation; SEM, standard error of the mean.

Strength and Thickness of the Eggshell

Hatching eggshell thickness was significantly higher on d 0, 1, 7, and 14 than posthatch eggshell thickness (P < 0.001). A significant positive correlation was found between eggshell thickness and strength (0.246) and eggshell weight and strength and thickness (0.392 and 0.483, respectively). However, these values were relatively low (Table 4).

Table 4.

Eggshell strength and thickness during incubation.

Item Eggshell strength (N) Eggshell thickness (mm)
D 0 35.89 ± 3.87 0.310a ± 0.006
D 1 35.55 ± 4.97 0.306a ± 0.005
D 7 37.43 ± 5.59 0.296a ± 0.004
D 14 34.78 ± 5.70 0.300a ± 0.004
Posthatch - 0.259b ± 0.006
SEM 0.60 0.003
P value 0.462 <0.001
Correlation
Eggshell thickness (mm) 0.247* -
Eggshell weight (g) 0.392* 0.483*
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a,b

Means within a column lacking a common superscript differ (P < 0.05); ±SD, standard deviation; SEM, standard error of the mean.

⁎

Correlation between features is statistically significant, P value <0.05.

Yolk’ Vitelline Membrane Strength

The strength of the egg yolk vitelline membrane was characterized by an intense, significant decrease (P < 0.001). These data were obtained only in fresh eggs and on the first day of incubation, where the difference was 0.05 N. The vitelline membrane strength decreased by over 58%. On the seventh day, the vitelline membrane of the egg yolk lacked strength (Figure 2).

Figure 2.

Figure 2

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Yolk’ vitelline membrane strength during incubation. a,bMeans lacking a common superscript differ (P < 0.05); ±SD, standard deviation; SEM, standard error of the mean.

Thick Albumen, Amniotic Fluid, and Yolk's pH

Changes in the pH of albumen, amniotic fluid, and yolk are presented in Table 5. Significantly higher pH was found in thick albumen collected from a fresh egg and on d 1 of incubation compared to subsequent days of embryogenesis. On d 7, thick albumen and amniotic fluid had a higher pH than on d 14. On the other hand, the pH of thick albumen on d 14 was significantly higher than the pH of amniotic fluid (P < 0.001). Yolk pH values tended to increase with incubation time. The yolk pH was significantly lower on d 0 and 1 than on the other days.

Table 5.

pH in thick albumen, amniotic fluid, and yolk during incubation.

Incubation (n = 18) Thick albumen and amniotic fluid pH Yolk pH
D 0 (t.a.) 9.20a ± 0.06 6.09c ± 0.05
D 1 (t.a.) 9.35a ± 0.07 6.07c ± 0.06
D 7 (t.a.) 8.15b ± 0.39 7.26b ± 0.31
D 7 (a.f.) 8.00b ± 0.21 -
D 14 (t.a.) 7.51c ± 0.14 8.13a ± 0.07
D 14 (a.f.) 6.89d ± 0.47 -
SEM 0.09 0.10
P value <0.001 <0.001
Correlation
Yolk vitelline membrane strength (N) −0.538* 0.086
Albumen pH - −0.129
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a–d

Means within a column lacking a common superscript differ (P < 0.05); ±SD, standard deviation; SEM, standard error of the mean; t.a., thick albumen; a.f., amniotic fluid.

⁎

Correlation between features is statistically significant, P value <0.05.

On the other hand, on d 7, the pH was lower than on d 14 (P < 0.001). Thus, there was a trend of decreasing pH in albumen and amniotic fluid and an increase in yolk pH. In addition, a significant negative correlation was found between vitelline membrane strength and albumen pH (−0.538).

Enzymatic Activity of Lysozyme in Thick Albumen and Amniotic Fluid

In fresh eggs and the first week (d 1 and 7), as well as on the 14th day of incubation in thick albumen, a similar enzymatic activity of lysozyme was demonstrated (P > 0.05). However, no lysozyme activity was found in amniotic fluid on d 7 of embryonic development (P < 0.001). Then, after another week (d 14), lysozyme activity increased by more than 25,000 U/mL (P < 0.001). At the same time, this value was significantly lower than on all days of incubation in thick albumen (P < 0.001) (Figure 3).

Figure 3.

Figure 3

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Viscosity, crude protein content, and lysozyme activity in thick albumen and amniotic fluid during incubation. a,bMeans lacking a common superscript differ (P < 0.05); ±SD, standard deviation; SEM, standard error of the mean; t.a., thick albumen; a.f., amniotic fluid.

Thick Albumen and Amniotic Fluid Viscosity

In the thick albumen, significantly higher viscosity was demonstrated on d 14 than on other days. Then, high viscosity was found on d 7 in thick albumen, while significantly lower viscosity was found in amniotic fluid on d 7 (P < 0.001). When considering the days of incubation, a decrease in viscosity was observed, followed by an increase on d 7 and d 14. Amniotic fluid viscosity increased significantly from d 7 to d 14 (P < 0.001) (Figure 3).

Crude Protein Content in Thick Albumen and Amniotic Fluid

The concentration of crude protein in thick albumen and the amniotic fluid is shown in Figure 3. Its significantly higher content was demonstrated on d 14 in thick albumen (t.a.). Significantly, the lowest concentration was found in the amniotic fluid (a.f.) on d 7 (P < 0.001). The difference between the minimum and maximum values was 32.44%. The crude protein content was decreased between each of the days: d 14 (t.a.) > d 7 (t.a.) > d 14 (a.f.) > d 0 (t.a.) > d 1 (t.a.) > d 7 (a.f.).

