Optimal dietary vitamin B1 content enhanced egg production, eggshell thickness, and serum antioxidant status in breeder geese

Article information

Anim Biosci. 2025;38(8):1746-1755

Publication date (electronic) : 2025 February 27

doi : https://doi.org/10.5713/ab.24.0751

Lin Dai ¹^,^a

, Baowei Wang ¹^,

, Qian Li ¹^,^a

, Mingai Zhang ¹

, Jing Zhang ²

, Bin Yue ³

, Min Kong ⁴

, Binghan Wang ⁵

, Wenlei Fan ¹^,

¹College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, China

²College of Food Science and Engineering, Qingdao Agricultural University, Qingdao, China

³College of Science and Information Science, Qingdao Agricultural University, Qingdao, China

⁴Institute of High-Quality Waterfowl, Qingdao Agricultural University, Qingdao, China

⁵Qingdao Huihe Biotechnology Co., Ltd., Qingdao, China

^*Corresponding Author: Baowei Wang, Tel: +86-532-58957780, E-mail: wangbw@qau.edu.cn

Wenlei Fan, Tel: +86-532-58977771, E-mail: fanwenlei@qau.edu.cn

aThese authors contributed equally to this work.

Received 2024 October 25; Revised 2024 November 19; Accepted 2025 January 24.

Abstract

Objective

This study aimed to evaluate the effects of vitamin B1 (VB1) supplementation on laying performance, egg quality, serum biochemical parameters, antioxidant status, and nutrient digestion in breeder geese.

Methods

A total of 150 geese (30 males and 120 females, aged 34 weeks) were randomly assigned to 6 dietary treatment groups, each 5 replicates of 5 birds (1 male and 4 females). The geese were fed a basal diet supplemented with 0 (control), 1, 2, 3, 4, or 5 mg/kg of VB1 for 10 weeks.

Results

VB1 supplementation had no significant effects on average feed intake, average egg weight, feed-to-egg ratio, egg shape index, eggshell strength, protein height, and Haugh unit (p>0.05). However, it increased egg-laying rate, eggshell thickness, and yolk color (p<0.05) in a quadratic manner, with the maximum values observed at 2 mg/kg VB1. Supplementing 2 mg/kg VB1 reduced serum aspartate transaminase activity (p<0.05), but did not affect serum alanine transaminase activity, lipid and protein concentrations (p>0.05). Serum glutathione peroxidase and total superoxide dismutase activities were enhanced by VB1 supplementation (p<0.05), while total antioxidant capacity and malondialdehyde concentration remained unchanged (p>0.05). Additionally, VB1 supplementation at 2 mg/kg increased crude ash digestibility, but did not affect the digestibility of ether extract, crude protein, calcium, and phosphorus.

Conclusion

Dietary supplementing VB1 improved egg-laying performance, egg quality, antioxidant status, and mineral absorption in breeder geese. The optimal dietary VB1 concentration ranged from 3.83 to 4.81 mg/kg for improving egg production and quality, while higher concentrations of 4.99 to 6.51 mg/kg were needed to boost serum antioxidant status.

Keywords: Antioxidation; Egg Production; Geese; Nutrient Digestion; Reproduction; Vitamin Requirement

INTRODUCTION

Geese (Anas cygnoides) are among the most economically important farm birds, with over 600 million reared commercially in China. However, their relatively low laying performance, with an egg-laying rate (LR) of less than 50% during the breeding season, poses a significance challenge to the growth of the goose industry [1–3]. This limitation has driven increasing interest in strategies to improve laying performance, with a particular focus on developing nutritionally balanced feed formulation.

