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A deep understanding of the dynamics and mechanisms of male reproductive tract development is necessary for adoption of either genetic techniques or environmental management practices for improving fertility and hatchability in poultry. However, compared with other poultry such as chickens and ducks, less is known about the age- and breed-related changes in the reproductive tract development of domestic goose ganders exhibiting relatively poor reproductive performance as well as the regulatory mechanisms. In the present study, by taking 2 Chinese domestic goose breeds (Sichuan White goose, SW and Gang goose, GE; Anser cygnoides) and one European goose breed (Landes goose, LD; Anser anser) as the experimental objects, we comprehensive analyzed the morphological, histological, and genome-wide transcriptomic variations in their testicular and external genital development during the period from hatching to sexual maturity. Results from histomorphological analysis demonstrated that the reproductive tract of all goose breeds developed in both age- and breed-dependent manners, and the left and right testis developed asymmetrically throughout posthatch development. The tenth week posthatch was a critical developmental stage for all goose ganders, because both the testicular and external genital histomorphological parameters significantly changed before and after this period. During the first 10 wk posthatch, the weight, organ index, or size of male reproductive organs developed more rapidly in SW than in LD, and so were the testicular parenchymal-to-interstitial ratio and the external genital lymphatic lumen diameter. However, the testicular seminiferous epithelium thickness, seminiferous tubule diameter, and Leydig cell number, as well as the external genital keratinized epithelium thickness were significantly higher in LD than in SW at 10 wk of age. Through comparative transcriptomics analysis and RT-qPCR validation, several pathways related to germ and somatic cell function, organ remodeling, and energy metabolism were thought to be responsible for the developmental variations in the early testicular development between Chinese and European domestic ganders, where 10 hub genes involved in the cell cycle, RNA polymerase II-dependent transcription, and mitotic cell division pathways might play essential roles. These data shed new light on the interbreed differences in the male goose reproductive tract development and the molecular mechanisms regulating male goose testicular functions and fertility.

Cathepsin D (CTSD) is known to be crucial for the degradation and utilization of yolk precursors in ovarian follicles. However, little is known about its expression profiles and physiological actions in avian ovarian cells. In this study, the intact coding sequence of the CTSDgene in geese was cloned for the first time, with a length of 1197 bp. It encoded a polypeptide of 398 amino acids (AA) consisting of a signal peptide and two conserved functional domains (i.e., A1_Propeptide and Cathepsin_D2). The AA sequence of goose CTSDhad > 96% similarities with the homologs of turkeys, chickens, and ducks. Results from real-time PCR showed that goose CTSDmRNA was present in all tissues examined, with higher levels in the adrenal gland, liver, heart, and reproductive organs. Furthermore, levels ofCTSDmRNA were much higher in goose granulosa layers than in the theca layers in any follicular category. Significantly, its expression remained almost unchanged in the theca layers throughout follicle development, while it increased gradually in the granulosa layers from 2-4 mm to F5 follicles but declined there after. These results suggested thatCTSDmay regulate goose ovarian follicle development through its actions on both the degradation and absorption of yolk precursors and granulosa cell apoptosis.