When analyzing Figure 3, a similar tendency in viscosity, crude protein content, and lysozyme activity in thick albumen and amniotic fluid was found. On d 7, the lowest values were noticed, whereas was the lack of lysozyme activity, almost lack of the crude protein, and the fluid was very liquefied.

Yolk and Yolk Sac Fatty Acid Content

Yolks from fresh hatching eggs (d 0) of broiler chickens characterized by significantly lower percentage content of C14:0, C14:1, C15:0, C16:0, C16:1, C17:1, C18:1n9t+C18:1n9c, C18:3n6 and MUFA and TFA groups (P < 0.001). After 14 d of incubation, the content of the yolk sac showed a significantly lower percentage of C17:0, C18:0, C21:0, C20:2, C20:4n6, C20:5n3, C24:0, C24:1n9, C22:6n3, and PUFA group acids (P < 0.001) (Table 6).

Table 6.

The fatty acid content in egg yolk and yolk sac during incubation.

Fatty acids (%) Incubation days
 SEM P value
D 0 ±SD D 14 ±SD
C14:0 0.34b 0.07 0.42a 0.07 0.012 0.001
C14:1 0.05b 0.01 0.09a 0.04 0.005 <0.001
C15:0 0.08b 0.02 0.12a 0.06 0.007 0.001
C15:1 0.02 0.01 0.02 0.01 0.002 0.222
C16:0 22.30b 1.01 24.66a 0.89 0.219 <0.001
C16:1 2.27b 0.58 3.16a 0.53 0.103 <0.001
C17:0 0.16a 0.02 0.14b 0.03 0.004 0.008
C17:1 0.10b 0.02 0.14a 0.02 0.003 <0.001
C18:0 12.32a 2.01 9.84b 0.80 0.284 <0.001
C18:1n9t+C18:1n9c 34.13b 3.95 38.22a 1.61 0.524 <0.001
C18:2n6c 16.67 0.89 16.55 2.23 0.242 0.819
C18:3n6 0.65b 0.20 0.79a 0.19 0.030 0.013
C18:3n3 0.24 0.03 0.25 0.02 0.003 0.738
C21:0 0.15a 0.03 0.07b 0.04 0.007 <0.001
C20:2 0.56a 0.11 0.34b 0.13 0.023 <0.001
C20:4n6 4.62a 1.17 2.57b 0.45 0.196 <0.001
C20:5n3 0.60a 0.10 0.40b 0.06 0.019 <0.001
C24:0 1.22a 0.22 0.68b 0.13 0.047 <0.001
C24:1n9 0.55a 0.12 0.33b 0.07 0.022 <0.001
C22:6n3 2.98a 0.86 1.19b 0.22 0.158 <0.001
SFA 36.57 2.95 35.92 1.48 0.337 0.344
MUFA 37.12b 4.42 41.95a 2.01 0.604 <0.001
PUFA 26.31a 1.67 22.08b 2.56 0.436 <0.001
TFA 34.13b 3.95 38.22a 1.61 0.524 <0.001
Open in a new tab
a,b

Means within a row lacking a common superscript differ (P < 0.05); ±SD, standard deviation; C14:0, myristic acid; C14:1n-5, myristoleic acid; C15:0, pentadecanoic acid; C15:1n-5, 10-pentadecenoic acid; C16:0, palmitic acid; C16:1n-7, palmitoleic acid; C17:0, margaric acid; C17:1n-7, heptadecanoic acid; C18:0, stearic acid; C18:1n-9t, elaidic acid; C18:1n-9, oleic acid; C18:2n-6cc, linoleic acid; C18:3n6, ɣ-linolenic acid; C18:3n-3, α-linolenic acid; C21:0, heneicosanoic acid; C20:2, eicosadienoic acid; C20:4n-6, arachidonic acid; C20:5n-3, eicosapentaenoic acids; C24:0, lignoceric acid; C24:1n-9, nervonic acid; C22:6n-3, docosahexaenoic acid; SFA, saturated fatty acids (C14:0; C15:0; C16:0; C17:0; C18:0; C21:0; C24:0); MUFA, monounsaturated fatty acids (C14:1n-5; C15:1n-5; C16:1n-7; C17:1n-7; C18:1n-9t; C18:1n-9; C24:1n-9); PUFA, polyunsaturated fatty acids (C18:2n-6cc; C18:3n-6; C18:3n-3; C20:2; C20:3n-3; C20:4n-6; C20:5n-3; C22:6n-3); and TFA, trans fatty acids (C18:1n-9t).

DISCUSSION

In the presented study, hatchability was 95.07%. The eggs were obtained from the meat-type hens aged 32 wk. In the study by Salahi et al. (2011), hatchability ranged from 94 to 99%. The eggs were obtained from a 45-wk-old breeding flock. Durmuş et al. (2021) analyzed hatchability from hatching eggs obtained from Ross 308 flocks at 30, 47, and 59 wk of age. The hatchability was 87.33, 75.33, and 73.33%. The hatching parameters in the presented study were high. Differences may depend on age, as well as origin and environmental conditions.