Vitamin B1 (thiamine, VB1) is essential for energy metabolism, neurological function, antioxidant defense, digestion, and cardiovascular health. Dietary VB1 levels have been shown to influence the development and health of various animal species [4]. VB1 deficiency can cause memory and learning impairments, immune suppression, blood-brain barrier dysfunction, and neurological disorders in animals [5–9]. In rats, VB1 deficiency has been linked to delayed sexual maturation, reduced reproductive capacity, and lactation failure [10]. In livestock, VB1 supplementation has been associated with improved reproductive performance. For example, supplementation of VB1, vitamin E, and selenium improved pregnancy rates in Merino ewes [11], highlighting VB1’s importance in reproductive system function. In chickens, appropriate levels of VB1 supplementation enhanced productive and slaughter performances [12]. Chen et al [13] demonstrated that a corn-soybean meal based diet sufficiently met the VB1 requirements of Longyan egg-laying ducks from 22 to 42 weeks of age. Moreover, our previous research determined the dietary VB1 requirements for growing geese: 5.60 mg/kg of feed for geese aged 0 to 4 weeks and 4.98 mg/kg for those aged 5 to 15 weeks [14].

Research on the dietary VB1 requirements for laying geese remains limited. In the absence of specific data, the Dale N [15] recommends using the requirements established for growing geese (2.0 mg/kg) and adult geese (1.8 mg/kg), with breeding geese often using nutrient estimates for chickens. To fill this knowledge gap, we supplemented diets with graded levels of VB1 and assessed its effects on laying performance, egg quality, serum biochemical parameters, antioxidant status, and nutrient digestion in laying Wulong geese. This study aims to determine the optimal dietary VB1 dosage for breeding geese, providing evidence-based recommendations for feed supplementation during the laying period.

MATERIALS AND METHODS

Birds, Management and Diet

The experiment was approved by the Animal Ethics Committee of Qingdao Agricultural University (IACUC, approval number: QAU20210319), in accordance with the Guidelines for Experimental Animals established by the Ministry of Science and Technology. A total of 150 Wulong breeder geese (30 males and 120 females), aged 34 weeks with an average body weight of 3±0.2 kg (mean±standard deviation), were obtained from the Breeding Center of Qingdao Agricultural University (Shandong, China), where the experiment was conducted. The geese were randomly assigned to 6 dietary treatments in a randomized complete block design, with egg production level as a blocking factor, using the Experimental Animal Allocation Procedure (EAAP). Geese were reared in a free-range system within fenced pens, with half of each pen covered by a shed containing feeders and cylindrical plastic water tanks. Each pen measured 12 m×1.24 m and was equipped with a wire floor inside the shed and a sand floor in the open yard. Geese were randomly allocated to 30 pens, each had 5 birds (1 male and 4 female). During the experimental period, geese had ad libitum access to feed and water and were housed outdoors under natural daylight conditions. Birds were fed twice daily, at 08:00 and 17:00. The basal diet was formulated according to Dale [15] recommendations to meet the nutrient requirements of breeder geese. The ingredient composition and nutrient content of the diet are presented in Table 1, with an analyzed VB1 level of 2.26 mg/kg.

Table 1

Composition and nutrient concentrations of the basal diet

Experimental design

The 30 pens of geese were randomly assigned to 6 dietary treatment groups, each with 5 replicate pens, and randomly allocated to the diets supplemented with 0 (control), 1, 2, 3, 4, or 5 mg VB1 per kg of diet. Each pen, containing 5 geese, served as a biologically experimental unit to minimize confounding factors such as social interactions, feed competition, and environmental influences on individual performance [16,17]. The experiment lasted for 10 weeks. This design was practically manageable, ensuring consistent experimental conditions (e.g., diet preparation, monitoring, and data collection) without overburdening resources.

Crystalline VB1 (purity 98%), purchased from Qingdao Puxing Biotechnology Co., Ltd. (Qingdao, China), was mixed into the vitamin premix and then incorporated into the final diet.

Laying performance and collection of blood sample

The feed consumption of birds in each pen was recorded daily to calculate the average daily feed intake (ADFI), average egg weight (AEW), LR, and feed-to-egg ratio (F/E). On the last day of the experiment, 2 female geese from each pen were randomly selected, fasted for 12 h, and had blood samples collected from the wing vein into coagulant tubes. The samples were centrifuged at 1,520×g at 4°C for 10 min to separate the serum, which was then stored at −20°C for analysis.

