The dynamic landscape of chromatin accessibility and active regulatory elements in the mediobasal hypothalamus influences the seasonal activation of the reproductive axis in the male quail under long light exposure.

BACKGROUND: In cold and temperate zones, seasonal reproduction plays a crucial role in the survival and reproductive success of species. The photoperiod influences reproductive processes in seasonal breeders through the hypothalamic-pituitary-gonadal (HPG) axis, in which the mediobasal hypothalamus (MBH) serves as the central region responsible for transmitting light information to the endocrine system. However, the cis-regulatory elements and the transcriptional activation mechanisms related to seasonal activation of the reproductive axis in MBH remain largely unclear. In this study, an artificial photoperiod program was used to induce the HPG axis activation in male quails, and we compared changes in chromatin accessibility changes during the seasonal activation of the HPG axis. RESULTS: Alterations in chromatin accessibility occurred in the mediobasal hypothalamus (MBH) and stabilized at LD7 during the activation of the HPG axis. Most open chromatin regions (OCRs) are enriched mainly in introns and distal intergenic regions. The differentially accessible regions (DARs) showed enrichment of binding motifs of the RFX, NKX, and MEF family of transcription factors that gained-loss accessibility under long-day conditions, while the binding motifs of the nuclear receptor (NR) superfamily and BZIP family gained-open accessibility. Retinoic acid signaling and GTPase-mediated signal transduction are involved in adaptation to long days and maintenance of the HPG axis activation. According to our footprint analysis, three clock-output genes (TEF, DBP, and HLF) and the THRA were the first responders to long days in LD3. THRB, NR3C2, AR, and NR3C1 are the key players associated with the initiation and maintenance of the activation of the HPG axis, which appeared at LD7 and tended to be stable under long-day conditions. By integrating chromatin and the transcriptome, three genes (DIO2, SLC16A2, and PDE6H) involved in thyroid hormone signaling showed differential chromatin accessibility and expression levels during the seasonal activation of the HPG axis. TRPA1, a target of THRB identified by DAP-seq, was sensitive to photoactivation and exhibited differential expression levels between short- and long-day conditions. CONCLUSION: Our data suggest that trans effects were the main factors affecting gene expression during the seasonal activation of the HPG axis. This study could lead to further research on the seasonal reproductive behavior of birds, particularly the role of MBH in controlling seasonal reproductive behavior.

The DNA methylation status of the serotonin metabolic pathway associated with reproductive inactivation induced by long-light exposure in Magang geese.

BACKGROUND: Domestic geese are seasonal breeders and have the lowest reproductive capacity among all poultry species. Magang geese is a topical short-day breeder, short photoperiod exposure stimulates its reproductive activity while long photoperiod inhibits. To explore epigenetic change that could influence reproductive activity, we performed whole genome bisulfite sequencing and transcriptome sequencing in the hypothalamus at three reproductive stages during long-light exposure in male Magang geese. RESULTS: A total number of 10,602 differentially methylated regions (DMRs) were identified among three comparison groups. We observed that the vast majority of DMRs were enriched in intron regions. By integrating the BS-sequencing and RNA-seq data, the correlation between methylation changes of CG DMRs and expression changes of their associated genes was significant only for genes containing CG DMRs in their intron. A total of 278 DMR-associated DEGs were obtained among the three stages. KEGG analysis revealed that the DMR-associated DEGs were mainly involved in 11 pathways. Among them, the neuroactive ligand-receptor interaction pathway was significantly enriched in both two comparisons (RA vs.RD and RD vs.RI); the Wnt signaling pathway, apelin signaling pathway, melanogenesis, calcium signaling pathway, focal adhesion, and adherens junction were significantly enriched in the RA vs. RI comparison. In addition, the expression level of two serotonin-metabolic genes was significantly altered during reproductive axis inactivation by the methylation status of their promoter region (TPH2) and intron region (SLC18A2), respectively. These results were confirmed by Bisulfite sequencing PCR (BSP), pyrosequencing, and real-time qPCR, indicating that serotonin metabolic signaling may play a key role in decreasing the reproductive activity of Magang geese induced by long-light exposure. Furthermore, we performed a metabolomics approach to investigate the concentration of neurotransmitters among the three stages, and found that 5-HIAA, the last product of the serotonin metabolic pathway, was significantly decreased in the hypothalamus during RI. CONCLUSIONS: Our study reveals that the methylation status of the serotonin metabolic pathway in the hypothalamus is associated with reproductive inactivation, and provided new insight into the effect of DNA methylation on the reproductive regulation of the hypothalamus in Magang geese.