Banaszewska et al. (2019) analyzed the quality of eggs obtained from the parent flock Ross 308, aged 30 and 60 wk. The results correspond to the results of this study in terms of morphological composition, as well as physicochemical characteristics (thick albumen height and Haugh unit). The authors found that the quantitative characteristics of eggs increased with the age of the hens, with a decrease in the proportion of albumen and an increase in the proportion of yolk. The egg-shape index in the presented study was, on average, 79.78%. Shaker et al. (2019) showed an egg-shape index of 76.29 to 79.32%. Eggs were obtained from hens at the age of 30 wk. Kontecka et al. (2012) showed that the shape index of eggs obtained from Cobb 500 hens at 31 wk was 75.60%. The authors showed that with the age of the hens, the shape index decreases (eggs are elongated). Shaker et al. (2019) showed similar correlations between egg traits as in the presented research.

A constant decrease characterized the egg weight loss, and no dependence on the temperature of the egg surface or the incubator's temperature was found. The temperature on the seventh day could be significantly lower due to the functioning of the incubator and the humidity, which was set at 55 to 60%. However, daily values that could fluctuate were not recorded. The evaporation of water from the egg may depend on the changing relative humidity inside the incubator (Van der Pol et al., 2013). As the authors indicate, a decrease in humidity can lead to an increase in the evaporation of water from the egg. In this study, the weight loss was lower than recommended, which may have been caused by too considerably humidity. As mentioned, no daily humidity parameters were recorded. However, high humidity alarms occurred during routine work, which was immediately reduced.

Van der Pol et al. (2013) also found the optimum EST to be 37.5°C to 38°C. In this study, it ranged from 37.68°C to 38.45°C. EST is essential for embryonic development, treated as the embryo's temperature. Hulet et al. (2007) analyzed EST's effect on broiler chickens' posthatch growth performance. The authors divided the eggs into incubators where the eggshell temperature was kept low (37.5°C), medium (38.6°C), and high (39.7°C). A high EST resulted in a higher weight of chicks at hatch. However, in the late-stage rearing period, the weight was lower. In addition, chicks incubated at high temperatures were less temperamental and did not take up feed and water well. The temperature of the developing embryo may depend on the temperature of the incubator and heat transfer and production on its own (Alasahan et al., 2016). Therefore, it can be concluded that these changes depend on round-the-clock environmental conditions in the incubator and the embryo development stage.

The thickness of the eggshell may depend on the age of the flock, where younger hens lay eggs with a thicker eggshell. During incubation, the eggshell thickness was reduced, regardless of the age of the hens (Gualhanone et al., 2012). In the presented study, no changes in eggshell thickness were found until the 14th day of incubation. However, the eggshell on d 21 (posthatch) was significantly thinner. According to the literature, 80% of the calcium required for skeletal formation (after d 10 of development) comes from the eggshell (approximately 47% of the weight of the eggshell). Gualhanone et al. (2012) observed these changes only after 13 d of incubation. Peebles et al. (2001) similarly found this after the 12th day of incubation.

After hatching, birds are characterized by rapid bone formation between the 4th and 18th day, and mineralization occurs between the 4th and 11th day. During incubation, calcium is released from the eggshell. It passes through the capillaries (via calcium-binding protein). Calcium is transported to the CAM (chorioallantoic membrane). The intensification by the CAM transport increases 20-fold from d 12 to 19, then decreases. It is correlated with the mineralization of the embryo skeleton (Torres and Korver, 2018; Halgrain et al., 2022).

Given the above, it is reasonable that significant changes in the thickness of the Ross 308 chicken eggshell and its strength were not observable during 14 d of incubation. In addition to the formation of the embryo's skeleton, the eggshell's thickness can affect the chicks' hatchability. Hatching eggs from broiler breeders with dead embryos had a thicker eggshell (Peebles and Brake, 1985).

The strength of the vitelline membrane decreased by more than 58% within 24 h of incubation. Thus, the incubation temperature certainly affects it (Kirunda and McKee, 2000). In addition, it was shown that the vitelline membrane's strength decreased with increasing albumen pH (negative correlation, r = −0.538). Kirunda and McKee (2000) showed similar values (r = −0.57). During incubation, a yolk sac is formed after a few days, which is liquefied and irregular and adjusts to the egg's space (Starck, 2021). Thus, the vitelline membrane disappears, which may explain its lack of strength on d 7 of incubation.

Bruggeman et al. (2006) analyzed the relation between CO2 and albumen pH. The pH initially increased, followed by an intense decrease in the eggs on incubation d 2, 6, 7, 8, and 10. It corresponds to the results of the presented study. Kouame et al. (2019) showed that the albumen pH in guinea fowl eggs decreased from 9.64 on d 0 to 8.54 on d 6 of incubation. Even storing eggs in CO2 resulted in a decrease in pH (Walsh et al., 1995). In eggs obtained from commercial flocks of Peterson × Minibro Shaver broilers at 32 wk of age, the pH of the albumen in fresh eggs was shown to be 8.08 and increased with storage time (up to 9.12 on d 8). During egg incubation, the following increase in albumen pH was also shown: 12 h—8.65, 24 h—8.95, 38 h—9.05, and 60 h—9.15 (Lapao et al., 1999). According to the authors, this is due to the buffering capacity of the albumen. In this research, on the first day of incubation, an increase (P > 0.05) of 0.15 units was also shown. The pH of the albumen from oviposition and during storage increases to alkaline pH, which favorably inhibits bacterial growth (Guyot et al., 2016). The cited authors described the albumen change during incubation, including pH variations and alternation of lysozyme activity. The albumen proteins are constantly transferred into the amniotic fluid. In addition, in the presented research, an inverse increase in the pH of the yolk was found. If the vitelline membrane weakens, the gradient changes, and fluid diffusion occurs between the albumen and yolk (Von Engelhardt et al., 2009). It may also explain the changes in the pH of the albumen, amniotic fluid, and yolk.

