Arginine shapes growth and mTOR pathway gene expression in Japanese quails (Coturnix japonica)

Abstract

Arginine is a crucial amino acid that regulates growth, metabolism, and cellular homeostasis through nutrient-sensing pathways such as the mechanistic target of rapamycin (mTOR). However, the mechanisms by which dietary arginine modulates mTOR signalling and associated gene networks during early development remain poorly understood in birds. Here, we tested the effects of dietary arginine levels (low, control, and high) on body mass, gene expression, and gene network connectivity in Japanese quail chicks (Coturnix japonica). Using 153 birds over a 14-day experiment, we show that post-hatch arginine restriction significantly impairs growth in both sexes, while gene expression and gene network connectivity responses to arginine availability are strongly sex-specific. Arginine supplementation in females led to coordinated upregulation of growth-related genes (IGF1, GHR), translation regulators (EIF4EBP1), lipid metabolism genes (FASN, FABP1), and oxidative defence (SOD2). In contrast, males showed more limited changes, primarily involving MTOR and autophagy-related gene ATG5. Gene network analysis revealed enhanced connectivity and centrality in females under arginine supplementation, suggesting a more extensive gene interaction response, while males showed sparser networks under restriction, with MTOR and IGF1 emerging as key regulatory hubs. These findings provide novel insights into the sensitivity of developing birds to early nutritional conditions and suggest that the mechanisms underlying growth, gene expression, and gene networking can differ between the sexes, even without sex differences in phenotypic traits. Our results highlight the importance of considering sex-specific gene regulatory architectures when evaluating nutritional effects on vertebrate development.

Introduction

Nutrition is a fundamental determinant of organismal development, shaping growth trajectories, metabolic efficiency, reproduction, and long-term health¹. In the postnatal period, the availability of specific nutrients can act as a cue that calibrates gene expression, resource allocation, and developmental priorities, a phenomenon referred to as nutritional programming². Amino acids, beyond their role as protein building blocks, serve as potent signalling molecules that activate nutrient-sensitive pathways regulating growth, immunity, metabolism, and cellular stress responses³.

One of the central signalling hubs linking nutrient status to anabolic processes is the mechanistic target of rapamycin (mTOR) signalling pathway, activated by specific amino acids, especially leucine, methionine, and arginine^4–6. The mTOR pathway integrates signals from nutrients, growth factors, and cellular energy levels to regulate protein synthesis and cell growth through downstream effectors such as eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) and autophagy gene⁷. This molecular pathway is highly conserved across animals and provides a mechanistic framework for understanding how diet influences physiology and performance⁸.

Among mTOR-regulating amino acids, arginine plays a particularly multifaceted role. Some vertebrate taxa, including birds and reptiles, have an incomplete urea cycle and cannot synthesise arginine endogenously, making its dietary intake critical^9,10. It also serves as a precursor for nitric oxide, creatine, and polyamines, linking it to vascular function, immune competence, and nutrient absorption^11,12. Arginine availability modulates mTOR activity and downstream gene expression in mammals, fish, and birds^13,14, with both deficiency and excess eliciting distinct, dose and context-dependent effects on growth and metabolism^13–16.

Sex-specific responses to nutritional signals are increasingly recognised as key components of developmental plasticity¹⁷. Males and females often diverge in their growth strategies, reproductive allocation, and metabolic priorities, especially during critical developmental windows when nutrients are scarce or imbalanced¹⁸. Research on fish, mice, and Japanese quail demonstrates that males and females exhibit distinct gene expression patterns in response to dietary and environmental factors, particularly in genes associated with mTOR and autophagy signalling^19–21. These differences are influenced by sex hormones and body size, highlighting the importance of considering both factors when studying growth and metabolic regulation^20,22.

Birds offer unique and underutilised opportunities for understanding the evolutionary basis of physiological plasticity. Their uricotelic nitrogen excretion, high metabolic rate, and rapid development present a contrast with mammalian models and enable comparative insights into how nutrient-sensing pathways like mTOR evolve and function under divergent life-history constraints. This makes avian systems particularly suitable for disentangling the roles of nutrient availability, sex, and developmental stage in shaping gene expression responses. Importantly, findings from bird models are increasingly being recognised for their relevance to broader ecological and evolutionary questions, as well as for informing biomedical research in areas like ageing, oxidative stress, and metabolic disease²³.

