ABSTRACT
Background
Chlorella vulgaris is rich in protein, beneficial fatty acids and antioxidants, making it a promising natural feed additive.
Objectives
This study evaluates the effects of dietary supplementation with the microalgae C. vulgaris on the productivity and egg quality of laying hens.
Methods
In a randomized design, 84 Lohmann Brown hens (47 weeks old) were divided into four groups: a control and three groups receiving 0.1%, 0.3%, or 0.5% C. vulgaris biomass in their feed over 8 weeks. Egg production, laying intensity, egg weight and composition were monitored.
Results
The 0.5% supplementation significantly increased egg production and laying intensity, without affecting egg weight. Eggs from microalgae‐supplemented hens showed largely stable protein, fat and energy content, while dose‐dependent trends were observed for cholesterol (increasing up to 275 mg/100 g in the highest dose) and the omega‐6/omega‐3 ratio (rising from 7.75:1 in CON to 16:1 in CV5).
Conclusions
These results indicate that C. vulgaris supplementation can improve egg yield and potentially enhance nutritional value, though care is needed regarding lipid profile changes. The study supports the use of microalgae as a sustainable feed additive in poultry diets, promoting productivity and contributing to environmentally friendly egg production.
Keywords: Chlorella vulgaris, egg composition, laying hens, productivity
Dietary supplementation with Chlorella vulgaris up to 0.5% improved laying hen productivity, increasing egg number and daily production rate without compromising egg weight. Egg nutritional composition remained largely unchanged, with only indicative trends in fatty acid ratio and cholesterol, suggesting potential for productivity enhancement without loss of egg quality.
1. Introduction
Nowadays, food quality and sustainable, environmentally friendly production are increasingly important for consumers (Martins et al. 2021; Coudert et al. 2020). As a result, the farming industry is looking for new, natural ways to provide animals with the nutrients they need (e.g., minerals) (Costa et al. 2024). One of the feed supplements that has been widely studied in recent years is various microalgae (Coudert et al. 2020). Microalgae are a very large and diverse group of photosynthetic unicellular eukaryotic organisms that mainly inhabit aquatic environments. Researchers often include Spirulina (Arthrospira or Limnospira), a prokaryote belonging to the phylum Cyanobacteria, also known as blue‐green algae, among microalgae (Thoré et al. 2023; Nowicka‐Krawczyk et al. 2019).
Microalgae are used as a feed and feed additives in aquaculture for fish, shrimps and bivalves (Ma and Hu 2024). Their use has also been explored in domestic animals: cattle, sheep, goats, pigs, rabbits, poultry and pets. When used as feed supplements, they can improve the health of animals (e.g., by enhancing the immune response), as well as improve productivity and the quality of animal products (Spínola et al. 2024; Madeira et al. 2017).
Chlorella vulgaris (CV) is a unicellular green microalga that grows in freshwater (Safi et al. 2014). Its chemical composition depends on environmental factors (it is affected by such factors as availability of nutrients, light intensity, pH and temperature) and extraction methods. Crude protein content can vary between 22.7% and 67.7% of dry matter, lipid content can range from 2.4% to 14.2%, as well as specific fatty acid profile varies significantly (Gadzama et al. 2025). C. vulgaris contains vitamins A, C, E and vitamin B complex; it is also a good source of potassium, phosphorus, calcium, magnesium and zinc (Gadzama et al. 2025; Wang et al. 2024; Safi et al. 2014).
Due to its high protein content, favourable fatty acid profile and richness in vitamins, minerals and bioactive compounds, C. vulgaris has been increasingly used as a feed supplement to improve productivity and product quality in poultry (Mendes et al. 2024; Maurício et al. 2023; Ru et al. 2020; Rani et al. 2018).
In poultry, microalgae supplementation has been associated with improved laying performance, egg quality and mineral bioavailability. Feeding them can also affect the nutritional value of eggs (e.g., by increasing omega‐3 fatty acids and iron, while reducing cholesterol and triglycerides) (Abdel‐Wareth et al. 2024). In ruminants and swine, the benefits of supplementation of microalgae include enhanced growth, immunity and nutrient utilization (Gadzama et al. 2025; Kusuma et al. 2025; Abdel‐Wareth et al. 2024; Kholif et al. 2020; Furbeyre et al. 2017). However, effects may vary depending on algal species, inclusion level, duration of supplementation and dose‐dependent effects on lipid metabolism have also been described (Kusuma et al. 2025; Gadzama et al. 2025; Van Nerom et al. 2024; Coelho et al. 2022).
