Evaluation of the Quality of Guinea Fowl (Numida meleagris) Eggs from Free-Range Farming Depending on the Storage Period and Age of Laying Hens

The aim of the study was to determine the changes occurring in the eggs of helmeted guinea fowl (Numida meleagris) from free-range farming in relation to the laying season and storage time. The experimental material consisted of 360 guinea fowl eggs, collected in the first, second and third laying seasons and stored for 7, 14 and 21 days. After each period, physical and physicochemical characteristics of the eggs were determined, as well as the basic chemical composition and mineral content of the albumen and yolk and the yolk fatty acid profile. The age of the guinea fowls affected certain physical parameters of the eggs. The egg weight, shape index and shell thickness increased with the age of the laying hens; however, a decrease in the proportion of shell in the egg was demonstrated. Storage time had a significant effect on egg weight, weight loss during storage and air cell height. Significant differences were found in the chemical composition of guinea fowl eggs depending on the age of the laying hens. Eggs obtained from older laying hens were characterized by higher yolk fat content and lower ash content, while the albumen contained higher water content and lower ash content. During the three-year laying period, changes were observed in the mineral composition of the eggs. The fatty acid profile underwent significant changes; however, no important differences were observed in the total content of SFA, MUFA, PUFA and n-6 fatty acids. Conversely, significant differences were found for n-3 acids and the n-6/n-3 ratio. Eggs in the first and second laying seasons exhibited the most favorable composition. The slow dynamics of changes occurring in successive laying seasons and egg storage time indicated that the raw material studied was safe and could be used by consumers


Introduction
There is a preconception among present-day consumers that alternative farming systems provide greater animal welfare and, consequently, healthier and safer products compared to conventional rearing [1][2][3].Hence, increasing attention is being paid to husbandry methods to satisfy consumer expectations regarding the quality of the final product, as well as the treatment of animals.Egg buying preferences have changed significantly over the past two decades.Currently, consumers are willing to pay more for eggs from hens kept in alternative systems, especially those from free-range farming [4].This stems from the belief in their better sensory and nutritional quality, as well as from the increasing importance of animal welfare for the modern consumer [5][6][7].
The economic significance of chickens and the large consumption of chicken eggs have led research efforts to primarily focus on evaluating the quality concerning various husbandry systems of hens of this species.Due to the fact that hens are typically kept for one laying season, studies have often been limited to analyzing a single laying period.These works have shown that husbandry systems, including free range, affect the quality characteristics of animal products [1,[8][9][10][11][12][13]. Free-range eggs were found to be richer in n-3 fatty acids, minerals, carotenoids and tocopherol [14], and they were characterized by a lower total cholesterol level and a better ratio of n-6 to n-3 fatty acids [1,11].In addition, hens with access to green runs laid eggs with a more intense yolk color [14].As already mentioned, chicken eggs are the most popular in the world for both direct consumption and processing.However, in some parts of the world, there is growing consumer interest in eggs from other poultry species.In Africa, guinea fowl and ostrich eggs are very popular, while in the Far East, quail and duck eggs are favored [15,16].In West African countries, guinea fowl are the second most common source of meat and eggs after chickens.In Europe, guinea fowl farming is most significant in France, Italy, Belgium and the Scandinavian countries [15].Guinea fowl can be raised using both intensive and alternative farming systems.Due to their ease of rearing, high resilience to environmental conditions and ability to last for many years, guinea fowls are wellsuited for semi-intensive farming.In Poland, they are mainly kept in the free-range system, for 2-3 laying seasons [17].The increasing costs of guinea fowl rearing, mainly due to rising feed prices and electricity, make single-season utilization of laying hens less profitable.Thus, extending their use to two or more production periods is advisable [16].According to Sokołowicz and Krawczyk [18], this results in spreading the rearing costs over a higher number of eggs laid during three laying periods, contributing to increased profitability of production.Guinea fowl eggs are considered a delicacy, with very good taste qualities [15,[19][20][21].Such food products are increasingly sought after in the food market by consumers who have grown tired of chicken eggs as a regular part of their daily diet.Guinea fowls may also gain popularity due to their natural way of rearing, which aligns with the expectations of modern consumers.A literature review on guinea fowl utilization has revealed a lack of comprehensive research results, particularly regarding the long-term maintenance of these birds.It is also unknown how the quality of eggs changes in subsequent laying seasons and how the free-range system affects their physical characteristics and chemical composition.Considering that information on the nutritional value of eggs is important for consumers, it was decided to evaluate the quality of eggs of guinea fowls kept in the free-range system, taking into account their morphological structure and chemical composition in three consecutive reproductive seasons and different storage periods.The aim of this study was comprehensive research on the influence of storage time and laying season on the quality of guinea fowl eggs.

