Effects of replacing Na selenite in laying hen feed with selenized glucose on production performance, egg quality, egg selenium content, microbial population, immunological response, antioxidant enzymes, and fatty acid composition

Abstract

This study aimed to explore the effects of selenized glucose (SeGlu) and Na selenite supplementation on various aspects of laying hens such as production performance, egg quality, egg Se concentration, microbial population, antioxidant enzymes activity, immunological response, and yolk fatty acid profile. Using a 2 × 2 factorial design, 168 laying hens at 27-wk of age were randomly divided into 4 treatment groups with 7 replications. Se source (Na selenite and SeGlu) and Se level (0.3 and 0.6 mg/kg) were used as treatments. When 0.3 mg SeGlu/kg was compared to 0.3 mg Na selenite/kg, the interaction findings revealed that 0.3 mg SeGlu/kg increased egg production percent and shell ash (P < 0.05). When compared to 0.3 mg Na selenite/kg, dietary supplementation with 0.3 and 0.6 mg SeGlu/kg resulted in an increase in albumen height, Haugh unit, and yolk color of fresh eggs (P < 0.05). SeGlu enhanced albumen height, Haugh unit, shell thickness (P < 0.01), albumen index, yolk share, specific gravity, shell ash (P < 0.05) of fresh eggs and shell thickness (P < 0.05) of stored eggs as compared to Na selenite. The interaction showed that 0.6 mg SeGlu/kg enhanced yolk Se concentration while decreasing malondialdehyde levels in fresh egg yolk (P < 0.05). SeGlu enhanced Se concentration in albumen and glutathione peroxidase activity in plasma (P < 0.05) as compared to Na selenite. 0.6 mg Se/kg increased lactic acid bacteria, antibody response to sheep red blood cells, and lowered ∑n-6 PUFA/ ∑n-3 PUFA ratio (P < 0.05). As a result, adding SeGlu to the feed of laying hens enhanced egg production, egg quality, egg Se concentration, fresh yolk lipid oxidation, and glutathione peroxidase enzyme activity.

INTRODUCTION

Modern laying hens, with greater productivity and metabolic rates, are more susceptible to the negative impacts of environmental elements such as, heat stress, mycotoxins and heavy metals in their feed, huge flock numbers, and oxidative stress (Wang et al., 2020; Ding et al., 2022). Oxidative damage, caused by reactive nitrogen species (RNS) or reactive oxygen species (ROS) leads to protein degradation, nucleic acid impairment, mitochondrial dysfunction, and lipid peroxidation (Zhang et al., 2017; Basiouni et al., 2023). The presence of these toxic compounds can cause oxidative stress, which can disrupt biological systems, decrease egg production, and increase susceptibility to diseases. To protect against oxidative damage induced by ROS and RNS, organisms utilize 2 antioxidant systems: non-enzymatic antioxidants and antioxidant enzymes (Bodnar et al., 2016; Surai and Kochish, 2019). Given the importance of nutrition in immune system function and disease resistance, there is an increased emphasis on the discovery of efficient antioxidant agents. Se, an important mineral with intrinsic antioxidant capabilities, has gotten a lot of attention in both human and animal nutrition for its capacity to increase immune responses and perform a variety of biological functions, including antioxidation (Dong et al., 2021). Notably, Se enzymes such as glutathione peroxidase (GSH-Px), thioredoxin oxidoreductase, and iodothyronine deiodinase play critical roles in antioxidant activity inside the human body. Se has been shown to be involved in a variety of critical biological functions, including thyroid hormone production, DNA synthesis, fertility, and immunological function (Qin et al., 2015; Huang et al., 2020). The concentrations of Se naturally present in plants vary depending on the plant species, the sampled plant part, the season of sampling, and the Se status of the soils in which they are cultivated (Surai, 2018). Plants absorb Se from the soil in the forms of selenite or selenate, synthesizing selenoamino acids in grain seeds. Selenomethionine constitutes the primary Se compound in grains, legumes, and soybeans. For instance, in corn, rice, wheat, and soy, selenomethionine comprises 45.5 to 82%, 54.9 to 86.5%, 50.4 to 81.4%, and 62.9 to 71.8% of total Se, respectively. While Se can be toxic for poultry at high doses (10–20 mg/kg), it is crucial to note that Se toxicity is typically observed only when the dose exceeds the physiological requirement by at least tenfold. Although data on this topic may sometimes be conflicting, doses of Se below 3 to 5 mg/kg of the diet are generally not associated with toxicity (Surai, 2002). Existing information indicates that the bioavailability of different Se sources, whether organic or inorganic, varies (Qin et al., 2015). Organic forms of Se, such as Se-enriched yeast, selenomethionine, and selenocysteine, are less harmful than inorganic forms such as selenite, selenate, and selenide. Furthermore, organic Se sources have a better transfer efficiency to milk, meat, and eggs, as well as improved bioavailability and the capacity to retain Se inside the body (Araujo et al., 2021; Chen et al., 2022). As a result, there is an increasing interest in adding organic Se into animal diets, which is leading to the creation of novel organic Se compounds. However, difficulties with synthesis and higher prices are now impeding the widespread use of organic Se (Khajeh Bami et al., 2022). As a result, determining the appropriate dietary form of Se for feeding poultry, especially laying hens, is critical.

Selenized glucose (SeGlu), a novel organic Se source, has developed. It is now possible to produce SeGlu on a large scale using the selenization process of glucose and sodium hydrogen selenide (Zhou et al., 2020). We hypothesized that mixing glucose with Se might potentially improve Se's biocompatibility and safety since glucose is quickly absorbed into the circulation and acts as an intermediate in cellular metabolism. As a result, scientists have been motivated to examine the unique features of SeGlu and investigate the possible benefits it might provide for animal health and production performance. According to one research in laying hens, SeGlu increased GSH-Px activity (Zhao et al., 2021b) and SeGlu improved antioxidant capacity in the oviduct, spleen, and liver (Zhao et al., 2021a). Another study showed that feeding rats SeGlu enhanced the serum metabolome by optimizing the gut microbiota, reducing the number of pathogenic bacteria, and improving the serum metabolome, particularly in certain antioxidants and sex hormones (Sun et al., 2023). Shokrinejad Gerdin et al. (2023) discovered that feeding SeGlu improves broiler performance, intestinal morphology, microbial population, and immunological response more effectively than Na selenite and Se yeast. According to Bai et al. (2022), the effects of SeGlu on serum malondialdehyde (MDA) concentration, serum antioxidant status, and Se deposition in Hu lamb muscle and organs were comparable to those of Se enriched yeast.

