Effect of spirulina (Arthrospira platensis) supplementation on growth performance, egg production, egg quality, fatty acid profile, and hematobiochemical indices in guinea fowl

Abstract

This study investigates the influence of dietary spirulina (SP) on growth performance, egg production, egg quality, fatty acid profile, and hemato-biochemical profile of guinea fowl. A Total of 120 guinea fowl (GF) at 12 weeks of age were randomly allocated into four groups with five replicates (male-female ratio = 2:4). Animals were provided with four different diets over the 12–30 week trial: a corn-based basal diet for the control group (T₀) and basal diets with 1 % (T₁), 2 % (T₂), and 3 % (T₃) SP for the other three groups. Growth and egg production were measured weekly. The physical parameters of the egg were examined at the 24th, 27th^, and 30th weeks, and the fatty acid profiles were assessed at the end of the experiment. Final body weight and weight of the 1st laid eggs were significantly higher in T₁ and T₂ groups, where FCR was significantly improved in the T2 group compared to the control (P < 0.05). Egg weight was significantly increased in T₁ and T₂ group and shell thickness improved (P < 0.05) in all SP-treated groups at 27th and 30th weeks, where the Haugh unit was lower in T₂ compared to control at 27th and 30th weeks. Egg yolk color was significantly higher at 2 % and 3 % SP at 27th week and all SP-treated groups at 30th week. Groups T₂ and T₃ revealed an increase in serum albumin (P = 0.001). In group T₂, serum high-density lipoprotein (HDL) has increased significantly, and total serum cholesterol decreased (P < 0.05). Monounsaturated fatty acids increased significantly, whereas the omega-6/omega-3 ratio decreased in T₂. Polyunsaturated fatty acids significantly increased in both T₁ and T₂ groups compared to the control. The atherogenic index decreased in T2, and the thrombogenic index decreased in all SP-treated groups compared with the control. In conclusion, supplementation of 2 % SP with the basal diet made optimal improvements in growth performance, biochemical profile, egg physical quality, and fatty acid profile of guinea fowl eggs.

Introduction

Poultry meat and eggs provide an economical source of high-quality animal protein in Bangladesh. Bangladesh produced 24.41 billion eggs and 8.95 million metric tons of meat in the fiscal year 2024–2025. The livestock and poultry sector constituted 1.81 % of the nation's GDP (BBS, 2024). In Bangladesh, chickens and ducks constitute the predominant types of poultry cultivated, while pigeons, quails, and turkeys are also reared in certain rural regions. Guinea fowl farming is not as common as farming other poultry species like chicken and duck. Nowadays, People have started raising guinea fowl for food, and the value of meat and eggs in Bangladesh can be increased by using natural superfoods to improve their production quality. Since antibiotic growth promoters were outlawed (Demir et al., 2003; El-Sabrout et al., 2023; Righi et al., 2021), more and more people are looking into natural feed additives as long-term options. From ancient times, certain types of seaweed have been used as food (Mesripour et al., 2019). Microalgae like spirulina and chlorella are nowadays used as therapeutic feed supplements that are rich in nutrients (Bondar et al., 2023). Spirulina is a type of blue-green algae that grows in spirals and is valued for its high content of protein, vitamins, phycocyanin, essential fatty acids, and minerals (Somchit et al., 2014; Eilam et al., 2023; Kiron et al., 2012; Spínola, Mendes, et al., 2024). Spirulina is a safe and non-toxic nutritional supplement since it has a lot of iron, protein, phosphorus, and all the essential and non-essential amino acids, resulting in the improvement of the health, growth, and reproduction of mammals and birds (Alaqil and Abbas, 2023). In the case of broiler chicken (Ross 308), Spirulina plantesis increases carcass weight (Elbaz et al., 2022) and decreases cooking loss (Spínola, Costa, et al., 2024). Incorporating 0.3 % Spirulina into the diet of Norfa laying hens significantly increased the number of eggs laid, total egg weight, and the overall pace of egg production from the 38th to the 46th week of age (Abou-Elkhair et al., 2018). Dietary supplementation with 2 % and 4 % fresh green algae in laying ducks reduced the feed conversion ratio (FCR), or the quantity of feed required to yield a specific number of eggs (Indarsih et al., 2015). The high protein content of algae may have significantly contributed to the increase in egg production (Madacussengua et al., 2025). Spirulina platensis augmented the pigmentation of yolks in laying hens (White Leghorn and Rhode Island Red), rendering it redder and yellower (Rey et al., 2021). Supplementation of spirulina also enhances the weight, height, and Haugh units of the eggs and albumen (Lohmann White), the color score of the yolks (Isa Brown), and the shape index of the eggs (H&N Brown Nick) (Madacussengua et al., 2025; Omri et al., 2019; Vui et al., 2024). Additionally, it elevated unsaturated fatty acids and lowered saturated fatty acids, with linolenic (ω−3) and oleic (ω−9) acids exhibiting the most pronounced responses in quail (Vejdani Nia et al., 2022). The use of microalgae in the production of broiler chickens, ducks, laying hens, geese, and quails has been well researched. That is why microalgae production has surged due to the accessibility of certain species, and their diverse array of functional components (Bondar et al., 2023; Ekýzoðlu et al., 2020; El-Hady et al., 2022).

However, there have not been any feeding trials with spirulina in guinea fowl in Bangladesh. This study aims to evaluate the growth performance, the quality and characteristics of guinea fowl eggs, and their lipid profile along with hematobiochemical indices to enable future researchers to generate healthy eggs for human consumption.

Materials and methods

Ethical approval

Experimental protocols describing the management and care of animals were reviewed and approved by the Animal Experimentation and Ethics Committee of Sylhet Agricultural University, Bangladesh, under approval number [AUP2023048].

Animals and housing

A total of 120 guinea fowl (Pearl guinea fowl) from the same flock of 12 weeks of age (were already sexed), of similar body weight (920±23 g), were randomly distributed into four groups (30 birds in 5 cages per treatment,6 birds/cage) for the experiment. The flock in each cage consisted of 2 males and 4 females of the same age and weight, and both sexed guinea fowl were reared together according to the following methods of Sarfo et al. (2019). The trial started when the birds were 12 weeks old, and laying started at 19 weeks of age, while our experiment continued until the 30th week of age (Sarfo et al., 2019). This actually gives 12 weeks of laying periods, since 19 weeks were included. Guinea fowl were housed in a temperature-controlled room, with a 16L:8D light regimen throughout the experimental period. The cage size was 124 cm (width) × 64 cm (length) × 40 cm (height), allowing free access to feed and water during the experimental period.

