Transcriptome and proteome revealed the differences in 3 colors of earlobe in Jiangshan Black-bone chicken.

The earlobe is a featherless, exposed thickening located beneath the ear canal of chickens, which plays a visual signaling role in age, performance, mental vitality, reproduction, and other aspects. However, despite its importance, there have been few studies on the color differences and formation mechanisms of chicken earlobes, particularly the structurally blue earlobes characteristic of the Jiangshan black-bone chicken. In this study, we explored the physiological mechanisms that may influence the formation of differently colored earlobes using 3 types of earlobes from Jiangshan black-bone chickens: light peacock green (Green group), dark peacock green (Blue group), and dark reddish purple (Black group). All 3 earlobe colors exhibited positive melanin Masson-Fontana staining, and the thickness of collagen fibers in the dermis decreased in the order of Green, Blue, and Black groups. A total of 1,953 differentially expressed genes (DEGs) were detected in the 3 earlobes through mRNA sequencing, among which the GO term "collagen trimer" was significantly enriched in DEGs between groups. Additionally, 716 differentially expressed proteins (DEPs) were identified in the 3 earlobes using 4D-DIA proteomics, with the term "collagen fibril organization" being significantly enriched in DEPs between the Green and Black groups. Integrated analysis of transcriptome and proteome data revealed that 12 DEGs and DEPs were commonly differentially expressed between the Green and Black groups, including the gene LUM (corneal keratan sulfate proteoglycan), which was significantly enriched in the "collagen fibril organization" GO term. In conclusion, our study suggests that LUM plays a crucial role in the formation of peacock green earlobes in Jiangshan black-bone chickens. The high level of LUM in peacock green (Green and Blue groups) may affect collagen nanostructures, leading to a stronger effect of melanin-supported dermal collagen on the production of non-iridescent structural colors through coherent scattering, resulting in a bright structural blue color in Jiangshan black-bone chickens. In contrast, the low expression of LUM in dark reddish purple (Black group) reduces the reflection of non-iridescent structural colors, making the earlobe color appear almost black, similar to melanin.

NTRK2 Promotes Sheep Granulosa Cells Proliferation and Reproductive Hormone Secretion and Activates the PI3K/AKT Pathway.

Neurotrophin receptor B (NTRK2), also named TRKB, belongs to the neurotrophic factor family. Previous studies have shown that NTRK2 is associated with high fertility in mammals. However, the molecular mechanism and regulatory pathway of this neurotrophic factor remain unclear. In this study, NTRK2 overexpression and NTRK2-siRNA were constructed to detect the effects of NTRK2 on the proliferation and hormone secretion of the ovarian granulosa cells (GCs) of sheep. We successfully isolated follicular phase granulosa cells in vitro from the ovaries of sheep in simultaneous estrus, and the immunofluorescence results confirmed that NTRK2 was expressed in the collected cells. Subsequently, the effect of NTRK2 on the proliferation of sheep granulosa cells was examined via cell transfection experiments. The results showed that the expression of CDK4 and CyclinD2 was significantly increased after NTRK2 overexpression, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). The EdU and CCK-8 assays showed that the proliferation rate of sheep GCs was significantly increased after NTRK2 overexpression, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). Moreover, NTRK2 significantly increased the expression of steroidogenesis-related genes, including steroidogenic acute regulatory protein (STAR) and hydroxy-δ-5-steroid dehydrogenase (HSD3B1), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1). The ELISA results showed that the secretion levels of E2 and P4 significantly increased after NTRK2 overexpression, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). Previous studies had confirmed that NTRK2 gene belongs to the PI3K-AKT signaling pathway and participates in the signaling of this pathway. This was demonstrated by protein-protein interaction analysis and NTRK2 belongs to the PI3K-AKT pathway. The modification of PI3K and AKT, markers of the PI3K-AKT pathway, via phosphorylation was increased after NTRK2 overexpression in the sheep GCs, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). Overall, these results suggest that the NTRK2 gene regulates the proliferation of GCs and the secretion of steroid hormones in sheep, and that it influences the phosphorylation level of the PI3K/AKT signaling pathway. These findings provided a theoretical basis and new perspectives for exploring the regulation of NTRK2 gene in the development of ovine follicles.

