Effects of immersing Japanese quail eggs in various doses of riboflavin on reproductive, growth performance traits, blood indices and economics

Abstract

This investigation aimed to evaluate the impact of immersion (IM) riboflavin treatment on the hatchability, production efficiency, and carcass characteristics of Japanese quail eggs. A total of 260 eggs of Japanese quail birds were used for hatching and were randomly divided into 4 treatments with 5 replicates (13 eggs/replicate) in a fully randomized design. Hatching eggs were immersed in riboflavin for 2 min before incubation. The experiment treatments were designed as follows: G1 control group with no treatment, G2 treated with 3 g/L vit. B2 (IM), G3 treated with 4 g/L vit. B2 (IM) and G4 were treated with 5 g/L vit. B2 (IM). After hatching, 128 Japanese quail chicks, aged 7 d, were randomly grouped into 4 treatment groups, with 32 birds in each group. When quails were given vitamin B2 via immersion, they demonstrated significant enhancements in live body weight, body weight gain, feed consumption, and feed conversion ratio at different stages compared to the control group. Compared to control and other groups, the carcass parameters of Japanese quails given a 4 g/L immersion solution showed a significant improvement (P < 0.05). Hatchability and fertility (%) were considerably raised by Vit.B2 treatments of 3, 4, and 5g; the group immersed in 5 g/L had the highest percentages compared to the other groups. Furthermore, treated chickens with all concentrations of vitamin B2 had significantly higher blood indices than the controls. During the exploratory phase (1–6 wk) of age, the highest returns were reported in G4 treated with 5g/L vit. B2 (IM). Treating Japanese quail eggs with different dosages of vitamin B2 by immersion may be recommended to improve their productive and reproductive performance, blood indices, carcass traits, and economic efficiency.

INTRODUCTION

Chicken farmers are increasingly focused on how to raise healthier and more productive chickens. This is important because chicken meat is a major source of protein for people around the world. In fact, it's predicted that by 2031, chicken will be the most popular type of meat, even more popular than beef and lamb combined (FAO, 2022). To preserve farm animals' general health and boost their productivity and capacity for reproduction, numerous feed additives were also added (Ashour et al., 2020; Swelum et al., 2020; Wasef et al., 2020; El-Tarabany et al., 2021; Salah et al.,2021 Abd El-Hack et al., 2022). Several studies (Alagawany et al., 2016, 2017; Khafaga et al., 2019; Saeed et al., 2019; Abdelnour et al., 2020; Youssef et al., 2023a, b; Abd Elzaher et al., 2023; Youssef et al., 2024) have suggested feed additives derived from natural sources as safer substitutes for antibiotics.

An essential B vitamin, riboflavin influences the synthesis of energy, development, and immune regulation in the metabolism of chickens. Redox processes and energy metabolism depend on it for the best possible development and growth. Riboflavin is necessary to synthesize Adenosine triphosphate (ATP) and convert tryptophan to niacin (Nemati et al., 2023; Shastak & Pelletier, 2023). Skeletal deformities, reduced growth, and immunological dysfunction can all be caused by riboflavin deficiencies. Dietary supplementation is necessary due to the variable absorption of riboflavin generated from plant sources (Gharibzahedi et al., 2023; Sarkar et al., 2024). The growth and resilience of chickens to oxidative stress are influenced by the antioxidant properties of riboflavin and its role in protein synthesis. Numerous investigations have shown that riboflavin is required for optimum hatchability (Ciudad-Mulero et al., 2023; Mahmudiono and Haliman, 2023). The coenzymes flavin adenine dinucleotide and flavin mononucleotide, which are required for numerous redox actions in significant metabolic processes like the mechanism of the antioxidant system and the production of energy, require riboflavin (Lienhart et al., 2013; Kanwal et al., 2024). Furthermore, insufficient dietary riboflavin led to low levels of riboflavin in the albumen and yolk, as well as decreased hatchability, egg production, and chicken weight, all of which were associated with embryonic death in laying hens (Shastak & Pelletier, 2023).

According to Cogburn et al. (2018) and Tang et al. (2019), insufficient energy-generating processes may cause a high death rate in riboflavin-deficient embryos, resulting in energy depletion and hazardous low blood sugar levels. Riboflavin plays an essential part in the antioxidant system. Its involvement in aerobic cell oxidation processes is particularly significant (Ciudad-Mulero et al., 2023). Glutathione disulfide is converted to reduced glutathione (GSH) by the enzyme glutathione reductase, which relies on flavin adenine dinucleotides.

Additionally, riboflavin serves as a coenzyme in the metabolic pathways involved in the metabolism of proteins and carbohydrates. In addition to its primary function, riboflavin inhibits bile salt hydrolase (BSH) and acts as an antioxidant. It can enhance FCR and chicken productivity (Lin, 2014; Geng et al., 2020). According to Ashoori and Saedisomeolia (2014), it can also function as an antioxidant, preserving tissues in the body. Requirements of riboflavin vary depending on the type of poultry and their age. Broilers generally need 5 to 8 mg/kg, while layers and breeders may require 6 to 15 mg/kg (Leiber et al., 2022). Also, higher levels are needed during periods of rapid growth or egg production. Also, the minimum amount to prevent deficiency symptoms is around 2.5 mg/kg, but this might not optimize performance. Studies suggest that somewhere between 3 and 5 mg/kg might be sufficient for some poultry under certain conditions, while others might benefit from higher levels (up to 8.5 mg/kg) (Mateos et al., 2020). Because Japanese quail eggs are small and speckled, immersing them with vitamin B2 is much better than using injections. More time and energy are wasted as a result of producing fewer chicks. This experiment examined the impact of immersion of Japanese quail eggs in vitamin B2 on the hatch, growth, and carcass characteristics.

