Microalgae (Chlorella vulgaris and Spirulina platensis) as a Protein Alternative and Their Effects on Productive Performances, Blood Parameters, Protein Digestibility, and Nutritional Value of Laying Hens’ Egg

Abstract

Protein is an essential nutrient for laying hens, playing a crucial role in egg production and supporting their overall health. An 8-week feeding trial was conducted on 120 Lohmann Brown laying hens (aged 38 weeks). The layers were assigned randomly to three groups and housed in cages (twenty replicates × two birds/cage). All groups were fed a corn–soybean meal basal diet (2750 kcal/kg metabolizable energy (ME) with 17.8% crude protein (CP)). In contrast to conventional diet (CON), the experimental groups were supplemented primarily at the expense of soybean meal with 2.0% Chlorella vulgaris (CV2%) and 2.0% Spirulina platensis (SP2%). Their high concentrations of chlorophyll a (5.56; 9.06 mg/g), chlorophyll b (0.88; 1.34 mg/g), and antioxidant activity expressed as 2,2-diphenyl-1-picrylhydrazyl (73.29; 81.27 DPPH% inhibition) improved egg yolk quality. At the end of the trial, eighteen eggs/group (six yolk samples/group, three eggs/sample) were collected to determine the egg quality and nutritional parameters (fatty acids profile, cholesterol, β-carotene, yolk color, and antioxidant capacity). To determine the activity of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH), and total antioxidant capacity (TAC), blood samples were collected at the end of the period. Microalgae inclusion increased (p < 0.05) the fatty acid content, ß-carotene concentration (p < 0.001), antioxidant capacity (p < 0.0001), and yolk color intensity (p < 0.001) significantly, especially the yolk redness a* color parameter, but without any significant results concerning cholesterol concentration. Boiling the eggs for 10 min significantly (p < 0.001) increased the b* color parameter on microalgae treatments. The supplementation of laying hens’ diet with microalgae positively influenced egg quality and nutritional properties.

1. Introduction

The protein source in a hen’s diet is of utmost importance as it directly influences the bird’s growth and health, egg quality, and overall production performances [1].

Soybean meal is considered a high-quality protein source, widely used in poultry diet formulation [2], which contains a balanced amino acid profile, crucial for egg production and overall growth. These amino acids contribute to the synthesis of proteins, enzymes, and hormones, supporting various physiological functions in hens [3]. Soybean protein can be compared to proteins found in meat, milk, and eggs. Among plant-based protein sources, soybean protein is widely regarded as having the highest biological value [4]. Alshelmani et al. [5] consider that the increasing competitiveness of feedstuffs for poultry nutrition presents a challenge to food security; therefore, ongoing efforts are made to explore alternative protein sources that can partially replace soybean meal in poultry diets.

Microalgae are being increasingly explored as a valuable and sustainable alternative in animal and poultry nutrition due to high protein content [6], and are primary sources of omega-3 fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [7]. Moreover, they are considered environmentally friendly due to their minimal impact on land and water resources [8]. Furthermore, microalgae contain bioactive compounds that exhibit antioxidant [9], antimicrobial, and immunomodulatory properties [10], thereby contributing to disease prevention and supporting the immune system.

Chlorella (Chlorella vulgaris) is a naturally single-celled green microalga considered as an alternative for partially replacing soybean meal included in poultry diets [11]. Previous studies found that Chlorella has a positive impact on egg production and quality, enhancing the intensity of the yolk color due to carotenoid transfer (canthaxanthin and β-carotene) [12]. Furthermore, it promotes the growth of lactic-acid-producing bacteria in the intestines and lowers the total cholesterol and triglyceride level concentration in serum and liver [13].

Spirulina (Sp. platensis), a blue-green spiral filamentous alga [14], is a natural product with high nutritional value and increased antioxidant potential. Its utilization improves production efficiency, egg production [15], and yolk redness, while also exhibiting favorable amino acid profiles and high digestibility [16]. Spirulina is recognized as a sustainable protein source with a low-impact environmental footprint that can vary significantly depending on factors such as the production system and regional climate [17].

Despite previous research on this topic, the results of previous experiments involving the inclusion of microalgae in poultry diets have generated inconsistent findings with regard to both poultry productivity and egg quality. As a result, our study aims to investigate the partial substitution of soybean meal in the diet of laying hens, and to examine the potential impacts and effects of chlorella and spirulina, both at equivalent inclusion levels, on these specific parameters.

2. Materials and Methods

2.1. Ethical Statement

The study was carried out at the Laboratory of Animal Physiology, National Research-Development Institute for Animal Biology and Nutrition (IBNA), Balotesti, Romania. The feeding, handling, and slaughtering procedures of the study were performed in accordance with Directive 2010/63/EU on the protection of animals used for scientific purposes, and the experimental procedures, according to an experimental protocol (No. 6252/27.10.2021), were approved by the Research Ethics Committee for Animal Production studies of IBNA.

2.2. Microalgae Purchase and Chemical Analyses

Microalgae chlorella and spirulina powder were purchased from the agri-food market. Triplicate analyses were conducted on samples of chlorella and spirulina powder to determine the following: dry matter (DM), ash, organic matter (OM), crude protein (CP), ether extract (EE), crude fiber (CF), and non-fermentable extractive substance (NFE); in vitro nutrient digestibility of protein, dry matter, and organic matter (DCP, DDM, DOM); and antioxidant activity and fatty acid profile.

Metabolizable energy (ME) of the microalgae was calculated using formula (1), according to [18] and cited by [19]:

ME (kcal/kg) = (35.3 × CP %) + (79.5 × EE %) + (40.6 × NFE %) + 199.0

(1)

2.3. Animals, Housing, and Experimental Diets

An eight-week feeding trial was conducted on 120 Lohmann Brown layers (38 weeks), individually weighed and assigned in 3 treatments (CON, CV2%, and SP2%). The layers were randomly placed in twenty replicates with 2 birds per treatment, housed in metabolic cages (50 cm width × 40 cm height × 50 cm length) under controlled environmental conditions monitored by a ViperTouch computer (16 h light/24 h; T = 23.08 ± 0.98 °C; H = 66.35 ± 5.68%). Each replicate was considered an experimental unit and performance parameters were evaluated per pen. The feed was administrated daily at 08:30 a.m. and water was available at all times. Throughout the experimental period, no vaccination treatment was applied to the birds.

The isocaloric and isonitrogenous three experimental treatments (in mash form) were formulated by a nutritional optimization program (HYBRIMIN^® Futter5) to meet the nutrient requirements for laying hens as given by [20]. All groups were fed a corn–soybean meal basal diet (17% crude protein and 2750 kcal ME/ kg feed) as follows: CON—a commercial diet without microalgae (chlorella or spirulina); CV2%—a control diet containing 2.0% chlorella powder; and SP2%—a control diet containing 2.0% spirulina powder, as shown in

Table 1. Ingredients and chemical composition of the diets (% as fed).

Ingredients, %	Experimental Diets
	Control (CON)	Chlorella Powder (CV2%)	Spirulina Powder (SP2%)
Corn	40.00	40.00	40.00
Wheat bran	22.49	23.72	23.60
Chlorella vulgaris	-	2.00	-
Spirulina platensis	-	-	2.00
Soybean meal	24.36	21.58	21.63
Vegetable oil	1.48	0.97	1.04
L-lysine HCl	-	0.01	0.01
DL–methionine	0.16	0.17	0.17
Calcium carbonate	8.83	8.84	8.84
Monocalcium phosphate	1.32	1.33	1.33
Salt	0.33	0.33	0.33
Choline premix	0.04	0.04	0.04
Vitamin–mineral premix *	1.00	1.00	1.00
Total	100.00	100.00	100.00
Calculated analysis (%) **
Metabolizable energy (Kcal/kg)	2.750.00	2.750.00	2.750.00
Crude protein	17.00	17.09	17.00
Lysine	0.87	0.80	0.80
Methionine+Cystine	0.73	0.71	0.71
Threonine	0.64	0.59	0.59
Calcium	3.90	3.90	3.90
Phosphorus	0.63	0.62	0.62

Where: CON, conventional diet; CV2%, conventional diet supplemented with 2% chlorella powder; SP2%, conventional diet supplemented with 2% spirulina powder; * 1 kg diet contains: = 11,000 IU/kg vit. A; 2000 IU/kg vit. D3; 27 IU/kg vit. E; 3 mg/kg vit. K; 2 mg/kg Vit. B1; 4 mg/kg vit. B2; 14.85 mg/kg pantothenic acid; 27 mg/kg nicotinic acid; 3 mg/kg vit. B6; 0.04 mg/kg Vit. B7; 1 mg/kg vit. B9; 0.018 mg/kg vit. B12; 20 mg/kg vit. C; 80 mg/kg manganese; 80 mg/kg iron; 5 mg/kg copper; 60 mg/kg zinc; 0.37 mg/kg cobalt; 1.52 mg/kg iodine; 0.18 mg/kg selenium. ** Calculated according to NRC [20].