In this context, we used the Japanese quail (Coturnix japonica) as a model to explore how dietary arginine availability during early post-hatch development influences body mass and hepatic expression of genes related to the mTOR pathway, lipid metabolism, autophagy, and oxidative stress. Quails are widely used in poultry research and serve as a tractable vertebrate model for developmental and molecular physiology due to their short generation time and well-characterised developmental stages^24,25. While earlier studies have linked arginine availability to mTOR activation in chicken²⁶, it remains poorly understood how dietary arginine orchestrates coordinated gene regulatory responses during early post-hatch development, particularly in relation to growth, metabolism, and oxidative balance. Our study addresses this gap by mapping how dietary arginine modulates these interconnected pathways in a growing vertebrate.

To investigate how arginine modulates metabolic and growth-related pathways, we examined a panel of genes representing distinct but interconnected physiological functions which represented core regulatory nodes of the mTOR pathway and its principal downstream processes^6,20,27–29. These genes provide an integrated view of how dietary arginine influences growth, metabolism, and stress responses through the mTOR pathway. MTOR and its downstream target, EIF4EBP1 (Eukaryotic translation initiation factor 4E binding protein), were included as core regulators of nutrient sensing and protein synthesis. Lipid metabolism was assessed through FASN (Fatty acid synthase), which drives de novo fatty acid synthesis, and FABP1 (Fatty acid binding protein 1), which facilitates intracellular fatty acid transport and utilisation. FASN and FABP1 were chosen as functional effectors of lipid synthesis and transport, both of which are highly responsive to dietary amino acid supply in avian species^30–32. We assessed autophagy through ATG5 (Autophagy-related 5), a key mediator of autophagosome formation during nutrient stress. Oxidative balance was gauged via SOD2 (Superoxide dismutase 2), a mitochondrial antioxidant enzyme involved in neutralising reactive oxygen species. SOD2 is localised mainly in mitochondria, the primary site of reactive oxygen species generation under nutrient and metabolic stress, where it protects the electron transport chain from oxidative damage caused by superoxide radicals^33–35. Finally, we included IGF1(insulin-like growth factor 1) and its upstream GHR (growth hormone receptor), central components of the somatotropic axis that regulate systemic growth and hepatic metabolism. Collectively, these genes provide insight into how dietary arginine shapes coordinated molecular responses related to growth, energy allocation, cellular maintenance, and stress resilience during early development.

Given the liver’s central role in nutrient sensing and arginine activation of the mTOR pathway, we hypothesised that dietary arginine levels modulate body mass and hepatic expression of mTOR-related genes in growing Japanese quails. Thus, by experimentally manipulating dietary arginine levels (low, control, and high) and analysing gene expression across eight functionally related genes in males and females, we aimed to test how early nutritional signals are integrated at the molecular level, and whether this integration differs between sexes.

Material and methods

Experimental animal management

The experiment was conducted at the Animal House of the Institute of Animal Science, Biotechnology, and Nature Conservation, University of Debrecen, Hungary. We collected 500 eggs from 95 female Japanese quails over five days and incubated them artificially in an industrial incubator (WQ-63 Model 2021 Version 2, AGROFORTEL, Budapest, Hungary) under standard conditions²⁸ About 350 healthy hatchlings were obtained and reared with ad libitum access to feed and water until they reached two weeks of age. Infrared lamps maintained the initial rearing temperature of 37 °C for four days. After that, it was lowered by 3 °C every four days until it reached 24 °C at the end of the two weeks. Relative humidity in the cage was from 60 to 65%. After the experiment started, the room was maintained at 24 ± 3 °C, 60–75% relative humidity, a 12:12 h light-dark cycle, and 20 lux light intensity³⁶. The quails were fed a grower diet formulated based on corn, wheat, corn germ meal, corn gluten meal, and fishmeal, containing 22% crude protein and 12.13 MJ/kg metabolizable energy (Table 1.).

Table 1.

Feed composition and calculated nutrient content of the experimental diets.

Ingredients	Treatments
	Control	Low arginine	High arginine
Corn	3.40	3.40	3.40
Wheat	30.00	30.00	30.00
Corn germ meal	39.19	39.19	39.19
Corn gluten meal	10.90	10.90	10.90
Fishmeal	3.74	3.74	3.74
Oil	8.08	8.08	8.08
Limestone	1.40	1.40	1.40
MCP	0.42	0.42	0.42
L-Lys	0.77	0.77	0.77
DL-Met	0.06	0.06	0.06
L-Thr	0.28	0.28	0.28
L-Trp	0.06	0.06	0.06
L-Ile	0.20	0.20	0.20
L-Arg	0.39	0	0.78
Inert (kaolin)	0.39	0.78	0
Salt	0.22	0.22	0.22
Premixa	0.50	0.50	0.50
Nutrient content (%)
ME (MJ/kg)	12.13	12.13	12.13
Crude protein	22.00	22.00	22.00
Lys	1.30	1.30	1.30
Met	0.50	0.50	0.50
Thr	1.02	1.02	1.02
Trp	0.22	0.22	0.22
Leu	2.16	2.16	2.16
Ile	0.98	0.98	0.98
Arg	1.25	0.94	1.56
Leu/Ile	2.20	2.20	2.20
Ca	0.80	0.80	0.80
P	0.56	0.56	0.56
non phytate P	0.30	0.30	0.30
Na	0.15	0.15	0.15