Experimental diets

Guinea fowls were fed a corn-based grower basal diet for 18 weeks. The experimental diets, in pellet form to meet the nutrient requirements of growing guinea fowls according to the (NRC, 1994) guidelines, were formulated following the formulation approach by Sarfo et al. (2019) showed in Table 1. The dietary treatments were a control basal diet without Spirulina or with 1.0 %, 2.0 %, and 3.0 % Spirulina. Spirulina was prepared as a powder form of a total of 500 mg (Arulina; 500 mg Spirulina; Aristopharma Ltd., Bangladesh) mixed and supplemented to the basal diet. The chemical composition of spirulina powder was done by triplicate, following the AOAC (2025) methods (Horwitz and Latimer, 2005). The measurements of dry matter (DM) via Soxhlet extraction using petroleum ether, crude fiber (CF), ash content by incineration at 600°C for 6 hours in a muffle furnace, crude protein (CP) calculated via Kjeldhal method, and moisture content by weight difference between fresh and oven-dried samples.

Table 1.

Composition of experimental basal diet for laying guinea fowls.

Ingredients, air dry basis (g/kg)	T0	T1	T2	T3
Maize	554.1	552.2	546.8	542.8
Soybean	100.1	97.5	94.5	91.5
Wheat bran	250.0	245.0	243	240
Spirulina	0	10	20	30
Fishmeal	41.1	41.1	41.1	41.1
Oyster shell	50.0	50.0	50.0	50.0
Dicalcium phosphate	2.6	2.6	2.6	2.6
Vitamin Premix1	1.0	1.0	1.0	1.0
Salt	1.0	1.0	1.0	1.0

Calculated chemical composition (g/kg)2
Crude protein	160.0	161.6	162.4	163.1
Methionine	2.4	2.3	2.3	2.3
Lysine	6.3	6.21	6.21	6.21
Crude Fiber	42.0	42.04	42.06	42.07
Metabolizable energy (kcal/kg)	2962.0	2968.4	2974.8	2979.8

Analyzed chemical composition (g/kg)
Crude protein	152.0	152.4	152.9	153.2
Ash	80.0	80	80	80
Fat	25.0	25	25	25
Crude fiber	67.0	67.1	67.1	67.2
Moisture	55.0	54.8	54.8	54.7
Dry matter	945.0	945.2	945.2	945..3
Carbohydrate	622.0	623	623	623

¹Premix Each 2.5 kg contains Vitamin A, 12,500,000 IU; Vitamin B1, 2,500 mg; Vitamin B2, 5,000 mg; Vitamin B6, 4,000 mg; Pantothenic acid, 12,500 mg; Vitamin D3, 2,500,000 IU; Vitamin E, 20,000 mg; Vitamin K3, 4,000 mg; Nicotinic acid, 40,000 mcg; Vitamin B12, 12,000 mcg; Folic acid 800 mg; Biotin 100 mg; Zinc 50,000 mg; Iron, 40,000 mg; Selenium 150 mg; Cobalt, 400 mg; Copper, 10,000 mg; Iodine 400 mg; Manganese, 60,000 mg; Di-Calcium Phosphate, 380 gm; DL-Methionine, 100,000 mg; l-Lysine, 60,000 mg; Zinc-bacitracin 4,000 mg; Anti-Oxidant 5,000 mg; Carrier (limestone), q. s. to make 2.5 kg (Square Premix Broiler, SQUARE PHARMACEUTICALS PLC, square Centre, 48, Mohakhali C/A, Dhaka1212, Bangladesh.

²Calculated values are based on already analyzed values of main feed ingredients (i.e., maize, soybean, fishmeal, wheat bran).

For the amino acid profile of spirulina powder, proximate analysis was done by using an automated amino acid analyzer (AminoSAAYA LA8080, Hitachi Crop., Japan), which was used for the determination of the composition of amino acids. For mineral analysis, colorimetric methods were used to assess phosphorus, and complexometric titration was used to quantify calcium. Zinc, iron, manganese, and magnesium were measured using atomic absorption spectrophotometry (AAS). The analyzed chemical composition and amino acid profile of spirulina are presented in Table 2.

Table 2.

Nutrient composition of spirulina.

Chemical composition (% on dry weight basis)
Dry matter	93.70
Crude fibre	3.50
Crude protein	60.36
Crude Fat	4.78
Moisture	6.30
Ash	7.62

Amino Acid (mg/g)
Aspartate	89.7
Threonine	48.3
Glutamate	112.2
Alanine	24.8
Valine	24.4
Metheonine	8.8
Isoleucine	17.98
Leucine	24.26
Tyrosine	17.2
Phenylalanine	20.1
Lysine	11.92
Histidine	15.65
Arginine	51.43

Mineral concentrations (mg/g)
Calcium	94.3
Phosphorus	167.9
Magnesium	9.5
Iron	21.98
Manganese	1.3
Zinc	2.8