MATERIALS AND METHODS

This experiment was approved under the ethical guidelines of Zagazig University's Institutional Animal Care and Use Committee (ZU-IACUC/2/F/56/2021), Zagazig, Egypt. The study was implemented over 9 wk throughout 2 distinct stages: the incubation stage (21 - 3 to 12 – 4 - 2023) and the growing stage (12 - 4 to 17- 5 - 2023) in the animal and poultry production department's farm at Zagazig University in Zagazig, Egypt.

Experimental Design and Management

A total of 260 eggs were used for hatching and were randomly divided into 4 treatments with 5 replicates (13 eggs/replicate). Hatching eggs were cleaned and sterilized as usual in the hatching process and then immersed in riboflavin for 2 min before incubation. The experiment treatments were designed as follows: G1 control group with no treatment, G2 treated with 3 g vit. B2/L (IM), G3 treated with 4 g vit. B2/L(IM) and G4 treated with 5 g vit. After hatching, 128 Japanese quail chicks, aged 7 d, were randomly grouped into 4 treatment groups, with 32 birds in each group. The birds' initial body weight (209.15 ± 2.46) was almost the same for all treatments. Four replicates, eight chicks in each repeat for each treatment, were reared for 42 d.

The basal feed was produced using National Research Council (NRC) 1994 and the feed conformation tables for animal and poultry feeds used in Egypt (2001) to satisfy the nutritional requirements of Japanese quail (Table 1). Every bird in the experiment received a continuous supply of fresh water and feed. Under the same management parameters, there was a 22-h light cycle and a 2-h dark period in the window houses containing the birds. The birds received medications when needed, such as anti-coccidiostats that are added to the quail chicks' feed to prevent coccidiosis only during the experiment, and an inspection of their health to ensure they were healthy.

Table 1.

Ingredients and chemical composition of the experimental basal diet.

Percentage (%)	Ingredients
Yellow corn	58.45
Soybean meal (44% CP)	25.80
Corn gluten meal (62% CP)	6.70
Vegetable oil	1.30
Dicalcium phosphate	1.10
Limestone	5.70
Common salt (NaCl)	0.34
Premix2	0.30
DL-Methionine	0.05
L-Lysine	0.06
Choline chloride	0.20
Total	100.00
Chemical analysis of diet (Calculated) 1
Crude protein (CP) %	23.00
ME (kcal/kg)	2890
Ca %	2.50
Av. Phosphorus %	0.35
L-Lysine %	1.00
DL-Methionine %	0.45
Methionine + Cyst %	0.80

¹According to Feed Composition Tables for Animal & Poultry Feedstuffs used in Egypt (2001).

²premix added to the 1 kg of diet including Vit. A 10,000 I.U; Vit. D3 2000 I.U; Vit. E 15 mg; Vit. K3 1 mg; Vit. B1 1mg; Vit. B2 5mg; Vit. B12 10 μg; Vit. B6 1.5mg; Niacin 30mg; Pantothenic acid 10mg; folic acid 1mg; Biotin 50 μg; Choline 300 mg; Zinc 50mg; Copper 4mg; Iodine 0.3 mg; Iron 30mg; Selenium 0.1mg; Manganese 60mg and Cobalt 0.1mg.

Growth Performance Parameters

Average Live Body Weight and Body Weight Gain

At the beginning of the study and then every 3 wk after that, each bird in each examination treatment was weighed individually to the nearest gram. Live body weight (LBW) was calculated by recording each treatment's distinct live body weights (g). Moreover, body weight gain (BWG) (g) was calculated using the 1 to 3 and 3 to 6 phase averages and the (1–6) wk age range.

Feed Consumption and Feed Conversion Ratio

The feed given weekly to each growing bird in each trial group was recorded and stated as a gram /bird/day within the study phases from 1, 3, and 6 wk of age. The feed (g)/weight gain (g) was used for every trial period to calculate the feed conversion ratio.

Carcass Traits

Following the 6-wk investigation, 3 quails from each trail group were randomly selected, and they were kept fasting all night with only access to water. The quails were individually weighed before being slaughtered following Islamic practice. After being bled, feathers were weighed, and birds were weighed as well. Following evisceration, the carcass (%), head, liver, and heart (giblets) were weighed. Baéza et al. (2022) state that the predicted weights of the head, giblets, and carcass were used to calculate the dressing percentage.