The objective of the present study was to investigate the replacement of Na selenite with SeGlu and its impact on production performance, egg quality, antioxidant enzymes activity, yolk lipid oxidation, intestinal microbial population, yolk fatty acid profile, immune response, and Se enrichment of eggs due to limited research on the use of SeGlu in the diet of laying hens and the lack of substitution with inorganic Se.

MATERIALS AND METHODS

Birds, Experimental Design, and Diets

A group of 168 Bovans white laying hens, aged 27 wk, were selected from a laying hen farm based on their similar laying performances and body weights. These hens were then divided into 4 treatment groups randomly, with each group having 7 replicates. Each replicate consisted of 6 birds. The arrangement followed a 2 × 2 factorial design, where the dietary factors were the source of Se (Na selenite and SeGlu) and the level of Se (0.3 and 0.6 mg/kg). The diets were formulated in accordance with the Bovans white nutrient management guidelines outlined in Table 1. Initially, a basic diet devoid of Se was prepared. Subsequently, predetermined doses of Se (either Na selenite or SeGlu) were added to the main diet. The SeGlu was prepared using the technique described by Zhou et al., (2020). A portion of sodium borohydride (NaBH₄) was gently added after the Se powder suspension in EtOH solution was cooled to −25°C. The remaining NaBH₄ was added at 15 to 17°C when the reaction started to drift toward equilibrium. Following the addition of glucose, the prepared NaHSe solution was agitated for 12 h. The EtOH medium was then recovered through distillation, and the resulting powder was dried for 80 h at 60 to 75°C and 0.1 atm of pressure. When finally obtained, the SeGlu product was a white powder. The dietary treatments were administered to the birds for a period of 12 wk, spanning from 27 to 39 wk of age. Before the actual feeding trial began, a pre-feeding period of 2 wk was implemented to gradually acclimate the birds to the transition from their regular commercial laying hen diet to the experimental diets. During the experiment, the birds were housed in cages, with each cage accommodating 2 birds. The cage size was length 45 cm, width 42 cm, height 50 cm in front and 48 cm in behind.

Table 1.

Composition and nutrient level of basal diet for laying hens.

Item	Amount
Ingredients, %
Corn	57.21
Soybean meal	29.43
Soybean oil	1.66
Dicalcium phosphate	1.69
Calcium carbonate	6.98
Oyster shell	2.00
Vitamin premix1	0.25
Mineral premix2	0.25
NaCl	0.39
DL-methionine	0.14
Calculated nutrient levels, %
Metabolizable energy, Kcal/kg	2798
Crude protein,	18.78
Linoleic acid	1.57
Calcium	4.00
Available phosphorus	0.42
Lysine	1.02
Methionine	0.44
Methionine + cystine	0.76
Analyzed selenium, mg/kg	0.05

¹Provided per kg of diet: vitamin A (retinol), 12000 IU; vitamin D3 (Cholecalciferol), 5000 IU; vitamin K3, 2.55 mg; thiamin, 3 mg; riboflavin, 7.5 mg; vitamin B6 (pyridoxine), 4.5 mg; vitamin B12 (cyanocobalamin), 0.02 mg; niacin, 51 mg; folic acid, 1.5 mg; biotin, 0.2 mg; pantothenic acid, 13.5 mg; choline chloride, 250 mg.

²Supplied per kg of diet: Mn, 120 mg; Cu, 16 mg; I, 1 mg; Fe, 40 mg; Zn, 100 mg.

They had unrestricted access to feed and water, while the environmental conditions were maintained at a relative humidity of 45 to 55%, a temperature of 23 ± 2°C, and an illumination schedule of 16 h of light and 8 h of darkness (30 lx). This research adhered to the animal welfare guidelines established by the Institute of Veterinary Research and Control in Kerman, Iran.

Production Performance

In order to determine production performance, daily records of egg production and egg weight were collected. Additionally, feed intake was measured on a weekly basis for each replicate. Egg mass was determined by multiplying the egg weight by egg production. Feed conversion ratio was calculated by dividing the total feed intake in grams to the total egg mass in grams. The number of abnormal eggs, including eggs weighing less than 50 g or more than 70 g, cracked eggs, shell-less eggs, or broken eggs were also recorded (Nguyen et al., 2021).

Egg Quality Parameters

Towards the end of the experiment, a total of 6 eggs were randomly selected from each replication for quality measurements. These measurements encompassed both external (shape index, specific gravity, shell thickness, and shell share) and internal (yolk share, albumen share, albumen height, albumen index, Haugh unit, and yolk color) characteristics. The egg shape index was measured using a 0.01 mm digital caliper, calculated as the ratio of its height to diameter. The specific gravity of the eggs was determined using the egg floating method in a saltwater solution (Van Emous, 2023).

To analyze shell thickness and shell share, the eggshell was separated from the albumen and yolk. The eggshell was then air-dried at room temperature for 24 h to determine shell weight, with shell share represented as a percentage of egg weight. The eggshell thickness was measured at 3 points without the eggshell membrane (top, bottom, and middle) using an electronic micrometer (Mitutoyo Corporation, Kanagawa, Japan), and the average value of these measurements was used (Nguyen et al., 2021).

To calculate the eggshell ash content, the eggshells were dried for 48 h at room temperature. After drying, the shells were weighed, ground, and subjected to calcination to determine ash content (Wang et al., 2020). The breaking strength of the eggshells was measured using a texture analyzer (STM-1, Santam Co., Tehran, Iran) following the procedure outlined by Ezazi et al. (2021). The eggs were placed horizontally in the device, and a 5 mm diameter probe was used. The texture analyzer operated at a speed of 100 mm/min, and the maximum force exerted on the shell (measured in Newtons) was recorded as shell strength, providing an indication of how much force the eggshells could withstand before breaking (Ahmadi et al., 2022).

Yolk share and albumen share were calculated as a percentage of egg weight. Albumen height was measured using a 0.01 mm digital caliper, while the albumen index was calculated as the ratio of its height to diameter. The Haugh unit was determined using the formula: Haugh unit = 100 * Log (Albumen height + 7.57 - 1.7 × Egg weight^0.37) (Haugh, 1937). The egg yolk color was assessed using the Roche scale, ranging from 1 (lightest) to 15 (darkest).

To study the effects of SeGlu on egg quality during storage, 6 eggs from each replicate were chosen and stored in plastic boxes in a cold room at 4°C (relative humidity at 45 to 55%) for 28 d (Kralik et al., 2021). After the storage period, the aforementioned parameters were assessed for the stored eggs. To calculate weight loss accurately, the difference between the egg weight before and after storage was multiplied by 100, providing the percentage of weight loss over the 28-d storage period

Se Content of Egg

Se content in the egg albumen and yolk was measured both before the start of the experiment and at the end of the period. To conduct this analysis, 6 eggs were chosen from each replication, broken, and separated into albumen and yolk fractions. The samples were dried at 65°C for 12 h, ground, and then digested using concentrated HNO3 (Sigma-Aldrich, St. Louis, MO). The mixture was transferred to a microwave digestion system (Milestone, Sorisole, Italy) with the following digestion procedures: 100, 140, 160, and 180°C for 3 min each, followed by 190°C for 15 min. Upon reaching a temperature below 50°C, the samples were transferred into a volumetric flask. Subsequently, the volumetric flask was filled to the mark with ultrapure water. Se content in the yolk and albumen was determined using an inductively coupled plasma mass spectrometer (Thermo Fisher Scientific, Waltham, MA), following the method described by Zhao et al. (2021a).

