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The low egg production of goose greatly limits the development of the industry. China possesses the most abundant goose breeds resources. In this study, genome resequencing data of swan goose (Anser cygnoides) and domesticated high and low laying goose breeds (Anser cygnoides domestiation) were used to identify key genes related to egg laying ability in geese and verify their functions. Selective sweep analyses revealed 416 genes that were specifically selected during the domestication process from swan geese to high laying geese. Furthermore, SNPs and Indels markers were used in GWAS analyses between high and low laying breed geese. The results showed that RTCB, BPIFC, SYN3, SYNE1, VIP, and ESR1 may be related to the differences in laying ability of geese. Notably, only ESR1 was identified simultaneously by GWAS and selective sweep analysis. The genotype of Indelchr3:54429172, located downstream of ESR1, was confirmed to affect the expression of ESR1 in the ovarian stroma and showed significant correlation with body weight at first egg and laying frequency of geese. CCK-8, EdU, and flow cytometry confirmed that ESR1 can promote the apoptosis of goose pre-hierarchical follicles ganulosa cells (phGCs) and inhibit their proliferation. Combined with transcriptome data, it was found ESR1 involved in the function of goose phGCs may be related to MAPK and TGF-beta signaling pathways. Overall, our study used genomic information from different goose breeds to identify an indel located in the downstream of ESR1 associated with goose laying ability. The main pathways and biological processes of ESR1 involved in the regulation of goose laying ability were identified by cell biology and transcriptomics methods. These results are helpful to further understand the laying ability characteristics of goose and improve the egg production of geese.

Libido plays a crucial role in influencing semen quality, yet the underlying regulatory mechanisms remain unclear. As a central axis in male goose reproduction, the hypothalamic-pituitary-testicular-external genitalia (HPTE) axis may contribute to the regulation of this process. In this study, we established a rating scale for goose libido based on average number of massages to erection (ANM) and the erection type, and evaluated semen quality across the entire flock. Correlation analyses showed that ANM was negatively correlated with sperm concentration (SC), acrosome integrity (AI), and semen quality factor (SQF), while positively correlated with morphological abnormal sperm (MAS) (P < 0.01). A comparison of semen quality and testicular histology between high libido (HG) and low libido (LG) groups showed that SC and SQF were significantly higher and MAS was lower in HG (P < 0.05). The lumen diameter of seminiferous tubules (LD) (P < 0.01) and the number of Sertoli cells (Sc) (P < 0.05) were also significantly greater in HG. Further, the number of spermatogonia (Sg) was significantly (P < 0.01) lower, and spermatocyte (Sp) and elongated spermatid (Se) were significantly higher in HG (P < 0.05). Through transcriptome sequencing (RNA-seq), we identified 98, 163, 2,474 and 400 differentially expressed genes (DEGs) in the hypothalamus, pituitary, testis and external genitalia, respectively. Gene Ontology (GO) analysis indicated that the term ''male gonad development'' was significantly enriched in the hypothalamus. Here, the expression of LHX9 was positively correlated with ANM, and negatively correlated with SC and SQF (P < 0.05). Additionally, WNT4 was positively correlated with ANM and MAS (P < 0.01), and negatively correlated with SC (P < 0.05), suggesting that LHX9 and WNT4 might serve as key upstream regulatory genes. Further analysis through Weighted Gene Co-Expression Network Analysis (WGCNA) showed that the yellow module (R = 0.89, P = 7e-09) was strongly associated with testicular development, with genes predominantly involved in male reproductive process. Based on these findings, we screened genes significantly correlated with LHX9 and WNT4 from the yellow module (|Cor |≥0.6, P < 0.05). These genes were significantly enriched in 8 pathways, primarily associated with metabolic processes, including drug metabolism - other enzymes, metabolism of xenobiotics by cytochrome P450, metabolic pathways, pyrimidine metabolism, glycerolipid metabolism, and riboflavin metabolism. Using the Maximal Clique Centrality (MCC) algorithm in the CytoHubba plug-in, SYCP3, DDX4, STRA8, AMH, MEIOB, CDT1, BCL2, PRIM1, and DLGAP5 were identified as hub genes. In conclusion, within the HPTE axis, libido might influence metabolism-related signaling pathways (mainly involving genes such as SYCP3, DDX4, STRA8, AMH, MEIOB, CDT1, BCL2, PRIM1, and DLGAP5) through LHX9 and WNT4 to regulate the development of the seminiferous tubules and germ cell number, ultimately affecting SC and MAS in geese. These findings offer practical insights into libido rating and shed light on the mechanisms by which libido regulates semen quality, potentially aiding in the improvement of goose breeding capacity.

Avian species exhibit a notable asymmetry in their reproductive systems, with the left ovary maturing into a functional organ and the right ovary degenerating. This asymmetry is widespread across bird species, yet the molecular underpinnings of right ovarian degeneration are not fully understood. In this study, we constructed a cellular atlas of the degenerating right ovary in geese through single-cell transcriptome sequencing (scRNA-seq), revealing distinct cellular compositions and gene expression profiles. Meanwhile, we identified variations in the rate of right ovarian degeneration among individuals, which may influence the gene expression profiles of the hypothalamic-pituitary-ovary (HPO) axis, particularly the related pathways involved in steroid hormone secretion and follicular development of the left ovary. Genomic sequencing combined with transcriptomic data, led to the identification of LHX9 and ARID5B as potentially associated with the rate of right ovarian degeneration in avian species. Our study provides a comprehensive cellular and molecular understanding of the degeneration process in the right ovary of geese. The identification of LHX9 and ARID5B as potential regulators of degeneration rate offers new insights into the genetic factors influencing avian reproductive asymmetry. These findings have implications for improving reproductive efficiency in geese and contribute to the broader understanding of the evolution of avian reproductive systems.

