Dietary fish oil, microalgae, and plant oils differentially influence egg quality, yolk fatty acid composition, nutritional lipid quality indices, and fatty acid transfer efficiency in laying hens

Abstract

This study evaluated the effects of different dietary omega-3 fatty acid sources on production performance, egg quality, yolk fatty acid composition, fatty acid transfer efficiency, nutritional lipid quality indices, and blood biochemical parameters in laying hens. A total of 175 Super Nick White laying hens (54 weeks of age) were randomly assigned to five dietary treatments for 14 weeks: a control diet containing 3% sunflower oil, 3% flaxseed oil, 3% hempseed oil, 3% fish oil, or 1.5% microalgae (Schizochytrium spp.) + 1.5% sunflower oil (MALG). Dietary treatments had no significant effects on feed intake, feed conversion ratio, egg production, egg weight, egg mass, or blood biochemical parameters (P > 0.05). However, egg quality traits were differentially affected by dietary oil source. Fish oil and MALG significantly improved Haugh unit, whereas hempseed oil reduced eggshell weight, shell ratio, and shell thickness but increased shell weight per unit surface area (P < 0.05). Yolk color characteristics were significantly altered, with MALG reducing Roche yolk color score, redness, and brightness (P < 0.05). Flaxseed oil markedly increased yolk α-linolenic acid, while fish oil and MALG significantly increased docosahexaenoic acid (DHA) concentrations (P < 0.05). Hempseed oil and MALG exhibited greater DHA transfer efficiency than fish oil, whereas fish oil showed the highest α-linolenic acid transfer rate (P < 0.05). Dietary lipid source also significantly influenced nutritional lipid quality indices. Flaxseed oil produced the lowest n-6/n-3 ratio and atherogenicity index, whereas hempseed oil yielded the highest PUFA/SFA ratio (P < 0.05). In conclusion, dietary omega-3 supplementation improved the nutritional quality of eggs without compromising laying performance or health status. Among the tested lipid sources, microalgae emerged as an effective strategy for enhancing yolk DHA while maintaining overall egg quality.

Introduction

The enrichment of poultry products with health-promoting nutrients has become an important strategy to improve the nutritional quality of foods for human consumption (Rafique et al., 2025; Sultan et al., 2025; Wahyuni et al., 2025). Among these nutrients, omega-3 polyunsaturated fatty acids (n-3 PUFA) have gained substantial attention because of their beneficial effects on cardiovascular health, immune function, neural development, and inflammatory regulation in humans (Hidayaturrahmah et al., 2025). Eggs are considered an ideal functional food for omega-3 enrichment due to their widespread consumption, high nutrient density, and the efficient transfer of dietary fatty acids into egg yolk lipids (Raza et al., 2016).

Conventional poultry diets are generally rich in omega-6 fatty acids but relatively deficient in omega-3 fatty acids, resulting in eggs with a high n-6:n-3 ratio (Kartikasari et al., 2024). Therefore, nutritional manipulation through dietary supplementation of omega-3-rich ingredients has emerged as an effective approach to modify yolk fatty acid composition and produce omega-3-enriched eggs (Sultan et al., 2015). Various dietary lipid sources such as flaxseed oil, fish oil, hempseed oil, and microalgae have been investigated for their potential to enhance the deposition of α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) in eggs (Abbas et al., 2022; Ghaderi‐Chaparabad et al., 2025).

Previous studies have demonstrated variable effects of omega-3-rich dietary oils on laying performance and egg quality. Some reports indicated improvements in yolk fatty acid composition without detrimental effects on feed intake, egg production, or egg weight, whereas others observed alterations in yolk color, shell characteristics, albumen quality, and lipid metabolism depending on the type and inclusion level of oil sources (Gao et al., 2022; Zaazaa et al., 2022). Flaxseed oil is recognized as a rich source of ALA, whereas fish oil and microalgae provide preformed long-chain omega-3 fatty acids, particularly EPA and DHA (Altaçlı et al., 2022). Hempseed oil has also attracted attention because of its balanced fatty acid profile and antioxidant components (Hossain et al., 2026). However, differences among these oil sources in terms of fatty acid transfer efficiency, egg quality characteristics, oxidative stability, and bird performance remain incompletely understood. Moreover, dietary inclusion of highly unsaturated fatty acids may influence egg sensory properties, yolk pigmentation, shell quality, lipid metabolism, and physiological responses in laying hens.

Microalgae have recently emerged as a promising sustainable source of long-chain omega-3 fatty acids for poultry feeding because they provide natural DHA without reliance on marine fish resources (Kumari et al., 2024). In addition to their nutritional value, microalgae contain bioactive compounds such as carotenoids, antioxidants, vitamins, and pigments that may influence egg quality and bird physiology (Panaite et al., 2023). Because commercially available microalgae products are often supplied as concentrated lipid-rich ingredients, they are commonly incorporated into poultry diets using conventional vegetable oils to facilitate uniform mixing, improve feed homogeneity, and maintain comparable dietary energy density among treatments. Sunflower oil is frequently used for this purpose because of its high oxidative stability, widespread availability, and compatibility with poultry feed formulations, while contributing minimal amounts of long-chain omega-3 fatty acids. Similarly, fish oil remains one of the most effective dietary sources for increasing yolk DHA concentration, although concerns related to oxidative stability and off-flavor development persist (Wen et al., 2023). Flaxseed oil and hempseed oil are plant-derived alternatives that may improve the omega-3 profile of eggs while maintaining production efficiency (El‐Zenary et al., 2023).

Although numerous studies have evaluated individual omega-3 sources, few have directly compared multiple plant- and marine-derived lipid sources under identical experimental conditions. Furthermore, direct comparisons require diets to be formulated with similar energy density and lipid inclusion levels so that any observed responses can be primarily attributed to differences in fatty acid composition rather than total dietary fat. Therefore, sunflower oil was included as the carrier oil in the microalgae treatment to standardize dietary lipid inclusion while allowing evaluation of the biological effects of the microalgal omega-3 source. Therefore, the present study evaluated the effects of sunflower oil, flaxseed oil, hempseed oil, fish oil, and microalgae supplementation on production performance, egg quality, yolk fatty acid composition, apparent fatty acid deposition, and blood biochemical parameters in laying hens.