Local chicken breeds exhibit abundant TCR-V segments but similar repertoire diversity.

The thymus-derived lymphocytes of jawed vertebrates have four T-cell receptor (TCR) chains that play a significant role in immunity. As chickens have commercial value, their immune systems require a great deal of attention. Local chicken breeds are an essential part of poultry genetic resources in China. Here, we used high-throughput sequencing to analyze the TCRα and TCRß repertoires and their relative expression levels in the native chicken breeds Baier Buff, Longyou Partridge, Xiaoshan, and Xianju. We found that TCR Vα and TCR Vß were expressed and included 17, 19, 17, and six segments of the Vα2, Vα3, Vß1, and Vß2 subgroups, respectively. V-J pairing was biased; Jα11 was utilized by nearly all Vα segments and was the most commonly used. Breed-specific V segments and V-J pairings were detected as well. The results of the principal coordinate analysis (PCoA) as well as the V-J pairing and CDR3 diversity analyses suggested that the four local chicken breeds did not significantly differ in terms of TCR diversity. Hence, they expressed not significant differentiation, and they are rich genetic resources for the development and utilization of immune-related poultry breeding.

Cell Heterogeneity Analysis Revealed the Key Role of Fibroblasts in the Magnum Regression of Ducks.

Duck egg production, like that of laying hens, follows a typical low-peak-low cycle, reflecting the dynamics of the reproductive system. Post-peak, some ducks undergo a cessation of egg laying, indicative of a regression process in the oviduct. Notably, the magnum, being the longest segment of the oviduct, plays a crucial role in protein secretion. Despite its significance, few studies have investigated the molecular mechanisms underlying oviduct regression in ducks that have ceased laying eggs. In this study, we conducted single-cell transcriptome sequencing on the magnum tissue of Shaoxing ducks at 467 days of age, utilizing the 10× Genomics platform. This approach allowed us to generate a detailed magnum transcriptome map of both egg-laying and ceased-laying ducks. We collected transcriptome data from 13,708 individual cells, which were then subjected to computational analysis, resulting in the identification of 27 distinct cell clusters. Marker genes were subsequently employed to categorize these clusters into specific cell types. Our analysis revealed notable heterogeneity in magnum cells between the egg-laying and ceased-laying ducks, primarily characterized by variations in cells involved in protein secretion and extracellular matrix (ECM)-producing fibroblasts. Specifically, cells engaged in protein secretion were predominantly observed in the egg-laying ducks, indicative of their role in functional albumen deposition within the magnum, a phenomenon not observed in the ceased-laying ducks. Moreover, the proportion of THY1+ cells within the ECM-producing fibroblasts was found to be significantly higher in the egg-laying ducks (59%) compared to the ceased-laying ducks (24%). Similarly, TIMP4+ fibroblasts constituted a greater proportion of the ECM-producing fibroblasts in the egg-laying ducks (83%) compared to the ceased-laying ducks (58%). These findings suggest a potential correlation between the expression of THY1 and TIMP4 in ECM-producing fibroblasts and oviduct activity during functional reproduction. Our study provides valuable single-cell insights that warrant further investigation into the biological implications of fibroblast subsets in the degeneration of the reproductive tract. Moreover, these insights hold promise for enhancing the production efficiency of laying ducks.

Metabolites assay offers potential solution to improve the rooster semen cryopreservation.