Lipid Oxidation

The oxidation of lipids in the yolks of fresh and stored eggs (28 d at 4°C) was measured using the TBARS value, which stands for thiobarbituric acid reactive substances. This value is calculated as milligrams of MDA per gram of egg yolk. To determine the lipid oxidation in yolk samples of fresh eggs (n = 6 per replication) and stored eggs (n = 6 per replication), the following steps were carried out: Two grams of yolk were mixed with 5 mL of 20% trichloroacetic acid and 4 mL of distilled water. The mixture was homogenized for 30 s using a blender. The homogenate was centrifuged at 1,000 × g for 20 min. The resulting supernatant was filtered. Two milliliters of the filtered solution were combined with 2 mL of thiobarbituric acid solution (0.02 M) in a test tube. The test tube was heated in boiling water for 20 min. After cooling, the absorbance of the colored product formed was measured at 532 nm using a microplate spectrophotometer (Epoch, Biotek, Winooski, VT). The lipid oxidation was reported as mg of MDA/kg of sample (Nemati et al., 2020).

Microbial Population (Lactic Acid Bacteria & Coliforms)

On the last day of the experiment, fecal samples were collected from 1 randomly chosen bird in each replicate (7 birds from each treatment). The samples were serially diluted in phosphate-buffered saline for lactic acid bacteria (LAB) and coliforms (COL) counts. The dilution concentration from 10⁻² to 10⁻⁵ for COL and from 10⁻³ to 10⁻⁶ for LAB counts were used. LAB counts were performed by plating the diluted samples on MRS agar and incubating them at 37°C for 48 h. Similarly, COL counts were conducted by plating the diluted samples on MacConkey agar and incubating them at 37°C for 24 h (Rezaeipour et al., 2022).

Immune Response

To evaluate the primary and secondary humoral immune response, 2 hens per replicate were injected with 1 mL of a 5% Sheep red blood cells (SRBC) suspension into the breast muscle. The second injection was administered to the same hens after 7 d to ensure a stable immune response during the second sampling. Seven d after the injections, blood samples (2 mL/hen) were collected from the brachial vein. The blood samples were centrifuged to separate the serum, which were then stored at −20°C until further analysis. The antibody titers, including total antibodies and IgG anti-SRBC antibodies (mercaptoethanol-resistant antibodies against SRBC), were measured using hemagglutination assays. In brief, for the assessment of total antibodies, 50 µL of PBS was placed in the first row of wells in a fresh 96-well V-bottom microtitration plate. Subsequently, 50 µL of serum (after inactivation at 56°C for 30 min) was added to the same wells. The plates were then sealed and incubated at 37°C for 30 min. Following incubation, 50 µL of PBS was added to the 11 remaining wells in each row. A 2-fold serial dilution of the samples was carried out on successive rows. Furthermore, 50 µL of a 2.5% SRBC suspension was added to each well, and the plates were sealed and incubated for an additional 30 min. The IgG antibody titers were assessed using a similar procedure as for total titers, with the exception that 50 µL of 2-mercaptoethanol-resistant was added to the first row of wells. Titers were determined by observing wells showing agglutination while holding plates over a lighted mirror. All antibody titers were reported as log2 of the reciprocal of the last dilution in which agglutination was observed. The IgM titer was determined by calculating the difference between the total antibody titer and the IgG titer (Khajeh Bami et al., 2022).

Antioxidant Enzymes

On the final day of the experiment, blood samples were collected from 2 birds per replicate through the brachial vein and transferred into sterilized tubes. The tubes were then centrifuged at 3,000 × g for 10 min at 4°C to separate the serum, which was subsequently frozen at −80°C for future analysis. The activity of enzymes, such as GSH-Px and superoxide dismutase (SOD), was assessed using a Randox Assay Kit (London, UK) according to the manufacturer's instructions. The activity of catalase (CAT) in the serum was determined using the Sinha (1972) method, which involves the use of hydrogen peroxide (H₂O₂) as a substrate. In this method, a mixture containing phosphate buffer, distilled water, and the necessary enzyme was prepared, to which 1 mL of H₂O₂ was added to initiate the reaction. The solution was then incubated at 37°C for 2 min. To terminate the reaction, 2.5 mL of the DDA reagent (potassium dichromate solution 5% and glacial acetic acid) was added (at a ratio of 1:3 v/v). Finally, the absorbance of the reaction mixture was measured at 570 nm (Rafeeinia et al., 2022).

Yolk Fatty Acid Profile

At the end of the experiment, 1 egg per replicate was collected and the yolks were stored at -20°C for further analysis. The fatty acid concentration in the yolks was determined using the Folch et al. (1957) extraction method. Gas chromatography (Agilent 7890A, Santa Clara, CA) was employed to separate the different fatty acids, with specific temperature settings and an internal standard used. The peaks corresponding to the fatty acids were identified and quantified based on their retention times and peak areas. The results were expressed as a percentage of the total fatty acids in the yolk (Ghasemi et al., 2022). In evaluating the proportions of specific fatty acids and their groups, we assessed indexes of egg lipid by calculating the following: atherogenicity index (AI) (Batkowska et al., 2021); peroxidizability index (PI) (Haraf et al., 2021); thrombogenic index (TI) (Araujo et al., 2021). The n-6 PUFA / ∑n-3 PUFA ratio calculated as (Linoleic (C18:2n-6) + (C18:3n-6) / α-linolenic (C18:3n-3) + Eicosatrienoic (C20:3n3)."

Statistical Analysis

The data were analyzed using the GLM procedure of Minitab (Minitab, 2019) in a completely randomized design with a 2 × 2 factorial arrangement of treatments. The main effects of Se source, which included Na selenite and SeGlufv, and Se level at 0.3 and 0.6 mg/kg diet, were assessed. To compare the means between treatments, pairwise comparisons were conducted using the Tukey test (P < 0.05). The normality of the data was assessed using the Shapiro–Wilk test. Experimental parameters were subjected to Spearman's correlation test for correlation analysis, and the software provided corresponding P values. The data were analyzed using the following model: Yijk = μ + Ai + Bj + ABij + eijk, where μ represents the mean, Ai denotes the effect of the source of Se, Bj signifies the effect of the level of Se, ABij accounts for the effect of the interaction between the source of Se and the level of Se, and eijk represents the random residual effect of the observation.