. A quantity of 500 g feed samples from each group were taken and analyzed by chemical composition as described previously for the microalgae samples. Following the manufacturing of the diets, the feed was packaged, appropriately labeled, and stored under optimal conditions, specifically in a cool environment, in preparation for the experimental procedures.

2.4. Laying Hens Performance

During the 8-week feeding trial, the performance parameters of the laying hens (daily feed intake (DFI; g/day/layer), feed conversion ratio (FCR; g feed/g egg), hen day egg production (HDEP; %), egg weight (EW; g), and egg size classification (%)) were monitored. At the initial and the final period, body weight (g/hen) was measured, and eggs were collected and weighed every day. Hen day egg production was calculated using the following formula [(100 × number of eggs laid)/(number of hens × days)] and classified according to the European Council Directive (2006). Data on feed intake and egg mass were used to calculate the feed conversion ratio (feed intake/egg mass; g/g). All performance parameters were determined for each replicate of treatment groups.

2.5. Nutrient Digestibility Trial

During the last week of the feeding trial (the 8th wk), 6 cages per group (2 birds per cage) were randomly selected from the digestibility trial to measure the apparent nutrient digestibility. For 5 days, both feed leftovers and excreta were collected and weighed daily to determine nutrient intake. During the balance period, fecal samples were stored in a refrigerator at a constant temperature of 4 °C. Finally, each sample was homogenized, and approximately 200 g samples were extracted and dried for 48 h at a constant temperature of 65 °C in an oven (ECOCELL Blueline Comfort, Nuremberg, Germany). After drying, the samples were ground (using a Grindomix GM 200 knife mill, Retsch, Germany) and analyzed for chemical composition. The values obtained from the laboratory chemical analysis were used to calculate the apparent digestibility of nutrients (DDM, DOM, DCP, DEE, and DNFE) as described earlier by [21] using the following formula:

$$\mathrm{Apparent} \mathrm{nutrient} \mathrm{digestibility} (\%)=\frac{\left(\mathrm{nutrient} \mathrm{intake} - \mathrm{nutrient} \mathrm{excreta}\right)}{\mathrm{nutrient} \mathrm{intake}}\times 100$$

(2)

2.6. Blood Collection and Analysis

On the final day of the experiment, approximately 3 mL of venous blood samples per birds were aseptically collected from 18 laying hens from the sub-axial region into 9 mL anticoagulant-free Vacutainers containing 14.3 U/mL of lithium heparin (Vacutest^®, Arzergrande, Italy). Further, these samples were used to determine the activity of blood antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH), and total antioxidant capacity (TAC). Blood samples were separated by centrifugation at 3000× g in a refrigerated centrifuge (Eppendorf Centrifuge 5430R; Eppendorf, Hamburg, Germany) for 25 min at 4 °C. Afterwards, the supernatant obtained from serum samples were carefully transferred to plastic vials and stored at −20 °C until the analysis.

2.7. Egg Quality Measurement

A total of 306 eggs were collected during the experiment. The collected eggs (18 eggs/group: 3 eggs/cage, 6 cages/lot; each cage representing a sample) were analyzed at the end of the experiment (2 months) to evaluate the impact of microalgae-based diets, specifically those containing chlorella and spirulina, on the fatty acid composition. The antioxidant profile of the yolk, as well as the internal and external quality parameters of the eggs, were determined at the end of the experiment (8 weeks) using a Digital Egg Tester DET-6500 (NABEL Co., Ltd., Kyoto, Japan). First, the eggs were weighed whole and then cracked, and the yolks were separated from the albumen and shell; every yolk was rolled onto a paper towel to remove any adherent albumen or chalazae membrane as described by [22]. Each egg component was weighed with a Kern scale (precision 0.001). The yolk color intensity was measured using the portable colorimeter 3 nh YS3020 (Shenzhen ThreeNH Technology Co., Ltd., Beijing, China), and the temperature and pH of yolk and albumen were measured using a portable pH meter (Five Go F2-Food, Greifensee, Switzerland) and Haugh unit. After measuring the internal and external quality parameters of the eggs, the yolk samples were dried for 48 h at a constant temperature of 65 °C in an oven (ECOCELL Blueline Comfort, Nuremberg, Germany) for further chemical analysis, such as the concentration of β-carotene (µg/g), total polyphenols (mg/g GAE), antioxidant activity (expressed as DPPH % inhibition and µM Trolox), fatty acid profile (g acid/100 g total FAME), and cholesterol concentration (g/egg).

To assess the yolk color stability after boiling, at the end of the experiment, 90 eggs were collected (10 eggs/group, 30 eggs/period) and boiled for 10, 15, and 20 min, respectively.

2.8. Chemical Analysis of Samples

2.8.1. Determination of In Vitro Digestibility of Nutrients

The in vitro digestibility of nutrients was determined following the method proposed by [23] and adapted for poultry as described by [21] using a Daisy Incubator (ANKOM Technology, Macedon, NY, USA) in a 2-step procedure: two successive incubations with pepsin and pancreatin. The samples were introduced into F57 bags (Ankom) and incubated in Daisy Incubator jars with 0.1 M phosphate pH 2.0 buffer with 0.3 g of pepsin (porcine, 2000 FIP U/g) per liter for 6 h at 39 °C. After draining the buffer and washing bags with slightly warm tap water, the next 0.04 M phosphate buffer pH 6.8 with 1 g of pancreatin (porcine, grade IV, reference Sigma P-1750) per liter was added to the jars. Incubation lasted for 18 h at 39 °C and finally the bags were dried in a forced draught oven at 65 °C for 48 h. The final weight after digestion of each bag was recorded for in vitro digestibility of dry matter calculation. Some of the bags was retained for nitrogen analysis and consequently for calculation of the in vitro digestibility of nitrogen. The remaining bags were subsequently subjected to incineration in a muffle furnace at a temperature of 550 °C for a duration of 5 hours. The resulting ash was utilized for the purpose of residue digestion and in vitro calculation of organic matter digestibility. The results were expressed as mean ± standard deviation of five replicate analyses.

2.8.2. Pigment Extraction from Spirulina platensis and Chlorella

To extract pigments from feed and dried Spirulina platensis and Chlorella, we used a combined method of sonication–solvent extraction followed by stirring on a magnetic stirrer [24]. Acetone solvent ratio was 1:100, w:v; sonication was performed for 30 min, and magnetic stirring was applied for 60 min. The extract was obtained by centrifugation (SIGMA 2-16KL refrigerated centrifuge) at 2599× g for 10 min. The resulting precipitate was extracted until no color was observed. Pigment extracts were then analyzed using a spectrum UV-Vis with wavelengths between 400 and 700 nm and absorbance at 470, 645, and 663 nm (JASCO V-670 spectrophotometer), in triplicate. The pigment levels, including chlorophyll a (Ca), chlorophyll b (Cb), and total carotenoids (Cc), were estimated with Equations (3)–(5). The results were reported, taking into account the dilution factor (DF) as mg/g DW (dry weight) for Spirulina platensis and Chlorella vulgaris powder and μg/g feed.

Ca = 11.24 × A₆₆₃ − 2.04 × A₆₄₅ × DF

(3)

Cb = 20.13 × A₆₄₅ − 4.19 × A₆₆₃ × DF

(4)

$$\mathrm{Cc}=\frac{\left(1000\times A_{470}-1.90\times \mathrm{C}a-63.14\times \mathrm{C}b\right)}{214}\times \mathrm{DF}$$

(5)

2.8.3. Measurement of Some Antioxidant Enzyme Activity and GSH in Blood Serum

The activity of superoxide dismutase (SOD) was determined following the method described by [25]. Blood serum was added to the assay mixture containing 66 mM phosphate buffer with a pH of 7.8, 0.1 mM EDTA, 5.7 M nitro blue tetrazolium (NBT), 9.9 mM L-methionine, and 2.5% (w/v) Triton X100 and riboflavin (0.01 mL of 4.4%, w/v) was finally added to initiate the reaction. NBT reduction was measured at 560 nm in a Jasco V-670. The activity of SOD was calculated in units of enzyme/mL.