In contrast to the later stages of follicle development, little is known about the characteristics and mechanisms associated with early folliculogenesis in avian species. The objectives of the present study were to examine and compare the histomorphological and molecular changes of primordial, primary, and secondary follicles from duck and goose ovaries during the first 6 post-hatching week. Morphological analysis showed that the length and width of both duck and goose ovaries increased steadily during weeks 1 to 5 but increased acutely at week 6, whereas a greater increment was observed in the ovarian length of ducks than that of geese during weeks 4 to 5. Furthermore, smaller diameters of the 3 categories of follicles were observed in ducks than those in geese at the first appearance, but they reached a similar size at week 6. More importantly, secondary follicles were found in the ovaries of ducks 1 wk earlier than in those of geese. These results indicated a more rapid growth rate for ovarian follicles in ducks than in geese during early post-hatching development. At the molecular level, it was found that the mRNAs encoding follicle stimulating hormone receptor (FSHR), anti-Müllerian hormone (AMH), B-cell leukemia/lymphoma 2, and cysteine-dependent aspartate specific protease 3 (CASPASE3) were ubiquitously expressed in all ovarian follicles of ducks and geese with different expression profiles in each follicular category during the first 6 post-hatching week. Notably, transcript levels of FSHR, AMH, and CASPASE3changed differently between ducks and geese during weeks 5 to 6, which was postulated to be one of the mechanisms inducing more rapid growth of ovarian follicles in ducks rather than in geese. In conclusion, our results revealed, for the first time, differences in early folliculogenesis, including the rate of growth of each follicular category and the timing of transition of primary to secondary follicles, between ducks and geese, and these differences could result from different expression profiles of FSHR, AMH, and CASPASE3during early post-hatching development.

There are great differences in physiological and biological functions between animals of different sexes. However, whether there is a consensus between sexes in duck intestinal development and microorganisms is still unknown. The current study used Nonghua ducks to estimate the effect of sex on the intestine by evaluating differences in intestinal growth indexes and microorganisms. The intestines of male and female ducks were sampled at 2, 5, and 10 wk from the duodenum, jejunum, ileum, and cecum. Then, the intestinal length and weight were measured, the morphology was observed with HE staining, and the intestinal content was analyzed by 16S rRNA sequencing. The results showed that male ducks have shorter intestinal lengths with higher relative weights/relative lengths. The values of jejunal villus height (VH)/crypt depth (CD) of female ducks were significantly higher at 2 wk, whereas the jejunal VH/CD was significantly lower at 10 wk. There was obvious separation of microorganisms in each intestinal segment of ducks of different sexes at the 3 time periods. The dominant phyla at different stages were Firmicutea, Proteobacteria, Bacteroidetes, and Actinobacteria. The duodenal Chao index at the genus level of male ducks was significantly higher at 10 wk than that of female ducks. Significantly different genera were found only in the jejunum, and the abundances of Escherichia_Shigella, Pseudomonas, Clostridium_sensu_stricto_1, Sphingomonas,and Desulfovibrioin male ducks were higher than those in female ducks, whereas the abundance of Rothiawas lower, and the abundance of viral infectious diseases, lipid metabolism, metabolism of terpenoids and polyketides, parasitic infectious diseases, xenobiotic biodegradation and metabolism, cardiovascular disease, and metabolism of other amino acids in male ducks were higher than that in female ducks, whereas gene folding, sorting and degradation pathways, and nucleotide metabolism were lower. This study provides a basic reference for the intestinal development and microbial symbiosis of ducks of different sexes.

AbstractLong non-coding RNAs (lncRNAs) have been essentially involved in all kinds of important biological processes, such as cell differentiation and development. In this study, we comprehensively profiled genome-wide lncRNAs of bovine skeletal muscle in two Chinese cattle breeds using RNA-seq approach. The longissimus dorsi samples were collected from three Charolais (CH) and three Tongjiang (TJ) adult cattle, respectively. Approximately 80 million reads were obtained per library, and by which we assembled 31,009 unique transcripts. We successfully identified a total of 4155 different lncRNAs in six muscle samples, consisting of 3204 intronic lncRNAs, 737 intergenic lncRNAs, 113 sense lncRNAs and 101 antisense lncRNAs. Among them, 32 lncRNAs were differentially expressed (Fold Change >1.0, FDR < 0.05) between CH and TJ. Among these lncRNAs, 1744 identified bovine lncRNAs were located adjacent to 1146 protein-coding genes (<100 kb upstream and downstream) and functionally enriched in organ development, gene regulation and signal transduction. Our results provide a catalogue of bovine skeletal muscle-related lncRNAs, which will complement the list of bovine lncRNAs and contribute to understanding of the molecular mechanism of underpinning muscle development in cattle.HighlightsThe first study to compare specialised beef cattle breed with Chinese local beef cattle breed on long non-coding RNA (lncRNA).The 4155 different lncRNAs in beef cattle genome were identified to complement the list of bovine lncRNAs.The 1146 protein-coding genes were found maybe in cis-regulatory relationships with lncRNAs to understand the molecular mechanism underpinning muscle development in cattle.