Research Material
The research material consisted of eggs from guinea fowl (Numida meleagris) raised in a free-range system.The experimental flock consisted of 35 female birds.Guinea fowls were fed with a complete pelleted feed mixture based on corn, wheat, soybean meal and barley (ME: 11.9 MJ/kg, total protein: 17%, fiber: up to 6%), formulated for this poultry species, considering the laying period (tables with the feed mixture composition are available in the Supplementary Materials).The birds had ad libitum access to feed and drinking water.The study was conducted during the first three laying seasons.The research was carried out in 2020-2022.Guinea fowl started the laying season at the turn of March/April, while the laying season ended in September.Eggs were collected for one week in each laying season in early July and stored at room temperature (20.0 ± 2.0 • C) until analysis.For analysis in each laying season, 120 eggs weighing between 42-48 g were selected, from which 40 eggs were randomly selected for each storage period (7, 14, 21 days).Selected physicochemical parameters, basic chemical composition, mineral content and fatty acid profile were determined in these samples.

Determination of Physicochemical Parameters
The weight of the eggs and their individual components (shell, albumen, yolk) were measured using a precise WPS 600/C/1 digital balance (Radwag, Radom, Poland), with an accuracy of 0.01 g.Weight loss during storage was determined using the same balance by subtracting the egg weight on the day of analysis from the egg weight on the day of laying.The length and width of the eggs, as well as the area and height of the thick albumen, and the height and diameter of the yolk, were determined using a 31C628 digital caliper (TOPEX, Warsaw, Poland), with an accuracy of 0.02 mm.The shape index (SI) was calculated according to the formula: SI = (egg width/egg length) × 100%.The height of the air cell was determined using a special mm scale for measuring the depth of the air cell, after outlining the air cell under an ovoscope.Shell thickness was measured using a YT-72305 micrometer with a digital display (YATO, Wrocław, Poland), with an accuracy of 0.002 mm.The albumen index (AI) was calculated using the following formula: PI = height of thick albumen/average width of thick albumen.The yolk index (YI) was calculated using the formula: yolk height/yolk diameter.Albumen and yolk pH were determined using a SG2 pH meter (Mettler-Toledo AG, Schwerzenbach, Switzerland).Yolk color was determined using the 16-point La Roche scale.

Determination of Basic Chemical Composition
The basic chemical composition of the eggs was determined using conventional methods.Water content was determined using a gravimetric method with an Entris 224I-1S analytical balance (Sartorius, Goettingen, Germany) and a DRY-Line 115 laboratory dryer (VWR, Quito, Ecuador).Protein content was determined via the Kjeldahl method using a KjelFlex K-360 distillation apparatus (Büchi, Flavil, Switzerland) after prior sample mineralization in a K-435 Digestion Unit mineralization apparatus (Büchi, Flavil, Switzerland).Fat content was determined using a gravimetric method with an Entris 224I-1S analytical balance (Sartorius, Goettingen, Germany) and a DRY-Line 115 laboratory dryer (VWR, Quito, Ecuador).The Soxhlet method was used for fat extraction.For this purpose, 3.0 g of yolk was weighed into a weighing vessel with an accuracy of 0.0001 g and dried in a laboratory dryer at 105 • C to constant weight.The sample was then quantitatively transferred to the extraction thimble.The thimble was weighed with an accuracy of 0.0001 g and then placed in a Soxhlet apparatus.Extraction was carried out for 12 h using diethyl ether (Chempur, Piekary Śl ąskie, Poland) as a solvent.After extraction, the samples were dried in Petri dishes in a fume hood until the solvent evaporated completely.Next, the thimbles were dried in a laboratory dryer at a temperature of 105 • C to constant weight, then transferred to a desiccator for stabilization, and then weighed again with an accuracy of 0.0001 g.The fat content was calculated from the difference in the mass of the thimble before and after extraction.

