
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00754-5
10.1016/j.psj.2024.104175
104175
METABOLISM AND NUTRITION
Supplemental L-arginine promotes hepatocyte proliferation and alters liver fatty acid metabolism in the late embryonic phase: an RNA-seq analysis
Chen Ziwei *†1
Zheng Xiaotong *†1
Shu Xin *†
Hua Guoying ‡
Zhu Runbang *
Sun Liumei *†
Chen Jianfei jfchen@just.edu.cn
*†2
⁎ Jiangsu Key Laboratory of Sericultural and Animal Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, China
† Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture and Rural Affairs, Sericultural Scientific Research Center, Chinese Academy of Agricultural Sciences, Zhenjiang 212100, China
‡ Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, China
2 Corresponding author: jfchen@just.edu.cn
1 These 2 authors contribute equally to this paper.

14 8 2024
11 2024
14 8 2024
103 11 10417531 5 2024
1 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The in ovo feeding (IOF) of L-arginine (L-Arg) to chick embryos is a viable method for improving early intestinal development, subsequently leading to an acceleration in growth rate during the posthatch stage. However, the liver, being the pivotal organ for energy metabolism in poultry, the precise effects and mechanisms of L-Arg on the liver development and metabolism remain unclear. To elucidate these, the present study injected 2 doses of L-Arg (10 mg/egg and 15 mg/egg) into the embryos of Hongyao chickens at 17.5 d of incubation, subsequently incubating them until d 19 for further analysis. IOF of 15 mg L-Arg/egg significantly increased the organ indices of liver and small intestine, as well as the duodenal villus height/crypt depth. RNA-Seq analysis of liver tissues showed that the metabolism of xenobiotics, amino acid metabolism, and the fatty acid metabolism were significantly enriched in L-Arg injection group. The core differentially expressed genes (DEGs) were primarily involved in cell proliferation and fatty acid metabolism. The CCK8 assays revealed that supplemental L-Arg significantly enhanced the proliferation of primary embryo hepatocytes and leghorn male hepatoma (LMH) cells. Upregulation of core DEGs, including HBEGF, HES4, NEK3, EGR1, and USP2, significantly promoted the proliferation of liver cells. Additionally, analysis of triglyceride and total cholesterol content, as well as oil red O staining, indicated that supplemental L-Arg effectively reduced lipid accumulation. Overall, L-Arg supplementation in late chick embryos may promote early liver and small intestine development by reducing liver lipid deposition and enhancing energy efficiency, necessitating further experimental validation. This study provides profound insights into the molecular regulatory network of L-Arg in promoting the development of chicken embryos. The identified DEGs that promote cell proliferation and lipid metabolism can serve as novel targets for further developing methods to enhance early development of chicken embryos.

Key words

L-arginine
in ovo feeding
RNA-seq
cell proliferation
liver metabolism
==== Body
pmcINTRODUCTION

L-arginine (L-Arg) is recognized as an indispensable amino acid for gestating mammals and developing chicks, playing a critical role in intestinal development, bone health, and neonatal growth (Castro and Kim, 2020; Liu and Kim, 2023; Fathima et al., 2024). However, Birds, lacking the key enzyme involved in Arg synthesis within the urea cycle, are unable to produce L-Arg endogenously (Khajali and Wideman, 2010). In nowadays, in ovo feeding (IOF) technology of bioactive substances plays an important role in improving the health and development of chicks (Givisiez et al., 2020; Reicher et al., 2022; Kpodo and Proszkowiec-Weglarz, 2023) and turkey poults (Foye et al., 2006, 2009). Consequently, the application of IOF of L-Arg has become an important research field in the realm of early nutritional supplementation in poultry.

Several studies have demonstrated that dietary supplementation of Arg in broiler chicks can enhance gastrointestinal tract development, stimulate gastrointestinal hormone secretion, increase lymphatic organ weight, boost posthatch immune response, and ultimately enhance growth performance (Tahmasebi and Toghyani, 2016; Gao et al., 2017a; Gao et al., 2017b; Gao et al., 2018a; Gao et al., 2018b; Subramaniyan et al., 2019; Nabi et al., 2022). Furthermore, IOF of 0.5% L-Arg has an improving effect on cecal microflora in broilers (Omidi et al., 2020). The combination of periodic low eggshell temperatures during incubation with L-Arg dietary supplementation has been found to significantly improve chick quality at hatch in broilers (Miri et al., 2022). Additionally, the intramuscular administration of N-carbamylglutamate during the late incubation phase was found to enhance the meat quality of broilers' pectoral muscles (Zhang et al., 2020a). Amniotic injections of N-acetylglutamate, a crucial substrate in de novo Arg synthesis, were shown to promote intestinal development and improve jejunal digestive function (Wang et al., 2023). In the case of FUNAAB-Alpha chickens, a breed indigenous to Nigeria, supplementing with Arg and raising the birds in outdoor runs resulted in significantly lower feed intake and improved feed conversion ratios (Odutayo et al., 2020). In layer chicks, prenatal supplementation of L-Arg demonstrated positive effects on early intestinal development, microbial colonization patterns, and host metabolism (Dai et al., 2020; Dai et al., 2021). In a slow growing chicken line, IOF of 1% L-Arg improved the expression of myogenic genes, and increased the antioxidant capacity of the breast muscle in the starter period (Lu et al., 2022a; Lu et al., 2022b). Collectively, administration of Arg into embryonic eggs has the potential to enhance intestinal development in chicks, consequently influencing various growth traits in later stages.

Current research on the impact of Arg on chicks has mainly focused on posthatching studies, with many indicating that developmental benefits are linked to early advantages. Early chicken embryo development relies heavily on the absorption of yolk material, particularly lipids. The liver, the main organ for lipid metabolism, undergoes significant improvement in both structure and function by the 8 d of embryonic development. It palys a crucial role in regulating energy metabolism and other processes in chicken (Leveille et al., 1968; Anderson and Hammes, 1985; Chen et al., 2021; Xu et al., 2023). Previous research found that Arg solution can improve energy reserves in the liver and muscles of chicken embryos (Yu et al., 2018). To gain a deeper insight into how L-Arg impacts growth and development in chick embryos, this study aimed to unravel the specific molecular mechanism by which Arg regulates energy metabolism and embryonic development, which is still largely unknown. Initially, we employed IOF of L-Arg technology to investigate its effects on the development of organ indices. Subsequently, the RNA-seq analysis, along with in vitro and in vivo experiments were employed to examine the molecular regulatory network of L-Arg in liver. The findings of this study are expected to enhance our understanding of how L-Arg influences growth and development in chick embryos.