Egg quality

Eggs laid during the last two weeks of the experiment were collected daily and stored at 16°C with 75% relative humidity. The egg quality analyses included egg weight, shape index, eggshell strength, eggshell thickness, albumen height, yolk color, Haugh unit, and yolk weight. All analyses were performed within 24 hours of collection. Egg length and width were measured using a digital caliper, and the egg shape index (ESI, %) was calculated as width/length. Yolk weight was expressed as a percentage of total egg weight. Eggshell thickness was measured at the two poles and the middle of the shell with a digital micrometer. The remaining analyses were performed using an Egg Analyzer (DET-6000; NABEL Co., Ltd., Kyoto, Japan) according to the manufacturer’s protocols. Detailed procedures are provided in the Supplement 1.

Measurement of biochemical parameters and antioxidant indices

Serum samples were analyzed for concentrations of triglyceride (TG), total cholesterol (T-CHO), albumin (ALB), total protein (TP), and malondialdehyde (MDA), the activity of aspartate transaminase (AST), alanine transaminase (ALT), glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), as well as total antioxidant capacity (T-AOC). All analyses were performed using commercial kits (Nanjing Jiangcheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocols. Detailed protocols are described in the Supplement 1.

Nutrient utilization

In the final week of the experiment, two female geese from each replicate pen were randomly selected and placed in individual metabolic cages. After a 24-hour fast, each bird was fed 120 g/day of the diet for 3 days. Fecal excreta were collected daily for 3 days, pooled by cage, and dried at 65°C to a consist weight. Both feed and fecal excreta samples were ground and analyzed for crude fat (ether extract), crude protein, crude ash, calcium (Ca), and phosphorus (P) following AOAC methods [18]. Ca and P contents were determined using inductively coupled plasma-optical emission spectrometry (Optima 8×00; PerkinElmer Inc., Alpharetta, GA, USA).

Statistical analysis

The effects of dietary VB1 supplementation were analyzed using one-way ANOVA in SPSS statistical software (version 20.0, SPSS, Chicago, IL, USA). For variables showing significant differences, Duncan’s multiple-range test was used to compare means between treatments. Polynomial contrasts were applied to evaluate linear and quadratic effects of VB1 levels. Data are presented as means with pooled standard error of the mean, and differences were considered statistically significant at p<0.05.

The estimation of the maximum responses to total dietary VB1 was conducted using a quadratic polynomial model [19,20] as follows:

Y=β0+β1×X+β2×X2,

where Y is the responsive variable, X represents dietary total VB1 concentration (mg/kg diet); β₀ is the intercept; β₁ stands for the linear coefficient; and β₂ as the quadratic coefficient. The total dietary VB1 concentration corresponding to the maximum response is calculated as VB1 = − β₁÷(2×β₂).

RESULTS

Egg-laying performance

As shown in Table 2, the VB1 level in the diet had a significant effect on LR (p<0.05), exhibiting a quadratic response (p< 0.05). The maximum LR was achieved at 2 mg/kg VB1 supplementation (Figure 1A), corresponding to a total of 4.14 mg/kg VB1 in the diet (Figure 1A). In contrast, there was no significant effect of VB1 supplementation on ADFI, AEW, and F/E (p>0.05).

Table 2

Effects of dietary vitamin B1 supplementation on production performance indexes in breeder geese

Figure 1

The effect of supplementing vitamin B1 in the diet on egg production rate (A), eggshell thickness (B), GSH-Px(C), and T-SOD (D). GSH-Px, glutathione peroxidase; T-SOD, total superoxide dismutase.

