Abstract

Background

   Induced molt is an effective measure to reduce the introduction cost,
   cope with the continuous rise of feed cost, and realize the prolonged
   rearing of laying hens as well. Vitamins are beneficial to the
   antioxidant capacity and reproductive performance of laying hens,
   however, studies on vitamin metabolism during fasting are rarely
   reported.

Results

   We analyzed the association between cecal metabolome and liver
   transcriptome of laying hens during molt. The results showed that 3009
   differences genes (FDR < 0.05), among which there were 62 differential
   genes related to vitamin metabolism. Eight core genes (ALDH1A1, CYP1A1,
   CYP1A4, AOX2P, AOX1, CYP3A7, BCAT1, CYP26B1) were obtained by protein
   network interaction (PPI). These genes were mainly enriched in
   Metabolic pathways, Retinol metabolism, Folate biosynthesis, One carbon
   pool by Folate, and Chemical carcinogenesis. After association analysis
   between these genes and cecal metabolites, a total of 176 differential
   metabolites were obtained. Among them, the metabolites with higher
   connectivity were Bifemelane, L-valine, Butyryl Fentanyl-D5 and
   Rimcazole.

Conclusion

   During fasting, vitamin A and vitamin E stored in the liver of laying
   hens are released in large quantities with the oxidative decomposition
   of lipids in the liver during fasting, which accelerates the metabolism
   of vitamin A in the liver. Folate and biotin may participate in the
   physiological remodeling of laying hens through epigenetic regulation.
   In addition, through association analysis, we constructed a data
   platform for vitamin metabolism-related pathway genes and cecal
   metabolites, laying a foundation for future research. However, whether
   the relationship between gene expression in the liver and metabolites
   in the cecum is bidirectional or unidirectional is still unclear and
   needs to be further studied.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12864-025-11730-7.

   Keywords: Induced molt, Vitamin, Liver, Cecum, Laying hens

Introduction

   The molting process is an inherent physiological phenomenon observed in
   avian species [[46]1]. Induced molt (IM) refers to the sudden stress
   given to chickens, so that they can quickly renew their feathers in a
   short period of time and recover their laying performance [[47]2,
   [48]3]. Because of the simple operation, low cost and good effect,
   fast-induced molting is one of the most common artificial molting
   methods in China [[49]4, [50]5]. In recent years, IM has been shown to
   remodel the ovarian function of laying hens [[51]6, [52]7], improve the
   semen quality of aged roosters [[53]8], and restore the luster of the
   feathers of laying hens at the late laying stage [[54]9], which has
   important production and economic value [[55]10].

   Vitamin is an indispensable nutrient for the body [[56]11], plays a
   significant role in the growth and development, metabolism and immunity
   of animals [[57]12, [58]13]. The metabolic activity of vitamins is
   mainly concentrated in the liver. Studies have shown that large amounts
   of fat-soluble vitamins and partially water-soluble vitamins are stored
   in the liver [[59]14, [60]15]. Vitamins have been shown to be important
   in improving the antioxidant capacity of liver tissue [[61]16].
   However, vitamin deficiency will reduce the immune function of
   hepatocytes and affect the normal physiological metabolism of the
   liver, thereby causing liver metabolic diseases [[62]17].

   As an essential nutrient, vitamins are also important for the
   maintenance of gut health. Studies have shown that vitamin A can
   mitigate the occurrence of intestinal inflammation by maintaining the
   integrity of intestinal cells and improving the intestinal barrier
   [[63]18–[64]20]. Similarly, vitamin E, a fat-soluble vitamin, also has
   a protective effect on intestinal structural proteins and tight
   junctions [[65]21]. In addition, studies have demonstrated that B
   vitamins (VB[2], VB[6]) can effectively improve the development of
   intestinal villi and increase the area of epithelial mucosa to maintain
   the integrity of the intestinal barrier [[66]22, [67]23].

