Comparison of Performance, Egg Quality, and Yolk Fatty Acid Profile in Two Turkish Genotypes (Atak-S and Atabey) in a Free-Range System

Simple Summary In recent years, consumers have shown increased interest in healthy and safe food produced under improved animal welfare standards. Therefore, production systems proving outdoor access have gained popularity, increasing the need for knowledge on genotypes suitable for free-range systems. This study aimed to investigate the suitability of two Turkish layer genotypes, Atak-S (brown) and Atabey (white), in a free-range system. We evaluated laying performance, egg quality parameters, and yolk fatty acid profile. The egg production was higher in Atabey than Atak-S, whereas the eggs from Atak-S hens tended to be heavier and had a stronger shell structure than eggs from Atabey hens. Furthermore, eggs from Atabey hens had improved egg yolk and albumen content compared to eggs from Atak-S hens. The total saturated fatty acid content in yolk was higher in Atabey eggs than in Atak-S eggs at 56 weeks of age, whereas a higher yolk color score was observed in Atak-S eggs than in Atabey eggs. Our results could help free-range egg producers to improve production, as well as satisfy consumer demands regarding egg quality in organic production. Abstract Consumer interest in buying eggs from animal welfare-friendly systems with outdoor access is increasing, leading to an increase in the need for knowledge on genotypes suitable for free-range systems. Two Turkish laying hen genotypes, Atak-S (brown, n = 210) and Atabey (white, n = 210), were reared in a free-range system from 19–72 weeks of age, and their suitability for the system was assessed based on laying performance, egg quality, and yolk fatty acid profile. Mean hen-day and hen-housed egg production were found to be higher in Atabey than Atak-S (p < 0.01). The brown eggs from Atak-S hens tended to be heavier than the white eggs from Atabey hens (p < 0.01). Brown eggs obtained from Atak-S hens had a stronger shell structure (p < 0.01), while white eggs from Atabey hens had higher mean yolk index, albumen index, and Haugh unit than brown eggs (p < 0.05). At 56 weeks of age, total saturated fatty acid content in yolk was higher in white eggs than in brown eggs (p < 0.01). These findings related to genotype could help free-range egg producers in their choices for more profitable production and for meeting consumer demands on egg quality and egg yolk fatty acid levels.


Introduction
Egg production is a major and significant component of animal production in Turkey, due to its high economic contribution (1.7 billion egg production in 2020) and its adaptability to new sectoral developments and consumer demands [1]. Consumers are now

Data Collection
To determine mean BW, hens (20% from each pen) were individually weighed weekly from 18 to 72 weeks of age. Daily feed intake (DFI) and egg weight (EW) were recorded on a weekly basis. The pens were monitored daily for egg production (EP) and mortality until the end of the experiment. Egg production was calculated by dividing the number of eggs daily collected by the number of hens on the same day. The efficiency of feed utilization (FCR) was calculated as the ratio between weekly feed intake and weekly EP multiplied by EW. Daily collected eggs were classified according to shell surface cleanliness into three categories, namely very dirty (shell surface >1 cm 2 dirty), dirty (shell surface <1 cm 2 dirty), and clean (no dirt on the shell), and as cracked eggs or floor eggs. Percentage values of the categories were calculated by dividing the number of

Data Collection
To determine mean BW, hens (20% from each pen) were individually weighed weekly from 18 to 72 weeks of age. Daily feed intake (DFI) and egg weight (EW) were recorded on a weekly basis. The pens were monitored daily for egg production (EP) and mortality until the end of the experiment. Egg production was calculated by dividing the number of eggs daily collected by the number of hens on the same day. The efficiency of feed utilization (FCR) was calculated as the ratio between weekly feed intake and weekly EP multiplied by EW. Daily collected eggs were classified according to shell surface cleanliness into three categories, namely very dirty (shell surface >1 cm 2 dirty), dirty (shell surface <1 cm 2 dirty), and clean (no dirt on the shell), and as cracked eggs or floor eggs. Percentage values of the categories were calculated by dividing the number of