Materials and methods

Animals and experimental design

A total of 175 commercial Super Nick White laying hens at 54 weeks of age were used in this study. All birds had been vaccinated against major infectious diseases during the chick and pullet phases, following a standard commercial vaccination program, and were previously fed balanced commercial diets formulated for pullets and laying hens. Prior to the start of the experiment, all hens were individually weighed and allocated to treatment groups to minimize variation in body weight and baseline performance among groups. A 14-day pre-experimental period was conducted using a control diet, during which egg production was recorded to ensure uniformity among groups in terms of body weight, egg production, and incidence of abnormal eggs. Birds showing consistently low egg production during this adaptation phase were replaced with healthy, actively laying hens from the same flock.

The hens were housed in individual wire laying cages (L45 × W32 × H35 cm) arranged in a four-tier battery system, with three cages per unit. Throughout the experimental period, hens were maintained under standard environmental conditions. The house temperature was maintained at 22 ± 2°C with a relative humidity of 60 ± 5%. A lighting schedule of 16 h light and 8 h dark was provided daily using artificial illumination to ensure a consistent photoperiod. The poultry house was naturally ventilated with supplemental mechanical ventilation when required to maintain adequate air exchange and minimize the accumulation of heat, moisture, and ammonia. Birds had ad libitum access to feed and fresh drinking water throughout the experiment. The experimental diets were provided in mash form and consisted of five dietary treatments: (1) control diet (3% sunflower oil (SFO), (2) 3% flaxseed oil (FLO), (3) 3% hempseed oil (HSO), (4) 3% fish oil (FO), and (5) 1.5% microalgae + 1.5% sunflower oil (MALG). The 1.5% microalgae + 1.5% sunflower oil combination was used to provide a balanced lipid inclusion (total 3%) while partially replacing plant oil with a rich source of long-chain n-3 fatty acids (EPA and DHA) from microalgae. Comparative overview of the major fatty acid profiles (%) of the oil sources used in the experimental diets are given in Table 1.

Table 1.

Comparative overview of the major fatty acid profiles (%) of the oil sources used in the experimental diets.

Fatty Acid (%)	Sunflower Oil	Flaxseed Oil	Hempseed Oil	Fish Oil	MALG
Linoleic acid (C18:2 n-6)	67	18	55	<5	<5
α-Linolenic acid (C18:3 n-3)	≤1	41	23	<5	<1
Oleic acid (C18:1 n-9)	29	19	16	9	<5
Palmitic acid (C16:0)	5	5	5	9	6
Stearic acid (C18:0)	3	3	2	6	<5
EPA (C20:5 n-3)	Not detected	Not detected	Not detected	10–18	Trace
DHA (C22:6 n-3)	Not detected	Not detected	Not detected	15	50
Omega-6: Omega-3 ratio	67:1	0.44: 1	2.3:1	<1	<1

MALG: 1.5% microalgae + 1.5% sunflower oil. 

This design allows evaluation of whether a reduced level of microalgae, combined with a stable carrier oil (sunflower oil), can efficiently improve fatty acid deposition in eggs while maintaining diet palatability, energy balance, and feed stability. Each treatment was evenly distributed across cage locations to minimize positional effects. Physical partitions were installed between feeders to prevent cross-feeding among adjacent cages, and each cage was equipped with one nipple drinker.

The birds were allocated in a completely randomized design, with five replicates per treatment. Each replicate consisted of a defined number of hens housed under uniform management conditions. The experimental period lasted for 14 weeks.

Ingredients and nutritional composition of basal diet

The experimental diets were formulated to meet the nutrient requirements of laying hens according to the National Research Council NRC (1994). All diets were isocaloric and isonitrogenic to ensure uniform nutrient supply across treatments. The sunflower oil, flaxseed oil, and hempseed oil were obtained from commercially available food-grade vegetable oil suppliers, while the fish oil (CCPA Turkey, 8 Eylül Mah. Ansızca Yolu Üzeri No:108/A, 35730 Kemalpaşa, İzmir, Türkiye) was provided as a purified feed-grade marine oil rich in EPA and DHA. The microalgae product used in this study was DHAgold® (Schizochytrium spp.; dsm-firmenich Animal Nutrition & Health, Wurmisweg 576, CH-4303 Kaiseraugst, Switzerland), a commercially available DHA-rich microalgal biomass intended for animal nutrition. According to the manufacturer's specifications, DHAgold® contains approximately 50% docosahexaenoic acid (DHA) of total fatty acids and is composed of approximately 93% dry matter, 18–20% crude protein, 40–45% crude fat, 15–20% crude fiber, 5–7% ash, and 2–4% moisture. In addition to DHA, the product contains natural carotenoids, antioxidants, and other bioactive compounds that contribute to its oxidative stability and nutritional value. Feed ingredients were ground using a 6-mm hammer mill and thoroughly mixed using a feed mixer (Kocamaz 500, İzmir, Türkiye). The ingredient composition and chemical analysis of the experimental diets are presented in Table 2, while the fatty acid composition of the experimental diets is presented in Table 3. Prepared feeds were sealed in airtight containers and stored in a cool, shaded area until use.

Table 2.

Composition of experimental diets.