RESULTS

Production Performance

The effect of dietary supplementation with different Se sources and levels on laying hen production performance is shown in Table 2. There was a significant interaction (P < 0.05) on egg mass and FCR; birds supplemented with 0.3 mg Na selenite/kg had lower egg mass and greater FCR than those supplemented with 0.6 mg Na selenite/kg (P < 0.05). Hens fed 0.3 mg SeGlu/kg had a greater percentage of egg production than hens fed 0.3 mg Na selenite/kg (P < 0.01). According to the Se source effects, hens fed diets enriched with Na selenite gained more weight (P < 0.05). There was no effect of the experimental treatments on egg weight, feed consumption, or the fraction of defective eggs.

Table 2.

Effect of Se source, Se level, and their interactions on laying performance.

Item	Production performance
	Egg production, %	Egg weight, g	Egg mass, g	Feed intake, g	FCR, (feed-to egg mass ratio)	Weight gain, (g/bird/d)	Abnormal egg, %
Selenium source (SeS)
Na selenite	93.29	56.89	53.14	99.44	1.880	1.008a	1.440
SeGlu	94.26	56.39	53.19	99.14	1.873	0.596b	1.274
SEM	0.895	0.345	0.688	0.873	0.020	0.124	0.307
Selenium level (SeL)
0.3 mg/kg	93.81	56.34	52.90	99.95	1.898	0.849	1.287
0.6 mg/kg	93.74	56.93	53.42	98.63	1.855	0.754	1.428
SEM	0.895	0.345	0.688	0.873	0.020	0.124	0.307
Interaction
Na selenite 0.3	91.45b	56.38	51.59b	99.54	1.936a	1.044	1.692
Na selenite 0.6	95.13ab	57.41	54.68a	99.34	1.825b	0.971	1.188
SeGlu 0.3	96.17a	56.31	54.21ab	100.37	1.860ab	0.655	0.881
SeGlu 0.6	92.34ab	56.46	52.16ab	97.91	1.885ab	0.538	1.667
SEM	1.266	0.487	0.973	1.235	0.028	0.175	0.435
P-values
SeS	0.449	0.303	0.959	0.812	0.877	0.028	0.704
SeL	0.952	0.229	0.594	0.286	0.114	0.593	0.746
SeS × SeL	0.004	0.372	0.010	0.363	0.027	0.903	0.142

Abbreviation: SeGlu, selenized glucose.

^a,bMeans with different superscript letters differ significantly in the same column (P < 0.05).

Egg Quality Parameters

The influence of experimental diets on egg quality measures is shown in Tables 3 and 4. Fresh egg albumen height, Haugh unit, and yolk color exhibited a significant interaction (P < 0.05); birds fed low Na selenite supplementation had lower height albumen, Haugh unit, and yolk color than those fed any other dietary treatment. Furthermore, the percentage of shell ash was substantially greater in treatments containing 0.3 mg SeGlu/kg than in treatments containing 0.3 mg Na selenite/kg (P < 0.05). When compared to Na selenite, SeGlu enhanced albumen height, Haugh unit, albumen index, yolk share, shell ash and specific gravity in fresh egg, and shell thickness in fresh and preserved egg (P < 0.05). Shape index, shell share, and egg weight loss were unaffected by the experimental treatments.

Table 3.

Effect of dietary supplementation with SeGlu and Na selenite on the internal quality of fresh and stored eggs.

Item	Albumen share, %		Albumen height, mm		Albumen index, %		Haugh unit		Yolk share, %		Yolk color
	Fresh	Stored	Fresh	Stored	Fresh	Stored	Fresh	Stored	Fresh	Stored	Fresh	Stored
Selenium source (SeS)
Na selenite	64.68a	63.00	7.841b	6.123	9.158b	6.798	87.74b	77.64	25.21b	26.64	7.274	7.179
SeGlu	64.06b	62.41	8.177a	6.332	9.566a	7.080	89.80a	79.17	25.80a	27.14	7.440	7.286
SEM	0.202	0.266	0.071	0.072	0.145	0.132	0.117	0.588	0.183	0.264	0.090	0.057
Selenium level (SeL)
0.3 mg/kg	64.52	62.82	7.684b	6.093a	9.025b	6.765	87.14b	77.54b	25.32	26.97	7.119b	7.202
0.6 mg/kg	64.22	62.59	8.334a	6.362b	9.700a	7.113	90.40a	79.27a	25.69	26.81	7.595a	7.262
SEM	0.202	0.266	0.071	0.072	0.145	0.132	0.117	0.588	0.183	0.264	0.090	0.057
Interaction
Na selenite 0.3	64.68	62.72	7.365b	5.956	8.677	6.682	85.31b	76.52	25.14	26.92	6.833b	7.119
Na selenite 0.6	64.68	63.28	8.316a	6.290	9.639	6.913	90.17a	78.77	25.28	26.36	7.714a	7.238
SeGlu 0.3	64.35	62.46	8.003a	6.230	9.372	6.847	88.97a	78.57	25.49	27.02	7.405a	7.286
SeGlu 0.6	63.76	62.36	8.351a	6.434	9.760	7.313	90.63a	79.77	26.10	27.26	7.476a	7.286
SEM	0.286	0.377	0.101	0.103	0.205	0.188	0.165	0.832	0.259	0.374	0.128	0.081
P-values
SeS	0.033	0.122	0.003	0.054	0.021	0.137	0.003	0.078	0.027	0.183	0.207	0.200
SeL	0.311	0.541	0.000	0.016	0.000	0.067	0.000	0.049	0.147	0.670	0.001	0.471
SeS × SeL	0.305	0.383	0.007	0.534	0.097	0.535	0.017	0.533	0.367	0.290	0.004	0.471

Abbreviation: SeGlu, selenized glucose.

^a,bMeans with different superscript letters differ significantly in the same column (P < 0.05).

Table 4.

Effect of dietary supplementation with SeGlu and Na selenite on egg quality indicators of fresh and stored.