MATERIALS AND METHODS

In Ovo Feeding Procedures

The animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Jiangsu University of Science and Technology (GQ20230302, Zhenjiang, China). Animal care and handling practices were followed by the IACUC guidelines.

Fertile eggs from 30-wk-old Hongyao chickens, a Chinese indigenous meat breed, were incubated in an automatic-controlled incubator (Ruitai Incubation Equipment Development Center, Dezhou, China), with a temperature set at 37.8°C and relative humidity maintained at 60% following standard hatchery protocols. IOF of L-Arg was administered at the 17.5 d embryo stage. A total of 45 fertile eggs of similar weight (P > 0.05) were randomly assigned to 3 groups. The blunt end of each egg was sanitized with 75% ethanol, a small hole was drilled in the eggshell above the air sac for the insertion of L-Arg into the amniotic fluid using a 27-gauge needle was used to through the small hole. The L-Arg solutions were freshly prepared and incubated for 2 h before injection. The IOF of L-Arg group was injected with 0.1 mL saline containing 10 mg or 15 mg L-Arg. All eggs were used for later parameter statistics and morphological evaluation experiments. Three eggs were randomly selected from saline injection group and 15 mg L-Arg injection group for RNA-Seq experiments, and 4 eggs were randomly selected from each group for reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR).

Samples Collection

At the embryonic age of 19, the liver and entire intestine were harvested and weighed to determine the organ index (organ weight/embryonic weight). Subsequently, approximately 1 cm segments from the middle of the duodenum were obtained and preserved in 10% neutral-buffered formalin for morphological assessments. The remaining samples were promptly frozen in liquid nitrogen and stored at −80 °C for subsequent analysis.

Intestinal Morphology

Following the completion of wash, dehydration, clarification, and paraffin embedding protocols for intestinal segments, serial sections were cut at 5 μm thickness, placed on glass slides, deparaffinized, rehydrated, and stained. Villus height and crypt depth were measured using Olympus IX7 microscopy (Olympus Co., Ltd., Tokyo, Japan).

RNA‑Seq Library Preparation and Sequencing

Total RNA was extracted from liver tissue or cells using RNAiso Plus Reagent (Takara Biomedical Technology Co., Ltd., Beijing, China), and its concentration and quality were measured with Nanophotometer N60 Touch (IMPLEN, Munich, Germany). RNA integrity was assessed with the RNA Nano 6000 Assay Kit on a Bioanalyzer 2100 system (Agilent Technologies, CA). mRNA sequence libraries were constructed using NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Inc., MA), and 150 bp paired-end reads were generated on an Illumina Novaseq 6000 platform.

Read Mapping

Raw reads were processed with fastp (v0.23.4) to remove adapters, poly-N regions, and low-quality reads, resulting in clean reads. Quality metrics (Q20, Q30, GC content) were calculated for the clean data. Clean reads were aligned to the chicken genome assembly (GRCg7b) using Hisat2 (v2.0.5). A reference genome index was created with Hisat2 (v2.0.5), and clean paired-end reads were aligned to this index. Mapped reads from each sample were assembled using String Tie (v1.3.3b) in a reference-based manner. Counts (v1.5.0-p3) were used to quantify reads mapped to genes, then fragments per kilobase of exon per million mapped fragments were calculated for each gene. Differential expression analysis between 2 groups (saline injection and L-Arg injection) was done using the DESeq2 R package (1.20.0).

Gene Ontology and Kyoto Encyclopedia of Genes and Genomes Analysis

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes (DEGs) was implemented by the clusterProfiler R package (3.8.1), in which gene length bias was corrected.

Reverse Transcription Quantitative Real-time Polymerase Chain Reaction

cDNA was synthesized using the HiScript II 1st Strand cDNA Synthesis Kit with gDNA wiper (Vazyme Biotech Co., Ltd, Nanjing, China). The cDNA was diluted to a 1:3 ratio and stored at −20 °C. The RT-qPCR was performed as our previously report (Chen et al., 2023), with TBP and WAC as internal reference genes. The gene-specific primers were designed using Primer Premier 5.0 software (Supplementary Table S1).

Cell Culture

Chicken primary embryo hepatocytes were isolated from 14-d incubated eggs using a previously described improved method (Zhang et al., 2020b). The liver was washed with cooled PBS, cut into small pieces with sterile scissors, and digested with 1% trypsin at 37 °C for 15 min. The cells were then filtered through cell strainers of different pore sizes (100um, 70um, and 40um) to obtain chicken embryo liver cells. The primary liver cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Shanghai, China) plus 1% penicillin/streptomycin at 37°C/5% CO2 in an incubator. Leghorn Male Hepatoma (LMH) cells were cultured in DMEM Mixture F-12 (DMEM/F12) (Gibco) plus 10% superfine fetal bovine serum (BioChannel Biotechnology Co., Ltd, Nanjing, China), 1% penicillin/streptomycin at 37°C/5% CO2 in an incubator.

Vector Construction and Transient Transfection

The CDS region of the gene was cloned by Phanta Max Master Mix (Vazyme), the primers information was listed in Supplementary Table S2. After sequencing by a Sanger method (Shangya Biotechnology Co., Ltd., Hangzhou, China), the overexpression vectors were constructed by homologous recombination method using ClonExpress Ultra One Step Cloning kit V2 (Vazyme). Transfections were performed with ExFect Transfection Reagent (Vazyme) as per the manuscript's guidelines.