Blood Parameters

At 42 d of age, 3 blood samples were obtained from the slaughtered birds in each experimental group. For every sample, 2 tubes were used: one with an anticoagulant agent and the other without an anticoagulant agent. Using the Davice and Lewis (1991) techniques, the unclotted blood samples were analyzed for the hematological picture of hemoglobin (Hb), white blood cells (WBCs), red blood cells (RBCs), packed cell volume (PCV), and platelets. The serum was frozen at -20 °C after separation to preserve it for biochemical examination. Diamond Diagnostics' commercial kits measured albumin, globulin, total protein, and creatinine (Peters et al., 1982). Also, unclotted blood samples were rapidly centrifuged at 3000 rpm for 15 min in order to test for alanine amino transferees (ALT) and aspartate amino transferees (AST) (Youssef et al., 2023c).

Fertility and Hatchability Traits

A total amount of 260 eggs from Japanese quail were used. Every treatment group's 65 eggs were divided into 5 replicates, each having 13 eggs. The eggs have been kept in a 15 to 18°C storage environment and 70% relative humidity. A photogrammetric fertility screen was performed on the 7th d.

Using the following formula, the percentage of viable eggs to total eggs was determined, or fertility (%):

$$\text{Fertile}\text{eggs}\left(\text{No}.\right)=\text{Fully}\text{set}\text{eggs}-\text{non}-\text{fertile}\text{eggs}.$$

$$\text{Fertility}(\%)\text{is}\text{calculated}\text{as}\left[\text{fertile}\text{eggs}\left(\text{No}.\right)/\text{Total}\text{set}\text{of}\text{eggs}\right]\times 100.$$

$$\text{Non}-\text{fertile}\text{eggs}(\%)\text{are}\text{calculated}\text{as}\left[\text{non}-\text{fertile}\text{eggs}\left(\text{No}.\right)/\text{fully}\text{set}\text{eggs}\left(\text{No}.\right)\right]\times 100.$$

Hatchability was evaluated in this way:

$$\text{Hatchability}\text{of}\text{the}\text{entire}\text{set}\text{of}\text{eggs}(\%)=\left[\text{chicks}\left(\text{No}.\right)/\text{eggs}\text{in}\text{the}\text{full}\text{set}\left(\text{No}.\right)\right]\times 100.$$

$$\text{Unhatched}\text{egg}(\%)=\left[\text{Total}\text{of}\text{unhatched}\text{eggs}/\text{Total}\text{of}\text{set}\text{eggs}\left(\text{No}.\right)\right]\times 100.$$

$$\text{Hatchability}\text{of}\text{fertile}\text{eggs}(\%)=\left[\text{chicks}\left(\text{No}.\right)/\text{fertile}\text{eggs}\left(\text{No}.\right)\right]\times 100.$$

Moreover, the percentage of pecked chicks was determined as follows after hatching:

$$\text{Pecked}\text{chicks}(\%)\text{are}\text{calculated}\text{as}\text{follows}:\left[\text{Pecked}\text{chicks}\left(\text{No}.\right)/\text{Total}\text{of}\text{set}\text{eggs}\left(\text{No}.\right)\right]\times 100.$$

Economic Efficiency (%)

We used an input/output analysis to see how using riboflavin to quails affected their economic efficiency (EE). This considered the cost of the riboflavin and the total revenue from quails. Economic efficiency is basically net income per unit of total cost. The next lines explain how we calculated both net revenue and EE in more detail below:

The cost of all yielded chicks - all feed expenses calculate net revenue.

EE equals net revenue/feed consumption price.

There were exclusions for labor, housing, medical care, and bird purchases because these costs were the same for every course of treatment.

Statistical Analysis

Data were analyzed as a completely randomized design, using the general linear model procedure of one-way analysis of variance in SAS software (SAS, 2004). The orthogonal polynomial contrasts were used to determine the linear (L) and quadratic (Q) effects of increasing concentrations of riboflavin for quails. The pen was used as the experimental unit for growth performance. Selected individual quails were considered as experimental units for the blood parameters, and carcass traits. Riboflavin effect was the single factor considered for analysis. Variability in the data was expressed as the pooled standard error of the mean (SEM). When the treatment effect was significant (p<0.05), means were separated using Duncan's various test procedures of SAS software (Duncan, 1955).

RESULTS AND DISCUSSION

Growth Performance Parameters

Live Body Weight and Body Weight Gain

Table 2 showed how immersion of Japanese quail eggs at different riboflavin concentrations affected the birds' overall body weight, weight gain, feed intake, and feed conversion ratio over the trial 6 wk. Data showing significantly (P < 0.05) enhanced LBW in birds treated with (3, 4, and 5g) vit. B2/l immersion at 1, 3, and 6 wk of age in comparison to the controls. The Japanese quails are handled with 5g vit. B2/L (IM) revealed the highest LBW (1,496.50 g). However, the control group revealed the lowest LBW (1,364.00 g). Table 2 demonstrates that birds received (3, 4, and 5g) of vitamin vit B2/L immersions; they enhanced significantly (P < 0.05) BWG during all trial phases in comparison to the control group. The third group of quails received 4 g of vitamin B2/L (IM), revealing the highest BWG (1,275.50 g). However, the control group revealed the lowest BWG (1,711.75g). These findings are consistent with a study of Florian et al. (2021), who showed that chickens given varying doses of vitamin B2 revealed the highest LBW and BWG relative to the control group.

Table 2.

Effect of immersion Japanese quail eggs with different levels of riboflavin on total body weight, body weight gain, feed consumption and feed conversion ratio from 1 to 6 wk of age.