   Fasting induces significant physiological changes in the liver and
   intestine of the body [[68]24–[69]28], and the interaction between the
   intestine and liver regulates the physiological metabolism of the body
   through the “gut-liver axis” [[70]29]. In this study, induced molting
   was implemented in aged laying hens. Subsequently, the correlation
   analysis of cecal metabolites and liver transcriptome data of laying
   hens investigate, through the “gut-liver axis”, we aim to reveal the
   regulatory mechanism, of induced molt on gut and liver of laying hens
   from the perspective of vitamin metabolism, and provided a theoretical
   basis for future nutritional regulation and special dietary design
   during IM of laying hens.

Materials and methods

Experimental design and sample collection

   Ninety Houdan chicken of 500 days of age (uniform body weight and 60%
   egg production rate) were selected for IM (Provided by Poultry
   Germplasm Resource Farm, Henan Agricultural University), referring to
   the method of Zhang et al. [[71]30] (Fig. [72]1, Supplementary Table
   [73]S1). In this context, F0 represents the period before fasting, F3
   indicates 3 days of fasting and water deprivation, followed by resuming
   drinking but continuing fasting until day 15, which is denoted as F15.
   R6, R16, and R32 represent 6, 16, and 32 days of resumed feeding,
   respectively. The process of resuming feeding involves a gradual
   reintroduction of feed. The procedure also involves changes to the
   light - dark cycle. During fasting, daily light exposure is reduced
   from 16 to 8 h. After resuming feeding, the daily light duration is
   increased by 0.5 h each day until it returns to 16 h per day. Fresh
   liver (n = 9) and cecal contents (n = 9) were collected at six critical
   periods and stored in an ultra-low temperature refrigerator (-80 ℃) for
   subsequent liver transcriptome sequencing (n = 3) and cecal contents
   metabolome sequencing (n = 3). The experimental chickens were
   euthanized by venous exsanguination.

Fig. 1.

   [74]Fig. 1
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   The procedures of induced molt (IM). F0 means before fasting, F3 means
   fasting and water deprivation for 3 days, F16 means fasting for 16
   days, but drinking water resumed at this time, R6, R16, R32 means food
   intake resumed for 6 days, 16 days, 32 days

Total RNA extraction and sequencing

   otal RNA extraction and sequencingTotal RNA was extracted using Trizol
   reagent kit (Invitrogen, Carlsbad, CA, USA), RNA quality was assessed
   on an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA,
   USA) and examined using RNase free agarose gel electrophoresis. When
   the samples were qualified, liver mRNA was enriched and disrupted.
   Subsequently, the fragmented mRNA was used as a template to synthesize
   and purify cDNA. The purified double-stranded cDNA was repaired, and
   the length of the fragment was selected (AMPure XP beads screened about
   200 bp cDNA). Then PCR amplification was performed, and AMPure XP beads
   were used again to purify PCR products, and finally the library was
   obtained.

   After passing the library examination, the liver tissues at different
   stages were sequenced using the HiSeq X-TEN sequencing platform.
   Low-quality reads containing more than 50% of bases with a quality
   score ≤ 20 were filtered out using fastp (version 0.18.0). Short
   fragments were compared with the ribosomal RNA (rRNA) database using
   the short fragment comparison tool Bowtie2 (version 2.2.8). Then remove
   the rRNA mapping. The remaining clean reads were further used for
   assembly and gene abundance calculations. To assess sample
   reproducibility and identify potential outliers, principal component
   analysis (PCA) was performed based on the expression levels of known
   genes across samples. Differential expression analysis was conducted
   using DESeq2, with differentially expressed genes (DEGs) identified
   based on significance thresholds (|log2FoldChange| > 1 and FDR < 0.05).

GO and KEGG enrichment

   GO (Gene Ontology) [[76]31] is an international standard classification
   system of Gene functions. It aims to establish a language vocabulary
   standard that is applicable to various species, defines and describes
   the functions of genes and proteins, and can be updated with further
   research. GO is divided into three parts: Molecular Function,
   Biological Process and Cellular Component. Kyoto Encyclopedia of Genes
   and Genomes (KEGG) [[77]32] is the main public path-related database.
   Pathway enrichment analysis identified significantly enriched metabolic
   pathways or signal transduction pathways in DEGs compared to the
   genome-wide background. We used GO and KEGG enrichment analysis to
   explore the changes in vitamin metabolic pathways in the liver of
   laying hens during the induced molt.

