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.

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.

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.

Inhibition of Japanese encephalitis virus proliferation by long non-coding RNA SUSAJ1 in PK-15 cells.

BACKGROUND: Japanese encephalitis virus is a mosquito-borne neurotropic flavivirus that causes acute viral encephalitis in humans. Pigs are crucial amplifier host of JEV. Recently, increasing evidence has shown that long non-coding RNAs (lncRNAs) play important roles in virus infection. METHODS: JEV proliferation was evaluated after overexpression or knockdown of lncRNA-SUSAJ1 using western blotting and reverse-transcription polymerase chain reaction (RT-PCR). C-C chemokine receptor type 1 (CCR1) was found to regulate the expression of lncRNA-SUSAJ1 by inhibitors screen. The expression of lncRNA-SUSAJ1 was detected using RT-PCR after overexpression or knockdown of transcription factor SP1. In addition, the enrichments of transcription factor SP1 on the promoter of lncRNA-SUSAJ1 were analyzed by chromatin immunoprecipitation. RESULTS: In this study, we demonstrated that swine lncRNA-SUSAJ1 could suppress JEV proliferation in PK-15 cells. We also found that CCR1 inhibited the expression of lncRNA-SUSAJ1 via the transcription factor SP1. In addition, knockdown of CCR1 could upregulated the expression of SP1 and lncRNA-SUSAJ1, resulting in resistance to JEV proliferation. CONCLUSIONS: These findings illustrate the importance of lncRNAs in virus proliferation, and reveal how this virus regulates lncRNAs in host cells to promote its proliferation.

Coenzyme Q10 ameliorates BPA-induced apoptosis by regulating autophagy-related lysosomal pathways.

Bisphenol A (BPA) is a widely distributed environmental endocrine disruptor. The accumulation of BPA has been proved that produce various toxic effects both on human and animals. However, the strategies to reduce the damage of BPA on the body and related mechanisms remain to be studied. Coenzyme Q10 (CoQ10), as a powerful antioxidant, is ubiquitous in many eukaryotic cells, which can improve the integrity of lysosomal membrane, lysosomal degradation function and promote autophagy. Here, we examined the ability of CoQ10 to alleviate oxidative stress and apoptosis in BPA-induced damages in C2C12 cells, and how to alleviate it. Our results showed that BPA treatment significantly reduced cell viability, increased the number of cell apoptosis and ROS production, decreased mitochondrial membrane potential, and inhibited the gene expression of mitochondria biogenesis. Moreover, we demonstrated that exposure to BPA increased expression levels of autophagy protein (LC3-II, p62), inhibited autophagy flux, and disrupted the acidic pH environment of lysosomes. Importantly, CoQ10 supplementation effectively restored these abnormalities caused by BPA. CoQ10 significantly decreased the apoptotic incidence and ROS levels, improved mitochondrial membrane potential. Moreover, CoQ10 improved lysosome function and enhanced autophagy flux. Taken together, our results indicate that CoQ10 supplementation is a feasible and effective way to promote the level of autophagy by improving lysosomal function, thereby reducing the apoptosis caused by BPA accumulation. This study aims to provide evidence for the role of CoQ10 in repairing BPA-induced cell damage in clinical practice.

T cell subset profile and inflammatory cytokine properties in the gut-associated lymphoid tissues of chickens during infectious bursal disease virus (IBDV) infection.

While infectious bursal disease virus (IBDV) mainly targets immature B cells and causes T cell infiltration in the bursa of Fabricius (BF) of chickens, the effect of IBDV infection on the properties of T cells and relevant cytokine production in avian gut-associated lymphoid tissues (GALTs) remains unknown. Here, we show that while the CD8+ T cell subset is not affected, IBDV infection decreases the percentage of CD4+ T cells in the cecal tonsil (CT), but not in esophagus tonsil, pylorus tonsil, and Meckel's diverticulum of GALTs, in contrast to BF and spleen, in which the proportion of CD4+ cells increases upon IBDV infection. Further, IBDV infection upregulates IFN-γ, IL-10, and the T cell checkpoint receptor LAG-3 mRNA expression in BF. In contrast, in CTs, IBDV infection significantly increases the production of IFN-ß and CTLA-4 mRNA, while no significant effect is seen in the case of IFN-γ, IL-10 and LAG-3. Together, our data reveal differential modulation of T cell subsets and proinflammatory cytokine production in different lymphoid tissues during the course of IBDV infection.