Endocrine and molecular regulation mechanisms of follicular development and egg-laying in quails under different photoperiods.

Photoperiod is a key environmental factor in regulating bird reproduction and induces neuroendocrine changes through the hypothalamic-pituitary-gonadal (HPG) axis. OPN5, as a deep-brain photoreceptor, transmits light signals to regulate follicular development through TSH-DIO2/DIO3. However, the mechanism among OPN5, TSH-DIO2/DIO3, and VIP/PRL in the HPG axis underlying the photoperiodic regulation of bird reproduction is unclear. In this study, 72 laying quails with 8-week-old were randomly divided into the long-day (LD) group [16 light (L): 8 dark (D)] and the short-day (SD) group (8 L:16 D), and then samples were collected on d 1, d 11, d 22, and d 36 of the experiment. The results showed that compared with the LD group, the SD group significantly inhibited follicular development (P < 0.05), decreased the P4, E2, LH, and PRL in serum (P < 0.05), downregulated the expression of GnRHR, VIP, PRL, OPN5, DIO2, and LHß (P < 0.05), reduced the expression of GnRH and TSHß (P > 0.05), and promoted DIO3, GnIH gene expression (P < 0.01). The short photoperiod downregulates OPN5, TSHß, and DIO2 and upregulates DIO3 expression to regulate the GnRH/GnIH system. The downregulation of GnRHR and upregulation of GnIH resulted in a decrease in LH secretion, which withdrew the gonadotropic effects on ovarian follicles development. Slow down of follicular development and egg laying may also arise from lack of PRL potentiation to small follicle development under short days.

Comparative transcriptomics in the hypothalamic-pituitary-gonad axis of mammals and poultry.

The hypothalamic-pituitary-gonad (HPG) axis is vital for reproductive activities in vertebrates. The large-scale comparative analyses of gene expression in the HPG axis across vertebrates have not been carried out yet. Here we collected 175 high-quality RNA-seq samples of hypothalamus, pituitary, ovary and testis from eight species (four mammals and four poultry) to compare transcriptome in the HPG axis, and to detect key pathways and related genes associated with reproduction. We demonstrated the distinguished difference in gene expression of the HPG axis between mammalian and avian species by a series of bioinformatics analysis, including gene differential expression, the phylogeny analysis of gene expression, and their functional annotations. We revealed two pathways, i.e., neuroactive ligand-receptor interaction and calcium signaling pathway, which play important roles in animal reproduction. In these two pathways, we detected 17 differentially expressed genes shared in 4 tissues, while 13, 27, and 27 were specifically differentially expressed genes in hypothalamus, pituitary and ovary, respectively. Our study on the comparative transcriptomics in the HPG axis across species will provide novel knowledge for exploring the molecular mechanism underlying reproductive traits in animals.

DNA Demethylation of Myogenic Genes May Contribute to Embryonic Leg Muscle Development Differences between Wuzong and Shitou Geese.

Skeletal muscle development from embryonic stages to hatching is critical for poultry muscle growth, during which DNA methylation plays a vital role. However, it is not yet clear how DNA methylation affects early embryonic muscle development between goose breeds of different body size. In this study, whole genome bisulfite sequencing (WGBS) was conducted on leg muscle tissue from Wuzong (WZE) and Shitou (STE) geese on embryonic day 15 (E15), E23, and post-hatch day 1. It was found that at E23, the embryonic leg muscle development of STE was more intense than that of WZE. A negative correlation was found between gene expression and DNA methylation around transcription start sites (TSSs), while a positive correlation was observed in the gene body near TTSs. It was also possible that earlier demethylation of myogenic genes around TSSs contributes to their earlier expression in WZE. Using pyrosequencing to analyze DNA methylation patterns of promoter regions, we also found that earlier demethylation of the MyoD1 promoter in WZE contributed to its earlier expression. This study reveals that DNA demethylation of myogenic genes may contribute to embryonic leg muscle development differences between Wuzong and Shitou geese.

Transcriptome analysis of pituitary gland revealed candidate genes and gene networks regulating the growth and development in goose.

