Carotenoids quantification in diets.

To quantify the carotenoids present in both types of feed (control and treated) and compare them, a modified procedure from the literature was adopted [21]. A calibration curve was first constructed using a sample of a standard carotenoid (lutein, Merck, 90% purity). A stock solution of lutein in acetone was prepared at a known concentration of 0.916 mg/mL. From this, successive dilutions in acetone were prepared. The absorbance of each solution was measured at 447 nm using a spectrophotometer (LANGE DR-3900) and plotted against the concentration of standard lutein (mg/mL). Both feed samples (control and treated) were finely pulverized using mortars. A precise mass of 1.060 g from each sample was weighed and placed inside two 50 mL plastic tubes. To each tube, 10 mL of an ethanol (1:1) solution was added to solubilize the carotenoids. The solutions were kept under magnetic stirring for 24 hours at room temperature. The next day, an aliquot of the supernatant solution was taken after centrifugation (6000 rpm for 5 minutes). The absorbance of each supernatant solution was measured at 447 nm and using the equation of the calibration line of standard lutein, the total carotenoids present in each feed were calculated as lutein equivalents (mg_LUTEIN/g_FEED). Results are reported in Supporting Information (S4 File).

Antioxidant activity of diets.

To evaluate the antioxidant capacity of each diet, the radical cation 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^•+) was employed, following the procedure used by Loganayaki et al. [22] and Marzorati et al. [7], with slight modifications. Initially, an aqueous solution containing 36.6 mg of ABTS^•+, and 6.5 mg of ammonium persulfate (Merck) was prepared in a 10 mL flask and left at room temperature in the dark for one day. The ABTS^•+ solution was then diluted in ethanol to achieve an absorbance of approximately 0.7 AU at 734 nm (approximately 1:75 v/v).

Subsequently, cuvettes containing 1.2 mL of the diluted ABTS^•+ solution were mixed with increasing concentrations of the previously prepared supernatant solutions extracted from the two feed samples. These cuvettes were kept in the dark for 1 hour, after which the absorbance was measured at 734 nm using a spectrophotometer. A blank cuvette devoid of any sample was used as reference. The percentage of remaining ABTS^•+ was calculated as: % remaining ABTS^•+ =  (A_{734 nm, 1 h, sample}/A_{734 nm, 1 h, blank}) ×  100%, and the results were graphically plotted against the concentration of the feed extract in the solution. Results are reported in Supporting Information (S5 Fig).

Animal welfare.

Animal welfare was assessed at the beginning, middle, and at the end of the trial (19^th, 38^th, and 51^st week of age). The European Welfare Quality^® Protocol [23], which evaluates the condition of feathers, keel bone, and footpads of the hens, was used. The assessment consisted in assigning a score ranging from 0 to 2 to the head, back, tail, cloaca, and comb, based on the severity of lesions:

• score 0: no damage;
• score 1: a damaged area of less than 5 cm or fewer than 3 lesions;
• score 2: a damaged area greater than 5 cm or more than 3 lesions.

For footpads, scores also ranged from 0 to 2:

• score 0: no lesions;
• score 1: presence of necrosis or epithelial proliferation or bumblefoot without dorsal swelling;
• score 2: dorsal swelling.

The keel bone was examined visually and by running fingers alongside and over it to the end of the bone. Scores assigned were:

• score 0: linear;
• score 1: slightly to moderately prominent keel, not sharp, with flat breast muscle;
• score 2: severely prominent keel, with depressed contour to breast muscle.

Additionally, each hen was weighed at each timepoint

Bone breaking strength.

At the end of the cycle, 3 right tibia per cage were sampled at the slaughterhouse to measure their breaking strength, serving as an indicator of adequate mineral supplementation and animal welfare [16]. The bones were previously frozen and then conditioned overnight at 20 °C. A TA.HDplus Texture Analyser (Stable Micro Systems Ltd., UK), equipped with a 500 N load-cell and controlled by the specific software Texture Exponent TEE32 (v. 3.0.4.0, Stable Micro Systems Ltd., UK), was utilized for the analysis.