Semen cryopreservation represents a promising technology utilized for preserving high-quality chicken varieties in husbandry practices. However, the efficacy of this methodology is significantly impeded by the diminished quality of sperm. Metabolites, as the end products of metabolic reactions, serve as indicators of biological processes and offer insights into physiological conditions. In this study, we investigaged the sperm quality and alteration in metabolic profiles during the cryopreservation of Longyou Partridge Chicken semen. Following artificial semen collection, four groups of semen samples were established based on four points of the cryopreservation process (Ⅰ, fresh semen; Ⅱ, semen added extender and chilled at 4 °C for 30 min; Ⅲ, semen added cryoprotectants; Ⅳ, semen gradient freezed and stored in liquid nitrogen). Semen cryopreservation has a negative effect on the percentage of sperm in a straight-line trajectory (LIN), has no significant effect on total motile sperms (TM) or the proportion of sperm with typical morphology (NM). Metabolites were identified using LC-MS technique and analyses including Principal Component Analysis (PCA), Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA), Univariate statistical analysis, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were employed to identify metabolites. A total of 2471 metabolites had been identified, with the majority of the list being made up of amino acids and their metabolites as well as benzene and substituted derivatives. Group II exhibits 882 metabolites with significantly elevated abundance relative to Group I, alongside 37 metabolites displaying decreased abundance. In Group III, 836 metabolites demonstrate notably augmented abundance compared to Group II, while 87 metabolites exhibit reduced abundance. Furthermore, Group IV showcases 513 metabolites with markedly heightened abundance in comparison to Group III, and 396 metabolites with decreased abundance. Specific metabolites such as 5-Hydroxylysine, Phosphocholine, and alpha-d-glucose-6-phosphate exhibited a progressive decline during the cryopreservation process, correlating with either dilution and chilling, cryoprotectant addition, or freezing. In conclusion, our investigation systematically examined the changes of seminal metabolome and sperm quality throughout the cryopreservation process of rooster semen.

Isolation and Proteomic Analysis of Extracellular Vesicles from Lactobacillus salivarius SNK-6.

The proteins carried by the extracellular vesicles of Lactobacillus salivarius SNK-6 (LsEVs) were identified to provide a foundation for further explorations of the probiotic activities of L. salivarius SNK-6. LsEVs were isolated from the culture media of L. salivarius SNK-6 and morphological analysis was conducted by scanning electron microscopy. Subsequent transmission electron microscopy and nanoparticle tracking analysis were performed to assess the morphology and particle size of the LsEVs. In addition, the protein composition of LsEVs was analyzed using silver staining and protein mass spectrometry. Finally, internalization of the identified LsEVs was confirmed using a confocal microscope, and enzyme-linked immunosorbent assay was employed to analyze the levels of inflammatory cytokines in LPS-challenged RAW264.7 cells. The results revealed that the membrane-enclosed LsEVs were spherical, with diameters ranging from 100-250 nm. The LsEVs with diameters of 111-256 nm contained the greatest amount of cargo. In total, 320 proteins (10-38 kD) were identified in the LsEVs and included anti-inflammatory molecules, such as PrtP proteinase, co-chaperones, and elongation factor Tu, as well as some proteins involved in glycolysis/gluconeogenesis, such as fructose-1,6-bisphosphate aldolase. Enrichment analysis showed these proteins to be related to the terms "metabolic pathway," "ribosome," "glycolysis/gluconeogenesis," "carbohydrate metabolism," and "amino acid metabolism." Furthermore, the LsEVs were internalized by host liver cells and can regulate inflammation. These findings confirm that LsEVs contain various functional proteins that play important roles in energy metabolism, signal transduction, and biosynthesis.

Comparative analysis of the hypothalamus transcriptome of laying ducks with different residual feeding intake.

Feed costs account for approximately 60 to 70% of the cost of poultry farming, and feed utilization is closely related to the profitability of the poultry industry. To understand the causes of the differences in feeding in Shan Partridge ducks, we compared the hypothalamus transcriptome profiles of 2 groups of ducks using RNA-seq. The 2 groups were: 1) low-residual feed intake (LRFI) group with low feed intake but high feed efficiency, and 2) high-residual feed intake (HRFI) group with high feed intake but low feed efficiency. We found 78 DEGs were enriched in 9 differential Kyoto Encyclopedia of Genes and Genome (KEGG) pathways, including neuroactive ligand-receptor interaction, GABAergic synapse, nitrogen metabolism, cAMP signaling pathway, calcium signaling pathway, nitrogen metabolism, tyrosine metabolism, ovarian steroidogenesis, and gluconeogenesis. To further identify core genes among the 78 DEGs, we performed protein-protein interaction and coexpression network analyses. After comprehensive analysis and experimental validation, 4 core genes, namely, glucagon (GCG), cholecystokinin (CCK), gamma-aminobutyric acid type A receptor subunit delta (GABRD), and gamma-aminobutyric acid type A receptor subunit beta1 (GABRB1), were identified as potential core genes responsible for the difference in residual feeding intake between the 2 breeds. We also investigated the level of cholecystokinin (CCK), neuropeptide Y (NPY), peptide YY (PYY), ghrelin, and glucagon-like peptide1 (GLP-1) hormones in the sera of Shan Partridge ducks at different feeding levels and found that there was a difference between the 2 groups with respect to GLP-1 and NPY levels. The findings will serve as a reference for future research on the feeding efficiency of Shan Partridge ducks and assist in promoting their genetic breeding.