Goose is important meat poultry and its growth and development has always been the focus of attention, but the regulation mechanisms of genes and gene network underlying growth and development of goose are still unclear. Three males of large-size Shitou goose and three males of small size Wuzong goose at 5 weeks of age were used for transcriptome analysis with deep sequencing. After slaughter, their pituitary gland was taken for RNA-seq. A total of 290 DEGs were identified by fold change ≥2 and false discovery rate (FDR) <0.05, where there were 148 upregulated genes and 142 downregulated genes in Shitou goose compared to Wuzong goose. Results also showed that the DEGs related to insulin signaling pathway could increase protein synthesis and fat production, and the interaction network of DEGs was mainly related to development, endocrine system, inflammatory diseases, tissue damage and abnormality. The DEGs involved in the growth and function of the pituitary organs may regulate the growth and development of the body by affecting the synthesis and secretion of pituitary hormones. The results of this study will help to understand the regulatory mechanism of goose growth and development.

The theory on and software simulating large-scale genomic data for genotype-by-environment interactions.

BACKGROUND: With the emphasis on analysing genotype-by-environment interactions within the framework of genomic selection and genome-wide association analysis, there is an increasing demand for reliable tools that can be used to simulate large-scale genomic data in order to assess related approaches. RESULTS: We proposed a theory to simulate large-scale genomic data on genotype-by-environment interactions and added this new function to our developed tool GPOPSIM. Additionally, a simulated threshold trait with large-scale genomic data was also added. The validation of the simulated data indicated that GPOSPIM2.0 is an efficient tool for mimicking the phenotypic data of quantitative traits, threshold traits, and genetically correlated traits with large-scale genomic data while taking genotype-by-environment interactions into account. CONCLUSIONS: This tool is useful for assessing genotype-by-environment interactions and threshold traits methods.

Identification of recombination in novel goose parvovirus isolated from domesticated Jing-Xi partridge ducks in South China.

Outbreaks of short beak and dwarfism syndrome (SBDS), caused by a novel goose parvovirus (NGPV), have occurred in China since 2015. This rapidly spreading, infectious disease affects ducks in particular, with a high morbidity and low mortality rate, causing huge economic losses. This study analyzed the evolution of NGPV isolated from Jing-Xi partridge duck with SBDS in South China. Complete genome sequences of the NGPV strains GDQY1802 and GDSG1901 were homologous with other GPV/NGPV and Muscovy duck parvovirus (MDPV) strains. Phylogenetic analysis showed that the NGPV isolated from mainland China was related to the Taiwan 82-0321v strain of GPV. In contrast to 82-0321v and the SDLC01 strain, which was first isolated from China, the two isolates showed no deletions in the inverted terminal repeat (ITR) region. Further, in these isolates, 24 amino acid sites of the replication protein were different compared to that of GPV live vaccine strain 82-0321v, and 12 sites were unique across all NGPV isolates. These isolates also showed differences in 17 amino acid sites of the capsid protein from that of 82-0321v, two of which were the same as those in MDPV. Recombination analysis identified the major parents of GDSG1901 and GDQY1802 as the NGPV-GD and NGPV-Hun18 strains, and the minor parents as the classical GPV 06-0329 and GPV LH strains, respectively. GDQY1802 and GDSG1901 are recombinant GPV-related parvovirus isolated from domesticated partridge duck. Recombination is evident in the evolution of NGPV, and as such, the use of live attenuated vaccines for NGPV requires further study.

Pathogenicity of snakehead vesiculovirus in rice field eels (Monopterus albus).

Snakehead vesiculovirus (SHVV) has caused mass mortality to cultured snakehead fish in China, resulting in enormous economic losses in snakehead fish culture. In this report, the whole genome of SHVV was sequenced. Interestingly, it shared more than 94% nucleotide sequence identity with Monopterus albus rhabdovirus (MoARV), which has caused great economic loss to cultured rice field eel (Monopterus albus). Therefore, the concern of cross-species infection of these viruses prompted us to investigate the susceptibility of rice field eel to SHVV infection. The results showed that rice field eel was susceptible to SHVV in both intracoelomical injection and immersion routes. Severe hemorrhage was observed on the skin and visceral organs of SHVV-infected rice field eels. Histopathological examination showed vacuoles in the tissues of infected liver, kidney and heart. Viral RNA or protein was detected in the tissues of infected fish by reverse transcription polymerization chain reaction (RT-PCR), in situ hybridization (ISH), or immunohistochemistry assay (IHC). Investigation of the epidemic of vesiculovirus in rice field eel as well as other co-cultured fish is invaluable for the prevention of vesiculovirus infection.