Egg quality

Egg quality results are presented Table 3. Dietary VB1 supplementation had significant effects on eggshell thickness (Figure 1B) (p<0.05) and egg yolk color (p<0.05), with a quadratic responsive fashion (p<0.05). The maximum value of eggshell thickness was reached at 2 mg/kg VB1 supplementation, corresponding to a total of 3.83 mg/kg VB1 in the diet. VB1 supplementation also had a significant effect on the egg yolk percentage (p<0.05). However, it had no significant effects on the egg weight, ESI, shell strength, protein height, and Haugh units (p>0.05).

Table 3

Effects of vitamin B1 supplementation on egg quality of breeder geese

Serum biochemical parameters

As shown in Table 4, dietary VB1 supplementation at a dose of 2 mg/kg significantly reduced serum aspartate transaminase activity (p<0.05). In contrast, serum concentrations of TGs, T-CHO, ALB, TP, and ALT activity were not affected by dietary VB1 supplementation (p>0.05).

Table 4

Effects of vitamin B1 supplementation on serum biochemical indicators in breeder geese

Antioxidant indicators

Serum antioxidant indicators are shown in Table 5. Dietary VB1 supplementation significantly enhanced the activity of GSH-Px (p<0.05) and T-SOD (p<0.05) in a quadratic response pattern (Figures 1C, 1D). The maximum GSH-Px activity occurred at 4 mg/kg VB1 supplementation, the corresponding to a total dietary VB1 concentration of 6.51 mg/kg, while the highest T-SOD activity was observed at 2 mg/kg VB1 supplementation, corresponding to 4.99 mg/kg VB1 in the diet. However, dietary VB1 supplementation had no significant effects on T-AOC activity and MDA concentration (p>0.05).

Table 5

Effects of vitamin B1 supplementation on antioxidant indicators in serum of breeding female geese

Nutrient digestibility

Table 6 shows that dietary VB1 supplementation increased crude ash digestibility (p<0.05) in a quadratic response pattern, with the highest digestibility observed at a dose of 2 mg/kg. However, it had no significant effects on digestibility of ether extract, crude protein, calcium, and phosphorus (p> 0.05).

Table 6

Effect of vitamin VB1 supplementation on nutrient utilization efficiency of breeder geese

Optimal dietary vitamin B1 concentration

Several measures showed a quadratic relationship with dietary VB1 concentration; therefore, quadratic polynomial analysis was performed to determine the optimal VB1 concentration associated with the maximum response for each measure. The results are presented in Table 7. The optimal total VB1 concentration in the diet was 4.14 mg/kg LR, 3.83 mg/kg for eggshell thickness, 4.81 mg/kg for yolk color, 4.36 mg/kg for egg yolk percentage, 6.51 mg/kg for GSH-Px activity, and 4.99 mg/kg for T-SOD activity, respectively.

Table 7

Quadratic polynomial¹⁾ response regressions with total dietary VB1 concentration (mg/kg diet) in breeder geese

DISCUSSION

VB1 supports poultry growth, energy metabolism, and offspring health [13,14,21]. Both deficiency and excess of VB1 can reduce productivity and cause metabolic disturbances, illness, and even mortality [22]. Research in ducks and chickens has highlighted the role of VB1 in improving production performance [23,24]. Our previous studies have shown its positive effects on the reproductive performance of geese [25]. In this study, we demonstrated its effects on breeder geese. We found dietary supplementation of 2 mg/kg of VB1 significantly enhanced LR in breeder geese. Previous studies found that VB1 deficiency can impair muscle function, leading to reduced egg production in poultry [26–28]. However, excessive VB1 supplementation also negatively affected egg production, as observed in the present study. These results are consistent with research on other B vitamins, such as riboflavin (B2) and pyridoxine (B6), which are crucial for poultry health [29,30]. Therefore, optimizing vitamin supplementation in poultry diets is essential for meeting nutritional requirements, improving production performance, and enhancing economic efficiency.