^aThe premix provided the following per kilogram of complete diet: 5000  IU vitamin A, 1000  IU vitamin D₃, 24.5 mg/kg vitamin E, 1 mg vitamin K₃, 0.75 mg vitamin B₁, 2.5 mg vitamin B2, 6 mg Ca-d-Pantothetane, 2 mg vitamin B₆, 10 ug vitamin B₁₂, 55 µg biotin, 12.5 mg niacin, 0.3 mg folic acid, 1500 mg choline chloride, 66 mg Zn, 9.6 mg Cu, 48.1 mg Fe, 66 mg Mn, 0.9 mg I, 0.21 mg Se, 60 µg Co. MCP: monocalcium phosphate; ME: metabolisable energy.

Experimental design and sampling

At two weeks of age, 153 unsexed, similar-sized Japanese quails were selected and randomly distributed to three treatment groups. The experiment followed a completely randomised design with three treatments and three replicates, each containing seventeen birds. The dietary treatments included a control group: a diet formulated per³⁷, a high arginine group (control diet supplemented with 25% arginine above the recommended amount (1.56%)), and a low arginine group (control diet restricted by 25% of arginine from the recommended amount (0.94%)) (Table 1.). The restriction or supplementation of arginine from the recommended level provides a balance between biological relevance and animal welfare by imposing a measurable nutritional stress without causing gross toxicity or severe deficiency. Several recent studies in poultry have used ±20–35% changes to investigate functional nutrients and amino acid effects^{20,27,29,38–40}. Quails were housed in groups in identical cages (40 cm × 50 cm × 40 cm; length × width × height) within the same facility to ensure consistent environmental conditions and were tagged individually with numbered plastic rings for individual body mass measurement. Feed was provided in specially designed feeders to minimise spillage. The trial was conducted for 14 days²⁷. Daily fresh feed was replaced between 08:00 and 09:00, and feed intake was recorded. The average daily feed intake was 13.53 ± 0.16g for low arginine, 14.47± 0.41g for control, and 14.65 ± 0.49g for high arginine groups. Feed intake was lowered in the low arginine groups compared with other groups. Live body weight was measured using a digital balance (± 0.1 g accuracy) on days 0 (initial), 7 (midpoint), and 14 (final) to assess body mass across treatments.

On day 14 of the trial, 48 birds (eight per treatment group of both male and female) were euthanised by cervical dislocation following sedation with midazolam (5 mg/mL; EGIS Pharmaceuticals PLC, Hungary) performed by professional veterinarians for tissue collection. Liver tissues were immediately collected, placed in sterile tubes, flash-frozen, transported to the laboratory, and stored at −80°C until analysis.

RNA extraction and purification

Total RNA was extracted from liver tissues using the peqGOLD Total RNA Kit (VWR, USA) with on-column DNase I digestion, following the manufacturer’s instructions. RNA concentration and purity were assessed using a Synergy HT Multi-mode Microplate Reader (BioTek Instruments, Winooski, VT, USA). Integrity was checked with a Qubit 4 fluorometer (Invitrogen™), and it was in the range of 7–10.

Complementary DNA (cDNA) was synthesised in a PCRmax AlphaThermal Cycler (Cole-Parmer Ltd., Vernon Hills, IL, USA) using the LunaScript® RT SuperMix Kit (New England Biolabs Inc., Ipswich, MA, USA) with 800 ng RNA, 5x LunaScript RT Supermix, and nuclease-free water in a 20 µL final volume. Thermal conditions included 25 °C for 2 min (annealing), 55 °C for 10 min (reverse transcription), and 95 °C for 1 min (inactivation). cDNA was diluted 10-fold and stored at −20°C for qPCR.