Accompanied by the accelerated growth rate of chickens, the quality of chicken meat has deteriorated in recent years. Wooden breast (WB) is a severe myopathy affecting meat quality, and its pathophysiology depends on gene expression and intercellular interactions of various cell types, which are not yet fully understood. We have performed a comprehensive transcriptomic and metabolomic atlas of chicken WB muscle. Our data showed a significant increase in the number of immune cells, WB muscle displayed a unique cluster of macrophages (cluster 11), distinct from the M1 and M2 macrophages. Regarding the myocytes, the most significant differences were the decrease in cell number and the intensification of fatty deposits. Satellite cells were involved in muscle repair and regeneration producing more collagen. Interestingly, the interaction network in the WB group was weaker compared to that in normal breast muscle. Additionally, we found six key differential metabolites across 22 pathways. When WB occurs, myocytes and endothelial cells undergo apoptosis, macrophages are activated and exert immune functions, satellite cells participate in muscle rebuilding and repair, and the content of metabolites undergoes significant changes. This cell transcriptome profile provides an essential reference for future studies on the development and remodeling of WB.

There is a gradual transition from water to dryland rearing of geese. In this study, we performed 16S rRNA sequencing (16S rRNA-seq) and transcriptome sequencing (RNA-seq) to reveal the effects of cage rearing (CR) and floor rearing (FR) systems on the microbial composition and transcriptome of the goose ileum. Through 16S rRNA-seq, Linear Discriminant Analysis Effect Size (LEfSe) analysis identified 2 (hgcI_clade and Faecalibacterium) and 14 (Bacteroides, Proteiniphilum, Proteiniclasticum, etc.) differential microbiota in CR and FR, respectively. The rearing system influenced 4 pathways including biosynthesis of amino acids in ileal microbiota. Moreover, we identified 1,198 differentially expressed genes (DEGs) in the ileum mucosa, with 957 genes up-regulated in CR and 241 genes up-regulated in FR. In CR, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed the significant enrichment (p

ABSTRACT: Artificial insemination (AI) technology has greatly promoted the development of the chicken industry. Recently, AI technology has also begun to be used in the duck industry, but there are some problems. Numerous researchers have shown that microbes colonizing in semen can degrade semen quality, and AI can increase the harmful microbial load in hen's reproductive tract. Different from the degraded external genitalia of roosters, drakes have well-developed external genitalia, which may cause drake semen to be more susceptible to microbial contamination. However, information on the compositions, sources, and effects of semen microbes on semen quality remains unknown in drakes. In the current study, high-throughput sequencing technology was used to detect microbial communities in drake semen, environmental swabs, cloacal swabs, and the spermaduct after quantifying the semen quality of drakes to investigate the effects of microbes in the environment, cloaca, and spermaduct on semen microbiota and the relationships between semen microbes and semen quality. Taxonomic analysis showed that the microbes in the semen, environment, cloaca, and spermaduct samples were all classified into 4 phyla and 25 genera. Firmicutes and Proteobacteria were the dominant phyla. Phyllobacterium only existed in the environment, while Marinococcus did not exist in the cloaca. Of the 24 genera present in semen: Brachybacterium, Brochothrix, Chryseobacterium, Kocuria, Marinococcus, Micrococcus, Rothia, Salinicoccus, and Staphylococcus originated from the environment; Achromobacter, Aerococcus, Corynebacterium, Desemzia, Enterococcus, Jeotgalicoccus, Pseudomonas, Psychrobacter, and Turicibacter originated from the cloaca; and Agrobacterium, Carnobacterium, Chelativorans, Devosia, Halomonas, and Oceanicaulis originated from the spermaduct. In addition, K-means clustering analysis showed that semen samples could be divided into 2 clusters based on microbial compositions, and compared with cluster 1, the counts of Chelativorans (P < 0.05), Devosia (P < 0.01), Halomonas (P < 0.05), and Oceanicaulis (P < 0.05) were higher in cluster 2, while the sperm viability (P < 0.05), total sperm number (P < 0.01), and semen quality factor (SQF) (P < 0.01) were lower in cluster 2. Furthermore, functional prediction analysis of microbes showed that the activities of starch and sucrose metabolism, phosphotransferase system, ABC transporters, microbial metabolism in diverse environments, and quorum sensing pathways between cluster 1 and cluster 2 were significantly different (P < 0.05). Overall, environmental/cloacal microbes resulted in semen contamination, and microbes from the Chelativorans, Devosia, Halomonas, and Oceanicaulis genera may have negative effects on semen quality in drakes by affecting the activities of starch and sucrose metabolism, phosphotransferase system, ABC transporters, and quorum sensing pathways that are associated with carbohydrate metabolism. These data will provide a basis for developing strategies to prevent microbial contamination of drake semen.

Abstract Background The development of asymmetric chick gonads involves separate developmental programs in the left and right gonads. In contrast to the left ovary developing into a fully functional reproductive organ, the right ovary undergoes gradual degeneration. However, the molecular mechanisms underlying the the degeneration of the right ovary remain incompletely understood. In the present study, we investigated the histomorphological and transcriptomic changes in the right ovary of ducks and geese during the the embryonic stage up to post-hatching day 1. Result Hematoxylin–eosin stainings revealed that the right ovary developed until embryonic day 20 in ducks (DE20) or embryonic day 22 in geese (GE22), after which it started to regress. Further RNA-seq analyses revealed that both the differentially expressed genes (DEGs) in ducks and geese right ovary developmental stage were significantly enriched in cell adhesion-related pathway (ECM-receptor interaction, Focal adhesion pathway) and Cellular senescence pathway. Then during the degeneration stage, the DEGs were primarily enriched in pathways associated with inflammation, including Herpes simplex virus 1 infection, Influenza A, and Toll-like receptor signaling pathway. Moreover, duck-specific DEGs showed enrichment in Steroid hormone biosynthesis, Base excision repair, and the Wnt signaling pathway, while geese-specifically DEGs were found to be enriched in apoptosis and inflammation-related pathways, such as Ferroptosis, Necroptosis, RIG-I-like receptor signaling pathway, and NOD-like receptor signaling pathway. These findings suggest that the degeneration process of the right ovary in ducks occurs at a slower pace compared to that in geese. Additionally, the observation of the left ovary of the geese varying degeneration rates in the right ovary after hatching indicated that the development of the left ovary may be influenced by the degeneration of the right ovary. Conclusion The data presented in this study provide valuable insights into the dynamic changes in histological structure and transcriptome during the degeneration of the right ovary in ducks and geese. In addition, through the analysis of shared characteristics in the degeneration process of the right ovary in both ducks and geese, we have uncovered the patterns of degradation and elucidated the molecular mechanisms involved in the regression of the right ovary in poultry. Furthermore, we have also made initial discoveries regarding the relationship between the degeneration of the right ovary and the development of the left ovary.