This study demonstrated that VB1 supplementation improved egg quality, such as increased eggshell thickness and yolk color, with the most pronounced effect on yolk pigmentation observed at a dose of 2 mg/kg. Conversely, excessive VB1 intake reduced eggshell thickness. Increased eggshell thickness is critical for minimizing egg breakage and embryonic mortality by preventing water loss, heat stress, and microbial invasion during incubation [31]. Previous studies have shown that varying dietary levels of VB1 can improve egg quality in breeder ducks [13]. Additionally, optimizing B-vitamin combinations has been shown to enhance overall egg quality [32], which aligns with our findings in this study. Comparisons with other vitamins, such as vitamin A and vitamin G, underscore the importance of a balanced intake of B vitamins is vital for maximizing egg quality and production efficiency in poultry [33]. While few studies have specifically address VB1’s role in egg quality, the broader importance of vitamin balance in maintaining egg quality is widely recognized [34–36] Further research is necessary to elucidate the specific mechanisms by which vitamins influence egg quality characteristics across different poultry species.

Serum biochemical parameters are significant indicators of the nutritional and health status of geese. This study found that VB1 supplementation had no significant effect on blood lipids and proteins, but it effectively regulated liver enzyme activities by reducing AST activity. Our results are align with previous studies in chickens [37]. In ducks, VB1 supplementation moderately affects blood protein levels, primarily due to its involvement in energy metabolism rather than direct regulation of protein synthesis [38]. In contrast, VB2, VB6, and VB12 exert more pronounced effects on lipid and protein metabolism, as they are more directly involved in coenzyme functions related to amino acid and fatty acid metabolism [39]. However, VB1 is primarily involved in energy production, making it less directly associated with lipid and protein regulation. Elevated AST activity is commonly linked to liver damage [40]. Studies in rats have demonstrated that VB1 deficiency negatively affects AST activity, disrupting energy metabolism and overall health [41–43]. Based on these findings, appropriate VB1 supplementation could improve liver health in geese without significant effects on its lipid and protein dynamics.

Antioxidant enzymes play a crucial role in mitigating oxidative stress, which can impair health and productivity in animals. This study demonstrated that appropriate VB1 supplementation enhanced serum GSH-Px and T-SOD activity in breeder geese, thereby enhancing their antioxidant status. VB1 is essential for the biosynthesis of antioxidant compounds [44], supporting overall antioxidant function in ducks [36], and enhancing antioxidant enzyme activity in fish by regulating cellular redox balance [45]. Ma et al. [46] found that supplementation of 200 mg/kg VB1 in a high-concentrate diet improved the total plasma antioxidant capacity in goats. These findings indicate that VB1 likely enhances antioxidant enzyme activity in geese by promoting the biosynthesis of antioxidant substances. Furthermore, studies on vitamins VB9 and VB12 suggest that combining these vitamins can have synergistic effects on improving egg production and overall health in poultry [47]. The commercial implications of these findings suggest that B-vitamins supplementation can reduce oxidative damage and optimize poultry health under farm conditions. However, a limitation of this study is its focus on geese, warranting further investigation across other poultry species to better understand the broader applicability of these findings.

The enhanced production performance of breeder geese may be resulted from improved nutrient digestion. In this study, dietary supplementation with 2 mg/kg of VB1 increased crude ash digestibility, indicating enhanced mineral absorption. Crude ash content, which reflects the total mineral content in poultry manure, serves as an indicator of mineral digestion and utilization [48]. Mohseni et al. [49] reported that dietary VB1 supplementation increased total body ash content in juvenile fish, suggesting improved mineral utilization efficiency. Similarly, VB1 levels were found to indirectly enhance nutrient utilization efficiency in broiler chickens [50]. VB1 interacts with proteins through non-coenzyme mechanisms, potentially modulating mineral utilization [8,51,52]. Research on other B vitamins, such as B2 and B6, further emphasizes their roles in improving mineral absorption and utilization in poultry [24,53]. These findings underscore the critical role of B-vitamins in optimizing mineral absorption across poultry species. However, limitations of this study include the absence of long-term performance data and the influence of varying environmental conditions, which may affect results. Future research should address these factors to enhance the practical application of vitamin supplementation in commercial poultry production.

