1. Introduction
In recent years, there has been an increased consumer demand for meat and egg products that focuses on animal welfare during production and product safety and quality [
1,
2,
3,
4]. As such, feeding forage or pasture intake in outdoor-based systems (conventional free-range systems or organic systems) for laying hens has gained tremendous attention in the poultry research field, as highlighted by certain reviews [
2,
3,
4,
5,
6,
7]. A considerable body of research has shown that in general, these systems delivered low egg production and a high feed conversion ratio (FCR), but they delivered high-quality and heavier eggs compared with conventional systems [
1,
8,
9,
10,
11,
12,
13,
14,
15,
16]. These findings resulted from reduced feed intake and diluted diets due to forage or pasture intake versus increased energy requirements of laying hens [
3,
17]. Moreover, information about feed intake from the outdoor areas is still scarce. There is no clear evidence that laying performance and egg quality properties are improved when hens are raised with free access to outdoor areas with vegetation, as has been reported previously [
11,
18]. Therefore, further studies are warranted.
White clover (
Trifolium repens L.) is a stoloniferous perennial plant capable of symbiotically fixing atmospheric nitrogen, resulting in high protein content and high nutritive value [
4,
19]. The chicory (
Cichorium intybus L) plant, a small perennial herbaceous aromatic herb, contains nutrients (carbohydrates, proteins, vitamins, and minerals) and bioactive phenolic compounds (inulin, esculin, terpenes, coumarins, and flavonoids) [
20]. These swards can increase herbage production and further improve herbage nutritive value [
19] and thus can contribute to a more sustainable production system. When hens are safely grazed on an area with white clover or chicory plants, the dietary amounts consumed during free-ranging contribute to the total feed intake to meet the nutrient needs of hens [
21]. These plants can also positively impact hen health and product quality [
4,
20] because of the increased intake of bioactive substances [
21].
Because free-range hens consume different levels of herbage in areas with various types of vegetation, their laying performances and egg qualities depend on herbage intake (HI) and forage species [
8,
9,
10,
11,
16]. Different herbage species, including white clover or chicory species, have been used in outdoor-based production systems of laying hens, but with contradictory results regarding laying rate and certain internal and external egg quality properties [
1,
3,
4,
10,
16]. Despite these observations, little information is available regarding the effects of the chicory and white clover species, used either as a single herb or a mixture in vegetated areas, on the laying performance and certain egg quality characteristics in layer hens fed a conventional diet. Fatty acid (FA) enrichment of the egg yolk is a key means to improve the nutraceutical properties of eggs [
22]. Some herbage species (grass, grass-clover, alfalfa, or chicory) consumed by free-range hens have enriched the FA content of egg yolk [
4,
9,
14,
16] depending upon the range utilization ratio [
4,
10]. However, limited studies related to outdoor-based systems of laying hens have focused on the association between FAs of the egg yolk resulting from FAs ingested via the HI [
4,
16].
It is possible that free access to chicory- and/or white clover-vegetated areas may improve egg production, feed intake, FCR, and egg quality characteristics (including the FA profiles, cholesterol content, and colour of the egg yolk) in layer hens fed with a conventional diet. In addition, it can provide knowledge about how the FA profiles of these species (as a single herb or a mixture) are distributed between the FAs of the egg yolks. Therefore, this study was designed (I) to test for distinct differences in the laying performance and egg quality characteristics in layer hens fed on a conventional diet with free access to the outdoor area, either vegetated or unvegetated with the chicory and/or white clover species, and (II) to characterize changes in the FA profile of the egg yolk produced by hens fed on a conventional diet with free access to the outdoor area to better understand the regulation process of FA transfer from the consumed herbage to the egg yolk. Accordingly, the main aims were to (I) compare the effects of free access to chicory- and/or white clover-vegetated areas or non-vegetated soil areas on the laying performance of layer hens fed with a conventional diet and on several egg quality traits, (II) to evaluate the reorganization of yolk FAs following the free access to outdoor areas with or without vegetation, and (III) to more clearly explain the contribution of the HI to the production of eggs with preferred FA attributes using a chemometric approach.
3. Results
No differences were found in the final body weight, laying rate, or egg weight (
Table 4). The C hens consumed more concentrate feed (
p < 0.05) and had a numerically higher FCR compared with the TR and MIX hens. The HIs of TR and MIX hens were higher than that of the CI birds (
p < 0.05). This HI amount numerically improved the FCR of the TR and MIX hens. The CI, TR, and MIX treatments did not affect either internal or internal egg quality characteristics, except for eggshell thickness (
Table 5). The C hens produced eggs with a thicker shell than the CI, TR, and MIX hens (
p < 0.05). However, this difference in the eggshell was not reflected in the cracked or broken egg ratio (
Table 4).
