## 3 Results and Discussion
### Assessment of Shelf Life of Eggs from Hens Fed with Supplemented Diet  
Control eggs and those from hens fed with a supplemented diet for four weeks (W4) were kept in paper boxes along 28 days, at room temperature, aiming at mimicking the storage conditions in the supermarket. Every 7 days, samples were collected and analyzed as previously described (cf 2.2). Egg protein quality (HU) was the only parameter that significantly decreased along time for both control and W4 eggs, although in a more pronounced way in control eggs (Figure 4). Nevertheless, no statistically significant differences (\(p>0.05\)) were observed for control and supplemented eggs with the same storage time. Several authors state that albumen height, a parameter used for HU calculation (cf 2.4), decreases with storage time as pH of egg white increases [46; 48]. Figure 4: Comparison of protein quality (Haugh units, HU) of control eggs and W4 eggs (from hens fed with 3% of _Nannochloropsis oceanica_ supplemented diet during the 4 weeks) along the storage time. * means statistically significant differences (\(p<0.05\)) between samples with different storage time (N = 12). Figure 3: Yolk colour of eggs from hens fed with 3% of _Nannochloropsis oceanica_ supplemented diet during the 4 weeks of the trial. * means statistically significant differences (\(p<0.05\)) (N = 12). The remaining physical parameters were constant during storage. Also, the amount of the fatty acids ALA, EPA and DHA did not significantly change during the storage period (\(p>0.05\)), demonstrating that eggs from hens fed with supplemented diet still preserve high levels of n-3 PUFA after 28 days (Table 3). The same pattern was observed for protein and lipids content conversely to the amount of ash that slightly increased throughout the storage. Several studies concerning the evaluation of eggs shelf life highlight distinct observations in nutritional and quality parameters. [PERSON] et al. [46] reported an increase in shell thickness, yolk colour intensity as well as in lipid oxidation, assessed by thiobarbituric acid reactive substance (TBARS) assay, in control and supplemented eggs with marigold extract. A decrease of carotenoids content in yolk eggs throughout storage time, both at room temperature and under refrigeration, was observed by [PERSON] et al. [49]. Another study showed a decrease of egg weight, HU value, and yolk colour in eggs stored for 3 weeks in refrigerator [50]. According to these authors, eggshell firmness does not change with storage period but depends on factors such as hen's age, genotype, and diet. In this study, a PCA was carried out aiming at providing an integrative approach of the influence of storage time on the nutritional composition and physical parameters of the control and of eggs from hens fed with the supplemented diet for 4 weeks (W4) (Figure 5). The first principal component PC1, representing 37.9% of sample variability, is mainly defined by the values of EPA, DHA, yolk colour and protein. Also representative of the variance explained by PC1 appears the total weight and the shell thickness, although with less weight to explain the observed pattern (shorter vectors). The second principal component PC2 explains 23.2% of the total variability and is more related to the egg protein quality (HU), lipids, ALA, ash contents and shell colour. The results obtained by the PCA allowed three groups to be distinguished (identified as I, II and III). Group I comprised W4 eggs and was mainly characterized by the higher levels of EPA, DHA and yolk colour. Comparing with control samples (groups II and III), W4 eggs were more homogeneous, suggesting higher stability of these samples during shelf life. Control samples distinguished from W4, by higher values of protein and of egg weight. Group II corresponded to 0 and 7 days of storage, whereas group III (shifted towards lower values of HU) contained the eggs with higher self-life (from 14 to 28 days).