Effects of N-carbamylglutamate on down yield and quality and thyroid transcriptome in breeding Huoyan geese

Abstract

The efficiency of goose down production depends on improvements in both yield and quality, and thyroid function plays a critical role in feather growth. Therefore, exploring nutritional strategies to regulate thyroid function for enhancing down traits is of great importance. N-carbamylglutamate (NCG), an amino acid supplement, has been shown to improve livestock and poultry production performance. However, its regulatory mechanism on down traits and thyroid function in Huoyan geese remains unclear. This study aimed to evaluate the effects of dietary NCG supplementation on down production performance in breeding Huoyan geese and to preliminarily elucidate its mechanism using thyroid transcriptome sequencing. A total of 240 two-day-old female Huoyan geese were randomly assigned to a control group (CON) and an NCG group (N), with six replicates per group and 10 geese per replicate. The experimental period lasted 293 days. At 295 days of age, blood, skin, and thyroid samples were collected. Down yield, cluster length, fiber diameter, thyroid index, and T4 concentration were measured, along with histomorphological and transcriptomic analyses. Results showed that the NCG group had significantly higher total down yield, chest cluster length, and 1000-cluster weight (P < 0.05), while fiber diameter was significantly lower (P < 0.05). The S/P ratio in skin follicles was significantly higher in the NCG group (P < 0.05). Thyroid follicle diameter and colloid resorption vacuole count were significantly increased (P < 0.05), while T4 concentration showed no significant change. Thyroid transcriptomic analysis identified 122 upregulated and 1663 downregulated genes. GO annotation highlighted that upregulated genes including CLIC5, WDR64, LMO1, and GPR158 were associated with cytoskeletal organization, transcriptional regulation, and signal transduction, whereas downregulated genes including COX5B and MAF1 were related to ribosome function, energy metabolism, and translation inhibition. KEGG enrichment analysis revealed significant enrichment in the MAPK signaling pathway, glutathione metabolism, and ribosome pathway. In conclusion, dietary 0.06% NCG improves down yield and quality in breeding Huoyan geese by promoting secondary follicle development, improving down structure, and synergistically activating thyroid function.

Introduction

The Huoyan goose (Anser cygnoides) is a distinguished local breed in China, primarily raised in Liaoning Province and its surrounding regions, and is well-known for its high down production, strong roughage tolerance, and robust adaptability. As one of the main by-products of goose farming, down is widely used in clothing, bedding, and outdoor products due to its light weight, thermal insulation, and softness, offering considerable economic value and market potential. With the growing demand for down products, improving down yield and quality has become an important research focus in poultry production. The growth and development of down are regulated by multiple factors, including genetics, nutrition, environment, and endocrine status, with follicle development and functional status directly determining down yield and quality. Therefore, employing nutritional strategies to promote follicle development and improve down structure is of great significance for enhancing the economic efficiency of the goose down industry.

N-carbamylglutamate (NCG) is an endogenous precursor for arginine synthesis and can be converted into N-acetylglutamate in the body, which in turn activates carbamoyl phosphate synthase I and promotes arginine synthesis. As a functional amino acid, arginine not only serves as an important substrate for protein synthesis but also acts as a precursor for nitric oxide (NO) and polyamine synthesis, participating in various physiological processes such as angiogenesis, cell proliferation, immune regulation, and hormone secretion (Blantz et al., 2000). Previous studies have shown that dietary NCG supplementation promotes capillary formation through its metabolite arginine and NO, thereby supporting hair follicle growth. For instance, Wang et al. reported that NCG upregulated VEGF expression by activating the PI3K/Akt signaling pathway, promoting vascular endothelial cell proliferation and capillary angiogenesis (Ma et al., 2020), which in turn improved hair follicle development and skin vascular density. However, the regulatory effects of NCG on down yield and quality in poultry, as well as the underlying mechanisms, remain largely unexplored.