Ingredients (g/kg)	Control	Flaxseed oil	Hempseed oil	Fish oil	MALG
Yellow corn	515.00	515.00	515.00	515.00	515.00
Wheat	43.97	43.97	43.97	43.97	43.97
Sunflower oil	30.00	-	-	-	15
Flaxseed oil	-	30.00	-	-	-
Hempseed oil	-	-	30.00	-	-
Fish oil	-	-	-	30.00	-
Microalgae (Schizochytrium spp.)	-	-	-	-	15.0
Soybean meal, 46	156.69	151.69	151.69	151.69	150.48
Vegetable oils	30.00	30.00	30.00	30.00	26.00
Salt, NaCl	3.19	3.19	3.19	3.19	3.20
Vitamin and mineral premix*	2.00	2.00	2.00	2.00	2.00
Di-calcium phosphate	4.80	4.80	4.80	4.80	4.30
Limestone	94.15	94.15	94.15	94.15	94.47
Methionine	0.55	0.55	0.55	0.55	0.55
Multi-enzyme	0.50	0.50	0.50	0.50	0.50
Wheat bran	59.15	59.15	59.15	59.15	58.5
Microalgae	0	0	0	0	15
Total	1000	1000	1000		1000
Analyzed Chemical composition
Dry matter, %	92.02	92.26	92.41	92.16	92.61
Crude protein, %	17.56	17.45	17.18	17.86	17.88
Crude fat, %	5.37	5.38	5.38	5.38	5.38
ADF	1.59	1.59	1.59	1.59	1.89
NDF	4.14	4.14	4.14	4.14	4.42
Ash, %	14.35	14.41	14.65	14.05	14.38
Calculated Chemical composition⁎⁎
Methionine, %⁎⁎	0.36	0.36	0.36	0.36	0.36
Lysine, %⁎⁎	0.77	0.77	0.77	0.77	0.77
Calcium, %⁎⁎	3.70	3.70	3.70	3.70	3.70
Available phosphorus, %⁎⁎	0.36	0.36	0.36	0.36	0.36
Metabolic Energy Kcal ME/kg⁎⁎⁎	2730	2730	2730	2730	2730

§: Oil type was changed according to treatment as shown top of column.

^⁎Vitamin-mineral premix per kilogram of the diet, Vitamin D3 2000 IU; Vitamin A, 15.000 IU; Vitamin E, 40.0 mg; Vitamin B1 (thiamine), 3.0 mg; Vitamin K, 5.0 mg; Vitamin B6, 5.0 mg; Vitamin B12, 0.03 mg; Vitamin B2 (riboflavin), 6.0 mg; Biotin, 0.1 mg; Niacin, 30.0 mg; Folic acid, 1.0 mg, Calcium d-pantothenate, 12 mg; Choline chloride, 400 mg, Iron, 35.0 mg; Manganese, 80.0 mg; Zinc, 50.0 mg; Iodine 2.0 mg; Cobalt, 0.4 mg; Copper, 5.0 mg; Selenium, 0.15 mg assures.

^⁎⁎Compositions are calculated according to NRC feedstuff tables (1994).

^⁎⁎⁎ME values calculated according to TSE (1994); MALG: 1.5% microalgae + 1.5% sunflower oil. 

Table 3.

Fatty acid composition of the experimental diets based on diet formulation and the reported fatty acid composition of the supplemental oil sources (% of total dietary fatty acids).

Fatty acid (% total FA)	Control	Flaxseed oil	Hempseed oil	Fish oil	MALG*
Palmitic acid (C16:0)	5.0	5.0	5.0	13.0	9.0
Stearic acid (C18:0)	3.0	3.5	2.5	3.0	2.5
Oleic acid (C18:1 n-9)	29.0	20.0	16.0	18.0	10.0
Linoleic acid (C18:2 n-6)	67.0	18.0	55.0	5.0	33.5
α-Linolenic acid (C18:3 n-3)	0.8	41.0	23.0	2.0	0.5
EPA (C20:5 n-3)	ND	ND	ND	12.0	Trace
DHA (C22:6 n-3)	ND	ND	ND	15.0	25.0
Total n-3 PUFA	0.8	41.0	23.0	29.0	25.5
Total n-6 PUFA	67.0	18.0	55.0	5.0	33.5
Estimated n-6:n-3 ratio	84:1	0.44:1	2.39:1	0.17:1	1.31:1

ND: Not detected.

Trace: <1% of total fatty acids.

MALG: Diet containing 1.5% Schizochytrium sp. microalgae and 1.5% sunflower oil.

Determination of feed consumption, and feed conversion ratio

Feed consumption was recorded per cage at 14-day intervals and calculated as the difference between feed offered and feed refused. Average daily feed intake per bird was calculated by dividing total feed intake by the number of feeding days (14) and the number of birds per cage. Feed conversion ratio (FCR) was calculated every two weeks as the ratio of feed intake (g) to egg mass (g). Egg mass was calculated using the following formula: egg mass = (egg production (%) × mean egg weight) / 100.

Evaluation of egg yield, egg weight, and internal quality traits

Egg production was recorded daily, and egg yield was expressed as percentage (%) for each 14-day period. Egg production percentage was calculated as: (total number of eggs produced per hen during each 14-day period ÷ 14) × 100. Egg mass per period was calculated as egg weight (g) × egg production (%).

For egg quality evaluation, a total of 24 eggs were collected during the last two days of each period (days 14, 28, 42, 56, 70, 84, and 98), resulting in 120 eggs in total. Egg weight was measured using a precision balance (±0.1 g). Internal quality parameters, including albumen height, Haugh unit, and yolk color (Roche scale), were determined using an Egg Analyzer® (Orka Food Technology, USA). Yolk color coordinates (L*, b*, and a*) were measured using a Minolta CR-400 colorimeter (Minolta Co., Japan) at 14-day intervals.

Determination of eggshell properties

Eggshells were separated from broken eggs, washed with tap water, and dried at 75°C for 24 h. Dried shells were weighed using a precision balance (±0.1 g). Eggshell percentage was calculated as: (shell weight ÷ egg weight) × 100. Eggshell thickness (µm) was measured using a digital micrometer (±0.01 mm) at three locations (sharp end, blunt end, and equator), and the mean value was used for analysis. Eggshell surface area (SSA) was calculated using the equation described by Carter (1976). Shell weight per unit surface area (USW, mg/cm²) was determined by dividing shell weight by egg surface area.