Compared with other microalgae, such as Spirulina (Limnospira spp.), C. vulgaris offers several competitive advantages, including higher protein content, a more balanced amino acid profile, better digestibility, broader commercial availability and the presence of bioactive compounds that can improve immunity and product quality (Gadzama et al. 2025; García‐Encinas et al. 2025; Panaite et al. 2023; Madeira et al. 2017; Safi et al. 2014).
The large‐scale use of microalgae is still limited by inefficient production methods and high costs. These costs are expected to decrease as production technologies improve and microalgae productivity increases. Costs also depend on cultivation methods and water sources; for example, small‐scale production for specialized uses is much more expensive than systems using wastewater and CO2 from industrial emissions. After production, microalgae are usually dried into a powder for easy addition to animal feed, which improves shelf life and makes transport and storage easier. However, drying methods such as freeze‐drying and spray‐drying are energy‐intensive, costly and raise sustainability concerns. For these reasons, scientists are encouraged to test using smaller amounts of microalgae in diets, which can reduce costs, increase efficiency and still provide nutritional benefits (Martins et al. 2021; Peng et al. 2018; Acién et al. 2012).
Different studies have used various doses of C. vulgaris (from 0.1% to 10%) in laying hen feed (Abdel‐Wareth et al. 2024; Madacussengua et al. 2024). For instance, Madacussengua et al. (2024) found that the addition of 2.5%, 5% and 10% C. vulgaris did not change egg production, while egg weight increased in birds fed 2.5% C. vulgaris. However, Kim et al. (2023) found that feeding 5 g/kg C. vulgaris did not change either the egg production or egg weight. Panaite et al. (2023) found that feeding 2% C. vulgaris to laying hens did not significantly change the cholesterol levels in the egg yolk, but did change the amount of fatty acid profile (e.g., feeding Chlorella significantly increased the omega‐3 fatty acids in the eggs). Similarly, Grigorova et al. (2006) found that feeding 2% and 10% of microalgae of the Chlorella genus to laying hens did not significantly change the cholesterol levels in the egg yolk, but did change the fatty acid content.
Given the inconsistent results and the economic aspects, it is important to clarify the optimal doses of microalgae that can be added to feed to obtain the desired results. In this study, we wanted to investigate the effect of different doses of the microalga C. vulgaris (0.1%, 0.3% and 0.5%) on the egg production, laying intensity, egg weight and the composition of eggs.
2. Material and Method
2.1. Birds, Experimental Design, Diets and Management
A total of 84 healthy 47‐week‐old Lohmann Brown laying hens were obtained from a commercial egg producer. The experiment was conducted in a completely randomized design, with the pen considered as the experimental unit. Laying hens were randomly divided into four dietary treatments (n = 21 hens per treatment): control (CON) and CV1, CV3 and CV5. Each treatment group was housed in a single‐floor pen, resulting in one pen per treatment. Hens were randomly allocated to pens at the start of the experiment to minimize selection bias.
Birds were fed a basal diet, and the CV1, CV3 and CV5 groups were supplemented with 0.1%, 0.3% or 0.5% C. vulgaris biomass, respectively. Dry biomass of C. vulgaris used in the poultry experiment was purchased from a commercial supplier (Buxtehude, Germany). The composition and nutritional value of the basal diet and C. vulgaris biomass are presented in Tables 1 and 2.
TABLE 1.
The composition and nutrient levels of laying hen basal diet.
| Item | Basal diet |
|---|---|
| Metabolized energy | 12.0 MJ/kg |
| Crude fat | 3.98% |
| Crude fibres | 5.31% |
| Crude proteins | 15.1% |
| Ash | 13.72% |
| Phosphorus | 0.5% |
| Lysine | 0.75% |
| Sodium | 0.16% |
| Calcium | 3.9% |
| Methionine | 0.39% |
| Additives (per kg) | Vitamin A—10,000 IU |
| Vitamin D3—3200 IU | |
| Vitamin E—75 mg | |
| Iron (ferrous sulphate monohydrate)—60 mg | |
| Zinc—100 mg | |
| Manganese (manganese (II) oxide)—100 mg | |
| Copper (copper (II) sulphate pentahydrate)—15 mg | |
| Iodine (calcium iodate anhydride)—1.8 mg | |
| Selenium (sodium selenite)—0.3 mg | |
| Butylated hydroxytoluene (E321)—9.71 mg | |
| Butylated hydroxyanisole—3.24 mg | |
| Propyl gallate (E310)—0.65 mg |
Ingredients: Corn, wheat, sunflower seeds, calcium carbonate, soybeans (GMO), molasses, rapeseed oil, monocalcium phosphate, sodium sulphate, sodium chloride.