Down is a specialized keratin structure derived from skin follicles, including primary follicles (PF) and secondary follicles (SF), with the density and development of secondary follicles being key factors determining down cluster yield and fineness. The process of follicle development is regulated by various hormones and signaling pathways, among which thyroid hormones (T3 and T4) play an important role in feather growth and molting. T4 is locally converted into T3 (Van Beek, 2011)and acts synergistically with prolactin (PRL) and IGF (Nozawa et al., 2025) to promote follicular cell proliferation (Hsieh et al., 2025). Thyroid hormones (TH) regulate follicle development by activating the expression of downstream genes such as SHH and BMP through their nuclear receptors (thyroid hormone nuclear receptors TRα1 and TRβ) (Contreras-Jurado et al., 2015). Studies have shown that N-carbamylglutamate (NCG), as an activator of carbamoyl phosphate synthase I, promotes arginine synthesis in animals and subsequently influences the secretion of various hormones (Peng et al., 2025). However, direct evidence regarding whether NCG affects down growth through the regulation of thyroid function is still lacking.

Based on the background described above, combined with the preliminary experimental results of our research group (0.06% NCG showed the best effect in improving the growth and down production performance of Huoyan geese) (Wang et al., 2026), this study used breeding Huoyan geese as the experimental model and supplemented the basal diet with 0.06% NCG to systematically evaluate its effects on down yield (down weight from the chest, abdomen, and back) and down quality (weight per 1000 clusters, down cluster length, and down fiber diameter). Additionally, thyroid histomorphological observation, thyroxine (T4) concentration measurement, and thyroid transcriptome sequencing were performed to explore the potential molecular mechanisms by which NCG regulates down growth. This study aims to provide a theoretical basis for the rational application of NCG in goose down production and to offer new insights into the endocrine mechanisms through which nutritional factors regulate follicle development.

Materials and methods

Experimental animals and design

The experiment was conducted at the Huoyan Goose Breeding Farm in Liaoning Province. A total of 240 two-day-old female Huoyan geese raised under the same conditions were randomly selected and divided into two groups, with six replicates per group and 20 geese per replicate. The geese were fed either a basal diet (CON group) or the basal diet supplemented with 0.06% NCG (N group). The basal diet was formulated based on the NRC Nutrient Requirements of Poultry (National Research Council, 1994), and its composition and nutrient levels are shown in Table 1. The experimental period lasted 293 days. During the brooding period, geese were raised on wire flooring with ad libitum access to feed and water. During the growing period, they were transferred to floor pens and subjected to restricted feeding, with feed provided twice daily (8:00 and 16:00) at a fixed amount (140 to 180 g per goose per day), adjusted according to body weight to control growth rate and prevent excessive fat accumulation. Pure NCG (purity≥99%) was purchased from Jiangxi Yuanchang Industrial Co., Ltd. The animal study protocol was approved by the Animal Care and Use Committee of Jinzhou Medical University (Ethics No. JZMULL2026131).

Table 1.

Composition and nutrient levels of the basal diet (dry matter basis).

Ingredients	Proportion (%)	Nutrient composition	Content (%)
Corn	59.00	Crude protein	15.50
Soybean meal	22.00	Metabolizable energy (cal/kg)	2,731,836
Pea hull	5.00	Calcium	2.56
Soybean oil	1.00	Phosphorus	0.65
Molasses	2.00	Salt	0.35
Limestone	6.00
Breeding goose premix	5.00
Total	100.00

Note: The premix provided per kilogram of diet: VA 15,000.00 IU, VD₃ 2,500.00 IU, VB₁ 1.80 mg, VB₂ 6.50 mg, VB₆ 3.25 mg, VE 20.00 mg, VK₃ 2.00 mg, biotin 75.00 μg, pantothenic acid 8.00 mg, niacin 28.00 mg, folic acid 0.80 mg, copper 4.00 mg, iron 50.00 mg, zinc 50.00 mg, manganese 80.00 mg, selenium 18.00 mg, iodine 1.30 mg. Nutrient levels were calculated values.