Determination of fatty acid composition of feed and egg yolk

Fatty acid (FA) profiles of diets, dietary oil sources and egg yolks were analyzed according to the Shimadzu application protocol. For FA analysis, eggs were collected during week 2, 4, 6, 8, 10, and 14. In each week, 40 eggs were sampled across all treatment groups (8 eggs per group). Two yolks from selected samples were pooled, cooked in boiling water (10 min), cooled, and stored at −20°C until analysis. For analysis, 1 g of egg yolk was placed in a 15 mL tube, mixed with 5 mL hexane, and vortexed for 3 min. The upper phase was transferred to a 5 mL tube, and 0.5 mL KOH was added. The mixture was vortexed for 5 min, and 0.5 mL of the final solution was transferred into a GC vial. Fatty acid composition was determined using gas chromatography (Shimadzu GC-2010 Plus, Japan) as described by Konca et al. (2019). Results were expressed as g FA/100 g total fatty acids. Fatty acid transfer efficiency from feed to egg was calculated as the ratio of FA content in eggs to FA content in feed for each experimental week.

Determination of the transfer rate of dietary fatty acids into egg yolk

The transfer rate (change ratio) of each fatty acid was calculated by dividing the concentration of a specific fatty acid detected in egg yolk by its corresponding concentration in the experimental diet, according to the following equation:

$$\text{Transfer}\text{rate}=\frac{\text{Egg}\text{yolk}\text{fatty}\text{acid}\text{concentration}}{\text{Dietary}\text{fatty}\text{acid}\text{concentration}}$$

Determination of nutritional lipid quality indices

To further evaluate the nutritional quality of egg yolk lipids, the n-6/n-3 polyunsaturated fatty acid ratio, PUFA/SFA ratio, and atherogenicity index (AI) were calculated from the fatty acid composition of egg yolk. The n-6/n-3 ratio was calculated as the sum of n-6 polyunsaturated fatty acids divided by the sum of n-3 polyunsaturated fatty acids. The PUFA/SFA ratio was calculated as the ratio of total polyunsaturated fatty acids to total saturated fatty acids. The AI was calculated according to the equation proposed by Ulbricht and Southgate (1991):

$$\text{AI}-\frac{C12:0+4(C14:0)+C16:0}{\sum \text{MUFA}+\sum \text{PUFA}}$$

Determination of blood components

At the end of the experiment, blood samples (10 mL) were collected from the wing vein of eight randomly selected hens per group. Serum was separated by centrifugation (5 min, 2500 rpm) and stored at −80°C in Eppendorf tubes until analysis. On the day of analysis, samples were thawed and analyzed for glucose, total protein, triglycerides, cholesterol, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) using an automated biochemical analyzer with commercial diagnostic kits (Biocheck, Inc, UK).

Statistical Analyses

The experiment was conducted using a completely randomized design. Prior to analysis, the assumptions of normality and homogeneity of variance were evaluated using the Shapiro–Wilk test and Levene’s test, respectively. The data satisfied these assumptions and were therefore considered suitable for parametric analysis. All data were analyzed using the one-way analysis of variance (ANOVA) procedure of SPSS statistical software (Version 25.0; IBM Corp., Armonk, NY, USA). When significant treatment effects were detected, differences among treatment means were separated using Duncan’s multiple range test. Statistical significance was declared at P < 0.05. Results are presented in the tables as means ± standard error of the mean (SEM).

Results

Table 4 summarizes the effects of dietary omega-3 fatty acid sources on production performance and egg quality traits in laying hens. Feed intake, feed conversion ratio, egg production, egg yield, egg weight, egg mass, and eggshell surface area were not significantly affected by dietary treatments (P > 0.05). However, significant differences were observed in several internal and external egg quality parameters. Haugh unit was significantly higher in the fish oil and microalgae groups compared with the hempseed oil group (P = 0.015). Eggshell weight, eggshell ratio, and eggshell thickness were significantly reduced in hens fed the hempseed oil diet compared with the other treatment groups (P = 0.010, P = 0.029, and P = 0.004, respectively). In contrast, eggshell per unit surface area was significantly higher in the hempseed oil group than in the remaining treatments (P = 0.015). Egg albumen height was significantly improved in the hempseed oil and microalgae groups compared with flaxseed oil and fish oil treatments (P = 0.020). Roche yolk color score, yolk redness (a*), and yolk brightness (L*) were significantly lower in the microalgae treatment compared with most other groups (P = 0.001), whereas yolk yellowness (b*) differed among treatments, with lower values observed in flaxseed oil and microalgae groups (P = 0.039).

Table 4.

Effect of different dietary oil sources on production performance and egg quality in laying hens.

	Control	Flaxseed oil	Hempseed oil	Fish oil	MALG	P value
Feed intake (g)	115.19±1.10	115.07±0.67	113.93±0.98	114.08±0.64	115.60±1.12	0.647
FCR	1.81±0.03	1.80±0.02	1.79±0.04	1.75±0.03	1.80±0.03	0.713
Egg production (%)	97.2 ± 0.11	97.9 ± 0.10	97.0 ± 0.12	98.1 ± 0.08	98.4 ± 0.05	0.582
Egg yield ratio (EY %)	97.15±0.76	97.89±0.73	97.07±0.83	98.07±0.57	98.29±0.36	0.615
Egg weight (EW)	65.91±0.76	65.62±0.62	66.31±0.76	66.83±0.68	65.76±0.82	0.777
Egg mass (EM)	64.01±1.14	64.22±0.75	64.43±1.35	65.55±1.11	64.63±1.20	0.889
Haigh unit	59.11±3.47bc	58.91±3.64bc	55.53±3.82c	66.11±2.44ab	68.70±1.09a	0.015*
Eggshell weight (g)	6.01±0.10a	6.07±0.09a	5.63±0.16b	6.06±0.10a	6.18±0.08a	0.010*
Eggshell ratio (ESR %)	9.32±0.11a	9.37±0.07a	8.98±0.21b	9.42±0.09a	9.48±0.05a	0.029*
Eggshell thickness (EST)	0.35±0.00a	0.34±0.00a	0.33±0.01b	0.35±0.00a	0.35±0.00a	0.004⁎⁎
Eggshell surface area (ESA)	76.37±0.62	76.14±0.51	76.70±0.62	77.13±0.55	76.24±0.67	0.772
Eggshell per unit (ES/U)	12.51±0.14b	12.43±0.09b	13.14±0.38a	12.33±0.11b	12.26±0.07b	0.015*
Egg albumen height (cm)	4.73±0.23ab	4.35±0.29b	5.12±0.12a	4.48±0.25b	5.20±0.14a	0.020*
Roche color scale (RRS)	9.46±0.06a	9.47±0.07a	9.57±0.10a	9.68±0.05a	8.93±0.07b	0.001⁎⁎
Egg yolk brightness (L*)	55.60±0.24a	54.50±0.22bc	54.15±0.33c	54.32±0.23c	55.11±0.26ab	0.001⁎⁎
Egg yolk redness (a*)	2.22±0.10a	2.19±0.08a	2.30±0.10a	2.41±0.08a	1.11±0.06b	0.001⁎⁎
Yellowness (b*)	38.19+0.39a	37.21+0.32b	38.21+0.27a	37.55+0.31ab	37.10+0.33b	0.039*