ABSTRACT: Age at first egg (AFE) has consistently garnered interest as a crucial reproductive indicator within poultry production. Previous studies have elucidated the involvement of the hypothalamic-pituitary-ovarian (HPO) and hypothalamic-pituitary-thyroid (HPT) axes in regulating poultry sexual maturity. Concurrently, there was evidence suggesting a potential co-regulatory relationship between these 2 axes. However, as of now, no comprehensive exploration of the key pathways and genes responsible for the crosstalk between the HPO and HPT axes in the regulation of AFE has been reported. In this study, we conducted a comparative analysis of morphological differences and performed transcriptomic analysis on the hypothalamus, pituitary, thyroid, and ovarian stroma between normal laying group (NG) and abnormal laying group (AG). Morphological results showed that the thyroid index difference (D-) value (thyroid index D-value=right thyroid index-left thyroid index) was significantly (P < 0.05) lower in the NG than in the AG, while the ovarian index was significantly (P < 0.01) higher in the NG than in the AG. Furthermore, between NG and AG, we identified 99, 415, 167, and 1182 differentially expressed genes (DEGs) in the hypothalamus, pituitary, thyroid, and ovarian stroma, respectively. Gene ontology (GO) analysis highlighted that DEGs from 4 tissues were predominantly enriched in the “biological processes” category. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that 16, 14, 3, and 26 KEGG pathways were significantly enriched (P < 0.05) in the hypothalamus, pituitary, thyroid, and ovarian stroma. The MAPK signaling pathway emerged as the sole enriched pathway across all 4 tissues. Employing an integrated analysis of the protein-protein interaction (PPI) network and correlation analysis, we found GREB1 emerged as a pivotal component within the HPO axis to regulate estrogen-related signaling in the HPT axis, meanwhile, the HPT axis influenced ovarian development by regulating thyroid hormone-related signaling mainly through OPN5. Then, 10 potential candidate genes were identified, namely IGF1, JUN, ERBB4, KDR, PGF, FGFR1, GREB1, OPN5, DIO3, and THRB. These findings establish a foundation for elucidating the physiological and genetic mechanisms by which the HPO and HPT axes co-regulate goose AFE.

ABSTRACT: Presently, excessive fat deposition is the main reason to limit the development of duck industry. In the production, the methods of restricted feeding (RF) were widely used to reduce the lipid deposition of ducks. The liver (L), abdominal adipose (AA), and subcutaneous adipose (SA) were the main tissues of lipid metabolism and deposition of ducks. However, the mechanisms of lipid metabolism and deposition of ducks under RF have not been fully clarified. In this study, in order to better understand the mechanisms of lipid metabolism and deposition in ducks under RF, a total of 120 male Nonghua ducks were randomly divided into a free feeding group (FF, n = 60) and RF group (RF, n = 60), then comparative transcriptomic analysis of L, AA, and SA between FF (n = 3) and RF (n = 3) ducks was performed at 56 d of age. Phenotypically, L, AA, and SA index of FF group was higher than that in RF group. There were 279, 390, and 557 differentially expressed genes (DEGs) in L, AA, and SA. Functional enrichment analysis revealed that ECM-receptor interaction and metabolic pathways were significantly enriched in L, AA, and SA. Lipid metabolism-related pathways including fatty acid metabolism, unsaturated fatty acid synthesis, and steroidogenesis were significantly enriched in AA and SA. Moreover, through integrated analysis weighted gene coexpression network (WGCNA) and protein-protein interaction network, 10 potential candidate genes involved in the ECM-receptor interaction and lipid metabolism pathways were identified, including 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), aldolase B (ALDOB), formimidoyltransferase cyclodeaminase(FTCD), phosphoenolpyruvate carboxykinase 1 (PCK1), tyrosine aminotransferase (TAT), stearoyl-CoA desaturase (SCD), squalene epoxidase (SQLE), phosphodiesterase 4B (PDE4B), choline kinase A (CHKA), and elongation of very-long-chain fatty acids-like 2 (ELOVL2), which could play a key role in lipid metabolism and deposition of ducks under RF. Our study reveals that the liver might regulate the lipid metabolism of abdominal adipose and subcutaneous adipose through ECM-receptor interaction and metabolic pathways (fatty acid metabolism, unsaturated fatty acid synthesis, and steroid synthesis), thus to reduce the lipid deposition of ducks under RF. These results provide novel insights into the avian lipid metabolism and will help better understand the underlying molecular mechanisms.