The activity of catalase (CAT) was determined by the classical method developed by [26]. CAT decomposes H₂O₂ (the substrate) and can be directly measured by decreased absorbance at 240 nm. Freshly prepared reagents prior to assays were phosphate buffer (66 mM, pH 7.0) and 30 mM H₂O₂ in a phosphate buffer. The final volume was 1 mL and the reaction was started by the addition of H₂O₂. To correct for any non-enzymatic reaction, a blank assay containing buffer instead of substrate was used. CAT activity is defined in specific units/mL.

The level of reduced glutathione (GSH) was measured according to the method described by [27] and was determined based on the reaction of GSH with 5,5′-dithiobis (2-nitrobenzoic acid). The resulting chromophore, TNB (5-thio-2-nitrobenzoic acid), has a maximum absorbance of 412 nm. The TNB formation rate is proportional to the sample GSH level. Blood serum was treated with 0.6% sulfosalicylic acid and centrifugated. The supernatant was added to the assay mixture containing 100 mM phosphate buffer with a pH of 7.5. A 3 mM stock solution of the DTNB reagent was prepared in phosphate buffer with a pH 7.5, and diluted to a final concentration of 10 μM. The reaction between GSH and DTNB was monitored at a wavelength of 412 nm using a Jasco UV/Vis V-670 spectrophotometer. The concentration of GSH in blood serum was calculated with the linear equation generated from a GSH standard curve.

Total antioxidant capacity (TAC) was analyzed by scavenging of DPPH (2,2-diphenyl-1-picrylhydrazyl) radical activity [28]. Blood serum proteins were removed with one volume of acetonitrile, incubated for 5 min and centrifugated for 10 min at 9000× g. Supernatant (25 μL) was added to the assay mixture containing 970 μL of methanol and 5 μL of 10 mM of DPPH radical methanolic solution. After 30 min, the absorbance was read at 517 nm by a Jasco UV/Vis V-670 spectrophotometer. In parallel, a negative control with 25 μL acetonitrile, instead of deproteinated blood serum was prepared. All determinations were performed in triplicate and the serum scavenging effect (Sc%) was calculated according to Equation (6).

$$\mathrm{Scavenging} \%=\frac{1-A_{517 sample}}{A{ }_{517 negativ control}}\times 100$$

(6)

2.8.4. Egg Yolk β-Carotene and Antioxidant Activity Determination

The β-carotene concentration of egg yolk was determined using spectroscopy method [29]. A quantity of 0.5 g of well-mixed egg yolk from each fresh or lyophilized form was taken in a 50 mL conical flask. First, 25 mL of acetone was added and the vortex was used to make a smooth paste. The solution was mixed well for 10 min and filtered (Whatman No. 1, Merck KGaA, Darmstadt, Germany). The remaining solid was re-extracted with another 20 mL of acetone using the vortex. The two filtrates were combined and the acetone extract was diluted to 50 mL. The egg yolk pigments expressed as μg β-carotene/g were measured at 450 nm wavelength (E1% 2500) using a JASCO V-670 spectrophotometer.

The total phenolic content of egg yolk samples was determined by the Folin–Ciocalteu colorimetric method [30]. The absorbance was recorded at 732 nm using a spectrophotometer (Jasco V-530, Japan Servo Co., Ltd., Tokyo, Japan). Gallic acid was used as standard solution. The total phenolic content is expressed as mg gallic acid equivalents (GAE)/ g of the sample on the basis of a standard curve of gallic acid.

The antioxidant capacity of egg yolk samples was measured using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity method described by [31]. The absorbance of the solution was measured at 517 nm with the help of a spectrophotometer (Jasco V-530, Japan Servo Co., Ltd., Japan). Trolox solution was used as standard. The results were expressed as mM Trolox equivalents (TE).

2.8.5. Egg Yolk Cholesterol Content and Fatty Acids Profile

The cholesterol content of dried yolk was determined using the gas chromatography (GC) method (AOAC, 1996) as described by [32]. The sample was saponified in a methanol–potassium hydroxide solution, extracted with petrol ether, concentrated using a rotavapor, and subjected to chloroform addition before being analyzed using a GC (Perkin Elmer Clarus-500, with a flame ionization detector). Separation was achieved using an HP-5 capillary column (30 m length, 0.32 mm internal diameter, 0.1 um film thickness), and the results were expressed as grams of cholesterol per whole egg. Fatty acid profile of dried yolk was determined as described by [21], using GC (Perkin ElmerClarus 500, Mass Spectrometer System) of fatty acid methyl esters (FAME) equipped with a flame ionization detector (FID) and a BPX70 capillary column (60 m × 0.25 mm ID, 0.25 μm film thickness). The column temperature was set at 5 °C/min⁻¹ ramped from 180 °C to 220 °C. The carrier gas was hydrogen (linear velocity 35 cm/s at 180 °C), and the split ratio was 1:100. The injector and detector temperatures were 250 °C and 260 °C, respectively. The results were expressed as g fatty acid per 100 g total fatty acids. The average amount of each fatty acid was used to calculate the sum of the total saturated (SFAs), total monounsaturated (MUFAs), and total polyunsaturated (PUFAs) fatty acids.

2.8.6. Color Measurement of Fresh and Boiled Eggs

Yolk color intensity was measured using a portable colorimeter as previously described by [32]. The yolk was separated from the albumen and subsequently positioned on a Petri dish (∅ = 50 mm) prior to measurement. The color parameters of L* (lightness), a* (red-green intensity), and b* (yellow-blue intensity) of the CIE-Lab system (Commission Internationale de l’Eclaraige) were determined by reflectance CIE—L* a* b* color coordinates. The instrument was calibrated with a white calibration before the measurements. All measurements were performed in triplicate.

2.9. Statistical Analysis

The results obtained from feed nutritional composition, apparent nutrient digestibility, laying hens’ performances, antioxidant enzyme activity, egg quality parameters, fatty acids, and yolk cholesterol content were analyzed using a randomized complete block design and the general linear model (GLM) procedures of SAS (Statistical Analysis System, Minitab version 17, SAS Institute Inc., Cary, NC, USA) considering a cage as an experimental unit, according to the following linear model:

Y_ij = µ + A_j + e_ij,

(7)

where Y_ij means value of trait (the dependent variable); µ, overall mean; A_j, the treatment effect; and e_ij, random observation error.

The effects of boiling time on fresh vs. boiled yolk color were analyzed to determine whether the factors studied (treatment and boiling time) affected the fatty acid concentration and yolk color of eggs for different time periods. The data obtained were analyzed by two-way ANOVA using the Tukey test, following the statistical model:

Y_ijk = µ + α_i + β_j + α_iβ_j + e_ijk

(8)

where Y_ijk = variable measured for the k^th observation of the i^th treatment and j^th feeding or boiling time; μ is the sample mean; α_i is the effect of the i^th treatment; βj is the effect of the j^th feeding or boiling time; αiβj is the interaction of the i^th treatment and j^th feeding or boiling time, and ε_ijk is the effect of error. The differences were highly significant when p < 0.001, significant if p < 0.05, and a tendency of influence was considered when p < 0.10.

The graphs for antioxidant enzyme activities were created using GraphPad Prism 9.1.2 software (GraphPad Software, La Jolla, CA, USA). Differences were considered significant when p < 0.05.

3. Results

3.1. Nutritional Value of Chlorella and Spirulina Powder

The nutritional values of the microalgae are presented within

Table 2. Proximate composition, antioxidant activity, and fatty acid profile of chlorella and spirulina.