Data collection

All the birds were weighed individually at the beginning and the end of the experiment to determine the live weight changes. Feed and water were provided ad libitum, and refusal was measured weekly. Egg production and weight were recorded daily. Daily feed intake, laying rate (number of laid eggs × 100/number of feeding days), and feed conversion ratio (feed intake/number of egg weights) were calculated per week. Eggs laid at the 24th, 27th, and 30th weeks were used for egg physical characteristics measurements. The external egg characteristics recorded included egg weight, egg dimensions (diameter, length, and width in millimeters), shell weight, and shell thickness. In contrast, the internal egg parameters included albumen weight, albumen height, yolk height, yolk weight, and Haugh unit. Egg yolk color was measured by using the color fan (yellowness towards redness). The egg diameter and shell thickness were measured using Digital Vernier Calipers. For internal egg quality traits, individual eggs were broken open on a flat white surface, and the egg content was carefully poured with attention to keep the vitelline membrane of the yolk intact. Fifteen eggs from each replicate, 75 eggs per group, were selected randomly and evaluated at the 24th, 27th, and 30th weeks. Egg quality was evaluated within 24 h after oviposition. On each evaluation date, a total of 300 eggs were assessed, and in total, 900 eggs were assessed for this experiment. The yolk and albumen were then carefully separated and placed in separate pre-weighed petri dishes. The difference between the yolk or albumen and the petri dish was determined. The heights of albumen and yolk were determined using the egg quality slide rule. The diameter of the thick albumen was also measured using a micrometer screw gauge with sensitivity to the nearest 0.01 mm. Shell thickness was measured from 3 different parts of the egg (equator, sharp end, and broad end) using a micrometer screw gauge (Ikeme et al., 1983). Haugh unit was calculated using albumen height and egg weight, using the formula:

$$\text{Haugh}\text{unit}=100\text{log}\left(H+7.57\mathrm{W}0.37\right),$$

where H = observed albumen height (mm) and W = observed egg weight (g) (Fasuyi and Ojo, 2012). Ten eggs were used for each replicate, totaling 50 eggs per treatment group for measuring the Haugh Unit.

Yolk index was calculated according to the formula:

$$\text{Yolk}\text{index}=\text{Yolk}\text{height}\left(\text{mm}\right)/\text{Yolk}\text{diameter}\left(\text{mm}\right).$$

Fatty acid profile of egg yolk

For the determination of the fatty acid profile of egg yolk of spirulina-supplemented guinea fowl, the methods described by Folch et al. (1957) were used. In terms of fat content separation from yolk and determination of fatty acid profiles, the procedure described by Şengül et al. (2025) was followed. Based on the obtained fatty acid data, saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), polyunsaturated fatty acid (PUFA), n-3, and n-6 fatty acids were calculated. Besides this, n-3/n-6 ratio was also calculated. Lipid quality indices, namely the atherogenicity index (AI) and thrombogenicity index (TI), were determined based on the equation given by Gao et al. (2020).

$$\text{Atherogenicity}\text{index}=\left(\frac{\mathrm{C}12:0+4\times \mathrm{C}14:0+\mathrm{C}16:0}{\sum \text{MUFA}+\sum \left(n-6\right)+\sum \left(n-3\right)}\right)$$

$$\text{Thrombogenicity}\text{index}=\left(\frac{\mathrm{C}14:0+\mathrm{C}16:0+\mathrm{C}18:0}{0.5\times \sum \text{MUFA}+0.5\times \sum \left(n-6\right)+3\times \sum \left(n-3\right)+\frac{\sum \left(n-3\right)}{\sum \left(n-6\right)}}\right)$$

The hypocholesterolemic to hypercholesterolemic (h/H) of fatty acid index of eggs of guinea fowl was calculated by the equation provided by Ulbricht and Southgate (1991).

$$\mathrm{h}/H=\left(\frac{\mathrm{C}18:1+\mathrm{C}18:2+\mathrm{C}18:3+\mathrm{C}20:4+\mathrm{C}20:5+\mathrm{C}22:5+\mathrm{C}22:6}{\mathrm{C}14:0+\mathrm{C}16:0}\right)$$

Hematological and biochemical study

For the hematological study, Blood samples were collected randomly from 3 birds (1 cock and 2 hens) from each replicate at 30 weeks of age. Blood samples were collected from the wing vein with 5 ml disposable syringe. From it, 2 ml was transferred into K2 EDTA tube, and the other 3 ml was kept without anticoagulant tube for serum separation. Complete Blood Count (CBC) was performed using a CBC analyzer (PE-7010VET, Shenzhen Prokan Electronics Inc, Shenzhen, China), which analyzed RBC (Red Blood Cell), hemoglobin, MCV (Mean Corpuscular Volume), MCH (Mean Corpuscular Hemoglobin), and MCHC (Mean Corpuscular Hemoglobin Concentration). The WBC (White Blood Cell) count included total Leukocytes, Platelets, Eosinophils, Neutrophils, Basophils, Lymphocytes, and Monocytes.

Blood samples were then centrifuged at 4°C for 15 minutes at 3,000 rpm, after which the serum was extracted and stored in an epindrop tube at −20°C. Serum biochemical markers measured included alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), alkaline phosphatase (ALP), albumin, Serum cholesterol (SC), triglycerides (TG), high-density lipoprotein cholesterol (HDL), and low-density lipoprotein cholesterol (LDL).

Statistical analysis

Experimental data were subjected to One-way ANOVA and Regression curve estimation (linear and quadratic) by using IBM SPSS Statistics 25 statistical package (SPSS Inc., Chicago, IL, USA). Significant differences among the treatments were determined using Tukey’s HSD test at P < 0.05. Graphs were formed by GraphPad Prism. Principal Component Analysis was carried out by using RStudio version 4.4.0 (packages “ggbiplot, devtools”).

Results

Laying performance

The Fig. 1 depicts the influence of spirulina powder (SP) supplementation at varying dietary inclusion levels (1 %, 2 %, and 3 %) on key performance traits, including average daily gain (ADG), final body weight (FBW), average egg weight (AEW), and feed conversion ratio (FCR), compared with the control group.

Fig. 1.

Effect of dietary spirulina supplementation on ADG, FBW, AEW, and FCR of guinea fowl. A = ADG (Average Daily Gain), B = FBW (Final Body Weight), C = AEW (Average Egg Weight), D = FCR (Feed Conversion Ratio), T₀ = Basal diet without spirulina, T₁, T_2, and T₃, guinea fowl fed a basal diet supplemented consequently with 1%, 2%, and 3% spirulina; SEM, pooled standard error of the means; g = gram, SP = Spirulina.

A,b,c- means in the same row with different letters are significantly different at P < 0.05.

For ADG (g), a trend to improvements was evident in birds supplemented with SP at 1 % and 2 % level of spirulina, where ADG reached approximately 5.6–5.8 g compared with 4.9 g in the control group. Conversely, 3 % SP inclusion showed a significant decline from the control group (4.6 g), suggesting a threshold effect beyond which growth efficiency was compromised.

