The role of riboflavin refeeding in reversing deficiency effects on duck reproductive performance and embryonic development

Abstract

Riboflavin (vitamin B₂) is essential for poultry reproduction and embryonic development. Riboflavin deficiency adversely affects egg hatchability and embryonic survival, but whether these effects are reversible remains unclear. This study aimed to assess the effect of riboflavin refeeding (RR) on reproductive performance, embryonic development, riboflavin status, antioxidant capacity, and hepatic metabolism in duck breeders and embryos. A total of 60 duck breeders (40 weeks, 3.45 ± 0.90 kg) were randomly assigned to two groups: a riboflavin-deficient (RD) group (n = 40) and a control (CON) group (n = 20). The RD group received a riboflavin-deficient diet (1.48 mg/kg), while the CON group was fed a riboflavin-sufficient (RF) diet (16.48 mg/kg) for 9 weeks. Following this period, the pigeons previously fed the riboflavin-deficient diet were randomly divided into two subgroups: one continued receiving the RD diet, and the other was refed a riboflavin-sufficient diet (RR) group. The RR and CON groups were maintained on the RF diet for an additional 5 weeks. Riboflavin deficiency reduced egg hatchability (P < 0.001), but hatchability was restored to near-normal levels within 1 week of refeeding (P > 0.05). Plasma and tissue riboflavin concentrations, along with hepatic flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) levels, were restored to CON levels after 5 weeks of refeeding (P > 0.05). RR improved hepatic antioxidant capacity (P < 0.05) including glutathione peroxidase (GPX), reduced glutathione (GSH) and glutathione reductase (GR), reduced lipid accumulation (P < 0.001) including total lipid (TL), triglycerides (TG), and free fatty acids (FFA), and restored liver histomorphology. Embryonic growth and development were also restored to levels comparable to those in riboflavin-sufficient conditions (P > 0.05). Metabolomic analysis revealed that RR partially restored metabolic balance related to protein, vitamin, and lipid metabolism. These findings indicated that RR over a 5-week period can effectively reverse the adverse effects of a 9-week deficiency. However, full restoration of antioxidant capacity and metabolic balance may require longer refeeding periods or higher doses. These findings highlight the crucial role of riboflavin in poultry health and provide insights for preventing and treating riboflavin deficiency.

1. Introduction

Riboflavin, an essential micronutrient, serves as a critical precursor for flavoproteins, which function as cofactors in the forms of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These flavoproteins play pivotal roles in various biochemical processes, including glucose metabolism (Lienhart et al., 2013; Powers, 2003), lipid metabolism (Sakurai et al., 1982), and redox reactions (Ashoori and Saedisomeolia, 2014). The liver is the primary organ for riboflavin storage, containing high riboflavin concentrations (Mosegaard et al., 2020). Plants and microorganisms can autonomously synthesize riboflavin, and ruminants produce it through microbial activity in their rumen (Bacher et al., 2000). In contrast, humans and non-ruminant animals cannot synthesize riboflavin or are insufficient to meet their physiological needs, thus requiring riboflavin intake from food sources (Fischer and Bacher, 2005; Northrop-Clewes and Thurnham, 2012). Riboflavin deficiency has been shown to adversely affect growth and health in both humans and animals, resulting in growth retardation (Bugiani et al., 2006), reduced antioxidant capacity (Ashoori and Saedisomeolia, 2014), and disrupted lipid metabolism (Gunanti et al., 2014).

Increasing evidence indicates that riboflavin is vital to sustaining maternal health during pregnancy and lactation, as well as supporting normal physical development of the progeny (Tang et al., 2019; Zhang et al., 2024). Previous studies have demonstrated that riboflavin deficiency in female rats reduced the birth weight of their offspring (Roth-Maier et al., 2010). In laying hens, riboflavin deficiency caused developmental delays in embryos, fatty liver, skin hemorrhages, and even death (Cogburn et al., 2018). Furthermore, the longer riboflavin deficiency persists, the higher the mortality in embryos and the lower the egg hatchability (Naber and Squires, 1993; Squires and Naber, 1993). Research into the mechanisms of riboflavin deficiency-impaired chicken embryo development has found metabolic abnormalities in the liver, particularly in fatty acid metabolism and vitamin metabolism, along with hepatotoxicity (Cogburn et al., 2018). Similar results were observed in our previous study where duck breeder riboflavin deficiency impaired embryonic growth and development, increased embryo mortality, and caused liver damage (Zhang et al., 2024). Proteomic analyses of duck embryo livers showed that differential proteins between riboflavin-sufficient and riboflavin-deficient (RD) groups were enriched in metabolic pathways, such as the tricarboxylic acid (TCA) cycle, β-oxidation of fatty acids, and mitochondrial respiratory chain (Tang et al., 2019). Additionally, our subsequent research revealed that riboflavin deficiency led to hepatic lipid accumulation of both duck breeders and embryos, impairing fatty acid β-oxidation (Zhang et al., 2021). Riboflavin re-supplementation has been shown to alleviate riboflavin deficiency-induced symptoms, such as low activity of complex I and glutathione reductase (GR) (Bernsen et al., 1993; Staal et al., 1975). In RD chicken embryos, riboflavin injection maintained normal survival and development at d 19. In contrast, a significant number of embryos from the RD group without riboflavin injection died between d 10 to 13 (Lee and White, 1996). Based on these findings, it was hypothesized that refeeding riboflavin to RD duck breeders may improve the symptoms in both the breeders and their embryos. This hypothesis remains unexamined in prior research. Therefore, the reversibility of riboflavin deficiency in duck breeders and duck embryos warrants further investigation.

In this study, a RD duck breeder model was established through the administration of a RD diet over 9 weeks. Subsequently, RR was provided. The study aimed to investigate the effects of RR on riboflavin deficiency in both duck breeders and embryos. Additionally, non-targeted metabolomics was used to explore the effect of riboflavin re-supplementation on hepatic metabolites in duck embryos.

2. Materials and methods

2.1. Animal ethics statement

The experimental procedures involving the birds in this study complied with the requirements of the Animal Ethics Committee of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (Beijing, China) under protocol number IAS2022-104, and were conducted in accordance with the animal care and use guidelines of the institution.