Genome-wide DNA methylation profiles of porcine ovaries in estrus and proestrus.

DNA methylation is an important epigenetic modification involved in the estrous cycle and the regulation of reproduction. Here, we investigated the genome-wide profiles of DNA methylation in porcine ovaries in proestrus and estrus using methylated DNA immunoprecipitation sequencing. The results showed that DNA methylation was enriched in intergenic and intron regions. The methylation levels of coding regions were higher than those of the 5'- and 3'-flanking regions of genes. There were 4,813 differentially methylated regions (DMRs) of CpG islands in the estrus vs. proestrus ovarian genomes. Additionally, 3,651 differentially methylated genes (DMGs) were identified in pigs in estrus and proestrus. The DMGs were significantly enriched in biological processes and pathways related to reproduction and hormone regulation. We identified 90 DMGs associated with regulating reproduction in pigs. Our findings can serve as resources for DNA methylome research focused on porcine ovaries and further our understanding of epigenetically regulated reproduction in mammals.

OGG1 regulates the level of symmetric dimethylation of histone H4 arginine-3 by interacting with PRMT5.

OGG1 is the first enzyme in the base excision repair pathway (BER) responsible for repairing 8-oxoguanine DNA lesions. Recent studies found that OGG1 may also be involved in epigenetic regulation. In this study, we focused on the roles of OGG1 in histone modification. First, to study the effects of OGG1 on histone modification, the protein levels of symmetric dimethylation of histone H4 arginine-3 (H4R3me2s) were determined by western blot analysis following the knockdown or overexpression of OGG1. Second, the molecular mechanisms by which OGG1 regulates H4R3me2s were assessed by co-immunoprecipitation (CO-IP) assays in mouse embryonic fibroblast (MEF) wild-type (WT) and Ogg-/- cells. Finally, to verify the regulation of H4R3me2s by OGG1 on specific genes, chromatin immunoprecipitation (CHIP) was performed on MEF WT and Ogg-/- cells. We found that OGG1 affects PRMT5 binding on histone H4 and the formation of H4R3me2s via PRMT5. The methylation level of H4R3me2s was dramatically decreased in MEF Ogg-/- cells compared to WT cells. Knockdown of OGG1 by siRNA led to a decrease in H4R3me2s, while overexpression of OGG1 increased the level of H4R3me2s. OGG1 also interacted with PRMT5 and histone H4, and the interaction between PRMT5 and histone H4 was reduced in MEF Ogg-/- cells. Our data not only illustrate the important roles of OGG1 in histone modification, but also reveal the mechanism by which OGG1 affects PRMT5 binding on H4R3 resulting in the symmetrical dimethylation of histone H4 arginine-3.

Transcriptome analysis of the livers of ducklings hatched normally and with assistance.

OBJECTIVE: "Hatchability" is an important economic trait in domestic poultry. Studies on poultry hatchability focus mainly on the genetic background, egg quality, and incubation conditions, whereas the molecular mechanisms behind the phenomenon that some ducklings failed to break their eggshells are poorly understood. METHODS: In this study, the transcriptional differences between the livers of normally hatched and assisted ducklings were systematically analyzed. RESULTS: The results showed that the clean reads were de novo assembled into 161,804 and 159,083 unigenes (≥200-bp long) by using Trinity, with an average length of 1,206 bp and 882 bp, respectively. The defined criteria of the absolute value of log2 fold-change ≥1 and false discovery rate≤0.05 were differentially expressed and were significant. As a result, 1,629 unigenes were identified, the assisted ducklings showed 510 significantly upregulated and 1,119 significantly down-regulated unigenes. In general, the metabolic rate in the livers of the assisted ducklings was lower than that in the normal ducklings; however, compared to normal ducklings, glucose-6-phosphatase and ATP synthase subunit alpha 1 associated with energy metabolism were significantly upregulated in the assisted group. The genes involved in immune defense such as major histocompatibility complex (MHC) class I antigen alpha chain and MHC class II beta chain 1 were downregulated in the assisted ducklings. CONCLUSION: These data provide abundant sequence resources for studying the functional genome of the livers in ducks and other poultry. In addition, our study provided insight into the molecular mechanism by which the phenomenon of weak embryos is regulated.