Western Blot Analysis

Total protein was extracted from cells using RIPA Lysis buffer with 1 mM phenylmethylsulfonyl fluoride. Fifteen μg of protein was separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Shanghai, China). The membranes were then probed with anti-Flag (dilution1:1,000, Cat No. 66008-4-Ig, Proteintech, Wuhan, China), and anti-β-Actin (dilution1:4,000, Cat No. ZB15001-HRP-100, Servicebio, Wuhan, China) antibodies overnight at 4°C. Followed by incubation with secondary anti-mouse-HRP antibodies (dilution1:4,000, Cat No. HRP-60008, Proteintech) for 1 h at room temperature. Chemiluminescence was detected using ECL reagent (Abbkine Scientific Co., Ltd, Wuhan, China) and blot bands were visualized with ChemiScope6100 (Clinx Science Instruments Co., Ltd., Shanghai, China).

Cell Counting Kit-8 Assay

The cell proliferation was determined by using the Cell Counting Kit-8 (CCK8) (APExBIO, Shanghai, China) assay. In brief, chicken embryo primary hepatocytes and LMH cells were cultured at 37 °C with 5% CO2 in a 96-well plate separately, after over expression of different vectors, about 26 h and 36 h later, 10 μL of CCK8 reagent were added per well and incubated at 37 °C for 2.5 h. Finally, measured the absorbance at 450 nm.

Triglyceride and Total Cholesterol Content Detection

Lipid content, including triglyceride (TG) and total cholesterol (TC), in liver tissue, hepatocytes, and LMH cells was assessed using commercial kits in accordance with the manufacturer's guidelines (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The protein concentration, determined by the BCA protein quantification kit (Vazyme), was utilized to normalize the TG and TC content. Absorbance readings were obtained using a BioTek Epoch 2 microplate auto-reader (BioTek, Winooski).

Oil Red O Staining of Liver Tissue and Liver Cells

The liver tissue was dehydrated, embedded in OCT embedding agent, sliced to 8 μm, stained with Oil Red O solution (Servicebio), differentiated in 60% isopropanol, stained with hematoxylin solution, and sealed with glycerin gelatin. Cells were stained with modified oil red O staining kit (Senbeijia Biotechnology, Nanjing, China), as directed by the manufacturer. Finally, observe and take pictures under a light microscope (Olympus).

Statistical Analysis

RT-qPCR data was analyzed using Bio-Rad CFX96 Manage software and exported to Microsoft Excel for further analysis. Gene expression levels were normalized using the 2−△△CT method (Schmittgen and Livak, 2008). The RT-qPCR data analysis for detecting the successful overexpression of DEGs in chicken primary embryo hepatocytes and LMH cells was utilized t-tests, while ANOVA with Tukey's multiple comparison test was used for all other statistical analyses. Statistical significance was set at P < 0.05.

RESULTS

Chicken Embryo Weight and Organ Index

In order to assess the impact of IOF of L-Arg on embryo chicks at 19 d of incubation, weight data for the egg, embryo, liver, and entire intestine were gathered. As shown in Table 1, there were no significant differences in weight between fertilized eggs and embryos across the various groups. However, IOF of a 15 mg L-Arg solution per egg led to a significant increase in both liver and intestinal index at 19 d of incubation. Additionally, the villus height/crypt depth in the duodenum of the 10 mg and 15 mg L-Arg groups were significantly greater (P < 0.001) compared to those of the saline injection group. These results indicated that L-Arg has a significant promoting effect on the development of chicken embryo liver and intestine.Table 1 Effects of in ovo feeding of L-arginine on the embryo weight and organ index at 19 d of incubation.

Table 1		Injection content of L-arginine		
Item	Saline	10 mg	15 mg	P-value	
Egg weight	39.2 ± 0.982	39.2 ± 0.989	38.4 ± 1.057	0.827	
Embryo weight	31.2 ± 0.524	31.5 ± 0.747	32.0 ± 0.672	0.788	
Liver index	0.0148 ± 0.000420b	0.0151 ± 0.000434ab	0.0164 ± 0.000365a	0.0311	
Intestine index	0.0136 ± 0.000714b	0.0161 ± 0.00149ab	0.0189 ± 0.00110a	0.0180	
Villus height/crypt depth	4.46 ± 0.139B	5.82 ± 0.168A	5.69 ± 0.117A	<0.001	
a,b Means within a row with no common superscripts differ significantly (P <0.05). A, B means within a row with no common superscripts differ significantly (P <0.01).

Identification and Functional Enrichment Analysis of DEGs

To understand how L-Arg injection affects early embryonic egg development, we analyzed liver tissues from 2 groups (15 mg L-Arg and saline injections) using RNA-Seq. The data generated clean reads ranging from 5.75 Gb to 7.82 Gb per library, with an average of 6.95 Gb and 6.22 Gb for the 2 groups. The quality of the data was high, with Q20 values above 96% and Q30 values above 92%. The other RNA-Seq data quality parameters are shown in Supplementary Table S3. Approximately 88.63% to 90.48% of reads in each library were uniquely mapped to the chicken reference genome, with average mapping rates of 89.67% and 89.65% for the saline and 15 mg L-Arg groups, respectively (Supplementary Table S4). The raw data in FASTQ format can be found in the NCBI SRA database under accession number PRJNA1084888.

After assembly, a total of 20,290 genes were identified. 719 DEGs were chosen based on |FoldChange| > 0 and P < 0.05, with 370 upregulated and 349 downregulated (Figures 1A, 1B). Details of the DEGs can be found in Supplementary Table S5. Functional enrichment analysis showed main GO terms including Lipid localization, Reactive oxygen species metabolic process, Cellular response to xenobiotic stimulus, Enzyme inhibitor activity, Iron ion binding, and Heme binding (Figure 1C). The DEGs were enriched in pathways such as Metabolism of xenobiotics by cytochrome P450, Retinol metabolism, Tryptophan metabolism, Phenylalanine metabolism, Alanine, aspartate and glutamate metabolism, and fatty acid degradation (Figure 1D). Core DEGs were chosen based on |Foldchange| > 1.5 and P adjust < 0.05 (Supplementary Table S6). The top ranked DEGs could be classified according to their gene symbol and function, mainly involved in promoting cell proliferation and regulating lipid metabolism (Table 2). The RNA-seq results revealed that L-Arg may affect the development and lipid metabolism in the liver of chick embryos.Figure 1 RNA-seq analysis of the embryo liver treated with L-arginine. (A) Volcano plot of differentially expressed genes (DEGs); (B) Heatmap of DEGs; (C) GO analysis of DEGs; (D) KEGG analysis of DEGs.