2.2. Birds and research design

A total of 60 laying duck breeders aged 40 weeks (Z7 strain of Pekin duck, Pekin Duck Breeding Center of China of Agriculture and Academy Sciences, Beijing, China), with uniform body weight (3.45 ± 0.90 kg) and reproductive performance, were randomly divided into 2 groups. A total of 60 duck breeders were randomly assigned to two groups. The riboflavin-deficient group (RD; n = 40) was fed a basal diet containing 1.48 mg/kg riboflavin, while the control group (CON; n = 20) received a riboflavin-supplemented diet containing 15 mg/kg added riboflavin, resulting in a total riboflavin content of 16.48 mg/kg in the diet. Upon completion of the 9 weeks dietary intervention, duck breeders in the RD group were randomly split into 2 groups (n = 20), one receiving riboflavin-deficient diet (RD group) and the other receiving riboflavin-sufficient diet (riboflavin refeeding [RR] group). The ducks were fed the treatment diets for 5 weeks. Except for riboflavin, the basal duck diet (Table 1) composition complied with the nutrient specifications for meat-type ducks set by the NY/T 2122-2012 standard in China (Ministry of Agriculture of the People’s Republic of China, 2012). The crude protein content of the diet was analyzed using the method 990.03 (AOA., 2012) procedure by Kjeldahl Apparatus (KDY-9820, Ruibang Technology Co., Beijing, China). Calcium and phosphorus in the feed were determined using the disodium sodium iron ethylenediaminetetraacetate (EDTA) method GB/T 6436-2018 (China National Standard, 2018a) and the optical emission complexometric titration method GB/T 6437-2018 (China National Standard, 2018b), respectively. Samples were analyzed via ICP-OES using the Agilent 5800 platform (Agilent Technologies Inc., Palo Alto, CA, USA). The contents of Met, Lys, Thr, Try, and Arg in the feed were analyzed with a Biochrom 30 analyzer (Biochrom Ltd., Cambridge, UK) according to GB 5009.124-2016 (China National Standard, 2016). The metabolizable energy was calculated according to the NY/T 2122-2012 (Ministry of Agriculture of the People’s Republic of China, 2012) standards for meat-type duck.

Table 1.

Ingredients and composition of riboflavin-deficient basal diet (g/kg, as-fed basis).

Ingredients	Content	Composition	Content
Corn	560.0	Metabolizable energy2, MJ/kg	11.48
Soybean (44 % crude protein)	238.0	Crude protein	193.00
Corn-gluten meal	100.0	Calcium	31.00
Limestone	70.0	Phosphorus	7.50
Dicalcium phosphate	15.0	Lys	10.40
Vitamin and trace mineral premix1	10.0	Met	3.20
Sodium chloride	3.0	Thr	7.50
DL-Met	1.0	Try	2.30
Lys hydrochloride	3.0	Arg	10.10
Total	1000.0	Riboflavin, mg/kg	1.48

¹Supplied per kg of total diet: Cu (CuSO₄·5H₂O), 10 mg; Fe (FeSO₄·7H₂O), 60 mg; Zn (ZnO), 60 mg; Mn (MnSO₄·H₂O), 80 mg; Se (NaSeO₃), 0.3 mg; I (KI), 0.2 mg; choline chloride, 1000 mg; vitamin A (retinyl acetate), 10,000 IU; vitamin D₃ (cholecalciferol), 3000 IU; vitamin E (DL-α-tocopheryl acetate), 20 IU; vitamin K₃ (menadione sodium bisulfate), 2 mg; thiamin (thiamin mononitrate), 2 mg; pyridoxine hydrochloride, 4 mg; cobalamin, 0.02 mg; calcium-D-pantothenate, 20 mg; nicotinic acid, 50 mg; folic acid, 1 mg; biotin, 0.2 mg.

²The metabolizable energy value was calculated according to the NY/T 2122-2012 (Ministry of Agriculture of the People’s Republic of China, 2012).

All laying duck breeders were individually under ad libitum feeding, each raised in a plastic cage of dimensions of about 1.0 m (length) × 0.8 m (breadth) × 0.6 m (height). Rice husk was used as a bedding material on the cage floor to maintain humidity and reduce egg breakage. Fifteen age-matched male ducks were introduced for mating with the laying breeders. Female duck breeders from the three groups were alternately allocated, with one male duck assigned to mate with every four females.

2.3. Productivity and reproductive performance

Body weights of laying duck breeders were recorded at both the start and end of the refeeding trial. During the duration of the experiment, eggs from each cage were collected, identified, and recorded. Eggs and related data were collected and recorded weekly, consistent with the typical 7 to 10 d egg formation cycle in ducks. This approach ensured that reproductive performance and biochemical measurements corresponded to each week of the feeding and refeeding periods. All eggs were collected, weighed, and maintained at 16 °C with 75% relative humidity until transferred to the egg-incubator (Yiai 12096, Qingdao Xingyi Electronic Equipment Co., Ltd., Qingdao, Shangdong, China). Replicate-based averages for egg production, egg weight, and yolk weight were determined during 49 to 54 weeks. During the 1st and 5th week of the experiment, 20 eggs from each replicate were subjected to incubation using a commercial incubator. The number of fertilization and hatched eggs were recorded to determine fertility and hatchability per replicate.

2.4. Sample acquisition

On d 1, d 3, week 2, and week 5 of the refeeding experiment, blood samples were drawn from the wing veins of all breeders and collected in sodium heparin-containing blood collection tubes, and samples were spun at 1520 × g for 15 min to isolate plasma. Blood samples were kept at −20 °C until analysis. Upon completion of the trial, yolk samples were obtained by breaking eight eggs per group, and subsequently stored at −20 °C for riboflavin determination.

After 5 weeks of refeeding, liver samples of 8 duck breeders from each group were obtained for subsequent analysis. At the 5th week of incubation, 8 duck embryos from each of four developmental stages embryonic age day (E13, E18, E22, and E26) per replicate were weighed.

Duck breeders and E18 duck embryos were sacrificed by carbon dioxide asphyxiation, and dissected to collect the liver samples. The liver samples were preserved in tissue fixative for preparation of hematoxylin and eosin (H&E)-stained sections, and the remaining samples were kept at −20 °C for later evaluation of riboflavin, oxidative status, and lipid profiles.

2.5. Riboflavin, FMN, and FAD content

High-performance liquid chromatography (HPLC; reversed-phase) was used to evaluate riboflavin concentrations across feed, plasma, liver, and yolk samples, based on previously outlined procedures (Batey and Eckhert, 1990; Tang et al., 2013). The processing of feed and plasma samples followed the method of Britton et al. (2003), and yolk and liver samples were prepared as per the tissue sample protocol (Batey and Eckhert, 1990). Peak identification and quantification were performed by retention time comparison to the standards (Sigma Aldrich, St Louis, MO, USA). The HPLC analysis was performed using a Waters 2595 high-performance liquid chromatograph coupled with a Waters 2475 fluorescence detector (Waters Corporation., Milford, MA, USA).

2.6. Antioxidant capacity

Liver tissues of duck breeders and embryos were homogenized in normal saline (1:9) under ice bath conditions. Samples were centrifuged at 1500 × g for 15 min, and the resulting supernatant was utilized for further examination. The malondialdehyde (MDA; A003-1) and glutathione (GSH; A006-2), the total superoxide dismutase (T-SOD; A001-1), total antioxidant capacity (T-AOC; A015-1), glutathione peroxidase (GPX; A005-1), and GR (A062-1) activities in liver homogenate supernatants were measured with analysis kits according to the manufacturer's instruction (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China), using a Gene microplate reader (BioTek Instruments, Winooski, VT, USA).

2.7. Hematoxylin and eosin staining

Fresh hepatic samples were immersed in 4% paraformaldehyde for over 24 h, and livers from laying ducks and embryos underwent sequential alcohol dehydration before being embedded in paraffin. After preparing 4- to 5-μm sections, H&E staining was performed and samples were observed using a 400 × magnification light microscope (BX51, Olympus Corp., Tokyo, Japan).