Items	Wks.	Immersion treated group, (g/L)				SEM	P- value1
		Control	3g	4g	5g		T	L	Q
Body weight (g)	1	192.25b	223.75a	215.00a	222.00a	2.95	0.004	0.110	0.033
	3	1,112.75d	1,169.00c	1,187.25b	1,197.50a	3.47	0.001	0.004	0.002
	6	1,364.0c	1,486.75b	1,490.50a	1,496.50a	1.47	0.004	0.411	0.353
Body weight gain (g)	1-3	920.50c	945.25b	972.25ab	975.50a	5.21	0.010	0.014	0.013
	3-6	251.25c	317.75a	303.25a	299.00b	3.55	0.043	0.046	0.044
	1-6	1171.75c	1263.00b	1275.50a	1274.50a	2.44	0.017	0.015	0.018
Feed consumption (g)	1-3	1849.60c	1857.72b	1860.70a	1865.63a	1.35	0.015	0.014	0.017
	3-6	1871.66c	1876.79ab	1879.03ab	1881.06a	1.36	0.018	0.015	0.019
	1-6	4575.46c	4591.52b	4596.12b	4607.13a	1.22	0.001	0.038	0.045
Feed conversion ratio (g)	1-3	2.02a	1.97b	1.92b	1.92b	1.88	0.004	0.002	0.006
	3-6	7.65a	5.93c	6.20b	6.30b	2.34	0.002	0.001	0.002
	1-6	3.91a	3.64b	3.61b	3.62b	1.67	0.015	0.016	0.018

^a,bMeans are bearing different letters in the same row differ significantly (P < 0.05, 0.001).

¹T, overall effects of treatments; L, linear effects of increasing riboflavin levels of quails; Q, quadratic effects of increasing riboflavin levels of quails.

Moreover, elevated vitamin B2 levels have strengthened tissues' resistance to toxic substances and other harmful diseases (Witten & Aulrich, 2018; Leiber et al., 2022). Riboflavin also produces RBCs and other cellular processes that give energy to the body. Additionally, it offers defense against gastrointestinal illnesses by maintaining and protecting the mucosal membranes (Lambertz et al., 2021). The results demonstrated that the LBW and BWG of the birds receiving vit. B2 were more significant than those observed in the control group. The active ingredients of vitamin B2, which has been regarded as a useful feed resource because of its wide variety of physiologically and nutritionally relevant compounds, may be related to the study's findings (Jing et al., 2019). During the first part of the 20th century, one important discovery was that riboflavin was known as the “vitamin G that promotes growth” (Shastak & Pelletier, 2023). This knowledge is based on riboflavin's involvement in several physiological functions directly connected to the domestic chicken species' general growth performance. By affecting important factors like enzyme activity, protein synthesis, and food consumption, riboflavin plays an essential function in the complicated system of biological interactions that result in the growth of chickens (Alagawany et al., 2021).

Furthermore, it has been demonstrated by Lambertz et al. (2021) that heat stress reduces feed consumption. Treatment with riboflavin has been shown to have several pharmacological effects that can enhance immunological function and physiological activity. Additionally, by encouraging these enzymatic processes, riboflavin indirectly enhances the efficiency of food absorption and digestion inside the avian gastrointestinal tract. Consequently, this leads to a higher absorption of nutrients and energy from the feed consumed, supplying the essential building blocks required for optimal development. The primary mechanism via which riboflavin promotes growth in a range of chicken species is the improvement of nutrient consumption (Cogburn et al., 2018). Riboflavin is a component of coenzymes essential for breaking down carbohydrates, fats, and proteins for energy production. This improved energy availability can potentially contribute to better growth. Also, riboflavin is involved in amino acid metabolism, which is necessary for building proteins required for muscle growth and tissue development (Mineva and Georgieva-Dimitrova, 2024).

Feed Consumption and Feed Conversion Ratio

The data in Table 2 revealed that the FC of quails treated with (3, 4, and 5g) vit. B2/l immersion at all trial phases was significantly elevated (P < 0.01) compared to the controls. Furthermore, the birds that received 5g of vitamin B2/l (IM) had the greatest FC levels (4,607.13 g). In contrast, the control group's feed consumption was the lowest (4,575.46g). Moreover, The FCR differed significantly between the treatment and control groups (P < 0.01). Compared to the control and other groups, the G3 treated with 4g vit B2/l (IM) had an excellent FCR score (3.61). However, in contrast, the control group received a poor score (3.91). Throughout the investigation, birds given 5g of vitamin B2/l (IM) revealed the best feed intake (4,607.13 g). The present results align with the research conducted by Leiber et al. (2022), who stated that laying hens consume significantly more feed-varying concentrations of riboflavin than the control group. In addition, Jing et al. (2017) stated that there was a significant difference in the FC between the Pekin ducks that consumed 10 mg of vitamin B2/kg of diet and the control group. Riboflavin is a crucial constituent for 2 coenzymes in energy synthesis: flavin mononucleotide (FMD) and flavin mononucleotide (FMN). A sufficient amount of riboflavin can guarantee that feed energy is utilized efficiently, thus boosting the feed conversion rate. Certain studies suggest that elevated riboflavin levels could improve the efficiency of protein utilization of laying hens, leading to increased growth and feed efficiency (Jaroensuk et al., 2023; Choi et al., 2024).