Figure 1

Table 2 Functional annotations on the most significantly differentially expressed genes.

Table 2Gene Symbol	Full Name	Function	
HBEGF	Heparin binding EGF like growth factor	Induces liver regeneration (Khai et al., 2006; Dao et al., 2018)	
HES4	Hes family bHLH transcription factor 4	Control cell proliferation (El Yakoubi et al., 2012)	
NEK3	NIMA related kinase 3	Cell cycle regulators (Tanaka and Nigg, 1999)	
EGR1	Early growth response 1	Promotes cell proliferation (Xu et al., 2022)	
IGFBP1	Insulin like growth factor binding protein 1	Promote embryo growth (Vaccaro et al., 2022)	
USP2	Ubiquitin specific peptidase 2	Promotes cell proliferation (Wei et al., 2023)	
AvBD8	Avian beta-defensin 8	Antibacterial peptides (Rengaraj et al., 2018)	
GSTT1	Glutathione S-transferase theta 1	Involved in the cellular detoxification and cell proliferation (Salinas and Wong, 1999; Lee et al., 2023)	
CPT1A	Carnitine palmitoyltransferase 1A	Mediated fat oxidation (Schlaepfer and Joshi, 2020)	
LB-FABP	Liver basic fatty acid binding protein	Mediated lipid deposition (Zhang et al., 2013)	
ENSGALG00010023727	/	Predicted IncRNA, function unknow	
ENSGALG00010026289	/	Predicted IncRNA, function unknow	
ENSGALG00010010285	/	Predicted IncRNA, function unknow	

RT-qPCR Verification of DEGs in vivo and in vitro

To validate the reliability of the RNA-Seq gene expression data, RT-qPCR was conducted on 12 selected DEGs: the gene with the most significant difference (SLC25A25), regulatory genes (HBEGF, HES4, NEK3, EGR1, USP2), antibacterial peptide gene (AvBD8), exogenous metabolic gene (GSTT1), lipid metabolism genes (LB-FABP, CPT1A, ACACB), and an IncRNA (ENSGALG00010023727) were also selected for gene expression validation. The results of the RT-qPCR analysis were consistent with the RNA-Seq data, suggesting the accuracy and repeatability of the RNA-Seq analysis (Figure 2). Additionally, the levels of gene expression were observed to fluctuate in response to varying doses of L-Arg injection, providing further evidence of L-Arg's ability to modulate the expression of DEGs (Figure 2).Figure 2 Verification of gene expression levels in liver tissue treated with different levels of L-arginine via a reverse transcription quantitative real-time PCR assay. Different lowercase letters indicate significant differences (P < 0.05). IncRNA-6289 represent ENSGALG00010026289.

Figure 2

Subsequent experiments were conducted on chicken primary embryonic hepatocytes with different concentrations of L-Arg. The expression of DEGs was also assessed after 24 h of incubation. The expression of HBEGF, NEK3, USP2, AvBD8, GSTT1, LB-FABP, IncRNA (ENSGALG00010023727), CPT1A, and ACACB genes can be regulated by L-Arg (Figure 3), while SLC25A25, HES4, and EGR1 were not significantly expressed. The majority of DEGs were found to be regulated by L-Arg in chicken primary embryonic hepatocytes, suggesting that L-Arg exhibits similar mechanisms of action both in vivo and in vitro.Figure 3 Verification of gene expression levels in primary embryo hepatocytes treated with different levels of L-arginine via a reverse transcription quantitative real-time PCR assay. Different lowercase letters indicate significant differences (P < 0.05). IncRNA-6289 represent ENSGALG00010026289.

Figure 3

Supplemental L-Arg Promoted Cell Proliferation Through DEGs

RNA-Seq and RT-qPCR results showed that L-Arg supplementation significantly affects the expression of key genes involved in cell proliferation. Treatment with different concentrations of L-Arg enhanced cell proliferation, with the most significant effect at 5 mM after 26 h (Figure 4A). To investigate the molecular mechanism of L-Arg promoting cell proliferation, key DEGs were overexpressed in primary hepatocytes. RT-qPCR results confirmed successful overexpression of all plasmids at 26hs and 36hs post-transfection, as shown in Figures 4B–G. However, western blot analysis did not detect any signal. CCK8 assay results indicated that overexpression of certain DEGs, specifically HBEGF, HES4, NEK3, EGR1, and USP2, significantly promoted cell proliferation in chicken primary embryonic hepatocytes at 26 h, while LB-FABP had no significant effect. USP2 had the most significant impact on cell proliferation compared to the empty vector control (Figure 4H).Figure 4 Effect of L-arginine on cell proliferation in primary embryo hepatocytes. (A) The effect of different L-arginine concentrations on cell proliferation. (B–G) represented the expression levels of differentially expressed genes (DEGs) were measured at various culture times post-transfection. (H) Overexpression of DEGs was found to affect cell proliferation, as shown by CCK8 assay results. Significant differences were denoted by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4

The supplementation of L-Arg at concentrations of 10, 20, and 30 mM demonstrated a significant impact on LMH cell proliferation, with 20 mM exhibiting the most favorable outcome (Figure 5A). The RT-qPCR analysis indicated successful overexpression of all genes in LMH cells (Figure 5B-G). Western blot results revealed successful overexpression of all plasmids except for EGR1 and USP2 (Supplementary Figure S1). In comparison to the pcDNA3.1-2×Flag vector, the CCK8 results showed that overexpression of HBEGF, HES4, NEK3, EGR1, and USP2 significantly enhanced the proliferation ability of LMH cells at 36hs (Figure 5H). These results suggested that L-Arg promotes liver development possibly through the regulation of DEGs.Figure 5 Effect of L-arginine on cell proliferation in LMH cells. (A) The effect of different L-arginine concentrations on cell proliferation. (B–G) represented the expression levels of differentially expressed genes (DEGs) were measured at various culture times post-transfection. (H) Overexpression of DEGs was found to affect cell proliferation, as shown by CCK8 assay results. Significant differences were denoted by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5