2.8. Hepatic total lipid, triglyceride, total cholesterol, and free fatty acids content

The total lipid (TL) in the liver was extracted using the chloroform-methanol mixture of 2:1 method described before (Wen et al., 2014). The hepatic lipid extract was dried to a constant weight by evaporation and was determined gravimetrically. Lipid extracts were resuspended in the chloroform-methanol mixture, and triglyceride (TG; A110-1), total cholesterol (T-CHO; A111-2), and free fatty acid (FAA; A042-2) contents in the suspension were determined with commercial kits following the instructions, procured from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China), using a Gene microplate reader.

2.9. Hepatic metabolism

2.9.1. Liquid chromatography-tandem mass spectrometry analysis

Metabolomic profiling was carried out with an Acquity I-Class PLUS UHPLC system coupled to a Xevo G2-XS QTof mass spectrometer (Waters Corp., Milford, MA, USA). The analysis used a 1.8-μm Acquity UPLC HSS T3 column (Waters Corp., Milford, MA, USA, 2.1 mm × 100 mm). In positive ion mode, mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile. For the negative ion mode, the mobile phases were identical. The loading quantity was 2 μL. The Xevo G2-XS QTOF mass spectrometer was used to acquire both primary and secondary mass spectra in MSe mode, managed by MassLynx V4.2 software.

2.9.2. Data cleaning and metabolite identification

The raw data from MassLynx V4.2 were analyzed using Progenesis QI, which was used for peak detection, alignment, and subsequent preprocessing. Metabolite identification was conducted by matching the features to entries in the METLIN online database and Biomark's in-house spectral library.

2.9.3. Metabolism data analysis

Total ion current normalization was applied, and both principal component analysis (PCA) and Spearman correlation were used to evaluate the consistency of the samples and the reliability of the data. Differential metabolites were identified based on fold change (FC) > 1.2, P-value <0.05, and variable importance in projection (VIP) value > 1. Pathway enrichment analysis was carried out using Kyoto Encyclopedia of Genes and Genomes (KEGG), Human Metabolome Database (HMDB), and LipidMaps databases. Orthogonal partial least squares discriminant analysis (OPLS-DA) was conducted using the R package ‘ropls’ with model validation by 200 permutations. KEGG pathway enrichment was evaluated via hypergeometric analysis.

2.10. Gene expression

Total RNA was extracted from duck embryo liver samples (n = 6 per group) using the TRIzol reagent (Invitrogen, Thermo Fisher Scientific Inc., Carlsbad, CA, USA) following the manufacturer's protocol. RNA purity and concentration were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA), and integrity was verified by agarose gel electrophoresis. Subsequently, 1 μg of total RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit with gDNA Eraser (Takara Bio Inc., Kusatsu, Japan). Quantitative real-time PCR (qRT-PCR) was performed using TB Green Premix Ex Taq II (Takara Bio Inc., Kusatsu, Japan) on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA). Each reaction was carried out in a 20 μL volume containing 10 μL of 2 × TB Green Master Mix, 0.4 μL of each primer (10 μmol/L), 2 μL of diluted cDNA, and 7.2 μL of nuclease-free water. The thermal cycling conditions were as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Melting curve analysis was performed to confirm primer specificity. The relative expression levels of target genes, including ACADS, ACADM, and ACADL were normalized to the expression of the housekeeping gene β-actin, and calculated using the 2^−ΔΔCt method. Primer sequences are listed in Table S1.

2.11. Calculation data analysis

Embryo liver index (%) = Embryo liver weight (g)/Embryo body weight (g) × 100.

Statistical analysis was performed using one-way ANOVA to compare the three treatment groups (n = 20 per group), using SAS software version 9.4 (SAS Institute Inc., Cary, NC, USA). The model used was:

Y = μ + α + ϵ,

where Y represents the dependent variable; μ is the overall mean; α denotes the effect of riboflavin status (RD, RR, and CON); and ϵ is the random error. Duncan's multiple range test was used for post-hoc comparisons. This method was selected due to its higher sensitivity in detecting mean differences when the number of groups is small (three treatments in this study), and the relatively large number of replicates per group (n = 20) provided sufficient power for comparison. Moreover, the exploratory nature of the study emphasized sensitivity over strict control of Type I error, making Duncan's test more appropriate than more conservative approaches such as Tukey's or Bonferroni corrections. Figure analysis was conducted using Image-Pro Plus (IPP 6.0) software. The data were presented as mean and standard error of the mean (SEM) and visualized with GraphPad Prism version 9.3. Statistical significance was set at P < 0.05, while a 0.05 < P < 0.10 indicated a trend.

3. Results

3.1. Effects of RR on reproductive performance of duck breeders

As shown in Table 2, dietary riboflavin concentration had no effect on body weight, feed intake, egg weight, yolk weight, egg production, and egg fertility in duck breeders (P > 0.05). However, the RD group egg hatchability was lower than the CON group (P < 0.001), an effect that was rapidly alleviated with subsequent riboflavin supplementation. Specifically, after 1 week of RR, egg hatchability in the RD group (3.80%) was lower than in the RR (76.22%) and CON (77.11%) groups (P < 0.001). Moreover, there was no difference observed between the RR and CON groups (P > 0.05). After 5 weeks of RR, egg hatchability in the RD group further decreased to 2.42%, which was significantly lower than in the RR (84.03%) and CON (89.05%) groups (P < 0.001), and no significant variation was found between latter two groups (P > 0.05).

Table 2.

Effects of riboflavin refeeding (RR) on reproductive performance of laying duck breeders.

Item	Groups1			SEM	P-value
	RD	RR	CON
Initial body weight, kg	3.38	3.52	3.45	0.199	0.352
Final body weight, kg	3.41	3.47	3.50	0.210	0.824
Feed intake, g/d	263.8	270.1	263.3	3.33	0.830
Egg
Weight, g	91.7	93.1	90.8	4.40	0.591
Yolk weight, g	31.8	32.5	31.5	1.80	0.500
Production, %	86.1	85.4	83.1	11.04	0.880
Fertility, %	87.0	95.4	87.7	9.32	0.240
Hatchability 1 week, %	3.80b	76.2a	77.1a	6.79	<0.001
Hatchability 5 week, %	2.42b	84.0a	89.1a	4.86	<0.001

RD = riboflavin deficient; CON = control; SEM = standard error of the mean.

Within a row, means without a common superscript letter differ at P < 0.05 (n = 20).

¹RD group, fed a diet containing 1.48 mg/kg riboflavin; RR group, initially fed the riboflavin-deficient diet and then switched to a riboflavin-sufficient diet containing 16.48 mg/kg riboflavin; CON group, fed the riboflavin-sufficient diet containing 16.48 mg/kg riboflavin throughout the experiment.

3.2. Effects of RR on riboflavin status of duck breeders

Dietary riboflavin supplementation on plasma riboflavin levels in duck breeders is presented in Table 3. It was observed that after 1 d of refeeding, plasma riboflavin concentration in the RR group rapidly increased, higher than the RD group (P < 0.001), but still lower than the CON group (P < 0.001). With the extension of re-supplement time, plasma riboflavin concentration in the RD group continued to decrease, while it continued to rise in the RR group, and the CON group remained stable. After 2 weeks of refeeding, plasma riboflavin concentration in the RR group remained higher than in the RD group (P < 0.001), but still lower than in the CON group (P < 0.001). After 5 weeks of refeeding, plasma riboflavin concentration in the RR group returned to levels similar to those in the CON group (P > 0.05), and both groups were higher than the RD group (P < 0.05).