TABLE 2.
The composition and nutrient levels of microalga Chlorella vulgaris.
| Item | Chlorella vulgaris (100 g microalgal biomass) |
|---|---|
| Energy value | 1448 kJ/326 kcal |
| Crude fat | 11.50 g |
| of which saturated fatty acids | 2.34 g |
| of which unsaturated fatty acids | 4.59 g |
| Carbohydrates | 17.3 g |
| of them sugars | 0.32 g |
| Dietary fibre | 10.68 g |
| Crude proteins | 60.20 g |
| Dry matter | 95.27 g |
| Total carotenoids | 0.24 ± 0.02 g |
| Chlorophyll A | 0.61 ± 0.10 g |
| Chlorophyll B | 0.28 ± 0.03 g |
| Total chlorophyll | 0.89 ± 0.13 g |
The experiment was conducted at the Clinical Research Centre of the Latvia University of Life Sciences and Technologies from 1 May to 18 July 2024. The adaptation period lasted 24 days, followed by a 56‐day experimental period (8 weeks). All laying hens were housed in floor pens (6 m2 each) and had free access to water and feed. Water was supplied using 10‐L plastic poultry drinkers, and feed was provided in 12‐L plastic feeders (150 g per hen per day).
The relative humidity during the study was 53 ± 6.6%, and the room temperature was 22.9 ± 1.9°C. The light regime followed the natural circadian rhythm in Latvia during May–July, with a minimum of 8 h of darkness per day. As the room had windows on only one wall, additional lighting was provided from 7:00 AM to 8:00 PM to ensure balanced illumination.
A limitation of the present study is that each dietary treatment was represented by a single floor pen, with the pen serving as the experimental unit. Consequently, production and performance data were analysed at the pen level, and pen‐level replication was not available. This limits the ability to fully account for potential pen‐related effects and reduces the statistical power to detect treatment differences. Therefore, the results should be interpreted with caution, and conclusions regarding the effects of C. vulgaris supplementation should be considered preliminary. Future studies should include multiple replicate pens per treatment to strengthen statistical inference and confirm the observed effects under commercial conditions.
2.2. Data and Sample Collection
Egg production was recorded daily for each group throughout the experiment. Starting 2 weeks after the initiation of microalga supplementation, the total egg weight was measured daily for each group. The nutritional composition of a batch of eggs was tested at an accredited laboratory (J.S. Hamilton Baltic) at the fourth and eighth weeks of the study. Fatty acid content was determined using an accredited gas chromatography test method (PN‐EN ISO 12966‐1:2015‐01; PN‐EN ISO 12966‐2:2017‐05; PN‐EN ISO 12966‐4:2015‐07). Other parameters that were measured and analysed included the following: cholesterol (PB‐75/GC ed. I of 20.01.2009), protein (PB‐116 ed. III of 11.08.2020), energy value (Regulation (EU) No 1169/2011 of the European Parliament and of the Council) and fat (PN‐A‐86509:1994).
2.3. Statistical Analysis
To account for the longitudinal nature of the data and the repeated measurements over the 8‐week experimental period, Mixed‐Effects Models were employed, where dietary group was treated as a fixed effect and time was included as a random effect. Generalized Linear Mixed Model (GLMM) with a Negative Binomial distribution and a log link function was used. Laying intensity and egg weight were analysed using Linear Mixed Models (LMM) with a Gaussian distribution. Residual normality and homoscedasticity were verified using visual plots.