Effect of Supplemental L-Arg on Lipid Metabolism

RNA-Seq and RT-qPCR results indicated that the addition of L-Arg has a significant impact on the expression of multiple key genes involved in fatty acid metabolism. This study examined the impact of L-Arg on lipid metabolism and found that injecting different concentrations of L-Arg reduced TG and TC levels in chicken embryonic liver tissue. The 15 mg L-Arg treatment group was most effective in reducing TG content (Figures 6A, 6B), as shown in Oil Red O staining results (Figure 6C). Subsequent in vitro culture experiments utilizing primary chicken embryo liver cells demonstrated that varying concentrations of L-Arg resulted in a significant reduction in TG and TC content. The most effective reduction in TG occurred after 24hs, while the most effective reduction in TC occurred after 32hs. (Figures 6D, 6E). The Oil red O staining results revealed a decrease in Oil red O content with increasing concentrations of L-Arg (Figure 6F). These findings suggested that L-Arg supplementation in chicken liver tissue and primary cells can effectively reduce lipid accumulation.Figure 6 Effect of L-arginine (L-Arg) on lipid content in chicken embryonic liver tissue and primary hepatocytes. (A), and (B) represent TG and TC content in chicken embryonic liver tissue after in ovo feeding (IOF) of L-Arg respectively. (C) Oil red O staining observations of liver tissues after IOF of L-Arg. (D), and (E) represent TG and TC content in chicken primary hepatocytes with different concentration of L-Arg with different culture time. (F) Oil Red O staining observations of primary hepatocytes with different concentration of L-Arg after culturing 24h. Significant differences were denoted by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 6

DISCUSSION

Embryo egg injection technology represents a significant advancement in the poultry industry. Previously research on IOF of L-Arg has demonstrated its ability to enhance early intestinal development and posthatch growth rate in chicks (Lu et al., 2022a; Nabi et al., 2022). While existing studies have primarily examined the effects of L-Arg on gut health, limited attention has been given to its impact on the liver, a crucial organ for energy metabolism. Injection of L-Arg has been shown to modulate liver energy metabolism in posthatch broilers (Yu et al., 2018). However, the specific molecular mechanisms and potential alterations in other metabolic pathways remain to be comprehensively elucidated. In this study, we hypothesized that IOF of L-Arg promotes the overall growth of chick embryos by enhancing liver metabolism and development.

Initially, we investigated the effects of IOF of 10 mg and 15 mg of L-Arg into embryonic eggs, with a control group receiving saline injections. The research results indicated that L-Arg significantly increased the total intestinal weight index and intestinal villus height/crypt depth of chicken embryos, as presented in Table 1. This observation underscores the promotive role of L-Arg on the development of intestinal tissues, which aligns with previous research (Dai et al., 2020; Dai et al., 2021). The impact on organ development was found to be greater with a dosage of 15 mg L-Arg per egg compared to 10 mg, suggesting that 15 mg may be the optimal dose. Furthermore, the study revealed a significantly higher liver index in the group of 15 mg L-Arg than in the control group and 10 mg injection group, which has not been previously reported. These findings suggested that IOF of L-Arg may promote the development of chicken embryonic liver tissue.

Thus, this research elucidated the impact of L-Arg perturbation on hepatic gene expression through RNA-Seq. To enhance the understanding of L-Arg's influence on liver metabolism, an initial DEGs screening criterion of | FoldChange | > 0 and P < 0.05 was employed. Subsequent functional enrichment analysis of the DEGs revealed significant enrichment in pathways such as exogenous substance metabolism, lipid metabolism, amino acid synthesis, and cellular circulation. Additionally, functional annotation of the most significant DEGs highlighted differences in cell proliferation, fatty acid synthesis, and metabolism as predominant features. Specially, HBEGF has been shown to induce liver regeneration (Khai et al., 2006; Dao et al., 2018). HES4 has the ability to regulate the proliferative properties of neural stem cells (El Yakoubi et al., 2012). NEK3 has been implicated in cell cycle regulation (Tanaka and Nigg, 1999), and both EGR1 and USP2 have been reported to promote cell proliferation (Xu et al., 2022; Wei et al., 2023). These DEGs suggest that L-Arg may play a role in promoting liver development. Moreover, the expression of IGFBP1 was significantly lower in the L-Arg group compared to the control group. Previous research has demonstrated that IGFBP1 can enhance the growth and development of chicken embryos, but the expression of IGFBP1 in muscles decreases during incubation (Vaccaro et al., 2022). This indicates that L-Arg may promote embryonic development, resulting in a decrease in the expression level of IGFBP1.

LB-FABP, a liver-specific fatty acid binding protein found in poultry and classified within the family of fatty acid binding proteins, plays a vital role in transporting fatty acids to the mitochondria for oxidation, a process essential for fatty acid metabolism (Newberry et al., 2003). CPT1A, acting as a mediator of fat oxidation, also contributes to this metabolic pathway (Schlaepfer and Joshi, 2020). The differential expression of LB-FABP and CPT1A in RNA-seq analysis suggests that L-Arg may exert an influence on fatty acid metabolism. Additionally, the transporter SLC25A25, responsible for the transportation of Zn2+ as an essential cofactor in enzyme metabolism, has been associated with ATP transport (Anunciado-Koza et al., 2011; Ma et al., 2022). SLC25A25 plays a critical role in cellular metabolism via ciliary transduction (Hofherr et al., 2018). The SLC25A25 was notably higher expressed in the group receiving L-Arg injection compared to the control group, showing a dose-dependent effect. The addition of L-Arg significantly increased the metabolic activity of chicken embryo liver. These findings suggest that L-Arg supplementation may impact cellular processes such as cell proliferation and liver lipid metabolism. Moreover, AvBD8 is identified as a chicken-derived antimicrobial peptide that exhibits high expression levels in the liver and intestines (Rengaraj et al., 2018). Additionally, it has been shown to induce immune responses through the activation of the MAPK signaling pathway (Hong et al., 2020). Notably, the administration of L-Arg significantly upregulated the expression of AvBD8 in the liver and hepatocytes, suggesting that IOF of L-Arg to embryonic eggs could potentially enhance the innate immunity of chicks, warranting further investigation.