Table 3.

Effects of riboflavin refeeding (RR) on riboflavin status of laying duck breeders.

Item	Groups1			SEM	P-value
	RD	RR	CON
Plasma riboflavin, μmol/L
1 d	1.30c	3.88b	4.44a	0.870	<0.001
3 d	1.24c	3.63b	4.38a	0.873	<0.001
2 week	1.17c	4.53b	5.08a	0.856	<0.001
5 week	0.92b	4.70a	4.18a	1.020	<0.001
Hepatic (5 week), μg/g
FAD	0.89b	1.31a	1.54a	0.354	0.001
FMN	3.64b	6.11a	5.93a	1.440	0.002
Riboflavin	3.09b	4.06a	4.01a	0.643	0.004
Egg yolk riboflavin (5 week), μg/g	3.04b	8.71a	8.27a	1.030	<0.001

RD = riboflavin deficient; CON = control; FMN = flavin mononucleotide; FAD = flavin adenine dinucleotide; SEM = standard error of the mean.

Within a row, means without a common superscript letter differ at P < 0.05 (n = 8).

¹RD group, fed a diet containing 1.48 mg/kg riboflavin; RR group, initially fed the riboflavin-deficient diet and then switched to a riboflavin-sufficient diet containing 16.48 mg/kg riboflavin; CON group, fed the riboflavin-sufficient diet containing 16.48 mg/kg riboflavin throughout the experiment.

Furthermore, changes in riboflavin status in the duck breeder livers and egg yolk are shown in Table 3. The results indicated that after 5 weeks of refeeding, liver riboflavin, FMN, and FAD concentrations in the RR group were not different from those in the CON group (P > 0.05), and both groups had higher concentrations than the RD group (P < 0.05). A similar trend was observed in egg yolk riboflavin concentrations, and no observable variation was found between the RR and CON groups (P > 0.05), and both groups showed higher riboflavin concentrations than the RD group (P < 0.001).

3.3. Effects of RR on antioxidant status, lipid content, and histomorphology of duck breeder liver

The role of dietary RR on liver antioxidant indices of duck breeders is shown in Table 4. The results indicated that after 5 weeks of refeeding, hepatic T-AOC activity in the RR group did not differ from the RD group (P > 0.05), and the two groups had lower hepatic T-AOC activity than the CON group (P = 0.024). Additionally, hepatic MDA content, GSH activity, GPX activity, and GR activity in the RR group were restored to levels similar to those in the CON group (P > 0.05), and GPX and GR activities were higher than those in the RD group (P < 0.05).

Table 4.

Effects of riboflavin refeeding (RR) for 5 weeks on hepatic antioxidant status and lipid content of laying duck breeders.

Item	Groups1			SEM	P-value
	RD	RR	CON
Antioxidant status
MDA, nmol/mg prot	1.59a	1.09b	1.06b	0.396	0.005
T-SOD, U/mg prot	7.11b	7.18b	8.47a	1.150	0.008
T-AOC, U/mg prot	0.57b	0.58b	0.73a	0.141	0.024
GSH, mg/mg prot	1.14b	1.33ab	1.46a	0.293	0.021
GPX, U/mg prot	38.1b	47.7a	48.8a	7.53	0.007
GR, U/mg prot	28.1b	41.7a	41.6a	2.27	<0.001
Lipid content
TL, %	22.60a	6.49b	7.55b	2.569	<0.001
TG, μmol/g	89.4a	51.0b	47.7b	12.88	<0.001
T-CHO, μmol/g	9.36b	15.3a	15.7a	2.14	<0.001
FFA, μmol/g	162.7a	116.7b	119.3b	8.66	<0.001

RD = riboflavin deficient; CON = control; prot = protein; MDA = malondialdehyde; T-SOD = total superoxide dismutase; T-AOC = total antioxidant capacity; GSH = reduced glutathione; GPX = glutathione peroxidase; GR = glutathione reductase; TL = total lipid; TG = triglycerides; T-CHO = total cholesterol; FFA = free fatty acids; SEM = standard error of the mean.

Within a row, means without a common superscript letter differ at P < 0.05 (n = 8).

¹RD group, fed a diet containing 1.48 mg/kg riboflavin; RR group, initially fed the riboflavin-deficient diet and then switched to a riboflavin-sufficient diet containing 16.48 mg/kg riboflavin; CON group, fed the riboflavin-sufficient diet containing 16.48 mg/kg riboflavin throughout the experiment.

The liver histological examination of duck breeders by H&E staining is shown in Fig. 1A–C. The results revealed significant white fat droplets in the liver of the RD group, while very few fat droplets were observed in the liver of the RR and CON groups. Subsequently, hepatic lipid content was measured, which is shown in Table 4. The results showed that the TL, TG, and FFA contents in the liver of the RD group were higher than those in the RR and CON groups (P < 0.001), with no difference between the RR and CON groups (P > 0.05). Moreover, hepatic T-CHO content in the RD group showed lower contents in the RR and CON groups (P < 0.001), and no difference between the latter two groups (P > 0.05).

Fig. 1.

Effects of riboflavin refeeding (RR) for 5 weeks on histological examination of liver. Hematoxylin and eosin (H&E) stained liver sections and hepatic fat duck breeders (A-C), and embryos (D-F). Black arrows indicate visible lipid droplets in hepatic tissue. RD group, fed a diet containing 1.48 mg/kg riboflavin; RR group, initially fed the riboflavin-deficient diet and then switched to a riboflavin-sufficient diet containing 16.48 mg/kg riboflavin; CON group, fed the riboflavin-sufficient diet containing 16.48 mg/kg riboflavin throughout the experiment. RD = riboflavin deficient; CON = control.

3.4. Effects of RR on growth and development of duck embryos

Effects of dietary RR on the growth and development of duck embryos are shown in Table 5, Table 6. The results indicated that dietary riboflavin levels did not affect egg weight, but decreased gradually with the increase in incubation days. After 5 weeks of refeeding, at E13 (RD vs. RR vs. CON = 4.44 vs. 5.24 vs. 5.26 g), E18 (RD vs. RR vs. CON = 14.9 vs. 19.2 vs. 21.3 g), E22 (RD vs. RR vs. CON = 30.7 vs. 39.7 vs. 39.0 g), and E26 (RD vs. RR vs. CON = 45.9 vs. 51.0 vs. 52.7 g), no differences were observed in embryo weight, embryo weight/egg weight ratio, total body length, trunk length, leg length, and chest width between the RR and CON groups (P > 0.05), while all of these parameters were higher than in the RD group (P < 0.05). At E22, influenced by the overall embryo weight, liver weight in the RD group was lower than that in the RR and CON groups (P < 0.05), with no differences between the latter two groups (P > 0.05). However, the liver index of embryos showed that at E13 (RD vs. RR vs. CON = 1.98% vs. 1.59% vs. 1.75%), E18 (RD vs. RR vs. CON = 2.34% vs. 1.60% vs. 1.64%), and E22 (RD vs. RR vs. CON = 1.98% vs. 1.79% vs. 1.74%), the RD group had a higher liver index than the RR and CON groups (P < 0.05), with no differences between the RR and CON groups (P > 0.05). At E26, the liver index in the RD group still showed a tendency to be higher than in the RR and CON groups (RD vs. RR vs. CON = 2.10% vs. 1.88% vs. 1.88%, P = 0.094).