Similarly, FBW (g) demonstrated a robust dose-responsive improvement (P < 0.001). The control group averaged 1550 g, whereas SP 1 % and 2 % groups attained significantly higher body weights (1620 g and 1690 g, respectively). However, FBW declined in the 3 % SP group (1570 g), mirroring the trend observed in ADG.

In terms of AEW (g), supplementation exerted a marked positive effect (P < 0.001). Control birds recorded an average egg weight of 38.5 g, while SP 1 % and 2 % groups exhibited a substantial increase (42.0 g and ∼44.5 g, respectively). Notably, 3 % SP inclusion resulted in a modest increase (39.5 g) that was not significantly superior to the control.

With regard to FCR, birds in the SP 2 % group achieved the most efficient feed utilization (1.9), significantly lower (P < 0.01) than the control (2.3) group. The 1 % group showed moderate improvement (2.1), whereas the 3 % group displayed no advantage (2.3), again confirming the optimal effect at 2 % supplementation. Collectively, these results highlight 2 % spirulina powder supplementation as the optimal inclusion level, enhancing growth performance, egg weight, and feed efficiency, whereas higher inclusion (3 %) may induce negative effects.

First laying parameters

Table 3 shows the weight of the first egg laid, age, and weight of birds at first egg among different treatment groups. The average age of birds at first egg was not different (P > 0.05) among treatments. However, the average weight of birds at first egg for birds in the SP 2 % treatment group was higher (P = 0.047) than the control group. Besides this, the weight of the first laid egg was higher in T₁ and T₂ dietary spirulina-supplemented groups from the control.

Table 3.

Average weight of age at first laying, age, and weight of bird at first laying, Weight of first egg laid (gm).

Parameters	Treatment groups				SEM	P Value
	T0	T1	T2	T3		ANOVA	Linear	Quadratic
Age of bird at first egg (day)	132.60	129.40	126.80	128.20	0.932	0.147	0.055	0.069
Weight of bird at first egg (gm)	1073.2b	1104.8ab	1143.7a	1090.8ab	9.522	0.047	0.298	0.040
Weight of first egg laid (gm)	26.5c	29.9ab	32.87a	27.89bc	0.667	0.001	0.221	0.001

SEM, pooled standard error of the means; T₀ = Basal diet without spirulina, T₁, T_2, and T₃, guinea fowl fed a basal diet supplemented consequently with 1%, 2%, and 3% spirulina; gm = gram.

^a,b,c - Means in the same row with different letters are significantly different at P < 0.05.

Egg physical characteristics

The physical characteristics of the eggs of guinea fowl at different time intervals are demonstrated in Table 4. Egg weight was significantly improved (P < 0.001) at all time points with SP 1 % and SP 2 % supplementation, with the greatest enhancement observed at 2 % inclusion of spirulina with the basal diet. By the 30th week, eggs from the SP 2 % group weighed 45.30 ± 0.32 g, markedly higher than the control (41.87 ± 0.31 g). In contrast, SP 3 % resulted in lower egg weights, comparable to control levels. Egg height and diameter remained largely unaffected, except for a significant improvement in egg diameter at the 27th week (P = 0.001), with SP 2 % showing the highest values.

Table 4.

Physical characteristics of the different treatment groups’ guinea fowl eggs at different time intervals.

Parameters	Time	Treatment Groups				SEM	P Value
		T0	T1	T2	T3		ANOVA	Linear	Quadratic
Egg Weight (gm)	24th week	35.75c	37.14ab	38.90a	35.15c	0.37	0.011	0.774	0.001
	27th week	38.32c	41.69b	44.70a	39.08c	0.49	0.001	0.239	0.001
	30th week	41.87c	43.47b	45.30a	40.93c	0.34	0.001	0.759	0.001
Egg height (mm)	24th week	38.83	38.92	38.82	38.96	0.11	0.968	0.791	0.959
	27th week	43.27	43.3	43.72	43.15	0.29	0.920	0.987	0.847
	30th week	46.73	46.85	47.02	46.63	0.14	0.800	0.914	0.669
Egg diameter (mm)	24th week	35.08	35.16	35.30	34.82	0.12	0.574	0.564	0.445
	27th week	36.92bc	37.39ab	38.04a	36.23c	0.16	0.000	0.365	0.001
	30th week	38.70	39.08	38.68	38.03	0.32	0.336	0.640	0.263
Albumen height (mm)	24th week	3.68	3.68	3.94	3.83	0.08	0.620	0.325	0.589
	27th week	4.29	4.50	4.44	4.52	0.04	0.262	0.106	0.213
	30th week	4.94	4.92	4.88	4.86	0.02	0.768	0.285	0.569
Haugh unit	24th week	68.88	68.06	66.49	69.22	0.46	0.145	0.889	0.51
	27th week	74.20a	72.69ab	70.79b	72.68ab	0.41	0.027	0.485	0.090
	30th week	76.10a	75.27ab	74.15b	75.95a	0.25	0.021	0.509	0.024
yolk weight(gm)	24th week	9.54	9.67	9.68	9.61	0.03	0.551	0.518	0.210
	27th week	9.65	9.69	10.01	9.35	0.09	0.103	0.491	0.146
	30th week	9.90	9.58	9.92	9.57	0.08	0.311	0.410	0.715
yolk height (mm)	24th week	14.85	15.05	15.34	15.01	0.08	0.271	0.334	0.210
	27th week	15.43	15.41	15.45	15.37	0.02	0.584	0.424	0.556
	30th week	15.68	16.00	16.08	15.81	0.08	0.332	0.527	0.178
yolk diameter (mm)	24th week	35.00	35.01	35.04	35.19	0.06	0.774	0.349	0.576
	27th week	35.80	36.00	35.75	36.05	0.07	0.436	0.477	0.739
	30th week	37.31	37.13	37.29	37.22	0.06	0.793	0.838	0.904
Yolk Index	24th week	0.42	0.42	0.42	0.42	0.001	0.918	0.536	0.781
	27th week	0.43	0.43	0.43	0.43	0.001	0.276	0.334	0.515
	30th week	0.42	0.42	0.42	0.42	0.001	0.980	0.934	0.982
Shell thickness (mm)	24th week	0.33b	0.33b	0.40a	0.35b	0.007	0.000	0.010	0.007
	27th week	0.33c	0.39b	0.43a	0.38b	0.006	0.000	0.003	0.001
	30th week	0.35c	0.41b	0.46a	0.40b	0.008	0.000	0.002	0.001
Shell Weight (gm)	24th week	4.41b	4.72ab	4.85a	4.50b	0.05	0.003	0.373	0.001
	27th week	4.47b	4.79ab	5.14a	4.56b	0.07	0.003	0.370	0.004
	30th week	4.49b	4.93ab	5.22a	4.89ab	0.07	0.007	0.035	0.003

SEM, pooled standard error of the means; T₀ = Basal diet without spirulina, T₁, T_2, and T₃, guinea fowl fed a basal diet supplemented consequently with 1%, 2%, and 3% spirulina, (n = 75).