Complete genome sequence analysis of the fish pathogen Flavobacterium columnare provides insights into antibiotic resistance and pathogenicity related genes.

We analyzed here the complete genome sequences of a highly virulent Flavobacterium columnare Pf1 strain isolated in our laboratory. The complete genome consists of a 3,171,081 bp circular DNA with 2784 predicted protein-coding genes. Among these, 286 genes were predicted as antibiotic resistance genes, including 32 RND-type efflux pump related genes which were associated with the export of aminoglycosides, indicating inducible aminoglycosides resistances in F. columnare. On the other hand, 328 genes were predicted as pathogenicity related genes which could be classified as virulence factors, gliding motility proteins, adhesins, and many putative secreted proteases. These genes were probably involved in the colonization, invasion and destruction of fish tissues during the infection of F. columnare. Apparently, our obtained complete genome sequences provide the basis for the explanation of the interactions between the F. columnare and the infected fish. The predicted antibiotic resistance and pathogenicity related genes will shed a new light on the development of more efficient preventional strategies against the infection of F. columnare, which is a major worldwide fish pathogen.

Co-infections of infectious spleen and kidney necrosis virus and Siniperca chuatsi rhabdovirus in Chinese perch (Siniperca chuatsi).

In spite of the quite common co-infections of viruses in the cultured fish, most of the previous studies have just simply focused on the infection of a single pathogen. In this report, we observed that about 13% of cultured Chinese perch have been co-infected by infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV). Furthermore, Chinese perch could co-infected by ISKNV and SCRV by intraperitoneally injection with the two viruses. Interestingly, we revealed that the two viruses could even co-infect a single cell of Chinese perch in vivo and a single Chinese perch brain cells (CPB) cell in vitro. The dynamic co-infected viruses loads in the different tissues of Chinese perch showed dependent. When CPB cells were infected with the same 10 MOI of SCRV and ISKNV, the replication of SCRV overwhelmed the replication of ISKNV. When the MOI of ISKNV (10 MOI) was 10,000 times of MOI of SCRV (0.001 MOI), the dynamic virus loads of the two viruses in CPB cells indicated that co-infections could synergistically stimulate both viruses replication at the late time points but not at early time points. The co-infections of ISKNV and SCRV in the cultured Chinese perch will shed a new light on the prevention of the viral diseases of Chinese perch. The development of multivalent vaccine which could be effective for preventing against the co-infections of the viruses is highly needed.

Growth Performance of and Liver Function in Heat-Stressed Magang Geese Fed the Antioxidant Zinc Ascorbate and Its Potential Mechanism of Action.

The aim of this study was to investigate the in vitro antioxidant activity of zinc ascorbate (AsA-Zn), its effects on the growth performance of and liver function in Magang geese under heat stress, and its potential mechanism. At AsA-Zn concentrations of 7.5, 15, 30, and 60 µmol/L, the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS·+) radical scavenging rate increased significantly by 120.85%, 53.43%, 36.12%, and 0.99%, respectively, compared with that of ascorbic acid (AsA), indicating that AsA-Zn had better antioxidant performance in vitro. In this study, Magang geese were divided into a control group (basal diet, CON) and experimental groups, who received the basal diet supplemented with 400 mg/kg AsA or 30 (AsA-Zn30), 60 (AsA-Zn60), or 90 (AsA-Zn90) mg/kg AsA-Zn. AsA-Zn supplementation considerably reduced the feed-to-gain ratio, whereas both AsA and AsA-Zn significantly increased the thymus index. Moreover, AsA-Zn supplementation improved serum protein levels, lipid metabolism, liver function, and antioxidant capacity while reducing hepatocyte vacuolar degeneration. Furthermore, supplementation with AsA-Zn60 significantly increased the total antioxidant capacity, glutathione peroxidase activity, and superoxide dismutase activity and decreased the malondialdehyde content in the serum, liver, and hepatic mitochondria (P < 0.05), with more pronounced effects in the AsA-Zn60 group. Moreover, supplementation with ASA-Zn regulated the Nrf 2 signaling pathway and significantly increased the expression of genes encoding antioxidant-related factors in the liver. In conclusion, AsA-Zn has good antioxidant activity, and AsA-Zn supplementation may improve the antioxidant capacity of heat-stressed geese and promote their growth. Supplementation with 30 mg/kg AsA-Zn is recommended.