Although a number of nongenetic factors have been reported to be able to modulate skeletal muscle phenotypes in meat-type birds, neither the underlying mechanisms nor the muscle group–specific phenotypic and molecular responses have been fully understood. In the present study, a total of 240 broiler ducks were used to compare the effects of floor raising system (FRS) and net raising system (NRS) on the physicochemical properties and global gene expression profiles of both breast and thigh muscles at the posthatching week 4 (W4), W8, and W13. Our results showed that compared with FRS, NRS generally induced higher pH, lower lightness (L∗) and yellowness (b∗), lower drip loss and cooking loss, and lower shear force in either breast or thigh muscles during early posthatching stages but subsequently showed less pronounced or even reverse effects. Meanwhile, it was observed that the raising system differently changed the myofiber characteristics depending on the muscle group and the developmental stage. Genome-wide transcriptome analysis showed that compared with FRS, NRS induced the most extensive gene expression changes in breast muscle (BM) at W4 but in thigh muscle (TM) at W13, suggesting the asynchronous molecular responses of BM and TM to the raising system and period. Most of differentially expressed genes in either BM or TM between NRS and FRS were enriched in the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes terms associated with regulation of muscle cellular functions, metabolic and contractile activities, and tissue remodeling, indicating similar molecular mechanisms principally responsible for the raising system-caused phenotypic changes in both muscle groups. Nevertheless, several crucial pathways (e.g., adipocytokine signaling, AGE-RAGE signaling, and apoptosis) and genes (e.g., ANO6, ACER2, UCP3, DTL, and TMEM120A) were tightly related to the muscle group–specific adaptive remodeling on different raising systems. These data could not only contribute to a better understanding of the molecular mechanisms behind meat quality but also provide novel insights into the molecular causes of the muscle group–specific adaptive remodeling in response to environmental stimuli.

Rearing system is a critical nongenetic factor influencing meat quality of ducks. In this study, a total of 360 birds were randomly allocated into floor rearing system (FRS) and net rearing system (NRS) to compare their effects on intramuscular fat (IMF) deposition, fatty acid composition, and related gene expression in muscles of Nonghua ducks. Sawdust bedding and stainless mesh bed were equipped in FRS and NRS, respectively. At the eighth week (8w) and 13th week (13w), the breast and thigh muscles of ducks were collected to determine the profiles of lipids composition and the expressions of lipid metabolism–related genes. The IMF content was higher in 13w-FRS than 8w-FRS and 8w-NRS in breast muscle, whereas it was higher in 13w-NRS than other groups in thigh muscle (P< 0.05). C16:1, C20:5(n-3) of muscles were higher in 8w-NRS than 8w-FRS, whereas C18:1(n-9)c, C18:2(n-6)c, Ʃ monounsaturated fatty acid (MUFA), and ƩMUFA/Ʃsaturated fatty acid (SFA) ratio of muscles were higher in 13w-NRS than 8w-FRS and 8w-NRS (P< 0.05). C22:6(n-3), C20:4(n-6) of breast muscle and C20:3(n-6) of thigh muscle were higher in 13w-NRS than 13w-FRS (P < 0.05). Fatty acids variation was studied by principal component analysis, exhibiting extensive positive loadings on principal components. SREBP1, ACADL, and FABP3were downregulated in breast muscle, whereas PPARαand ELOVL5were upregulated in thigh muscle of NRS ducks at 13w. Principal components were extensively correlated with lipids composition parameters, and principal components of breast muscle 1 and principal components of thigh muscle 1 were correlated with SREBP1and PPARα, respectively (P< 0.05). In conclusion, with increasing age, FRS enhanced IMF deposition in breast muscle, and the same promotion in thigh muscle was because of NRS. The variation of fatty acids in muscles was uniform, and the change of single fatty acid was unable to distinguish NRS and FRS. However, as NRS downregulated SREBP1, ACADLand FABP3in breast muscle and upregulated PPARα and ELOVL5in thigh muscle, NRS could improve nutrient value and meat quality by increasing ƩMUFA, ƩMUFA/ƩSFA ratio, and important PUFA levels. Therefore, NRS was more recommended than FRS for Nonghua ducks during week 8 to 13 posthatching.