Effects of dietary supplement of ε-polylysine hydrochloride on laying performance, egg quality, serum parameters, organ index, intestinal morphology, gut microbiota and volatile fatty acids in laying hens.

BACKGROUND: ε-polylysine hydrochloride (ε-PLH) is a naturally occurring antimicrobial peptide extensively utilized in the food and medical industries. However, its impact on animal husbandry remains to be further explored. Therefore, the present study aimed to determine the effect of ε-PLH on laying hens' health and laying performance. RESULTS: Dietary supplementation with ε-PLH to the diet significantly increased average egg weight during weeks 1-8. Meanwhile, compared with the control group, supplementation with ε-PLH decreased the feed egg ratio during weeks 9-12 and egg breakage rate during weeks 9-16 ，whereas it increased eggshell strength during weeks 1-4 and 13-16 . The ε-PLH 0.05% group increased yolk percentage during weeks 5-8 and yolk color during weeks 1-4 . Furthermore, ε-PLH supplementation significantly increased the concentrations of total protein, albumin, globulin and reproductive hormones estradiol, as well as decreased interleukin-1 beta and malondialdehyde in the serum. Compared with the control group, supplementation with 0.05% ε-PLH significantly increased the relative abundance of Cyanobacteria and Gastranaerophilales and decreased the abundance of Desulfovibrio and Streptococcus in the cecum microbiota. In addition, ε-PLH 0.1% supplementation also increased acetic acid content in the cecum. CONCLUSION: Dietary supplementation with ε-PLH has a positive impact on both productive performance and egg quality in laying hens. Furthermore, ε-PLH can also relieve inflammation by promoting the immunity and reducing oxidative damage during egg production. ε-PLH has been shown to improve intestinal morphology, gut microbial diversity and intestinal health. © 2023 Society of Chemical Industry.

Japanese encephalitis virus perturbs PML-nuclear bodies by engaging in interactions with distinct porcine PML isoforms.

Promyelocytic leukemia (PML) protein constitutes an indispensable element within PML-nuclear bodies (PML-NBs), playing a pivotal role in the regulation of multiple cellular functions while coordinating the innate immune response against viral invasions. Simultaneously, numerous viruses elude immune detection by targeting PML-NBs. Japanese encephalitis virus (JEV) is a flavivirus that causes Japanese encephalitis, a severe neurological disease that affects humans and animals. However, the mechanism through which JEV evades immunity via PML-NBs has been scarcely investigated. In the present study, PK15 cells were infected with JEV, and the quantity of intracellular PML-NBs was enumerated. The immunofluorescence results indicated that the number of PML-NBs was significantly reduced in JEV antigen-positive cells compared to viral antigen-negative cells. Subsequently, ten JEV proteins were cloned and transfected into PK15 cells. The results revealed that JEV non-structural proteins, NS2B, NS3, NS4A, NS4B, and NS5, significantly diminished the quantity of PML-NBs. Co-transfection was performed with the five JEV proteins and various porcine PML isoforms. The results demonstrated that NS2B colocalized with PML4 and PML5, NS4A colocalized with PML1 and PML4, NS4B colocalized with PML1, PML3, PML4, and PML5, while NS3 and NS5 interacted with all five PML isoforms. Furthermore, ectopic expression of PML isoforms confirmed that PML1, PML3, PML4, and PML5 inhibited JEV replication. These findings suggest that JEV disrupts the structure of PML-NBs through interaction with PML isoforms, potentially leading to the attenuation of the host's antiviral immune response.