Parameters	Chlorella Powder	Spirulina Powder
	$\overline{\mathit{X}} \pm {\mathit{s}}_{ \overline{\mathit{X}}}$
Proximate composition *
Calculated metabolizable energy (ME), MJ/kg	9.77 ± 0.41	9.68 ± 0.25
Crude protein (CP), %	51.06 ± 0.35	67.02 ± 0.04
Dry matter (DM), %	94.68 ± 0.80	92.70 ± 0.62
Organic matter (OM), %	88.33 ± 0.75	87.75 ± 0.50
Ether extract (EE), %	3.56 ± 0.50	0.48 ± 0.03
Crude fiber (CF), %	0.49 ± 0.09	0.19 ± 0.11
Non-fermentable extractive substance (NFE), %	33.22 ± 0.39	20.06 ± 0.07
Ash, %	6.35 ± 0.25	4.95 ± 0.18
In vitro nutrient digestibility **
Digestible crude protein (DCP), %	96.59	96.71
Digestible dry matter (DDM), %	99.56	99.05
Digestible organic matter (DOM), %	99.59	99.12
Antioxidant activity *
Chlorophyll a, mg/g	5.56 ± 1.08	9.06 ± 0.79
Chlorophyll b, mg/g	0.88 ± 0.20	1.34 ± 0.34
Carotenes, mg/g	1.52 ± 0.19	1.68 ± 0.31
Total polyphenols, mg/g GAE	1.16 ± 0.16	1.35 ± 0.05
Antioxidant capacity (DPPH % inhibition)	73.29 ± 2.93	81.27 ± 1.60
Antioxidant capacity (µM Trolox)	15.49 ± 3.87	16.78 ± 2.47
Fatty acids (g FAME/100 g Total FAME) *
Caproic (C 6:0)	0.65 ± 0.05	0.16 ± 0.02
Caprilic (C 8:0)	0.25 ± 0.02	7.73 ± 0.65
Capric (C 10:0)	1.20 ± 0.10	-
Lauric (C 12:0)	0.07 ± 0.001	0.68 ± 0.06
Miristic (C 14:0)	0.96 ± 0.08	0.88 ± 0.07
Pentadecanoic (C 15:0)	0.11 ± 0.001	-
Palmitic (C 16:0)	27.25 ± 2.32	34.71 ± 3.12
Stearic (C 18:0)	2.51 ± 0.21	6.87 ± 0.55
Heneicosanoic (C 21:0)	0.17 ± 0.01	-
Behenic (C 22:0)	0.60 ± 0.05	-
∑ SFA	33.77 ± 3.00	51.03 ± 4.34
Pentadecenoic (C 15:1)	0.05 ± 0.004	-
Palmitoleic (C 16:1)	0.19 ± 0.02	4.87 ± 0.46
Heptadecenoic (C 17:1)	0.08 ± 0.007	-
Oleic cis (C 18:1)	8.73 ± 0.72	22.25 ± 2.14
∑ MUFA	9.05 ± 0.19	27.12 ± 1.3
Linoleic cis (C 18:2n6)	14.13 ± 1.20	16.84 ± 1.43
Linolenic γ (C 18:3n6)	0.16 ± 0.01	-
Linolenic α (C 18:3n3)	37.37 ± 3.18	4.41 ± 0.39
Octadecatetraenoic (C18:4n3)	0.73 ± 0.06	-
Eicosadienoic (C20:2n6)	0.33 ± 0.03	-
Eicosatrienoic (C20:3n6)	3.61 ± 0.31	-
Arachidonic (C20:4n6)	0.21 ± 0.01	0.60 ± 0.05
∑ PUFA	56.56 ± 4.80	21.85 ± 1.94
∑ PUFA n-3	38.10 ± 3.65	4.41 ± 0.41
∑ PUFA n-6	18.45 ± 1.77	17.44 ± 1.48
∑ PUFA n-6/∑ PUFA n-3	0.48 ± 0.05	3.95 ± 0.35
Other fatty acids	0.62 ± 0.05	-

Where: ∑SFA, sum of saturated fatty acid; ∑MUFA, sum of monounsaturated fatty acid; ∑P sUFA, sum of polyunsaturated fatty acid; * Mean ± standard deviation of three replicate analyses; ** Mean ± standard deviation of five replicate analyses.

. Both microalgae present a high nutrient content, with a high concentration of easily digestible proteins, metabolizable energy, and a low level of cellulose. Compared to chlorella, spirulina is characterized by a higher antioxidant capacity, with a significantly higher content of chlorophylls a and b. The concentration of carotenoids is high in both chlorella and spirulina. Chlorophylls are green pigments found in plants and algae. Chlorella has a chlorophyll a content of 5.56 mg/g and chlorophyll b content of 0.88 mg/g. Spirulina platensis has a higher chlorophyll content compared to chlorella, with chlorophyll a at 9.06 mg/g and chlorophyll b at 1.34 mg/g. It is known that carotenes are orange or red pigments that serve as powerful antioxidants. Chlorella contains 1.52 mg/g of carotenes and spirulina has a slightly higher carotene content at 1.68 mg/g. Polyphenols are compounds found in plants and algae known for their antioxidant properties and potential health benefits. The total polyphenol content of chlorella was assessed to be 1.16 mg/g of total polyphenols, while spirulina has a slightly higher content of 1.35 mg/g of total polyphenols. DPPH is a common method used to evaluate the antioxidant activity of substances. Trolox is a synthetic antioxidant used as a standard for comparison. Chlorella exhibited an antioxidant capacity of 73.29% inhibition of DPPH (2,2-diphenyl-1-picrylhydrazyl) and an antioxidant capacity of 15.49 µM Trolox. On the other hand, spirulina demonstrated a higher antioxidant capacity compared to chlorella, with 81.27% inhibition of DPPH and an antioxidant capacity of 16.78 µM Trolox. Both chlorella and spirulina exhibited antioxidant activity, with spirulina generally showing higher values in terms of chlorophyll content, carotenes, total polyphenols, and antioxidant capacity. These findings suggest that spirulina demonstrated stronger antioxidant properties compared to chlorella.

Concerning the fatty acid concentrations, spirulina had a higher content of total SFAs at 51.03 g FAME/100 g Total FAME compared to chlorella, which had a total SFA content of 33.77 g FAME/100 g Total FAME. The monounsaturated fatty acids (MUFAs) of chlorella contained 9.05 g FAME/100 g Total FAME, while spirulina contained a higher content of MUFA at 27.12 g FAME/100 g Total FAME. MUFAs are considered to be healthier fats compared to SFAs. Polyunsaturated fatty acids (PUFAs) include both omega-3 and omega-6 fatty acids. Chlorella has a higher total PUFA content of 56.56 g FAME/100 g Total FAME compared to spirulina (21.85 g FAME/100 g Total FAME). The data presented suggest that chlorella has a higher content of total PUFAs, particularly PUFA n-3, compared to spirulina. On the other hand, spirulina has higher contents of SFAs, MUFAs, and PUFA n-6. The nutritional value and potential health benefits of these microalgae may be influenced by their fatty acid composition. Overall, the fatty acid profile indicates that chlorella contains a significantly higher amount of polyunsaturated fats (PUFAs), with over 67% being represented by omega-3 polyunsaturated fats, particularly α-linolenic acid (ALA).

3.2. Nutritional Value of the Experimental Diets

The nutritional content of the diets is presented in

Table 3. Nutritional compounds of the feed (% as fed).

Parameters	Dietary Treatments			SEM	p-Value
	CON	CV2%	SP2%
Antioxidant activity
Chlorophyll a, µg/g	10.31 c	344.76 b	383.22 a	5.41	<0.0001
Chlorophyll b, µg/g	2.77 b	40.93 a	38.92 a	1.44	<0.0001
Carotenes, µg/g	31.45 b	84.92 a	81.70 a	1.08	<0.0001
Total polyphenols, mg/g GAE	1.49	1.67	1.64	0.177	0.933
Antioxidant capacity (DPPH % inhibition)	32.00 c	57.57 b	67.23 a	1.23	<0.0001
Antioxidant capacity (µM Trolox)	5.19	6.25	5.79	0.403	0.620
Fatty acid composition (% of total fat)
ΣSFA	29.03 a	19.72 b	22.44 b	0.893	0.001
ΣMUFA	47.42 a	37.06 b	40.12 ab	2.06	0.030
∑ PUFA, from which:	23.16 b	42.95 a	37.11 a	2.36	0.003
Σ n-3 PUFA	1.03 b	1.43 a	1.21 ab	0.06	0.007
∑ n-6 PUFA	22.14 b	41.52 a	35.90 a	2.32	0.003
∑ n-6/∑ n-3	21.58 b	29.18 a	29.47 a	1.22	0.006

Where: CON, conventional diet; CV2%, conventional diet supplemented with 2% chlorella powder; SP2%, conventional diet supplemented with 2% spirulina powder; n = 5 samples per group; SEM, standard error of the mean; ^abc Mean values within a row with different letters are significantly different at p ≤ 0.05. Abbreviations: ΣSFA, sum of saturated fatty acids; ∑MUFA, sum of monounsaturated fatty acid; ΣPUFA, sum of polyunsaturated fatty acids; Σ n-3 PUFA = C18:3n-3 + C18:4n-3; Σ n-6 PUFA = C18:2n-6 + C20:2n-6 + C20:3n-6 + C20:4n-6.