Geese, descendants of migratory birds, have preserved the distinct reproductive and lipid metabolism traits of their wild ancestors. Therefore, compared to other poultry, geese have lower egg production ability and a more sensitive susceptibility to fatty liver. Recent research underscores the impact of lipid metabolism disorders on female reproductive health. In this context, we observed reproductive disorders (RD) and lipid metabolism anomalies in certain geese populations. This study systematically elucidated the differences between RD and normal geese at various levels, including genomics, transcriptomics, bile acid metabolomics, and microbiomics, revealing the crucial role of microorganisms. Our study provides a thorough examination of the ovarian anatomical, histological, and transcriptomic profiles between normal and RD geese. Genomic analyses pinpoint mutations in genes associated with bile acid metabolism, highlighting their potential role in RD pathogenesis. The genomic discoveries are substantiated by precise bile acid assays and ileum transcriptome analyses, which expose a significant disruption in bile acid absorption, activation of FXR, and an increase in serum chenodeoxycholic acid (CDCA) concentrations within RD geese. Notably, 16S rRNA sequencing uncovers significantly greater beta diversity in the ileum microbiota of RD geese as compared to the normal group. Both Wilcoxon rank sum test and LEfSe analyses highlighted a marked increase in Romboutsiaabundance in RD geese. Experimental cultivation of microbiota with CDCA supplementation confirms the impact of CDCA on Romboutsia lituseburensis(R. lituseburensis) proliferation. Gavage experiments with R. lituseburensiselucidates its involvement in primary follicle reduction via immune-mediated pathways. Collectively, our multi-faceted analysis unravels the intricate involvement of Romboutsiain goose RD, offering insights from genetic, physiological, and microbial dimensions. Our findings not only deepen our understanding of the etiology of RD in geese but also suggest potential avenues for therapeutic interventions targeting bile acid metabolism and the modulation of specific microbiota components.

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 GREB1emerged 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.

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. Firmicutesand Proteobacteriawere the dominant phyla. Phyllobacteriumonly existed in the environment, while Marinococcusdid not exist in the cloaca. Of the 24 genera present in semen: Brachybacterium, Brochothrix, Chryseobacterium, Kocuria, Marinococcus, Micrococcus, Rothia, Salinicoccus, and Staphylococcusoriginated from the environment; Achromobacter, Aerococcus, Corynebacterium, Desemzia, Enterococcus, Jeotgalicoccus, Pseudomonas, Psychrobacter, and Turicibacteroriginated from the cloaca; and Agrobacterium, Carnobacterium, Chelativorans, Devosia, Halomonas, and Oceanicaulisoriginated 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 Oceanicaulisgenera 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.

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.