Sample collection

At the end of the 293-day experimental period (at which time the geese were 295 days of age, and all samples described below were collected at this same age) , blood was collected from the brachial vein using sterile syringes(Randomly select 6 geese per group). The samples were centrifuged at 3,000 r/min for 15 min, and the supernatant was collected, aliquoted, and stored at −80°C. Subsequently, the experimental geese were euthanized by intravenous injection of 1 cc of Euthasol. After collecting down samples from the chest, abdomen, and back, both thyroid glands were quickly removed and weighed. The left thyroid gland was immediately frozen in liquid nitrogen and stored at −80°C for transcriptome analysis, while the right thyroid gland was fixed in 4% paraformaldehyde for histomorphological analysis.

Determination of down parameters

Down yield from the chest, abdomen, and back was measured according to the NY/T 823-2020 Terms and Measurement Methods for Poultry Production Performance (NY/T 823-2009, 2009). The weight per 1,000 down clusters was recorded, down cluster length was measured using a vernier caliper, and down fiber diameter was examined under a microscope.

Histomorphological analysis

Skin and thyroid samples were fixed, dehydrated, cleared, and embedded in paraffin before sectioning. Routine hematoxylin and eosin (H&E) staining was performed for follicle density calculation and thyroid morphological observation. For follicle density, 10 random fields of view were selected under a 10x objective lens, and the numbers of primary follicles (PF) and secondary follicles (SF) per unit area were counted.

Determination of thyroxine T4 concentration

Plasma T4 concentration was measured using a competitive ELISA kit (Nanjing Jiancheng Bioengineering Institute), and the procedure was carried out according to the manufacturer's instructions.

Thyroid transcriptome sequencing

Total RNA was extracted from thyroid samples using Trizol reagent, and RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The constructed libraries were evaluated and tested on the Agilent Bioanalyzer 2100 system, and then subjected to PE150 paired-end transcriptome sequencing on the Illumina NovaSeq 6000 platform. After quality control of the raw data using fastp software, clean reads were aligned to the reference genome (Anser cygnoides domesticus, ASM263433v1) using HISAT2, and gene expression levels were quantified using FPKM. Differentially expressed genes between groups were screened using DESeq2 with the criteria of |log2 (Fold Change)| > 1 and adjusted P value (padj) <0.05. Subsequently, GO functional annotation and KEGG pathway enrichment analysis were performed on the identified differentially expressed genes to determine their biological functions and metabolic pathways.

qRT-PCR validation

To verify the accuracy of the transcriptome sequencing data obtained by RNA-seq, the expression levels of CLIC5, WDR64, LMO1, RPL15, GSS, and COX5B were examined using real-time quantitative PCR (qRT-PCR). Total RNA was extracted from thyroid tissue samples, and cDNA was synthesized using a total RNA reverse transcription kit (Takara, Dalian, China). Real-time PCR was performed on a QuantStudio™ 5 system using SuperStar Blue Universal SYBR Master Mix (Takara). The optimized cycling conditions were as follows: initial denaturation at 94°C for 5 minutes, followed by 40 cycles of denaturation at 94°C for 15 seconds and annealing/extension at 55°C for 15 seconds. Each sample was analyzed in triplicate to ensure accuracy. The relative expression levels of target genes were calculated using the 2⁻ΔΔCt method, with β-actin used as the internal reference for normalization. Primers used to amplify the target genes were designed based on sequences available in the GenBank database (see Supplementary Table 1).

Statistical analysis

All data in this study are presented as mean ± SD from at least six independent replicate experiments. Data on down production performance and morphological parameters were organized using Excel 2010 and analyzed by independent t-test using SPSS 19.0. Results are presented as mean ± standard deviation, and differences were considered significant at P < 0.05.