Values bearing different superscripts in a row differ significantly (P < 0.01); MALG: 1.5% microalgae + 1.5% sunflower oil. 

Table 5 shows the effects of dietary omega-3 fatty acid sources on egg yolk fatty acid composition at day 98. Myristic acid, stearic acid, arachidonic acid, and eicosapentaenoic acid concentrations were not significantly influenced by dietary treatments (P > 0.05). Palmitic acid was significantly higher in the fish oil group and lower in the flaxseed oil group compared with the other treatments (P = 0.001). Palmitoleic acid was significantly increased in hens fed flaxseed oil compared with the control, hempseed oil, and microalgae groups (P = 0.038). Oleic acid content was significantly reduced in the hempseed oil group relative to the control, flaxseed oil, and microalgae treatments (P = 0.048). Linoleic acid was significantly elevated in the hempseed oil group and reduced in flaxseed and fish oil groups (P = 0.007). Alpha-linolenic acid content was markedly increased in the flaxseed oil treatment, followed by the hempseed oil group, whereas the control and microalgae groups showed the lowest values (P = 0.001). Docosahexaenoic acid concentration was significantly higher in fish oil and microalgae-fed hens compared with the control group (P = 0.001).

Table 5.

Effect of omega-3 fatty acid oil sources on fatty acids (FAs) composition of egg yolk on d 98.

	Oil sources
Fatty acids (%)	Control	Flaxseed oil	Hempseed oil	Fish oil	MALG	P value
(C14:0) Myristic acid	0.37±0.01	0.49±0.14	0.36±0.03	1.55±1.10	0.64±0.18	0.450
(C16:0) Palmitic acid	26.58±0.25ab	23.57±0.19c	25.31±0.24b	27.60±0.95a	26.07±0.41ab	0.001⁎⁎
(C16:1) Palmitoleic acid	2.63±0.09b	3.29±0.15a	2.66±0.05b	2.97±0.31ab	2.56±0.11b	0.038*
(C18:0) Stearic acid	7.37±0.04	8.55±0.62	8.62±0.27	10.02±1.76	8.23±0.40	0.338
(C18:1n9c) Oleic acid	43.12±0.41a	44.20±1.55a	39.37±0.39b	41.84±1.83ab	44.00±0.52a	0.048*
(C18:2n6c) Linoleic acid	18.65±0.37ab	11.03±3.12c	20.20±0.31a	13.65±1.72bc	16.65±0.41ab	0.007⁎⁎
(C18:3n3) a-Linolenic acid	0.26±0.03d	7.04±0.26a	2.04±0.09b	0.75±0.02c	0.40±0.05cd	0.001⁎⁎
(C20:4n6) Arachidonic acid	0.84±0.03	1.21±0.74	0.86±0.06	0.73±0.06	0.74±0.03	0.846
(C20:5n3) Eicosapentaenoic acid	0.12±0.04	0.08±0.03	0.06±0.04	0.08±0.01	0.07±0.01	0.601
(C22:6n3) Docosahexaenoic acid	0.08±0.00c	0.56±0.09b	0.53±0.08b	0.83±0.08a	0.67±0.02ab	0.001⁎⁎

Values bearing different superscripts in a row differ significantly (P < 0.01); MALG: 1.5% microalgae + 1.5% sunflower oil. 

Table 6 presents the transfer efficiency of dietary fatty acids from feed into egg yolk. Significant differences among treatments were observed for palmitic acid, alpha-linolenic acid, and docosahexaenoic acid transfer rates (P = 0.015, P = 0.001, and P = 0.026, respectively). The transfer rate of alpha-linolenic acid was significantly greater in the fish oil group compared with flaxseed oil, hempseed oil, and microalgae treatments. Docosahexaenoic acid transfer efficiency was significantly enhanced in the hempseed oil and microalgae groups compared with the fish oil treatment. No significant treatment effects were detected for myristic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid, or eicosapentaenoic acid transfer rates (P > 0.05).

Table 6.

Effect of omega-3 fatty acid oil sources on the transfer rate of dietary omega-3 fatty acids into egg yolks.

	Change ratio, egg FA/feed FA
Fatty Acids	Control	Flaxseed oil	Hempseed oil	Fish oil	MALG	P value
(C14:0) Myristic Acid	15.38	1.63	1.50	0.86	1.03	0.056
(C16:0) Palmitic acid	3.09	2.81	2.45	1.89	1.92	0.015*
(C16:1) Palmitoleic acid	24.82	10.43	9.03	1.38	4.63	0.075
(C18:0) Stearic acid	2.88	2.04	2.12	2.74	2.09	0.984
(C18:1n9c) Oleic acid	1.19	1.95	1.82	1.31	1.57	0.212
(C18:2n6c) Linoleic acid	0.33	0.55	0.33	0.40	0.32	0.656
(C18:3n3) a-Linolenic acid	0.40	0.14	0.29	0.53	0.15	0.001⁎⁎
(C20:4n6) Arachidonic acid	1.81	7.30	3.67	0.96	2.88	0.055
(C20:5n3) Eicosapentaenoic acid	9.50	4.00	1.33	0.08	2.25	0.057
(C22:6n3) Docosahexaenoic acid	2.13	4.00	6.22	0.27	5.55	0.026*

Values bearing different superscripts in a row differ significantly (P < 0.01); MALG: 1.5% microalgae + 1.5% sunflower oil. 