Riboflavin also helps with digestion and nutrition absorption by encouraging the growth of good gut bacteria and the reduced FCR results from improved feed nutrient use (Gan et al., 2020). Riboflavin is an electron transport chain component and is also necessary for energy production and cellular respiration. Appropriate riboflavin levels support Pheasant growth and performance, which improves FCR through efficient energy synthesis (Akinyemi & Adewole, 2021; Ding et al., 2024). The scientists used Western blotting methods to assess specific liver-resident proteins. It is possible that hypovitaminosis B2 could block the oxidation of fatty acids and the electron transport chain's (ETC) advancement in the mitochondria, which could result in hepatic lipid accumulation and growth inhibition. This is supported by the tissue's lowered concentrations of particular proteins, which were mostly connected to these mechanisms (Rousseaux & Bolon, 2013).

Carcass Traits

The impacts of (3, 4, and 5) g vit. B2/l immersion on the carcass traits of Japanese quail was displayed in Table 3. The data shows that the quail carcass traits were administered with (3, 4, and 5) g vit. B2/l immersion was significantly improved (P < 0.5) compared to the control group. Birds given 4g vitamin B2/l (IM) showed the best results for dressing weight percentage (78.26%) (Q, P = 0.035) and carcass weight percentage (75.52%) in comparison to the other groups (Q, P = 0.043). Our data support the idea that changing the riboflavin levels did not significantly alter the other carcass characteristic parameters. However, birds given 4g vitamin B2/l (IM) showed better results for giblet weight and empty body weight than other treatment groups (Q, P = 0.031, 0.038, respectively)

Table 3.

Effect of immersion Japanese quail eggs with different levels of riboflavin on carcass characteristics from 6 wk of age.

Items	Immersion treatment group				SEM	P- value1
	Control	3g	4g	5g		T	L	Q
Body weight before slaughter	178.00	169.33	183.33	185.33	2.11	0.067	0.070	0.052
Body weight after slaughter	173.66	164.66	180.33	181.00	2.98	0.097	0.76	0.092
Blood weight	4.33ab	4.66a	3.00c	4.33ab	1.67	0.043	0.040	0.047
Body weight after feathers removed	157.86	157.60	164.80	169.30	3.88	0.074	0.62	0.051
Feathers weight	15.80a	7.06c	15.53a	11.70b	5.16	0.034	0.037	0.029
Head weight	7.80	8.63	8.50	8.836	2.44	0.076	0.092	0.094
Non-giblets weight	6.36	5.90	7.60	6.86	2.97	0.063	0.071	0.67
Giblets weight	4.83	4.60	5.23	4.53	1.83	0.077	0.084	0.031
Liver weight	3.30	3.10	3.60	3.23	0.95	0.068	0.071	0.066
Heart weight	1.53	1.50	1.63	1.30	0.87	0.142	0.133	0.099
Testes weight	1.639b	3.60a	1.06b	1.16b	1.05	0.033	0.029	0.047
Leg weight	4.30	4.13	4.03	3.76	2.11	0.178	0.135	0.175
Gizzard weight	3.83	2.93	3.53	3.40	1.86	0.214	0.201	0.174
Empty body weight	131.86	125.80	138.33	121.50	2.35	0.068	0.047	0.038
Carcass weight %	74.04ab	74.280ab	75.52a	65.54c	2.74	0.014	0.046	0.043
Dressing weight %	76.76ab	77.01ab	78.26a	67.98c	2.58	0.003	0.049	0.035

^a,bMeans bearing different letters in the same row differ significantly (P < 0.05, 0.001).

¹T, overall effects of treatments; L, linear effects of increasing riboflavin levels of quails; Q, quadratic effects of increasing riboflavin levels of quails.

Hot carcass % = [Empty body weight including head wt. / Pre-slaughter weight] × 100.

Dressed % = [Hot carcass wt. + Giblets weight (liver+ heart weights)] / Pre-slaughter weight × 100.

Moreover, the information suggests that riboflavin supplementation in Japanese quails does not negatively impact the carcass characteristics. These findings indicate that the carcass traits of Japanese quails are not negatively affected by riboflavin supplementation. The comparable results align with a previous study conducted by Leiber et al. (2022), who assumed that Japanese quails fed a diet supplemented with (2.5 or 4) mg of vitamin B2 per kg might have noticed improvements in carcass features and reduced visceral and shoulder fat. In contrast, different riboflavin treatments did not affect the growth status, general health, or carcass quality of 1- to 6-wk-old Japanese quails. Riboflavin aids tissue growth, feather development, and energy synthesis. Insufficiency can lead to delayed growth, abnormal feather development, and immune system dysfunction. In addition, studies on different chicken ideal dietary riboflavin concentrations have improved growth rate and FCR in broilers, indirectly affecting carcass yield. Excessive supplementation could not yield further benefits (Tang et al., 2023). In order to protect birds from illnesses and infections that could impair their health and growth, riboflavin boosts their immune systems, which helps them withstand infections and diseases and produce higher-quality carcasses (Yogeswari et al., 2024). Riboflavin deficiency can hinder muscle growth and development. This could potentially affect carcass weight and meat yield. Also, B2 is involved in the stress response. Deficient quail might be more susceptible to stress during pre-slaughter handling, potentially impacting meat quality (Işık and Çiçek, 2024).