Results

Effects of NCG on back down yield and down quality in breeding Huoyan geese

Total down weight, chest down weight, chest weight per 1,000 down clusters, and chest down cluster length were significantly higher in the N group than in the CON group (P < 0.05). Down weight and weight per 1,000 clusters in the back and abdomen were higher in the N group compared with the CON group, but the differences were not significant (P > 0.05) (Fig. 1A, B). Down fiber diameter in the chest, abdomen, and back was significantly lower in the N group than in the CON group (P < 0.05) (Fig. 1C).

Fig. 1.

Effects of NCG on down yield and down quality in breeding Huoyan geese.

Note: A: Down yield and weight per 1,000 clusters in the two groups; B: Down cluster length in the chest, abdomen, and back in the two groups; C: Down fiber diameter in the chest, abdomen, and back in the two groups. The same letters indicate no significant difference (P > 0.05), while different letters indicate significant differences (P < 0.05),n = 6.

Effects of NCG on thyroid weight and organ index in breeding Huoyan geese

The thyroid index was calculated as the percentage of thyroid weight relative to body weight. The results showed that body weight, thyroid weight, and organ index were higher in the N group than in the CON group, but the differences were not significant (P > 0.05) (Fig. 2A-2B). T4 concentration in the N group was also not significantly different from that in the CON group (P > 0.05) (Fig. 2C).

Fig. 2.

Effects of NCG on thyroid weight, organ index, and T4 concentration in breeding Huoyan geese.

Note: A: Body weight and thyroid weight in the two groups; B: Thyroid organ index in the two groups; C: T4 hormone concentration in the two groups, n = 3.

Effects of NCG on thyroid and skin histomorphology in breeding Huoyan geese

Follicle diameter and the number of colloid resorption vacuoles (A functional morphological marker of thyroid follicular epithelial cells endocytosing stored colloid from the follicular lumen for subsequent enzymatic release of thyroid hormones (T3/T4).) in the thyroid were significantly higher in the N group than in the CON group (P < 0.05) (Fig. 3A-3C). Primary follicle density was highest in the CON group and was significantly higher than that in the N group (P < 0.05). Secondary follicle density was highest in the N group and was significantly higher than that in the CON group (P < 0.05). The S/P ratio was highest in the N group and was significantly higher than that in the CON group (P < 0.05) (Fig. 4A-4C).

Fig. 3.

Effects of NCG on thyroid histomorphology in breeding Huoyan geese.

Note: (A) Thyroid follicle diameter; (B) Number of vacuoles; (C) HE staining of sections from the CON and N groups (4 ×, 10 ×). In Figure C: FO: follicle; SF: small follicle; MF: medium follicle; LF: large follicle; white arrows: colloid resorption vacuole; black arrows: colloid (CO). Data are presented as mean ± standard deviation. Different lowercase letters indicate significant differences (P < 0.05), n = 6.

Fig. 4.

Effects of NCG on skin histomorphology in breeding Huoyan geese.

Note: (A) Number of primary follicles, number of secondary follicles, and S/P ratio; (B) HE staining of sections from the CON and N groups (4 ×, 10 ×). In Figure B: APM: arrector pili muscle; FME: feather medullary epithelium; SF: secondary follicle. Data are presented as mean ± standard deviation. Different lowercase letters indicate significant differences (P < 0.05), n = 6.

Effects of NCG on thyroid transcriptomics in breeding Huoyan geese

Transcriptome sequencing quality

Among the six cDNA libraries constructed, the average raw reads were higher than 43,838,417.33. Quality control results showed that the proportion of clean reads in all samples exceeded 98.50%, Q20 and Q30 values were above 96%, and GC content was above 43.75% (Supplementary Table 2).