Trend analysis

   Trend analysis is an important method in the study of gene expression
   patterns [[78]33, [79]34]. To investigate the expression patterns of
   differentially expressed genes (DEGs), the expression data of each
   sample (ordered by treatment sequence) were normalized to 0,
   log2(v1/v0), log2(v2/v0), and subsequently clustered using the Short
   Time-series Expression Miner (STEM) software. The parameters for STEM
   analysis were configured as follows: (1) the maximum unit change in
   model profiles between time points was set to 1; (2) the maximum number
   of output profiles was set to 20 (with similar profiles being merged);
   and (3) the minimum fold change ratio of DEGs was set to no less than
   2.0.

Metabolome identification and integration analysis

   Team-based database of differential metabolites in the cecum, we
   combined multivariate statistical analysis of VIP value of OPLS-DA and
   univariate statistical analysis of t-test P value to screen for
   significantly different metabolites between different comparison groups
   [[80]35]. The thresholds for a significant difference are: VIP ≤ 1 in
   the OPLS-DA model and the T-test was P < 0.05. In this study, Pearson
   correlation coefficient was used to measure the correlation between the
   two variables, representing the strength of the co-variability of the
   two variables, and the value range was [-1, + 1]. Pearson’s coefficient
   of gene expression and metabolite abundance was calculated to evaluate
   the correlation between genes and metabolites. Pearson correlation
   coefficients were calculated for metabolome and transcriptome data
   integration. Gene and metabolite pairs were ranked in the descending
   order of absolute correlation cofficients. Finally, Cytoscape (version
   3.8.2) was used to visualize the data.

Results

Liver vitamin metabolic pathways were significantly enriched during fasting
in laying hens

   After passing the quality inspection, clean reads were used for all
   further analyses (Supplementary Table [81]S2). The relationship between
   samples in different periods and the number of differentially expressed
   genes were analyzed. (Fig. [82]2-A, B). The results showed that there
   were small between-individual differences and large between-group
   differences in each stage of IM. As can be seen in the Figure, the
   samples from the F0 and R32 groups have a strong correlation. This
   suggests that the F0 group exhibited no significant differences when
   compared to the flocks both after 32 days of resumed rearing and prior
   to the initiation of the experiment. A total of 3009 differential genes
   (FDR < 0.05) were screened.

Fig. 2.

   [83]Fig. 2
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   Transcriptome analysis. (A) Principal component analysis of samples.
   (B) Number of differential genes between comparison groups. Enrichment
   analysis of genes associated with vitamin metabolism during IM. (C)
   KEGG pathway analysis. (D) GO enrichment analysis

   Constructing a set of differentially related genes related to vitamin
   metabolism, then using KEGG enrichment analysis (Supplementary Table
   [85]S3), we found that pathways related to vitamin metabolism were
   strongly significant at different times during the IM. Subsequently, we
   generated the gene set for genes related to vitamin metabolism, and the
   gene set was subjected to enrichment analysis again. The results of GO
   enrichment analysis (Fig. [86]2-D) showed that the enrichment
   significance (P < 0.05) Top 5 GO entries were small molecule metabolic
   process, oxidation-reduction process, cofactor metabolic process, and
   drug metabolic process, pteridine-containing compound metabolic
   process. KEGG pathway enrichment analysis (P < 0.05) was performed on
   the differentially expressed genes in each period, and the top 20
   significantly enriched KEGG signaling pathways were selected. As shown
   in (Fig. [87]2-C), genes related to vitamin metabolism were mainly
   enriched in metabolic pathways, retinol metabolism, folate
   biosynthesis, one carbon pool by folate, and nicotinate and
   nicotinamide metabolism.