^a,b,c - Means in the same row with different letters are significantly different at P < 0.05.

Albumen height showed no significant differences across groups, although slight numerical improvements were observed with SP supplementation. However, the Haugh unit, a key indicator of albumen freshness and quality, However, the Haugh unit decrease at both the 27th (P = 0.027) and 30th weeks (P = 0.021). The SP 2 % group recorded a significantly lower Haugh unit at week 27. Yolk weight, height, diameter, and index did not differ significantly across treatments (P > 0.05), remaining relatively stable over time.

Shell thickness and shell weight exhibited highly significant differences (P < 0.001 and P < 0.01, respectively). At the 30th week, SP 2 % produced the thickest shells (0.46 ± 0.01 mm), a notable increase from the control (0.35 ± 0.01 mm). Similarly, shell weight was highest in the SP 2 % group (5.22 ± 0.22 g) compared to the control (4.49 ± 0.13 g). These results indicate a clear improvement in shell quality with moderate spirulina inclusion.

Egg yolk color

Fig. 2 shows the effect of dietary supplementation with spirulina powder (SP) at different inclusion levels (1 %, 2 %, and 3 %) on yolk color scores at three time points (24th, 27th, and 30th weeks of age) compared to a control group. At the 24th week, no statistically significant difference was observed among treatments (P > 0.05), with yolk color values ranging between 6.2 and 7.2. By the 27th week, significant improvements were detected (P < 0.01), where SP-supplemented groups, particularly at 2 % and 3 % inclusion, exhibited higher yolk color values (7.8–8.0) relative to the control group (6.7). The effect was most prominent at the 30th week (P < 0.001), where all SP-supplemented groups displayed consistently elevated yolk pigmentation (8.2–8.5) compared to the control (6.6).

Fig. 2.

Egg yolk color at different time points across treatment groups in guinea fowl. T₀ = Basal diet without spirulina, T₁, T_2, and T₃, guinea fowl fed a basal diet supplemented consequently with 1%, 2%, and 3% spirulina; SEM, pooled standard error of the means; ns = not significant.

A,b,c- means in the same row with different letters are significantly different at P < 0.05.

Fatty acid profile of guinea fowl egg

The outcome of dietary supplementation of spirulina powder (SP) at different inclusion levels (1 %, 2 %, and 3 %) on the egg yolk fatty acid profile of guineafowl compared to the control is demonstrated in Table 5. MUFA increases significantly (P < 0.05) in the T₂ groups, where PUFA increased significantly in the T₁ and T₂ groups of guinea fowl eggs. Similarly, the unsaturated to saturated fatty acid (PUFA: SFA) ratio increased significantly (P < 0.001) in the T₁ and T₂ groups compared to controls. The omega-6 to omega-3 (n-6/n-3) ratio declined significantly (P < 0.05), reaching its lowest value in the group T₂ (5.713) compared to the control T₀ (7.823). The atherogenic index was also shown in reduced (P = 0.003) in 2 % spirulina treated group, though it remains slightly above 0.5. The other parameters are shown in Table 6.

Table 5.

Fatty acid composition and lipid indicators in the egg yolk of guinea fowl fed with spirulina.

Parameters	Treatment Groups				SEM	P Value
	T0	T1	T2	T3		ANOVA	Liner	Quadratic
C12:0	0.090	0.087	0.081	0.089	0.003	0.871	0.836	0.772
C14:0	1.565	1.486	1.472	1.538	0.026	0.621	0.708	0.390
C14:1	0.092	0.117	0.112	0.096	0.004	0.041	0.856	0.017
C15:0	0.135	0.124	0.129	0.132	0.004	0.609	0.555	0.391
C16:0	26.55	26.10	24.85	26.40	0.263	0.058	0.497	0.120
C16:1	0.565	0.570	0.595	0.625	0.013	0.400	0.082	0.211
C17:0	0.165	0.180	0.160	0.147	0.008	0.629	0.345	0.472
C17:1	0.265	0.288	0.306	0.273	0.006	0.152	0.512	0.087
C18:0	14.25	13.90	13.25	13.85	0.157	0.145	0.203	0.132
C18:1t9	1.08c	1.13b	1.21a	1.19a	0.016	>0.001	>0.001	>0.001
C18:1c9	31.50	32.29	32.88	31.78	0.206	0.055	0.463	0.032
C18:1c11	0.205a	0.250ab	0.295a	0.226ab	0..012	0.029	0.352	0.024
C18:2t	0.226b	0.227ab	0,289a	0.207ab	0.011	0.026	0.975	0.203
C18:2	16.10	17.00	17.37	16.82	0.195	0.107	0.156	0.040
C18:3 Omega 3	0.43c	0.98ab	1.15a	0.80b	0.084	>0.001	0.089	>0.001
C20:0	0.120	0.127	0.138	0.119	0.003	0.221	0.786	0.189
C20:1	0.248b	0.262b	0.293a	0.257ab	0.006	0.014	0.020	0.82
C20:2	0.235	0.312	0.278	0.262	0.012	0.153	0.691	0.154
C20:3	0.263b	0.295ab	0.344a	0.284ab	0.011	0.058	0.301	0.063
C20:4	0.165	0.191	0.227	0.156	0.019	0.053	0.911	0.059
C20:5 Omega 3	0.050b	0.073ab	0.79a	0.062ab	0.004	0.032	0.277	0.009
C22:1	0.045	0.049	0.051	0.055	0.001	0.299	0.046	0.152
C22:2	0.135	0.187	0.168	0.212	0.011	0.097	0.034	0.118
C22:6 Omega 3	1.71b	1.79ab	2.05a	1.77ab	0.050	0.051	0.358	0.117
SFA	42.875	42.005	40.081	42.286	0.422	0.075	0.353	0.110
MUFA	34.001b	34.960ab	35.758a	34.515ab	0.239	0.028	0.296	0.018
PUFA	19.319b	21.070a	21.972a	20.579ab	0.317	0.002	0.100	0.001
PUFA/SFA	0.451c	0.502b	0.548a	0.487bc	0.011	>0.001	0.129	0.001
n-6/n-3	7.823a	6.474ab	5.713b	6.825ab	0.262	0.008	0.113	0.003
n-3/n-6	0.127b	0.156ab	0.175a	0.146ab	0.006	0.012	0.183	0.006
AI	0.616a	0.573ab	0.533b	0.592a	0.010	0.003	0.225	0.004
TI	1.311a	1.174b	1.061c	1.217b	0.027	>0.001	0.115	>0.001
h/H	1.831b	1.957ab	2.112a	1.898b	0.035	0.003	0.272	0.008