Skeletal characteristics are important to the growth and development of poultry. In feeding management, constant free feeding (FF) of poultry may lead to imbalance between bone development and weight gain. Feed restriction (FR), to a certain extent, is one way to solve this problem. However, the effect of feed restriction on poultry bone development needs further elucidation at the molecular level. Therefore, in the present study, we investigated the effects of different levels of feed restriction (60% FR, 70% FR, 80% FR, and FF) on the sternum development of ducks at 7 and 8 wk old. In the seventh wk, with increasing feed restriction, the values of traits including body weight, breast muscle weight, sternal weight, keel length, and calcified keel length decreased. However, in the eighth wk, the sternum weight and keel length of ducks treated with 60% FR were unexpectedly higher than those of FF individuals, indicative of catch-up growth. Then, we conducted RNA-seq and metabolomic analysis on sterna from 7- and 8-wk-old FF and 60% FR ducks. The results identified multiple differentially expressed genes (DEGs) associated with sternum development that were influenced by feed restriction. Among them, we found that the mRNA expression levels of the chondroitin sulfate synthase 3(CHSY3) and annexin A2(ANXA2) which are involved in glycosaminoglycan biosynthesis and bone mineralization, had smaller changes over time under FR treatment than under FF treatment, implying that the FR treatment to a certain extent prevented the premature calcification and prolonged the development time of duck sternum. In addition, the metabolomic and integrative analyses revealed that several antiaging-related metabolites and genes were associated with sternal catch-up growth. Pyrimidine metabolism was identified as the most significant pathway in which most differential metabolites (DMs) between FF and 60% FR were enriched. The results from integrative analysis revealed that the content and expression of 4-aminobutyric acid (GABA) and its related genes showed relatively higher activity in the 60% FR group than in the FF group. The present study identifies multiple biomarkers associated with duck sternum development that are influenced by feed restriction and suggests the potential mechanism of feed restriction-associated duck sternal catch-up growth.

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 aureusinfection pathway were identified in testis, 2 genes (including BUB1Band 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.

Although miR-27b-3p has been evidenced to regulate the proliferation, apoptosis, and differentiation of a variety of mammalian cell types, its actions and mechanisms on ovarian cell steroidogenesis remains largely unknown in both mammalian and avian species. In this study, we aimed to determine the expression profiles of miR-27b-3p in granulosa cell layers during goose ovarian follicle development and to reveal its actions on estrogen (E2) secretion of goose granulosa cells as well as the underlying regulatory mechanisms. It was observed that miR-27b-3p was ubiquitously expressed throughout follicle development but exhibited much higher levels in hierarchical- than in prehierarchical follicles. In cultured granulosa cells from the fourth through second largest preovulatory (F4-F2) follicles of goose, up- and downregulation of miR-27b-3p by using its mimic and inhibitor significantly decreased and increased E2secretion, respectively. Meanwhile, the mRNA levels of STARand CYP19A1were significantly reduced while those of CYP11A1and 3βHSDwere elevated in the mimic-transfected granulosa cells. By comparison, downregulation of miR-27b-3p enhanced the mRNA levels of STARbut had no significant effects on those of CYP19A1, CYP11A1, and 3βHSD. Results from bioinformatic prediction and luciferase reporter assay demonstrated that CYP1B1was a downstream target of miR-27b-3p. Although the siRNA-mediated downregulation of CYP1B1did not significantly change E2secretion by goose granulosa cells, it reduced the mRNA levels of STARand CYP19A1as well as those of LKB1and AMPKα, which are involved in the AMPK signaling pathway. Taken together, these data suggest that miR-27b-3p plays an inhibitory role in E2secretion by goose F4-F2 granulosa cells, at least in part, by targeting CYP1B1through the AMPK signaling pathway.

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.