The traverse speed was set at 20 mm/min, and a wsxc deformation was applied to the center of the bones, using a support span distance of 60 mm. The results are expressed as the mean and standard deviation of breaking load (N). Additionally, considering variations in bone dimensions, the results were standardized based on minimum diameter, maximum diameter, and support span distance [24].

Performance.

Egg production and percentage of discarded eggs were recorded daily.

The egg production was calculated with the following formula:

The percentage of discarded eggs was calculated with the following formula:

Egg quality.

Weight.

During the trial, every month 3 eggs per cage were collected. The total weight of each egg and the weight of its components (eggshell, albumen, and yolk) were measured using a digital balance. The weight of the albumen (g) was calculated by subtracting the combined weight of the yolk and eggshell (g) from the total egg weight (g).

Yolk colour.On the same eggs, the yolk colour was measured using a DSM-Firmenich digital colorimeter (Digital YolkFan™), which objectively assigns a score on the Roche scale. A lens reads the yolk colour positioned on a flat white surface and communicates the result to a mobile phone application.

Antioxidant activity of the yolk.

The determination of antioxidant activity in the yolks was conducted at the 39^th week using 16 eggs, and at the 51^st week using 8 eggs. According to Omri et al. [25], approximately 3 g of yolk mass was weighed for each sample and transferred quantitatively into 50 mL plastic tubes containing 10 mL of acetone. After manual shaking for about 1 minute, the tubes were centrifuged at 6000 rpm for 10 minutes. The supernatant was separated and tested for its antioxidant activity. The same procedure previously applied for determining the antioxidant activity of the feed was performed. EC₅₀ values were determined and compared.

Eggshell thickness. To assess the influence of calcium source on eggshell thickness, measurements were taken at three points on the acute pole using a digital caliber, and the mean of these three measurements was calculated.

Eggshell breaking strength. At the 39^th and 51^st weeks, the breaking strength of 10 eggs per cage was measured using an Instron dynamometer (model 3365, Instron Italia, Turin, Italy) operated with “Bluehill Universal” software. A 100 N load cell was mounted on the instrument, connected to a probe with a plate diameter of 2.5 cm. The traverse speed was set to 20 mm/min, and deformation was applied to the equator of the egg, up to a maximum of 1 mm, positioning the egg along its major axis. Results were expressed as mean ±  standard deviation in terms of breaking load (N).

Eggshell ultrastructure (SEM).

The ultrastructure of the eggshell was analyzed using 8 samples (4 controls +  4 treated) collected at the 42^nd week and 6 samples (3 controls +  3 treated) collected at the 51^st week. These samples were taken from eggs previously subjected to mechanical testing to explore potential mechanical-structural correlations.

Analyses were conducted on the inner side of the eggshell at the acute pole (area of maximum curvature). Eggshell caps, approximately 1 cm² each, were rinsed in distilled water, boiled in 0.5 M NaOH for 10 minutes, thoroughly rinsed again with distilled water, and air-dried. Subsequently, they were mounted on stubs, gold-sputtered, and observed under a Scanning Electron Microscope (FE-SEM SIGMA Zeiss). Five random photos were taken for each eggshell sample.

Purity and magnesium content of eggshell.

Conventional powder X-ray diffraction technique (XRD) was used to characterize the crystalline structure and the composition of eggshells that are made essentially of calcium carbonate (about 95%), in the form of calcite, containing small amount of magnesium (until 3%). The breaking strength results were used to select the most different eggshells for this analysis. The eggshell material was washed by immersion in ethanol for 20 minutes and dried in oven at 40 °C overnight. The whole eggshell was then finely grounded in a mortar and a small portion was deposited on a flat sample holder of glass. Ambient X-ray powder diffraction patterns were collected on a series of samples using a Miniflex-600 diffractometer (Rigaku, Japan) with Cu K_α (λ =  1.540598 Å) radiation over the angular range of 10–60° (2θ), with an incremental step size of 0.02° (2θ) and a counting time of 1 s × step^-1. Preliminary phase identification was performed by comparing the experimental data to that stored in reference databases (COD and PDF-2) [26,27]. The spectra were then refined and analysed to quantify the magnesium content, as described in the Supporting Information (S6 File).