Principal component analysis results

In the boxplot of the JN and JC groups, the expression levels of replicate samples within each group showed a concentrated trend in the box, with small fluctuations in the interquartile range, while the medians and interquartile ranges between groups were similar, indicating good within-group repeatability and no significant overall expression bias between groups (Fig. 5A). The correlation coefficients within the JN and JC groups were high, and expression differences between groups were observed, indicating high data reliability (Fig. 5B). The two groups of samples were completely separated along the PC1 axis, with replicate samples within each group clustering tightly. The cumulative contribution of PC1 and PC2 accounted for 59.39% of the variance, confirming significant overall differences in gene expression profiles between the two groups (Fig. 5C). A total of 19,272 co-expressed genes were identified between the two groups, with 1,226 genes uniquely expressed in the control group and 604 genes uniquely expressed in the treatment group (Fig. 5D). Differentially expressed genes in the JN group were predominantly downregulated and shown in blue, while corresponding genes in the JC group were mostly upregulated and shown in red. Samples from the two groups each formed separate branches, with highly consistent expression profiles within groups, indicating that the treatment factor caused substantial differences in gene expression patterns (Fig. 5E).

Fig. 5.

Effects of NCG on thyroid transcriptomics.

Note: A: Boxplot showing the overall distribution of samples in each group; B: Clustering heatmap of samples between groups (red: upregulation; blue: downregulation); C: Principal component analysis (PCA) plot of the C group and N group; D: Venn diagram; E: Clustering heatmap of differentially expressed genes between the two groups; F: Volcano plot of differentially expressed genes between the two groups (red: significantly upregulated genes; green: significantly downregulated genes; criteria: 1.5 ≤ |log₂FC| ≤ 2, padj < 0.01),n = 3,The same applies below..

Differentially expressed genes

A total of 1,785 differentially expressed genes were identified, including 122 significantly upregulated genes and 1,663 significantly downregulated genes (Fig. 5F). Using the criteria of 1.5 ≤ |log2 (Fold Change)| ≤ 2 and P < 0.01, selected differentially expressed genes are shown in Table 2.

Table 2.

Selected differentially expressed genes.

Gene	log2FC	pvalue	FDR	regulation
GPR158	1.5736	0.000074383374	0.00262232131	up
CLIC5	1.9621	0.000493921359	0.01154490432	up
LMO1	1.6631	0.001826015634	0.03325219292	up
ST8SIA6	1.7097	0.002555130499	0.04302388986	up
WDR64	1.654	0.008574344952	0.10918212532	up
GSS	−1.8343	0.000000001371	0.000000450003	down
MAF1	−1.577	0.000000290326	0.000031353349	down
COX5B	−1.9078	0.000003589155	0.000223510065	down

GO functional analysis of differentially expressed genes

To characterize the functional features of differentially expressed genes, GO functional annotation was performed. The annotated genes were mainly enriched in basic functional categories, including intracellular organelles and membrane-bound organelles (cellular component), cation binding and nucleic acid binding (molecular function), as well as macromolecule metabolism, cellular nitrogen compound metabolism, and biosynthesis (biological process) (Fig. 6A).

Fig. 6.

Effects of NCG on GO annotation and functions of differentially expressed genes in the thyroid.

Note: A: GO annotation plot showing the top 20 significantly enriched terms and the corresponding number of genes, colored by GO category; B: GO biological process enrichment bubble plot (x-axis: enrichment factor, y-axis: pathway name, bubble size: number of genes, color: padj, with red indicating lower padj and blue indicating higher padj).

To further explore the potential functions of differentially expressed genes, GO enrichment analysis was conducted using a significance threshold of P < 0.05. The results showed that differentially expressed genes were significantly enriched in cellular nitrogen compound biosynthesis (GO:0044271), organic substance metabolism and biosynthesis (GO:0009059), translation and ribosomal function (GO:0006412), as well as signaling receptor binding and regulation of molecular activity (Fig. 6B).

KEGG enrichment results of differentially expressed genes

Based on the GO functional analysis, KEGG pathway annotation and enrichment analysis were performed to further investigate the signaling pathways involved in differentially expressed genes.