Quail-specific, intron-spanning primers were designed using Oligo 7 software (version 7.6) and checked for target identity via the Primer BLAST web based tool (NCBI, https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on January 28, 2025) (Table 2.). qPCR was performed with AriaMx Real-Time PCR System (Agilent Technologies, Santa Clara, CA, USA) using HOT FIREPol® EvaGreen® qPCR Mix Plus (Solis BioDyne, Teaduspargi, Estonia) according to the manufacturer’s protocol. Each reaction included 5x HOT FIREPol® EvaGreen® qPCR Mix Plus Solis BioDyne, Teaduspargi, Estonia, 8 ng cDNA template, 200 nM of each primer, and distilled water in a 10 µL final volume. Reactions were run in duplicates using 96-well plates (Sorenson 2633), with calibrators included to control for interplate variation, and no template controls were included for each primer. Cycling conditions were 95 °C for 12 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 20 s, and 75 °C for 20 s, with melt curve analysis to confirm specificity.

Table 2.

Primer design for the target and reference gene.

Gene	Primer sequences (5´ < ---- > 3´) Forward/reverse	Gene bank accession no.	Amplicon Length (bp)	Melting temperature (Tm)
MTOR	F: CCG AAG CAT TGA ATT GGC CCT R: CAT CTC TCA AAG GCA GCG GAC C	XM_015882433.2	116	F:61.57 R: 63.50
IGF1	F: CAC TAT GCG GTG CTG AGC TGG TT R: ATC CCC TTG TGG TGT AAG CGT CT	XM_015867574.2	118	F: 65.42 R: 63.80
GHR	F: GGC ACT GGT CTG TGT GAA TGA CT R: CCA GCT CAG GTG ATC TGC ACT T	XM_032441512.1	89	F: 62.93 R: 62.58
FASN	F: TCA GCC CGA ACC TCC GCC AT R: ATG CCT GCA ATC ACC ACG TCT	XM_015879647.2	72	F: 66.24 R: 62.67
FABP1	F: AGT CCC ATG AGA ACT TTG AGC CTT R: CTG GAT CTG TTC ATC AGG AAG CCC	XM_015862881.2	62	F: 62.04 R: 63.03
EIF4EBP1	F: ACC AGC CCA ATT GTG GAG GAG TT R: CTC AGG GCA CGT GCT TTA GAT GT	XM_015883175.1	120	F: 64.29 R: 63.03
ATG5	F: ATA GTG GAT TTC GGT ACA TCC CA R: TCC TCC AGA AGC AAT TGG TCG	XM_015858735.1	95	F: 59.03 R: 60.34
SOD2	F: ACA GCA AAC ACC ACG CCA CCT R: AGC GAC ACC TGA GCT GTA ACA TC	XM_015858046.1	100	F: 65.38 R: 62.77
RPL19	F: CAT CGG TAA GAG GAA GGG T R: ACG TTG CCC TTG ACC TTC AG	XM_015885843.1	162	F: 55.80 R: 60.54

MTOR Mechanistic target of rapamycin, IGF1 Insulin-like growth factor 1, GHR, Growth hormone receptor, FASN Fatty acid synthase, FABP1 Fatty acid binding protein 1, EIF4EBP1 Eukaryotic translation initiation factor 4E binding protein, ATG5 Autophagy-related 5, SOD2 Superoxide dismutase 2, RPL19 Ribosomal protein L19.

Reference gene stability was assessed for RPL19, GAPDH, and RN18S using NormFinder, BestKeeper, and Delta Ct algorithms. RPL19 was identified as the most stable and used for normalisation^41,42. Relative expression of mechanistic target of rapamycin (MTOR), insulin-like growth factor 1 (IGF1), growth hormone receptor (GHR), fatty acid synthase (FASN), fatty acid binding protein 1 (FABP1), eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1), autophagy-related 5 (ATG5), and antioxidant gene superoxide dismutase (SOD2) was quantified as fold change using the 2^−ΔΔCt method⁴³. Log-transformed expression values were used for statistical analysis.

Statistical analysis

All statistical analyses were performed using R v. 4.2.2⁴⁴. Data visualisation was conducted using the ‘ggplot2’ package (version 3.4.3).