Data Collection
To determine mean BW, hens (20% from each pen) were individually weighed weekly from 18 to 72 weeks of age. Daily feed intake (DFI) and egg weight (EW) were recorded on a weekly basis. The pens were monitored daily for egg production (EP) and mortality until the end of the experiment. Egg production was calculated by dividing the number of eggs daily collected by the number of hens on the same day. The efficiency of feed utilization (FCR) was calculated as the ratio between weekly feed intake and weekly EP multiplied by EW. Daily collected eggs were classified according to shell surface cleanliness into three categories, namely very dirty (shell surface > 1 cm 2 dirty), dirty (shell surface < 1 cm 2 dirty), and clean (no dirt on the shell), and as cracked eggs or floor eggs. Percentage values of the categories were calculated by dividing the number of dirty/cracked/floor eggs by the total number of eggs. Mean BW, DFI, EP, EW, and FCR were calculated for each 6-week period between 19 and 72 weeks of age.
A total of 30 eggs from each genotype were randomly sampled to define external and internal egg quality parameters at 24, 40, 56, and 72 weeks of age. The measurements were performed 24 h after the eggs were laid. After weighing the eggs with ±0.01 g precision, the length and width of the eggs were measured by using a digital caliper with 0.01 mm precision (Mitutoyo, 300 mm, Neuss, Germany). The measured values were used to calculate the egg shape index with a formula of (egg width/egg length) × 100 [18]. Eggshell breaking strength (kg/cm 2 ) was determined by using an eggshell force reader machine (Egg Force Reader, Orka Food Technology, Israel). The eggs were broken to obtain the albumen and yolk, and then the yolk weight was measured with ±0.01 g precision.
The eggshells were cleaned by washing process and then put in an oven at 105 • C (Nüve FN-500, Ankara, Turkey) for drying process for 24 h. Then, the eggshell weight was determined with ±0.01 g precision. Albumen weight was calculated by subtracting yolk and shell weight from total egg weight. The ratios of albumen, eggshell, and yolk were given as a percentage of EW. Eggshell thickness was measured at three different points of the eggshell, specifically air cell, sharp end, and equator region, by using a digital caliper with ±0.01 mm precision. The eggshell thickness was given as the average of three values measured for these points.
At 56 weeks of age, 12 yolk and albumen samples were randomly selected from each genotype and analyzed for dry matter (method number 934.01), ash (method number 942.05), protein (method number 954.01), and lipid (method number 920.39) content, according to AOAC (2006) [17].
To determine the fatty acid profile, 12 yolk samples from each genotype were randomly obtained at 56 weeks of age. Yolk fatty acid analysis was performed using a gas chromatography method developed by Folch et al. [22]. The fatty acid profile of the egg yolk was expressed as a percentage of total fatty acids [23].

Statistical Analysis
The parametric data for performance parameters (BW, EP, DFI, EM, and FCR) for each genotype (Atak-S and Atabey) were analyzed with the mixed model procedure in the statistical analysis software SAS (version 9.4, 2012, Cary, NC, USA). A completely randomized, repeated measures design on a weekly basis was used for performance parameters, and the mean values for each parameter were calculated for consecutive 6-week periods for the whole production period. For performance parameters, egg external and internal quality parameters, and egg chemical composition, the main effects (G = effect of genotypes and A = effect of age) and the combined effect (G × A interaction) were determined. Significant differences between means were compared using the Tukey test. Analyses of percentage data were conducted after arcsine square root transformation of the data. The total mortality data were analyzed using chi-square tests to determine differences between the genotypes. The effects of genotype on egg chemical composition and yolk fatty acid profile were subjected to the t-test procedure in SAS (version 9.4, 2012, Cary, NC, USA). Differences were considered statistically significant at p ≤ 0.05.

Results
Mean egg production and egg weight for the brown (Atak-S) and white (Atabey) layer genotypes during their production life in the free-range system are presented in Table 2. A significant genotype × age interaction was found for hen-day and hen-house EP (p < 0.01). At 19-24 weeks of age, Atak-S hens showed higher hen-day and hen-house EP levels (56.4% and 55.4%, respectively) than Atabey hens (40.4% and 40.2%, respectively) (p < 0.01). However, Atabey hens reached a peak level at 25-30 weeks of age, with an EP level of 90.1% on a hen-day basis and 89.2% on a hen-house basis (p < 0.01). The laying performance of Atabey hens decreased to below 80% after 54 weeks of age, whereas Atak-S hens showed a rapid decline to a similar EP level after 37 weeks of age. Egg weight showed significant differences arising from genotype × age interactions. Table 2. Mean egg production values and mean egg weight for two Turkish laying hen genotypes (Atak-S and Atabey) in a free-range system.