KEGG annotation results showed that differentially expressed genes were widely distributed across pathway categories such as "transport and catabolism", "signal transduction", "cell motility", and "cellular community in eukaryotes", with the MAPK signaling pathway, focal adhesion, and cytoskeleton regulation-related pathways containing the largest number of genes (Fig. 7A). KEGG enrichment analysis revealed that differentially expressed genes were significantly enriched in pathways related to cell junction and cytoskeleton regulation (such as tight junction and focal adhesion), immune and infection-related pathways, as well as metabolic pathways including carbohydrate metabolism, amino acid metabolism, and nitrogen metabolism (Fig. 7B).

Fig. 7.

Effects of NCG on KEGG annotation and enrichment of differentially expressed genes in the thyroid.

Note: A: KEGG annotation plot showing the top 20 significantly enriched pathways and the number of genes, colored by pathway category; B: KEGG pathway enrichment bubble plot (x-axis: enrichment factor, y-axis: pathway name, bubble size: number of genes, color: padj, with red indicating lower padj and blue indicating higher padj).

Validation of differentially expressed genes by qRT-PCR

To verify the reliability of the RNA-Seq results, the transcript levels of three significantly upregulated genes (CLIC5, WDR64, LMO1) and three significantly downregulated genes (RPL15, GSS, COX5B) in the NCG group were quantitatively analyzed using qPCR. The data showed that the transcript levels of CLIC5, WDR64, and LMO1 in the thyroid of the NCG treatment group were significantly increased (P < 0.05), while the expression levels of RPL15, GSS, and COX5B were significantly decreased (P < 0.05) (Fig. 8). These results were consistent with the transcriptome analysis data, confirming the reliability and accuracy of the transcriptome data.

Fig. 8.

Validation of differentially expressed gene expression by qPCR.

Discussion

Effects of NCG on down yield and quality

To elucidate the impact of NCG on the yield and quality of down from adult geese, this study employed 295-day-old open-eyed geese as experimental subjects. At this age, geese have completed the full transition from juvenile to adult feathers, with secondary follicles largely developed and down structure stabilized, making it the optimal period for evaluating down characteristics (Ageenko, 2022).

The textile value and market positioning of down are determined by both yield and quality. Poultry feathers are produced by primary follicles, while down feathers are generated by secondary follicles, and the yield of feathers and down is determined by follicle density (Kozak, 2011). A higher S/P ratio (the ratio of secondary follicles to primary follicles) indicates more secondary follicles, resulting in greater down production. In this study, secondary follicle density and the S/P ratio were significantly higher in the NCG group than in the control group, indicating that NCG effectively promoted secondary follicle development. This finding is consistent with previous studies in mammals, in which arginine, as a substrate for NO synthesis, improved follicle microcirculation either directly or indirectly by upregulating vascular endothelial growth factor (VEGF). Additionally, its metabolite NO acts directly on follicle cells by regulating the follicle cycle, inhibiting inflammation and apoptosis, and affecting key signaling pathways such as Wnt/β-catenin, thereby creating an internal environment conducive to cell growth, differentiation, and survival.

Furthermore, the increase in down cluster length and the decrease in down fiber diameter observed in this study further indicate that NCG not only increased down yield but also improved down structure, which may be related to the regulation of keratin synthesis and arrangement within the follicles (Huang et al., 2019). Gianni et al. reported that keratinocytes can produce large amounts of structural keratin, and arginine supplementation can affect their terminal differentiation process (Baratto et al., 2018). The increase in weight per 1,000 clusters and the increase in down cluster length are closely associated with enhanced keratin synthesis (Alibardi, 2025), while the reduction in down fiber diameter can improve the softness and loft of down, thereby enhancing its thermal insulation properties and quality (Shi et al., 2024). Lukas Jenni et al. demonstrated that nutritional factors influence feather keratin structure and fiber diameter (Lukas et al., 2020). As NCG is metabolized to arginine, arginine metabolites such as proline serve as precursors for collagen synthesis (Shi et al., 2024). Prolyl hydroxylase (PHD), which is produced during NCG metabolism, can indirectly affect serum VEGF levels (Li et al., 2014), thereby influencing follicle density and ultimately affecting down fiber diameter.