The RNA-Seq analysis suggested that L-Arg may impact cell proliferation and lipid metabolism processes. RT-qPCR was utilized for validation of DEGs identified through RNA-seq, with successful validation observed in vivo (Figure 2). Specifically, the genes HBEGF, NEK3, USP2, AvBD8, GSTT1, LB-FABP, IncRNA-6289, CPT1A, and ACACB in primary liver cells were found to be regulated by L-Arg, influencing processes such as cell proliferation, immunity, cellular lipid metabolism, and extracellular substance metabolism. However, some DEGs: SLC25A25, HES4, and EGR1 were not fully validated, indicating some small discrepancies in the effects of L-Arg between in vivo and in vitro settings. So, both in vivo and in vitro experiments were conducted in this study.

During the experimental verification of L-Arg's ability to cell proliferation, CCK8 assays revealed a dose-dependent increase in cell proliferation rates in both primary hepatocytes and LMH cells upon L-Arg treatment. Overexpressing DEGs like HBEGF, HES4, NEK3, EGR1, and USP2 further boosted cell proliferation, indicating that L-Arg promotes cell growth may be achieved by upregulating these genes. During overexpression, all genes were effectively overexpressed in primary embryo hepatocytes and LMH cells, with a higher level of expression observed in LMH cells. However, the corresponding protein expression was not observed in primary liver cells, potentially due to the absence of serum in the culture medium. In LMH cells, only EGR1 and USP2 were not detected, which could be attributed to the FLAG tag being obscured by the target protein at the C-terminus or being cleaved post-translation, thus hindering the binding of the anti-Flag antibody. This issue may be resolved by considering the placement of the FLAG tag at the N-terminus.

Through RNA-Seq analysis, this study posited that L-Arg may mitigate lipid deposition in a manner not conventionally governed by genes like PPARA (Nematbakhsh et al., 2021), but rather by pivotal lipid metabolism genes such as CPT1A, LB-FABP, and ACACB. Furthermore, during the experimental verification of L-Arg's ability to lipid metabolism, the IOF of L-Arg resulted in a significant decrease in TG and TC levels in the liver, as evidenced by Oil Red O staining. This finding aligns with prior research that L-Arg supplementation may be beneficial in reducing adipose tissue accumulation in poultry (Fouad et al., 2013; Castro and Kim, 2020), thereby suggesting the extensiveness of L-Arg's role in regulating lipid metabolism. During the later stages of chicken embryo development, adding L-Arg reduces liver lipid synthesis, directing energy towards protein synthesis and organ development, particularly in the digestive organs like the liver and small intestine. The early development of these organs is advantageous for the posthatch growth of chicks as it enhances their ability to digest and absorb nutrients, ultimately promoting overall growth. Moreover, a variety of differentially expressed lncRNAs were discovered through RNA-Seq analysis, and as of now, their functions remain unannotated. Additional studies are warranted to explore the involvement of these lncRNAs in lipid metabolism and cell proliferation induced by L-Arg supplementation, given their lengthy sequences and the potential challenges associated with constructing overexpression vectors.

In conclusion, the IOF of L-Arg significantly elevated the organ indices of both the liver and intestine in chick embryos. Considering the liver's pivotal role as the primary organ for energy metabolism in poultry, L-Arg may promote chicken embryo development by regulating the expression of genes related to cell proliferation and lipid metabolism within the liver. The core differential gene network identified here provides insights into the mechanisms of L-Arg's role during the late stage of chick embryos. Our study establishes a theoretical foundation for further improving chicken production efficiency through the application of IOF with L-Arg.

DISCLOSURES

The authors declare no conflict of interest.

Appendix Supplementary materials

Image, application 1

Image, application 2

ACKNOWLEDGMENTS

This work was supported by the Natural Science Foundation of China (32302725), the Natural Science Foundation of Jiangsu Province (BK20220648), the Postgraduate Research & Practice Innovation Program of Jiangsu Province (SJCX24_2575), and the Natural Science Research of Jiangsu Higher Education Institutions of China (24KJB230001). We would like to express our gratitude to Instrumental Analysis Center, Jiangsu University of Science and Technology for providing essential instruments in this study. We thank Professor Zhiliang Gu from Changshu Institute of Technology, Changshu, China, for providing LMH cells.

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104175.
==== Refs
REFERENCES