Construction of a core set of differentially expressed genes

   The STEM software was used to analyze the differentially expressed
   genes in the vitamin metabolism pathway (Fig. [88]3-A), and four
   significant modules were screened out, profile18, profile10, profile11
   and profile9. The expression of genes in profile18, profile10 and
   profile11 increased first and then decreased. However, the expression
   of genes in profile9 showed a completely opposite trend. Based on the
   STRING database [89]https://cn.string-db.org/, we performed protein
   network interactions (PPI) for differential genes related to vitamin
   metabolism, and the results are shown in Fig. [90]3-B, with darker
   colors indicating higher degree of vitamin-related proteins. The genes
   with connectivity from high to low were ALDH1A1, CYP1A1, CYP1A4, AOX2P,
   AOX1, CYP3A7, BCAT1, and CYP26B1. Through comparative analysis, it can
   be found that ALDH1A1 gene interacts with the most genes and has the
   strongest connectivity.

Fig. 3.

   [91]Fig. 3
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   The expression trend and protein network interaction analysis of genes
   related to vitamin metabolism during induced molt. (A) Trend analysis.
   (B) Protein network interaction analysis (PPI), the darker the gene,
   the higher the degree value. (C) The expression trends of genes with
   higher degree values

   Subsequently, transcriptome data were used to analyze the expression
   levels of some key genes in the vitamin metabolism pathway. As shown in
   Fig. [93]3-C, the expression levels of CYP1A1, CYP1A4 and CYP3A7 had
   the same changing trend. With the extension of fasting time, the
   expression levels gradually increased, reached the maximum on the 16th
   day of fasting, and R6 decreased sharply. The expression levels of
   CYP26B1, AOX2P, AOX1, and BCAT1 gradually increased with the extension
   of fasting time, and reached the highest level on the 6th day after
   resuming feed intake, then gradually decreased when feed intake was
   stable. The expression of ALDH1A1 peaked on the 16th day of fasting and
   remained high until the 6th day of feed intake. The expression of
   ALDH1A1 decreased when feed intake stabilized.

Analysis of liver vitamin metabolism

   During fasting, the expression of ADH, ALDH1A1, and AOX was
   significantly up-regulated (Fig. [94]4), which promoted the conversion
   of all-trans retinol (vitamin A) to retinoic acid. Among them,
   all-trans retinoic acid is further catalyzed by CYP family to generate
   bioactive retinoids (Fig. [95]5). However, the conversion of retinoic
   acid to retinoyl glucuronide was inhibited during fasting. In folate
   metabolism, tetrahydrofolate can produce 5, 10-methylene-THF under the
   catalysis of MTHFD1L and MTHFD2, which inhibits the conversion of
   5,10-methylenetetrahydrofolate to 5-Methyltetrahydrofolate. In vitamin
   B6 metabolism, the expression of PDXK was significantly up-regulated
   during fasting, which promoted the production of pyridoxal phosphate
   and participated in body metabolism. For riboflavin metabolism, FAD is
   converted to FMN, which, catalyzed by ENPP3, subsequently produces
   riboflavin by FMN catalyzed by ACP5. Notably, this process, during the
   fasting period of moult induction in laying hens, is promoted. HLCS
   (holocarboxylase synthetase) catalyzes the binding of biotin to
   carboxylases and histones. This protein plays an important role in
   gluconeogenesis, fatty acid synthesis, and branched-chain amino acid
   catabolism. The expression of this enzyme was significantly upregulated
   during fasting and was involved in the conversion of Biotin to
   biotinyl-5’-AMP and biotin-carboxyl-carrier protein.

Fig. 4.

   [96]Fig. 4
   [97]Open in a new tab

   Expression of genes involved in vitamin metabolism

Fig. 5.

   [98]Fig. 5
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   Analysis of hepatic vitamin metabolic pathways during IM

Integrated analysis of transcriptome and metabolome

   In the previous study, we performed metabolomics sequencing of cecal
   contents from laying hens during five key periods (F0, F3, F16, R6,
   R32)(Supplementary Figure [100]S1-[101]S5) [[102]36]. We correlated all
   the differential genes and metabolome data related to vitamin
   metabolism. As shown in Fig. [103]6, the correlation between these
   genes and metabolites was greater than 0.88 (positive correlation and
   negative correlation). By analyzing the degree value through
   connectivity, The top 20 were CYP3A5, CYP1A1, BCO1, CBR3, AOX2, PANK3,
   TYMS, AOX1, CYP26B1,
   N’2-benzylidene-5-hex-1-ynylfuran-2-carbohydrazide, and bifemela,
   respectively ne, L-valine, butyrylfentanyl-d5, rimcazole,
   N4-phenyl-3,5-dimethylisoxazole-4-sulfonamide, NAMPTP1, colchicoside,
   isorhamnetin, leukotriene E4, hexanoylglycine.