Table 7 shows that dietary omega-3 oil sources significantly affected the nutritional lipid quality indices of egg yolk. The n-6/n-3 ratio differed among treatments (P = 0.043), with the flaxseed oil group exhibiting the lowest ratio, followed by the hempseed oil and fish oil groups, which did not differ from each other. The MALG treatment showed an intermediate ratio, whereas the control group had the highest value. The PUFA/SFA ratio was also significantly influenced by dietary treatment (P = 0.027). The hempseed oil group exhibited the highest PUFA/SFA ratio, followed by the control and flaxseed oil groups, which were statistically similar. The MALG treatment showed an intermediate ratio, whereas the fish oil group had the lowest value. Similarly, the AI was significantly affected by dietary oil source (P = 0.036). The fish oil treatment exhibited the highest AI, whereas the flaxseed oil group showed the lowest value. The control, hempseed oil, and MALG treatments displayed intermediate values, with the control and hempseed oil groups not differing significantly from the MALG treatment.

Table 7.

Nutritional lipid quality indices of egg yolk as affected by dietary omega-3 oil sources.

Index	Control	Flaxseed oil	Hempseed oil	Fish oil	MALG	P value
n-6/n-3 ratio	42.37±4.35a	1.59±0.98d	8.01±1.2c	8.66±1.4c	15.25±2.37b	0.043
PUFA/SFA ratio	0.58±0.02b	0.61±0.01b	0.69±0.02a	0.41±0.03d	0.53±0.03c	0.027
Atherogenicity index	0.69±0.01b	0.58±0.03c	0.72±0.01b	0.91±0.02a	0.68±0.04b	0.036

Values with different superscript in a column differ significantly (P < 0.01); Abbreviations: MALG = 1.5% microalgae + 1.5% sunflower oil.

Table 8 shows the effects of dietary omega-3 fatty acid oil sources on blood biochemical parameters of laying hens. Serum glucose, triglycerides, cholesterol, total protein, AST, and ALT concentrations were not significantly affected by dietary treatments (P > 0.05).

Table 8.

Effect of omega-3 fatty acid oil sources on blood parameters (BP) of laying hens.

Parameters	Control	Flaxseed oil	Hempseed oil	Fish oil	MALG	P value
Glucose, mg/dL	227.75±5.35	227.88±6.02	225.38±4.19	230.13±4.35	223.63±2.12	0.878
Triglyceride, mg/dL	951.18±164.15	1073.75±52.02	1256.75±50.01	1253.43±170.41	1120.00±101.25	0.183
Cholesterol, mg/dL	122.40±18.75	110.44±17.52	121.76±15.62	143.35±16.33	93.83±9.51	0.284
Protein, mg/dL	170.63±7.62	184.75±8.24	179.75±6.27	191.75±15.11	181.00±7.85	0.630
AST, IU/L	2.88±0.62	3.040±0.55	3.03±0.67	4.16±0.79	3.21±0.56	0.546
ALT, IU/L	6.12±0.21	5.59±0.20	5.83±0.18	5.99±0.12	5.63±0.10	0.131

Values with different superscript in a column differ significantly (P < 0.01); MALG: 1.5% microalgae + 1.5% sunflower oil. 

Discussion

The present study demonstrated that supplementation of different omega-3 fatty acid-rich oil sources did not significantly affect feed intake, feed conversion ratio, egg production, egg weight, or egg mass of laying hens, indicating that the inclusion levels used in the current experiment were well tolerated and did not impair productive performance. Similar findings have been reported in laying hens fed flaxseed oil, fish oil, or microalgae-based diets, where moderate dietary inclusion of omega-3 sources maintained normal production performance due to adequate dietary energy balance and nutrient digestibility (Javed et al., 2025; Madacussengua et al., 2025; Idowu et al., 2026). The absence of adverse effects on performance may also indicate that the experimental diets remained isocaloric and isonitrogenic, thereby preventing metabolic disturbances associated with altered lipid supplementation.

Although productive performance remained unaffected, several egg quality parameters responded significantly to dietary oil source. Haugh unit and albumen height were improved particularly in fish oil and microalgae-fed hens, suggesting enhanced albumen quality and protein stability (Anam et al., 2025; Javed et al., 2025; Madacussengua et al., 2025). This improvement may be related to the antioxidant properties of omega-3 fatty acids and bioactive compounds present in marine-derived products, which may protect albumen proteins from oxidative deterioration and improve protein metabolism (Usturoi et al., 2025). However, oxidative stability parameters, such as thiobarbituric acid reactive substances (TBARS), peroxide value, or antioxidant status, were not determined in the present study. Therefore, the proposed relationship between dietary omega-3 supplementation and oxidative protection remains speculative and should be interpreted with caution. Future studies should incorporate oxidative stability measurements to better elucidate the mechanisms underlying the observed improvements in egg quality. Similar improvements in Haugh unit following dietary omega-3 supplementation have been previously observed in hens receiving DHA-rich diets (Ma et al., 2026). Nevertheless, previous reports have only partially compared marine-derived DHA sources with plant-derived omega-3 oils regarding internal egg quality responses.