In total, 11 FAs containing two trace FAs (> 0.1% weight) were identified. These two FAs, lauric acid and myristic acid, are not shown in
Table 6. Significant differences were found among treatments in terms of both total FAs, except for
n-3 FAs, and individual FAs, except for linoleic acid and linolenic acid (
Table 6). Among the treatments, the lowest value of palmitic acid was recorded for the MIX group (
p < 0.05). The TR and MIX hens produced yolk with higher stearic acid than the C and CI hens (
p < 0.05). The behenic acid content of the yolk was lower in the eggs from the CI hens than those from the C and TR birds (
p < 0.05). The eggs from the MIX hens displayed lower arachidic acid content compared with the other treatments (
p < 0.05). In the samples of the C eggs, saturated FAs (SFA) constituted about 38.2% of the total amount of FAs. This percentage was at the same level for the CI and TR groups but was decreased for the MIX group (
p < 0.05). The most abundant of the saturated FAs was palmitic acid, constituting about 26.8% of the total FA profile. The C hens produced yolk with higher palmitoleic acid levels compared with the TR and MIX hens (
p < 0.05). The oleic acid content of the yolk was higher in the CI eggs than in those from the TR and MIX birds (
p < 0.05). The C hens produced yolks that contained more monounsaturated fatty acids (MUFA) compared with the yolks of the TR hens (
p < 0.05). The eggs from the C hens displayed higher arachidonic acid content compared with the CI, TR, and MIX hens (
p < 0.05). The polyunsaturated fatty acid (PUFA) content of the yolks from the MIX hens was higher than that from the C and TR hens (
p < 0.05). The corresponding value of the TR hens was higher than that of the C hens (
p < 0.05). Among the PUFAs, the most abundant acid was linoleic acid, but it was not significantly different between all investigated groups (
p = 0.155). The total
n-6 FA content for eggs from the C hens was lower than that from the other treatment hens (
p < 0.05). The
n-6 to
n-3 ratio for eggs from the CI hens was higher than that from the hens undergoing other treatments (
p < 0.05). The corresponding ratios of the C and TR hens were higher than that of the MIX hens (
p < 0.05). No differences were found in the cholesterol content of the egg yolk (
p > 0.05).
The most significant PCs generated from the yolk FA data and their statistical loadings in the present study are depicted in
Figure 1. The first and second PCs, i.e., PC1 and PC2, accounted for 70% of the variability in the data set from the four outdoor-based systems and had the highest eigenvalues, which were 6.44 and 1.35, describing 58.5% and 12.2% of the total variance, respectively (
Figure 1a). Although FAs were distributed in all quadrants of the PCA, the loadings (or scores) corresponding to the PCs indicated high contributions from three groups. Therefore, from the PCA, three main FA profiles may be identified based on natural groupings in the PC2 versus PC1 plot. Group 1 was composed of FAs with positive loadings for PC1 and PC2 (stearic (0.950 and 0.133) and linolenic acids (0.707 and 0.596)). Group 2 included FAs with positive loadings for PC1 and negative loadings for PC2 (palmitic (0.989 and −0.039), behenic (0.908 and −0.062]), arachidonic (0.646 and −0.463), and lauric (0.586 and −0.281]) acids). Group 3 was composed of FAs with negative loadings for PC1 (oleic (−0.946 and −0.259), palmitoleic (−0.827 and −0.435), linoleic (−0.816 and 0.480), arachidic (−0.370 and 0.270), and myristic (−0.303 and 0.340) acid variables). Based on the correlation matrix loadings (≥ 0.75 and positive factor loadings) of the variables, stearic, palmitic, and behenic acids contributed most strongly to PC1, while linolenic, arachidonic, and lauric acids contributed less strongly.
Figure 1b depicts the score plots of FAs in the yolk from all treatments, generated by comparing the groups based on PC1 and PC2. The FAs of groups 1 and 2 were strongly related to the TR and MIX treatments, while group 3 was related to the C and CI treatments. The position of yolk samples in the score plot was consistent with the vegetation type in the outdoor area and also demonstrates that PC1 described the FA distribution between the studied herbages. The TR and MIX treatments were characterized by high values for stearic, palmitic, and behenic acids, while the CI group was characterized by high myristic and arachidic acids. The C group was somewhat in the middle and less tightly clustered than either of the other groups. Therefore, PC1 separated the TR and MIX samples from the CI samples. PC2 did not enable the C samples to be separated from the CI and MIX treatments, which were associated with FAs from groups 1 and 2, respectively.