Abstract As one of the promising next-generation probiotics (NGPs), Akkermansia muciniphila, a well-known mucin-degrading bacterium, has been proven to be closely related to the metabolic diseases of its human host. However, the role of A. muciniphila in the host’s intestinal health remains ambiguous. Here, we comprehensively summarize and discuss the characteristics, the distribution, and the colonization of A. muciniphila in the human gastrointestinal tract (GIT). We propose that the application of A. muciniphila as a biomarker for longevity, for diagnostics and prognostics of intestinal diseases, or for intestinal health should be cautiously considered. Precise dietary regulation can mediate the treatment of intestinal diseases by altering the abundance of A. muciniphila. Although the beneficial role of A. muciniphila and its component in intestinal inflammation has been discovered, in gnotobiotic mice with specific gut microbiota, certain genotype, and colorectal cancer, or in animal models infected with a specific pathogen, A. muciniphila may be related to the occurrence and development of intestinal diseases. Genomic analysis, emphasizing the strain-level phylogenetic differences of A. muciniphila, indicates that a clear description and discussion of each strain is critical before its practical application. Our review provides much needed insight for the precise application of A. muciniphila.

Objective This study was conducted to clone and compare the molecular characteristics of the deiodinase 2 (DIO2) gene between Sichuan White geese and Landes geese, and to analyze the association between polymorphisms of the DIO2 gene and head dimensions in Tianfu meat geese. Methods The coding sequence of the DIO2 gene was cloned by polymerase chain reaction and vector ligation and aligned by DNAMAN software. A total of 350 Tianfu meat geese were used to genotype the polymorphisms of the DIO2 gene and measure the head dimensions. Association analysis between the polymorphisms of the DIO2 gene and head dimensions was carried out. Results An 840-bp coding sequence of the DIO2 gene was obtained and comparison analysis identified four polymorphic loci between Sichuan White geese and Landes geese. Further analysis showed that the dominant alleles for the four polymorphic loci were G, G, A, and T and the frequency of the heterozygous genotype was higher than that of the homozygous genotype in Tianfu meat geese. Compared to that in the population of non-knob geese of Tianfu meat geese, the head dimensions in the population of knob geese were significantly higher except for nostril height. However, in the non-knob geese, beak width 1, beak width 2, nostril length, cranial width 1, and maxillary length had significant differences among different genotypes or haplotypes/diplotypes. Conclusion These results suggested that polymorphisms of the DIO2 gene could be considered molecular markers to select larger heads of geese in the population of non-knob geese.

Abstract Background Egg production is one of the most important economic traits in the poultry industry. The hypothalamic-pituitary–gonadal (HPG) axis plays an essential role in regulating reproductive activities. However, the key genes and regulatory pathways within the HPG axis dominating egg production performance remain largely unknown in ducks. Results In this study, we compared the transcriptomic profiles of the HPG-related tissues between ducks with high egg production (HEP) and low egg production (LEP) to reveal candidate genes and regulatory pathways dominating egg production. We identified 543, 759, 670, and 181 differentially expressed genes (DEGs) in the hypothalamus, pituitary, ovary stroma, and F5 follicle membrane, respectively. Gene Ontology (GO) analysis revealed that DEGs from four HPG axis-related tissues were enriched in the "cellular component" category. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the neuroactive ligand-receptor interaction pathway was significantly enriched based on DEGs commonly identified in all four HPG axis-related tissues. Gene expression profiles and Protein–Protein Interaction (PPI) network were performed to show the regulatory relationships of the DEGs identified. Five DEGs encoding secreted proteins in the hypothalamus and pituitary have interaction with DEGs encoding targeted proteins in the ovary stroma and F5 follicle membrane, implying that they were these DEGs might play similar roles in the regulation of egg production. Conclusions Our results revealed that neuroactive ligand-receptor interaction pathway and five key genes(VEGFC, SPARC, BMP2, THBS1, and ADAMTS15) were identified as the key signaling pathways and candidate genes within the HPG axis responsible for different egg production performance between HEP and LEP. This is the first study comparing the transcriptomic profiles of all HPG axis-related tissues in HEP and LEP using RNA-seq in ducks to the best of our knowledge. These data are helpful to enrich our understanding of the classical HPG axis regulating the egg production performance and identify candidate genes that can be used for genetic selection in ducks.

ABSTRACT: The meat and egg of goose is one of the main components of human food supply. The improvement of goose egg production is particularly important for the increasing human population. However, limited information is available about the effective molecular markers and mechanisms of egg production in goose. In this study, we jointly utilized the data of genome resequencing in different egg production Sichuan white goose and transcriptome at different follicle development stages to identified the molecular markers and mechanisms of egg production. The coefficient of variation of individual egg production in Sichuan white goose population is 0.42 to 0.49. Fifty individuals with the highest (laying 365 days egg number, LEN365 = 79–145) and 50 individuals with the lowest (LEN365 = 8–48) egg production were divided into high and low egg production groups. Based on whole-genome sequencing data of the selected samples, 36 SNPs (annotation novel.12.470, CELF2, ATP1A1, KCNJ6, RAB4A, UST, REV3L, DHX15, CAVN2, SLC5A9, Cldn5, MRPS23, and Tspan2) associated with the LEN365 were identified, involving multiple pathways such as metabolism and endocrinology. Notably, 5 SNPs located in the exon9 of ATP1A1 were identified by GWAS analysis. The association analysis with LEN365 showed the phenotypic variance explained of this haplotype consisting of 5 SNPs is 20.51%. Through transcriptome data analysis, we found the expression of ATP1A1 in the granular layers was increased in the stage of small yellow follicle to large yellow follicle (LYF) and LYF to F5, while decreased in F2 to F1. For the first time, we report the haplotype region formed by 5 SNPS on exon9 of ATP1A1 is associated with egg production in goose and involved in follicle selection and maturation processes.