Table 5.

Effects of riboflavin refeeding (RR) on growth performance of duck embryos.

Item	Groups1			SEM	P-value
	RD	RR	CON
Egg weight, g
E13	88.8	88.5	88.7	8.25	0.998
E18	85.4	83.2	82.0	3.94	0.237
E22	83.7	83.4	84.3	6.84	0.963
E26	84.2	83.2	84.3	8.21	0.953
Embryo body weight, g
E13	4.4b	5.2a	5.5a	0.55	0.002
E18	14.9b	19.2a	21.3a	3.28	0.003
E22	30.7b	39.7a	39.0a	3.06	<0.001
E26	45.9b	51.0ab	52.7a	5.11	0.034
Egg weight/embryo weight, g/g
E13	5.0b	6.0a	6.2a	0.82	0.013
E18	17.4b	23.2a	26.0a	3.54	<0.001
E22	36.7b	47.6a	46.4a	3.11	<0.001
E26	54.6b	61.3a	62.8a	4.62	0.004
Embryo liver weight, g
E13	0.087	0.084	0.095	0.0135	0.195
E18	0.325	0.307	0.348	0.0424	0.124
E22	0.606b	0.708a	0.678a	0.0534	0.006
E26	0.952	0.955	0.991	0.0884	0.586
Embryo liver index, %
E13	1.98a	1.59b	1.75b	0.226	0.006
E18	2.34a	1.60b	1.64b	0.343	0.003
E22	1.98a	1.79b	1.74b	0.127	0.002
E26	2.10	1.88	1.88	0.219	0.094

RD = riboflavin deficient; CON = control; SEM = standard error of the mean; E = embryonic age day.

Within a row, means without a common superscript letter differ at P < 0.05 (n = 8).

¹RD group, fed a diet containing 1.48 mg/kg riboflavin; RR group, initially fed the riboflavin-deficient diet and then switched to a riboflavin-sufficient diet containing 16.48 mg/kg riboflavin; CON group, fed the riboflavin-sufficient diet containing 16.48 mg/kg riboflavin throughout the experiment.

Table 6.

Effects of riboflavin refeeding (RR) on the body size of duck embryos (mm).

Item	Groups1			SEM	P-value
	RD	RR	CON
Body length
E13	56.6b	60.4ab	60.8a	2.99	0.014
E18	79.6b	89.7a	89.7a	6.92	0.016
E22	102.7b	111.6a	111.1a	6.89	0.029
E26	120.2	123.8	127.4	5.52	0.077
Trunk length
E13	26.7b	29.1ab	30.7a	2.57	0.014
E18	40.8b	43.2ab	46.4a	3.68	0.020
E22	51.7	55.5	54.3	4.79	0.267
E26	57.4	60.0	62.9	4.05	0.055
Leg length
E13	22.7b	26.9a	26.0a	1.85	<0.001
E18	39.3b	47.9a	45.8a	4.88	0.016
E22	61.3b	69.9a	68.8a	5.79	0.034
E26	82.4b	91.1a	90.7a	5.39	0.019
Breast width
E13	10.2b	12.2a	11.7a	0.91	<0.001
E18	13.5b	15.4a	15.7a	1.71	0.040
E22	19.5b	21.4ab	22.3a	2.10	0.024
E26	21.6	21.9	22.8	1.49	0.237

RD = riboflavin deficient; CON = control; SEM = standard error of the mean; E = embryonic age day.

Within a row, means without a common superscript letter differ at P < 0.05 (n = 8).

¹RD group, fed a diet containing 1.48 mg/kg riboflavin; RR group, initially fed the riboflavin-deficient diet and then switched to a riboflavin-sufficient diet containing 16.48 mg/kg riboflavin; CON group, fed the riboflavin-sufficient diet containing 16.48 mg/kg riboflavin throughout the experiment.

3.5. Effects of RR on hepatic riboflavin status of duck embryos

The effect of RR on riboflavin status in duck embryo livers is shown in Table 7. The results revealed that after 5 weeks of refeeding, riboflavin, FMN, and FAD concentrations in the embryos from the RR group were not different from those in the CON group (P > 0.05), and both groups had higher concentrations than the RD group (P < 0.001).

Table 7.

Effects of riboflavin refeeding (RR) for 5 weeks on riboflavin status, antioxidant status, and lipid content of duck embryos.

Item	Groups1			SEM	P-value
	RD	RR	CON
Riboflavin status, μg/g
FAD	0.70b	2.07a	2.01a	0.159	<0.001
FMN	1.44b	5.80a	6.08a	0.704	<0.001
Riboflavin	2.40b	4.02a	4.02a	0.173	<0.001
Antioxidant status
MDA, nmol/mg prot	3.05a	1.62b	1.44b	0.286	<0.001
T-SOD, U/mg prot	5.51c	7.08b	7.93a	0.831	<0.001
T-AOC, U/mg prot	0.89c	1.41b	1.52a	0.092	<0.001
GSH, mg/mg prot	1.14b	1.52a	1.54a	0.123	<0.001
GPX, U/mg prot	37.4b	49.0a	48.4a	5.44	<0.001
GR, U/mg prot	22.6b	38.8a	38.1a	2.57	<0.001
Lipid content
TL, %	8.65a	6.70b	6.69b	0.841	<0.001
TG, μmol/g	412a	220b	215b	51.7	<0.001
T-CHO, μmol/g	810a	504b	524b	71.1	<0.001
FFA, μmol/g	267a	177b	177b	29.7	<0.001

RD = riboflavin deficient; CON = control; FMN = flavin mononucleotide; FAD = flavin adenine dinucleotid; MDA = malondialdehyde; prot =protein; T-SOD = total superoxide dismutase; T-AOC = total antioxidant capacity; GSH = reduced glutathione; GPX = glutathione peroxidase; GR = glutathione reductase; TL = total lipid; TG = triglycerides; T-CHO = total cholesterol; FFA = free fatty acids; SEM = standard error of the mean.

Within a row, means without a common superscript letter differ at P < 0.05 (n = 8).

¹RD group, fed a diet containing 1.48 mg/kg riboflavin; RR group, initially fed the riboflavin-deficient diet and then switched to a riboflavin-sufficient diet containing 16.48 mg/kg riboflavin; CON group, fed the riboflavin-sufficient diet containing 16.48 mg/kg riboflavin throughout the experiment.

3.6. Effects of RR on antioxidant capacity, lipid content, and histomorphology of duck embryo liver

Effects of dietary RR on the hepatic antioxidant parameter in the liver of duck embryos are shown in Table 7. The results indicated that after 5 weeks of refeeding, the hepatic MDA content of embryos in the RR group was close to that in the CON group (P > 0.05), with both groups being higher than the RD group (P < 0.001). The activities of GSH, GPX, and GR in the liver were higher in the RR group than in the RD group (P < 0.05), with no differences between the RR and CON groups (P > 0.05). Furthermore, the activities of T-SOD and T-AOC were higher in the RR group compared to the RD group (P < 0.001), but still lower than in the CON group (P < 0.001).