Cunningham (1974) showed a decrease in lysozyme activity during the first 12 d of incubation of chicken eggs. Shbailat and Abuassaf (2018) analyzed the diffusion route of duck egg albumen into the egg yolk, simultaneously specifying the type of activated proteases. The study used lysozyme as a reference protein to track egg albumen transfer by measuring its activity. It was shown that lysozyme activity appeared on the 15th day in the extra-embryonic fluid, on the 17th—in the amniotic and intestinal fluid, and on the 19th—in the yolk. It was summarized that the main route of diffusion of the egg albumen into the yolk is the amniotic cavity and the lumen of the embryo's intestine. Shbailat and Safi (2015) reported similar research and conclusions in hatching turkeys' eggs. In this case, lysozyme activity was also detected on the 15th day in the extra-embryonic and amniotic fluid, the 16th day in the intestinal fluid, and the 17th day in the yolk. It is in line with the present study's assumptions. After a week of embryogenesis, no lysozyme activity was detected, and on the 14th day of incubation, the activity increased significantly. Thus, the antibacterial effect of lysozyme is guaranteed during embryonic development. Fang et al. (2012) hypothesized that the increase in pH and liquefaction of thick albumen probably limited the antimicrobial properties of albumen proteins. However, these were replaced by pH unfavorable to bacterial growth in the early stages of incubation. The egg albumen antimicrobial defense system is activated under early incubation conditions. During incubation, the ovotransferrin concentration in the egg albumen increased, and initially, the lysozyme concentration decreased and increased (slightly). Lysozyme activity in egg albumen decreased on d 2 of incubation (Fang et al., 2012). The mechanism underlying lysozyme transfer can be explained that after 12 d of incubation, egg albumen proteins are massively transferred into the amniotic sac, where they are, consequently, absorbed orally by the embryo as a source of amino acids to support its rapid growth until hatching. This process highly impacts the protein concentration of the amniotic fluid (Da Silva et al., 2019). Thus, the lysozyme, as an enzymatic protein, is also transferred.

The albumen and amniotic fluid viscosity study corresponds to the pilot research (Biesek et al., 2023). A significant increase in albumen viscosity was demonstrated on the 7th and 14th day of incubation, while the viscosity of the amniotic fluid was significantly the lowest. The higher the water content, the lower the albumen viscosity (Caner and Yüceer, 2015). In the eggs of Ross 308 and Cobb 500 chickens, the viscosity of the amniotic fluid was analyzed on the 17th day of incubation. The authors described heating increased viscosity (Omede et al., 2017). The viscosity depends on the ovomucin-lysozyme complex. It was shown that the concentration of ovomucin was 4 times higher in thick albumen than in liquefied albumen (Caner and Yüceer, 2015). This mechanism was also confirmed in the present research, where the viscosity and activity of lysozyme decreased in the amniotic fluid. As mentioned, the viscosity of the thick albumen or amniotic fluid depends on the flow of substances between the egg's extra-embryonic structures and the water's evaporation (i.e., environmental conditions). Based on this, it can be concluded that the appropriate viscosity is conducive to maintaining the embryo in the initial incubation period but also retains microorganisms. On the other hand, a higher viscosity of albumen or amniotic fluid may cause impeded gas exchange and sorption of fluids into the embryo's body, which will increase mortality (Mayes and Takeballi, 1983; Tona et al., 2001).

Since pH, lysozyme activity, or the viscosity of thick albumen (or amniotic fluid) are features of a protective nature for the embryo, it can be suggested that on d 7, when the amniotic membrane is filled with a fluid with a low activity of biologically active substances, attention should be paid to the safe and hygienic handling of eggs at the hatchery, including during candling. This is because the embryos in the egg are exposed to microbes, while exposition increases during the extra-embryonic structure formation (Hincke et al., 2019).

Omede et al. (2017) showed that the crude protein content in the amniotic fluid from the 17th day of incubation of hatching eggs of broiler chickens was 11.4 to 12.5%. In the present research, this level of protein was found in thick albumen in fresh eggs and after the first day of incubation. The amniotic fluid (on d 7) decreased, and on d 14 an intensive increase in crude protein content. It is another aspect that confirms the flow of nutrients between the structures in the egg. If the protein decreases in the following days, it indicates its sorption into the embryo's organism. Baggott (2009) found that the sero-amniotic connection is formed near the 12th day of embryo development and enables protein transfer from albumen and water to the amniotic fluid. In the present research, it is concluded that the crude protein content is related to the viscosity as well as the activity of the lysozyme. Carinci and Manzoli-Guidott (1968) found a similar conclusion. The assumptions coincide with the findings of the proteomics study of Meng et al. (2019). As stated by the cited authors, a dynamic flow of ovalbumin from egg albumen to egg yolk was observed on d 14 of incubation in fertilized eggs but not in unfertilized eggs. This points to an undisclosed pathway of egg protein (peptide) absorption. A relation between the antibacterial activity of proteins, their concentration, or albumen viscosity is suggested. Guyot et al. (2016) found that the activity of egg proteins gradually decreases in the first half of incubation due to a change in specific antimicrobial proteins. It may be embryo-related and offset by increased protein concentration in the egg albumen. Thus, intensive egg protection is provided, but new structures gradually take over these functions during incubation, which the presented results may confirm.