Identification of Differentially Expressed Genes and microRNAs in the Gray and White Feather Follicles of Shitou Geese.

The Shitou goose, a highly recognized indigenous breed with gray plumage originating from Chaozhou Raoping in Guangdong Province, China, is renowned for being the largest goose species in the country. Notably, during the pure breeding process of Shitou geese, approximately 2% of the offspring in each generation unexpectedly exhibited white plumage. To better understand the mechanisms underlying white plumage color formation in Shitou geese, we conducted a comparative transcriptome analysis between white and gray feather follicles, aiming to identify key genes and microRNAs that potentially regulate white plumage coloration in this unique goose breed. Our results revealed a number of pigmentation genes, encompassing TYR, TYRP1, EDNRB2, MLANA, SOX10, SLC45A2, GPR143, TRPM1, OCA2, ASIP, KIT, and SLC24A5, which were significantly down-regulated in the white feather follicles of Shitou geese. Among these genes, EDNRB2 and KIT emerged as the most promising candidate genes for white plumage coloration in Shitou geese. Additionally, our analysis also uncovered 46 differentially expressed miRNAs. Of these, miR-144-y may play crucial roles in the regulation of feather pigmentation. Furthermore, the expression of novel-m0086-5p, miR-489-y, miR-223-x, miR-7565-z, and miR-3535-z exhibits a significant negative correlation with the expression of pigmentation genes including TYRP1, EDNRB2, MLANA, SOX10, TRPM1, and KIT, suggesting these miRNAs may indirectly regulate the expression of these genes, thereby influencing feather color. Our findings provide valuable insights into the genetic mechanisms underlying white plumage coloration in Shitou geese and contribute to the broader understanding of avian genetics and coloration research.

Transcriptome and lipidome integration unveils mechanisms of fatty liver formation in Shitou geese.

Geese evolved from migratory birds, and when they consume excessive high-energy feed, glucose is converted into triglycerides. A large amount of triglyceride deposition can induce incomplete oxidation of fatty acids, leading to lipid accumulation in the liver and the subsequent formation of fatty liver. In the Chaoshan region of Guangdong, China, Shitou geese develop a unique form of fatty liver through 24 h overfeeding of brown rice. To investigate the mechanisms underlying the formation of fatty liver in Shitou geese, we collected liver samples from normally fed and overfed geese. The results showed that the liver size in the treatment group was significantly larger, weighing 3.5 times more than that in the control group. Extensive infiltration of lipid droplets was observed in the liver upon staining of tissue sections. Biochemical analysis revealed that compared to the control group, the treatment group showed significantly elevated levels of total cholesterol (T-CHO), triglycerides (TG), and glycogen in the liver. However, no significant differences were observed in the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are common indicators of liver damage. Furthermore, we performed a combined transcriptomic and lipidomic analysis of the liver samples and identified 1,510 differentially expressed genes (DEGs) and 1,559 significantly differentially abundant metabolites (SDMs). The enrichment analysis of the DEGs revealed their enrichment in metabolic pathways, cellular process-related signaling pathways, and specific lipid metabolism pathways. We also conducted KEGG enrichment analysis of the SDMs and compared them with the enriched signaling pathways obtained from the DEGs. In this study, we identified 3 key signaling pathways involved in the formation of fatty liver in Shitou geese, namely, the biosynthesis of unsaturated fatty acids, glycerol lipid metabolism, and glycerophospholipid metabolism. In these pathways, genes such as glycerol-3-phosphate acyltransferase, mitochondrial (GPAM), 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2), diacylglycerol O-acyltransferase 2 (DGAT2), lipase, endothelial (LIPG), lipoprotein lipase (LPL), phospholipase D family member 4 (PLD4), and phospholipase A2 group IVF (PLA2G4F) may regulate the synthesis of metabolites, including triacylglycerol (TG), phosphatidate (PA), 1,2-diglyceride (DG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). These genes and metabolites may play a predominant role in the development of fatty liver, ultimately promoting the accumulation of TG in the liver and leading to the progression of fatty liver.