SEM, pooled standard error of the means; T₀ = Basal diet without spirulina, T₁, T₂ and T₃, guinea fowl fed a basal diet supplemented consequently with 1%, 2% and 3% spirulina; C12:0 (Lauric acid), C14:0 (Myristic acid), C14:1 (Myristoleic acid), C15:0 (Pentadecanoic acid), C16:0 (Palmitic acid), C16:1 (Palmitoleic acid), C17:0 (Heptadecanoic acid), C17:1 (Heptadecenoic acid), C18:0 (Stearic acid), C18:1t9 (Elaidic acid), C18:1c9 (Oleic acid), C18:1c11 (Cis-vaccenic acid), C18:2t (trans-Octadecadienoic acid), C18:2 (cis,cis-9,12-Octadecadienoic acid), C18:3 (α-Linolenic acid (ALA)), C20:0 (Arachidic acid), C20:1 (Gondoic acid), C20:2 (cis-11,14-Eicosadienoic acid), C20:3 (Dihomo-γ-linolenic acid (DGLA)), C20:4 (Arachidonic acid), C20:5 (EPA (Eicosapentaenoic acid)), C22:1 (Erucic acid), C22:2 (Docosadienoic acid), C22:6 (Docosahexaenoic acid), SFA (Saturated Fatty Acids), MUFA (Monounsaturated Fatty Acids), PUFA (Polyunsaturated Fatty Acids), AI (Atherogenicity Index), TI (Thrombogenic Index), h/H (Hypocholesterolemic/Hypercholesterolemic Ratio); (n = 15).

^a,b,c - Means in the same row with different letters are significantly different at P < 0.05.

Table 6.

Hematological indices of the impact of dietary supplementation of spirulina on guinea fowl.

Parameters	Treatment groups					P Value
	T0	T1	T2	T3	SEM	ANOVA	Linear	Quadratic
WBC (10^9/L)	448.3	451.2	461.36	473.0	3.64	0.053	0.329	0.318
Neu (10^9/L)	152.73	152.51	155.3	155.9	1.36	0.770	0.819	0.964
Lym (10^9/L)	228.75b	229.63b	234.7ab	245.19a	2.25	0.022	0.380	0.343
Mon (10^9/L)	19.28	19.36	20.68	19.79	0.21	0.055	0.629	0.620
Eos (10^9/L)	44.19	44.36	44.46	45.35	0.42	0.802	0.798	0.696
Bas (10^9/L)	5.35	5.37	5.59	5.49	0.53	0.150	0.692	0.931
RBC (m/µL)	2.49b	2.57ab	2.71a	2.50b	0.03	0.029	0.80	0.461
HGB (g/dL)	19.47b	19.62b	22.38a	20.35ab	0.39	0.017	0.151	0.276
MCV (fL)	128.2	122.96	118.74	129.52	1.59	0.045	0.715	0.115
MCH (pg)	78.00	76.34	80.78	78.18	1.34	0.737	0.526	0.654
MCHC (g/dL)	60.80	62.04	68.20	60.40	1.23	0.078	0.760	0.439
HCT (%)	0.32	0.32	0.32	0.32	0.004	0.952	0.569	0.823

SEM, pooled standard error of the means; T₀ = Basal diet without spirulina, T₁, T₂ and T₃, guinea fowl fed a basal diet supplemented consequently with 1%, 2% and 3% spirulina; WBC= White bold cell, Neu= Neutrophil, Lym= Lymphocyte, Mon= Monocyte, Eos= Eosinophil, Bas= Basophil, RBC= Red Blood Cell, HGB= Hemoglobin, MCV= Mean Corpuscular Volume, MCH= Mean Corpuscular Hemoglobin, MCHC= Mean Corpuscular Hemoglobin Concentration, HTC= Hematocrit, (n = 15).

^a,b,c - Means in the same row with different letters are significantly different at P < 0.05.

Principal component analysis

Fig. 3 illustrates the principal component analysis (PCA) of individual fatty acid profiles in the egg yolk fat of spirulina-treated guinea fowl, where the positive side of PC1 (the dominant axis) contains Omega 3, C18:3, and unsaturated fatty acid content, whereas saturated and trans fats like C22:0, C18:2t, and C20:0 are in the negative side for the control treatment.

Fig. 3.

Principal component analysis of egg yolk of different treatment groups. T₀ = Basal diet without spirulina, T₁, T_2, and T₃, guinea fowl fed a basal diet supplemented with 1%, 2%, and 3% spirulina.

Hematological parameters

The impact of the administration of dietary spirulina on the hematological indices of guinea fowl at 30 weeks of age is shown in Table 7. In comparison to the control group, SP 3 % shows a trend to higher (P = 0.053) in WBC and Lymphocyte, where SP 2 % exhibits higher levels of RBC, HGB, and lower levels of MCV compared to controls. Notable alterations were not observed in other hematological parameters.

Table 7.

Serum biochemical indices of different treatment groups of guinea fowl.