Determination of Mineral Content
The content of selected mineral components (P, K, Ca, Na, Mg, Zn, Si, Fe, Cu, Ba, Sr, Mn, Al, Se, Cr, Pb, Cd) was determined using inductively coupled plasma optical emission spectrometry (ICP-OES).The measurements were conducted using an Optima 2000DV instrument (Perkin Elmer, Waltham, MA, USA) following prior mineralization in a Speedwave Xpert microwave oven (Berghof GmbH, Eningen, Germany) equipped with a continuous temperature and pressure control system.For mineralization, 700.0 mg of frozen sample was weighed and placed in the mineralization vessel.Next, 9.0 mL of 69% HNO 3 (Supelco, Bellefonte, PA, USA) and 1.0 mL of 30% H 2 O 2 (Supelco, Bellefonte, PA, USA) were added.The mixture was carefully stirred and left for 15 min before sealing the vessels, then mineralization was carried out.Standard solutions (0.1; 0.5; 1.0; 2.0; 5.0; 10.0; 50.0; 150.0 mg/L) of the tested elements were prepared using the TraceCert ® multi-element standard (Sigma-Aldrich, St. Louis, MO, USA).Yttrium (Y) solution (Merck, Darmstadt, Germany) in HNO 3 concentration was used in the mineralizates and standard solutions as an internal standard.The basic validation parameters of the analytical method used were estimated.Mineralized solutions were diluted directly before analysis; the blank was demineralized water used for dilution, 4.0 mL of 69% HNO 3 , yttrium solution and spectral buffer-(1% Cs in 2% HCl).The measurement results were analyzed based on standard curves plotted for each of the elements.The correlation coefficient for each curve was above 0.99.The average recovery for the samples was 96-98%.All analyses were performed in triplicate.

Determination of Fatty Acid Profile in Egg Yolks-GC-MS Method
The analyses were conducted according to PN-EN ISO 12966-2 [22].The first step involved preparing the samples for analysis.For this purpose, frozen (-82 • C) samples of egg yolks weighing approximately 1.0 g were placed in screw-cap 7.5 mL vials of amber glass with a Teflon seal (Supelco, Bellefonte, PA, USA).For extraction, 5.0 mL of a mixture of chloroform (Chempur, Piekary Śl ąskie, Poland) and methanol (Merck, Darmstadt, Germany) in a volume ratio of 2:1 (Folch's mixture) was added to each vial.The samples were then sealed under a stream of nitrogen 5.0 (Air-Liquide, Kraków, Poland) and vigorously shaken for 1 h using a Vibramax 100 laboratory shaker (Heidolph, Schwabach, Germany).To separate the chloroform phase from non-lipid residues, the vials were centrifuged using a Z 205A centrifuge (Hermle, Gosheim, Germany) for 20 min at 2000 RPM.The next step was lipid hydrolysis.The chloroform layer was transferred to 4.0 mL amber glass vials (Supelco, Bellefonte, PA, USA); then, the vials were sealed with Mininert ® Valves (Supelco, Bellefonte, PA, USA), allowing the addition of derivatizing reagents in an inert gas atmosphere.Chloroform was evaporated from the extracts with a stream of nitrogen, after which 400.0 µL of 0.5 M KOH solution in methanol was added to the dry residue and heated for 30 min in a heating block at 80 • C. The next step was esterification.After cooling to room temperature, 500.0µL of 14% boron trifluoride (BF 3 ) solution in methanol (Sigma-Aldrich, St. Louis, MO, USA) was added to the vials and incubated at 80 • C for 30 min.For more efficient extraction of fatty acids methyl esters (FAME), 1.0 mL of saturated NaCl solution and 2.0 mL of isooctane (POCH, Gliwice, Poland) were added into the cooled vials, as an extractant.The mixture was then shaken for an hour and left for 30 min to allow phase separation.The octane layer was transferred to separate vials containing approximately 0.5 g of anhydrous sodium sulfate, and then the vials were sealed under a nitrogen stream.The dried FAME extracts were placed in vials and subjected to chromatographic analysis.The fatty acid profile of the yolk samples was determined via gas chromatography coupled with a mass spectrometry (GC-MS) using a Clarus 600 gas chromatograph combined with a Clarus 600T mass spectrometer (Perkin Elmer, Waltham, MA, USA).The instrument was equipped with a TC-80 column with a length of 60 m, 0.25 mm inner diameter and a stationary phase film thickness of 0.25 µm (GL Sciences, Tokyo, Japan).For the analyses, a standard mixture of 37 fatty acids-FAME mix C4-C24 (Sigma-Aldrich, St. Louis, MO, USA)-was used.The samples were analyzed in triplicate.