Effects of NCG on thyroid histomorphology

NCG can produce nitric oxide (NO) through metabolism. As a potent vasodilator, NO increases thyroid blood flow, improves nutrient and oxygen supply, and thereby supports the synthetic and secretory functions of follicular epithelial cells (Robertson et al., 2006). Thyroid hormones (T3 and T4) are key endocrine signals that regulate the feather growth cycle and keratin gene expression (Péczely et al., 2011). In this study, thyroid follicle diameter and the number of colloid resorption vacuoles (1.7 times that of the control group in the NCG group) were significantly increased. An increase in follicle diameter generally reflects an enhanced capacity for thyroid hormone synthesis and storage, while an increase in colloid resorption vacuoles indicates greater activity in the release of thyroid hormones into the bloodstream (Shi et al., 2016). These histological features collectively indicate a highly active state of thyroid hormone synthesis and release, which is consistent with the results reported by Amit Singh Vishen et al. (Singh et al., 2021). However, studies by Ridgway et al. have shown that when blood thyroid hormone levels rise, the body maintains hormonal stability through the negative feedback regulatory mechanism of the hypothalamic-pituitary-thyroid (HPT) axis, keeping circulating T4 concentration relatively constant . Therefore, although NCG induced significant changes in thyroid morphology and functional activity, peripheral blood T4 concentration may remain unchanged. In summary, the enhancement of thyroid function may influence follicle activity through circulatory or local effects, providing endocrine support for down growth.

Potential molecular mechanisms of NCG in regulating thyroid function

In this study, thyroid transcriptome sequencing revealed 1,785 differentially expressed genes between the NCG treatment group and the control group, including 122 upregulated genes and 1,663 downregulated genes. These genes were significantly enriched in pathways related to ribosome function, protein translation, cytoskeletal organization, the MAPK signaling pathway, and various metabolic pathways, all of which are closely associated with follicle development and feather formation. Among the significantly upregulated genes, multiple genes were closely related to cytoskeletal organization, cell morphology maintenance, and signal transduction.

Among the upregulated genes, CLIC5, a member of the chloride intracellular channel protein family, is involved in the connection between the cytoskeleton and the plasma membrane and plays a key role in maintaining the polarity and morphology of basal cells in hair follicles (Salles et al., 2014). As an activator of endogenous arginine synthesis, NCG promotes arginine production, which generates nitric oxide through the action of nitric oxide synthase, subsequently activating soluble guanylate cyclase and promoting the conversion of GTP to cGMP (Wang et al., 2018), thereby enhancing CLIC5 expression. Its upregulation may enhance the structural stability of dermal papilla cells and their interaction with the surrounding matrix, providing support for the proliferation and differentiation of secondary follicles (Berryman and Bretscher, 2000). WDR64 contains WD repeat domains and is involved in the assembly of various protein complexes; it may influence follicle morphology through interactions with other cytoskeletal proteins (Zhang et al., 2024), and its upregulation may promote secondary follicle growth. Together, these two genes synergistically optimize the cytoskeletal structure of follicle cells, resulting in finer and softer down fibers.

LMO1 functions not only as a transcriptional regulator but also as a cytoskeleton-associated protein that directly binds to keratin fibers under mechanical stress, thereby regulating hair follicle stem cells (Kim et al., 2024). NCG activates the MAPK pathway through its metabolites, and activated ERK directly initiates gene transcription by phosphorylating the transcription factor Elk-1, subsequently upregulating LMO1 expression (Gao et al., 2022). Its upregulation may promote secondary follicle development, consistent with the increased S/P ratio observed in skin histological analysis.