The nutritional parameters of eggs (cholesterol, fatty acids and proximate composition) were analysed using two‐way ANOVA or mixed models to evaluate the effects of the dietary group (baseline vs. supplemented) and the sampling period (time: Week 4 vs. Week 8). In accordance with the study objectives, the primary focus was placed on comparing each supplemented group (CV1, CV3, CV5) against the CON group as the baseline. Subsequently, the influence of dosage levels and temporal changes was assessed. For all models, estimated marginal means (EMMs) were calculated. Statistical data analysis was performed using Jamovi software, and statistical significance was set at p < 0.05, with results between p ≥ 0.05 and p < 0.10 discussed as trends.
3. Results
3.1. Egg Production and Laying Intensity
The statistical analysis revealed that supplementation with CV significantly influenced egg production parameters. The GLMM showed a significant overall effect of group on egg counts (χ 2 = 9.25, df = 3, p = 0.026). The group receiving the highest dose, CV5, showed a significant increase in egg production compared to the CON group (p = 0.021, OR = 1.099). However, no significant differences were observed between CV1 (p = 0.405) or CV3 (p = 0.574) and the control group.
The LMM indicated a significant effect of supplementation on laying intensity (F(3, 165) = 22.5, p < 0.001). Both CV3 (mean = 0.843, p = 0.002) and CV5 (mean = 0.908, p < 0.001) significantly outperformed the CON group (mean = 0.792). Laying intensity in the CV1 group (mean = 0.792) did not differ from the control (p = 0.992).
Further comparisons between supplemented groups showed that the CV5 dose was significantly more effective than CV1 in terms of both egg counts (p = 0.003, OR = 1.14) and laying intensity (p < 0.001). The CV5 group also showed higher laying intensity compared to the CV3 group (p < 0.001), suggesting a dose‐dependent improvement in productivity.
3.2. Egg Mean Weight
The influence of CV supplementation on egg weight was less pronounced than its effect on production volume. The omnibus test for the LMM suggested a marginal overall group effect on egg mean weight (F(3, 129) = 2.73, p = 0.047). However, post hoc comparisons did not identify significant differences between any of the supplemented groups and the CON group (p > 0.05). The EMM for the control group was 65.2 g, compared to 64.4 g for CV1, 63.8 g for CV3 and 67.4 g for CV5 (Table 3).
TABLE 3.
Productivity metrics.
| Parameter | CON (Control) | CV1 (0.1%) | CV3 (0.3%) | CV5 (0.5%) | p‐value |
|---|---|---|---|---|---|
| Egg count |
16.6 [15.7, 17.6] |
16.1 [15.1, 17.1] |
17.0 [16.0, 18.1] |
18.3* [17.2, 19.4] |
0.026 |
| Laying intensity |
0.792 [0.764, 0.820] |
0.792 [0.764, 0.820] |
0.843* [0.815, 0.870] |
0.908* [0.880, 0.936] |
< 0.001 |
| Egg weight (g) |
65.2 [63.0, 67.4] |
64.4 [62.3, 66.6] |
63.8 [61.6, 65.9] |
67.4 [65.2, 69.5] |
0.047 |
Note: Data are presented as estimated marginal means [95% confidence interval]. * indicates a significant difference (p < 0.05) compared to the CON group.
3.3. Temporal Influence
The analysis of productivity metrics through LMM revealed varying degrees of temporal influence. Time, included as a random factor, accounted for a substantial portion of the variance in laying intensity (explaining approximately 26.5% of the variance; marginal R 2 = 0.171, conditional R 2 = 0.436) and egg mean weight (ICC = 0.257, indicating that 25.7% of the variance was attributable to the time factor). Conversely, for the total egg count, the influence of time was negligible, with the marginal and conditional R 2 values remaining identical at 0.037.
3.4. Nutritional Composition of Eggs
The chemical analysis of eggs at Weeks 4 and 8 revealed that most nutritional parameters remained relatively stable, although some dose‐dependent trends were observed.
EPA and DHA values are expressed as g per 100 g of egg. Values reported as ‘< 0.1’ indicate concentrations below the limit of quantification of the analytical method.
EPA and DHA were detected at very low levels, close to or below the limit of quantification, and therefore, small numerical differences should be interpreted with caution. Although no statistically significant differences were observed among groups, changes in the omega‐6/omega‐3 ratio and cholesterol content are discussed as descriptive trends rather than definitive nutritional effects. The absence of confidence intervals and the low concentrations of long‐chain n‐3 fatty acids limit the strength of nutritional interpretations.