To evaluate the effects of treatment, sex, and their interaction on body mass over the 14-day trial period, we fitted linear mixed models (LMMs) using the ‘lmer’ function from the ‘lme4’ package⁴⁵. Treatment, sex, and time effects were included as fixed factors, while individual cages were treated as random effects. The best-fitting model was selected using Akaike’s Information Criterion corrected (AICc)⁴⁶. The significance of fixed effects was assessed using the ‘lmerTest’ package (version 3.1.3) to compute p-values using two-way ANOVA⁴⁷. Post-hoc pairwise comparisons of means were performed using estimated marginal means (via the ‘emmeans’ package) with Tukey’s HSD test at a significance level of p < 0.05^47,48. For gene expression analysis, linear models were employed to examine the effects of treatment, sex, and their interaction on the expression of target genes. In a gene network connectivity analysis, degree centrality measures how many direct connections a gene has, emphasising the importance of connections with high-degree genes (hubs) to the overall network function. Betweenness centrality reflects a gene’s role as a bridge, indicating how often it lies on the shortest paths between other genes. In contrast, closeness centrality assesses how close a gene is to all other genes in the network, providing insight into the efficiency of information flow through that gene. P-values were calculated for the Benjamini–Hochberg False Discovery Rate (FDR) method. However, gene co-expression networks were constructed based on correlation strength rather than statistical significance. In this network, genes are represented as nodes, and nodes are connected when there is a high level of co-expression between the corresponding genes⁴⁹ and using the igraph package in R.

Results

Arginine restriction reduces body mass in both sexes

Arginine restriction led to a significant, time-dependent reduction in body mass in both male (treatment × time: F_{4, 134} = p <0.001) and female (treatment × time: F_4,156 = 10.72, p < 0,001) quails. During the first week, the treatments had no effect on body mass. However, by the second week, birds on the low-arginine (LA) diet weighed less than those in the control and high-arginine (HA) groups (females: all p < 0.001, Fig. 1a; males: LA vs control: p = 0.003, LA vs HA: p < 0.001, Fig. 1b).

Fig. 1.

Effect of dietary arginine manipulation on body mass of (a) female and (b) male Japanese quail in a two-week treatment period. The low-arginine groups showed significantly lower body weight compared to the control and high-arginine groups in both sexes at the second week of the experimental period. Data are means ± SEM. Different letters indicate significant differences at a given time point at p ≤ 0.05. LA: low arginine, CON: control, HA: high arginine.

Sex-specific effects of dietary arginine on mTOR pathway gene expression

Dietary arginine manipulation significantly altered gene expression in the mTOR signalling pathway, with distinct sex-specific responses (Fig. 2). Arginine restriction significantly upregulated the expression of MTOR (p = 0.002; Fig. 2a) and FABP1 (p < 0.001; Fig. 2e) in males, while EIF4EBP1 (p < 0.001; Fig. 2f) in both sexes. In females, arginine restriction reduced the expression of fatty acid-related FASN (p = 0.032; Fig. 2d) and FABP1 (p < 0.001; Fig. 2e) while elevating antioxidant SOD2 (p < 0.001; Fig. 2h). On the contrary, arginine supplementation upregulated several targets, including the growth-related genes IGF1 (p = 0.016; Fig. 2b) and GHR (p < 0.001; Fig. 2c) and also SOD2 (p < 0.001; Fig. 2h) in females, the lipid transport gene FABP1 (p = 0.012; Fig. 2e) in males, and EIF4EBP1 in both sexes (males: p = 0.022; females: p = 0.016; Fig. 2f).

Fig. 2.

Sex-specific gene expression. (a) MTOR, mechanistic target of rapamycin. (b) IGF1, insulin-like growth factor 1; (c) GHR, growth hormone receptor; (d) FASN, fatty acid synthase; (e) FABP1, fatty acid binding protein 1; (f) EIF4EBP1, eukaryotic translation initiation factor 4E binding protein 1; (g) ATG5, autophagy-related 5; (h) SOD2, superoxide dismutase 2. Data are means ± s.e.m from 8 birds per group and were analysed using ANOVA. Different letters indicate significant differences at a given time point at p ≤ 0.05. LA: low arginine, CON: control, HA: high arginine.

Gene expression exhibited notable sex-specific variation across treatments, particularly in genes associated with growth, metabolism, and autophagy (Fig. 2). Arginine restriction in female birds showed higher expression of FABP1 (p < 0.001; Fig. 2e) and ATG5 (p = 0.008; Fig. 2g), while IGF1 (p = 0.005; Fig. 2b), FABP1 (p < 0.001; Fig. 2e), and ATG5 (p < 0.001; Fig. 2h) in the arginine supplementation diet and during control conditions; only MTOR and FABP1 were different (Fig. 2a, e).