Şahan et al. (2014) assessed changes in the fatty acid composition of the yolk and yolk sac (d 18) of hatching eggs from broiler breeders aged 36 and 53 wk. It was shown that palmitic, stearic, oleic, and linoleic acids had the highest share in fresh yolk (from 13.02 to 29.24%). On the other hand, from 1.24 to 7.04% were palmitoleic and arachidonic acids. Other fatty acids accounted for less than 1%. In the yolk sac, a higher share of myristic, palmitoleic, and oleic acids was found, and a lower share of heptadecanoic, stearic, linoleic, and arachidonic acids. It was concluded that the absorption of fatty acids by the embryo during incubation is selective. Yilmaz-Dkimen and Şahan (2009) studied the correlations between age, yolk fatty acid content, and incubation results of broiler breeders. Significant relations were demonstrated, and it was concluded that hatchability may depend on the content of fatty acids in the egg yolk as nutrients available to embryos. Peebles et al. (2000) also found significant changes in the fatty acid profile in broiler-hatching egg yolk. It was found that differences in the fatty acid profile of the yolk during early incubation may affect the subsequent weight of the embryo. Yolk lipids are the only source of essential fatty acids for the chick. Thus, its level in the yolk and changes during incubation may indicate demand (Cherian, 2022). Deficiencies can affect the incorporation of fatty acids (especially PUFA) into tissues, lipid metabolism, and pre-/posthatch growth and development of chicks. The present study also showed a lower PUFA content after 14 d of incubation, corresponding to the abovementioned studies. It indicates individual acids, including those from the PUFA group. Kucharska-Gaca et al. (2023) also showed changes in the content of fatty acids, including PUFA, on different days of incubation of goose eggs, depending on the age of the flock and the laying period. The above confirms the selective use of fatty acids by embryos. The increase in the content of some acids, including MUFA, may result from the proportions between all analyzed fatty acids. A study by Duan et al. (2013) showed that fertilized hen eggs had higher lipid and MUFA levels and lower cholesterol and PUFA content up to the ninth day of development than unfertilized eggs. Wang et al. (2023) performed a quantitative lipidomic analysis of hen egg yolk during its formation. It has been found that lipid transport and accumulation are dynamically adjusted during egg yolk formation. The transport and timing of different types of lipid molecules are different, which may explain the fatty acid profile results. Changes in FA content during egg incubation may be an essential clue for ovo technology and support of developing embryos. As Schaal (2008) indicated, providing exogenous fatty acids and an antioxidant would modify the state of the above substances and lipids of the chicken embryo and may promote increased hatchability.

In conclusion, the embryogenesis of Ross 308 broiler chickens is a complex process characterized by many changes in morphological components and the formation of new structures with protective and nutritional functions. It was shown that the eggshell is thinning in the last week of incubation, which may indicate the embryo's skeleton formation. The vitelline membrane is reduced in favor of the yolk sac formation. In addition, its disappearance affects the physicochemical properties and mixing of the albumen with the yolk. Fatty acids were used selectively, which was suggested by decreasing some FA, including PUFA. When analyzing changes in the enzymatic activity of lysozyme, viscosity, and crude protein content, it is concluded that these features are interrelated. Low values or absence on d 7 of embryogenesis in the amniotic fluid indicate its sterile nature. In addition, the increase in these features in the following days in the amniotic fluid suggests a flow of nutrients, which are also protective, from the thick albumen. Thus, on the seventh day of development, the embryo is surrounded by sterile amniotic fluid, and this may be a critical moment, apart from the change of the breathing process (from the blood vessels to the allantoic membrane) when there is a high possibility of embryo death. The results also suggest the importance of amniotic fluid in transferring nutrients from morphological components to the chick embryo.

ACKNOWLEDGMENTS

This research was funded by the Bydgoszcz University of Science and Technology as part of the subsidy BN-WHiBZ-1/2023, and it is a continuation grant No. DNM 5/2022 of Scientific Activities of Young Scientist program (Działania Naukowe Młodych), where the pilot study was conducted.

The author thanks Dorota Wejnerowska, Ph.D., and Izabela Narloch, M.Eng., from the Department of Food Analytics and Environmental Protection, for assistance with the fatty acid composition analyses. Thanks to Sebastian Wlaźlak, M.Eng., from the Department of Animal Breeding and Nutrition, for technical assistance during incubation.

DISCLOSURES

The author declares no conflict of interest.