Polysaccharide of Atractylodes macrocephala Koidz alleviates LPS-induced proliferation, differentiation inhibition and excessive apoptosis in chicken embryonic myogenic cells.

BACKGROUND: Lipopolysaccharide (LPS) can induce systemic inflammation and affect the growth and development of poultry. As a kind of traditional Chinese medicine, polysaccharide of Atractylodes macrocephala Koidz (PAMK) can effectively improve the growth performance of animals and improve the immunity of animal bodies. OBJECTIVES: The purpose of this study was to investigate the effects of PAMK on LPS-induced inflammatory response, proliferation, differentiation and apoptosis of chicken embryonic myogenic cells. METHODS: We used chicken embryonic myogenic cells as a model by detecting EdU/MYHC immunofluorescence, the expression of inflammation, proliferation, differentiation-related genes and proteins and the number of apoptotic cells in the condition of adding LPS, PAMK, belnacasan (an inhibitor of Caspase1) or their combinations. RESULTS: The results showed that LPS stimulation increased the expression of inflammatory factors, inhibited proliferation and differentiation, and excessive apoptosis in chicken embryonic myogenic cells, and PAMK alleviated these adverse effects induced by LPS. After the addition of belnacasan (inhibitor of Caspase1), apoptosis in myogenic cells was inhibited, and therefore, the number of apoptotic cells and the expression of pro-apoptotic genes Caspase1 and Caspase3 were increased. In addition, belnacasan inhibited the increased expression of inflammatory factors, inhibited proliferation, differentiation and excessive apoptosis in chicken embryonic myogenic cells induced by LPS. CONCLUSIONS: This study provides a theoretical basis for further exploring the mechanism of action of PAMK and exogenous LPS on chicken embryonic myogenic cells and lays the foundation for the development and application of green feed additives in animal husbandry industry.

The DNA methylation status of the vitamin A signaling associated with testicular degeneration induced by long-day photoperiods in Magang geese.

Magang geese are typical short-day breeders whose reproductive behaviors are significantly influenced by photoperiod. Exposure to a long-day photoperiod results in testicular regression and spermatogenesis arrest in Magang geese. To investigate the epigenetic influence of DNA methylation on the seasonal testicular regression in Magang geese, we conducted whole-genome bisulfite sequencing and transcriptome sequencing of testes across 3 reproductive phases during a long-day photoperiod. A total of 250,326 differentially methylated regions (DMR) were identified among the 3 comparison groups, with a significant number showing hypermethylation, especially in intronic regions of the genome. Integrating bisulfite sequencing with transcriptome sequencing data revealed that DMR-associated genes tend to be differentially expressed in the testes, highlighting a potential regulatory role for DNA methylation in gene expression. Furthermore, there was a significant negative correlation between changes in the methylation of CG DMRs and changes in the expression of their associated genes in the testes. A total of 3,359 DMR-associated differentially expressed genes (DEG) were identified; functional enrichment analyses revealed that motor proteins, MAPK signaling pathway, ECM-receptor interaction, phagosome, TGF-beta signaling pathway, and calcium signaling might contribute to the testicular regression process. GSEA revealed that the significantly enriched activated hallmark gene set was associated with apoptosis and estrogen response during testicular regression, while the repressed hallmark gene set was involved in spermatogenesis. Our study also revealed that methylation changes significantly impacted the expression level of vitamin A metabolism-related genes during testicular degeneration, with hypermethylation of STRA6 and increased calmodulin levels indicating vitamin A efflux during the testicular regression. These findings were corroborated by pyrosequencing and real-time qPCR, which revealed that the vitamin A metabolic pathway plays a pivotal role in testicular degeneration under long-day conditions. Additionally, metabolomics analysis revealed an insufficiency of vitamin A and an abnormally high level of oxysterols accumulated in the testes during testicular regression. In conclusion, our study demonstrated that testicular degeneration in Magang geese induced by a long-day photoperiod is linked to vitamin A homeostasis disruption, which manifests as the hypermethylation status of STRA6, vitamin A efflux, and a high level of oxysterol accumulation. These findings offer new insights into the effects of DNA methylation on the seasonal testicular regression that occurs during long-day photoperiods in Magang geese.