Gene network analysis

Gene co-expression network analysis revealed that dietary arginine availability modulates the overall structure and efficiency of molecular interactions in the liver, with marked sex-specific differences (Fig. 3, Table 3.). Arginine supplementation increased gene connectivity and integration, particularly in females, as shown by higher degree and closeness centrality values (Fig. 3c). Key regulatory genes such as EIF4EBP1 and ATG5 became more central under high-arginine diets in females, suggesting coordinated upregulation of growth and cellular maintenance pathways. In contrast, males display more variable responses across diets. Notably, mTOR and IGF1 exhibit higher betweenness and centrality in arginine-restricted groups (Fig. 3d), indicating their pivotal role in maintaining network function under nutrient restriction. The control group exhibited more balanced, intermediate overall connectivity in both sexes (Fig. 3b, e), suggesting a baseline level of regulatory coordination that is either enhanced or disrupted by dietary manipulation.

Fig. 3.

Gene network analysis. (a) female: low arginine treatment, (b) female: control treatment, (c) female: high arginine treatment, (d) male: low arginine treatment, (e) male: control treatment, (f) male: high arginine treatment. MTOR, mechanistic target of rapamycin; IGF1, insulin-like growth factor 1; GHR, growth hormone receptor; FASN, fatty acid synthase; FABP1, fatty acid binding protein 1; EIF4EBP1, eukaryotic translation initiation factor 4E binding Protein 1; ATG5, autophagy-related 5; SOD2, superoxide dismutase. Each gene is represented as a node, and significant correlations between gene expression values (r > 0.3) are represented as edges. The node size (ranges from 0 - 7; where 0 degree is no connectedenss whereas 7 degree is high connectedness) reflects degree of centrality which indicating how many direct connections a gene has, emphasising the importance of connections with high-degree genes (hubs) to the overall network function, while the node colour represents betweenness centrality, with yellow indicating high and red indicating low centrality reflects how often it lies on the shortest paths between other genes. Closeness centrality assesses how close a gene is to all other genes in the network, providing insight into the efficiency of information flow through that gene. The edge thickness corresponds to the strength of the correlation between genes.

Table 3.

The effect of treatments and sex on centrality measures.

Gene	Sex	Degree			Betweenness			Closeness
		LA	CON	HA	LA	CON	HA	LA	CON	HA
MTOR	Female	6	3	7	0	0	0	0.178	0.147	0.207
IGF1	Female	1	4	7	0	0	0	0.148	0.148	0.231
GHR	Female	6	6	7	0	4	0	0.203	0.204	0.177
FASN	Female	5	3	7	0	0	0	0.196	0.143	0.199
FABP1	Female	6	2	7	0	3	0	0.202	0.183	0.194
EIF4EBP1	Female	6	3	7	3	0	0	0.261	0.173	0.233
ATG5	Female	6	6	7	0	5	1	0.194	0.199	0.233
SOD2	Female	6	5	7	6	3	0	0.214	0.185	0.200
MTOR	Male	6	6	3	3	2	1	0.271	0.207	0.213
IGF1	Male	6	2	5	4	0	6	0.261	0.143	0.272
GHR	Male	7	4	2	0	0	0	0.226	0.203	0.156
FASN	Male	6	6	4	0	0	2	0.209	0.196	0.235
FABP1	Male	5	4	2	0	0	0	0.237	0.165	0.158
EIF4EBP1	Male	4	4	3	1	0	0	0.221	0.164	0.187
ATG5	Male	5	5	3	0	3	3	0.226	0.208	0.213
SOD2	Male	5	7	0	0	6	0	0.199	0.267	Na

LA low arginine, CON control, HA high arginine, Na, not applicable.

Discussion

The study demonstrated that early post-hatch dietary arginine availability affects growth and gene expression in Japanese quail. Arginine restriction led to growth retardation in both sexes, indicating that adequate dietary arginine is essential for normal development^50,51. However, gene expression responses showed clear sex-specific patterns. In the present study, although arginine supplementation did not enhance growth significantly, it triggered changes in gene expression, particularly related to growth regulation and oxidative stress. By separating the effects of arginine restriction and supplementation, we clarified how changes in dietary arginine influence growth-related molecular pathways. These findings indicate that grower quails are sensitive to early nutritional conditions and suggest differences in metabolic regulation between the sexes, even without visible differences in body mass.

The somatotropic GHR-IGF1-mTOR axis tightly regulates avian growth, with IGF1 serving as a reliable marker of growth potential due to its role in promoting protein synthesis and muscle development^52,53. In our study, arginine supplementation increased hepatic IGF1 and GHR in females (Fig. 2b, c), while males showed no significant changes, which aligns with the notion that male hepatic IGF1 and GHR expression are less responsive to environmental factors⁵⁴. Despite elevated IGF1 and GHR in females, body mass did not differ significantly between sexes under arginine supplementation conditions, suggesting a potential lag between transcriptional activation and physical growth²⁸. Under arginine restriction, IGF1 and GHR expression remained unchanged in both sexes, consistent with the observed growth retardation. These results indicate that arginine supplementation enhances IGF1 and GHR expression primarily in females, potentially amplifying growth signalling, while arginine deficiency suppresses growth without altering gene expression^28,55,56. The sex-specific IGF1 response highlights females exhibiting greater molecular sensitivity to dietary arginine, which could support growth under nutrient-rich conditions⁵⁷.