REFERENCES

  1. Alasahan S., Akpinar G.C., Canogullari S., Baylan M. The impact of eggshell colour and spot area in Japanese quails: I. eggshell temperature during incubation and hatching results. R. Braz. Zootec. 2016;45:219–229. [Google Scholar]
  2. Baggott G.K. Development of extra-embryonic membranes and fluid compartments. Avian Biol. Res. 2009;2:21–26. [Google Scholar]
  3. Banaszewska D., Biesiada-Drzazga B., Ostrowski D., Drabik K., Batkowska J. The impact of breeder age on egg quality and lysozyme activity. Turk. J. Vet. Anim. Sci. 2019;43:583–589. [Google Scholar]
  4. Benton C.E., Jr., Walsh T.J., Brake J. Effects of presence of a blastoderm on albumen height and pH of broiler hatching eggs. Poult. Sci. 2001;80:955–957. doi: 10.1093/ps/80.7.955. [DOI] [PubMed] [Google Scholar]
  5. Biesek J., Wlaźlak S., Adamski M. The biological value of hatching eggs of broiler chickens in the early-mid incubation period based on physiochemical and morphologic features. Poult. Sci. 2023;102 doi: 10.1016/j.psj.2023.102689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bruggeman V., De Smit L., Tona K., Everaert N., Witters A., Debonne M., Degraeve P., Garain P., Decuypere E. EPC 2006 – 12th European Poultry Conference, Verona, Italy, 10-14 September 2006, pp.paper 260, ref. 12. 2006. Changes in albumen pH due to higher CO2 concentrations during the first ten days of incubation. [Google Scholar]
  7. Caner C., Yüceer M. Efficacy of various protein-based coating on enhancing the shelf life of fresh eggs during storage. Poult. Sci. 2015;94:1665–1677. doi: 10.3382/ps/pev102. [DOI] [PubMed] [Google Scholar]
  8. Carinci P., Manzoli-Guidott L. Albumen absorption during chick embryogenesis. J. Embryol. Exp. Morph. 1968;20:107–118. [PubMed] [Google Scholar]
  9. Castilla A.M., Van Dongen S., Herrel A., Francesch A., Martínez de Aragón J., Malone J., Negro J.J. Increase in membrane thickness during development compensates for eggshell thinning due to calcium uptake by the embryo in falcons. Sci. Nat. 2009;97:143–151. doi: 10.1007/s00114-009-0620-z. [DOI] [PubMed] [Google Scholar]
  10. Cherian G. Hatching egg polyunsaturated fatty acids and the broiler chick. J. Anim. Sci. Biotechnol. 2022;13:98. doi: 10.1186/s40104-022-00757-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Congjiao S., Junnian L., Ning Y., Guiyun X. Egg quality and egg albumen property of domestic chicken, duck, goose, turkey, quail, and pigeon. Poult. Sci. 2019;98:4516–4521. doi: 10.3382/ps/pez259. [DOI] [PubMed] [Google Scholar]
  12. Cunningham F.E. Changes in egg white during incubation of the fertile egg. Poult. Sci. 1974;53:1561–1565. doi: 10.3382/ps.0531561. [DOI] [PubMed] [Google Scholar]
  13. Da Silva M., Dombre C., Brionne A., Monget P., Chesse M., De Pauw M., Mills M., Combes-Soia L., Labas V., Guyot N., Nys Y., Réhault-Godbert S. The unique features of proteins depicting the chicken amniotic fluid. Mol. Cell. Proteom. 2019;18:S174–S190. doi: 10.1074/mcp.RA117.000459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ding P., Liu H., Tong Y., He X., Yin X., Yin Y., Zhang H., Song Z. Developmental change of yolk microbiota and its role on early colonization of intestinal microbiota in chicken embryo. Animals. 2022;12:16. doi: 10.3390/ani12010016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Duan X., Li M., Wu F., Yang N., Nikoo M., Jin Z., Xu X. Postfertilization changes in nutritional composition and protein conformation of hen egg. J. Agric. Food Chem. 2013;61:12092–12100. doi: 10.1021/jf403099q. [DOI] [PubMed] [Google Scholar]
  16. Durmuş M., Kurşun K., Baylan M., Kutlu H.R. The effect of flock age on hatching results and chick quality in ross 308 broiler. Turk. J. Agric. – Food Sci. Technol. 2021;9:362–367. [Google Scholar]
  17. Fang J., Ma M., Jin Y., Qiu N., Huang Q., Sun S., Geng F., Guo L. Liquefaction of albumen during the early incubational stages of the avian embryo and its impact on the antimicrobial activity of albumen. J. Food Agric. Environ. 2012;10:423–427. [Google Scholar]
  18. Gualhanone A., Furlan R.L., Fernandez-Alarcon M.F., Macari M. Effect of breeder age on eggshell thickness, surface temperature, hatchability and chick weigh. Braz. J. Poult. Sci. 2012;14:9–14. [Google Scholar]
  19. Guyot N., Réhault-Godbert S., Slugocki C., Harichaux G., Labas V., Helloin E., Nys Y. Characterization of egg white antibacterial properties during the first half of incubation: a comparative study between embryonated and unfertilized eggs. Poult. Sci. 2016;95:2956–2970. doi: 10.3382/ps/pew271. [DOI] [PubMed] [Google Scholar]