Statistical Analysis
The results were statistically analyzed using the Statistica 13.1 PL software package (IBM Corp.; SPSS Statistics for Windows; Version 23.0; 2016).Two-way ANOVA was used to determine if two different factors, laying season and storage time and laying season × storage time, have an effect on a measured variable.Least squares means were obtained using the Tukey test.The significance was calculated at a 5% confidence level.

Determination of Physicochemical Parameters
Tables 1 and 2 present a comparison of the physical and physicochemical characteristics of eggs during the three-year laying period of guinea fowls, considering different storage periods.Laying season had a significant effect (p < 0.05) on: egg weight, albumen weight, yolk weight, shell percentage, yolk percentage, yolk pH, albumen pH and albumen index.On the other hand, storage time significantly (p < 0.05) affected egg weight, albumen weight, shell percentage, albumen percentage, yolk percentage, albumen pH, yolk color, yolk index, albumen index and air cell height.Significant interactions between the laying season and egg storage time (p < 0.05) were observed for albumen pH, yolk height, yolk index, and yolk pH.a,b -Statistically significant differences (p ≤ 0.05) for the laying season are marked with different letters in the subscript; d-f -statistically significant differences (p ≤ 0.05) for the storage time are marked with different letters in the subscript; a-e -statistically significant differences (p ≤ 0.05) for interaction type of laying season × storage time are marked with different letters in the superscript.

Determination of Basic Chemical Composition
Table 3 presents the results of the basic chemical composition of guinea fowl egg albumen depending on the laying season and storage time.The water content in egg albumen varied from year to year (from 86.7 to 89.1%) and depended only on the laying season (p < 0.05).No significant differences were found in the protein content (11.0%), for either laying season or storage period (p > 0.05).The ash content significantly decreased (p < 0.05) with successive laying seasons, from 0.73% in the first season to 0.58% in the third season.Interactions of laying season and egg storage time (p < 0.05) were found for both water and ash content.The chemical composition of egg yolks is shown in Table 4.In the three laying seasons studied, there were no significant differences in the water content in the yolk.However, significant changes were observed during storage (p < 0.05)-with increasing storage time, the water content also rose (47.3% to 49.0%).There were no significant differences observed between protein content and laying season or storage time (p > 0.05).The highest fat content was determined in egg yolks in the third laying season (31.6-33.3%),with an average value of 31.9% in all yolk samples tested.A tendency towards an increase in the level of this component was observed with the age of guinea fowls (p < 0.05).The lowest mean ash content was determined in the yolk in the second laying season, 1.66%, while the highest was in the first, 1.93% (p < 0.05).The ash content decreased significantly (p < 0.05) with storage time, from 1.8% to 1.7%, respectively.Interactions of laying season and egg storage time (p < 0.05) were observed only for fat content.