As a downstream effector of IGF1, mTOR integrates arginine availability to regulate protein synthesis and growth, linking dietary inputs to the GHR-IGF1 axis. Arginine activates mTOR through several mechanisms^4,58–60. In our study, male quails in the arginine restriction group showed significantly increased MTOR expression, potentially compensating for limited arginine to sustain protein synthesis, though insufficient to prevent growth retardation. Because IGF1 levels in males remained stable across diets, the rise in MTOR under arginine restriction likely reflects an IGF1-independent compensatory response to low intracellular arginine. However, females displayed higher basal MTOR expression under the control diet than males (Fig. 2a), suggesting a sex-specific regulatory pattern which might be hormonal factors^{19,60,61,61–63}. Thus, females may maintain a stable mTOR activity through hormonal modulation, regardless of dietary differences. indicate that arginine modulates the GHR-IGF1-mTOR axis differently by sex.

Fatty acid metabolism, crucial for energy storage and reproductive preparation, is regulated by arginine through mTOR, influencing lipogenic gene expression⁶⁴. In our study, the expression of FASN, a central lipogenetic enzyme, was downregulated in females under the arginine-restricted diet, while males showed no significant differences across treatments (Fig. 2d). This suggests that arginine deficiency impairs lipid synthesis more prominently in females, potentially exacerbating growth retardation by limiting energy reserves and reflecting sex-related metabolic differences. To complement this, we also analysed FABP1, which facilitates intracellular fatty acid transport and metabolism. FABP1 was expressed at higher levels in females than males across all treatments (Fig. 2e). However, specifically, males in both arginine-restricted and supplemented groups showed moderately elevated expression compared to the control diet group. This consistent sex-specific expression may reflect differences in lipid metabolic regulation, potentially influenced by early hormonal activity, as females exhibit higher lipid mobilisation and storage capacity^65–68. These sex-specific patterns indicate that females prioritise lipid transport and storage, potentially driven by IGF1 and mTOR signalling under arginine-supplemented conditions, supporting growth despite stable mTOR expression. In males, FABP1 upregulation aligns with mTOR activation under arginine restriction, reflecting a compensatory mechanism to maintain lipid metabolism during growth limitation²⁰.

Downstream of mTOR, EIF4EBP1 regulates translation and metabolic processes critical for growth and energy homeostasis, further mediating arginine’s effects on the GHR-IGF1-mTOR axis⁶⁹. In our study, EIF4EBP1 expression was significantly upregulated in both sexes under arginine restriction and supplementation (Fig. 2f). Unlike IGF1, mTOR, and FABP1, EIF4EBP1 showed no sex-specific differences, suggesting a conserved response to arginine availability. Under arginine restriction, EIF4EBP1 upregulation in both sexes likely inhibits translation initiation, contributing to growth retardation by limiting protein synthesis, consistent with reduced body mass. In contrast, under high-arginine diets, EIF4EBP1 upregulation may fine-tune translation to support metabolic adaptation, particularly in females. This bidirectional 4EBP1 response aligns with mTOR activation in males under low arginine and IGF1-driven signalling in females under high arginine, reinforcing arginine’s role in modulating growth through the GHR-IGF1-mTOR axis⁷⁰, which indicates that 4EBP1 acts as a key regulator of arginine-mediated metabolic responses.

In addition to translation regulation, mTOR modulates autophagy via ATG5, an essential gene that recycles cellular components during nutrient stress, influencing growth and metabolic adaptation^71,72. In our study, female quails exhibited significantly higher ATG5 expression than males under both arginine-restricted and supplemented groups, indicating a sex-specific response to arginine availability. In females, ATG5 upregulation under arginine restriction likely enhances cellular recycling to mitigate nutrient stress, contributing to growth retardation alongside EIF4EBP1 and FASN responses. Under arginine-supplemented diets, female ATG5 upregulation may support metabolic flexibility, complementing IGF1 and FABP1 to sustain growth potential²⁷. The inverse relationship between mTOR activation and autophagy⁷³ explains these patterns: male mTOR upregulation under low arginine suppresses ATG5, while female IGF1-driven mTOR signalling under high arginine allows ATG5 expression to persist.