Porcine promyelocytic leukemia protein isoforms suppress Japanese encephalitis virus replication in PK15 cells.

BACKGROUND: Promyelocytic leukemia protein (PML) is a primary component of PML nuclear bodies (PML-NBs). PML and PML-NBs play critical roles in processes like the cell cycle, DNA damage repair, apoptosis, and the antiviral immune response. Previously, we identified five porcine PML alternative splicing variants and observed an increase in the expression of these PML isoforms following Japanese encephalitis virus (JEV) infection. In this study, we examined the functional roles of these PML isoforms in JEV infection. METHODS: PML isoforms were either knocked down or overexpressed in PK15 cells, after which they were infected with JEV. Subsequently, we analyzed the gene expression of PML isoforms, JEV, and the interferon (IFN)-ß signaling pathway using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Viral titers were determined through 50% tissue culture infectious dose (TCID50) assays. RESULTS: Our results demonstrated that the knockdown of endogenous PML promoted JEV replication, while the overexpression of PML isoforms 1, 3, 4, and 5 (PML1, PML3, PML4, and PML5) inhibited JEV replication. Further investigation revealed that PML1, PML3, PML4, and PML5 negatively regulated the expression of genes involved in the interferon (IFN)-ß signaling pathway by inhibiting IFN regulatory factor 3 (IRF3) post-JEV infection. CONCLUSIONS: These findings demonstrate that porcine PML isoforms PML1, PML3, PML4, and PML5 negatively regulate IFN-ß and suppress viral replication during JEV infection. The results of this study provide insight into the functional roles of porcine PML isoforms in JEV infection and the regulation of the innate immune response.

Macroevolution of avian T cell receptor C segments using genomic data.

All jawed vertebrates have four T cell receptor (TCR) chains expressed by thymus-derived lymphocytes that play a significant role in animal immune defense. However, avian TCR studies have been limited to a few species, although their co-functional major histocompatibility complexes (MHCs) have been studied for decades, showing various copy numbers and polymorphisms. Here, using public genome data, we characterized the copy numbers, the phylogenic relationship and selection of T cell receptor complex (TCR-C) segments, and the genomic organization of TCR loci across birds. Various numbers of C segments were found in the TCRα/TCRδ, TCRß, and TCRγ loci, and phylogenetic analysis reflected both ancient gene duplication events (two Cß segments and Cδ segments divergent into CδI and CδII) and contemporary evolution (lineage-specific and species-specific characteristics). Most passerines lack CδII segments and a second TRD locus, except Meliphagidae and Maluridae. A relatively stable structure was verified in four TCR loci of birds, except for the arrangement of V segment groups. In this study, we explored the phylogenetic relationships of TCR-C segments across avians for the first time. We inferred gene duplication and loss events during the evolution process. The finding of diverse TCR germline repertoires provides a better understanding of the immune systems of birds.

Dynamic distribution of gut microbiota in posthatching chicks and its relationship with average daily gain.

The colonization and development of gut microbiota are essential for the health and growth of chicks after hatching. However, the colonization and prevalence of gut microbiota have not been well characterized, and knowledge of which microbes and their relationship with average daily gain in chicks is still limited. This study characterized the dynamic succession of microbiota in the intestinal tract of chicks and investigated its relationship with daily weight gain. A total of 121 fecal samples across 7 time points from d 0 to 10 posthatching were collected from 19 chicks randomly selected from 1,950 chicks. Using 16S rRNA gene sequencing examined microbial composition of fecal samples. The observed species index of alpha diversity increased with age, gradually achieving stability at 3 d of age. The microbiota of chicks after hatching was primarily Clostridium_sensu_stricto_1 (34.49%), and its relative abundance diminishes with age. In contrast, Lactobacillus had a low relative abundance in the first 2 d after hatching and gradually increased with age. Predicted functional capacities found that the microbiota of early-stage posthatching (d 0 and 1 after hatching) was involved in metabolism, including amino acid metabolism, metabolism of cofactors and vitamins, and nitrogen metabolism. However, at the later stage posthatching (from d 3-10 after hatching), the intestinal microbial function was involved in carbohydrate metabolism, amino acid metabolism, cell growth and death, and methane metabolism. It was identified that 47 operational taxonomic units were associated with average daily gain of chicks, 12 of which were annotated with Lactobacillus and significantly positive associated with average daily gain. In addition, Clostridium_sensu_stricto_1 was significantly negatively associated with average daily gain. Taken together, we characterized the dynamic successions of intestinal microbiota in hatching chicks. The intestinal microbiota of chicks has an impact on the host average daily gain. Our findings should be instrumental in improving local chick production.