ABSTRACT: Libido can affect the semen quality of male, and the sperm motility in semen quality parameters is a reliable index to evaluate the fertility of male. In drakes, the sperm motility is gradually acquired in testis, epididymis, and spermaduct. However, the relationship between libido and sperm motility in drakes has not been reported and the mechanisms of testis, epididymis, and spermaduct regulating the sperm motility of drakes are unclear. Therefore, the purpose of the present study was to compare the semen quality of drakes with libido level 4 (LL4) and libido level 5 (LL5), and tried to identify the mechanisms regulating the sperm motility in drakes by performing RNA-seq in testis, epididymis, and spermaduct. Phenotypically, the sperm motility of drakes (P < 0.01), weight of testis (P < 0.05), and organ index of epididymis (P < 0.05) in the LL5 group were significantly better than those in LL4 group. Moreover, compared with the LL4 group, the ductal square of seminiferous tubule (ST) in testis was significantly bigger in the LL5 group (P < 0.05), and the seminiferous epithelial thickness (P < 0.01) of ST in testis and lumenal diameter (P < 0.05) of ductuli conjugentes/dutus epididymidis in epididymis were significantly longer in the LL5 group. In transcriptional regulation, in addition to KEGG pathways related to metabolism and oxidative phosphorylation, lots of KEGG pathways associated with immunity, proliferation, and signaling were also significantly enriched in testis, epididymis, and spermaduct, respectively. Furthermore, through the integrated analysis of coexpression network and protein–protein interaction network, 3 genes (including COL11A1, COL14A1, and C3AR1) involved in protein digestion and absorption pathway and Staphylococcus aureus infection pathway were identified in testis, 2 genes (including BUB1B and ESPL1) involved in cell cycle pathway were identified in epididymis, and 13 genes (including DNAH1, DNAH3, DNAH7, DNAH10, DNAH12, DNAI1, DNAI2, DNALI1, NTF3, ITGA1, TLR2, RELN, and PAK1) involved in Huntington disease pathway and PI3K-Akt signaling pathway were identified in spermaduct. These genes could play crucial roles in the sperm motility of drakes with different libido, and all data the present study obtained will provide new insights into the molecular mechanisms regulating sperm motility of drakes.

Abstract Background All birds reproduce via internal fertilization, but only ~3% of male birds possess the external genitalia that allows for intromission. Waterfowl (e.g., duck and goose) are representatives of them, and the external genitalia development of male geese is directly related to mating ability. Notably, some male geese show abnormal external genitalia development during ontogenesis. However, until now little is known about the molecular mechanisms of the external genitalia development in goose. In the present study, comparative transcriptomic analyses were performed on the hypothalamus, pituitary gland, testis, and external genitalia isolated from the 245-day-old male Tianfu meat geese showing normal (NEGG, n = 3) and abnormal (AEGG, n = 3) external genitals in order to provide a better understanding of the mechanisms controlling the development of the external genitalia in aquatic bird species. Results There were 107, 284, 2192, and 1005 differentially expressed genes (DEGs) identified in the hypothalamus, pituitary gland, testis and external genitalia between NEGG and AEGG. Functional enrichment analysis indicated that the DEGs identified in the hypothalamus were mainly enriched in the ECM-receptor interaction pathway. The ECM-receptor interaction, focal adhesion, and neuroactive ligand-receptor interaction pathways were significantly enriched by the DEGs in the pituitary gland. In the testis, the DEGs were enriched in the neuroactive ligand-receptor interaction, cell cycle, oocyte meiosis, and purine metabolism. In the external genitalia, the DEGs were enriched in the metabolic, neuroactive ligand-receptor interaction, and WNT signaling pathways. Furthermore, through integrated analysis of protein-protein interaction (PPI) network and co-expression network, fifteen genes involved in the neuroactive ligand-receptor interaction and WNT signaling pathways were identified, including KNG1, LPAR2, LPAR3, NPY, PLCB1, AVPR1B, GHSR, GRM3, HTR5A, FSHB, FSHR, WNT11, WNT5A, WIF1, and WNT7B, which could play crucial roles in the development of goose external genitalia. Conclusions This study is the first systematically comparing the hypothalamus, pituitary gland, testis, and external genitalia transcriptomes of male geese exhibiting normal and abnormal external genitals. Both bioinformatic analysis and validation experiments indicated that the neuroactive ligand-receptor interaction pathway could regulate the WNT signaling pathway through PLCB1 to control male goose external genitalia development.

ABSTRACT: Egg weight is an important indicator of egg phenotypic traits, which directly affects the economic benefits of the poultry industry. In the present research, laying ducks were classified into high egg weight (HEW) and light egg weight (LEW) groups. To reveal the underlying mechanism that may be responsible for the egg weight difference, the integrated analysis of transcriptomes and serum metabolomics was performed between the two groups. The results showed extremely significant differences (P < 0.01) in the total egg weight at 300 d, and average egg weight between the HEW and LEW groups. 733, 591, 82, and 74 differentially expressed genes (DEGs) were identified in the liver, magnum, F1, and F5 (hierarchical follicles) follicle membrane, respectively. The candidate genes were screened further from the perspective of forming an egg. In terms of egg yolk formation, the functional analysis revealed fatty acid metabolism-related pathways account for 36% of the liver's top pathways, including fatty acid biosynthesis, folate biosynthesis, fatty acid metabolism, and glycerol lipid metabolism pathways. FASN gene was identified as the key candidate gene by comprehensive analysis of gene expression and protein-protein interaction (PPI) network. In the follicle membrane, the DEGs were mainly enriched in protein processing in the endoplasmic reticulum, and MAPK signaling pathway, and HSPA2, HSPA8, BAG3 genes were identified as crucial candidate genes. In terms of egg white formation, the functional analysis revealed protein metabolism-related pathways account for 40% of the magnum's top pathways, which includes protein processing in the endoplasmic reticulum pathway. HSP90AA1 and HSPA8 genes were identified as key candidate genes. In addition, the integrated transcriptomic and metabolomic analysis showed that arginine and proline metabolism pathways could contribute to differences in egg weight. Thus, we speculated that the potential candidate genes, regulatory pathways, and metabolic biomarkers mentioned above might be responsible for the egg weight difference. These findings might provide a theoretical basis for improving the egg weight of ducks.