. The antioxidant activity characterized by chlorophyll a is significantly higher (p < 0.0001) in the SP2% group compared to the CV2% and CON groups, but the CV2% group is also significantly higher (p < 0.0001) compared to the CON group. Chlorophyll b was found in significantly higher (p < 0.0001) concentrations in both the CV2% and SP2% groups compared to the CON group. The antioxidant capacity was significantly higher (p <0.0001) in the SP2% group compared to the CV2% and CON groups, but the CV2% group also registered a significantly higher (p < 0.0001) antioxidant capacity compared to the CON group. The ΣSFA was significantly higher (p < 0.001) in the CON group compared to the SP2% and CV2% groups. Additionally, ΣMUFA concentration was significantly higher (p < 0.03) in the CON group compared to the CV2% group. The ∑ PUFA concentration was observed to be significantly higher (p < 0.003) in the SP2% and CV2% groups compared to the CON group. The highest value concentration of Σ n-3 PUFA (p < 0.007) was noticed in the CV group compared to the SP2% and CON groups. Concerning the ∑ n-6 PUFA, a statistically higher concentration was determined for the CV2% and SP2% groups compared to the CON group. The ∑ n-6/∑ n-3 ratio was highly significant for the SP2% and CV2% groups compared to the CON group.

3.3. Production Performances

The production performance values are shown within

Table 4. Performances of laying hens fed with Ch. vulgaris and Sp. platensis.

Parameters	Dietary Treatments			SEM	p-Value
	CON	CV (2%)	SP (2%)
Initial body weight (g/layer)	1561.30	1599.17	1556.88	23.23	0.580
Final body weight (g/layer)	1670.68	1681.52	1643.96	23.63	0.688
Daily feed intake (g/day/layer)	112.78	112.40	113.46	0.441	0.608
Feed conversion ratio (g feed/g egg)	2.04 b	2.17 a	1.92 c	0.024	<0.0001
Egg weight (g)	59.78 b	62.42 a	62.44 a	0.110	<0.0001
Hen day egg production (%)	94.61 ab	93.32 b	95.56 a	0.412	0.046
Egg classification *, %
“XL” (>73 g), extra large	0.15 b	2.99 a	2.08 a	0.350	<0.0001
“L” (63–73 g), large	22.87 b	38.50 a	39.65 a	1.01	<0.0001
“M” (53–63 g), medium	71.22 a	57.321 b	56.50 b	1.03	<0.0001
“S” (<53 g), small	5.76 a	1.19 b	1.77 b	0.346	<0.0001

Where: CON, conventional diet; CV2%, conventional diet supplemented with 2% chlorella powder; SP 2%, conventional diet supplemented with 2% spirulina powder; * C.E Regulation no. 852/2004 on the general rules of food hygiene with subsequent amendments and completions and Directive 2000/13 / C.E.; SEM, standard error of the mean. ^abc Mean values within a row with different letters are significantly different at p ≤ 0.05.

. There were no significant differences concerning the initial or final body weight (p = 0.580; p = 0.688) of hens in the CON, CV2%, and SP2% groups. The feed conversion ratio (g feed/g egg) registered the optimum value in the SP2% group compared to CON and CV2% (p < 0.0001). There was no significant difference in the daily feed intake among the dietary treatments (p = 0.608). The mean daily feed intake was 112.78 g/day/layer for CON, 112.40 g/day/layer for CV2%, and 113.46 g/day/layer for SP2%. A highly significant difference (p < 0.0001) was noticed in the feed conversion ratio between all groups. There was a highly significant difference (p < 0.0001) concerning egg weight among the dietary treatments. The mean egg weight was 59.78 g for CON, 62.42 g for CV2%, and 62.44 g for SP2%. There was a significant difference (p = 0.046) in the hen day egg production among the dietary treatments; the highest percentage was registered for the SP2% group (95.56%) compared to CON and CV2%. There were significant differences in the percentages of eggs classified into different sizes (XL, L, M, and S) categories among the dietary treatments (p < 0.0001). The data show that the proportions of eggs in each size category varied among the treatment groups, with significant differences observed for all categories (p < 0.0001). The CV2% group presented a higher percentage of eggs classified as “M” (medium) compared to the SP2% and CON groups. This suggests that chlorella supplementation might have influenced egg size distribution, potentially leading to a significantly higher proportion of medium-sized eggs (p < 0.0001). The SP2% group showed a significantly higher (p < 0.0001) percentage of eggs classified as “L” (large) compared to the CV2% and CON groups.

3.4. Serum Antioxidant Status

The effect of experimental diets CV2% and SP2% on the blood antioxidant enzymes SOD, CAT, GSH, and TAC activity is presented within Figure 1. A significant increase (p < 0.001; p < 0.0001) of SOD concentration in serum of laying hens fed with microalgae was noticed compared to the CON group. Also, a highly significant difference (p < 0.001) was registered between experimental groups, where SP2% was characterized by an increased antioxidant activity which assumes a high antioxidant status of this group. The chlorella and spirulina powder diet supplementations improved the enzymatic activity of CAT, increasing (p < 0.01) its serum concentration value significantly compared to CON. There were no differences (p ≥ 0.05) noticed between the two experimental groups.

GSH serum concentration significantly increased (p < 0.0001) in the SP2% and CV2% groups compared to CON. Also, significant statistically differences (p < 0.05) were observed between the experimental groups. The same trend was noticed for total antioxidant capacity value from serum, but with highly statistical differences (p < 0.001) between the two experimental groups.

3.5. Digestibility Trial

The coefficients of apparent nutrient digestibility of laying hens fed with chlorella and spirulina powder diets are presented within

Table 5. Apparent nutrient digestibility (%) of laying hens fed with Ch. vulgaris and Sp. platensis powder diets.

Parameters	Dietary Treatments			SEM	p-Value
	CON	CV2%	SP2%
Digestible dry matter (DDM),%	73.17	71.29	72.61	0.835	0.288
Digestible organic matter (DOM), %	73.05	70.52	71.62	0.806	0.112
Digestible crude protein (DCP), %	85.81	85.20	84.85	0.607	0.539
Digestible crude fat (DCF), %	90.58 a	87.82 b	89.07 ab	0.740	0.052
Digestible non-fermentable extractive substance (DNFE), %	68.60	66.27	67.59	0.768	0.128

Where: CON, conventional diet; CV2%, conventional diet supplemented with 2% chlorella powder; SP2%, conventional diet supplemented with 2% spirulina powder; n = 6 samples per group; SEM, standard error of the mean. ^ab Mean values within a row having different letters are significantly different at p ≤ 0.05.

. The supplementation of the conventional diet with chlorella or spirulina powder had no effect on the digestibility of dry matter, organic matter, crude protein, or digestible NFE because there were no significant differences (p > 0.05) between the CON diet and algae-supplemented diets. We registered only a tendency for crude fat digestibility to decrease just for chlorella powder.

3.6. Nutritional Egg Quality Parameters

Table 6. Effect of dietary Ch. vulgaris and Sp. platensis powder in laying hens’ diets on egg yolk nutrients and external and internal egg quality parameters.