Drp1 regulated PINK1-dependent mitophagy protected duck follicular granulosa cells from acute heat stress injury.

The mitochondrial quality control system is crucial in maintaining cellular homeostasis during environmental stress. Granulosa cells are the main cells secreting steroid hormones, and mitochondria are the key organelles for steroid hormone synthesis. The impact of the mitochondrial quality control system on granulosa cells' steroid hormone synthesis and survival under heat stress is still unclear. Here, we showed that acute heat stress induces mitochondrial damage and significantly increases the number of mitophagy-like vesicles in the cytoplasm of duck ovary granulosa cells at the ultra-structural level. Meanwhile, we also found heat stress significantly increased mitochondrial fission and mitophagy-related protein expression levels both in vivo and in vitro. Furthermore, by confocal fluorescence analysis, we discovered that LC3 was distributed spot-like manner near the nucleus in the heat treatment group, and the LC3 spots and lysosomes were colocalized with Mito-Tracker in the heat treatment group. We further detected the mitophagy-related protein in the cytoplasm and mitochondria, respectively. Results showed that the PINK1 protein was significantly increased both in cytoplasm and mitochondria, while the LC3-Ⅱ/LC3-Ⅰ ratio increase only occurred in mitochondrial. In addition, the autophagy protein induced by acute heat treatment was effectively inhibited by the mitophagy inhibitor CysA. Finally, we demonstrated that the alteration of cellular mitophagy by siRNA interference with Drp1 and PINK1 inhibited the steroid synthesis of granulosa cells and increased cell apoptosis. Study provides strong evidence that the Drp1 regulated PINK1-dependent mitophagy pathway protects follicular granulosa cells from acute heat stress-induced injury.

Effects of stocking density on the homeostasis of uric acid and related liver and kidney functions in ducks.

OBJECTIVE: Stocking density (SD) is an important issue in the poultry industry, which is related to the production performance, intestinal health and immune status. In the present study, the effects of SD on the metabolism and homeostasis of uric acid as well as the related functions of the liver and kidney in ducks were examined. METHODS: A total of 360 healthy 56-day-old Shan-ma ducks were randomly divided into the low stocking density (n = 60, density = 5 birds/m2), medium stocking density (n = 120, density = 10 birds/m2) and high stocking density groups (HSD; n = 180, density = 15 birds/m2). Samples were collected in the 3rd, 6th, and 9th weeks of the experiment for analysis. RESULTS: The serum levels of uric acid, lipopolysaccharide and inflammatory cytokines (interleukin-1ß [IL-1ß], IL-8, and tumor necrosis factor-α [TNF-α]) were increased significantly in the HSD group. Serious histopathological lesions could be seen in both the livers and kidneys in the HSD group in the 9th week. The mRNA expression levels of inflammatory cytokines (IL-8 and TNF-α) and related pathway components (toll-like receptor 4, myeloid differentiation primary response gene 88, and nuclear factor-κB) were increased significantly in both the livers and kidneys in the HSD group. The mRNA expression levels of enzymes (adenosine deaminase, xanthine oxidase, phosphoribosyl pyrophosphate amidotransferase, and phosphoribosyl pyrophosphate synthetase 1) related to the synthesis of uric acid increased significantly in the livers in the HSD group. However, the mRNA expression level of solute carrier family 2 member 9, which plays an important role in the excretion of uric acid by the kidney, was decreased significantly in the kidneys in the HSD group. CONCLUSION: These results indicated that a higher SD could cause tissue inflammatory lesions in the liver and kidney and subsequently affect the metabolism and homeostasis of uric acid, and is helpful for guiding decisions related to the breeding and production of ducks.

Effect of immunization against OPN5 on the reproductive performance in Shan Partridge ducks under different photoperiods.