Histological examination of liver sections stained with H&E showed that in the RD group, small and medium-sized lipid droplets were observed in the liver, accompanied by initial signs of hydropic degeneration in hepatocytes and destruction of the hepatic lobular structure (Fig. 1D–F). In contrast, the liver morphology in the RR and CON groups was similar, with healthy hepatocytes. The lipid content in the liver of duck embryos was measured as shown in Table 7, and the results revealed that TL, TG, and FFA were higher in the liver of the RD group than in the RR and CON groups (P < 0.001), with no differences between the latter two groups (P > 0.05). Consistent with the findings in the duck breeders, T-CHO content in the liver of the RD group was lower than that in the RR and CON groups (P < 0.001), with no differences between the RR and CON groups (P > 0.05).

3.7. Effects of RR on hepatic metabolism of duck embryo

As a whole, 367 metabolites were significantly altered between the RD and CON groups, 173 between the RD and RR groups, and 145 between the RR and CON groups. The top ten metabolites for each comparison group are listed in Table S2. As shown in Fig. 2, dietary riboflavin levels affected the hepatic metabolomics profile in duck embryos. The PCA score plots (Fig. 2A–C) revealed that the RD group was separated from the RR and CON groups, while the RR and CON groups were relatively close to each other. Additionally, the clustering heatmap (Fig. 2G) indicated notable variations in metabolite levels between the RD group and the other two groups, with the RR and CON groups showing more similarity. A total of 69 metabolites were enriched between the RD and CON groups (P < 0.05, Fig. 2G). Among these, 61 metabolites were downregulated and 8 were upregulated in the RD group compared to the CON group. In the comparison between the RD and RR groups, 36 metabolites were enriched (P < 0.05, Fig. 2H), with 31 metabolites downregulated and 5 upregulated in the RD group compared to the RR group. For the RR and CON groups, 28 metabolites were enriched (P < 0.05, Fig. 2I), with 16 metabolites downregulated and 12 upregulated in the RR group as opposed to the CON group.

Fig. 2.

Effects of riboflavin refeeding (RR) for 5 weeks on hepatic metabolism in duck embryos. (A-C) Principal component analysis (PCA) plots show group separation based on liver metabolite profiles: (A) RD vs. CON, (B) RD vs. RR, and (C) RR vs. CON. (D-F) Hierarchical clustering heatmaps of differentially expressed metabolites between groups. Red represents upregulation and blue indicates downregulation. (G-I) Volcano plots of differential metabolites: red points (upregulated), blue points (downregulated), and grey (no significant). (J-L) Enrichment of metabolic pathways. Color intensity reflects the significance of enrichment. RD group, fed a diet containing 1.48 mg/kg riboflavin; RR group, initially fed the riboflavin-deficient diet and then switched to a riboflavin-sufficient diet containing 16.48 mg/kg riboflavin; CON group, fed the riboflavin-sufficient diet containing 16.48 mg/kg riboflavin throughout the experiment. RD = riboflavin deficient; CON = control; FMN = flavin mononucleotide; FAD = flavin adenine dinucleotide; Dim = dimension; GABAergic synapse = gamma-aminobutyric acid-ergic synapse.

Further analysis of the metabolic pathways enriched in each comparison group showed the following: between the RD and CON groups (Fig. 2J), differential metabolites were predominantly enriched in protein digestion and absorption (P = 0.005), vitamin digestion and absorption (P = 0.010), terpenoid backbone biosynthesis (P = 0.011), proximal tubule bicarbonate reclamation (P = 0.013) and glycerophospholipid metabolism (P = 0.008). Between the RD and RR groups (Fig. 2K), the differential metabolites were primarily enriched in protein digestion and absorption (P = 0.003), mineral absorption (P = 0.003), vitamin digestion and absorption (P < 0.001), thiamine metabolism (P = 0.012), and sphingolipid metabolism (P = 0.091). Between the RR and CON groups (Fig. 2L), the differential metabolites were enriched in primary bile acid biosynthesis (P = 0.008), thiamine metabolism (P = 0.015), protein digestion and absorption (P = 0.015), phosphonate and phosphinate metabolism (P = 0.015), and neuroactive ligand–receptor interaction (P = 0.015). Additionally, analysis of differential metabolites among the three groups revealed common changes and trends (Table 8). Specifically, when comparing the RD group to both the CON and RR groups, the concentrations of Pro–Gly, Ile–Tyr, riboflavin, FMN, and FAD were all lower in the liver, while the concentration of Ile–Ser was higher in the RD group. After riboflavin supplementation, except for oleic acid, which remained higher in the RD group as opposed to the CON group, all other common differential markers were restored to levels similar to those in the CON group.

Table 8.

Effects of riboflavin refeeding (RR) for 5 weeks on hepatic metabolism of duck embryos¹.

Item	CON	RD	Fold change	P-value
Pro–Gly	0.003	0.002	−1.63	0.028
Ile–Ser	0.002	0.003	1.38	0.015
Oleic acid	0.035	0.052	1.14	0.008
Ile–Cys	0.102	0.069	−1.48	0.031
Ile–Tyr	0.115	0.079	−1.45	0.035
Riboflavin	0.041	0.014	−2.90	<0.001
FMN	0.010	0.007	−1.50	0.007
FAD	0.076	0.055	−1.36	0.023
Item	RR	RD
Pro–Gly	0.004	0.002	−1.78	0.020
Ile–Ser	0.0026	0.0034	1.33	0.015
Ile–Cys	0.099	0.070	−1.43	0.032
Ile–Tyr	0.111	0.079	−1.40	0.047
Riboflavin	0.038	0.014	−2.71	<0.001
FMN	0.010	0.007	−1.52	0.007
FAD	0.076	0.056	−1.37	0.022
Item	CON	RR
Oleic acid	0.147	0.167	1.13	0.021

RD = riboflavin deficient; RR = riboflavin refeeding; CON = control; FMN = flavin mononucleotide; FAD = flavin adenine dinucleotid.

Within a row, means without a common superscript letter differ at P < 0.05 (n = 8).

¹RD group, fed a diet containing 1.48 mg/kg riboflavin; RR group, initially fed the riboflavin-deficient diet and then switched to a riboflavin-sufficient diet containing 16.48 mg/kg riboflavin; CON group, fed the riboflavin-sufficient diet containing 16.48 mg/kg riboflavin throughout the experiment.

3.8. Effects of RR on gene expression of duck embryo

The effects of RR on the hepatic mRNA expression of β-oxidation-related genes in duck embryos were shown in Fig. 3. The results revealed that after 5 weeks of refeeding, the expression levels of ACADS and ACADM in the RR group were not different from those in the CON group (P > 0.05), and both were higher than those in the RD group (P < 0.001). In contrast, the expression of ACADL in the RR group remained significantly lower than in the CON group (P < 0.001), although it was elevated compared to the RD group (P < 0.001).