Identification of the differentially expressed genes in the leg muscles of Zhedong white geese (Anser cygnoides) reared under different photoperiods.

Light is a factor affecting muscle development and meat quality in poultry production. However, few studies have reported on the role of light in muscle development and meat quality in geese. In this experiment, 10 healthy 220-day-old Zhedong white geese were reared for 60 d under a long photoperiod (15L:9D, LL) and short photoperiod (9L:15D, SL). The gastrocnemius muscles were collected after slaughter to evaluate muscle fiber characteristics and meat color, and RNA-seq analysis. The results showed that compared to the LL group, the SL group had large muscle fiber diameter and cross-sectional area, few muscle fibers per unit area, high meat color a* value, and low L* value at 24 h postmortem. On comparing the 2 groups, 70 differentially expressed genes (DEGs) were identified. Compared to the SL group, the LL group had 25 upregulated and 45 downregulated genes. Gene Ontology (GO) enrichment analysis showed that these DEGs were mainly involved in cell, cell part, binding, cellular processes, and single-organism processes. Several significantly enriched athways were identified in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, such as the calcium and PI3k-Akt signaling pathways. The expression of five randomly selected DEGs was verified using quantitative real-time PCR, and the results were consistent with the RNA-seq data. This study provides a theoretical basis for studying the molecular mechanisms by which light affects muscle development and meat color in geese.

LncRNA-SUSAJ1 Activates the ER Stress Pathway Inhibiting JEV Proliferation by Promoting PK15 Cells Apoptosis.

BACKGROUND: Epidemic encephalitis B is a common zoonosis that threatens both pigs and humans. Effective prevention and control of epidemic encephalitis B is difficult. The cellular defence mechanism is closely related to the body's resistance to viral invasion. Long non-coding RNAs (lncRNAs) are involved in regulating various cellular activities. We previously found that lncRNA-SUSAJ1 could inhibit the proliferation of Japanese encephalitis virus (JEV). However, the mechanism underlying this suppression remains unclear. METHODS: We performed Western blotting and quantitative reverse-transcription polymerase chain reaction (RT-qPCR) analyses, as well as mitochondrial membrane potential, flow cytometry, terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL), RNA pull-down, and RNA immunoprecipitation assays. RESULTS: JC-1 cationic dye staining showed that lncRNA-SUSAJ1 promoted the depolarisation of mitochondrial membrane potential; H2DCFDA probe staining showed that lncRNA-SUSAJ1 enhanced the level of reactive oxygen species in PK15 porcine kidney cells. qRT-PCR and Western blotting revealed the expression levels of associated mRNAs and proteins, and the TUNEL and flow cytometry assays detected cell apoptosis. Their results showed that lncRNA-SUSAJ1 promoted the expression of pro-apoptotic genes and inhibited the expression of anti-apoptotic genes. RNA pull-down experiments using biotin-labelled lncRNA-SUSAJ1 showed colocalisation between lncRNA-SUSAJ1 and the 70 kDa heat shock protein (Hsp70). lncRNA-SUSAJ1 also activated unfolded protein response-related pathways, regulated protein degradation, and promoted apoptosis via the endoplasmic reticulum stress response, thereby inhibiting viral replication. CONCLUSIONS: The findings of this study provide insight into the specific molecular mechanism of lncRNA-SUSAJ1 resistance to viral proliferation by promoting cell apoptosis, clarify the antiviral effect of lncRNA-SUSAJ1 on JEV.

Comparative analysis of the follicular transcriptome of Zhedong white geese (Anser Cygnoides) with different photoperiods.