Blood Parameters

Table 4 presents the blood parameters of Japanese quail following the experiment, illustrating the impact of (3, 4, and 5) g of vitamin B2/l immersion treatment. When Japanese quail eggs were immersed in (3, 4, and 5g) of vitamin B2/L, show significant (P < 0.05) enhancements in blood indices in comparison to the control group. The finding indicates that the Japanese quails who received 5g of vitamin B2/L (IM) revealed the most positive hemoglobin(20g/dl), platelets (11.5U/L), total protein (5g/dL), albumin (2g/dL), globulin (3g/dL), RBCs (3.41 × 10⁶), WBCs (209 × 10³), MCH (61.80pg), MCV (132.78Fi), MCHC (41g/dL), ALT (9.43U/L), and AST (173U/L) levels when compared to the other groups. However, A / G ratio was higher in all treatments than 3g vitamin B2/L (IM) group (L, P = 0.011). These results are aligned with those presented by Goel et al. (2013), and El-Kholy et al. (2019), who found that Japanese quails supplemented with vitamin B2 revealed significantly increased RBC, albumin, serum total protein, PCV, Hb, and RBC levels (P < 0.05) than the control group.

Table 4.

Effect of immersion Japanese quail eggs with different levels of riboflavin on blood traits from 6 wk of age.

Items	Immersion treated group				SEM	P- value1
	Control	3g	4g	5g		T	L	Q
Total protein (g/dL)	3.38c	4.30b	`4.63b	5.00a	2.67	0.010	0.009	0.054
Albumin (g/dL)	1.45b	1.45b	1.90a	2.00a	2.96	0.043	0.038	0.037
Globulin (g/dL)	1.93c	2.85ab	2.73b	3.00a	4.27	0.001	0.001	0.000
A/G ratio	0.76	0.51	0.70	0.67	1.85	0.214	0.011	0.223
Hemoglobin (g/dL)	15.30c	18.65b	19.70ab	20.00a	6.38	0.000	0.001	0.001
RBC's (×106)	2.13d	2.57c	2.97b	3.41a	1.22	0.000	0.000	0.001
Platelets (U/L)	6.70c	10.60ab	11.20a	11.50a	5.32	0.024	0.038	0.047
WBC's (×103)	137.50c	187.50b	202.50a	209.00a	7.34	0.024	0.028	0.022
PCV (%)	35.65b	44.70a	45.60a	47.15a	7.54	0.031	0.029	0.032
MCH (Pg)	59.35b	59.60a	57.75b	61.80a	3.25	0.038	0.034	0.029
MCV (Fi)	94.49d	102.35c	117.99b	132.78a	2.99	0.010	0.011	0.009
MCHC (g/dL)	33.20b	39.05a	39.50a	41.00a	3.33	0.010	0.017	0.014
Creatinine (mg/dL)	0.46	0.48	0.47	0.45	1.07	0.111	0.107	0.384
ALT U/L	6.83b	9.00a	9.25a	9.43a	2.80	0.001	0.004	0.003
AST U/L	137.40c	159.30b	169.55a	173.00a	4.62	0.000	0.051	0.050

^a,bMeans bearing different letters in the same row differ significantly P < 0.05, 0.001).

¹T, overall effects of treatments; L, linear effects of increasing riboflavin levels of quails; Q, quadratic effects of increasing riboflavin levels of quails.

Vitamin B2 levels were shown to be significantly raised at all doses by Kitakoshi et al. (2007). These results are also in accordance with those reported by El-Kholy et al. (2019), who found that when Japanese quails with vitamin B2, comparing their blood levels to the control group, they revealed a significant (P < 0.05) elevation in total protein, albumin, and globulin. Blood parameters are typically linked to an individual's health. Hens under heat stress exhibit several physiological symptoms, including changed leukocyte counts and, more prominently, distinct lymphocytopenia and heterophilia. These conditions were previously considered realistic markers of stress (Bin-Jumah et al., 2020). For birds to maintain the health of their red blood cells, they must consume adequate riboflavin. A deficit may cause anemia, characterized by a drop in hemoglobin levels, red blood cell count, and oxygen-carrying ability. Moreover, riboflavin's role in metabolism and generation of energy may affect red blood cell development and synthesis (Aljaadi et al., 2023).

The action of riboflavin in DNA and RNA formation and cell proliferation may affect the production and function of white blood cells (Kiruba et al., 2023). Although the bird's body attempts to retain vitamin B2, daily consumption is required. Vitamin B2 is one of the B vitamins that support several cellular processes that provide the body energy and aid in producing red blood cells (Cogburn et al., 2018). According to the current investigation, vitamin B2 did not significantly (Q, P=0.384) raise creatinine levels at 42 d of age compared to the controls. Following the findings of Kitakoshi et al. (2007), increased AST and ALT levels were observed in response to riboflavin administration.