ABSTRACT: The importance of thyroid-related genes has been repeatedly mentioned in the transcriptome studies of poultry with different laying performance, yet there are few systematic studies to unravel the regulatory mechanisms of the thyroid-ovary axis in the poultry egg production process. In this study, we compared the transcriptome profiles in the thyroid and ovarian stroma between high egg production (GP) and low egg production (DP) ducks, and then revealed the pathways and candidate genes involved in the process. We identified 1,114 and 733 differentially expressed genes (DEGs) in the thyroid and ovarian stroma, separately. The Gene Ontology (GO) analysis showed that a total of 504 and 189 GO terms were identified in the thyroid and ovarian stroma (P < 0.05). Three common GO terms were identified from the top 5 GO terms with the highest significant level in two tissues, including extracellular space, calcium ion binding, and integral component of plasma membrane. The enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that 15 and 14 KEGG pathways were significantly (P < 0.05) enriched in the thyroid and ovarian stroma, respectively. And, there were 8 common pathways, including neuroactive ligand-receptor interaction, calcium signaling pathway, ECM-receptor interaction, PPAR signaling pathway, melanogenesis, wnt signaling pathway, vascular smooth muscle contraction, and cytokine-cytokine receptor interaction. Notably, the neuroactive ligand-receptor interaction pathway was the most significantly enriched by the DEGs both in the thyroid and ovarian stroma. The interaction among DEGs enriched in the neuroactive ligand-receptor interaction and ECM-receptor interaction suggested that the thyroid may regulate ovarian development by these genes. Through integrated analysis of the protein-protein interaction (PPI) network and KEGG pathway maps, 9 key DEGs (PTH, THBS2, THBS4, CD36, ADIPOQ, ACSL6, PRKAA2, CRH, and PCK1) were identified, which could play crucial roles in the thyroid to regulate ovarian function and then affect egg-laying performance between GP and DP. This study serves as a basis to explore the molecular mechanism of the thyroid affecting ovarian function and egg production in female ducks and may help to identify molecular markers that can be used for duck genetic selection.

Currently, FRS and CRS are the two predominant dryland rearing systems in the goose industry. However, the effects of these two systems on goose growth performance and health, as well as the underlying mechanisms, have not been fully clarified. Thus, this study aimed to compare growth performance and immune status, as well as investigate the genome-wide transcriptomic profiles of spleen in geese, between CRS and FRS at 270 d of age. Phenotypically, the body weight and body size traits were higher in geese under FRS, while the weight and organ index of spleen were higher in geese under CRS (p < 0.05). Noticeably, the bursa of Fabricius of geese under FRS was degenerated, while that under CRS was retained. At the serum level, the immune globulin-G (IgG) and interleukin-6 (IL-6) levels were higher in geese under CRS (p < 0.05). At the transcriptomic level, we identified 251 differentially expressed genes (DEGs) in the spleen between CRS and FRS, which were mainly enriched in scavenger receptor activity, inflammatory response, immune response, neuroactive ligand–receptor interaction, phenylalanine metabolism, ECM receptor interaction, calcium signaling pathway, phenylalanine, tyrosine, and tryptophan biosynthesis, regulation of actin cytoskeleton, and MAPK signaling pathways. Furthermore, through protein–protein interaction (PPI) network analysis, ten candidate genes were identified, namely, VEGFA, FGF2, NGF, GPC1, NKX2-5, FGFR1, FGF1, MEIS1, CD36, and PAH. Further analysis demonstrated that geese in CRS could improve their immune ability through the “phenylalanine metabolism” pathway. Our results revealed that the FRS improved growth performance, whereas the CRS improved goose immune function by increasing levels of IL-6 and IgG in serum. Moreover, the phenylalanine metabolism pathway could exert positive effects on immune function of geese under CRS. These results can provide reliable references for understanding how floor and cage rearing systems affect goose growth performance and immune capacity.

(1) Background: The role of estrogen receptor gene 1 (ESR1) in female reproduction and lipid metabolism has been extensively investigated. However, its contribution to lipid metabolism during the development of poultry follicles remains unclear. (2) Methods: This study aimed to explore the function of ESR1 via overexpressing (ESR1ov) and interfering (ESR1si) with its expression in pre-hierarchical granulosa cells (phGCs) and hierarchical granulosa cells (poGCs). (3) Results: We successfully cloned and obtained an 1866 bp segment of the full-length CDS region of the Sichuan white goose ESR1 gene. In phGCs of the ESR1ov and ESR1si groups, there were no significant changes compared to the control group. However, in poGCs, the ESR1ov group exhibited decreased lipid deposition, triglycerides, and cholesterol compared to the control group, while the ESR1si group showed increased lipid deposition, triglycerides, and cholesterol. The expression of APOB and PPARα was significantly reduced in the ESR1ov group compared to the ESR1ov-NC group. Moreover, significant changes in the expression of ACCα, DGAT1, SCD, CPT1, and ATGL were observed between the ESR1si and ESR1si-NC group. (4) Conclusions: These findings shed light on the function and molecular mechanism of ESR1 in lipid metabolism in goose poGCs, providing a better understanding of the physiological process of goose follicular development.