4. Discussion
In the present study, free access to chicory- and/or white clover-vegetated areas decreased the concentrate FI of hens and contributed to the production of eggs with preferred FA attributes without affecting laying performance. These results did not confirm the suggestion that outdoor-based systems cause low egg production, high FCR, less weight uniformity, and high mortality compared with conventional production systems [
1,
8,
9,
10,
12,
13,
14]. However, free access to permanent or temporary vegetated areas did not negatively affect laying performance [
8,
9,
11] of the hens, despite increased egg and yolk weights [
11,
16]. In addition, some studies [
9,
11,
16] indicated that vegetation type in outdoor areas was a potential contributor to nutrient content, including FAs, as well as certain quality parameters of eggs. These inconsistencies between our results and results reported previously may be attributed to environmental conditions, the strain and age of hens, the level of dilution of diets due to forage or pasture intake, grazed outdoor area, and the change in the energy requirements of hens depending on laying rate [
3,
8,
11,
17].
Although it is difficult to describe the supply of nutrients to the hen from the HI in outdoor-based systems [
4], in general, the HI contributes to the nutrition of laying hens [
3,
4,
21]. Laying hens can consume 2–57 g of dry matter per day from herbage [
29,
30], depending on the forage species or vegetation type [
31], age and genotype of the hens, rearing conditions, etc. [
30]. Although the hens with access to the TR- and MIX-vegetated areas consumed more herbage compared with those in the CI-vegetated area, the HI of hens in the present study were within this range. These differences between treatments may be related to the fact that the nutritive value of the outdoor area changed according to both vegetation type [
32] and macro-invertebrates living in the outdoor area [
4,
10]. Indeed, the white clover, a leguminous species, and the mixture of chicory and white clover displayed relatively high crude protein and low neutral detergent fibre content compared with chicory, which belongs to the Asteraceae family. Unfortunately, the contribution of macro-invertebrates to the diet of hens was not quantified in our study.
The data relating to the final body weight suggests that despite a reduction in the concentrate feed intake, the HI promoted weight gain in the hens kept under the same rearing conditions. This may show that there were no severe effects of the change in digestibility due to fibre content of plant species on the performance of herbage-consuming hens, as Iqbal et al. also revealed [
17,
32]. Our result regarding the HI supports the idea that poultry with access to pasture often consume herbage in quantities resulting in diet dilution and reduced concentrate feed intake [
3,
32]. It is expected that the decreased concentrate feed intake of hens that access the vegetated area results in the production of fewer eggs. However, the present HI might be used to cover the production demands of the vegetated area versus the non-vegetated area. In addition, the forage consumed from TR- or MIX-vegetated areas caused a numerically lower FCR compared with that in the other treatments. Mohammed et al. [
10] noted that when the additional consumed forage was considered, an increase in FI was used to cover the high production demands and was subsequently utilized for producing more eggs. As such, our results indicate that establishing the amount and the nutritional value of herbage to be ingested is very important to manipulate the nutritional quality of the egg [
9].
The HI might have changed the mineral content [
15] and the calcium to available phosphorus ratio of the diet [
3,
32] and reduced intestinal calcium uptake because feed ingredients from plant sources are inadequate in meeting the requirements for calcium and phosphorus [
33]. Therefore, the decreased eggshell thickness in the vegetated area groups, a major concern of the egg industry, could be associated with the consumed forage. High eggshell thickness is associated with high eggshell breaking strength [
17,
34]. However, the decrease in eggshell thickness in the hens consuming herbage was not reflected in the eggshell strength and cracked egg ratio. This finding, which concurs with Samiullah et al. [
35], indicates that the decrease in eggshell thickness was not severe enough to have an impact on this parameter. This means that similarly sellable eggs are available for all treatments. When using chicory and white clover in outdoor-based systems, the producers could add extra vitamin D to the conventional diet and provide a free-choice calcium source to overcome the eggshell issues.
The results regarding the studied egg quality properties support the findings of studies that indicated no negative effects of the production system on these properties [
12,
16]. The Haugh unit, a measure of egg protein quality based on the albumin height, and egg yolk colour, an important commercial characteristic, are both important properties of egg quality, along with other parameters such as eggshell thickness and strength. In contrast to results reported previously [
4,
15,
17,
35], the HI of hens did not have a significant effect on the Haugh unit and egg yolk colour. This discrepancy among the studies might be due to differences in the housing type (deep or floor litter, cage, or enrich cage), vegetation type (grass, legume, other forbs, or their mixture), the range utilization percentage, the environmental conditions, and the age and strain of hens used [
14,
17,
35,
36]. Differences in environmental temperature between the free-range and conventional systems resulted in differences in egg weight [
5] and Haugh units [
14]. Therefore, we observed a similar egg weight and Haugh unit among the treatments due to the similar environmental temperature because all hens had free access to the outdoors. This similarity in the egg weights was similarly reflected in internal egg quality parameters, as reported by Ketta and Tůmová [
34].