Hen-House Egg Production (%) Egg Weight (g)
Genotype The effects of genotype on BW, DFI, and FCR are presented in Table 3. The results indicated that as the hens aged, DFI increased in both genotypes, with a corresponding increment in BW. As expected, BW was found to be higher in Atak-S than Atabey (2087.3 g vs. 1497.4; p < 0.01). Atak-S hens tended to consume more feed than Atabey hens. Mean DFI was found to be higher in Atak-S (117.2 g) than in Atabey (109.8 g) (p < 0.01). Based on the difference in BW, DFI and FCR differed significantly between two genotypes during the whole experimental period. Higher FCR between 19 and 72 weeks of age was observed in Atabey than in Atak-S (2.48 vs. 2.54; p = 0.001). Mortality between 19 and 72 weeks of age was 11.0% in Atak-S and 8.1% in Atabey, although no significant difference was found (chi-square value = 0.995, p = 0.319). Table 3. Mean body weight, daily feed intake, and feed conversion rate (FCR) for two Turkish laying hen genotypes (Atak-S and Atabey) in a free-range system. The proportions of eggs with different degrees of shell cleanness differed between the genotypes (Figure 3). The proportions of very dirty and dirty eggs were found to be higher for Atabey (27.8% and 39.4% respectively) than for Atak-S (18.2% and 34.2%, respectively) (p < 0.001), whereas the ratio of clean eggs was higher for Atak-S than Atabey (47.6% vs. 32.8%; p < 0.001). Higher percentages of cracked and floor eggs were observed for Atabey (6.2% and 12.0%, respectively) than Atak-S (4.6% and 5.4%, respectively) (p < 0.05) (Figure 4). the genotypes (Figure 3). The proportions of very dirty and dirty eggs were found to be higher for Atabey (27.8% and 39.4% respectively) than for Atak-S (18.2% and 34.2%, respectively) (p < 0.001), whereas the ratio of clean eggs was higher for Atak-S than Atabey (47.6% vs. 32.8%; p < 0.001). Higher percentages of cracked and floor eggs were observed for Atabey (6.2% and 12.0%, respectively) than Atak-S (4.6% and 5.4%, respectively) (p < 0.05) (Figure 4).  An effect of genotype on egg composition was found ( Table 4). The interaction (genotype x age) was significant for EW (p < 0.01), yolk ratio, and albumen ratio (p < 0.05), but not for shell ratio. Yolk ratio was found to be higher at 56 and 72 weeks of age in brown (Atak-S) eggs and at 40, 56, and 72 weeks of age in white (Atabey) eggs. The albumen ratio showed a decline with hen age for both Atak-S and Atabey (p < 0.01). The shell ratio was only affected by hen genotype (p < 0.01). Table 4. Mean egg weight and egg content for two Turkish laying hen genotypes (Atak-S and Atabey) in a free-range system.  An effect of genotype on egg composition was found ( Table 4). The interaction (genotype × age) was significant for EW (p < 0.01), yolk ratio, and albumen ratio (p < 0.05), but not for shell ratio. Yolk ratio was found to be higher at 56 and 72 weeks of age in brown (Atak-S) eggs and at 40, 56, and 72 weeks of age in white (Atabey) eggs. The albumen ratio showed a decline with hen age for both Atak-S and Atabey (p < 0.01). The shell ratio was only affected by hen genotype (p < 0.01).

Main Factors Egg Weight (g) Yolk Ratio (%) Albumen Ratio (%) Shell Ratio (%)
An effect of genotype on egg exterior quality parameters was also observed ( Table 5). A significant interaction (genotype × age) was observed for egg shape index (p = 0.027) and eggshell thickness (p < 0.01). The highest mean value of shape index was observed for Atak-S eggs at 24 weeks of age (79.7%). For both Atak-S and Atabey eggs, eggshells tended to be thinner at 72 weeks of age (0.347 and 0.320 mm) than at earlier ages, more so for Atabey than Atak-S eggs. On the other hand, breaking strength was affected by genotype and age. Atak-S eggs had a stronger eggshell structure (3.429 g/cm 2 ) than Atabey eggs (2.982 g/cm 2 ) (p < 0.01). As hen age increased, the breaking strength of the eggs showed a deterioration, with the mean value for all hens decreasing from 3.398 g/cm 2 at 24 weeks of age to 2.890 g/cm 2 at 72 weeks of age (p < 0.01). Table 4. Mean egg weight and egg content for two Turkish laying hen genotypes (Atak-S and Atabey) in a free-range system.
Higher mean values of yolk index, albumen index, and Haugh unit were observed for Atabey eggs (44.4%, 12.3%, and 92.4, respectively) compared with Atak-S eggs (43.1%, 10.9%, and 88.5, respectively). Yolk index had the highest mean value at 56 weeks of age (46.9%) and declined to 43.7% at 72 weeks of age (Table 6). Furthermore, higher yolk color score was observed in Atak-S eggs than in Atabey eggs (12.9 vs. 12.2; p < 0.01). Yolk color score was highest at 40 and 56 weeks of age and then started to decrease after 56 weeks (p = 0.016). On the other hand, albumen index and Haugh unit started to decrease after 40 weeks of age in both genotypes. Table 6. Mean values of interior egg quality parameters in two Turkish laying hen genotypes (Atak-S and Atabey) in a free-range system.