The mean omega‐6/omega‐3 ratio was calculated, and in the control group, it was the lowest—7.75:1, whereas in the treatment groups this ratio was higher (10.66:1 in group CV1, 10.33:1 in group CV3 and 16:1 in group CV5).
Regarding cholesterol, the CV5 group exhibited a notable increase in cholesterol levels (mean = 275.0 mg/100 g) compared to the CON group (mean = 213.5 mg/100 g), representing a 28.8% increase. In contrast, the lowest dose (CV1) resulted in a slight reduction in cholesterol (196.5 mg/100 g). Omega‐3 fatty acid content showed a downward trend with increasing CV supplementation, decreasing from 0.2 g/100 g in the control group to 0.1 g/100 g in the CV5 group.
The protein content remained consistent across groups, ranging from 12.0 to 12.6 g/100 g. Similarly, fat content (8.85– 9.35 g/100 g) and energy values (563–585.5 kJ/100 g) did not show significant deviations from the control group (p > 0.05).
A two‐way ANOVA indicated that while the group effect was nonsignificant for most nutrients due to the limited sample size of composite samples, the factor of time approached significance for protein content (F(1, 3) = 6.82, p = 0.079), suggesting minor fluctuations in egg composition as the laying period progressed (Table 4).
TABLE 4.
Change of nutritional composition of eggs.
| Parameter | CON (Baseline) | CV1 (0.1%) | CV3 (0.3%) | CV5 (0.5%) |
Trend / Change (CV5 vs. CON) |
|---|---|---|---|---|---|
| Cholesterol (mg/100 g) |
213.5 [156.3, 270.7] |
196.5 [0.0, 533.2] |
216.0 [0.0, 546.4] |
275.0 [198.8, 351.2] |
+28.8% |
| Protein (g/100 g) |
12.2 [9.7, 14.7] |
12.6 [12.6, 12.6] |
12.0 [6.9, 17.1] |
12.4 [7.3, 17.5] |
+1.6% |
| Fat (g/100 g) |
9.3 [9.3, 9.3] |
9.3 [8.6, 9.9] |
8.9 [5.7, 12.0] |
9.4 [4.9, 13.8] |
+0.5% |
| Energy (kJ/100 g) |
578.5 [534.0, 623.0] |
574.5 [568.2, 580.9] |
563.0 [474.1, 651.9] |
585.5 [337.7, 833.3] |
+1.2% |
| SFA (g/100 g) |
2.75 [2.11, 3.39] |
2.75 [2.11, 3.39] |
2.65 [2.01, 3.29] |
2.80 [1.53, 4.07] |
+1.8% |
| MUFA (g/100 g) |
4.60 [3.33, 5.87] |
4.70 [3.43, 5.97] |
4.35 [2.44, 6.26] |
4.65 [1.47, 7.83] |
+1.1% |
| PUFA (g/100 g) |
1.65 [1.01, 2.29] |
1.75 [0.0, 3.66] |
1.70 [1.70, 1.70] |
1.70 [1.70, 1.70] |
+3.0% |
| Omega‐3 (g/100 g) |
0.20 [0.20, 0.20] |
0.15 [0.0, 0.79] |
0.15 [0.0, 0.79] |
0.10 [0.10, 0.10] |
−50.0% |
| Omega‐6 (g/100 g) |
1.55 [0.91, 2.19] |
1.60 [0.33, 2.87] |
1.55 [0.91, 2.19] |
1.60 [1.60, 1.60] |
+3.2% |
| Omega‐9 (g/100 g) |
4.05 [3.41, 4.69] |
4.15 [3.51, 4.79] |
3.85 [1.94, 5.76] |
4.15 [0.97, 7.33] |
+2.5% |
| EPA (g/100 g) | < 0.10 | < 0.10 | < 0.10 | < 0.10 | NA |
| DHA (g/100 g) | < 0.10 | < 0.10 | < 0.10 | < 0.10 | NA |
Note: Values are expressed as mean [95% confidence interval]. The 95% CI was calculated based on measurements at Weeks 4 and 8. Lower CI bounds were adjusted to 0 where calculation resulted in negative values due to limited sample size.
3.5. Observed Power of Analysis
To evaluate the reliability of the statistical inferences, an observed power (post hoc power) analysis was performed for the primary productivity metrics based on the observed effect sizes, sample sizes (N = 84) and an alpha level of 0.05.