Parameters	Treatment groups				SEM	P value
	T0	T1	T2	T3		ANOVA	Linear	Quadratic
ALT (U/L)	32.4a	29.14ab	25.49b	28.20ab	0.79	0.010	0.18	0.006
AST (U/L)	206.89	196.60	193.70	191.20	2.33	0.069	0.011	0.028
Alkaline Phosphate (U/L)	54.29	53.20	54.60	55.40	0.93	0.890	0.585	0.770
Creatinine (mg/dl)	0.48	0.47	0.44	0.47	0.01	0.583	0.626	0.512
BUN	12.55	11.75	10.29	11.75	0.41	0.296	0.314	0.244
S. Cholesterol (mg/dl)	168.60a	154.60ab	143.20b	150.20b	2.83	0.003	0.005	0.001
Triglyceride (mg/dl)	55.60	49.20	46.40	50.20	1.91	0.413	0.279	0.232
HDL	31.40b	34.40ab	40.00a	37.78a	1.02	0.006	0.004	0.005
LDL	104.60	89.20	87.60	96.00	2.62	0.073	0.253	0.028
S. Bilirubin	1.09	1.02	1.15	1.18	0.028	0.227	0.139	0.245
Total Protein	45.08b	50.75ab	53.04a	47.46ab	0.95	0.007	0.284	0.002
Albumin	17.32c	20.27bc	24.06a	21.37ab	0.68	0.001	0.006	0.001
Globulin	27.76	30.45	28.98	26.09	0.71	0.151	0.314	0.074

SEM, pooled standard error of the means; T₀ = Basal diet without spirulina; T₁, T₂ and T₃, guinea fowl fed a basal diet supplemented consequently with 1%, 2% and 3% spirulina; ALT= Alanine Aminotransferase, AST= Aspartate Aminotransferase, BUN= Blood Urea Nitrogen, HDL= High-Density Lipoprotein, LDL= Low-Density Lipoprotein; S. Bilirubin= Serum Bilirubin, (n = 15).

^a,b,c - Means in the same row with different letters are significantly different at P < 0.05.

Biochemical parameters

The administration of spirulina supplementation with a basal diet caused notable changes in the serum biochemical parameters of different treatment groups. A considerable reduction in ALT (P = 0.01) in group T₂ and serum cholesterol levels in group T₂ and T₃ was experienced compared to the control group. In contrast, both HDL and albumin levels demonstrated a significant elevation in the T2 and T3 groups compared to the control. Total protein levels was improves significantly in the 2 % SP group (P = 0.006) compared to the control. Nonetheless, no substantial alterations (P < 0.05) were observed in other serological indices among all groups.

Discussion

The present study examined the effects of spirulina supplementation on growth performance, egg production, egg quality, fatty acid profile, and hematobiochemical indices of guinea fowl. Average daily gain is higher in the T1 and T2 groups; spirulina supplement affects (P < 0.05) the daily gain of guinea fowl hens. Dietary supplementation of 1 % and 2 % Spirulina has a positive effect on the final body weight of guinea fowl. These results partially agree with those reported by Abou-Elkhair et al. (2018), who found that inclusion levels of 0.1 %, 0.2 %, and 0.3 % spirulina increased hens’ final weight from 1222 g (0 %) to 1227 g (0.1 %), 1238 g (0.2 %), and 1253 g (0.3 %), respectively. A 2.5 % Spirulina-supplemented diet increased egg weight for the Lohmann White laying hen (Omri et al., 2019). In this study, 1 % and 2 % dietary supplementation of spirulina significantly increased (P < 0.05) egg weight in guinea fowl. Due to the high protein content, spirulina supplementation increases egg weight (Omri et al., 2019). In the present study, Dietary supplementation with 2 % spirulina decreased the feed conversion ratio of guinea fowl hens, similar to the study by Mariey et al. (2012), who found that spirulina decreased the feed conversion ratio of hens. Dogan et al. (2016) also reported that dietary inclusion of 0.5 %, 1 %, and 2 % spirulina did not affect the laying rate, feed conversion ratio, and egg weight of Japanese quail.

Microalgae contain a high quantity of calcium, which increases the egg shell strength and quality. In Hy-line hens fed with spirulina (2 %) supplemented feed, the shell quality was improved (Tufarelli et al., 2021a). In the present study, shell weight and shell thickness increased significantly in 2 % spirulina treatment group. This may occur due to increased calcium content in the guinea fowl egg. Saeid, A. et al. (2016) showed that Spirulina maxima is rich in copper and Iron, which improves the strength of eggshells and reduces cracked eggs. In our Study, egg height, Albumen height, Yolk weight, Yolk height, Yolk diameter, yolk index, are not significantly affected by the Spirulina supplementation. Omri et al. (2019) showed that albumen height is not affected by the algae supplementation. Also, Vui et al. (2024) found that albumen weight, yolk weight, egg shape index, and yolk index were not significantly increased by the algae supplement. In Laying hen (Lohmann White), Spirulina platensis affects the haugh unit, as shown in this study, where it was shown that 2 % and 3 % spirulina decreases the haugh unit, which depends on albumen height and egg weight of guinea fowl (Omri et al., 2019). Haugh Unit range 60 to 71 is good for egg quality in the case of guinea fowl (Djitie, K. et al., 2024). According to Rayan et al. (2022) An equal or greater than 72 Haugh unit means fresh and very firm albumen, which is seen 3 % spirulina treated group in the present study.

Algae contain high levels of carotenoids, which are the main factor in improving egg parameters such as egg color (Alfaia et al., 2021). Yolk color is the leading indicator of egg choice by consumers, but fatty acids and cholesterol do not change consumer preference (Englmaierová et al., 2013). In general, golden to yellow tones are considered more attractive. However, this varies across countries (Baiao et al., 1999). As laying hens cannot synthesize these pigments, carotinoid-rich feed is supplied to hens (Kljak et al., 2021). Feeding spirulina supplemented feed increased the yolk color measured by yolk color fan as shown in this study which is similar with Zahroojian et al. (2013) who demonstrate that algae feeding positively affected the egg yolk color in which 1.5 %, 2 %, and 2.5 % spirulina increased the egg yolk color score from 10.55 (1.5 %) and 11.43 (2 %) to 11.66 (2.5 %) compared to the control (1.55). 0.1 %, 0.15 %, and 0.2 % spirulina increased the egg yolk color score (RYCF) from 6.3 (0.1 %) and 6.7 (0.15 %) to 7.6 (0.2 %), as reported by Mariey et al. (2012). Park et al. (2015) reported that in the case of laying hen, 0.5 % and 1 % marine microalgae (schizochytrium) increased the egg yolk color score after 6 weeks of treatment, with a mean value of 9 and 8.8, respectively, compared to 8.7, corresponding to the control group. Additionally, Anderson et al. (1991) assessed the impact of adding 0.25 %, 0.5 %, 1.2 %, and 4 % spirulina to the diet, resulting in increased egg yolk color on days 2 and 23 of treatment.

