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.
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:
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).
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).
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).
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).
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).
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.
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.
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