Evidences have found important effects of breeds/strains on the content of amino acids (AAs) which is an important substrate for protein synthesis and contributes greatly to meat quality. Therefore, the objective of the present study was to compare the AAs content and protein synthesis-related genes expression levels in breast muscle of native breed (Jianchang duck (J)), hybrid strains (BH1, BH2, and MC♂ × (BGF2♂ × GF2♀)♀ (MC)), and commercial breed (Cherry Verry duck). Results showed that a total of 17 AAs (TAAs) was detected from breast muscle among 5 duck breeds/strains including 11 essential AAs (EAAs). Among these AAs, the contents of Proline, Threonine, Glutamine, Serine, Methionine, Phenylalanine, Histidine, and Cysteine were significant difference among 5 duck breeds/strains. The contents of EAAs, TAAs, and flavor AAs were higher in breast muscle of J and BH2 than those in other duck breeds/strains, and the ratio of EAAs/TAAs was higher in breast muscle of BH2. Furthermore, the expression levels of eukaryotic translation initiation factor 4E-binding protein 1, mammalian target of rapamycin, and proton-coupled amino acid transporter 1 were the highest in breast muscle of BH2, and that of solute carrier family 38 member 2 was the highest in breast muscle of J. Meanwhile, principal component analysis results showed that principal component 1 of BH1, principal component 3 of BH2, and principal component 2 of MC were positively corelated with EAAs/TAAs, and principal component 1 was positively correlated with flavor AAs and EAAs. In conclusion, compared to BH1, MC, and Cherry Verry duck, AA content was higher in breast muscle of BH2 and J, which might be associated with the higher expression levels of mammalian target of rapamycin, eukaryotic translation initiation factor 4E-binding protein 1, and proton-coupled amino acid transporter 1 in breast muscle of BH2 and solute carrier family 38 member 2 in breast muscle of J. The comparative analysis of AA content in breast muscle among different duck breeds/strains could provide an important basis for improving the nutritional value of duck meat in the breeding process.

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 0.42-0.49. Fifty individuals with the highest (laying 365 days egg number, LEN365=79-145) and fifty 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, five SNPs located in the exon9 of ATP1A1were identified by GWAS analysis. The association analysis with LEN365 showed the phenotypic variance explained of this haplotype consisting of five SNPs is 20.51%. Through transcriptome data analysis, we found the expression of ATP1A1in 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 five SNPS on exon9 of ATP1A1is associated with egg production in goose and involved in follicle selection and maturation processes.

Semen quality is the most important indicator in evaluating drake fecundity. At present, the low semen quality has become a major factor restricting the development of artificial insemination (AI) technology in ducks. Numerous studies have indicated that seminal plasma proteins play a crucial role in semen quality, but the mechanism of seminal plasma proteins regulating semen quality of drakes remains unclear. Thus, the objective of this study was to identify seminal plasma proteins associated with semen quality by comparing the seminal plasma proteomic profile of drakes with high-quality semen (HQS) and low-quality semen (LQS). Using a label-free MS-based method, a total of 745 seminal plasma proteins were identified. Of these, 55 differentially expressed proteins (DEPs) were identified (40 up-regulated and 15 down-regulated). Gene Ontology (GO) analysis showed that the DEPs were mainly enriched in transmembrane transport, extracellular matrix structural constituent, transferase activity, transferring acyl groups other than amino-acyl groups, transmembrane transporter activity, and integral component of membrane (P< 0.05). Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis indicated that the DEPs were significantly enriched in apoptosis, tyrosine metabolism, glycerophospholipid metabolism, and sulfur metabolism pathways (P< 0.05). Moreover, through protein-protein interaction (PPI) network analysis, eight potential candidate proteins were identified, including P19140 (Alpha-enolase), R0KUV7 (Calreticulin), R0K3X3 (Solute carrier family 2, facilitated glucose transporter member 5), R0L6V0 (Proteasome subunit beta), R0JKW0 (Cytochrome c), R0JMC5 (Tubulin alpha chain), R0LCK1 (Cathepsin C), and R0JUP6 (Cathepsin D), which could play crucial roles in semen quality. Notably, further analysis demonstrated that key protein P19140 (Alpha-enolase) might can control the semen quality of drakes by regulating the expression of proteins related to apoptosis pathway. This study is the first systematically comparing the seminal plasma proteome of drakes exhibiting high and low semen quality. These results provide novel insights into the mechanisms regulating semen quality of drakes.