The LMM for laying intensity yielded a high F‐ratio (F = 22.5), resulting in an observed power of > 0.99. This indicates that the study was highly sensitive to the effects of C. vulgaris supplementation on egg production rate, providing a near‐certain probability of detecting the observed differences.
For the average egg weight, where the group effect was marginally significant (p = 0.047, F = 2.73), the observed power was approximately 0.65. This suggests that while a significant effect was detected, the variance associated with the random factor of time and the relatively small effect size of the microalgae on weight reduced the statistical sensitivity for this specific parameter compared to laying intensity.
The GLMM demonstrated a significant effect for the highest dose group (CV5, p = 0.021). The observed power for this count‐based model remained adequate to support the conclusion that 0.5% CV supplementation significantly increases total egg production.
For laboratory‐based nutritional parameters, the observed power was limited by the small number of composite samples. Consequently, these results are interpreted as experimental trends rather than definitive population estimates.
4. Discussion
The Novel Foods Regulation (EU) 2015/2283 mandates a pre‐market safety assessment for all novel foods before they can be sold in the EU. Foods not widely consumed in the EU prior to 15 May 1997 are classified as ‘novel’, with this date serving as the reference point for determining whether a safety evaluation is required. Microalgae with a confirmed history of consumption in the EU before 1997 are exempt from this assessment (European Parliament 2015). This includes commonly used commercial species such as Spirulina (Cyanobacteria), Chlorella (Chlorophyta) and Aphanizomenon flos‐aquae (Cyanobacteria). Microalgae‐based products can be marketed either as whole cells, offering diverse nutritional profiles, or as high‐value extracts containing polyunsaturated fatty acids (PUFA), essential amino acids, enzymes, vitamins, carotenoids and polysaccharides (Matos et al. 2017).
Different types of microalgae, such as Spirulina, Chlorella and others, are widely used in laying hen diets to enhance egg production and quality (Selim et al. 2018; Kalia and Lei 2022; An et al. 2014). In our study, we observed that the inclusion of 0.5% C. vulgaris biomass (CV5) significantly increased egg production 18.3 [17.2, 19.4] compared to the control group (CON) 16.6 [15.7, 17.6] (p = 0.021, Exp(B) = 1.099). These results align with findings from other studies. For example, Kim and Kang (2015) found that higher levels of C. vulgaris (2.5%, 5% and 7.5%) positively influenced egg production, with no effect on egg weight. Zheng et al. (2012) also observed that feeding laying hens aged 80 weeks with fermented C. vulgaris (CBT) at 0, 1.000 or 2.000 mg/kg for 42 days led to a significant increase in egg production (p < 0.05), while egg weight remained unchanged. These findings are consistent with ours, as we did not observe significant differences (p > 0.05) in mean egg weight between the control group and the groups receiving 0.1%, 0.3% or 0.5% C. vulgaris. However, the heaviest eggs were produced by hens in the CV5 group (67.4 [65.2, 69.5] g), and the lowest in the CV3 group (63.8 [61.6, 65.9] g). In our study, we observed that laying performance improved with increasing dose of C. vulgaris; the eggs themselves did not significantly differ in size between the experimental groups and the control group. In other words, the hens did not start laying more eggs at the expense of making their eggs smaller. The eggs remained the same weight, so the improvement was real and not due to a reduction in quality or size. A comparable outcome was reported by Halle et al. (2009), where dietary supplementation with 0.25%, 0.5% or 0.75% C. vulgaris had no effect on egg weight. Interestingly, Kor and Mohamadi (2015) found that although laying performance was not significantly affected by adding 100, 200 or 400 ppm C. vulgaris to drinking water, mean egg weight improved with increasing levels of the microalga.