  20. Halgrain M., Bernardet N., Crepeau M., Même N., Narcy A., Hincke M., Réhault-Godbert S. Eggshell decalcification and skeletal mineralization during chicken embryonic development: defining candidate genes in the chorioallantoic membrane. Poult. Sci. 2022;101 doi: 10.1016/j.psj.2021.101622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hamburger V., Hamilton H.L. A series of normal stages in the development of the chick embryo. J. Morphol. 1951;88:49–92. [PubMed] [Google Scholar]
  22. Hegab I.M., Hanafy A.M. Effect of egg weight on external and internal qualities, physiological and hatching success of Japanese quail eggs (Coturnix coturnix japonica) Braz. J. Poult. Sci. 2019;21 eRBCA-2018-0777. [Google Scholar]
  23. Hincke M.T., Da Silva M., Guyot N., Gautorn J., McKee M.D., Guabiraba-Brito R., Réhault-Godbert S. Dynamics of structural barriers and innate immune components during incubation of the avian egg: critical interplay between autonomous embryonic development and maternal anticipation. J. Innate Immun. 2019;11:111–124. doi: 10.1159/000493719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hulet R., Gladys G., Hill D., Meijerhof R., El-Shiekh T. Influence of egg shell embryonic incubation temperature and broiler breeder flock age on posthatch growth performance and carcass characteristics. Poult. Sci. 2007;86:408–412. doi: 10.1093/ps/86.2.408. [DOI] [PubMed] [Google Scholar]
  25. Kirunda D.F.K., McKee S.R. Relating quality characteristics of aged eggs and fresh eggs to vitelline membrane strength as determined by a texture analyzer. Poult. Sci. 2000;79:1189–1193. doi: 10.1093/ps/79.8.1189. [DOI] [PubMed] [Google Scholar]
  26. Kontecka H., Nowaczewski S., Sierszuła M.M., Witkiewicz K. Analysis of changes in egg quality of broiler breeders during the first reproduction period. Ann. Anim. Sci. 2012;12:609–620. [Google Scholar]
  27. Kouame Y.A.E., Nideou D., Kouakou K., Tona K. Effect of guinea fowl egg storage duration on embryonic and physiological parameters, and keet juvenile growth. Poult. Sci. 2019;98:6046–6052. doi: 10.3382/ps/pez264. [DOI] [PubMed] [Google Scholar]
  28. Kucharska-Gaca J., Adamski M., Biesek J. The fatty acid profile in the yolk and yolk sac from incubated goose eggs depends on the breeder age and laying period. Br. Poult. Sci. 2023;64:518–528. doi: 10.1080/00071668.2023.2193287. [DOI] [PubMed] [Google Scholar]
  29. Lapao C., Gama L.T., Soares M.Chaveiro. Effects of broiler breeder age and length of egg storage on albumen characteristics and hatchability. Poult. Sci. 1999;78:640–645. doi: 10.1093/ps/78.5.640. [DOI] [PubMed] [Google Scholar]
  30. Lavelin I., Meiri N., Pines M. New insight in eggshell formation. Poult. Sci. 2000;79:1014–1017. doi: 10.1093/ps/79.7.1014. [DOI] [PubMed] [Google Scholar]
  31. Marzec A., Michalczuk M., Damaziak K., Mieszkowska A., Lenart A., Niemiec J. Correlations between vitelline membrane strength and selected physical parameters of poultry eggs. Ann. Anim. Sci. 2016;16:897–907. [Google Scholar]
  32. Mayes F.J., Takeballi M.A. Microbial contamination of the hen's egg: a review. J. Food Protect. 1983;46:1092–1098. doi: 10.4315/0362-028X-46.12.1092. [DOI] [PubMed] [Google Scholar]
  33. Meng Y., Sun H., Qiu N., Geng F., Zhu F., Li S., Huo Y. Comparative proteomic analysis of hen egg yolk plasma proteins during embryonic development. J. Food Biochem. 2019;43:e13045. doi: 10.1111/jfbc.13045. [DOI] [PubMed] [Google Scholar]
  34. Merck, 2022. Sigma Aldrich. Protocol of enzymatic assay of lysozyme (EC 3.2.1.17). Accessed July 2023. https://www.sigmaaldrich.com/PL/pl.
  35. Nakage E.S., Cardozo J.P., Pereira G.T., Queiroz S.A., Boleli I.C. Effect of temperature on incubation period, embryonic mortality, hatch rate, egg water loss and partridge chick weight (Rhunchotus rufescens) Braz. J. Poult. Sci. 2003;5:131–135. [Google Scholar]
  36. Omede A.A., Bhuiyan M.M., Islam A.F., Iji P.A. Physico-chemical properties of late-incubation egg amniotic fluid and a potential in ovo feed supplement. Asian-Australas. J. Anim. Sci. 2017;30:1124–1134. doi: 10.5713/ajas.16.0677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Peebles E.D., Brake J. Relationship of eggshell porosity to stage of embryonic development in broiler breeders. Poult. Sci. 1985;64:2388–2391. [Google Scholar]
  38. Peebles E.D., Doyle S.M., Zumwalt C.D., Gerard P.D., Latour M.A., Boyle C.R., Smith T.W. Breeder age influence embryogenesis in broiler hatching eggs. Poult. Sci. 2001;80:272–277. doi: 10.1093/ps/80.3.272. [DOI] [PubMed] [Google Scholar]
  39. Peebles E.D., Gardner C.W., Brake J., Benton C.E., Bruzual J.J., Gerard P.D. Albumen height and yolk and embryo compositions in broiler hatching eggs during incubation. Poult. Sci. 2000;79:1373–1377. doi: 10.1093/ps/79.10.1373. [DOI] [PubMed] [Google Scholar]
  40. Polish Committee for Standardization. 2015. PN-EN ISO 8968-1:2014-03. Milk and milk products - determination of nitrogen content - Part 1: Kjeldahl's principle and calculation of crude protein [in Polish]. Accessed August 2023. https://www.pkn.pl/