Main Factors Yolk Index (%) Yolk Color Albumen Index (%) Haugh Unit
Genotype Atak-S (brown) 43 The effects of genotype on egg chemical composition and yolk fatty acid profile at 56 weeks of age are shown in Table 7. The dry matter, ash, and fat contents in yolk were found not to differ between two genotypes, whereas a higher content of protein of yolk was observed in Atabey eggs (p = 0.031). The dry matter content of albumen was significantly higher in Atabey eggs than in Atak-S eggs (88.2% vs. 86.6%; p = 0.009), whereas a higher protein content of albumen was observed in Atak-S eggs (13.6% vs. 11.9%; p = 0.009). The saturated fatty acid profile of yolk, except C15:0, was significantly affected by genotype, and the total saturated fatty acid content of yolk was found to be higher in Atabey eggs (35.4%) than in Atak-S eggs (32.1%) (p < 0.01). The unsaturated fatty acid profile of yolk, except C18:1c, C18:2c, and C20:3n6, was significantly affected by genotype, whereas the totals of UFAs, MUFAs, and PUFAs were found not to differ between the genotypes. Higher ratios of UFAs/SFAs, MUFAs/SFAs, and PUFAs/SFAs were observed in Atak-S eggs than in Atabey eggs (p < 0.01). Table 7. Chemical composition and yolk fatty acid profile in eggs from two Turkish laying hen genotypes (Atak-S and Atabey) in a free-range system (56 weeks of age).