Egg production.

The deposition curve is presented in Fig 1, showing the percentage of deposition each week during the trial for both the treated and control group, compared to the Hy-Line Brown standard. Initially, production increased exponentially during the first period, but both groups were slightly below the hybrid standard. The peak was reached around the 24^th week, after which production stabilized until the 51^st week, when a physiological decrease occurred. The control group maintained higher production compared to the treated group, following the Hy-Line standard. In contrast to these findings, Kismiati et al. [44] reported an increase in egg production with the use of biologically derived calcium from eggshells at 7.5%. Similarly, Alm et al. [33] observed higher production in hens fed with 8% calcium from oyster shells, although not statistically significant. Saunders-Blades et al. [18] and Lee et al. [45] found no differences in egg production among hens fed different calcium sources, such as oyster shells. Additionally, Cufadar et al. [13], Frontng and Bergquist [46], Gongruttananun [47], and Islam and Nishibori [20] supported these findings, indicating no adverse effects on performance using biological sources of calcium.

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Fig 1. Egg production.

https://doi.org/10.1371/journal.pone.0314981.g001

Discarded eggs.

The treated group exhibited a lower percentage of discarded eggs compared to the control (2.10% vs 2.38%), suggesting a potential positive effect of S-Ca on this parameter. However, other factors can influence the percentage of discarded eggs, such as the age of the animals, their health status, and environmental conditions [48].

Weight.

The egg weight is a critical parameter used to assess its quality [49] and determines its commercial classification. Typically, it ranges between 50–70 g and correlates with the weight of the hen; thus, as hens age, eggs tend to increase in both size and weight [50,51]. The results of the statistical analyses of egg and component weights are presented in Table 4. Data were analyzed by GLM considering treatment and time as factor together with their interaction. During the trial (time factor), the whole egg weight, albumen, and yolk weight slightly changed significantly (see Table 4), as expected. For this reason, the mean weights in treated and control group were reported as marginal mean with 95% confidence interval, which took into account time variation. Time and treatment interaction for the whole egg, albumen and yolk was not significant (F₇;₃₆₈ =  0.5; p =  0.835. F₇;₃₆₈ =  0.4; p =  0.902. F₇;₃₆₈ =  0.5; p =  0.856), confirming that the significant effect of the treatment was not affected by time. However, eggshell weights did not significantly differ between the two groups, even if there was a time effect (F₇;₃₆₈ =  8.4; p <  0.001) and time and treatment interaction (F₇;₃₆₈ =  3.2; p =  0.002).

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Table 4. Marginal mean with 95% confidence interval of eggs and components weight (g).

https://doi.org/10.1371/journal.pone.0314981.t004

As indicated in Table 4, the whole egg, albumen and yolk of the treated group were significantly lighter than those of the control group, which contrasts with the findings of Kismiati et al. [44], who reported significantly higher egg weights by adding calcium from eggshells at 7.5%. However, various factors influence egg weight, including breed, age, temperature, and high laying intensity, which negatively correlates with egg size [49–52]. Despite being statistically significant, the observed differences are slight and, most of all, both groups’ eggs belong to the same commercial category (size M).

Yolk colour.

Yolk colour is typically measured on the Roche scale, ranging from 1 to 15 [53]. In this study, the higher score frequencies were 9 in both treated and control group (respectively, 45% and 50%). The second colour score more frequent was 10 (respectively, 27% and 22%). Frequencies distribution in observed colour (from 7 to 12) were not significantly different in the two groups (Likelihood Ratio =  2.74; df =  5; p =  0.741). Yolk colouration is an important parameter because it influences consumers’ perception of egg quality worldwide. Consumers generally prefer yolks with intense colours [54], which depend on the accumulation of carotenoids obtained through diet or synthesized by the animals [53]. Since Marzorati et al. [7] identified carotenoids (echinenone, astaxanthin, and β-carotene) in sea urchin waste, this analysis aimed to determine whether the experimental feed affected final yolk colour compared to the control feed.