Determination of Mineral Content
The mineral profile of egg albumen in relation to the laying season and storage time is presented in Tables 5 and 6.The average micronutrient contents (Table 5) for the three laying seasons were as follows: 0.09-0.17mg Zn/kg, 3.20-4.32mg Si/kg, 0.20-0.38mg Fe/kg, 0.22-0.27mg Cu/kg, 0.012-0.015mg Ba/kg, 0.12-0.16mg Sr/kg, 0.020-0.026mg Mn/kg, 0.15-0.27mg Al/kg, 0.11-0.12mg Se/kg, 0.34-0.35mg Cr/kg, 0.059-0.06mg Pb/kg and 0.0048-0.0054mg Cd/kg.The average contents of macronutrients (Table 6) in the three laying seasons were as follows: 102-104 mg P/kg, 1441-1451 mg K/kg, 42.7-47.8mg Ca/kg, 1632-1965 mg Na/kg and 114-119 mg Mg/kg.Interactions of laying season and egg storage time (p < 0.05) were found for the content of the following elements: Fe, Cu, Ba, Mn, Pb, Cd, P and Na.a-c -Statistically significant differences (p ≤ 0.05) for the laying season are marked with different letters in the subscript; d,e -statistically significant differences (p ≤ 0.05) for the storage time are marked with different letters in the subscript; a-e -statistically significant differences (p ≤ 0.05) for interaction type of laying season × storage time are marked with different letters in the superscript.
Tables 7 and 8 present the contents of selected elements in the egg yolks of guinea fowls according to the laying season and storage time.The research results indicated that the average contents of trace elements (Table 7) in the three laying seasons were as follows: 44.8-45.2mg Zn/kg, 3.67-5.11mg Si/kg, 3.12-3.19mg Fe/kg, 2.01-2.13mg Cu/kg, 2.97-4.03mg Ba/kg, 1.81-2.22mg Sr/kg, 0.83-1.0mg Mn/kg, 0.71-0.81mg Al/kg, 0.54-0.74mg Se/kg, 0.36-0.37mg Cr/kg, 0.024-0.025mg Pb/kg and 0.0024-0.0032mg Cd/kg.The average macronutrient contents (Table 8) were as follows: 7018-7487 mg P/kg, 1259-1302 mg K/kg, 1938-2033 mg Ca/kg, 511-1965 mg Na/kg and 140-155 mg Mg/kg.Interactions of laying season and egg storage time (p < 0.05) were found for the content of the following elements: Zn, Si, Ba, Se, Pb and Na.a-c -Statistically significant differences (p ≤ 0.05) for the laying season are marked with different letters in the subscript; d,e -statistically significant differences (p ≤ 0.05) for the storage time are marked with different letters in the subscript; a-c -statistically significant differences (p ≤ 0.05) for interaction type of laying season × storage time are marked with different letters in the superscript.