Discussion
This study showed that Atabey hens had a higher EP level in a free-range system than Atak-S hens. Between 19 and 24 weeks of age, Atabey hens displayed better laying performance, with approximately 3.2% and 3.8% higher hen-day and hen-house EP compared with Atak-S hens. According to the standard performance data for Atak-S and Atabey given in the management and performance guide [24], under optimum management standards in cage systems, hen-day and hen-house EP are 83.3% and 82.4%, respectively, for Atak-S, and 83.9% and 82.8%, respectively, for Atabey. This is better laying performance than observed in our free-range system and may be attributed to differences between the cage system and free-range system in terms of stress level, nutrition, physical activity level, and varying environmental conditions, especially during pasturing of laying hens in the free-range system [25]. Egg formation is an energy-requiring activity, so the observed decline in EP level could be attributable to the hens in this study spending energy for physical activity while outdoor ranging.
Hens of both genotypes produced heavier eggs with increasing age, confirming previous findings of [26] for Italian genotypes and [27] for conventional and traditional genotypes (Lohmann Brown vs. Banat Naked-Neck). In the present study on a free-range system with outdoor access, the hens tended to show reduced egg weight and EP level. This is consistent with the findings of Samiullah et al. [28], Miao et al. [29], and Campbell et al. [30]. In contrast, some studies have found no significant differences in EW for eggs obtained from free-range systems and conventional unenriched cage systems [31][32][33]. Compared with standard values given in the performance guide for the Atabey and Atak-S genotypes, lower BW of hens and higher DFI were recorded in this study. Our findings are consistent with previous results reported by Mugnai et al. [10], Lampkin [34] and Küçükyılmaz et al. [35]. Those studies attributed higher feed intake in organic systems with outdoor access to increased physical activity, including walking and foraging behaviors, which also increased the energy requirement of laying hens. The higher percentages of cracked and floor eggs observed for Atabey hens in the present study could be related to their lower body weight and higher motor activity than Atak-S hens. In an earlier study, a higher percentage of cracked and broken eggs was observed for Atak-S hens (0.44%) than for a white laying hen genotype (Lohmann-LSL) (0.31%) in an organic system [35].
It is known that different commercial laying hen genotypes produce different sizes of eggs, and consequently, the weights and ratios of yolk, albumen, and shell can differ [36]. Our results support findings of Küçükyılmaz et al. [35] of higher yolk ratio in Lohmann-LSL hens and higher albumen ratio in Atak-S hens in organic systems. The increase in yolk ratio and decline in albumen ratio observed with increasing hen age confirm previous findings of Van den Brand et al [31], Rizzi and Chiericato [37], Tůmová and Ledvinka [38], and Zita et al. [39].
Egg exterior characteristics, namely egg shape index, eggshell breaking strength, and shell thickness, are important quality parameters in commercial handling and transport [36,40]. In the present study, egg quality parameters were affected by both genotype and age. A higher shape index was observed for Atak-S eggs, confirming the findings of Küçükyılmaz et al. [35] and Onbaşılar et al. [41]. On the other hand, it was found that egg shape index decreased as hen age increased in both genotypes. Similarly, Onbaşılar et al. [41] found that egg shape index showed a decline from 78.13% at 20 weeks of age to 75.03% at 70 weeks of age. This change could be related to older hens producing longer eggs due to some anatomical structural changes to the shape of the pelvic bones with aging in laying hens [41]. However, Tůmová et al. [42] observed no significant effect of hen genotype on egg shape index. We found higher breaking strength and shell thickness for brown Atak-S eggs, whereas Küçükyılmaz et al. [35] reported higher eggshell breaking strength and thickness for white Lohmann-LSL eggs (4.225 g/cm 2 and 409.17 µ, respectively) than for brown Atak-S eggs (3.715 g/cm 2 and 388.56 µ, respectively) in organic systems.
Our study showed that hen genotype affected yolk index, confirming the findings of Zita et al. [39] and Ledvinka et al. [43]. Similar to our results, Leyendecker et al. [44] found a higher Haugh unit for eggs obtained from white hens than for eggs obtained from brown hens. Bozkurt and Tekerli [45], Ledvinka et al. [43], and Kraus and Zita [46] indicated that yolk index decreased with hen age. Our results relating to yolk color support the findings of Ledvinka et al. [43], who indicated that yolk color was significantly affected by hen age, and of Kraus and Zita [46], who found a significant effect of hen genotype on yolk color.
Observed changes in albumen index and Haugh unit by hen genotype and age are similar to the previous findings of Tůmová et al. [38], Zita et al. [39], Bozkurt and Tekerli [45], and Kraus and Zita [46]. Our data indicated a possible relationship between albumen index and Haugh unit. Similarly, Kraus and Zita [46] found that when albumen index increased, Haugh unit also showed an increment. Further, Molnár et al. [47] observed a decline of 0.38 in Haugh unit for each week after 60 weeks of age. However, other studies have found no significant effects of genotype on Haugh unit [37].
The observed changes in protein content of the eggs by genotype could be related to differences in DFI and EP level between Atak-S and Atabey birds. Küçükyılmaz et al. [35] also found a higher protein level in brown eggs than in white eggs. Some studies have found that hen genotype affects yolk fatty acid content [48][49][50]. Our results showed that brown (Atak-S) eggs had a lower content of SFAs than white (Atabey) eggs (p < 0.01), confirming the findings of Ayerza and Coates [51]. On the other hand, it is known that the content of n-3 PUFAs in egg yolk can be enhanced in hens consuming fresh vegetables, insects, and worms [52]. The higher content of C18:3n observed in Atabey eggs could be related to more activity and foraging behavior of this genotype compared with Atak-S. Likewise, Hammershøj and Johansen [53] reported that the fatty acid content of egg yolk is largely influenced by the amount of grasses and herbs consumed by the hens in free-range systems.

Conclusions
In conclusion, the findings in this study are of practical significance for consumers and for Turkish producers employing alternative egg production systems such as free-range and organic production. This study examined the suitability of local brown and white egg genotypes (Atak-S and Atabey) for free-range systems. Atabey hens showed a higher egg production rate with better feed utilization, as well as better yolk, albumen, and eggshell quality, than Atak-S hens. However, the Atak-S hens gave higher egg weight, darker yolk color, stronger shell structure, and a lower level of SFAs in yolk. These findings on genotype effects could help producers in their choices for profitable production and for meeting consumer demands on egg quality and egg yolk fatty acid levels. The findings could contribute to more sustainable poultry production by improving the use of safe and more natural production systems and increase customer satisfaction.  Data Availability Statement: All data sets collected and analyzed during the current study are available from the corresponding author on fair request.