The upregulation of angiogenesis-related genes GPR158 and ST8SIA6 provided a foundation for the increase in secondary follicles. GPR158 is a G protein-coupled receptor involved in the regulation of the MAPK signaling pathway, which is one of the core pathways regulating cell proliferation, differentiation, and apoptosis. KEGG enrichment analysis also showed significant enrichment of the MAPK signaling pathway, which is consistent with previous findings regarding the role of the MAPK pathway in mediating hair follicle development (Diao et al., 2023). ST8SIA6 encodes a sialyltransferase involved in the synthesis of cell surface glycoconjugates, and its upregulation may promote angiogenesis and intercellular communication by altering the surface characteristics of cells in the follicle microenvironment (Li et al., 2022).The synergistic action of these two genes improved the nutrient supply to follicles, providing energy and material support for secondary follicle proliferation.

Among the downregulated genes, COX5B (cytochrome c oxidase subunit 5B) is a component of mitochondrial respiratory chain complex IV, and its downregulation may reflect a shift in cellular energy metabolism (Ding et al., 2025). MAF1 is an inhibitor of RNA polymerase III and is involved in translational regulation; its downregulation may relieve the inhibition of ribosome biogenesis, thereby promoting protein synthesis, which is consistent with the downstream enrichment of ribosome-related pathways (Zhang et al., 2024). GSS encodes glutathione synthetase, and its downregulation may reflect an adaptive adjustment of cellular redox status. Nitric oxide can regulate the activity of nuclear factor erythroid 2-related factor 2 (Nrf2), affecting the expression of antioxidant enzyme systems (He and Hewett, 2025); simultaneously, cells may enhance other antioxidant pathways such as the thioredoxin system, thereby downregulating glutathione synthesis to reduce energy consumption.

Proposed pathways of NCG in regulating down growth and study limitations

Based on the above results, this study proposes that the promoting effect of NCG on down growth may be achieved through the following pathways. On one hand, after entering the body, NCG promotes skin capillary formation through the arginine-NO pathway, directly improving the nutrient supply to follicles. On the other hand, NCG may influence the hypothalamic-pituitary-thyroid axis to activate thyroid function, altering the expression of genes related to cytoskeleton, signal transduction (such as the MAPK pathway), and energy metabolism within thyroid tissue, thereby indirectly regulating follicle development and differentiation through the circulatory system. However, this study has certain limitations, such as the lack of measurement of thyroid hormone levels and deiodinase activity in skin tissue, and the inability to directly verify changes in related signaling pathways at the follicle level. Future studies may employ tissue-specific gene knockout or inhibitor treatments in skin tissue to further clarify the direct evidence of NCG regulating follicle development through the thyroid.

Conclusion

Dietary supplementation with 0.06% NCG significantly improved down yield and quality in breeding Huoyan geese. The underlying mechanism may be associated with the promotion of secondary follicle development, activation of thyroid function, and regulation of the expression of genes related to the cytoskeleton, MAPK signaling pathway, and ribosomes in the thyroid. In summary, adding 0.06% NCG to the diet can enhance the overall down performance of Huoyan geese through the synergistic regulation of multiple pathways, thereby increasing down yield and improving down quality. This study provides a theoretical basis and technical support for related research.

Ethics approval and consent to participate

All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of Jinzhou Medical University (approval code: JZMULL2026131).

Competing interest

The authors declare that they have no competing interests.

Funding

This research was funded by the Liaoning Provincial Science and Technology Plan Joint Project Nos. 2025-MSLH-254 and 2024-MSLH-144; Liaoning Provincial Science and Technology Special Commissioner Action Special Plan No. 2025JH5/10400041; Commissioned Special Project for the Construction of the Liaoning Innovation Team of the National Modern Agricultural Industrial Technology System (Broiler Industry), 2025.

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CRediT authorship contribution statement

Meiqi Wang: Writing – review & editing, Writing – original draft, Investigation. Meng Sun: Investigation, Formal analysis. Di Han: Investigation, Formal analysis. Sida Lei: Investigation, Formal analysis. Huacheng Xu: Investigation, Formal analysis. Xiaobing Peng: Investigation, Formal analysis. Chunqiang Wang: Investigation, Formal analysis. Wei Ma: Writing – review & editing, Supervision, Methodology, Conceptualization.