Item	Shape index, %		Specific gravity		Shell share, %		Shell thickness, mm		Shell ash, %	Weight loss, %	Shell strength, N/cm2
	Fresh	Stored	Fresh	Stored	Fresh	Stored	Fresh	Stored	Fresh	Stored	Fresh
Selenium source (SeS)
Na selenite	76.66	76.80	1.146b	1.116	10.10	10.36	0.375b	0.380b	83.60b	3.108	38.10
SeGlu	77.25	77.07	1.148a	1.118	10.14	10.45	0.385a	0.392a	84.88a	2.960	39.13
SEM	0.280	0.229	0.000	0.000	0.073	0.093	0.002	0.003	0.448	0.082	0.757
Selenium level (SeL)
0.3 mg/kg	76.84	76.76	1.147	1.115b	10.17	10.44	0.371b	0.383	83.80	3.141	36.96b
0.6 mg/kg	77.06	77.11	1.148	1.119a	10.08	10.37	0.389a	0.390	84.68	2.928	40.27a
SEM	0.280	0.229	0.000	0.000	0.073	0.093	0.002	0.003	0.448	0.082	0.757
Interaction
Na selenite 0.3	76.60	76.56	1.146	0.114	10.18	10.37	0.364	0.376	82.44b	3.239	35.81
Na selenite 0.6	76.72	77.04	1.146	0.119	10.03	10.36	0.386	0.385	84.75ab	2.978	40.40
SeGlu 0.3	77.09	76.96	1.147	0.117	10.16	10.52	0.378	0.390	85.16a	3.043	38.12
SeGlu 0.6	77.40	77.18	1.150	0.119	10.13	10.38	0.392	0.394	84.61ab	2.878	40.14
SEM	0.397	0.324	0.000	0.001	0.104	0.132	0.003	0.005	0.634	0.116	1.071
P-values
SeS	0.143	0.408	0.021	0.176	0.709	0.521	0.008	0.024	0.046	0.217	0.349
SeL	0.591	0.284	0.184	0.002	0.406	0.590	0.000	0.177	0.169	0.080	0.005
SeS × SeL	0.805	0.694	0.184	0.362	0.557	0.607	0.243	0.634	0.027	0.685	0.241

Abbreviation: SeGlu, selenized glucose.

^a,bMeans with different superscript letters differ significantly in the same column (P < 0.05).

Se Concentration of Egg

The Se concentrations in albumen and egg yolk are summarized in Figure 1. The amounts of Se in albumen and egg yolk varied significantly as a result of the Se dietary treatments. There was a significant interaction (P < 0.05) on yolk Se concentration; birds supplemented with 0.6 mg SeGlu or Na selenite/kg had higher Se concentration than those supplemented with 0.3 mg SeGlu or Na selenite/kg. The main effect demonstrated that birds fed with SeGlu or 0.6 mg Se/kg had higher Se concentrations in their albumen (P < 0.05). Furthermore, 0.6 mg/kg Se increased the concentration of Se in the yolk compared to 0.3 mg Se/kg (P < 0.01). For the Se concentration of albumen, there was no significant relationship between Se source and Se level.

Figure 1.

Effects of dietary Se source and Se level on Se concentration of albumen and egg yolk in laying hens. (A) Se concentration of egg yolk before starting the experiment. (B) Se concentration of albumen before starting the experiment. (C) Se concentration of egg yolk in the end of experiment. (D) Se concentration of albumen in the end of experiment. SS: Na selenite, SG: Selenized Glucose. ^a-cValues without the same lowercase letter are significantly different (P < 0.05).

Lipid Oxidation of Egg Yolks

The findings of lipid oxidation in fresh and stored egg yolks (mg MDA/g yolk) are shown in Figure 2. On MDA of fresh yolk, there was a significant interaction (P < 0.01); birds provided low Na selenite supplementation had greater MDA than those fed any other dietary treatment. MDA decreased significantly in fresh yolks by increasing the Se level from 0.3 to 0.6 mg/kg (P < 0.01). MDA in stored yolks was unaffected by the various treatments.

Figure 2.

Effects of dietary Se source and Se level on lipid oxidation of fresh and stored egg yolk in laying hens. (A) MDA concentration of fresh egg yolk. (B) MDA concentration of stored egg yolk. SS: Na selenite, SG: Selenized Glucose. ^a-cValues without the same lowercase letter are significantly different (P < 0.05).

Microbial Population

The effect of dietary Se source and Se level on the microbial population is shown in Table 5. The main effect of Se level demonstrated that 0.6 mg Se/kg increased LAB population compared to 0.3 mg Se/kg (P < 0.05). The experimental treatments had no influence on the population of COL and LAB/COL ratio. For the microbial population, there was no relationship between Se source and Se level.

Table 5.

Effect of SeGlu and Na selenite on fecal microbiota (log cfu/g) of laying hens.

	Feeding treatment
fecal microbiota, log cfu/g	Se source (SeS)		Se level (SeL)		SEM	P-values
	Na selenite	SeGlu	0.3 mg/kg	0.6 mg/kg		SeS	SeL	SeS × SeL
Coliforms	5.418	5.221	5.166	5.473	0.199	0.491	0.286	0.112
Lactic acid bacteria	6.825	6.934	6.627b	7.132a	0.146	0.603	0.022	0.677
Lactic acid bacteria/Coliform	1.280	1.348	1.308	1.320	0.050	0.356	0.870	0.364

Abbreviation: SeGlu, selenized glucose.

^a,bMeans with different superscript letters differ significantly in the same row (P < 0.05).

Immune Response

Table 6 shows the influence of Se source and Se level on hen humoral response. The findings of the primary and secondary responses revealed that feeding hens with 0.6 mg Se/kg enhanced total antibody titers against SRBC compared to 0.3 mg Se/kg (P < 0.05). For the immunological response, there was no significant relationship between Se source and Se level.

Table 6.

Effect of SeGlu and Na selenite on primary and secondary immune response (log₂) of laying hens.

Item	Antibody response to sheep red blood cells
	Total antibody		IgG		IgM
	Primary response	Secondary response	Primary response	Secondary response	Primary response	Secondary response
Selenium source (SeS)
Na selenite	8.286	9.250	5.107	5.857	3.179	3.393
SeGlu	9.179	9.464	5.500	5.821	3.679	3.643
SEM	0.327	0.241	0.480	0.329	0.344	0.281
Selenium level (SeL)
0.3 mg/kg	8.071b	9.000b	4.750	5.750	3.321	3.250
0.6 mg/kg	9.393a	9.714a	5.857	5.929	3.536	3.786
SEM	0.327	0.241	0.480	0.329	0.344	0.281
Interaction
Na selenite 0.3	7.214	8.857	4.429	5.929	2.786	2.929
Na selenite 0.6	9.357	9.643	5.789	5.786	3.571	3.857
SeGlu 0.3	8.929	9.143	5.071	5.571	3.857	3.571
SeGlu 0.6	9.429	9.786	5.929	6.071	3.500	3.714
SEM	0.462	0.341	0.679	0.465	0.487	0.397
P-values
SeS	0.059	0.533	0.565	0.939	0.310	0.532
SeL	0.006	0.041	0.109	0.703	0.662	0.183
SeS × SeL	0.082	0.835	0.714	0.493	0.246	0.327

Abbreviation: SeGlu, selenized glucose.