Fatty acid composition contributes greatly to the nutritional value of meat, and breeds/strains are important factors affecting the composition of fatty acid. Recently, few studies have focused on the fatty acid composition in breast muscle of different duck breeds. Therefore, the objective of the present study was to compare the fatty acid composition and lipid metabolism-related genes expression in breast muscle of Jianchang duck (J), Cherry Verry duck (CV) and 3 crossbred strains (BH1, BH2 and MC♂ × (BGF2♂  × GF2♀)♀ (MBG)). Our results showed that the breast muscle of J had the highest contents of C22:1(n−9) but the lowest ratios of Ʃ-omega 6 (Ʃn−6)/Ʃ-omega 3 (Ʃn−3), Ʃ-mono-unsaturated fatty acid (ƩMUFA)/Ʃ-saturated fatty acid (ƩSFA) and Ʃ-polyunsaturated fatty acid (ƩPUFA)/ƩSFA. The ƩPUFA/ƩSFA ratio was higher in breast muscle of MBG than in that of BH2 and CV, and the contents of C22:1(n-9), ƩMUFA and ƩPUFA were higher in BH1 than in BH2 and CV. Furthermore, the mRNA levels of SCD1, FADS2, ELOVL2,and ELOVL5were significantly higher in MBG (P< 0.05), while those of FASD1and ACACAwere significantly higher in BH1 than in BH2 and CV (P< 0.05). Principal component analysis showed that fatty acids variation exhibited extensive positive loading on principal components (PCs). Correlation analysis showed that PC1 and PC3 of BH1, as well as PC1 of MBG were correlated with the mRNA levels of ACACAand FABP3, respectively. Thus, it could be concluded that the breast muscles of MBG and BH1 have better fatty acid composition, which was closely related to the increased expression levels of SCD1, FADS2, ELOVL2,and ELOVL5genes in MBG but FADS1and ACACAin BH1. Moreover, these results also showed that crossbreeding could optimize the composition of fatty acid in breast muscle of ducks.

Early research in our lab indicated that the effect of glucose, fructose and sucrose on the levels of triacylglycerol, and inflammatory factor was significantly different, and it is speculated that the regulatory mechanism of lipid deposition by different type of sugar in the liver is different. In order to explore lipid deposition difference mediated by different types of sugar (glucose, fructose, and sucrose) in goose fatty liver formation, this experiment was performed from cell culture, overfeeding experiment, and transcriptome analysis at 3 levels. Cell culture experiment results indicated that the levels of intracellular triglyceride, total cholesterol, and lipid content of fructose and sucrose treatment were significantly higher than those of glucose treatment (P< 0.05). In slaughter performance, the liver weight, the ratio of liver weight to body weight, feed conversion ratio (liver weight/feed consumption) were better in sucrose overfeeding group (P< 0.05). In addition, the liver of the sucrose overfeeding group contained a lot of unsaturated fatty acids, especially (n-3) polyunsaturated fatty acids (P< 0.05). Transcriptome analysis shown that the peroxisome proliferators-activated receptor (PPAR) signaling pathway is highly enriched in the fructose and sucrose overfeeding groups; cell cycle, and DNA replication pathways were highly enriched in the glucose overfeeding group. In conclusion, due to the decrease of lipids outward transportation and the anti-inflammation of unsaturated fatty acids, fructose, and sucrose have better ability to induce steatosis in goose fatty liver formation.