Furthermore, Faisal et al. (2008) stated that heat stress may have induced elevated liver enzymes (ALT and AST), which may have resulted in some liver damage in birds and mammals. FAD, a cofactor for ALT and AST, is produced from riboflavin. Additionally, ALT promotes the transfer of an amino group from alpha-ketoglutarate to glutamate and pyruvate from alanine. Furthermore, as the transfer moves forward, FAD participates by absorbing and releasing hydrogen atoms. Its redox characteristics make it easy to take and give electrons, making the transfer of amino groups easier (Olfat et al., 2022). The liver plays a crucial role in metabolism, and numerous enzymes are involved in these processes. Adequate riboflavin ensures these enzymes have the cofactors they need for proper function, potentially impacting processes like detoxification, nutrient breakdown, and energy production (Kumar et al., 2024). Many enzymes involved in breaking down toxins and harmful molecules require FMN or FAD to function. These enzymes might be responsible for neutralizing free radicals, conjugating toxins for excretion, or metabolizing drugs. Without enough riboflavin, these enzymes become less efficient, potentially leading to a buildup of toxins in the body (Zhu et al., 2024).

Fertility and Hatchability Parameters

The hatchability and fertility percentages of immersion-treated Japanese quail eggs (3, 4, and 5) g vit. B2/l are displayed in Table 5. The results demonstrated that Japanese quail eggs treated with (3, 4, and 5) g vit. B2/l immersion revealed a significantly increased fertility percentage (Q, P = 0.034) compared to the controls. The eggs that received the maximum dosage of 5g of vitamin B2/l (IM) had the highest fertility rate (96.93%). Conversely, the control group's fertility percentage (81.54%) was the lowest. The percentage of unhatched eggs in the group treated with (3, 4, and 5g) vit. B2/L immersion significantly elevated (P < 0.05) in comparison to the control group. Additionally, vitamin B2 in 3, 4, and 5 g/L immersions is administered to Japanese quails, and the percentage of infertile eggs was significantly decreased (P < 0.05), and G4 revealed the lowest result (3.03). Moreover, all eggs were treated with (3, 4, and 5g) vit. B2/L immersion had significantly elevated (P < 0.05) hatchability of total egg percentage in comparison to the control group, and G4 showed the highest results (84.62%), Followed by the G3 that received 4g vit. B2/L (IM) (78.47%). On the other hand, within all the groups of eggs, the control group had the lowest hatchability of total egg percentage values (69.23%) (Table 5). Furthermore, G4, which received 5g of vitamin B2/L (IM), had the highest hatchability percentage of fertile eggs (87.31%) compared to the other groups, while G2, treated with 3g of vitamin B2/L (IM), had the lowest readings (77.18%).

Table 5.

Effect of immersion Japanese quail eggs with different levels of riboflavin on fertility and hatchability parameters.

Items	Immersion treated group (g/l)				SEM	P- value1
	Control	3g	4g	5g		T	L	Q
Fertility %	81.54b	93.85a	93.85a	96.93a	1.88	0.004	0.047	0.034
Un-hatched egg (%)	6.40b	10.80a	4.80c	6.40ab	2.64	0.031	0.055	0.042
Pecked chicks	6.20	10.80	12.20	6.20	2.91	0.521	0.048	0.033
Non-Fertile eggs	18.47a	6.16b	6.22b	3.08c	4.27	0.000	0.172	0.097
Hatchability of total eggs, %	69.23c	72.31bc	78.47ab	84.62a	2.55	0.002	0.050	0.047
Hatchability of fertile eggs %	84.85ab	77.18c	83.59ab	87.31a	2.81	0.021	0.165	0.173
Chick's weight, g	87.71d	102.91c	116.63b	126.88a	4.08	0.000	0.043	0.038
Chick's weight, %	77.68c	86.08b	88.67b	91.21a	3.64	0.000	0.028	0.033

^a,bMeans bearing different letters in the same row differ significantly (P < 0.05, 0.001).

¹T, overall effects of treatments; L, linear effects of increasing riboflavin levels of quails; Q, quadratic effects of increasing riboflavin levels of quails.

Furthermore, the eggs that were treated with 5g of vitamin B2/L (IM) showed the best results in terms of chick weight (g, %) (126.88g and 91.21%). However, the weight (g, %) of the chicks in the control group was the lowest (87.71g and 77.68%, respectively). Nevertheless, birds given 3 or 4 g vitamin B2/L (IM) showed higher results for pecked chicks than other treatment groups (L, Q, P = 0.048, 0.033 respectively).

According to Florian et al. (2021) and Leiber et al. (2022), laying hens fed 4 mg vitamin B2/kg food and 3.1 mg vit. B2/kg diet, respectively, had a considerably greater (P < 0.05) fertility percentage than the control group. Moreover, the same findings were noted by Tang et al. (2019) and Zhang et al. (2020), who reported that during the different experimental phases, the fertility percentage of female ducks fed with 10 and 3 mg vitamin B2/kg diet, respectively, significantly improved. Furthermore, riboflavin is an antioxidant that protects against reactive oxygen species (ROS) damage to cells.