^a,bMeans with different superscript letters differ significantly in the same column (P < 0.05).

Antioxidant Enzymes

The impact of dietary supplementation with different Se sources and doses on antioxidant enzyme activity in laying hens is shown in Table 7. Dietary SeGlu enhanced GSH-Px activity as compared to Na selenite (P < 0.05). Furthermore, Se levels influenced GSH-Px and CAT activity (P < 0.01), with 0.6 mg Se/kg enhancing GSH-Px and CAT activity. For the antioxidant enzymes, there was no significant interaction between the Se source and the Se level.

Table 7.

Effect of SeGlu and Na selenite on antioxidant enzymes activity of laying hens.

	Feeding treatment
Antioxidant enzymes	Se source (SeS)		Se level (SeL)		SEM	P-values
	Na selenite	SeGlu	0.3 mg/kg	0.6 mg/kg		SeS	SeL	SeS × SeL
GSH-Px (U/L)	106.00b	115.21a	70.96b	150.25a	2.882	0.033	0.000	0.638
SOD (U/ml)	215.30	225.10	210.00	230.40	7.253	0.347	0.059	0.449
CAT (KU/L)	57.89	60.23	55.27b	62.84a	1.824	0.373	0.007	0.073

Abbreviations: SeGlu, selenized glucose; GSH-Px, glutathione peroxidase; SOD, superoxide dismutase; CAT, catalase.

^a,bMeans with different superscript letters differ significantly in the same row (P < 0.05).

Yolk Fatty Acid Profile

The fatty acid content of egg yolks is shown in Table 8. By raising the Se content in the diets, the concentration of Stearic acid (C₁₈) and Eicosatrienoic acid (C_20:3n3) in the yolk was raised (P < 0.05). Furthermore, the Se level influenced the ∑ n-6/∑ n-3 PUFA ratio and Myristoleic acid (C_14:1). Feeding 0.6 mg Se/kg to laying hens resulted in a reduction in the ∑ n-6/∑ n-3 PUFA ratio (P < 0.01) and Myristoleic acid (P < 0.05). There was no significant relationship between Se source and Se level for the yolk fatty acid. Experimental treatments had no significant effect on PI, AI, and TI.

Table 8.

Effect of SeGlu and Na selenite on fatty acid profile in eggs (%).

	Feeding treatment
Fatty acid, %	Se source (SeS)		Se level (SeL)		SEM	P-values
	Na selenite	SeGlu	0.3 mg/kg	0.6 mg/kg		SeS	SeL	SeS × SeL
Myristic (C14)	0.851	0.902	0.876	0.877	0.039	0.378	0.378	0.430
Palmitic (C16:0)	29.118	29.172	29.044	29.246	0.478	0.937	0.768	0.872
Heptadecanoic (C17:0)	0.153	0.144	0.150	0.147	0.019	0.726	0.905	0.490
Stearic (C18:0)	9.462	9.638	9.233b	9.867a	0.201	0.543	0.036	0.260
∑ SFA	39.586	39.857	39.304	40.139	0.492	0.701	0.243	0.729
Myristoleic (C14:1)	0.067	0.056	0.081a	0.042b	0.012	0.528	0.042	0.868
Palmitoleic (C16:1)	3.872	3.676	3.906	3.642	0.143	0.344	0.206	0.165
Heptadecenoic (C17:1)	0.045	0.059	0.058	0.046	0.012	0.454	0.525	0.224
Oleic (C18:1)	39.070	38.892	38.958	39.004	0.548	0.820	0.820	0.240
∑ MUFA	43.056	42.684	43.005	42.735	0.531	0.624	0.723	0.386
α-linolenic (C18:3n-3)	0.342	0.384	0.377	0.349	0.043	0.498	0.657	0.468
Eicosatrienoic (C20:3n3)	2.054	2.103	1.938b	2.219a	0.078	0.663	0.019	0.621
∑ n-3 PUFA	2.397	2.488	2.316	2.569	0.105	0.544	0.101	0.502
Linoleic (C18:2n-6)	14.873	14.884	15.279	14.478	0.673	0.990	0.408	0.314
γ-Linolenic (C18:3n-6)	0.087	0.085	0.094	0.078	0.019	0.951	0.554	0.319
∑ n-6 PUFA	14.960	14.970	15.374	14.556	0.671	0.992	0.397	0.298
∑ n-6/∑ n-3 PUFA	6.406	6.070	6.775a	5.701b	0.255	0.363	0.007	0.085
PI	20.92	21.10	21.18	20.84	0.781	0.871	0.765	0.466
AI	0.539	0.546	0.538	0.547	0.013	0.710	0.605	0.855
TI	1.089	1.091	1.083	1.096	0.024	0.954	0.718	0.977

Abbreviations: SeGlu, Selenized Glucose; PI, peroxidizability index; AI, atherogenicity index; TI, thrombogenicity index.

^a,bMeans with different superscript letters differ significantly in the same row (P < 0.05).

Correlation Analysis

Tables 9 and 10 show the correlation between the parameters. There is a significant positive correlation between egg quality parameters (albumen index, albumen height, Haugh unit, yolk color, shell thickness, and shell strength) with antioxidant enzymes and egg Se content (P < 0.01) and a significant negative correlation with MDA of egg yolk (P < 0.05). The findings demonstrate a substantial positive relationship (P < 0.05) between the Se content of yolk and albumen and antioxidant enzymes. The results show a strong positive correlation between MDA of yolk and n-6/n-3 ratio, as well as a significant negative correlation between Se concentration of yolk and GSH-Px activity (P < 0.05). There is a positive relationship (P < 0.05) between the LAB population and the GSH-Px enzyme. The results show a significant positive correlation between total antibody and GSH-Px activity (P < 0.05). The findings show a strong inverse relationship between the n-6/n-3 ratio and the Se concentrations of yolk and albumen, as well as the GSH-Px enzymes (P < 0.05).

Table 9.

Correlation between egg quality parameters and antioxidant status.

	Albumen index	Albumen height	Haugh unit	Yolk color	Shell thickness	Shell strength
Albumen index		0.895⁎⁎	0.915⁎⁎	0.452⁎	0.384⁎	0.470⁎
Albumen height			0.979⁎⁎	0.541⁎⁎	0.492⁎⁎	0.502⁎⁎
Haugh unit				0.501⁎⁎	0.458⁎	0.461⁎
Yolk color					0.497⁎⁎	0.447⁎
Shell thickness						0.488⁎⁎
Shell strength
MDA	−0.393⁎	−0.461⁎	−0.413⁎	−0.651⁎⁎	−0.541⁎⁎	−0.205
GSH-Px	0.613⁎⁎	0.751⁎⁎	0.695⁎⁎	0.571⁎⁎	0.694⁎⁎	0.498⁎⁎
SOD	0.166	0.254	0.225	0.311	0.323	0.131
CAT	0.504⁎⁎	0.474⁎	0.452⁎	0.406⁎	0.419⁎	0.589⁎⁎
yolk Se	0.584⁎⁎	0.668⁎⁎	0.592⁎⁎	0.636⁎	0.691⁎⁎	0.540⁎⁎
Albumen Se	0.695⁎⁎	0.739⁎⁎	0.703⁎⁎	0.645⁎⁎	0.582⁎⁎	0.504⁎⁎

Abbreviations: MDA, malondialdehyde; GSH-Px, glutathione peroxidase; SOD, superoxide dismutase; CAT, catalase.