In the current research, an 18-week trial with different percentages of spirulina with a basal diet showed a significant increase in MUFA and PUFA in the 2 % SP-treated groups of guinea fowl eggs. A similar result was found by Panaite et al. (2023), where 2 % SP supplementation with diet increases MUFA in laying hens; however, PUFA decreased significantly in 2 % SP-treated groups. This discrepancy may be due to the species variation or the length of spirulina supplementation. Spirulina is a rich source of polyunsaturated fatty acids and improves lipid metabolism, which may enhance the bioavailability of essential fatty acids for incorporation into egg yolk. Omega-6 (n-6): omega-3 (n-3) decreases in egg yolk of the 2 % spirulina-treated groups significantly. These findings corroborate previous findings of Panaite et al. (2023), where 2 % SP decreases n-6/n-3 value significantly. However, Rey et al. (2021) found that 1 % and 3 % SP added to the diet increases n-6:n-3 in the egg yolk of White Leghorn and Rhode Island Red. This alteration may be due to species variation or time variation of spirulina supplementation. PUFA/SFA significantly increased in 1 % and 2 % dietary SP-treated groups, which supports the result with Michalak et al. (2020), where 120 days of supplementation of spirulina significantly enhanced PUFA/SFA; however, 30 days, 60 days, or 90 days of supplementation do not enhance it in a significant way. Atherogenic index (AI) and thrombogenic index (TI), which are the indicators of cardiovascular health risk, were reduced significantly in T₂ group. This may be due to the addition of spirulina to guinea fowl food can decrease egg yolk levels of arachidonic acid (AA) by modifying fatty acid metabolism, increasing omega-3 storage, and altering the omega-6/omega-3 ratio, reducing anti-inflammatory activity, and reducing AI and TI.

The primary outcomes of this study indicated a significant increase in lymphocyte, RBC, and HGB in the 2 % SP-treated groups among guinea fowl. Hassan et al. (2022) stated that spirulina supplementation increased lymphocyte percentages in broiler, suggesting a potential enhancement of immune function. Dahi et al. (2024) stated that, spirulina supplementation improves the hemoglobin level and blood RBC in broilers.

In this study, dietary supplementation of 2 % SP significantly reduced a significant level of ALT compared to the control groups. This study aligns with the findings of Tufarelli et al. (2021b), where spirulina supplementation (2 % in diet) decreases serum ALT concentration in laying hens. Decreasing the level of ALT and AST indicates the hepatoprotective action of spirulina due to its antioxidant properties. The liver is the main metabolic organ of the body, and more severe liver damage releases higher amounts of liver enzymes (Tufarelli et al., 2021a). The decreased level of AST and ALT indicates less damage to hepatocytes (Jamil et al., 2015). However, EL-Gogary et al. (2023) reported that spirulina supplementation with a basal diet had no significant effect on ALT levels in broilers under normal conditions. This difference may be due to species variation or the length of treatment with spirulina. This experiment also shows that 2 % and 3 % dietary supplementation of spirulina exhibits a decrease in serum cholesterol level. However, HDL is increased in T2 and T3 groups, which indicates supplementation of 2 % and 3 % spirulina with basal feed increases the blood lipid profile of guinea fowl. Similar results were found with the experiment of El-Hady et al. (2022) and Tufarelli et al. (2021), where 2 % to 6 % of supplementation of spirulina with a basal diet decreases serum cholesterol level. Low cholesterol is beneficial for both poultry and human beings because it helps poultry for overall health and reduces the risk of high-fat-related diseases in humans who consume it (Alabi et al., 2021).

Basal diet with 2 % SP significantly increases the level of total serum protein and albumin compare to the control group. Spirulina is known as a high-protein-rich supplement that has an effect on serum protein levels in guinea fowl. An increased level of albumin indicates a high albumin level in the eggs of guinea fowl. A similar result is shown in Mahmoud et al. (2025) and Omar et al. (2022) where spirulina supplementation in the broiler diet increases serum total protein and albumin levels. Elevated serum protein levels, including albumin, suggest improved nutritional status and protein metabolism, which are essential for growth and immune responses. Spirulina's rich amino acid profile supports these physiological functions (Ogbuewu and Mbajiorgu, 2025).

Conclusions

This current study investigated that dietary supplementation of Spirulina in guinea fowl hen increase the egg weight, Shell thickness, shell weight, and yolk color, and decreases the FCR in hens. Spirulina supplementation with a basal diet improves the lipid profile of guinea fowl eggs, AI index, and TI index, which will reduce the rate of heart disease in humans. Mainly 2 % supplementation is more effective in the case of guinea fowl laying performance. Further study needed to evaluate the meat quality along with the gut health of guinea fowl treated with spirulina in its basal diet.

CRediT authorship contribution statement

Papri Rani Dey: Writing – review & editing, Writing – original draft, Visualization, Software, Methodology, Formal analysis, Data curation. Md. Ashiqur Rahman: Writing – review & editing, Writing – original draft, Visualization, Software, Methodology, Formal analysis, Data curation. Uttama Dey: Writing – review & editing, Software. Mahmuda Sultana: Writing – review & editing. Md. Fauzul Anam Fahim: Writing – review & editing. Md. Nazim Uddin: Writing – review & editing, Validation, Supervision, Investigation, Funding acquisition, Conceptualization.

Disclosures

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Papri Rani Dey reports financial support was provided by Government of the People’s Republic of Bangladesh Ministry of Science and Technology. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.