Fig. 3.

Effects of riboflavin refeeding (RR) for 5 weeks on hepatic expression of fatty acid β-oxidation-related genes in duck embryos. (A) ACADS, (B) ACADM, and (C) ACADL. Different lowercase letters above columns represent significant differences among treatments at P < 0.05 (n = 6). RD group, fed a diet containing 1.48 mg/kg riboflavin; RR group, initially fed the riboflavin-deficient diet and then switched to a riboflavin-sufficient diet containing 16.48 mg/kg riboflavin; CON group, fed the riboflavin-sufficient diet containing 16.48 mg/kg riboflavin throughout the experiment. RD = riboflavin deficient; CON = control.

4. Discussion

Previous earlier research indicated that riboflavin deficiency adversely affected egg hatchability, embryonic growth and development, and liver health, as well as increased embryo mortality (Zhang et al., 2021, 2020). Additionally, riboflavin deficiency caused abnormalities in hepatic lipid metabolism in both maternal and offspring (Zhang et al., 2021). Moreover, through systematic in vivo and in vitro experiments, the results demonstrated that riboflavin regulates hepatocyte apoptosis through the endoplasmic reticulum stress-CHOP signaling pathway (Zhang et al., 2022, 2024). In a previous study, the results also examined the temporal distribution of embryo mortality during incubation and found that deaths primarily occurred during the early embryonic stage (Tang et al., 2019). Nevertheless, it remains uncertain whether RR can effectively reverse the negative effects induced by riboflavin deficiency. Therefore, this study focused on examining the impact of RR on reproductive performance, embryonic growth, riboflavin concentration, antioxidant status, and hepatic metabolism, and to determine whether RR could alleviate the negative effects of riboflavin deficiency in both maternal and embryo stages. The results will provide scientific evidence for the prevention and treatment of riboflavin deficiency, and offer new insights into the essential role of riboflavin in the growth, development, and reproduction of poultry.

Previous studies have demonstrated that differential riboflavin concentrations can similarly affect reproductive performance in both chickens and ducks. In hens, a RD diet for less than 8 weeks did not affect egg production (Squires and Naber, 1993). In the previous research, the results observed no significant changes in body weight, egg weight, egg production, or egg fertility in duck breeders fed different riboflavin concentration diets (Zhang et al., 2020). The present study confirmed these findings, as no significant effects on body weight, egg weight, egg production, or egg fertility were observed after 9 weeks of riboflavin deficiency followed by 5 weeks of RR. However, egg hatchability was found to be highly sensitive to riboflavin concentrations. In both chickens and ducks, hatchability decreased as the duration of riboflavin deficiency increased, dropping to between 0 and 10% after 8 to 9 weeks of deficiency. In contrast, under riboflavin-sufficient conditions, hatchability remained above 80% (Squires and Naber, 1993; Zhang et al., 2020). In this study, RR for 1 week rapidly increased hatchability from 3.80% to 76.22%, nearly reaching the hatchability observed under normal riboflavin-sufficient conditions (77.11%).

Plasma and tissue riboflavin concentrations are sensitive indicators of riboflavin concentration in the body. Prior research has established that riboflavin deficiency reduced riboflavin concentrations in plasma, egg yolk, and liver, as well as decreased FMN and FAD concentrations in the liver (Tang et al., 2019; Zhang et al., 2020). In the current study, RR resulted in a rapid increase in plasma riboflavin levels within 1 d, although the levels remained lower than those in the CON group. After 5 weeks of RR, plasma, egg yolk, and liver riboflavin concentrations, as well as FMN and FAD concentrations in the liver, returned to normal riboflavin-sufficient levels. These findings highlight the rapid absorption and low storage capacity of riboflavin, as well as its fast turnover rate in the body.

Riboflavin plays a crucial role during pregnancy and the egg-laying period in female animals. In chickens, riboflavin deficiency causes developmental defects in the head, beak, and feathers of embryos, leading to reduced body weight (Cogburn et al., 2018; Romanoff and Bauernfeind, 1942). Previous studies also revealed that riboflavin deficiency in duck breeders led to poor embryonic growth, with a high mortality rate during the mid-embryonic stage (Tang et al., 2019; Zhang et al., 2024). It has been reported that riboflavin injected into RD chicken embryos allowed them to survive, whereas control embryos (injected with saline) died between d 10 to 13 of incubation (Lee and White, 1996). In this study, RR in duck breeders resulted in the restoration of embryonic body weight and size indicators, bringing them to levels observed under riboflavin-sufficient conditions. This indicates that RR can mitigate the developmental impairments caused by riboflavin deficiency in duck embryos, allowing them to develop and hatch normally.

Many antioxidant enzymes in the redox reaction system are flavoproteins. For example, GR uses FAD as a cofactor to neutralize reactive oxygen species (ROS) and maintain redox balance (Dringen et al., 2000; Schclz et al., 1982). Moreover, riboflavin deficiency has been verified to reduce the activity of SOD and CAT, reducing overall antioxidant status (Ashoori and Saedisomeolia, 2014; Tumkiratiwong et al., 2003). In previous studies, it was found that riboflavin deficiency decreased the activity of antioxidant enzymes (GSH, T-SOD, and T-AOC) in plasma and egg yolk, while increasing lipid peroxidation markers such as MDA levels (Zhang et al., 2020). Building upon these findings, the current study showed that RR partially restored hepatic antioxidant function. Specifically, hepatic MDA levels were reduced, and both GSH content and GPX activity were restored to levels comparable to the control group. However, the activities of T-SOD and T-AOC in the liver of both duck breeders and embryos remained significantly lower than in the CON group. This observation suggests that short-term riboflavin supplementation was insufficient to fully restore total antioxidant capacity. One plausible explanation is that antioxidant enzymes such as SOD may undergo structural oxidative damage during deficiency, and their recovery may require more time for transcription reactivation or protein turnover. In addition, T-AOC reflects the combined capacity of various enzymatic and non-enzymatic antioxidants. While the GSH-GPX system appeared to recover, other components such as SOD, catalase, or low-molecular-weight antioxidants may not have been fully re-established within the refeeding period. Therefore, full recovery of antioxidant homeostasis may require prolonged riboflavin supplementation. Further studies are warranted to investigate whether higher doses or longer refeeding duration could promote more complete antioxidant recovery, particularly for parameters such as T-SOD and T-AOC. Similar dose-dependent effects on antioxidant status have been reported in other species, such as rabbits supplemented with olive leaf extracts.