The reduction in eggshell weight, eggshell ratio, and shell thickness in the hempseed oil group may indicate altered mineral utilization or lipid-mediated interference with calcium metabolism. High levels of unsaturated fatty acids may influence intestinal absorption of minerals through soap formation with calcium ions, thereby reducing calcium availability for shell formation (Mulet-Cabero and Wilde, 2023). In addition, the consistent reduction in several shell quality traits observed in the hempseed oil treatment suggests that this dietary lipid source may have influenced eggshell mineralization rather than causing random variation in shell characteristics. Eggshell formation is a complex physiological process requiring coordinated calcium transport, carbonate deposition, and matrix protein synthesis in the shell gland. Alterations in dietary fatty acid composition may indirectly influence these processes by modifying lipid metabolism, mineral utilization, or the availability of calcium required during the period of shell calcification. Although the precise mechanisms were not investigated in the present study, alterations in membrane lipid composition of shell gland epithelial cells or changes in calcium transport efficiency have also been proposed as potential pathways through which dietary polyunsaturated fatty acids may influence shell mineralization. Conversely, the higher eggshell weight per unit surface area observed in the hempseed oil treatment suggests that although shell thickness declined, shell density or shell structural compactness may have increased. This finding indicates that shell quantity and shell microstructure may not respond identically to dietary lipid manipulation. A thinner shell does not necessarily indicate poorer shell compactness, as changes in the organization of the calcified matrix may partially compensate by increasing shell density. Nevertheless, the simultaneous decline in shell thickness and shell weight suggests that the overall shell-forming process was affected by hempseed oil supplementation. Previous studies evaluating the effects of plant-derived omega-3 oils on eggshell quality have reported inconsistent findings, with some observing no changes and others reporting slight reductions in shell thickness depending on dietary inclusion level, fatty acid composition, and calcium supply. Such variation indicates that the response of shell characteristics to dietary lipids is influenced by interactions between fatty acid profile, mineral nutrition, and the physiological status of laying hens. These results indicate that different omega-3 sources may distinctly influence shell mineralization processes. Comparative information regarding the influence of hempseed oil relative to fish oil and microalgae on shell characteristics remains partly explored in the existing literature.

Dietary oil source also significantly affected yolk color characteristics. The lower Roche color score, redness, and brightness observed in the microalgae group were unexpected because several studies using Schizochytrium spp. or other microalgae have reported neutral or positive effects on yolk pigmentation, largely attributed to the presence of carotenoids and other pigment-associated compounds. The discrepancy between the present findings and previous reports may be related to differences in microalgal strain, inclusion level, dietary formulation, or the intrinsic pigment composition of the microalgae product used. In particular, Schizochytrium spp. is primarily utilized as a DHA source and generally contains lower concentrations of carotenoid pigments than other commercially used microalgae species. Consequently, its contribution to yolk pigmentation may be limited. Unlike carotenoid-rich microalgae such as Spirulina, Chlorella, and Haematococcus, which contain substantial amounts of lutein, zeaxanthin, or astaxanthin, Schizochytrium biomass is characterized by a high lipid and DHA content but comparatively low concentrations of yolk-coloring carotenoids. Therefore, supplementation with Schizochytrium does not necessarily increase pigment deposition into egg yolks despite its high nutritional value as an omega-3 source. Furthermore, because the corn inclusion level remained identical among all experimental diets, the observed reduction in yolk pigmentation cannot be attributed to differences in dietary xanthophyll intake. Instead, the results suggest that differences in the fatty acid composition of the dietary lipid sources may have influenced the intestinal absorption, transport, and deposition efficiency of lipid-soluble carotenoids already present in the basal diet. Highly unsaturated lipid sources may alter micelle formation and lipid metabolism, thereby modifying carotenoid bioavailability and yolk deposition efficiency. In contrast, the relatively higher yellowness values observed in the control and hempseed oil groups may therefore reflect more efficient deposition of carotenoids derived from the basal diet rather than increased pigment intake (Sopian et al., 2025; Elmi et al., 2026). Changes in yolk color due to dietary lipid source have been widely documented and are closely associated with carotenoid content and fat-mediated pigment absorption efficiency (Zurak et al., 2022). Taken together, the present findings indicate that the influence of microalgae on yolk pigmentation depends not only on its inclusion level but also on the specific microalgal species and its carotenoid profile. Future studies comparing DHA-rich and carotenoid-rich microalgae under identical dietary conditions would help clarify their respective contributions to egg yolk pigmentation and nutritional quality.

One of the most important findings of the study was the marked alteration in egg yolk fatty acid composition following dietary omega-3 supplementation. Flaxseed oil substantially increased α-linolenic acid deposition in egg yolk because flaxseed oil is naturally rich in this fatty acid and serves as a direct precursor for omega-3 metabolism (El‐Zenary et al., 2023). Hempseed oil also increased α-linolenic acid content, although to a lesser extent than flaxseed oil, likely due to its more balanced omega-6 to omega-3 fatty acid ratio. Fish oil and microalgae supplementation markedly increased DHA concentrations in egg yolk, confirming their effectiveness as direct sources of long-chain omega-3 fatty acids. These findings partly support previous observations demonstrating that marine-derived lipid sources are more efficient than plant oils in enriching eggs with DHA because they provide preformed long-chain omega-3 fatty acids that bypass hepatic elongation and desaturation pathways (Kralik et al., 2024). In the present study, the reported "transfer efficiency" values should be interpreted as relative indices of fatty acid enrichment based on the ratio of egg yolk fatty acid concentration to dietary fatty acid concentration, rather than as true biological transfer efficiencies. Because feed intake, egg mass, and total fatty acid output were not incorporated into the calculation, these values provide a comparative estimate of fatty acid deposition among dietary treatments rather than absolute transfer efficiencies. However, direct simultaneous comparison of fish oil, microalgae, flaxseed oil, and hempseed oil for fatty acid transfer efficiency and yolk DHA deposition has remained limited in the review of literature.

The observed differences in DHA deposition efficiency among the oil sources can be directly interpreted in light of their contrasting fatty acid profiles. Microalgae oil, characterized by a very high DHA content (≈50%) and minimal competing fatty acids, and hempseed oil, with a more balanced omega-6:omega-3 ratio and moderate α-linolenic acid content, both demonstrated improved DHA deposition in yolk. In contrast, fish oil, despite containing substantial levels of both EPA (10–18%) and DHA (∼15%), showed comparatively lower DHA transfer efficiency. The comparatively lower DHA deposition observed in the fish oil treatment may be associated with differences in the fatty acid composition and utilization of the dietary lipid sources. However, the present study did not investigate the physiological mechanisms responsible for these differences. Therefore, explanations involving differential intestinal absorption, fatty acid metabolism, transport, or competition between EPA and DHA remain speculative and cannot be confirmed by the present data. Further studies evaluating these physiological processes are required to clarify the mechanisms underlying the observed differences in yolk DHA deposition. These interpretations should be viewed cautiously because the calculated values represent relative deposition indices rather than absolute biological transfer efficiencies. Future studies incorporating feed intake, egg production, egg mass, and total fatty acid output would provide a more comprehensive assessment of nutrient transfer efficiency. Such comparative observations among hempseed oil, fish oil, and microalgae regarding DHA transfer efficiency have only been partially addressed in previous reports.

