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ABSTRACT: The size of the initial primordial follicle pool in the ovary depends on primordial follicle formation, which determines the female reproductive lifespan. However, the molecular regulation of primordial follicle formation in chickens remains unclear. In this study, the left ovaries of chickens were collected at 2 d posthatch (dph), 5.5 dph, and 10.5 dph to examine the formation of primordial follicles. Single-cell mRNA sequencing (scRNA-seq) and spatial transcriptomic analysis were performed to explore the ovarian microenvironment and identify regulatory pathways involved in the formation of primordial follicles in chickens. Histomorphological analysis of chicken ovary tissues revealed the presence of germ cell cysts at 1 dph, which began to disintegrate at 2 dph. Primordial follicles appeared at 5.5 dph and continued to develop into larger-diameter follicles. scRNA-seq and spatial transcriptomic analysis revealed 24 cellular clusters involved in chicken primordial follicle formation. The metabolic pathway of steroid hormone synthesis was found in pregranulosa and pretheca cells. Histological analysis showed that chicken ovaries did not form primordial follicles after the inhibition of the steroid hormone synthesis pathway by simvastatin or tamoxifen. In addition, mRNA transcriptomic and bioinformatics analyses revealed that GREB1 was a downstream gene of the steroid hormone synthesis pathway during the formation of chicken primordial follicles. This study provides a valuable foundation for investigating primordial follicle formation in avian species and optimizing their reproductive performance.

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.

(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 Despite their important functions and nearly ubiquitous presence in cells, an understanding of the biology of intracellular lipid droplets (LDs) in goose follicle development remains limited. An integrated study of lipidomic and transcriptomic analyses was performed in a cellular model of stearoyl-CoA desaturase (SCD) function, to determine the effects of intracellular LDs on follicle development in geese. Results Numerous internalized LDs, which were generally spherical in shape, were dispersed throughout the cytoplasm of granulosa cells (GCs), as determined using confocal microscopy analysis, with altered SCD expression affecting LD content. GC lipidomic profiling showed that the majority of the differentially abundant lipid classes were glycerophospholipids, including PA, PC, PE, PG, PI, and PS, and glycerolipids, including DG and TG, which enriched glycerophospholipid, sphingolipid, and glycerolipid metabolisms. Furthermore, transcriptomics identified differentially expressed genes (DEGs), some of which were assigned to lipid-related Gene Ontology slim terms. More DEGs were assigned in the SCD-knockdown group than in the SCD-overexpression group. Integration of the significant differentially expressed genes and lipids based on pathway enrichment analysis identified potentially targetable pathways related to glycerolipid/glycerophospholipid metabolism. Conclusions This study demonstrated the importance of lipids in understanding follicle development, thus providing a potential foundation to decipher the underlying mechanisms of lipid-mediated follicle development.

Abstract Background In animals, many factors affect the small intestinal function and cecal microorganisms, including body weight and genetic background. However, whether paternal weight impacts the small intestinal function and cecal microorganisms remains unknown to date. The current study used Nonghua sheldrake to estimate the effect of paternal weight on the intestine of the offspring by evaluating differences in small intestinal morphology, digestive enzyme activity, and cecal microorganisms between the offspring of male parents with high body weight (group H) and that of male parents with low body weight (group L). Results The results of the analysis of small intestinal morphology showed that the villus height of the jejunum of group H ducks was higher than that of group L ducks, and the difference was significant for ducks at 10 weeks of age. Moreover, the villus height/crypt depth of the duodenum in group H significantly exceeded that of group L at a duck age of 2 weeks. The amylase activity in the jejunum content of group H exceeded that of group L at 5 and 10 weeks of age. Furthermore, the proportion of the Firmicutes to Bacteroidetes was significantly higher in group H (duck age of 2 weeks). Among the genera with a relative abundance exceeding 1%, the relative abundances of genera Desulfovibrio, Megamonas, Alistipes, Faecalibacterium, and Streptococcus observed in group H were significantly different between group H and group L. Conclusions For the first time, this study identifies the effect of paternal weight on offspring small intestinal function and cecal microorganisms. Consequently, this lays a foundation for further research on the relationship between male parents and offspring intestinal function.

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: 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.