^⁎P < 0.05.

^⁎⁎P < 0.01.

Table 10.

Correlation between antioxidant status and immune response.

Item	yolk Se	Albumen Se	n-6/n-3	GSH-Px	CAT
MDA	−0.518⁎⁎	−0.463⁎	0.459⁎	−0.484⁎⁎	−0.330
Yolk Se		0.760⁎⁎	−0.450⁎	0.781⁎⁎	0.436⁎
Albumen Se			−0.533⁎⁎	0.750⁎⁎	0.695⁎⁎
n-6/n-3				0.750⁎⁎	0.695⁎⁎
Total Ig				0.422⁎	0.208
LAB				0.454⁎	0.067

Abbreviations: MDA, malondialdehyde; GSH-Px, glutathione peroxidase; CAT, catalase; LAB, lactic acid bacteria.

^⁎P < 0.05.

^⁎⁎P < 0.01.

DISCUSSION

In our study, 0.6 mg Na selenite/kg improved egg mass and FCR more than 0.3 mg Na selenite/kg. Furthermore, 0.3 mg SeGlu/kg in the diets of laying hens resulted in a significant increase in egg production when compared to 0.3 mg Na selenite/kg diet. SeGlu is currently not commonly used in animal production. These findings are consistent with those of Nassef et al. (2020), who discovered that feeding 0.4 mg selenomethionine/kg to laying quails improved egg production. Another study found that feeding laying hens inorganic Se and Se yeast had no influence on the number of defective eggs (Muhammad et al., 2021). Mohammadsadeghi et al. (2023) found that Se-chitosan in the diet increased egg production and egg mass when compared to Na selenite. These findings suggest that various factors, including the source and dose of Se, the duration of the trial, and the individual breed or species of the layers, might influence hen production performance. The control of Se uptake, metabolism, and tissue distribution in animals remains a mystery. SeGlu's excellent reaction can be due to its effectiveness and better bioavailability when compared to Na selenite. One significant advantage of organic Se for laying hens is its capacity to prolong egg production at peak production. Even moderate stress can have a negative impact on productivity in a commercial egg production process. As an antioxidant, Se can assist reduce stress and sustain persistent high egg production throughout the peak of the production cycle (Surai et al., 2018). Se is required for the GSH-Px enzyme to operate properly, which protects cells from harm caused by oxidized low-density lipoprotein (Liu et al., 2020; Zhang et al., 2021). In our investigation, SeGlu increased the antioxidant enzyme activity in laying hens as compared to Na selenite. This increase in antioxidant enzyme activity may help to increase egg production.

The findings of this study showed that using SeGlu and greater amounts of Se supplementation in the diet enhanced the quality attributes of albumen, yolk, and shell in fresh eggs while also efficiently maintaining the quality of eggs during storage. Previous research has demonstrated that varying amounts of organic Se have a favorable influence on a variety of egg quality indexes. Organic Se supplementation (Se yeast and bacterial organic Se) has been shown to improve shell strength (Muhammad et al., 2021), albumen height (Qiu et al., 2021), Haugh unit and albumen height of hen's eggs (Nemati et al., 2020), and yolk color (Wang et al., 2022). These findings emphasize the favorable effect of organic Se on key elements of egg quality. In contrast to the results of this study, Zhao et al. (2021b) revealed that dietary SeGlu had no effect on the quality of fresh eggs and enhanced the Haugh unit of eggs stored for 2 wk. When oxygen penetrates the egg through holes in the shell, it oxidizes the yolk and albumen. This oxidative process causes albumen protein degradation, including the breakdown of the lysozyme ovomucin complex (Mahmoud et al., 1996; Sahin et al., 2002). The oxidation processes within eggs can cause a decrease in viscosity and dilution of the albumen. Se, on the other hand, has been shown to efficiently block these oxidation processes inside eggs (Fasiangova et al, 2017). Furthermore, the GSH-Px enzyme's activity can reduce the amount of protein and lipid oxidation during storage, resulting in enhanced egg quality (Pappas et al., 2005). Se supplementation can increase the activity of the GSH-Px enzyme in eggs, and this increase is frequently connected to Se levels in the diet (Pan et al., 2011). Increased GSH-Px activity protects the eggshell and interior contents from free radical damage (Payne et al., 2005; Pan et al., 2011). Increased dietary Se levels, as well as the usage of SeGlu instead of Na selenite, resulted in increased Se concentrations in eggs, according to our findings. Higher Se concentration in eggs is linked to higher efficiency in integrating Se into glutathione peroxidase, which provides stronger protection against free radical damage (Lu et al., 2020). The current study found a substantial positive association between Se level in the yolk and albumen and antioxidant enzyme activity. Furthermore, a strong negative connection was seen between the Se content of the yolk and albumen and the MDA level. Higher Se levels in the yolk and albumen appear to contribute to better antioxidant capability and decreased lipid peroxidation, eventually improving egg quality (Lu et al., 2020). Furthermore, the study found that raising dietary Se levels and using SeGlu resulted in a reduction in MDA levels in fresh egg yolk. This decrease in MDA, a sign of lipid peroxidation, supports greater egg quality preservation.

Our findings showed that increasing the Se dosage and incorporating SeGlu into the diet improved the color of fresh yolks. The color of the eggs is due to xanthophyll pigments (oxycarotenoids) derived from the hen's diet. These pigments may lose their color intensity when subjected to oxidation (Mohiti-Asli et al., 2008). The positive correlation between yolk color, antioxidant enzymes, and yolk Se content, as well as the negative correlation with MDA, provides strong support for the role of Se in protecting yolk colors. Shell strength and shell thickness were improved by increasing the level of Se and shell ash, and shell thickness was improved with dietary SeGlu. By controlling organic matrix formation, Se supplementation increases egg shell quality, especially shell strength (Surai et al., 2018). An increase in Se levels resulted in an increase in shell thickness in this research. This increase in shell thickness can be ascribed to the improvement in shell strength. Table 9 shows a positive association between shell thickness and shell strength, which confirms this relationship and verifies the notion that increasing shell thickness contributes to increased shell strength.