One possible explanation is that the antioxidants present in the sea urchin waste were degraded during the heat treatment required for drying (see Materials and methods paragraph), resulting in lower quantities than previously identified by Marzorati et al. [7]. Alternatively, these antioxidants may not be well absorbed in the intestine. The values obtained in both groups are lower than the consumer preference standard (12–14), indicating that no additional pigments were added to the diets.

Antioxidant activity of the yolks.

EC₅₀ value was calculated for yolks of both treated and control samples, at two different timepoints, half and end of the experimental trials. The results expressed as value±SD are reported in Table 5.

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Table 5. Results from ABTS^�+ antioxidant activity.

https://doi.org/10.1371/journal.pone.0314981.t005

The antioxidant activity of egg yolks from both the treated and control group appears to be similar, with no statistical difference observed neither at the halfway (p =  0.805), nor at the end of the trial (p =  0.08), indicating that including sea urchin waste into the diet does not affect the production of carotenoids in egg yolks, which are responsible for their antioxidant activity.

Interestingly, there is a notable difference in antioxidant activity between the yolks sampled midway through the experiment (exhibiting a higher EC₅₀, indicative of lower antioxidant activity) and those sampled at the end (showing higher antioxidant activity). The ambient temperature, light conditions, and humidity were maintained constant throughout the trial, excluding their influence. Previous studies have noted a decline in egg quality with increasing age of the hens, attributed to reduced antioxidant capacity in the liver and decreased formation of yolk precursors [55–58]. Stress could also be a contributing factor, as stressed hens may exhibit lower performance and reduced feed intake [45]. However, the welfare evaluation conducted in this study indicated that the hens were in a well state of welfare, with few lesions reported.

To gain further insights, additional studies incorporating blood tests and behavioural assessments are warranted.

Eggshell thickness.

As previously mentioned, eggshell thickness is a parameter used to evaluate egg quality [59]. In the present study, eggshell thickness was analyzed with GLM considering treatment and time as factor, together with their interaction. This parameter significantly varied during the trial (F_7;368 =  13.5; p <  0.001), however the eggs from treated group remained thicker than eggs from the control group (F_1;368 =  41.8; p <  0.001). Furthermore, there was an interaction between time and treatment (F_1;368 =  4.5; p <  0.001). The marginal means with 95% confidence interval for the treated and control group were respectively 0.38 mm (0.376–0.385) and 0.36 mm (0.354–0.364). The thickness of the treated group was slightly higher than values reported in the literature (0.28–0.36 mm) [52,60]. When eggshell thickness exceeds 0.34 mm, the opacity of the cuticle is more than 27.5%, enhancing eggshell antibacterial efficiency up to 98% [61]. Additionally, Ledvinka et al. [52] reported that thinner eggshells increase permeability, contributing to economic losses and risk of bacterial infection. Thus, an increase in eggshell thickness correlates with improved antibacterial protection [52,61]. The results obtained contrast with findings from Swiatkiewicz et al. [62] and Lee et al. [63], who did not observe differences in eggshell thickness when changing calcium sources. However, magnesium present in sea urchin waste (even in small quantities) could have positively influenced eggshell thickness, as observed in the study by Kim et al. [12].

Eggshell breaking strength.

In the study, to investigate whether the biogenic source of calcium affects eggshell breaking strength, quasi-static compression was applied to the equatorial zone, which represents the weakest and most uniform part of the egg, providing a more accurate estimate of the eggs’ resistance [64–66]. The results of t-test for eggshell breaking strength are reported in Table 6.

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Table 6. Breaking strength.

https://doi.org/10.1371/journal.pone.0314981.t006

The results of the breaking strength test show no significant difference between the two groups, consistent with findings from Swiatkiewicz et al. [17] and Lee et al. [63], who reported that calcium source does not influence breaking strength. However, Guinotte and Nys [42] found an improvement in egg breaking strength in hens supplemented with ground seashells and oyster shell.

The outcomes obtained at the 39^th week are comparable to those reported by Leyendecker et al. [41] and Koreleski and Swiatkiewicz [67]. A decrease in breaking strength was observed with increasing age of the animals, in line with several other studies [67–69].