Due to the demand for modern goose production and the pressure of environmental protection, the rearing systems of geese are changing from traditional waterside rearing to intensive rearing systems such as floor rearing (FR) and cage rearing (CR) systems. However, little is known about the effects of different rearing systems on goose intestinal functions and cecal microbial composition. Therefore, this study aimed to compare intestinal histomorphology and cecal microbial composition differences in geese reared under CR and FR at 270 d of age. Histomorphological analysis showed that the ileal villus height (VH) to crypt depth (CD) ratio was significantly greater in CR than in FR (P< 0.001). Taxonomic analysis showed that the dominant bacteria of cecal microorganisms in both rearing systems were roughly similar, with Bacteroidota, Firmicutes, Fusobacteriota, and Proteobacteria being the dominant phyla while Bacteroides, Fusobacterium, and uncultured_bacterium_o_Bacteroidales being the dominant genera. Differentially abundant taxa between CR and FR were also identified using Linear Discriminant Analysis Effect Size (LEfSe) analysis (P< 0.05, LDA score > 3.5). Megamonasand Anaerobiospirillumwere significantly enriched in the CR group at the genus level, while uncultured_bacterium_f_Rikenellaceae and Sutterellawere significantly enriched in the FR group. Notably, we found that the relative abundance of uncultured_bacterium_f_Rikenellaceae was significantly negatively correlated with the ileal VH and VH/CD (P< 0.05). The relative abundance of Megamonasand Anaerobiospirillumwere significantly negatively correlated with abdominal fat weight and relative abdominal fat weight (P< 0.01), whereas that of Sutterellawas significantly positively correlated with abdominal fat weight and relative abdominal fat weight (P< 0.01). Furthermore, PICRUSt2 analysis indicated that the lipid metabolism pathways of cecal microorganisms were lower enriched in CR than in FR. In conclusion, compared with FR, the CR significantly changed goose ileal histomorphological characteristics and cecal microbial composition, thereby affecting goose physiological functions and production performance.

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_32456and LOC106031011were 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 LAMC3during 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.

Ornithine decarboxylase (ODC) plays an indispensable role in the process of polyamine biosynthesis. Polyamines are a pivotal part of living cells and have diverse roles in the regulation of cell proliferation and apoptosis, aging and reproduction. However, to date, there have been no reports about ODC regulating follicular development in goose ovaries. Here, we constructed ODCsiRNA and overexpression plasmids and transfected them into goose primary granulosa cells (GCs) to elucidate the effects of ODCinterference and overexpression on the polyamine metabolism, hormone levels, cell apoptosis and proliferation of granulosa cells. After interfering with ODCin GCs, the mRNA and protein levels of ODCand the content of putrescine were greatly decreased (P< 0.05). When ODCwas overexpressed, ODCmRNA and protein levels and putrescine content were greatly increased (P< 0.05). The polyamine-metabolizing enzyme genes ornithine decarboxylase antizyme 1 (OAZ1) and spermidine / spermine-N1-acetyltransferase (SSAT) were significantly increased, and spermidine synthase (SPDS) was significantly decreased when ODCwas downregulated (P< 0.05). OAZ1, SPDSand SSATwere significantly increased when ODCwas upregulated (P< 0.05). In addition, after interference with ODC, progesterone (P4) levels in the culture medium of GCs increased greatly (P< 0.05), while the overexpression of ODCcaused the P4 level to decrease significantly (P< 0.05). After ODCdownregulation, granulosa cell activity was significantly reduced, the apoptosis rate was significantly increased, and the BCL-2 / BAX ratio was downregulated (P< 0.05). Under ODCoverexpression, the activity of GCs was notably increased, the apoptosis rate was significantly reduced, and the BCL-2 / BAX protein ratio was upregulated (P< 0.05). Our study successfully induced ODC interference and overexpression in goose ovarian GCs, and ODC regulated mainly putrescine content in GCs with a slight influence on spermidine and spermine. Moreover, ODC participated in the adjustment of P4 levels in the culture medium of GCs, promoted granulosa cell proliferation and inhibited granulosa cell apoptosis.

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 CTSKtogether supported that, duck sternal ossification was highly similar to typical endochondral ossification. Furthermore, continuously high expression of MMP13and SPARCand their significant (P< 0.05) co-expression with COL2A1, COL10A1, COL1A2,and CTSKsuggested the importance of MMP13and SPARCin 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.

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 significantly 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.