Abstract Background During domestication, remarkable changes in behavior, morphology, physiology and production performance have taken place in farm animals. As one of the most economically important poultry, goose owns a unique appearance characteristic called knob, which is located at the base of the upper bill. However, neither the histomorphology nor the genetic mechanism of the knob phenotype has been revealed in geese. Results In the present study, integrated radiographic, histological, transcriptomic and genomic analyses revealed the histomorphological characteristics and genetic mechanism of goose knob. The knob skin was developed, and radiographic results demonstrated that the knob bone was obviously protuberant and pneumatized. Histologically, there were major differences in structures in both the knob skin and bone between geese owing knob (namely knob-geese) and those devoid of knob (namely non-knob geese). Through transcriptome analysis, 592 and 952 genes differentially expressed in knob skin and bone, and significantly enriched in PPAR and Calcium pathways in knob skin and bone, respectively, which revealed the molecular mechanisms of histomorphological differences of the knob between knob- and non-knob geese. Furthermore, integrated transcriptomic and genomic analysis contributed to the identification of 17 and 21 candidate genes associated with the knob formation in the skin and bone, respectively. Of them, DIO2 gene could play a pivotal role in determining the knob phenotype in geese. Because a non-synonymous mutation (c.642,923 G > A, P265L) changed DIO2 protein secondary structure in knob geese, and Sanger sequencing further showed that the AA genotype was identified in the population of knob geese, and was prevalent in a crossing population which was artificially selected for 10 generations. Conclusions This study was the first to uncover the knob histomorphological characteristics and genetic mechanism in geese, and DIO2 was identified as the crucial gene associated with the knob phenotype. These data not only expand and enrich our knowledge on the molecular mechanisms underlying the formation of head appendages in both mammalian and avian species, but also have important theoretical and practical significance for goose breeding.

miRNAs are critical for steroidogenesis in granulosa cells (GCs) during ovarian follicular development. We have previously shown that miR-202-5p displays a stage-dependent expression pattern in GCs from goose follicles of different sizes, suggesting that this miRNA could be involved in the regulation of the functions of goose GCs; therefore, in this study, the effects of miR-202-5p on lipid metabolism and steroidogenesis in goose hierarchical follicular GCs (hGCs), as well as its mechanisms of action, were evaluated. Oil Red O staining and analyses of intracellular cholesterol and triglyceride contents showed that the overexpression of miR-202-5p significantly inhibited lipid deposition in hGCs; additionally, miR-202-5p significantly inhibited progesterone secretion in hGCs. A bioinformatics analysis and luciferase reporter assay indicated that Acyl-CoA synthetase long-chain family member 3 (ACSL3), which activates long-chain fatty acids for the synthesis of cellular lipids, is a potential target of miR-202-5p. ACSL3 silencing inhibited lipid deposition and estrogen secretion in hGCs. These data suggest that miR-202-5p exerts inhibitory effects on lipid deposition and steroidogenesis in goose hGCs by targeting the ACSL3 gene.

ABSTRACT: Low fertilization rate is the main reason to limit the development of artificial insemination (AI) technology in ducks. However, the libido of male livestock has been confirmed to be related to semen quality and fertilization rate, and we found that the libido of drakes was different. Thus, the research on the libido of drakes may be the key to further develop and apply AI technology. In this research, we established the first scoring standard for libido evaluation in drakes based on the performance of drakes during training period. Phenotypically, the body weight of high libido group was lighter than that of the other groups, while the weight of testis and epididymis in the high libido group was higher than that in the low libido group. Furthermore, we constructed the first expression profile of hypothalamus, pituitary, testis, and epididymis of drakes with high or low libido. There were 2, 1822, 214, and 892 differentially expressed genes (DEGs) in hypothalamus, pituitary, testis, and epididymis. The expression and sequence of Translocation Associated Membrane Protein 2 (TRAM2) were different in high and low libido drakes, indicating that it may be a candidate gene related to drake's libido. The estrogen, prolactin, and oxytocin signaling pathways were all activated in the pituitary of the low libido group. Meanwhile, the metabolic and oxidative phosphorylation pathways were enriched by DEGs in pituitary, testis and epididymis. Our research reveals that the difference in metabolic may cause changes in body weight of drakes, resulting in altered hormone levels and oxidative phosphorylation of gonad, which negatively affects libido and spermatogenesis in drakes. These results provide novel insights into the avian libido and will help better understand the underlying molecular mechanisms.

Semen volume is an important factor in artificial insemination (AI) of ducks. In drakes, seminal plasma that is produced by the epididymis determines the semen volume. However, the mechanism of epididymis regulating semen volume of drakes remains unclear. Therefore, the aim of the present study was to preliminarily reveal the mechanism regulating the semen volume through comparing the epididymal histomorphology and mRNA expression profiles between drakes with high-volume semen (HVS) and low-volume semen (LVS). Phenotypically, drakes in the HVS group produced more sperm than drakes in the LVS group. In addition, compared with the HVS group, the ductal square of ductuli conjugentes (DC) and dutus epididymidis (DE) in epididymis was significantly smaller in the LVS group, and the lumenal diameter and epithelial thickness of DC/DE were significantly shorter in the LVS group. In transcriptional regulation, 72 different expression genes (DEGs) were identified from the epididymis between HVS and LVS groups. Gene Ontology (GO) analysis indicated that the DEGs were mainly related to hormone secretion, neurotransmitter synthesis/transport, transmembrane signal transduction, transmembrane transporter activity, and nervous system development (p < 0.05). Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis showed that the DEGs were significantly enriched in pathways associated with hormone and neurotransmitter transmission (p < 0.05). In addition, further analysis of the top five pathways enriched by KEGG, nine key candidate genes (including SLC18A2, SNAP25, CACNA1B, GABRG2, DRD3, CAMK2A, NR5A1, and STAR) were identified, which could play a crucial role in the formation of semen. These data provide new insights into the molecular mechanism regulating semen volume of drakes and make feasible the breeding of drakes by semen volume.