According to the findings of this study, using 0.6 mg SeGlu/kg or Na selenite in the diet of laying hens increased the Se concentration in the yolk. Furthermore, the SeGlu and the highest amount of Se were responsible for the increase in albumen Se concentration. Zhao et al. (2021a) found that SeGlu enhanced Se content in the liver, spleen, and oviduct of laying hens as compared to Na selenite groups. Another study found that 0.6 mg/kg Se-chitosan in the meal enhanced Se concentration in the albumen and yolk compared to Na selenite (Mohammadsadeghi et al. 2023). Eggs are regarded as one of the most precious food products. The efficiency of Se deposition in yolk, albumen, or other sections is determined by the concentration and form of Se in the diet (Surai and Fisinin, 2014). The Se content of eggs increases as dietary Se levels rise (Mohiti-Asli et al., 2008; Liu et al., 2020; Qiu et al., 2021). An explanation has been proposed regarding the impact of the type of Se source on the accumulation of Se in tissues and eggs. Organic Se, with an absorption rate of approximately 85 to 95% of the total consumed Se, demonstrates higher absorption compared to inorganic Se, which absorbs around 40 to 70% depending on whether it is in the form of selenite or selenate. Organic Se that is not utilized in protein synthesis is retained in the body's tissues, while inorganic Se is rapidly eliminated from the body through various routes, notably urine (Hu et al., 2012; Fasiangova et al., 2017).

According to the current research, an increase in diet Se level was associated with a proportional rise in the population of LAB. Shokrinejad Gerdin et al. (2023) reported that broiler chickens fed supplemental SeGlu or Se yeast diets had lower intestinal COL counts than Na selenite and the number of intestinal LAB and LAB/COL ratios was higher in SeGlu-fed birds than in Se yeast and Na selenite-fed birds. Another study found that feeding Se-chitosan to broilers decreased COL while increasing the LAB/COL ratio in the ileum when compared with Na selenite (Khajeh Bami et al., 2022). Dietary Se levels can influence the diversity of gut flora. As an antioxidant, Se decreases oxidative stress and stimulates the growth of helpful bacteria while being poisonous to other bacterial species (Mohammadsadeghi et al. 2023). The capacity of LAB to consume Se from their environment may explain their enhanced development and activity (Araz et al., 2008; Zhang et al., 2009). Furthermore, our data demonstrated a favorable relationship between the LAB population and the PSH-Px enzyme.

According to the findings of the study, increasing dietary Se levels resulted in an increased total antibody response to SRBC throughout both the primary and secondary immunological responses. These findings are consistent with those of Mohammadsadeghi et al. (2023), who discovered that using 0.6 mg/kg of Se-chitosan in the feed improved overall antibody response and IgM against SRBC in laying hens. In contrast to the current findings, Shokrinejad Gerdin et al. (2023) found that feeding SeGlu to broilers enhanced overall anti-SRBC titer, IgG, and IgM titers more than Se yeast and Na selenite. Se is necessary for immunological function in both humans and animals, playing a role in prostaglandin metabolism, neutrophil activation, and T and B lymphocyte proliferation (Avery and Hoffmann, 2018). GSH-Px activity has been proven in studies to improve the immunological response by improving antibody production in old laying hens and shielding neutrophils from free radicals (Ebeid, 2011). Se deficiency impairs both humoral (lowering IgG and IgM antibody levels) and cellular immunity (Suchy et al., 2014). The results of the study show a favorable and substantial relationship between total antibody response and GSH-Px enzyme.

The experiment's findings demonstrated that laying hen food supplemented with SeGlu resulted in higher activity of the GSH-Px enzyme when compared to diets with Na selenite. Increasing the quantity of Se in the diet was also linked to increased activity of the GSH-Px and CAT enzymes. Our findings are consistent with those of Zhao et al. (2021b), who found that SeGlu treatment increased egg GSH-Px activity. Furthermore, giving SeGlu to laying hens increased GSH-Px activity in the spleen and oviduct compared to Na selenite (Zhao et al., 2021a). According to Mohammadsadeghi et al. (2023), using Se-chitosan in the diet of laying hens increased GSH-Px and SOD activity comparer to Na selenite. Chickens are subjected to significant environmental, nutritional, and biological stresses in industrial chicken rearing environments. To counteract the impact of these stressors, it is crucial to regulate the antioxidant defense system through nutritional interventions (Surai and Kochish, 2019). The biological antioxidant defense system, comprised of enzymes like GSH-Px, SOD, and CAT, serves as the initial defense against oxidative stress in cells (Rajashree et al., 2014). Se deficiency significantly decreases GSH-Px activity in the blood plasma of laying hens (Surai, 2002), and there exists a direct correlation between Se concentration and GSH-Px enzyme activity in blood or body tissues (Petrovic et al., 2006).

According to the findings of this study, increasing dietary Se levels resulted in a decrease in the n-6/n-3 ratio. Unfortunately, no research has been conducted to investigate the effect of SeGlu on the fatty acid content of yolk. Mohammadsadeghi et al. (2023) reported that the use of Se-chitosan in the diet of laying hens reduced the n-6/n-3 ratio compared to Na selenite, and with increasing Se levels in the diet, the ratio of n-6/n-3 decreased. Kralik et al. (2013) discovered that adding organic Se to broiler feed reduced the n-6/n-3 ratio in thigh muscles. Similarly, Gurbuz et al. (2012) found that supplementing with organic Se and flax seed increased omega-3 fatty acid levels in egg yolk. According to the researchers, Se likely regulates prostaglandin synthesis by increasing the activity of the GSH-Px enzyme involved in arachidonic acid metabolism (Pappas et al., 2005). Additionally, a higher Se content in the yolk can help preserve lipid quality by preventing oxidation and inhibiting the degradation of certain unsaturated fatty acids (Mohiti Asli et al., 2008; Tufarelli et al., 2016). The correlation analysis in this study revealed significant associations between various parameters. The n-6/n-3 ratio exhibited a negative correlation with the Se concentration of yolk and GSH-Px enzyme, while showing a positive correlation with the MDA concentration of yolk. Notably, the diet containing 0.6 mg Se/kg, compared to 0.3 mg Se/kg, resulted in increased Se concentration of yolk and GSH-Px enzyme activity, while reducing the MDA concentration in the yolk. Based on these findings, it can be inferred that the higher Se concentration of 0.6 mg Se/kg likely contributed to a decreased n-6/n-3 ratio by improving the antioxidant status and reducing the MDA concentration in the yolk.

CONCLUSIONS

In conclusion, our findings showed that supplementing laying hen diets with SeGlu, an alternate source of organic Se, has a favorable influence on egg production. SeGlu also increased egg quality, Se concentration in eggs, GSH-Px enzyme activity, and reduced MDA levels in fresh yolk.