Fig. 6.

   [104]Fig. 6
   [105]Open in a new tab

   The association analysis between liver differentially expressed genes
   related to vitamin metabolism and cecal metabolites during IM

   Among them, CYP3A5 gene expression decreased significantly during the
   fasting period, and gradually increased to the pre-fasting level with
   the recovery of food intake Fig. [106]7. On the contrary, CYP1A1, BCO1,
   CBR3, AOX2, PANK3, TYMS, AOX1, and CYP26B1 genes were significantly
   up-regulated at the beginning of fasting, and their expression levels
   decreased to pre-fasting levels after resumption of feeding
   (Fig. [107]4). The content of highly connected cecal metabolites, such
   as bifemelane, L-valine, butyrylfentanil-D5, rimcazole, isorhamnetin,
   leukotriene E4, decreased significantly during fasting (Fig. [108]7),
   and as the diet was supplied, finally, the content returned to the
   level before fasting. The content of colchicoside increased
   significantly during the fasting period and gradually decreased to the
   pre-fasting level with the recovery of food intake.

Fig. 7.

   [109]Fig. 7
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   Trend of differential metabolites in cecal contents during IM

Discussion

Enhanced metabolism of fat-soluble vitamins is beneficial to maintain liver
homeostasis for laying hens during fasting

   Fasting is accompanied with the lack of energy, as well as vitamins,
   which has a very serious impact on the normal physiological metabolism
   of laying hens. In a previous study, our team found that during
   fasting, lipids stored in the liver of hens were decomposed by a large
   amount of oxidation to provide energy for the body, and the expression
   of genes related to lipid synthesis was reduced [[111]30]. In the
   present study, based on transcriptome data, we found that fasting also
   accelerated vitamin metabolism in the liver of laying hens. Some of
   these vitamins are derived from liver storage and the other from
   follicular reabsorption in the laying hens [[112]6]. A research report
   on human health has indicated that during periods of fasting, the
   concentration of fat-soluble vitamins in the blood tends to rise. This
   phenomenon is attributed to the breakdown of fat tissues that occurs
   while fasting, which releases stored fat-soluble vitamins into the
   bloodstream [[113]37].

   Vitamin A (one of the fat-soluble vitamins) is an essential vitamin for
   the body [[114]38]. Vitamin A refers to a large group of substances
   containing retinol structure and possessing its biological activity,
   such as retinol, retinaldehyde, retinoic acid, retinyl ester complex
   and its metabolites. As the main site of lipid metabolism in laying
   hens, liver stores a large amount of lipids and 95% of vitamin A in the
   body is also stored in liver [[115]39–[116]41]. In order to maintain
   the normal physiological metabolism of the body, the lipid in the liver
   is rapidly mobilized after the liver glycogen is consumed. At this
   time, the fat-soluble vitamins stored in the liver are released in
   large quantities accompanied by lipid oxidation. In this study, hepatic
   CYP1A1 gene was significantly up-regulated during fasting, which also
   demonstrated the existence of lipid oxidation in the liver. As a large
   amount of vitamin A was released, it accelerated the metabolism of
   vitamin A in the liver, and BCO1 (β-carotene-15,15’ -oxygenase), which
   is a key gene in the process of vitamin A metabolism, was also
   significantly up-regulated.