Parameters	Dietary Treatments			SEM	p-Value
	CON	CV2%	SP2%
Nutrition quality of egg yolk
β-carotene, (µg/g)	30.77 b	38.82 a	38.25 a	0.432	<0.0001
Total polyphenols (mg/g GAE)	0.534	0.598	0.574	0.027	0.271
Antioxidant capacity (DPPH% inhibition)	16.84 b	25.14 a	28.15 a	0.869	<0.0001
Antioxidant capacity (µM Trolox)	0.74 b	0.79 a	0.82 a	0.019	0.033
External and internal egg quality parameters
Egg weight (g), of which:	61.28	61.53	61.82	0.389	0.617
albumen white (g)	37.00	37.76	37.38	0.352	0.320
egg yolk (g)	16.37	15.65	16.26	0.242	0.087
eggshell (g)	7.91	8.12	8.17	0.137	0.358
Albumen pH (value)	8.62 ab	8.44 b	8.80 a	0.069	0.002
Yolk pH (value)	6.51	6.38	6.46	0.093	0.625
t° albumen (°C)	19.10 a	18.57 ab	17.79 b	0.258	0.003
t° yolk (°C)	19.99 a	19.12 b	19.92 a	0.134	0.011
White height, mm	10.96	11.74	11.48	0.292	0.168
Haugh units (value)	102.92	106.04	104.82	1.150	0.163
Yolk color, (value)	4.06 c	8.11 b	11.06 a	0.113	<0.0001

Where: CON, conventional diet; CV2%, conventional diet supplemented with 2% chlorella powder; SP2%, conventional diet supplemented with 2% spirulina powder; n = 6 samples per group (3 eggs/ sample); SEM, standard error of the mean. ^abc Mean values within a row having different letters are significantly different at p ≤ 0.05.

presents the effect of dietary chlorella and spirulina powder on laying hens’ egg nutrients and external/internal quality parameters. The microalgae dietary supplementation influenced the nutritional egg quality positively. The β-carotene concentration registered a highly significant increase (p < 0.0001) in experimental diets, the results being positively correlated with the concentration of chlorophylls a and b and carotene of both dietary microalgae. The same observation could be noted for both antioxidant capacity expressed as DPPH% inhibition (p < 0.0001) and µM Trolox (p < 0.033).

Concerning the egg quality parameters, no effects (p > 0.05) of chlorella or spirulina powder were observed on egg weight or its components (albumen, yolk, and shell). The albumen pH was influenced by the presence of algae compared to the CON diet: lower value for chlorella diet, and higher value for spirulina diet. The yolk pH and the albumen height were not influenced by the dietary treatment. The Haugh unit, the most widely accepted indicator of internal egg quality, had an increasing tendency in eggs obtained from algae supplementation diets, especially for the chlorella diet. The egg yolk color in groups fed diets with chlorella or spirulina were significantly higher (p < 0.0001) compared to the CON diet.

3.7. Yolk Cholesterol Content and Fatty Acid Profile

Table 7. Fatty acid composition in total lipids of egg yolks (average values/group).

	Experimental Groups
Parameters	CON	CV2%	SP2%	SEM	p-Value		CON	CV2%	SP2%	SEM	p-Value
Yolk fat (% DM)	29.454	29.570	30.412	0.771	0.640
Cholesterol (mg col/egg)	224.00	173.00	192.00	0.013	0.061
Fatty acid content	g FAME/100 g Total FAME						mg fatty acid/egg
Miristic C14:0	0.308 b	0.346 a	0.346 a	0.008	0.005		4.716	5.063	5.266	0.196	0.167
Pentadecanoic C15:0	0.060	0.140	0.060	0.052	0.399		0.932	2.182	0.918	0.760	0.423
Palmitic C16:0	25.44	26.14	26.05	0.242	0.121		389.9	380.2	395.0	13.500	0.738
Heptadecanoic C17:0	0.128 b	0.128 b	0.145 a	0.003	0.001		1.974 ab	1.869 b	2.227 a	0.093	0.043
Stearic C18:0	10.09	10.11	9.61	0.232	0.256		154.71	146.93	145.79	5.990	0.533
∑SFA	36.03	36.87	36.22	0.409	0.339		552.2	536.3	549.2	19.20	0.825
Miristioleic C14:1	0.079 c	0.092 b	0.108 a	0.003	<0.0001		1.217 b	1.345 b	1.645 a	0.066	0.001
Pentadecenoic C15:1	0.100	0.101	0.417	0.184	0.394		1.53	1.46	6.35	2.810	0.392
Palmitoleic C16:1	4.37	3.98	4.34	0.421	0.765		65.58	57.95	65.86	5.170	0.488
Heptadecenoic C17:1	0.069 b	0.075 b	0.089 c	0.003	0.002		1.056 b	1.095 b	1.358 a	0.070	0.016
Oleic C18:1	36.09 b	36.80 ab	37.59 a	0.406	0.050		552.8	535.8	570.1	20.100	0.499
Erucic C22 (1n9)	0.121 a	0.120 a	0.100 b	0.004	0.002		1.854 a	1.456 b	1.819 a	0.077	0.004
Nervonic C24 (1n9)	0.277	0.279	0.272	0.006	0.640		4.246	4.063	4.128	0.161	0.724
∑MUFA	41.11	41.43	42.94	0.508	0.050		628.3	603.2	651.2	20.4	0.280
Linoleic C18:2	15.66 a	14.27 b	13.97 b	0.108	<0.0001		239.94 a	207.67 b	211.94 ab	8.320	0.031
Linolenic γ C18:3n6	0.126 b	0.134 ab	0.138 a	0.002	0.0007		1.931	1.944	2.089	0.080	0.326
Linolenic α C18:3n3	0.218 b	0.319 a	0.233 b	0.017	0.001		3.343 b	4.651 a	3.541 b	0.283	0.011
Eicosadienoic C20 (2n6)	0.229 b	0.232 b	0.269 a	0.007	0.002		3.515	3.389	4.084	0.190	0.046
Eicosatrienoic C20 (3n6)	0.290 a	0.257 b	0.289 a	0.005	<0.0001		4.451 a	3.743 b	4.393 ab	0.178	0.023
Eicosatrienoic C20 (3n3)	0.252 b	0.234 b	0.274 a	0.006	0.001		3.866 ab	3.401 b	4.154 a	0.172	0.023
Arachidonic C20 (4n6)	3.613 a	3.548 a	3.178 b	0.093	0.010		55.420	51.570	48.230	2.34	0.129
Docosatetraenoic C22 (4n6)	1.501 a	1.318 b	1.537 a	0.043	0.005		23.01 a	19.16 b	23.32 a	1.01	0.019
Docosapentaenoic C22 (5n3)	0.071 b	0.109 a	0.071 b	0.003	<0.0001		1.087 b	1.593 a	1.073 b	0.054	<0.0001
Docosahexaenoic C22 (6n3)	0.691 a	1.056 b	0.632 b	0.021	<0.0001		10.591 b	15.357 a	9.595 b	0.483	<0.0001
∑PUFA	22.65 a	21.45 b	20.59 c	0.183	<0.0001		347.2	312.5	312.4	12.30	0.102
∑Ω3	1.23 b	1.72 a	1.21 b	0.024	<0.0001		18.887 b	25.001 a	18.364 b	0.784	<0.0001
∑Ω6	21.42 a	19.76 b	19.39 b	0.168	<0.0001		328.270	294.059	287.475	11.5	0.053
∑Ω6/Ω3	17.40 a	11.51 c	16.06 b	0.228	<0.0001		266.27 a	167.44 b	243.36 a	8.59	<0.0001
Other fatty acids	0.207	0.214	0.245	0.025	0.517		3.164	3.108	3.749	0.393	0.460

Where: CON, conventional diet; CV2%, conventional diet supplemented with 2% chlorella powder; SP (2%), conventional diet supplemented with 2% spirulina powder; n = 6 samples per group (3 eggs/sample); SEM, standard error of the mean; ^abc Mean values within a row with different letters are significantly different at p < 0.05.

presents the effects of chlorella and spirulina powder dietary inclusion on egg yolk cholesterol content and fatty acid profile. There were no statistical differences (p = 0.061) concerning cholesterol content groups. When examining the fatty acid composition, there were variations observed among the different experimental groups depending on how the results were reported. For the health of consumers, it is important that results are be expressed as mg fatty acids/egg. Concerning the total SFAs (p = 0.825) and the total MUFAs (p = 0.280), there were no differences registered. Regarding polyunsaturated fatty acids (PUFAs), expressed as mg fatty acids/egg, there were no differences (p = 0.102) between groups, even though when expressed as g FAME/ 100 g Total FAME, both CV2% and SP2% groups led to a decrease in the overall amount of PUFAs compared to the control group. The highest (p < 0.0001) concentration of omega-3 was observed in the chlorella group (1.72 g FAME/100 g Total FAME; 25.001 mg fatty acids/egg) compared to spirulina (1.21 g FAME/100 g Total FAME; 18.364 mg fatty acids/egg) and control groups (1.23 g FAME/100 g Total FAME; 18.887 mg fatty acids/egg), which influenced the Ω6/Ω3 ratio; highly significantly (p < 0.0001) lower for the chlorella group.