Discussion
Guinea fowl eggs are considered a delicacy and a dietetic product due to their low cholesterol level, low calorie count and high vitamin A content [15,20].In terms of protein content, guinea fowl eggs are distinguished by its slightly higher albumen content compared to other bird species [16].Research indicates that the qualitative characteristics of eggs change during successive laying stages [8,23].A number of changes affecting egg quality can also occur during their storage [24][25][26][27][28]. Egg weight, yolk and albumen weight and shell thickness are important features influencing egg quality [25].The average weight of guinea fowl eggs from the three laying seasons was 45.5 g; only the first season showed a statistically lower weight of 43.4 g.Published reports indicate that the weight of guinea fowl eggs ranges from 38 to 45 g [16].According to many authors [29][30][31], egg weight is significantly influenced by the age of the laying hens through its effect on increasing the proportion of yolk in the overall egg weight, while simultaneously decreasing the egg's albumen content.Storage time and temperature is another factor affecting egg quality.It should be noted that in eggs, as in almost all biological systems, changes in one characteristic entail transformations that affect other qualitative features of the raw material.In the case of eggs, the primary change over time is a loss of their mass.This variability is continuous and linear, and importantly, it has been consistently observed regardless of changes in egg storage temperature or the use of protective substances [31][32][33].During three weeks of storage at room temperature, the average weight loss of eggs was 0.63 g in the first laying season and 0.62 g in the second and third seasons.Water evaporation through the shell into the surrounding environment is accompanied by its internal movement from the albumen through the vitelline membrane into the yolk, resulting in an increase in its volume.The average yolk weight was 15.5 g and its value increased with each subsequent laying season.Storage time had a significant effect on yolk weight only in the first laying season.A similar relationship was confirmed for albumen weight, with an average value of 23.1 g.Meanwhile, the proportion of yolk increased with storage time, while the albumen percentage decreased.A similar trend was demonstrated in the study by Menezes et al. [34].Guinea fowl eggs are known for having thicker and more durable shells compared to chicken eggs, which allows for longer storage periods [9].Natural aging of an egg leads to biophysical and chemical changes in its contents [26,35].It has been observed that alterations in egg quality are associated with temperature and storage time [25,34,36].According to Silversides and Scott [37], the age of the laying hens also affects the quality of the eggshell, including its thickness, whose average value was 0.52 mm and increased with successive laying seasons, while its average weight and percentage were 6.90 g and 15.2%, respectively.Shell percentage was significantly affected by both laying season (p < 0.01) and storage time (p < 0.05).In contrast, Van den Brand et al. [5] found no influence of hen age on eggshell thickness but observed only a decrease in egg shape index.In the present study, the average egg shape index during three years was 76.6%.According to some researchers, the egg shape index is influenced by the egg's weight-as the weight increases, the eggs take on an elongated shape, resulting in a lower egg shape index [35].Another factor influencing egg quality is freshness, which is assessed based on characteristics such as pH and air cell size.The present study found that the pH value of the albumen slightly increased with storage time, while it decreased with each subsequent year; its average value was 9.28.Wilkanowska and Kokoszy ński [38] observed a similar pH value of the egg albumen, i.e., 9.01.In contrast, the average pH value of the yolk was 6.14.The highest yolk pH was recorded in the third laying season-6.27.Wilkanowska and Kokoszy ński [38] compared white and grey guinea fowl eggs and found significant differences between varieties-the average yolk acidity was 6.13.When evaluating the yolk color during the present three-year experiment, it was found to be consistent across all seasons of guinea fowl farming, averaging 10.0 points on a 15-point La Roche scale.A similar color was recorded by Wilkanowska and Kokoszy ński [38], while more intense yolk coloration, 14.6 points, was obtained by Banaszewska et al. [36].The differences can be explained by different diets of guinea fowls, as yolk color is related to the content of natural pigments in the feed.The average depth of the air cell during the experiment was 1.91 mm, and its value increased with longer egg storage (p < 0.05).No significant differences were found in the depth of the air cell between successive laying seasons (p > 0.05).Higher albumen height and smaller spreading area indicate better quality, a finding confirmed in our own study.Moreover, significant influence of both laying season and egg storage time on albumen height, spread and albumen index was observed (p < 0.05).Wilkanowska and Kokoszy ński [38] demonstrated an albumen height of 4.7 mm (white guinea fowl) and 6 mm (grey guinea fowl), resulting in an average albumen index of 7.67.The average yolk index was 36% and decreased with prolonged egg storage (p < 0.05).The laying season did not have a significant impact on this characteristic.Wilanowska and Kokoszy ński [38] reported a higher yolk index, the value of which was 38.83.