CONCLUSION

This study demonstrates that VB1 supplementation enhanced egg-laying performance, egg quality, antioxidant status, and mineral absorption in breeder geese. The optimal dietary total VB1 concentration ranged from 3.83 to 4.81 mg/kg for improving egg production and quality, while higher concentrations of 4.99 to 6.51 mg/kg were required to boost serum antioxidant status. Future research should explore the long-term effects of VB1 on reproduction and overall health in geese, as well as investigate potential interactions between VB1 and other vitamins to optimize poultry productivity and health.

Notes

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

AUTHORS’ CONTRIBUTION

Conceptualization: Wang B.

Data curation: Li Q.

Formal analysis: Dai L.

Methodology: Zhang M, Zhang J.

Software: Dai L, Wang B.

Validation: Dai L, Yue B.

Investigation: Dai L, Li Q, Kong M.

Writing - original draft: Dai L, Li Q.

Writing - review & editing: Dai L, Wang B, Li Q, Zhang M, Zhang J, Yue B, Kong M, Wang B, Fan W.

FUNDING

This study was supported by China Agriculture Research System of MOF and MARA (CARS-42-14) and Key Technology Research and Development Program of Shandong (2021CXGC010805).

ACKNOWLEDGMENTS

Not applicable.

DATA AVAILABILITY

Upon reasonable request, the datasets of this study can be available from the corresponding author.

ETHICS APPROVAL

DECLARATION OF GENERATIVE AI

No AI tools were used in this article.

SUPPLEMENTARY MATERIAL

Supplementary file is available from: https://doi.org/10.5713/ab.24.0751

Supplement 1. Effects of dietary vitamin B1 on laying performance, egg quality, serum biochemicals, antioxidant capacity, and nutrient digestion ability of geese.

ab-24-0751-Supplementary-1.pdf

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Items	Dry matter basis (%)
Ingredients
Corn	60.00
Soybean meal	23.50
Wheat bran	3.00
Chrysanthemum pedicel powder	3.00
Soybean oil	2.00
Limestone	6.00
Ca (HCO₃)₂	1.50
NaCl	0.30
Trace elements premix¹⁾	0.50
Vitamin premix¹⁾	0.10
Methionine	0.10
Nutrient concentrtions²⁾
ME (MJ/Kg)	11.48
CP	16.22
CF	4.05
Ca	2.64
P	0.35
Lys	0.67
Met + Cys	0.57
Trp	0.19
Thr	0.61
Cl	0.14
VB₁ (mg/kg)	2.26

Items	Treatments¹⁾ (VB1 mg/kg)						SEM	p-value

	0 (CON)	1	2	3	4	5		ANOVA	Linear	Quadratic
ADFI (g/d)	181.44	181.63	193.29	183.36	184.22	175.06	2.57	0.521	0.529	0.137
AEW (g)	122.89	126.27	128.03	126.30	125.53	125.68	0.81	0.650	0.567	0.173
F/E	5.59	5.15	5.09	5.49	5.91	5.83	0.14	0.456	0.190	0.250
LR (%)	27.76^bc	29.82^b	33.62^a	27.81^bc	26.34^c	25.44^c	0.59	<0.001	0.001	<0.001