3.8. The Effect of Dietary Chlorella and Spirulina Powder on Egg Yolk Color in Fresh and Boiled Eggs

Table 8. Effects of chlorella and spirulina powder on egg yolk color in fresh and boiled eggs.

Yolk Color Parameter		L*	a*	b*
CON	fresh yolk	45.76 f	1.321 e	14.03 f
	10 min boiling time	79.94 e	−1.389 g	19.32 ef
	20 min boiling time	104.33 c	−3.836 h	23.46 de
	30 min boiling time	125.97 a	−5.514 i	25.65 d
CV2%	fresh yolk	43.82 f	3.156 d	17.74 ef
	10 min boiling time	76.56 e	0.883 e	33.14 c
	20 min boiling time	100.05 cd	0.073 ef	51.60 a
	30 min boiling time	122.15 a	−1.070 fg	48.21 a
SP2%	fresh yolk	40.49 f	4.924 c	14.18 f
	10 min boiling time	74.86 e	6.414 ab	32.16 c
	20 min boiling time	95.11 d	7.601 a	39.83 b
	30 min boiling time	116.46 b	5.591 bc	49.31 a
Main effect
Treatment	CON	88.99 a	−2.354 c	20.61 c
	CV2%	85.65 b	0.761 b	37.68 a
	SP2%	81.73 c	6.132 a	33.87 b
	SEMtreatment	0.583	0.151	0.653
Boiling time	fresh yolk	43.36 d	3.134 a	15.318 d
	10 min boiling time	77.12 c	1.969 b	28.205 c
	20 min boiling time	99.83 b	1.279 c	37.897 b
	30 min boiling time	121.53 a	−0.331 d	41.457 a
	SEMboiling time	0.682	0.177	0.764
p-Value
Treatment		<0.0001	<0.0001	<0.0001
Boiling time		<0.0001	<0.0001	<0.0001
Treatment × Boiling time		0.293	<0.0001	<0.0001

Where: CON, conventional diet; CV2%, conventional diet supplemented with 2% chlorella powder; SP 2%, conventional diet supplemented with 2% spirulina powder; SEM, standard error of the mean. ^a–i Mean values within a column with different letters are significantly different at p ≤ 0.05.

presents the effects of chlorella and spirulina powder dietary inclusion on egg yolk color in fresh and boiled eggs. The treatment factor (CON, CV2%, SP2%) exhibited a significant effect on the L*, a*, and b* parameters. The p-values registered for all three parameters indicated a highly significant difference (p < 0.0001) between the treatments. The boiling time factor (fresh yolk, 10, 20, and 30 min.) also had a highly significant effect (p < 0.0001) on the L*, a*, and b* parameters, which indicated that the boiling time had a significant impact on the egg yolk color. The interaction between treatment and boiling time showed highly significant (p < 0.0001) effects on the a* and b* parameters. However, the interaction effect was not statistically significant for the L* parameter (p = 0.293). The L* parameter represents the lightness of the color, and therefore higher values indicate lighter or brighter colors, while lower values indicate darker colors. Comparing the values across different treatments and boiling times, it can be observed that as the boiling time increased, the L* values showed a tendency to increase as well. This suggests that the egg yolks become lighter in color as they are boiled for a longer duration. The darkest color values were noticed for SP2%, followed by CV2% (p < 0.0001). The a* parameter values (red-green color axis, positive values indicate redness, negative values indicate greenness) varied across different treatments and boiling times. The highest value for a* parameters was noticed on SP2%, followed by CV2% (p < 0.0001), compared to CON. However, there was no consistent trend concerning boiling time. The interaction effect between treatment and boiling time was highly significant (p < 0.0001), indicating that the combination of treatment and boiling time influenced the red-green color component. Positive values indicate more yellowness, while negative values indicate more blueness. Similar to the a* parameter, the b* values also varied across treatments and boiling time. The b* parameter (yellow-blue color) registered the highest value on CV2%, followed by SP2% (p < 0.0001), compared to CON. The interaction effect between treatment and boiling time was highly significant (p < 0.0001), suggesting that the combination of treatment and boiling time affects the yellow-blue color component.

4. Discussion

From a nutritional point of view, the two microalgae (chlorella and spirulina) are considered food additives with high biological value due to their nutrient concentration. The results of our study analyses strongly indicate that spirulina shows higher antioxidant properties, carotenoid levels, polyphenols, and a superior DPPH inhibition, when compared to chlorella, which suggests that spirulina has a greater capacity to combat oxidative stress. On the other hand, the microalgae proximal composition showed that chlorella had a higher concentration of PUFA, particularly omega-3 fatty acids and a higher omega-3 content, and lower ∑ PUFA n-6/∑ PUFA n-3 ratio compared to spirulina.

Other authors confirm that the microalgae contain the highest protein value with an excellent essential amino acid profile [33,34], bioactive compounds, PUFA fatty acids, polysaccharides, volatile and phenolic compounds, vitamins, sterols, and natural pigments [35]. The high levels of carotenoids and fatty acids, especially α-linolenic, are associated with health benefits and nutrition [36]. The microalgae utilization in animal feed improves productive performance, the immune system, antioxidant activity, and tissue regeneration [35]. Other authors [37] found a concentration of 3.291 mg/L chlorophyll a, 1.174 mg/L chlorophyll b, 4.466 mg/L total chlorophyll, and 0.919 mg/L carotenoids in blue-green algae spirulina. Abou-El-Souod et al. [38] stated that chlorella possesses chloroplasts that contain green photosynthetic pigments called chlorophylls a and b. Utilizing the process of photosynthesis, it exhibits rapid growth and multiplication by utilizing carbon dioxide, water, sunlight, and a minimal amount of minerals. Similar findings to our results have been reported in other studies investigating the antioxidant activity and fatty acid composition of spirulina and chlorella. Khan et al. [39] found that spirulina exhibited significantly higher antioxidant activity compared to the control group. The presence of active compounds such as phycocyanin and beta-carotene in spirulina contributed to its strong antioxidant potential. In a study, Stunda-Zujeva et al. [40] stated that phycocyanin is the main antioxidant of spirulina, offering various uses for health benefits, although care should be taken in terms of the antioxidant activity, which fluctuates. Numerous studies have highlighted the higher antioxidant capacity and beneficial fatty acid profiles, including higher concentrations of omega-3 polyunsaturated fatty acids, in both microalgae species compared to control groups. These fatty acids are known for their beneficial effects on human health, including cardiovascular health and anti-inflammatory properties. Another study by [41] investigated the fatty acid profiles of microalgae species and found that both spirulina and chlorella exhibited higher concentrations of omega-3 fatty acids, particularly ALA, compared to the control group. They also noted that these microalgae species had lower levels of saturated fatty acids, contributing to a more desirable fatty acid composition. Other researchers [42] evaluated the fatty acid composition of spirulina and highlighted its high content of gamma-linolenic acid (GLA), an omega-6 fatty acid with anti-inflammatory properties.

Our research revealed that adding chlorella and spirulina to the laying hens’ diet at a 2% inclusion rate did not have a significant impact on initial or final body weight. Nevertheless, the group supplemented with spirulina demonstrated enhanced feed conversion efficiency, larger eggs, and higher rates of egg production compared to the control and chlorella groups. This suggests that dietary supplementation with spirulina could have more pronounced positive effects on egg production efficiency and size uniformity, with practical benefits for egg producers and consumer health. These findings are similar to those of other studies which studied different microalgae sources and inclusion levels and noticed an improved production parameter when including microalgae in poultry diets due to the high protein content, essential amino acids, vitamins, and minerals present in spirulina and chlorella. Additionally, the presence of certain bioactive compounds and antioxidants in microalgae may have positive effects on production performance. Mariey et al. [43] included four levels of spirulina powder (0, 0.10, 0.15, or 0.20%) in laying hens’ diet and registered an improved egg production rate, daily egg mass, and feed conversion ratio compared to those of the control group. Shanmugapriya and Saravanababu [44] tested spirulina on broilers and found a significant increase in body weight. Other studies have [45] supplemented the basal diet of laying hens raised under a chronic hot ambient temperature with spirulina powder (0.15 mg/kg diet) and seleno-methionine (0.10 mg/kg diet). The obtained results indicated that dietary spirulina and organic selenium showed improved productive performance under heat stress. In contrast, the chlorella supplementation at varying dosages of 2.5 g, 5.0 g, or 7.5 g per kg feed, in both spray-dried and bullet-milled/spray-dried forms, did not result in any impact on laying intensity, egg weight, daily egg mass production, or feed conversion. However, it was observed that the treatment groups exhibited an increase in yolk weight and an improvement in egg quality [46].