Photoperiod is an important environmental factor that influences seasonal reproduction behavior in birds. Birds translate photoperiodic information into neuroendocrine signals through deep brain photoreceptors (DBPs). OPN5 has been considered candidate DBPs involved in regulating seasonal reproduction in birds. We found that OPN5 could mediate light to regulate the follicle development in ducks. In this study, we further verified the effect of OPN5 on follicular development in Shan Partridge ducks by immunizing against the extracellular domain (ECD) of OPN5. We investigated the specific regulatory mechanism of photoperiod mediated by OPN5 on the reproductive activity of ducks. The trial randomly divided 120 Shan Partridge ducks into 3 groups with different treatments: the immunization of OPN5 group was done at d0, d15, d30, and d40 with 1 mL of vaccine containing OPN5 protein (thus containing 1, 1, 0.5, and 0.5 mg of OPN5-KLH protein), and the control group (CS and CL groups) was injected at the same time with the same dose of OPN5-uncontained blank vaccine. The group of CS (900 lux), OPN5 (600 lux), and CL (600 lux) lasted for 40 d in 12 L:12 D photoperiods, respectively. Then, the groups of CS, OPN5, and CL subsequently received 12 L:12 D, 12 L:12 D, and 17 L:7 D light treatments for 33 d, respectively. The ducks were caged in 3 constant rooms with the same feeding conditions for each group, free water, and limited feeding (150 g per duck each day). Duck serum and tissue samples were collected at d 40, d 62, and d 73 (n = 12). It was found that before prolonged light, the group of immunization (group OPN5) and the group of strong light intensity (group CS) were higher than the group of CL in egg production. Subsequent to prolonged light, the group CL in egg production rose about the same as the group immunization, while the strong light group (group CS) was lower. Group OPN5 increased the ovarian index of ducks, and both the immunization of group OPN5 and group CL (extended light) increased the thickness of the granular layer and promoted the secretion of E2, P4, LH, and PRL hormones. Compared with group CS, group CL and OPN5 increased the mRNA level and protein expression of OPN5 in the hypothalamus on d 62 and d 73 (P < 0.05). The gene or protein expression patterns of GnRH, TRH, TSHß, DIO2, THRß, VIP, and PRL were positively correlated with OPN5, whereas the gene expression patterns of GnIH and DIO3 were negatively correlated with OPN5. The results showed that immunization against OPN5 could activate the corresponding transmembrane receptors to promote the expression of OPN5, up-regulate the expression of TSHß and DIO2, and then regulate the HPG axis-related genes to facilitate the follicular development of Shan Partridge ducks. In addition, in this experiment, prolonging the photoperiod or enhancing the light intensity could also enhance follicle development, but the effect was not as significant as immunizing against OPN5. Our results will offer beneficial data and more supportive shreds of evidence in favor of elucidating the role of OPN5 in relation to photoperiods and reproduction.

Comparative Transcriptome Analyses of Leg Muscle during Early Growth between Geese (Anser cygnoides) Breeds Differing in Body Size Characteristics.

Goose is an important poultry commonly raised for meat. The early growth performance of geese significantly influences their market weight and slaughter weight, affecting the poultry industry's economic benefits. To identify the growth surge between the Shitou goose and the Wuzong goose, we collected the early growth body traits from 0 to 12 weeks. In addition, we investigated the transcriptomic changes in leg muscles at the high growth speed period to reveal the difference between the two geese breeds. We also estimated the growth curve parameters under three models, including the logistic, von Bertalanffy, and Gompertz models. The results showed that except for body length and keel length, the best-fitting model between the body weight and body size of the Shitou and Wuzong was the logistic model. The growth turning points of Shitou and Wuzong were 5.954 and 4.944 weeks, respectively, and the turning point of their body weight was 1459.01 g and 478.54 g, respectively. Growth surge occurred at 2-9 weeks in Shitou goose and at 1-7 weeks in Wuzong goose. The body size traits of the Shitou goose and Wuzong goose showed a trend of rapid growth in the early stage and slow growth in the later stage, and the Shitou goose growth was higher than the Wuzong goose. For transcriptome sequencing, a total of 87 differentially expressed genes (DEGs) were identified with a fold change ≥ 2 and a false discovery rate < 0.05. Many DEGs have a potential function for growth, such as CXCL12, SSTR4, FABP5, SLC2A1, MYLK4, and EIF4E3. KEGG pathway analysis identified that some DEGs were significantly enriched in the calcium signaling pathway, which may promote muscle growth. The gene-gene interaction network of DEGs was mainly related to the transmission of cell signals and substances, hematological system development, and functions. This study can provide theoretical guidance for the production and breeding management of the Shitou goose and Wuzong goose and help reveal the genetic mechanisms underlying diverse body sizes between two goose breeds.