   More and more studies have shown that vitamin A not only plays an
   important role in maintaining the immune function and anti-oxidation of
   animals, but also regulates the expression of lipid metabolism-related
   genes and their signaling pathways, and the number of adipocytes
   [[117]42–[118]44]. Vitamin A can cause fat catabolism by promoting
   oxidative phosphorylation of adipocytes. At the same time, vitamin A
   can promote the decomposition of fatty acids in the liver, inhibit the
   production of fat, and promote the expression of uncoupling protein
   1(Ucp1) [[119]45, [120]46]. It is well known that white adipose tissue
   is a storage site for energy, which can be mobilized and dissipated by
   thermogenesis in UCP1-expressing adipocytes, and both pathways are
   regulated by ALDH1A1 [[121]47].

   Previous studies have shown that aldehyde dehydrogenase 1A1(ALDH1A1)
   plays an important role in the metabolism of vitamin A [[122]48,
   [123]49]. ALDH1A1 has a high affinity for all-trans and 9-cis
   retinaldehyde and can oxidize it to retinoic acid (RA). In addition,
   ALDH1A1 has an esterase activity and can effectively oxidise aldehydes
   (such as 4-hydroxynonenal acid, hexanal and malondialdehyde) derived
   from lipid peroxidation [[124]50]. In a mouse study, ALDH1A1 was found
   to prevent oxidative stress damage to the lens and cornea by oxidizing
   aldehydes derived from lipid peroxidation to corresponding acids
   [[125]50]. In addition, ALDH1A1 can protect the nerve from 3,
   4-dihydroxyphenylacetaldehyde injury through its oxidative effect,
   suggesting that ALDH1A1 gene plays a protective role in brain nerve
   [[126]51]. Therefore, during fasting, vitamin A metabolism not only
   plays an important role in maintaining the antioxidant capacity and
   immune function of the body, but also plays a positive role in
   providing energy for lipid oxidation.

   Intestinal microorganisms are important factors that determine the
   physiology and health of the host. The digestive tract of birds is
   colonized with rich and stable microorganisms to maintain intestinal
   homeostasis and digestion and absorption of nutrients, among which the
   cecum is the most developed part of birds. Vitamins, as micronutrients,
   play an important role in cell metabolism, but the host cannot
   synthesize them endogenously or in sufficient amounts. Retinoic acid
   (RA) plays an important role in host immunity and cell metabolism.
   Recent studies [[127]51] have shown that intestinal microorganisms can
   convert dietary vitamin A into its active metabolite retinoic acid,
   which plays a role in host metabolism. In this study, cecal RA content
   was significantly increased during the induction of fasting, and the
   authors believe that the increase in cecal RA at this time may be due
   to the release of fat-soluble vitamin A from the liver, which is not
   the result of cecal microbial transformation. Because the morphology
   and microflora of the gut are greatly altered after prolonged fasting,
   studies have shown that the abundance of beneficial bacteria in the gut
   decreases significantly during fasting, and it seems difficult to use
   this pathway to convert vitamin A into the active metabolite RA
   [[128]52].

The physiological remodeling induced by fasting May be related to epigenetic
regulation

   At present, the association between epigenetic regulation and
   nutritional phenotypes has also been confirmed in animal individual
   genes and genome-wide nutritional epigenomic studies [[129]53–[130]55].
   A large number of experiments have shown that nutrients in feed affect
   the growth and development of animals and changes in their phenotypes.
   Appropriate and reasonable supply can promote the growth and
   development of animals and improve their performance [[131]56].
   Excessive or insufficient supply of nutrients can lead to abnormal
   physiological changes in the body, and induce the occurrence and
   development of diseases.

   Previous studies have found that primordial follicles are activated and
   the number of primordial follicles increases during fasting in laying
   hens [[132]6]. Notably, 16 differentially methylated genes (DMGs) that
   regulate cellular senescence, immunity and development have been
   identified during molting fasting and redevelopment [[133]7]. In
   addition, five hypermethylated DMGs (DSTYK, NKTR, SMOC1, SCAMP3, and
   ATOH8) that inhibited the expression of DEGs were identified. They
   epigenetically modified DEGs, resulting in rapid shutdown and restart
   of reproductive function and re-increase of egg production rate in
   laying hens. This study demonstrated that epigenetic modifications can
   regulate gene expression during fasting induced molt.