Supplementation of C. vulgaris affected laying intensity, with higher values observed in the CV3 (0.843 [0.815, 0.870]) and CV5 (0.908 [0.880, 0.936]) groups compared to the control (0.792 [0.764, 0.820]). In contrast, hens in the CV1 group had a laying intensity of 0.792 [0.764, 0.820], which was identical to the control group, indicating no improvement at the lowest supplementation level. The CV5 dose resulted in the highest laying intensity and outperformed both CV1 and CV3, indicating a clear dose‐dependent improvement in laying performance. Many other authors have the same observations about the impact of C. vulgaris on laying intensity. For example, Halle et al. (2009) in the 8‐month‐long study noticed a high level of laying intensity throughout the whole period, ranging from 93% to 96%, and the authors did not find any statistical difference among groups. The authors concluded that the laying intensity was at a very high level, and this proves that microalga C. vulgaris at levels 0.25%, 0.5% or 0.75% did not cause any adverse effects. Similar results were observed by Madacussengua et al. (2024) in their 16‐week study involving forty‐eight 19‐week‐old commercial laying hens, supplemented with 2.5%, 5% and 10% C. vulgaris. The study found no significant differences in laying intensity among the treatment and control groups. Laying intensity remained high across all groups, ranging from 96.9% in the group receiving 2.5% C. vulgaris to 98.6% in both the control group and the group receiving 10% C. vulgaris. Also, Kim et al. (2023) with 21‐week‐old laying hens in a 4‐week‐long study with adding 0.5% of C. vulgaris to the basal diet did not find a significant difference in laying intensity.
Regarding the composition of eggs in the control group and those groups which additionally received C. vulgaris microalga biomass to the basal diet at levels 0.1%, 0.3% and 0.5%, we did not notice any significant difference between the tested parameters. Omega‐3 fatty acid content decreased with increasing supplementation, from 0.2 [0.20, 0.20] g/100 g in the control to 0.1 [0.10, 0.10] g/100 g in the CV5 group. Protein (12.0–12.6 g/100 g), fat (8.85–9.35 g/100 g) and energy content (563–585.5 kJ/100 g) were not significantly affected.
Recent reviews and meta‐analyses indicate that the effects of microalgae supplementation on egg fatty acid composition are highly variable and depend on algal species, inclusion level, lipid profile of the biomass and basal diet composition. In particular, green microalgae C. vulgaris and cyanobacteria Spirulina/Limnospira differ in their lipid composition and metabolic utilization, which can lead to inconsistent effects on n‐3 deposition and omega‐6/omega‐3 ratios (Kusuma et al. 2025; Abdel‐Wareth et al. 2024; Madacussengua et al. 2025; Lemahieu et al. 2013; Thapa 2020). These findings suggest that changes in egg fatty acid profiles should be interpreted in the context of overall dietary lipid inputs rather than attributed solely to microalgae supplementation (Spínola et al. 2024).
In our study, a clear trend was observed in the ratio of omega‐6 to omega‐3 fatty acids. This ratio was lowest in the control group (7.75:1) and progressively higher in the experimental groups, ranging from 11.75:1 in CV1 and CV3 to 16:1 in CV5. Overall, these results indicate a trend toward an increased omega‐6 to omega‐3 ratio with the addition of C. vulgaris to the diet of laying hens. It has been studied that early humans ate foods with a ratio of omega‐6 to omega‐3 essential fatty acids of approximately 1:1, while Western diets have a ratio of 15:1–16.7:1, up to 20:1 and more. Western diets are deficient in omega‐3 fatty acids and have an excess of omega‐6 fatty acids. The optimal ratio of these fatty acids is approximately 5:1 (Bishehkolaei et al. 2024; Simopoulos 2002).
A higher omega‐6/omega‐3 ratio in the blood is associated with an increased risk of mortality from various causes, such as cancer and cardiovascular disease. These findings highlight the need to balance omega‐6 and omega‐3 intake in the diet to promote better health and reduce the risk of chronic diseases (Simopoulos 2002).
When comparing the obtained omega‐6/omega‐3 ratios with the recommended values for human nutrition (5:1), a significantly elevated ratio was observed in all experimental groups. Even in the control group (CON), where the ratio was the lowest at 7.75:1, it exceeded the recommended maximum by about 1.5 times. In the CV1, CV3 (11.75:1) and CV5 (16:1) groups, the ratio was approximately two to three times higher than the optimal range.
Although omega‐9 fatty acids, especially oleic acid, are not classified as essential, they are nevertheless necessary for cardiovascular health. Oleic acid can contribute to the reduction of low‐density lipoprotein cholesterol levels while maintaining high‐density lipoprotein cholesterol, and therefore, improved omega‐9 content may be a desirable criterion for evaluating egg quality. Eggs with a high proportion of omega‐9 complement the content of healthy fatty acids (Schwingshackl and Hoffmann 2014).