The liver is a primary organ for riboflavin storage, and riboflavin is critical for maintaining liver function (Mosegaard et al., 2020). In this study, the liver exhibited a highly sensitive response to dietary riboflavin concentrations. Riboflavin deficiency in both duck breeders and embryos led to liver enlargement and lipid degeneration. Notably, adult duck breeder livers exhibited more lipid droplets than embryonic livers across all groups, which may reflect physiological lipid reserves associated with reproductive metabolism, as opposed to the tightly regulated lipid utilization in developing embryos. The reduced T-CHO observed in the RD group may be related to riboflavin's critical role as a precursor of FMN and FAD, cofactors for enzymes involved in cholesterol biosynthesis. Riboflavin deficiency can impair the activity of these flavoproteins, leading to decreased cholesterol synthesis. The restoration of T-CHO levels after RR likely reflects recovery of these enzymatic pathways, consistent with previous reports on the importance of riboflavin in lipid metabolism (Tang et al., 2019; Zhang et al., 2021). These changes were associated with the downregulation of the flavoproteins ACADS, ACADM, and acyl-CoA dehydrogenase family member 9 (ACAD9), which are involved in lipid metabolism (Zhang et al., 2021). Consistent with prior findings, the present study further demonstrated that riboflavin deficiency caused excessive lipid accumulation in the livers. However, this phenomenon can be mitigated by riboflavin re-supplementation. First, after RR, the riboflavin status in the liver reached the level observed under riboflavin-sufficient conditions. Subsequently, hepatic lipid contents were restored to normal levels.

To gain deeper insight, it was compared the liver metabolites among the three treatment groups. Overall, the CON and RR groups showed similar metabolic profiles, while significant differences were observed between the RR and RD groups. RR improved the metabolic disorders induced by riboflavin deficiency, especially in terms of antioxidant capacity and lipid metabolism. However, short-term RR was insufficient to completely restore metabolic parameters to baseline levels, indicating that the recovery process may require a longer duration or higher re-supplementation doses. Moreover, RR restored several metabolic pathways, emphasizing its critical role in maintaining metabolic balance and health, particularly in protein metabolism, vitamin metabolism, and lipid metabolism. Metabolomic pathway enrichment analysis revealed that differential metabolites between RD and both RR/CON groups were significantly associated with protein digestion and absorption, vitamin digestion and absorption, and glycerophospholipid metabolism. Riboflavin is a precursor of FMN and FAD, which serve as essential cofactors in numerous redox and dehydrogenase reactions. These include pathways involved in amino acid catabolism, mitochondrial energy metabolism, and vitamin interconversion (Powers, 2003). The decreased levels of dipeptides such as Pro–Gly and Ile–Tyr in the RD group suggest impaired protein turnover or mitochondrial enzymatic activity. Additionally, alterations in thiamine metabolism pathways indicate possible crosstalk between riboflavin and other B vitamins, which share overlapping coenzyme functions (Said and Mohammed, 2006). The enrichment of glycerophospholipid and terpenoid biosynthesis pathways further highlights the role of riboflavin in maintaining membrane structure and oxidative balance (Ashoori and Saedisomeolia, 2014). Together, these findings demonstrate that riboflavin deficiency disrupts key aspects of hepatic amino acid, vitamin, and lipid metabolism, and these disturbances are partially reversible upon refeeding. However, the changes in oleic acid concentrations suggested that short-term riboflavin supplementation had not fully restored lipid metabolic balance. Specifically, oleic acid levels remained elevated in the RR group compared to the CON group, indicating that certain aspects of hepatic lipid metabolism-particularly involving monounsaturated fatty acids, which may recover more slowly than others. Oleic acid is a major monounsaturated fatty acid involved in membrane phospholipid composition, energy storage, and cellular signaling. Elevated hepatic oleic acid levels have been associated with altered fatty acid desaturation, oxidative stress, and lipotoxicity in previous study (Zeng et al., 2020). Riboflavin plays a critical role in β-oxidation and lipid homeostasis via its function as a precursor for flavin cofactors required by acyl-CoA dehydrogenases (Zhang et al., 2021). To further explore the molecular basis for the persistent lipid imbalance, the results examined the hepatic expression of three key flavoprotein genes involved in mitochondrial β-oxidation: ACADS, ACADM, and ACADL. The present results showed that ACADS and ACADM expression levels were fully restored after refeeding, whereas ACADL, which is responsible for long-chain fatty acid oxidation, remained significantly lower than in the control group. This partial restoration of β-oxidation pathways at the transcription level is consistent with the accumulation of oleic acid, a long-chain monounsaturated fatty acid, in the RR group. These findings imply that although RR can reverse major metabolic disruptions, the persistent impairment in hepatic antioxidant indices despite restored hatchability suggests that the recovery of oxidative balance lags behind that of embryonic viability. This is likely due to the time required for restoring redox-sensitive enzymatic systems and repairing accumulated oxidative damage, particularly in hepatic tissues. Thus, a longer refeeding duration or higher riboflavin dose may be required to re-establish the full metabolic equilibrium. This has broader implications for the design of riboflavin-based interventions aimed at correcting hepatic lipid disorders during embryonic development. Moreover, the persistence of sub-clinical metabolic impairments, despite apparent recovery in some physiological indices, raises concern that long-term production traits such as reproductive performance or offspring quality may still be negatively affected if early deficiencies are not promptly and fully corrected.

Consistent with the metabolomic results, direct biochemical measurements using HPLC revealed that hepatic riboflavin, FMN, and FAD concentrations in the RR group returned to levels comparable to those in the CON group after 5 weeks of refeeding, while remaining higher than in the RD group. These findings support the pathway enrichment results related to vitamin digestion and absorption, and reflect the restored availability of riboflavin-derived cofactors essential for metabolic homeostasis. Furthermore, the improvement in antioxidant indices (e.g., T-SOD, GSH, GPX, and GR activities) and the normalization of hepatic lipid content (TL, TG, FFA, and T-CHO) in the RR group align with metabolomic signatures indicating partial recovery of redox and lipid metabolism pathways. Notably, the persistent elevation of hepatic oleic acid levels in the RR group, despite biochemical and transcriptomic recovery of most β-oxidation components, highlights the slower restitution of long-chain fatty acid metabolism, in agreement with the delayed recovery of ACADL expression. Thus, the integration of metabolomics, targeted HPLC quantification, and classical biochemical assays provides a coherent picture of the progressive, but incomplete, reversal of riboflavin deficiency-induced hepatic metabolic disruptions.

5. Conclusions

This study investigated the effects of RR on reproductive performance, embryo development, riboflavin status, antioxidant capacity, and liver metabolism in duck breeders and embryos. Riboflavin deficiency significantly reduced egg hatchability, embryo growth, hepatic antioxidant capacity, and disrupted liver metabolism. Refeeding with riboflavin effectively restored egg hatchability (from 3.80% to 77.11%), plasma and liver riboflavin levels, and improved embryo body weight and size. It also enhanced hepatic antioxidant capacity and partially normalized metabolic profiles. These findings demonstrate that RR alleviates the physiological and metabolic impairments caused by riboflavin deficiency in duck breeders and embryos.

Credit Author Statement

Bo Zhang: Writing – original draft, Data curation. Jie Wei: Investigation, Formal analysis. Junting Cao: Conceptualization. Dongyan Zhang: Methodology. Guangnan Xing: Investigation. Yating Li: Methodology. Ming Xie: Methodology. Zhengkui Zhou: Resources. Shuisheng Hou: Writing – review & editing, Supervision. Jing Tang: Writing – review & editing, Supervision.

Declaration of competing interest

We declare that there are no financial or personal relationships with any individuals or organizations that could improperly influence our work. Additionally, we have no professional or personal interests in any product, service, or company that could affect the content of this manuscript.

Appendix A. Supplementary data

The following is the Supplementary data to this article.