The basic chemical composition of an egg determines its nutritional value.During the three laying seasons, the average values of the following components were determined in the albumen: water-87.5%,protein-11%, and ash-0.66%.The highest water content was found in the last laying season-89.1%.There was a significant effect of laying season on water and ash content (p < 0.05), while storage time significantly affected only ash percentage.In the study by Teodorescu et al. [39], the average water content in guinea fowl egg albumen was 74.6%, while the protein content was 10.5%.In the present study, the average water content in the yolk was 48.8%, the protein content was 16.6%, the fat content was 31.9% and the ash content was 1.75%.Significant differences were observed for storage time-the water content increased with storage time (from 47.3% to 49.0%), while the ash content decreased.No significant differences were found between the protein content in the yolk and laying season or storage time (p > 0.05).The highest fat content was determined in egg yolks in the third laying season (31.6-33.3%),with an average value of 31.9% in all yolk samples examined.An increase in the level of this component was observed with the age of guinea fowls (p < 0.05).A similar content of basic chemical components in the yolks of gray guinea fowl (Numida Meleagris) was obtained by Teodorescu et al. [39].
Mineral metabolism is an extremely important factor determining metabolic changes occurring in laying hens' bodies, associated also with egg production.Metabolic disorders involving these components often occur in laying hens undergoing production intensification [40].Due to the limited reports on the mineral composition of guinea fowl eggs, the most important components, which appear to be significant for poultry health and the quality of products obtained from them, were analyzed.The mineral composition of eggs can be modified, for example, through the selection of housing conditions and feeding [41][42][43].Studies on chicken egg demonstrated that its content is particularly rich in ions of such elements as phosphorus, chlorine, potassium, sodium, sulfur, calcium, magnesium and iron.Ions of zinc, fluorine, bromine, iodine, copper, manganese, arsenic, boron, barium, chromium, aluminum, silicon, lithium, molybdenum, lead, rubidium, sele-only from low consumption of EFA but also from an improper ratio of n-6 to n-3 fatty acids [21,46].The increase in the consumption of n-6 fatty acids in recent years is attributed to the advancement of modern technologies in food production, such as agronomy and the feed industry, involving introduction of cereal grains rich in n-6 fatty acids into animal feed.This has led to an unfavorable ratio of n-6 to n-3 fatty acids.Egg yolks are a rich source of lipids, constituting approximately 64% of their dry mass, including phospholipids, which make up one-third of the lipid fraction [47].The composition of fatty acids contained in egg lipids, such as saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA), is of great interest from a human health perspective.For example, insufficient intake of PUFA n-3, especially docosahexaenoic acid (DHA, C22:6 n-3), negatively affects brain growth and functional parameters in infants [48].Long-chain fatty acids are not found in phospholipids isolated from plant sources; therefore, the phospholipids present in egg yolks may be a component of functional food.In the current study, the fatty acid profile was determined in the yolk samples collected during the analysis (Table 9).The fatty acid profile present in the lipids of the egg yolk changed during the three-year laying period.Egg yolks of guinea fowls contained on average 20.3% SFA, 50.2% MUFA and 29.4% PUFA.The results concerning the fatty acid profile content in guinea fowl eggs are varied.The literature indicates that guinea fowl egg yolks are a rich source of EFA from both the n-3 and n-6 groups, whose amount depends mainly on the genotype, age of the laying hens, origin, and nutrition [38,[49][50][51].The percentage of fatty acids determined in the present study was similar to the data presented by Polat et al. [49].A study by Mohsenpur et al. [52] showed that the content of SFA in the yolk was 38.14%, MUFA 38.71% and PUFA 22.09%.Meanwhile, Bondoc et al. [51] reported the following fatty acid contents: 39.47% SFA, 34.47% MUFA and 17.30% PUFA.Similar PUFA profiles were demonstrated by Kouassi et al. [50].The products of EFA metabolism from the n-3 and n-6 groups significantly affect cellular biochemical processes, yet their different chemical structure determines varying biological activity.Therefore, it is crucial to properly balance the amounts of both groups of fatty acids in the diet [46].Achieving optimal health benefits requires the proper quantitative composition and appropriate proportions of ingested fatty acids, making it important from a nutritional standpoint to determine their relative ratio.According to literature data, the ratio of n-6 to n-3 fatty acids should be in the range 4-6:1 [53].In the present research, the most favorable ratio was calculated for the yolks of guinea fowl eggs in the first and second laying seasons (averaging 17.5-17.7:1).Egg yolks in the third laying season showed significantly higher values of the analyzed indicator, averaging 24.6:1.In a study by Bondoc et al. [51], the n-6/n-3 fatty acids ratio in the egg yolks of guinea fowls was 31.71, while for other poultry species it was as follows: turkeys-57.06;quails-64.66;chickens-26.14.The relatively high proportion of EFA and relatively favorable n-6/n-3 ratio found in the present study indicate the high nutritional value of guinea fowl eggs.On the other hand, in a study by Oguntona and Hughes [54], oleic acid (C18:1) was the main unsaturated fatty acid in guinea fowl egg yolks, while palmitic acid (C16:0) was the main saturated fatty acid.Overall, 49% of the fatty acids belonged to SFA, while 51% were UFA.There was no significant difference (p > 0.05) in the fatty acid profile in eggs collected during the three laying periods (at week 8, 12 and 16).