Anderson V.E. Hammes G.G. Distribution of reaction intermediates on chicken liver fatty acid synthase Biochemistry-Us 24 1985 2147 2154
Anunciado-Koza R.P. Zhang J. Ukropec J. Bajpeyi S. Koza R.A. Rogers R.C. Cefalu W.T. Mynatt R.L. Kozak L.P. Inactivation of the mitochondrial carrier SLC25A25 (ATP-Mg2+/Pi Transporter) reduces physical endurance and metabolic efficiency in mice J. Biol. Chem. 286 2011 11659 11671 21296886
Castro F.L.D.S. Kim W.K. Secondary functions of arginine and sulfur amino acids in poultry health: Review Animals (Basel) 10 2020 2106 33202808
Chen J. Hua G. Han D. Zheng X. Dong X. Wang S. Long J. Zheng Z. Wang A. Wang J. Wang X. Deng X. An EAV-HP insertion in the promoter region of SLCO1B3 has pleiotropic effects on chicken liver metabolism based on the transcriptome and proteome analysis Sci. Rep. 11 2021 7571 33828143
Chen J. Chen Z. Zhang J. Zhuang W. Zheng X. Screening of reliable reference genes for the normalization of RT-qPCR in chicken gastrointestinal tract Poult. Sci 102 2023 103169
Dai D. Wu S. Zhang H. Qi G. Wang J. Dynamic alterations in early intestinal development, microbiota and metabolome induced by in ovo feeding of L-arginine in a layer chick model J. Anim. Sci. Biotechno. 11 2020 19
Dai D. Zhang H. Qiu K. Qi G. Wang J. Wu S. Supplemental L-arginine improves the embryonic intestine development and microbial succession in a chick embryo model Front. Nutr. 8 2021 692305
Dao D.T. Anez-Bustillos L. Adam R.M. Puder M. Bielenberg D.R. Heparin-binding epidermal growth factor-like growth factor as a critical mediator of tissue repair and regeneration Am. J. Pathol. 188 2018 2446 2456 30142332
El Yakoubi W. Borday C. Hamdache J. Parain K. Tran H.T. Vleminckx K. Perron M. Locker M. Hes4 controls proliferative properties of neural stem cells during retinal ontogenesis Stem Cells 30 2012 2784 2795 22969013
Fathima S. Al Hakeem W.G. Selvaraj R.K. Shanmugasundaram R. Beyond protein synthesis: the emerging role of arginine in poultry nutrition and host-microbe interactions Front. Physiol. 14 2024 1326809
Fouad A.M. El-Senousey H.K. Yang X.J. Yao J.H. Dietary L-arginine supplementation reduces abdominal fat content by modulating lipid metabolism in broiler chickens Animal 7 2013 1239 1245 23472611
Foye O.T. Uni Z. Mcmurtry J.P. Ferket P.R. The effects of amniotic nutrient administration, “in ovo feeding” of arginine and/or β-hydroxy- betβ-methyl butyrate (hmb) on insulin-like growth factors, energy metabolism and growth in turkey poults Int. J. Poult. Sci. 5 2006 309 317
Foye O.T. Ashwell C. Uni Z. Ferket P.R. The effects of intra-amnionic feeding of arginine and/or ß-hyroxy-ß-methylbutyrate on jejunal gene expression in the turkey embryo and hatchling Int. J. Poult. Sci. 8 2009 437 445
Gao T. Zhao M.M. Zhang L. Li J.L. Yu L.L. Lv P.A. Gao F. Zhou G.H. Effects of in ovo feeding of L-arginine on the development of lymphoid organs and small intestinal immune barrier function in posthatch broilers Anim. Feed Sci. Tech. 225 2017 8 19
Gao T. Zhao M. Zhang L. Li J. Yu L. Lv P. Gao F. Zhou G. Effect of in ovo feeding of L-arginine on the hatchability, growth performance, gastrointestinal hormones, and jejunal digestive and absorptive capacity of posthatch broilers1 J. Anim. Sci. 95 2017 3079 3092 28727112
Gao T. Zhao M.M. Li Y.J. Zhang L. Li J.L. Yu L.L. Gao F. Zhou G.H. Effects of in ovo feeding of L-arginine on the development of digestive organs, intestinal function and post-hatch performance of broiler embryos and hatchlings J. Anim. Physiol. An. N. 102 2018 e166 e175
Gao T. Zhao M. Zhang L. Li J. Yu L. Gao F. Zhou G. In ovo feeding of L-arginine regulates intestinal barrier functions of posthatch broilers by activating the mTOR signaling pathway J. Sci. Food Agr. 98 2018 1416 1425 28771730
Givisiez P. Moreira F.A. Santos M. Oliveira H.B. Ferket P.R. Oliveira C. Malheiros R.D. Chicken embryo development: metabolic and morphological basis for in ovo feeding technology Poult. Sci. 99 2020 6774 6782 33248593
Hofherr A. Seger C. Fitzpatrick F. Busch T. Michel E. Luan J. Osterried L. Linden F. Kramer-Zucker A. Wakimoto B. Schütze C. Wiedemann N. Artati A. Adamski J. Walz G. Kunji E.R.S. Montell C. Watnick T. Köttgen M. The mitochondrial transporter SLC25A25 links ciliary TRPP2 signaling and cellular metabolism Plos Biol. 16 2018 e2005651
Hong Y. Lee J. Vu T.H. Lee S. Lillehoj H.S. Hong Y.H. Chicken avian beta-defensin 8 modulates immune response via the mitogen-activated protein kinase signaling pathways in a chicken macrophage cell line Poult. Sci. 99 2020 4174 4182 32867961
Khai N.C. Takahashi T. Ushikoshi H. Nagano S. Yuge K. Esaki M. Kawai T. Goto K. Murofushi Y. Fujiwara T. Fujiwara H. Kosai K. In vivo hepatic HB-EGF gene transduction inhibits Fas-induced liver injury and induces liver regeneration in mice: A comparative study to HGF J. Hepatol. 44 2006 1046 1054 16466829
Khajali F. Wideman R.F. Dietary arginine: metabolic, environmental, immunological and physiological interrelationships World Poult. Sci. J. 66 2010 751 765
Kpodo K.R. Proszkowiec-Weglarz M. Physiological effects of in ovo delivery of bioactive substances in broiler chickens Front. Vet. Sci. 10 2023 1124007
Lee E. Moon J. Ko J. Park S. Im G. GSTT1 as a predictive marker and enhancer for osteogenic potential of human adipose-derived stromal/stem cells J. Bone Miner. Res. 38 2023 1480 1496 37537994
Leveille G.A. O'Hea E.K. Chakbabarty K. In vivo lipogenesis in the domestic chicken Exp. Biol. Med. 128 1968 398