The structure of the eggshell also influences breaking strength [60]. Rodriguez-Navarro et al. [70] demonstrated a correlation between eggshell strength and crystallographic texture, where about 40% of the variance in shell strength could be attributed to differences in crystal orientation. Ketta and Tůmová [60] reported a positive correlation between eggshell thickness and breaking strength. In contrast, similar to our findings, Ahmadi and Rahimi [71] showed that increased eggshell thickness does not necessarily lead to higher breaking strength, as it is influenced by eggshell construction quality, including ultrastructure and the arrangement of calcite crystals. Therefore, in this study, the ultrastructure was also evaluated (see below). The absence of differences in bone and eggshell breaking strength, alongside a significant increase in eggshell thickness, seem to confirm that calcium derived from sea urchin waste does not negatively affect performance or animal welfare.

Ultrastructure.

The ultrastructural organization of the mammillary layer of the eggshell was analyzed using a Scanning Electron Microscope (SEM) to investigate whether supplementing with sea urchin waste had any structural impact on the eggshell and its potential correlation with mechanical performance. As previously mentioned, sea urchin skeletons are primarily composed of calcium carbonate, with a small amount of magnesium carbonate [7]. The presence of magnesium carbonate could positively influence eggshell quality in terms of weight, strength, and thickness, as demonstrated by Belkameh et al. [72] through increased magnesium supplementation in laying hens’ diets. Additionally, differences in the origins of calcium carbonate could affect its intestinal assimilation and thus its bioavailability. Saunders-Blades et al. [18] reported better eggshell quality with oyster shell supplementation compared to limestone, while Solomon [73] described various structures within the mammillary layer, including Type A and B mammillary bodies. These are cup- or cone-shaped protrusions with an irregular (“fibrous”) cap located on the inner side of the eggshell, connecting it to the egg membranes. Fusion of the fibrous caps of the mammillary bodies can influence the formation of the palisade layer and alter pore distribution. Park and Sohn [74] noted that low-density mammillary bodies weaken the eggshell’s resistance.

SEM observations of eggshells collected at the 42^nd week revealed no significant differences between the control and treated group. However, in some samples of the treated group, there were greater dimensional variations in the mammillary bodies and more frequent zones of fusion between their fibrous caps (Fig 2). Nevertheless, these minor differences did not affect the mechanical resistance of the eggshell, as no differences were found in breaking strength. No differences were observed between the two groups in the eggshells collected at the 51^st week (data not shown). Therefore, it is possible to conclude that the supplementation with sea urchin waste did not affect the ultrastructure (mammillary layer) of the eggshells, thus further confirming the outcomes from the mechanical tests.

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Fig 2. SEM image of the eggshell.

https://doi.org/10.1371/journal.pone.0314981.g002

The figure shows the inner side of the eggshell (42^nd week) with its characteristic cup-shaped mammillary bodies (arrows). No remarkable difference or anomalies can be observed between control (A) and treated sample (B), except for a larger amount of areas of fusion of the fibrous cap (arrowhead).

Purity and magnesium content.

Considering that sea urchin skeleton contains also magnesium, the X-ray diffraction (XRD) analysis was performed on selected samples to determine the purity and crystalline composition of eggshells. All spectra exhibit a single-phase pattern corresponding to the rhombohedral calcite crystal structure with the diffraction peaks slightly shifted to higher angles indicating shrinkage of the crystal lattice. The reduction of the lattice parameters can be explained by the random incorporation of Mg^2 + cations. The molar Mg-content was calculated as described in the Supporting information (S6 File). The absolute values are not significant, but the trends indicated by the data can be felt to be meaningful to evaluate the behaviour of the system.

It was found that the magnesium content varies between 2.5 and 3.0 mol% (2.72 ± 0.15 mean±MAD) for the control samples and between 1.8 and 3.1 mol% (2.46 ± 0.47 mean±MAD) for the treated samples.

The values are spread out over a relative narrow range in both series of samples. The mean values of the two sets are quite similar although the values of the mean absolute deviation (MAD) indicate a greater variability and spread of the data relative to the treated samples.

These results suggest that there is no important effect of diet on Mg content in eggshells. However, the one-factor testing results showed that the difference in behavioural response might not be statistically significant (p =  0.297).