Items	Treatments¹⁾ (VB1 mg/kg)						SEM	p-value

	0 (CON)	1	2	3	4	5		ANOVA	Linear	Quadratic
Egg weight (g)	119.72	130.76	132.10	130.17	122.45	120.78	1.70	0.090	0.504	0.008
Egg shape index	1.45	1.44	1.43	1.44	1.43	1.43	0.00	0.600	0.109	0.917
Eggshell strength (kg)	4.95	4.95	4.96	4.95	4.96	4.93	0.00	0.625	0.387	0.290
Eggshell thickness (mm)	0.49^abc	0.51^ab	0.52^a	0.48^bc	0.48^bc	0.46^c	0.01	0.006	0.004	0.027
Protein height (mm)	15.19	14.61	14.63	14.57	15.46	14.87	0.12	0.183	0.714	0.198
Yolk color	3.13^c	3.53^ab	4.11^a	3.65^ab	3.62^ab	3.24^bc	0.08	0.002	0.370	0.006
Haugh Unit	118.01	117.67	118.52	117.20	118.26	117.53	0.18	0.304	0.593	0.776
Egg yolk percentage	33.30^abc	35.59^ab	36.43^a	35.80^ab	32.66^bc	32.28^c	0.47	0.016	0.084	0.003

Items	Treatments¹⁾ (VB1 mg/kg)						SEM	p-value

	0 (CON)	1	2	3	4	5		ANOVA	Linear	Quadratic
TG (mmol/L)	2.34	2.50	2.59	2.83	2.57	1.30	0.23	0.499	0.330	0.115
T-CHO (mmol/L)	3.63	3.76	4.28	4.30	4.22	3.60	0.27	0.949	0.838	0.355
ALB (g/L)	36.70	37.35	37.49	38.26	37.17	38.44	0.70	0.985	0.574	0.915
TP (g/L)	52.69	56.30	63.07	56.83	54.04	57.66	2.03	0.790	0.788	0.433
AST (U/L)	17.73^a	14.98^ab	13.49^b	16.63^ab	15.28^ab	15.68^ab	0.41	0.047	0.409	0.057
ALT (U/L)	10.60	10.81	8.09	9.68	9.48	9.24	0.48	0.660	0.376	0.490

Items	Treatments¹⁾ (VB1 mg/kg)						SEM	p-value

	0 (CON)	1	2	3	4	5		ANOVA	Linear	Quadratic
T-AOC (mg/mL)	0.31	0.29	0.41	0.38	0.36	0.31	0.18	0.309	0.592	0.072
GSH-Px (μmol/L)	697.28^b	696.27^b	839.39^a	843.76^a	850.95^a	847.17^a	29.27	0.034	0.037	0.026
T-SOD (U/mL)	546.32^b	665.83^ab	842.88^a	736.71^ab	663.10^ab	662.25^ab	26.65	0.028	0.310	0.005
MDA (nmol/mL)	3.28	3.06	1.79	3.15	3.66	4.17	0.25	0.125	0.123	0.051

Parameters	Regression equation	VB1 for maximum response (mg/kg)	p-value	R²
Egg laying rate	Y = −0.6407X²+5.304X+19.60	4.14	0.001	0.6141
Eggshell thickness	Y = −0.004286X²+0.0328X+0.4435	3.83	0.027	0.7524
Yolk color	Y = −0.1132X²+1.088X+1.263	4.81	0.006	0.7988
Egg yolk percentage	Y = −0.5227X²+4.561X+26	4.36	0.003	0.8150
GSH-Px	Y = −9.957X²+129.6X+433.6	6.51	0.026	0.8439
T-SOD	Y = −28.65X²+286X+57.31	4.99	0.005	0.7001
Crude ash	Y = −1.738X²+17.82X−14.31	5.13	0.001	0.7668

Items	Treatments¹⁾ (VB1 mg/kg)						SEM	p-value

	0 (CON)	1	2	3	4	5		ANOVA	Linear	Quadratic
Ether extract (%)	74.93	75.21	78.58	80.32	75.80	76.47	0.74	0.228	0.448	0.076
Crude protein (%)	71.68	76.15	84.09	82.73	74.06	78.60	1.48	0.090	0.337	0.039
Crude ash (%)	18.35^c	21.39^bc	34.48^a	28.96^ab	29.98^ab	23.21^bc	1.38	0.001	0.042	0.001
Calcium (%)	20.32	24.20	26.54	24.31	22.57	21.59	0.96	0.519	0.969	0.076
Phosphorus (%)	49.28	48.57	49.47	53.66	44.58	49.83	1.46	0.594	0.844	0.754