   As one of the important water-soluble vitamins, folic acid is mainly
   stored in the liver [[134]57, [135]58]. Folate is mainly involved in
   one-carbon metabolism and is an important methyl donor [[136]59]. In
   this study, the expression of MTHFD1L and MTHFD2 was significantly
   upregulated during fasting. This facilitated the conversion of folate
   to 5,10-methylenetetrahydrofolate. However, the expression of MTHFR
   decreased first and then increased, reaching the lowest level at F3
   (the third day of fasting). Further conversion,
   5,10-methylenetetrahydrofolate to 5-Methyltetrahydrofolate, was
   inhibited. In addition, our team previously found that during fasting,
   a large number of follicles in the abdominal cavity of laying hens are
   reabsorbed to serve the body metabolism [[137]6]. Studies have shown
   that 5-Methyltetrahydrofolate is the main storage form of folate in
   yolk [[138]60]. As follicles are reabsorbed, MTHFR expression is
   suppressed to keep 5-Methyltetrahydrofolate balance. However, adequate
   methyl donors may be closely related to the physiological remodeling of
   tissues in laying hens. Similarly, it has been shown that biotin can
   also undergo epigenetic modification through biotinylation [[139]61].

Water-soluble vitamins are critical for later intestinal health care

   Studies have shown that gut microbes can synthesize B and K vitamins de
   novo, but only in small amounts [[140]62]. In this study, because the
   diet was stopped, the source of raw materials used by microorganisms to
   synthesize B vitamins and K vitamins was cut off, resulting in a
   significant decrease in the content of B vitamins (niacin, folate,
   pantothenic acid, pyridoxine, riboflavin) and vitamin K in cecal
   contents (Fig. [141]7). However, B vitamins play an important role in
   body metabolism. Thiamine (vitamin B1), as a coenzyme in cells, is
   involved in glucose metabolism and lipid metabolism in the body
   [[142]63]. Once the poultry lack thiamine will appear appetite loss,
   leg weakness, “star-gasting posture”, cause intestinal disease, and
   even death. Riboflavin and pyridoxine can also act as coenzymes in the
   body and are important for carbohydrate, protein, and fat metabolism.
   In addition, as an important B vitamin, folic acid is of great
   significance for maintaining animal health and normal physiological
   metabolism [[143]57, [144]64], and is an essential substance for
   intestinal microorganisms [[145]65]. The deficiency of B vitamins
   during fasting had a dramatic effect on the physiological metabolism of
   laying hens. During the induced moulting fasting period, although
   nutrients cannot be provided to laying hens in the form of diet, we can
   add vitamins through drinking water to maintain intestinal health and
   body immune function of laying hens.

   It is well known that the “gut-liver axis” is of great significance to
   the health of the body [[146]66]. In this study, we correlated the
   differentially expressed genes in the liver with metabolites in the
   cecum of laying hens, and constructed a data platform for genes
   involved in vitamin metabolism and cecum metabolites to explore the
   interaction between liver and intestine. For example, we found that the
   metabolites with strong correlation with BCO1 gene were: bifemelane,
   hexanoylcarnitine, hexanoylglycine, isorhamnetin, kynurenine, L-valine,
   octyl hydrogen phthalate, rimcazole. We believe that these metabolites
   are important for the physiological metabolic regulation of the body.
   However, whether the regulatory relationship between these metabolites
   and genes is one-way regulation or two-way interaction remains to be
   explored.

Conclusion

   In this study, we found that during IM, hepatic vitamin metabolism is
   accelerated during fasting, which is beneficial for maintaining body
   harmony and tissue remodeling for laying hens. In addition, RA and
   vitamin E contents increased in the cecal contents of laying hens
   during the fasting period, narrowing the content of partial
   water-soluble vitamins. It is suggested that in production practice,
   these water-soluble vitamins may be in addition to drinking water
   during the fasting period of peeling to maintain body health. More
   importantly, a correlation database of liver differentially expressed
   genes with cecal metabolites was built to facilitate further
   exploration of the interactions between gut microbiota and host through
   the “gut-liver axis”.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [147]Supplementary Material 1^ (1.4MB, docx)

Acknowledgements