Because the egg yolk colour depends mainly upon the diet, the differences in the yolk colour of eggs produced in an outdoor-based system are attributed to the herbage materials consumed by the hens [
4,
10,
14,
29,
37]. Horsted et al. [
29] reported that hens that consumed chicory leaves produced eggs with a darker yolk colour than those consuming a grass-clover pasture. Our results did not confirm these notions since our study failed to prove a positive effect of the herbage consumed by hens on the yolk colour. However, the results regarding egg yolk colour are consistent with those of Yilmaz Dikmen et al. [
38], who found that the influence of the rearing system on the colour of the yolk was insignificant. These discrepancies may result from the chemical and physical properties of egg yolk, the production cycle of the hens, and the varieties and maturity stage of the herbages used [
1,
14,
37]. Moreover, the HI levels in the present study were probably not high enough to significantly affect the egg yolk colour [
4]. Our similar results, namely the similar yolk colour of eggs from hens with free access to the vegetated or unvegetated outdoor area, was probably due to hens in all groups receiving a conventional diet containing ingredients with high pigments [
37].
The consumption of the white clover and chicory-clover mixture had a larger effect than either alone on the levels of MUFA and PUFA, which play a crucial role among unsaturated fatty acids, mainly due to their physiological functions [
25]. The results of the PCA confirmed previous findings on the FA content of the yolk from eggs produced by feeding forage or pasture intake in outdoor-based systems [
9,
14,
36]. Corrales-Retana et al. [
22] suggested that any change in the FA profiles of the yolk due to the diet of the hens is mainly linked to the lipid fraction, which represents the reserve lipid component of the cell. Walczak et al. [
25] noted that the metabolism of FAs is dependent on the relative cellular FA composition, rather than the absolute concentration. These observations may explain why the PCA grouped the FAs of the yolk differently and the mutual correlations between FA profiles of the egg yolk modified by the FAs ingested via the HI. Mierliță [
15] noted that the FA profile of egg yolk was characterized by a relatively low SFA and high MUFA and PUFA proportions, as reported herein. Moreover, findings relating to the FA profiles of the yolk were similar to previous reports [
15,
22,
39] in which the yolk FA contents were maximized when the relevant FA was higher in the diet, reflecting a higher concentration of FAs in fresh herbage. Although the PUFA content of chicory species (61.79%) was higher than that of white clover (37.68%), the eggs from CI and TR treatments had similar contents of PUFA (22.43% and 22.17%, respectively). This may be due to the lower chicory intake (13.7 g/hen/day) than white clover intake (18.0 g/hen/day), relatively low consumption of both herbages [
1,
4], and the differences in the quantity and digestibility of non-starch polysaccharides found in these herbages [
32]. Hammershøj and Johansen [
4] noted that a high HI (i.e., ≥ 50 g/hen/day) is estimated to be necessary for beneficial effects on egg qualities such as FA content and egg yolk colour. The egg yolk is a rich source of the
n-3 and
n-6 acid family, the amount of which depends mainly on the genotype and age of the laying hens [
25]. These findings show a significant dietary effect, as also revealed previously [
22,
39,
40], and explain why there were progressive changes in the
n-6 to
n-3 ratio in egg yolk with altered linolenic acid and arachidonic acid concentrations. The score plots generated between PC1 and PC2 segregated the free-range vegetation type based on FA composition. Indeed, PC1 indicates that FAs from the TR and MIX groups had a greater effect on the FA profiles of the egg yolks compared with the CI group. Our results indicate that the CI and MIX vegetations contributed to the production of eggs with preferred FA attributes because a low
n-6 to
n-3 ratio in the human diet is favourable [
22,
25]. As such, white clover, and especially a mixture of chicory and white clover, may be successfully employed by farmers to provide more information on the variation in vegetation types than is possible with experimental data alone.
The cholesterol content of eggs is affected by many factors, such as the productivity, strain and age of the hens, housing system, season, etc. [
9,
41,
42]. Indeed, studies have found higher levels of cholesterol in eggs produced by hens kept under free-range conditions [
9,
41]. This was not confirmed in our study. However, the result related to the cholesterol level of the egg yolk is consistent with that of previous reports [
15,
36] in which the rearing system (in cages vs. under free-range conditions) did not affect the cholesterol content of the egg yolk. These inconsistencies may be related to the housing system, strain and age of the hens, vegetation type, and HI level in the outdoor area [
4,
9,
42]. Moreover, all groups of our study had eggs with similar albumen and yolk proportions, which could imply a similar cholesterol level [
10].