Beyond autophagy, mTOR regulates oxidative stress responses via SOD2, a mitochondrial antioxidant enzyme that protects cells from reactive oxygen species during growth¹². In our study, females exhibited significant SOD2 upregulation under restricted and supplemented diets (Fig. 2i), while males showed no treatment effects. Despite this female-specific response, overall sex differences were not statistically significant, indicating a conserved role for SOD2 in both sexes. In females, elevated SOD2 under arginine restriction likely enhances antioxidant defence during growth retardation, complementing ATG5 and EIF4EBP1. Under high-arginine diets, SOD2 upregulation complements IGF1, FABP1, and ATG5, potentially protecting against reactive oxygen species to sustain growth potential. These suggest that arginine modulates SOD2 expression via the GHR-IGF1-mTOR axis, with females showing greater sensitivity that may contribute to metabolic resilience^35,74.

In this study, gene network analysis further elucidates these sex-specific responses, with betweenness centrality highlighting key hubs. Under arginine restriction, the female network (Fig. 3a) showed high SOD2 and EIF4EBP1 betweenness, reflecting their roles in antioxidant defence and translation regulation despite growth challenges, while the male network (Fig. 3d) was sparse with mTOR and IGF1 as central hubs, aligning with a focus on protein synthesis and growth signalling. Under arginine supplementation, the female network (Fig. 3c) exhibited high EIF4EBP1 and ATG5 betweenness, consistent with enhanced translation and autophagy to support metabolic adaptation, whereas the male network (Fig. 3f) emphasised IGF1, indicating limited responsiveness in growth signalling. The denser female networks across treatments underscore their greater molecular integration, supporting the hypothesis that arginine modulates the GHR-IGF1-mTOR axis to drive sex-specific growth and metabolic adaptations. These patterns are influenced by sex and nutrient availability^6,27. However, gene network connectivity in poultry is highly complex, involving multiple interacting genes that regulate key traits^75,76.

In conclusion, this study reveals the critical role of dietary arginine in shaping growth, acting through the GHR-IGF1-mTOR signalling axis. Arginine limitation impairs body mass across sexes, disrupts network connectivity, particularly in males, where sparse networks with MTOR and IGF1 hubs suppress growth potential. In contrast, in females, arginine supplementation induced changes in gene expression, which refers to molecular-level regulation rather than phenotypic gain, including the upregulation of growth-related (IGF1, GHR), translation (EIF4EBP1), autophagy (ATG5), and oxidative defence (SOD2) genes. These molecular shifts, together with denser female networks centred on SOD2 and ATG5, suggest greater female resilience in integrating nutritional signals. Consistent EIF4EBP1 upregulation across sexes highlights its central role in linking arginine availability to metabolic regulation. Overall, these findings provide new insight into how early nutritional conditions shape sex-specific metabolic response trajectories in birds. Examining the GHR–IGF1–mTOR axis in additional tissues and over extended periods could shed light on arginine’s variable effects and guide refined nutritional strategies.

Author contributions

Conceptualisation: M.G, G.K.R., Á.Z.L.; L.C., Methodology: M.G., G.K.R., B.C., G.G., R.K., C.S., Á.Z.L.; L.C., Software: M.G., G.K.R., Á.Z.L.; Validation: M.G., Á.Z.L.; L.C., Formal analysis: M.G.; Investigation: M.G., Resources: M.G., Á.Z.L.; L.C., Data curation: M.G.; Writing - original draft: M.G.; Writing - review & editing: M.G., G.K.R., F.N.A., E. M.A. D.M.K., S.F.N., B.C., G.G., R.K., C.S., Á.Z.L.; L.C., Visualization: M.G., G.K.R.,Á.Z.L.; Supervision: Á.Z.L.; L.C., Project administration: Á.Z.L.; L.C., Funding acquisition: Á.Z.L., L.C.

Funding

The study was supported by the National Development, Research and Innovation Office, Hungary (K139021). M.G., F.N.A., E.M.A., and D.M.K. are supported by a Stipendium Hungaricum Scholarship from Tempus Public Foundation for PhD studies.

Data availability

All data supporting the findings of this study are available within the paper and its Supplementary Information.

Competing interests

The authors declare no competing interests.

Ethical approval

The experiment was conducted in compliance with the EU Directive 2010/63/EU on the protection of animals used for scientific purposes. Approval was obtained from the Ethical Committee for Animal Use at the University of Debrecen, Hungary (Protocol No. 5/2021/DEMAB). All procedures adhered to institutional guidelines and regulations. The reporting of this study follows the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.