Conclusions
Summarizing the results obtained it can be concluded that 21 days storage time of guinea fowl eggs was characterized by small dynamics of undesirable changes.It was demonstrated that it significantly influenced only egg weight loss and air cell height during storage.On the other hand, the age of the guinea fowls affected certain physical parameters and the chemical composition of the eggs.The egg weight, shape index and shell thickness increased with the age of the laying hens.A decrease in the shell percentage was observed in successive laying seasons.Moreover, significant differences were found in the chemical composition of guinea fowl eggs depending on the age of the laying hens.Eggs obtained from older birds had a higher yolk fat content and lower ash content, while older eggs showed higher water content in the albumen and lower ash content.During the three-year laying period, alterations were observed in the mineral composition of the eggs.It should be noted that the fatty acid profile underwent significant changes; however, there were no important alterations observed in the total content of SFA, MUFA, PUFA and n-6 fatty acids.Significant differences were found for n-3 acids and the n 6/n-3 ratio, which was most favorable in the first and second laying seasons.It should be emphasized that none of the observed changes disqualify the tested guinea fowl eggs as valuable food products.

Table 1 .
Physical characteristics of eggs during three-year laying seasons of guinea fowls.
a,b -Statistically significant differences (p ≤ 0.05) for the laying season are marked with different letters in the subscript; d-f -statistically significant differences (p ≤ 0.05) for the storage time are marked with different letters in the subscript; a-e -statistically significant differences (p ≤ 0.05) for interaction type of laying season × storage time are marked with different letters in the superscript.

Table 2 .
Physical characteristics of egg content during three-year laying seasons of guinea fowls.

Table 3 .
Basic chemical composition of guinea fowl egg albumen.
a-c -Statistically significant differences (p ≤ 0.05) for the laying season are marked with different letters in the subscript; d,e -statistically significant differences (p ≤ 0.05) for the storage time are marked with different letters in the subscript; a-e -statistically significant differences (p ≤ 0.05) for interaction type of laying season × storage time are marked with different letters in the superscript.

Table 4 .
Comparison of chemical composition of guinea fowl egg yolk during three laying seasons.

Table 4 .
Cont.Statistically significant differences (p ≤ 0.05) for the laying season are marked with different letters in the subscript; d,e -statistically significant differences (p ≤ 0.05) for the storage time are marked with different letters in the subscript; a-c -statistically significant differences (p ≤ 0.05) for interaction type of laying season × storage time are marked with different letters in the superscript.

Table 5 .
Trace element content (mg/kg wet tissue) in guinea fowl egg albumen.
a-c -Statistically significant differences (p ≤ 0.05) for the laying season are marked with different letters in the subscript; d,e -statistically significant differences (p ≤ 0.05) for the storage time are marked with different letters in the subscript; a-d -statistically significant differences (p ≤ 0.05) for interaction type of laying season × storage time are marked with different letters in the superscript.

Table 7 .
Trace element content (mg/kg wet tissue) in guinea fowl egg yolk.
a-c -Statistically significant differences (p ≤ 0.05) for the laying season are marked with different letters in the subscript; d-f -statistically significant differences (p ≤ 0.05) for the storage time are marked with different letters in the subscript; a-d -statistically significant differences (p ≤ 0.05) for interaction type of laying season × storage time are marked with different letters in the superscript.

Table 9 .
Cont.Statistically significant differences (p ≤ 0.05) for the laying season are marked with different letters in the subscript; d,e -statistically significant differences (p ≤ 0.05) for the laying season are marked with different letters in the subscript; a-e -statistically significant differences (p ≤ 0.05) for interaction type of laying season