The laying performance of geese is mainly determined by follicular development and atresia, while follicular status is regulated by photoperiod. To understand the effect of photoperiod on the development of goose follicles, artificial light was used to change the photoperiod. In this study, ten healthy 220-day-old Zhedong white geese (Anser Cygnoides) with similar body weights and similar reproductive start times were reared for 60 days under long photoperiod (15 L:9 D) and short photoperiod (9 L:15 D) artificial light with the intensity controlled at 30 lux, and follicles were collected. Follicle development was analyzed by observing the morphology of follicle tissue, the localization of autophagosomes and autolysosomes, and the expression levels of apoptosis-related protein factors. Small white follicles (SWFs) were selected for RNA sequencing and bioinformatics analysis of the transcriptome. Under a long photoperiod, microtubule-associated protein 1 light chain 3 (LC3) and Caspase-3 were expressed in the granulosa cell layer and oocytes, respectively. LC3 and Caspase-3 protein expression was increased in SWF and large white follicles (LWFs), and there were more autophagosomes and autolysosomes in granulosa cells. RNA-seq found 93 differentially expressed genes (DEGs) in the short-photoperiod group, including 55 upregulated DEGs and 38 downregulated DEGs, distributed in 37 gene ontology categories. Kyoto Encyclopedia of Genes and Genomes-enriched signaling pathways revealed 5 pathways enriched in upregulated DEGs, including protein digestion and absorption, ECM-receptor interaction and regulation of lipolysis in adipocytes, and 4 pathways enriched in downregulated DEGs, such as fatty acid biosynthesis. Ten differentially expressed genes related to extracellular matrix and fatty acid metabolism (THBS2, COL12A1, MRC2, TUBA, COL1A1, COL11A1, HSPG2, FABP, MGLL, and OLAH) may be involved in the photoperiod regulation of follicle development in Zhedong white geese. The differentially expressed genes screened in this study will provide new ideas to further understand the molecular mechanism underlying photoperiod-mediated regulation of follicle development in Zhedong white geese.

A Specific microRNA Targets an Elongase of Very Long Chain Fatty Acids to Regulate Fatty Acid Composition and Mitochondrial Morphology of Skeletal Muscle Cells.

Recently, miR-22 has been suggested to be an important microRNA (miRNA) affecting meat quality. Studies have shown that muscle fatty acid composition and mitochondrial function are closely related to meat quality. The regulatory mechanism of miR-22 on skeletal muscle fatty acid composition and mitochondrial function is not well characterized. Therefore, we aimed to explore the effects of miR-22 on fatty acid composition and mitochondrial function in C2C12 cells. Here, it demonstrate that elevated expression of miR-22 significantly repressed fatty acid elongation and mitochondrial morphology in C2C12 myoblasts, while the knockdown of miR-22 showed opposite results. Furthermore, miR-22 targets the elongase of very long chain fatty acids 6 (ELOVL6) and represses its expression in muscle cells. Knockdown of ELOVL6 mimicked the effect of miR-22 on fatty acid composition and mitochondrial function, while overexpression of ELOVL6 restored the effects of miR-22. These findings indicate that miR-22 downregulates the elongation of fatty acids and mitochondrial morphology by inhibiting ELOVL6 expression in muscle cells, which may provide some useful information for controlling muscle lipid accumulation and mitochondrial function in livestock in the future.

A polymorphism in porcine miR-22 is associated with pork color.

MicroRNAs (miRNAs) are posttranscriptional regulators that play key roles in meat color regulation. Changes in miRNA expression affect their target mRNAs, leading to multifunctional effects on biological processes and phenotypes. In this study, a G > A mutation site located upstream of the precursor miR-22 sequence in Suhuai pigs was significantly correlated with the meat color parameter a*(redness) of the porcine longissimus dorsi (LD) muscle. AA genotype individuals had the highest average meat color a* value and the lowest miR-22 level. When G > A mutation was performed in the miR-22 overexpression vector, miR-22 expression significantly decreased. Considering that Ca2+ homeostasis is closely related to pig meat color, our results further demonstrated that ELOVL6 is a direct target of miR-22 in pigs. The effects of miR-22 on skeletal muscle intracellular Ca2+ were partially caused by the suppression of ELOVL6 expression.