  41. Reijrink I.A.M., Meijerhof R., Kemp B., van den Brand H. The chicken embryo and its microenvironment during egg storage and early incubation. Worlds’ Poult. Sci. J. 2008;64:581–598. [Google Scholar]
  42. Şahan U., Ipek A., Sozcu A. Yolk sac fatty acid composition, yolk absorption, embryo development, and chick quality during incubation in eggs from young and old broiler breeders. Poult. Sci. 2014;93:2069–2077. doi: 10.3382/ps.2013-03850. [DOI] [PubMed] [Google Scholar]
  43. Salahi A., Mousavi S.N., Foroudi F., Khabisi M.M., Norozi M. Effects of in ovo injection of butyric acid in broiler breeder eggs on hatching parameters, chick quality and performance. Global Vet. 2011;7:468–477. [Google Scholar]
  44. Schaal T.P. Oregon State Univ. Univ. Honors College; Corvallis, OR: 2008. The effects of in ovo feeding of fatty acids and antioxidants on broiler chicken hatchability and chick tissue lipids. [Google Scholar]
  45. Shaker A.S., Mustafa N.A., Ameen Q.A., Saadullah M.A., Ramadan A.A., Aziz S.B.R. Effect of hen oviposition time on some egg characteristics. J. Anim. Poult. Prod. Mansoura Univ. 2019;10:171–174. [Google Scholar]
  46. Shbailat S.J., Abuassaf R.A. Transfer of egg white proteins and activation of proteases during the development of Anas platyrhynchos domestica embryo. Acta Biol. Hungar. 2018;69:72–85. doi: 10.1556/018.68.2018.1.6. [DOI] [PubMed] [Google Scholar]
  47. Shbailat S.J., Safi H.M. Transfer of egg white proteins with reference to lysozyme during the development of Meleagris gallopavo (Galliformes: Phasianidae) Ital. J. Zool. 2015;82:349–357. [Google Scholar]
  48. Starck J.M. Morphology of the avian yolk sac. J. Morphol. 2021;282:959–972. doi: 10.1002/jmor.21262. [DOI] [PubMed] [Google Scholar]
  49. Takashi A., Masahiro O., Shigeru H. Migration of chicken egg-white protein ovalbumin-related protein X and its alteration in heparin-binding affinity during embryogenesis of fertilized egg. Poult. Sci. 2019;98:5100–5108. doi: 10.3382/ps/pez335. [DOI] [PubMed] [Google Scholar]
  50. Tona K., Bamelis F., Coucke W., Bruggeman V., Decuypere E. Relationship between broiler breeder's age and egg weight loss and embryonic mortality during incubation in large-scale conditions. J. Appl. Poult. Res. 2001;10:221–227. [Google Scholar]
  51. Tona K., Onagbesan O., Bruggeman V., Mertens K., Decuypere E. Effects of turning duration during incubation on embryo growth, utilization of albumen, and stress regulation. Poult. Sci. 2005;84:315–320. doi: 10.1093/ps/84.2.315. [DOI] [PubMed] [Google Scholar]
  52. Torres, C. A., and D. R. Korver. 2018. Influences of trace mineral nutrition and maternal flock age on broiler embryo bone development. 97:2996-3003. [DOI] [PubMed]
  53. Van der Pol C.W., van Roovert-Reijrink I.A.M., Maatjens C.M., van den Brand H., Molenaar R. Effect of relative humidity during incubation at a set eggshell temperature and brooding temperature posthatch on embryonic mortality and chick quality. Poult. Sci. 2013;92:2145–2155. doi: 10.3382/ps.2013-03006. [DOI] [PubMed] [Google Scholar]
  54. Veldsman Lm., Kylin H., Bronkhorst P., Engelbrecht I., Bouwman H. A method to determine the combined effects of climate change (temperature and humidity) and eggshell thickness on water loss from bird eggs. Environ. Geochem. Health. 2019;42:781–793. doi: 10.1007/s10653-019-00274-x. [DOI] [PubMed] [Google Scholar]
  55. Von Engelhardt N., Henriksen R., Groothuis T.G.G. Steroids in chicken egg yolk: metabolism and uptake during early embryonic development. Gen. Comp. Endocrinol. 2009;163:175–183. doi: 10.1016/j.ygcen.2009.04.004. [DOI] [PubMed] [Google Scholar]
  56. Walsh T.J., Rizk R.E., Brake J. Effects of temperature and carbon dioxide on albumen characteristics, weight loss, and early embryonic mortality of long stored hatching eggs. Poult. Sci. 1995;74:1403–1410. [Google Scholar]
  57. Wang Y., Sunwoo H., Cherian G., Sim J.S. Fatty acid determination in chicken egg yolk: a comparison of different methods. Poult. Sci. 2000;79:1168–1171. doi: 10.1093/ps/79.8.1168. [DOI] [PubMed] [Google Scholar]
  58. Wang Y., Wang J., Li H., Xiao Y., Widyanti Harlina P., Geng F. Quantitative lipidomic analysis of chicken egg yolk during its formation. J. Sci. Food Agric. 2023;103:3997–4005. doi: 10.1002/jsfa.12354. [DOI] [PubMed] [Google Scholar]
  59. Willems E., Decuypere E., Buyse J., Everaert N. Importance of albumen during embryonic development in avian species, with emphasis on domestic chicken. World's Poult. Sci. J. 2014;70:503–518. [Google Scholar]
  60. Yilmaz-Dkimen B., Şahan U. The relationship among age, yolk fatty acids content, and incubation results of broiler breeders. Poult. Sci. 2009;88:185–190. doi: 10.3382/ps.2008-00068. [DOI] [PubMed] [Google Scholar]

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