Landes geese and Sichuan White geese are two important genetic materials for commercial goose breeding. However, the differences in the male reproductive capacity between these two breeds and the potential molecular mechanisms and associated key genes have not been reported to date. The present study compared the testicular histology and mRNA–long non-coding RNA (lncRNA) expression patterns to reveal the differences in male reproductive performance between Sichuan White geese and Landes geese, as well as to explore the underlying molecular mechanisms. Histological results showed that the testicular organ index, semen volume, and long diameter of seminiferous tubules of Landes geese were significantly larger than those of Sichuan White geese. Analyses of mRNA-lncRNA expression profile showed that compared with Sichuan White geese, a total of 462 differentially expressed mRNAs (DEGs) (173 up-regulated and 289 down-regulated) and 329 differentially expressed lncRNAs (DE lncRNAs) (280 up-regulated, 49 down-regulated) were identified in Landes geese. Among these DEGs, there were 10 spermatogenesis-related and highly expressed (FPKM > 10) DEGs. Except for SEPP1, all of these DEGs were significantly up-regulated in the testes of Landes geese. Functional enrichment analysis indicated that the pathway related to metabolism progress and phosphoinositol signal is vitally responsible for differences in male reproductive performance between Landes geese and Sichuan White geese. These results show that compared with Sichuan White geese, the spermatogenesis in the testis of Landes geese was more active, which may be mainly related to the inositol phosphate signal. These data contribute to a better understanding of the mechanisms underlying different male reproductive performances between Landes geese and Sichuan White geese. This knowledge might eventually provide a theoretical basis for improving male reproductive performance in geese.

miR-26a is associated with sperm metabolism and can affect sperm motility and apoptosis. However, how miR-26a affects sperm motility remains largely unknown. Our previous study indicated that the PDHX gene is predicted to be a potential target of miR-26a, which is responsible for pyruvate oxidative decarboxylation which is considered as a key step for connecting glycolysis with oxidative phosphorylation. In this study, we first reported a potential relationship between miR-26a and PDHX and their expressions in fresh, frozen-thawed, and epididymal boar sperm. Then, sperm viability and survival were determined after transfection of miR-26a. mRNA and protein expression level of PDHX in the liquid-preserved boar sperm after transfection were also determined by RT-qPCR and Western Blot (WB). Our results showed that expression level of PDHX was significantly increased during sperm transit from epididymal caput to corpus and cauda. Similarly, expression of PDHX was significantly higher (P < 0.05) in fresh sperm as compared to epididymal cauda and frozen-thawed sperm. However, the expression of miR-26a in epididymal corpus sperm was significantly higher (P < 0.05) than that of caput and cauda sperm. Furthermore, after transfection of boar sperm with miR-26a mimic and inhibitor under liquid storage, the lowest and highest sperm viability was observed in miR-26a mimic and inhibitor treatment (P < 0.05), respectively. The protein levels of PDHX, after 24 and 48 h of transfection of miR-26a mimics and inhibitor, were notably decreased and increased (P < 0.05), respectively, as compared to negative control (NC) group. In conclusion, the novel and enticing findings of our study provide a reasonable evidence that miR-26a via PDHX, a link between glycolysis and oxidative phosphorylation, could regulate the glycometabolic pathway which eventually affect boar sperm viability and survival.

Peroxisome proliferator activated receptor-alpha (PPARα) and Egl nine homolog 3 (EGLN3) play critical roles in facilitating the adaptation to a hypoxic environment. However, the relationship between EGLN3 and PPARα variants and hypoxic adaptation remains poorly understood in Tibetan chickens. To better understand the effects of genetic variation, we sequenced exons of PPARα and EGLN3 in 138 Lowland chickens (LC) from 7 breeds that were located in Emei, Miyi, Shimian, Wanyuan, Pengxian, and Muchuan in the Sichuan province, and Wenchang in the Hainan province (altitudes for these locations are below 1800 meters). Total 166 Tibetan chickens (TC) from 7 subpopulations that were located in Shigatse, Lhoka, Lhasa, Garze, Aba, Diqing and Yushu in the Tibetan area were also sequenced (altitudes greater than 2700 meters). One single-nucleotide polymorphism (rs316017491, C > T) was identified in EGLN3 and was shared by TC and LC with no significant difference for allele frequencies between them (P > 0.05). Six single-nucleotide polymorphisms (SNP1, A29410G; SNP2, rs13886097; SNP3, T29467C; SNP4, rs735915170; SNP5, rs736599044; and SNP6, rs740077421) including one non-synonymous mutation (SNP2, T > C) were identified in PPARα. This is the first report of SNP1 and SNP3. There was a difference between TC and LC for allele frequencies (P