Phosphoglycerate dehydrogenase positively regulates the proliferation of chicken muscle cells.

Phosphoglycerate dehydrogenase (PHGDH) is the rate-limiting enzyme in the serine synthesis pathway. However, the regulatory role of PHGDH in muscle development is unclear. We report that the expression of PHGDH increased significantly during proliferation of chicken skeletal muscle satellite cells. Knockdown of PHGDH by an siRNA suppressed myoblast proliferation, whereas overexpression of PHGDH enhanced muscle cell proliferation. Furthermore, PHGDH promoted the expression of Forkhead box protein M1 (FoxM1). Knockdown of FoxM1 by an siRNA attenuated the proliferation of chicken muscle cells, whereas its overexpression significantly promoted proliferation. Additionally, siRNA-PHGDH inhibited pcDNA3.1-FoxM1-induced FoxM1 expression in chicken muscle cells. Moreover, PHGDH inhibition overcame the stimulation by pcDNA3.1-FoxM1 of cell cycle-related gene expression. We propose that PHGDH accelerates chicken muscle cell proliferation by increasing FoxM1 expression.

Reconstructing Macroevolutionary Patterns in Avian MHC Architecture With Genomic Data.

The Major Histocompatibility Complex (MHC) is a hyper-polymorphic genomic region, which forms a part of the vertebrate adaptive immune system and is crucial for intra- and extra-cellular pathogen recognition (MHC-I and MHC-IIA/B, respectively). Although recent advancements in high-throughput sequencing methods sparked research on the MHC in non-model species, the evolutionary history of MHC gene structure is still poorly understood in birds. Here, to explore macroevolutionary patterns in the avian MHC architecture, we retrieved contigs with antigen-presenting MHC and MHC-related genes from available genomes based on third-generation sequencing. We identified: 1) an ancestral avian MHC architecture with compact size and tight linkage between MHC-I, MHC-IIA/IIB and MHC-related genes; 2) three major patterns of MHC-IIA/IIB unit organization in different avian lineages; and 3) lineage-specific gene translocation events (e.g., separation of the antigen-processing TAP genes from the MHC-I region in passerines), and 4) the presence of a single MHC-IIA gene copy in most taxa, showing evidence of strong purifying selection (low dN/dS ratio and low number of positively selected sites). Our study reveals long-term macroevolutionary patterns in the avian MHC architecture and provides the first evidence of important transitions in the genomic arrangement of the MHC region over the last 100 million years of bird evolution.

Molecular Cloning of Alternative Splicing Variants of the Porcine PML Gene and Its Expression Patterns During Japanese Encephalitis Virus Infection.

Promyelocytic leukemia (PML) protein is a crucial component of PML-nuclear bodies (PML-NBs). PML and PML-NBs are involved in the regulation of various cellular functions, including the antiviral immune response. The human PML gene can generate several different isoforms through alternative splicing. However, little is known about the porcine PML alternative splicing isoforms and their expression profiles during Japanese encephalitis virus (JEV) infection. In the present study, we cloned seven mature transcripts of porcine PML, all of which contained the same N-terminal sequence but differed in the C-terminal sequences due to alternative splicing. These seven transcripts encoded five proteins all of which had the RBCC motif and sumoylation sites. Amino acid sequence homology analysis showed that porcine PML-1 had relatively high levels of identity with human, cattle, and goat homologs (76.21, 77.17, and 77.05%, respectively), and low identity with the mouse homolog (61.78%). Immunofluorescence analysis showed that the typical PML-NBs could be observed after overexpression of the five PML isoforms in PK15 cells. Quantitative reverse transcription PCR (RT-qPCR) analysis showed significant upregulation of PML isoforms and PML-NB-associated genes (Daxx and SP100) at 36 and 48 h post-infection (hpi). Western blotting analysis indicated that the PML isoforms were upregulated during the late stage of infection. Moreover, the number of PML-NBs was increased after JEV infection. These results suggest that porcine PML isoforms may play essential roles in JEV infection.