In a study conducted by Omri et al. [47], laying hens at 44 weeks of age were fed with diets containing 1.5% and 2.5% spirulina for a period of 6 weeks. The results indicated that the inclusion of 2.5% spirulina in the diet significantly increased egg weight. However, no significant effects were observed on other productive parameters, including dietary treatment, duration of the diet, or their interaction.

Concerning the antioxidant enzyme activity, the results obtained in our study showed that chlorella and spirulina dietary addition exhibited significant improvements in blood antioxidant enzyme activities (SOD, CAT, GSH) and total antioxidant capacity (TAC). Moreover, the increased serum levels of GSH and TAC in both experimental diets demonstrate and support the idea that the microalgae-supplemented diets positively influenced the hens’ antioxidant status compared to the control group. The main antioxidant enzymes, such as SOD, CAT, and GSH, protect the organism against oxidative stress [48], improving the poultry immune system [49]. CAT is one of the most important antioxidant enzymes which mitigates oxidative stress via the catalysis of hydrogen peroxide [50]. Park et al. [51] obtained the same linearly increased GPx and SOD enzymes in broilers fed with spirulina and explained that this was due to the fact that spirulina contains antioxidants such a β-carotene, tocopherol, selenium, polypeptide pigment, or phenolic acids. Wu et al. [52] suggested that spirulina has stronger antioxidant capabilities than chlorella, which is probably due to the higher content of phenolic compounds.

Utilization of microalgae in laying hens’ diet had no effect concerning the apparent digestibility coefficients. Our results are similar to those of [53], who reported that the incorporation of green seaweed (Ulwa spp.) meal between 20 and 35 g/kg in Boschveld hens’ diets did not alter apparent nutrient digestibility.

Additional research [19] indicated that the inclusion of brown seaweed meal derived from (Ecklonia maxima) into the diet of Boschveld cockerels did not have a significant impact on the digestibility of dry matter, organic matter, crude protein, and fiber. This result was observed despite the seaweed inclusion rate ranging from 2 to 8 g/kg.

In our experiment, we obtained a high β-carotene content and increased antioxidant capacity of the yolk, which represents indicators of an improved egg quality, with potential health-promoting effects for consumers. Omri et al. [47] observed no effect (p > 0.05) on total cholesterol concentration when using spirulina (1.5% and 2.5%) in laying hen diets.

The dietary microalgae supplementation had no influence on egg quality parameters (egg weight and its components). Similar results on egg weight were observed by [46] using chlorella supplementation in laying hens (26-week-old) diets. Other authors, such as [54], used chlorella supplementation in Hy-Line brown laying hens, aged 70 weeks, without any effects on egg weight, but registered the highest Haugh units when supplementing diets with 2.4% liquid chlorella in their study.

Our data indicate that the dietary treatments of chlorella and spirulina influenced the yolk coloration, spirulina having a more pronounced effect on enhancing red color. Additionally, longer boiling times result in darker and lower/more negative values for a* (greenish-gray ring) and higher/more positive values for b*.

In other studies [34,46,54], both chlorella- and spirulina-supplemented diets were confirmed to increase the color of yolk by lutein dosing.

The intensity of yolk color can vary depending on the types and concentrations of carotenoids consumed by the laying hens. Englmaierová et al. [55], using chlorella at 12.5 g/kg, noticed a significant intensification of the yellowness of fresh yolk. In the case of boiled eggs, a statistically significant increase in redness was observed. Conversely, an extension of the boiling duration to 10 min resulted in an increase in lightness and a concomitant reduction in yolk coloration.

The L* value for fresh yolk indicates that the color of the fresh yolk has a moderately bright appearance. As the boiling time increased, the L* values also increased. The L* value for the 10 min. boiling indicated that the boiled yolk became significantly brighter (p ≤ 0.0001) compared to the fresh yolk. The L* value increased progressively for the 20 min. and 30 min. boiling times, respectively; yolks became lighter as they were boiled for longer durations. The differences in L* values between the boiling time highlight the effect of heat exposure on the lightness of the yolks. This change in lightness can be attributed to structural and chemical transformations that occur during the cooking process, causing the denaturation of the proteins and altering the protein molecules. As a result, the yolks appear brighter or lighter in color [56]. According to Muñoz-Miranda and Iñiguez-Moreno [57], marine biopigments can be categorized into three main groups: chlorophylls, carotenoids, and phycobiliproteins. The rich carotenoid concentration of the pigments zeaxanthin, xanthophylls, and β-carotene offer different greenish, green, golden, red, and brown colors of algae [58]. Other authors [59] tested, in a short-term study, the effects of 1% and 3% spirulina supplementation on color, nutritional value, and stability of yolk. A decreased luminosity and increased redness (p = 0.0001) and yellowness (p = 0.0103) were observed for 1% supplementation, after only 15 experimental days, meaning that the high carotenoid levels present in spirulina are efficiently absorbed by the laying hens’ gastrointestinal tract [60].

Dietary supplementation with chlorella at 1% and 2% levels on Hisex Brown laying hens aged 56 weeks revealed a significant increase in total carotenoid deposition by 46% and 119% for the 1% and 2% chlorella groups, respectively. This increase was accompanied by a significant improvement in the yolk egg color, as evidenced by the Roche Fan Yolk Color grade, which registered 5.0 and 6.1 for the 1% and 2% chlorella groups, respectively, compared to 4% for the control group (p < 0.001). These findings suggest that chlorella dietary supplementation can enhance the carotenoid content and improve the color of yolks in laying hens.

Omri et al. [47] obtained increases in egg yolk redness from 1.33 (C) to 12.67 (1.5% spirulina) and 16.19 (2.5% spirulina), and a significant yellowness (b*) reduction parameter from 62.1 (C) to 58.17 (1.5% spirulina) and 55.87 (2.5% spirulina). Overall, the yolks from the experimental diets were highly significantly (p < 0.0001) darker, exhibited a stronger red color, and had reduced yellowness compared to the yolks from the control group. Other studies [43] tested 0.1%, 0.15%, and 0.2% spirulina in laying hens’ diet and observed increasing yolk color scores (RYCF) of 6.3, 6.7, and 7.6, respectively. Similarly, supplementation levels of 1.5%, 2%, and 2.5% of spirulina were tested by [61] and obtained significant yolk intensifications of 10.55, 11.43, and 11.66, respectively, compared to the control.

The present study also investigated the effectiveness of microalgae in enhancing the fatty acid composition of eggs, specifically through the increased presence of docosahexaenoic acid (DHA). The process of enriching eggs with omega-3 polyunsaturated fatty acids (n-3 PUFA) from dietary sources is gradual and requires time. However, achieving sufficient enrichment of eggs with these beneficial fatty acids is economically significant for the industry. The n-6/n-3 PUFA ratio reflected diet composition, with the ratio being lower for the eggs of the hens fed microalgae. Some studies consider that many salt and fresh-water microalgae, including spirulina, contain high concentrations of n3-long-chain polyunsaturated fatty acids (PUFA) (25–38%), including α-linoleic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), which are anti-inflammatory and cardiovascular- and brain-protective [39,62]. Microalgae, due to their high concentrations of n-3 PUFA, present an exceptional n-6/n-3 PUFA ratio [39,62]. Studies have demonstrated that laying hens fed with microalgae-enriched diets produce DHA-enriched eggs [63,64,65].

5. Conclusions

In conclusion, the supplementation of laying hens’ diet with chlorella and spirulina at a concentration of 2% each has demonstrated several positive effects on egg quality and nutritional content. This study has revealed significant improvements in egg weight, size, yolk intensity color, beta carotene content, and antioxidant capacity. Furthermore, the incorporation of chlorella has led to a noteworthy increase in omega-3 polyunsaturated fatty acids, resulting in a substantial reduction in the omega-6/omega-3 ratio. As a lower omega-6/omega-3 ratio is widely recognized for its potential benefits to human health and overall well-being, these findings have important implications beyond poultry production. While these results showcase the promising potential of chlorella and spirulina as valuable dietary supplements for laying hens, it is important to acknowledge that further research is necessary to comprehensively evaluate their capabilities in partially substituting costly protein sources in laying hens’ diets. By doing so, these microalgae could contribute to more sustainable and economically viable feed ingredients within poultry production systems.