Liu G. Kim W.K. The functional roles of methionine and arginine in intestinal and bone health of poultry: review Animals (Basel) 13 2023 2949 37760349
Lu P. Morawong T. Molee A. Molee W. Influences of L-arginine in ovo feeding on the hatchability, growth performance, antioxidant capacity, and meat quality of slow-growing chickens Animals (Basel) 12 2022 392 35158714
Lu P. Morawong T. Molee A. Molee W. L-arginine alters myogenic genes expression but does not affect breast muscle characteristics by in ovo feeding technique in slow-growing chickens Front. Vet. Sci. 9 2022 1030873
Ma T. Zhao L. Zhang J. Tang R. Wang X. Liu N. Zhang Q. Wang F. Li M. Shan Q. Yang Y. Yin Q. Yang L. Gan Q. Yang C. A pair of transporters controls mitochondrial Zn(2+) levels to maintain mitochondrial homeostasis Protein Cell 13 2022 180 202 34687432
Miri B. Ghasemi H.A. Hajkhodadadi I. Khaltabadi F.A. Effects of low eggshell temperatures during incubation, in ovo feeding of L-arginine, and post-hatch dietary guanidinoacetic acid on hatching traits, performance, and physiological responses of broilers reared at low ambient temperature Poult. Sci. 101 2022 101548
Nabi F. Arain M.A. Bhutto Z.A. Shah Q.A. Bangulzai N. Ujjan N.A. Fazlani S.A. Effect of early feeding of L-arginine and L-threonine on hatchability and post-hatch performance of broiler chicken Trop. Anim. Health Pro. 54 2022 380
Nematbakhsh S. Pei Pei C. Selamat J. Nordin N. Idris L.H. Abdull Razis A.F Molecular regulation of lipogenesis, adipogenesis and fat deposition in chicken Genes-Basel 12 2021 414 33805667
Newberry E.P. Xie Y. Kennedy S. Han X. Buhman K.K. Luo J. Gross R.W. Davidson N.O. Decreased hepatic triglyceride accumulation and altered fatty acid uptake in mice with deletion of the liver fatty acid-binding protein gene J. Biol. Chem. 278 2003 51664 51672 14534295
Odutayo O.J. Sogunle O.M. Adeyemi O.A. Sonibare A.O. Influence of in ovo arginine feeding on hatching traits and post-hatch performance of FUNAAB-Alpha chickens in two housing types Trop Anim. Health Pro. 52 2020 2349 2357
Omidi S. Ebrahimi M. Janmohammadi H. Moghaddam G. Rajabi Z. Hosseintabar Ghasemabad B. The impact of in ovo injection of L-arginine on hatchability, immune system and caecum microflora of broiler chickens J. Anim. Physiol. An. N. 104 2020 178 185
Reicher N. Melkman-Zehavi T. Dayan J. Wong E.A. Nutritional stimulation by in-ovo feeding modulates cellular proliferation and differentiation in the small intestinal epithelium of chicks Anim. Nutr. 8 2022 91 101 34977379
Rengaraj D. Truong A.D. Lillehoj H.S. Han J.Y. Hong Y.H. Expression and regulation of avian beta-defensin 8 protein in immune tissues and cell lines of chickens Asian Austral. J. Anim. 31 2018 1516 1524
Salinas A.E. Wong M.G. Glutathione S-transferases–a review Curr. Med. Chem. 6 1999 279 309 10101214
Schlaepfer I.R. Joshi M. CPT1A-mediated fat oxidation, mechanisms, and therapeutic potential Endocrinology 161 2020 z46
Schmittgen T.D. Livak K.J. Analyzing real-time PCR data by the comparative C(T) method Nat. Protoc. 3 2008 1101 1108 18546601
Subramaniyan S.A. Kang D.R. Park J.R. Siddiqui S.H. Ravichandiran P. Yoo D.J. Na C.S. Shim K.S. Effect of in ovo injection of L-arginine in different chicken embryonic development stages on post-hatchability, immune response, and myo-D and myogenin proteins Animals (Basel) 9 2019 357 31207968
Tahmasebi S. Toghyani M. Effect of arginine and threonine administered in ovo on digestive organ developments and subsequent growth performance of broiler chickens J. Anim. Physiol. An. N. 100 2016 947 956
Tanaka K. Nigg E.A. Cloning and characterization of the murine Nek3 protein kinase, a novel member of the NIMA family of putative cell cycle regulators J. Biol. Chem. 274 1999 13491 13497 10224116
Vaccaro L.A. Porter T.E. Ellestad L.E. The effect of commercial genetic selection on somatotropic gene expression in broilers: a potential role for insulin-like growth factor binding proteins in regulating broiler growth and body composition Front. Physiol. 13 2022 935311
Wang J. Xu C. Song Z. Wu S. Wang J. Qi G. Zhang H. Improvement in growth performance and digestive function from amniotic injections of N-acetylglutamate in broile chickens J. Sci. Food Agr. 103 2023 6966 6974 37312006
Wei C. Zhao X. Zhang H. Wang L. USP2 promotes cell proliferation and metastasis in choroidal melanoma via stabilizing Snail J. Cancer Res. Clin. Oncol. 149 2023 9263 9276 37199836
Xu Y. Wang S. Cao X. Yuan Z. Getachew T. Mwacharo J.M. Haile A. Lv X. Sun W. The effect of EGR1 on the proliferation of dermal papilla cells Genes-Basel 13 2022 1242 35886025
Xu Z. Li M. Lu W. Wang L. Zhang Y. Chicken xenobiotic receptor upregulates the BCRP/ABCG2 transporter Poult. Sci. 102 2023 102278
Yu L.L. Gao T. Zhao M.M. Lv P.A. Zhang L. Li J.L. Jiang Y. Gao F. Zhou G.H. In ovo feeding of L-arginine alters energy metabolism in post-hatch broilers Poult. Sci. 97 2018 140 148 29077951
Zhang Q. Shi H. Liu W. Wang Y. Wang Q. Li H. Differential expression of L-FABP and L-BABP between fat and lean chickens Genet. Mol. Res. 12 2013 4192 4206 24114214
Zhang F. Wang J. Zhang H. Wu S. Lin J. Qi G. Effect of amniotic injection of N-carbamylglutamate on meat quality of broilers Animals (Basel) 10 2020 576 32235422
Zhang Y. Huang J. Li X. Fang C. Wang L. Identification of functional transcriptional binding sites within chicken Abcg2 gene promoter and screening its regulators Genes-Basel 11 2020 186 32050731