Regarding the fatty acid content in eggs, interesting results were observed in a study conducted by Panaite et al. (2023). It was an 8‐week study on 120 Lohmann Brown laying hens (38 weeks old), supplementing their diet with 2% C. vulgaris powder. The total amounts of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) did not differ significantly between the groups. However, PUFA were significantly lower in the C. vulgaris group compared to the control. Additionally, the omega‐6 to omega‐3 ratio was significantly lower in the C. vulgaris group (11.51) than in the control group (17.40).
The variations in cholesterol and energy value between groups were also not statistically significant. A dose‐dependent effect was observed for cholesterol, with the CV5 group showing higher levels (275.0 [198.8, 351.2] mg/100 g) compared to the control (213.5 [156.3, 270.7] mg/100 g), while CV1 resulted in a slight reduction (196.5 [0.0, 533.2] mg/100 g). A similar trend, in which cholesterol content in eggs did not differ significantly, was reported by Panaite et al. (2023) in the study mentioned above. In the literature, the average cholesterol content in one egg is estimated to be around 200 mg; however, this may vary depending on the breed of bird, feed and the conditions of keeping laying hens (Usturoi et al. 2025; Fuller et al. 2015). Historically, dietary guidelines recommended limiting cholesterol intake to 300 mg per day (Vergara et al. 2021). Higher daily egg consumption may increase cardiovascular disease risk by raising low‐density lipoprotein cholesterol level in the blood and also increasing the ratio of low‐density to high‐density lipoprotein cholesterol (Li et al. 2020). Thus, the cholesterol content in eggs remains important in assessing both product quality and nutritional value. In our study, although the increase in cholesterol levels in the treatment groups was not statistically significant, it was consistently higher than in the control group. This trend may have meaningful nutritional or health implications, warranting further investigation with additional measurements in a longer study.
5. Conclusions
Overall, this study indicates that C. vulgaris microalgal biomass shows potential to enhance laying hen productivity and increase egg weight. The highest productivity, with a higher number of eggs and daily production rate and the heaviest eggs, was observed in the treatment group that received the highest dose of C. vulgaris (0.5%) in addition to the basal diet. Although egg weight did not differ significantly between groups, this suggests that the increased egg production and higher number of eggs did not occur at the expense of egg quality (weight), which is a positive aspect from a production perspective. The inclusion of C. vulgaris in the diet at levels up to 0.5% did not result in significant changes in the nutritional parameters of the eggs. However, a trend toward an increased mean omega‐6 to omega‐3 ratio and cholesterol content was observed with increasing doses of C. vulgaris. These findings should be interpreted as indicative trends rather than definitive effect. Further long‐term studies are recommended to determine the full potential of this microalga to enhance productivity, egg weight and egg composition, with greater focus on fatty acid and cholesterol profiles to support the production of eggs with an improved fat composition for human consumption.
Author Contributions
Sintija Jonova: methodology, data curation, investigation, formal analysis, visualization, writing – original draft. Alma Plivca: investigation, formal analysis, writing – review and editing. Anete Freiberga: conceptualization, methodology, investigation, formal analysis, writing – review and editing. Dace Gorbacevska: methodology, investigation, formal analysis, writing – review and editing. Agris Ilgazs: methodology, investigation, resources, writing – review and editing. Sabine Eglite: conceptualization, methodology, formal analysis, writing – review and editing. Maksims Zolovs: data curation, formal analysis, visualization. Oto Jekabs Apse: methodology, resources, writing – review and editing. Pavels Semjonovs: conceptualization, validation, supervision, funding acquisition, project administration, writing – review and editing. Aija Ilgaza: conceptualization, methodology, supervision, funding acquisition, project administration, writing – review and editing.
Funding
This study was supported by the Project ‘Development of a plant feed additive for strengthening poultry immunity and increasing the nutritional value of eggs with omega‐3 fatty acids’ (Grant No.: 22‐00‐A01612‐000015), the EAFRD, the Ministry of Agriculture and the Rural Support Service of Latvia.
Ethics Statement
The animal study was approved by the Food and Veterinary Service (Latvia) (Pilot project permit No. 152/2024 ‘The effect of feeding microalgae on the health, productivity and production quality of laying hens and broiler chickens’). The study was conducted in accordance with the local legislation and institutional requirements.
Conflicts of Interest
The authors declare no conflicts of interest.
Acknowledgements
The authors have nothing to report.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.