ABSTRACT: It is well established that the endocrine system plays a pivotal role in preparing the avian embryos for the abrupt switch from chorioallantoic to pulmonary respiration during the critical embryo-to-hatchling transition. However, as the master gland of the endocrine system, there has been little research focusing on the molecular mechanisms controlling the development and function of the pituitary gland during the peri-hatch period in birds. In the present study, we aimed to determine the genome-wide mRNA and miRNA transcriptome profiles of the pituitary during the embryo-to-hatchling transition period from embryonic day 22 (E22) to post-hatching day 6 (P6) in the goose (Anser cygnoides). Of note, expression of Anser_cygnoides_newGene_32456 and LOC106031011 were significantly different among these 4 stages (i.e., E22, E26, P2, and P6). Meanwhile, the neuroactive ligand-receptor interaction pathway was significantly enriched by the DEGs commonly identified among three pairwise comparisons. At the miRNA transcriptome level, there were not commonly identified DE miRNAs among these 4 stages, while the 418 of their predicted target genes were mutually shared. Both the target genes of DE miRNAs in each comparison and these 418 shared target genes were significantly enriched in the ECM-receptor interaction and focal adhesion pathways. In the predicted miRNA-mRNA interaction networks of these 2 pathways, novel_miRNA_467, novel_miRNA_154, and novel_miRNA_340 were the hub miRNAs. In addition, multiple DE miRNAs also showed predicted target relationships with the DEGs associated with extracellular matrix (ECM) components. Among them, expression of novel_miR_120, tgu-miR-92-3p, and novel_miR_398 was significantly negatively correlated with that of LAMC3 (laminin subunit gamma3), suggesting that these miRNAs may regulate pituitary tissue remodeling and functional changes through targeting LAMC3 during development. These identified DE mRNAs and miRNAs as well as their predicted interaction networks involved in regulation of tissue remodeling and cellular functions were most likely to play critical roles in facilitating the embryo-to-hatchling transition. These results provide novel insights into the early developmental process of avian pituitary gland and will help better understand the underlying molecular mechanisms.

ABSTRACT: As the largest single bone, avian sterna are very different from those of mammals in terms of morphology and functions. Moreover, years of artificial selection in poultry led to incomplete sternal ossification at slaughter age, which may cause diseases, sternal injury, and restriction to breast muscle growth. However, in living birds, studies have rarely described the ossification pattern and underlying mechanisms of the sterna. Here, we examined the pattern (timeline, ossification centers, ossification directions, weekly changes of different parts, quantified differences in ossification degree among sexes and parts) and developmental changes (histological structure, gene expression) of postnatal duck sternal ossification. Direct observation and alcian blue and alizarin red staining of whole sterna samples revealed that, duck sterna mainly ossified during 5 to 9 wk old with five ossification centers. These centers and their ossification directions were different from and more complex than the previously studied birds. The weekly changes of sterna and the quantitative analysis of ossification-related traits showed that ossifications in the three parts of duck sterna (sternum body, keel, posterolateral processes) were mutually independent in space and time, meanwhile, the male duck sterna were more late-maturing than the female. The results of hematoxylin-eosin, alcian blue, and toluidine blue stainings and the expression levels of COL2A1, COL10A1, COL1A2, and CTSK together supported that, duck sternal ossification was highly similar to typical endochondral ossification. Furthermore, continuously high expression of MMP13 and SPARC and their significant (P < 0.05) co-expression with COL2A1, COL10A1, COL1A2, and CTSK suggested the importance of MMP13 and SPARC in duck sternal ossification. Taken together, our results may be helpful for the understanding of avian sternal ossification and the improvement of the performance and welfare of poultry from a new perspective.

The lower egg production of geese (20~60 eggs per year) compared with chicken and duck limits the development of the industry, while the yolk weight and fatty liver susceptibility of geese was higher than that of other poultry. Therefore, the relationship between lipid metabolism and the laying performance of geese remains to be explored. Phenotypically, we observed that the liver fat content of the high-, low-, and no-egg production groups decreased in turn, while the abdominal fat weight increased in turn. For transcriptional regulation, the KEGG pathways related to lipid metabolism were enriched in all pairwise comparisons of abdominal fat and liver through functional analysis. However, some KEGG pathways related to inflammation and the circadian rhythm pathway were enriched by DEGs only in abdominal fat and the liver, respectively. The DEGs in ovarian stroma among different groups enriched some KEGG pathways related to ovarian steroidogenesis and cell adhesion. Our research reveals that lipid metabolism regulated by the circadian rhythm of the liver may directly or indirectly affect ovarian function through the inflammation and hormone secretion of abdominal fat. These results offer new insights into the regulation mechanisms of goose reproductive traits.

To date, research on poultry egg production performance has only been conducted within inter or intra-breed groups, while those combining both inter- and intra-breed groups are lacking. Egg production performance is known to differ markedly between Sichuan white goose (Anser cygnoides) and Landes goose (Anser anser). In order to understand the mechanism of egg production performance in geese, we undertook this study. Here, 18 ovarian stromal samples from both Sichuan white goose and Landes goose at the age of 145 days (3 individuals before egg production initiation for each breed) and 730 days (3 high- and low egg production individuals during non-laying periods for each breed) were collected to reveal the genome-wide expression profiles of ovarian mRNAs and lncRNAs between these two geese breeds at different physiological stages. Briefly, 58, 347, 797, 777, and 881 differentially expressed genes (DEGs) and 56, 24, 154, 105, and 224 differentially expressed long non-coding RNAs (DElncRNAs) were found in LLD vs. HLD (low egg production Landes goose vs. high egg production Landes goose), LSC vs. HSC (low egg production Sichuan White goose vs. high egg production Sichuan white goose), YLD vs. YSC (young Landes goose vs. young Sichuan white goose), HLD vs. HSC (high egg production Landes goose vs. high egg production Sichuan white goose), and LLD vs. LSC (low egg production Landes goose vs. low egg production Sichuan white goose) groups, respectively. Functional enrichment analysis of these DEGs and DElncRNAs suggest that the “neuroactive ligand–receptor interaction pathway” is crucial for egg production, and particularly, members of the 5-hydroxytryptamine receptor (HTR) family affect egg production by regulating ovarian metabolic function. Furthermore, the big differences in the secondary structures among HTR1F and HTR1B, HTR2B, and HTR7 may lead to their different expression patterns in goose ovaries of both inter- and intra-breed groups. These results provide novel insights into the mechanisms regulating poultry egg production performance. [ABSTRACT FROM AUTHOR]