Stressors like heat and crowding may increase ROS production in quail. Akinyemi & Adewole (2021) state that adequate levels of riboflavin can improve reproduction and alleviate stress. Its antioxidant properties depend on riboflavin's active participation in the glutathione redox chain. Glutathione is a potent tripeptide antioxidant that is necessary to protect cells from harm caused by ROS. Riboflavin plays a role in this pathway by promoting the action of glutathione reductase. This enzymatic mechanism encourages reduced glutathione to regenerate from an oxidized state. Maintaining cellular redox equilibrium is crucial for mitigating oxidative stress and primarily relies on this dynamic mechanism (Silva-Araújo et al., 2023; Shastak & Pelletier, 2023). Hatchability and reproductive performance are vital components of the poultry business that significantly influence the efficacy of egg production and the general success of chicken farming. (Baker et al., 2023). Vitamin B2 increases the body's vitality, hatchability %, reproductive rate, and chick body weight in hatching birds, according to Hocking et al. (2013).

The digestive system's mucous membranes protect and preserve it from diseases that could damage it (Witten and Aulrich, 2018, 2019). Additionally, riboflavin keeps egg white albumen firmly and structurally stable. Consequently, optimum egg quality may indirectly impact hatching success even when not directly interrelated to fertility (Khillare & Chilkhalikar, 2022).

Furthermore, riboflavin is a part of numerous vital metabolic processes necessary for a properly developing embryo. It supplies energy to the developing embryo by acting as a coenzyme in the electron transport chain. Deficiency might cause abnormal development, growth retardation, and decreased energy production (Shastak & Pelletier, 2023). The synthesis of albumen, which provides the developing embryo protection and essential nutrients, is aided by riboflavin. Sufficient vitamin B2 may increase the quality of the eggs and the viability of the embryos (Mirani et al., 2023). These findings are in line with the results of Florian et al. (2021) and Leiber et al. (2022), who discovered that when hens were fed 3.1 mg of riboflavin per kg of diet and 4 mg of vitamin B2 per kg of feed, significantly (P < 0.05) improve in the weight of chicks and the hatchability of the entire egg set (%) and fertile eggs percentage in comparison to the control group.

The percentage of hatchability eggs and the percentage of set hatchability eggs improved significantly (P < 0.05) in female ducks given 10 and 3 mg of riboflavin/kg of diet during the various trial intervals, according to Zhang et al. (2020) data. A riboflavin deficiency may impact reproduction in many ways, all involving complicated physiological mechanisms. Hypovitaminosis B2 is associated with riboflavin's action in energy metabolism and can cause a metabolic disorder (Cogburn et al., 2018). Since riboflavin is a coenzyme that creates FAD and FMN, it plays a role in the oxidative phosphorylation process, which is necessary for the production of ATP, the primary energy unit of cells (Balasubramaniam & Yaplito-Lee, 2020). Inadequate vitamin B2 levels may affect the increased energy demands of reproductive activities, including follicle progress, egg production, ovulation, and the viability and growth of the embryo. This is because low vitamin B2 levels can obstruct energy production (Shastak & Pelletier, 2023; Pirgozliev et al., 2024).

Economic Efficiency

Table 6 presents the economic efficiency (EE) for birds treated by immersion at (3, 4, and 5) g vit. B2/l. According to the data, the EE for birds treated with immersion (3, 4, and 5) g vit. B2/L was significantly (P < 0.05) elevated than the control group at the end of the research period (one to 6 wk of age). The 5g vit achieved the highest EE result. B2/L (IM) group. However, following the 1- to 6-wk trial period, the control group and G2 were treated with 3g vit. B2/L (IM) revealed the minimum EE levels. The results showed that the EE of Japanese quails preserved by immersion at all levels was significantly more than that of the control group. The results reported align with the finding of Arijeniwa et al. (1996); they found that the laying hens fed 3, 4, and 5 g vitamin B2 / kg diet showed the maximum EE compared to the control groups. Shastak and Pelletier (2023) also suggested that a lack of riboflavin could cause deformities and mortality in quail chicks. Supplementation can ensure that chicks survive and thrive, decreasing losses and increasing the overall productivity of the laying flock.

Table 6.

Effect of immersion Japanese quail eggs with different levels of riboflavin on economic efficiency from 1 to 6 wk of age.

Items	Control	3g (IM)	4g (IM)	5g (IM)
Total number chicks	45.00	47.00	51.00	55.00
Price/chick ($)	0.11	0.11	0.11	0.11
Total revenue chick ($)	4.95	5.17	5.61	6.05
Egg number	65.00	65.00	65.00	65.00
Price/Egg ($)	0.053	0.053	0.053	0.053
Total cost/ egg ($)	3.445	3.445	3.445	3.445
Fixed egg ($)	0.013	0.072	0.096	0.12
Total cost ($)	3.458	3.517	3.541	3.565
Net revenue ($)	1.492	1.653	2.069	2.485
Economic efficiency (EE)	43.14	47.00	58.42	69.70

Total revenue hen ($) = Number chicks × Price/chick ($).

Total cost/ egg ($) = Egg Number × Price/Egg ($).

Total cost ($) = Total cost/ egg ($) + Fixed bird ($).

Net revenue ($) = Total revenue chick - Total cost/egg ($).

Economic efficiency (EE) = Net revenue ($) / Total cost ($) × 100.

CONCLUSIONS

The study showed that adding various quantities of riboflavin to Japanese quail eggs by immersion may improve the birds' growth performance, blood and carcass traits, and cost-effectiveness over all periods analyzed. Furthermore, immersing Japanese quail eggs in vitamin B2 at a concentration of 5g/l improved their blood biochemical parameters, hatchability, and financial effectiveness.