The calculated nutritional lipid quality indices further demonstrated that dietary omega-3 oil sources substantially influenced the nutritional value of egg yolk lipids. The marked reduction in the n-6/n-3 ratio, particularly in the flaxseed oil treatment, reflects the effective enrichment of egg yolk with omega-3 fatty acids and a more balanced fatty acid profile for human consumption (Usturoi et al., 2025). The higher ratio observed in the control treatment reflects the predominance of omega-6 fatty acids in conventional eggs and is consistent with previous reports demonstrating that dietary supplementation with omega-3-rich oils effectively reduces the n-6/n-3 ratio in enriched eggs (Cherian, 2017; Villora et al., 2025).

The PUFA/SFA ratio also differed among dietary treatments, indicating that the source of dietary lipid influenced the balance between unsaturated and saturated fatty acids in egg yolk. Higher PUFA/SFA ratios are generally considered beneficial because they are associated with improved lipid quality and are recommended for reducing the risk of cardiovascular disease in humans (Usturoi et al., 2025). The relatively higher ratio observed in the hempseed oil treatment suggests that this oil promoted a more favorable balance between polyunsaturated and saturated fatty acids, whereas the lower ratio in the fish oil treatment reflected its comparatively higher proportion of saturated fatty acids (Kaçar et al., 2025). These findings demonstrate that although marine-derived oils are highly effective sources of long-chain omega-3 fatty acids, plant-derived omega-3 oils may provide additional advantages in improving the overall fatty acid balance of egg lipids.

The AI provides an integrated estimate of the potential effect of dietary lipids on atherosclerosis by considering the relative proportions of pro-atherogenic saturated fatty acids and protective unsaturated fatty acids. Lower AI values are considered nutritionally preferable because they indicate a lower proportion of saturated fatty acids associated with cardiovascular risk (Assempoor et al., 2025). In the present study, flaxseed oil produced the lowest AI, suggesting the greatest improvement in lipid quality, whereas the fish oil treatment exhibited the highest AI, primarily because of its relatively greater contribution of saturated fatty acids despite its superior enrichment of DHA. These findings highlight that evaluation of omega-3-enriched eggs should not rely solely on DHA concentration but should also consider comprehensive nutritional lipid quality indices that better reflect the overall health value of egg lipids. Nevertheless, increasing the concentration of long-chain polyunsaturated fatty acids, particularly DHA, may also increase the susceptibility of egg lipids to oxidative deterioration during storage because these fatty acids are highly prone to lipid peroxidation. Oxidative stability is an important quality attribute of omega-3-enriched eggs, as lipid oxidation may adversely affect shelf life, flavor, and nutritional value. Since oxidative stability indices, such as thiobarbituric acid reactive substances (TBARS), peroxide value, or antioxidant capacity, were not evaluated in the present study, the potential effects of the different omega-3 oil sources on lipid oxidation could not be determined. Therefore, future studies should integrate oxidative stability assessments with fatty acid enrichment to provide a more comprehensive evaluation of the nutritional quality and storage characteristics of omega-3-enriched eggs.

The lack of significant differences in blood biochemical parameters, including glucose, triglycerides, cholesterol, AST, and ALT, suggests that dietary supplementation with omega-3-rich oils did not adversely affect metabolic health or liver function of laying hens. These findings indicate that the experimental diets were physiologically safe and did not induce hepatic stress or lipid metabolism disorders. Previous studies have similarly reported that moderate inclusion of omega-3 fatty acid sources does not negatively influence serum biochemical indices in laying hens when diets are nutritionally balanced. Nevertheless, comparative evaluation of multiple marine- and plant-derived omega-3 lipid sources on serum biochemical responses under identical dietary and management conditions has been only partially investigated in the available literature.

An important limitation of the present study is that oxidative stability indices, including TBARS, peroxide value, and antioxidant status, were not evaluated. Because fish oil and microalgae are rich in highly unsaturated fatty acids that are susceptible to lipid oxidation, assessment of oxidative stability would have provided additional insight into the quality and stability of omega-3-enriched eggs. Consequently, the present findings are limited to production performance, egg quality, fatty acid deposition, and blood biochemical responses, while the oxidative stability of the enriched eggs remains to be established in future studies.

Conclusion

In conclusion, dietary supplementation with different omega-3 fatty acid-rich oil sources did not adversely affect laying performance or blood biochemical parameters. Fish oil and microalgae were the most effective dietary sources for increasing egg yolk docosahexaenoic acid (DHA), whereas flaxseed oil produced the greatest enrichment of α-linolenic acid and the lowest n-6/n-3 ratio. Hempseed oil improved the PUFA/SFA ratio but was associated with reduced eggshell quality. Moreover, dietary oil source significantly influenced the relative deposition of selected fatty acids into egg yolk, demonstrating differences in enrichment efficiency among marine- and plant-derived omega-3 sources. These findings indicate that the choice of dietary omega-3 source should be based on the desired nutritional objective, whether maximizing long-chain omega-3 enrichment, improving overall lipid quality, or balancing egg quality characteristics. Future studies should also evaluate oxidative stability, sensory quality, and storage characteristics of omega-3-enriched eggs to complement the nutritional benefits observed in the present study.

Funding

This study was financially supported by the scientific research projects units of Erciyces University, with the project No FYL-2023-13011.

Ethical statement

The protocol for animal use was reviewed and approved Erciyes University Institutional Animal Care and Use Committee (protocol 03.05.2023, no 23/101).

Disclosures

No potential conflict of interest was reported by the author(s).

Data availability

The relevant data are provided in the paper. The data of the current experiment can be obtained from corresponding author when needed.