High-throughput sequencing reveals differential expression of miRNAs in prehierarchal follicles of laying and brooding geese.

Broodiness is the primary factor influencing egg production in geese, in which several genes and miRNAs participate. Detailed spatiotemporal profiles of miRNAs encompassing follicle development levels, however, are lacking. In this study, we collected preovulatory follicles (classified as small white follicles, large white follicles, and small yellow follicles) from brooding and laying geese and aimed to analyze microRNA (miRNA or miR) during folliculogenesis. High-throughput sequencing and bioinformatics analysis were used to identify the miRNAs involved in follicle development. The let7 family, miR-10 family, and miR-143 family were abundant in these libraries, and they have been suggested to play a housekeeping role during folliculogenesis. Joint comparisons revealed 23 upregulated and 21 downregulated miRNAs (in at least two comparisons of follicles during brooding and laying, P < 0.1) in the laying stage. Unlike reproduction pathways reported for ovaries, GO and KEGG analysis suggested pathways for cell apoptosis and proliferation, such as the regulation of actin cytoskeleton, endocytosis, axon guidance, pathways in cancer, tight junctions, focal adhesion, the MAPK signaling pathway, cytokine-cytokine receptor interactions, and the Wnt signaling pathway in folliculogenesis. This study revealed the miRNAs that were directly involved in follicular atresia, and our results added to the understanding of the functional involvement of miRNAs during specific stages of follicle development.

miR-124 attenuates Japanese encephalitis virus replication by targeting DNM2.

BACKGROUND: Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes acute viral encephalitis in humans. Pigs are important amplifier hosts of JEV. Emerging evidence indicates that host microRNAs (miRNAs) play key roles in modulating viral infection and pathogenesis. However, mechanistic studies delineating the roles of miRNAs in regulating host-JEV interactions remain scarce. RESULTS: In this study, we demonstrated that miR-124 inhibited JEV replication in porcine kidney epithelial PK15 cells. Furthermore, using bioinformatics tools, we identified dynamin2 (DNM2), a GTPase responsible for vesicle scission, as a target of miR-124. Small interfering RNA (siRNA) depletion studies inicated that dynamin2 was required for efficient JEV replication. We also demonstrated that upregulation of miR-124 expression corresponded to decreased expression of its target, DNM2, in the JEV-infected PK15 cells. CONCLUSIONS: Overall, these results suggest the importance of miR-124 in modulating JEV replication and provide a scientific basis for using cellular miRNAs in anti-JEV therapies.

Goose broodiness is involved in granulosa cell autophagy and homeostatic imbalance of follicular hormones.

Broodiness is observed in most domestic fowls and influences egg production. The goose is one of the most important waterfowls, having strong broody behavior. However, whether autophagy and follicular internal environment play a role in the broodiness behavior of goose is unknown. In this report, we analyzed the follicular internal environment and granulosa cell autophagy of goose follicles. The results show that the contents of hormones, including prolactin (PRL), progesterone (P4), and estradiol (E2), increased in broody goose follicles. Most importantly, the level of granulosa cell autophagy in broody goose follicles was elevated, detected by electron microscopy and western blotting. Also, the expressions of positive regulators of autophagy, including miR-7, miR-29, miR-100, miR-181, PRLR, LC3, p53,Beclin1, Atg9, and Atg12, were up-regulated and the expressions of negative regulators of autophagy, including miR-34b and miR-34c, were down-regulated in broody goose follicles. Our results suggest that goose broodiness is involved in increased granulosa cell autophagy and homeostasis imbalance of internal environment in the follicles. This work contributes to our knowledge of goose broodiness and may influence egg production.

The Influence of Increasing Roughage Content in the Diet on the Growth Performance and Intestinal Flora of Jinwu and Duroc × Landrace × Yorkshire Pigs.

The Jinwu pig (JW) is a hybrid breed originating from the Chinese indigenous Jinhua pig and Duroc pig, boasting excellent meat quality and fast growth rates. This study aimed to verify the tolerance of JW to roughage, similar to most Chinese indigenous pigs. In this research, two types of feed were provided to JW and Duroc × Landrace × Yorkshire pigs (DLY): a basal diet and a roughage diet (increasing the rice bran and wheat bran content in the basal diet from 23% to 40%) for a 65-day experimental period. The roughage diet showed an increasing trend in the feed conversion ratio (F/G), with a 17.61% increase in feed consumption per unit weight gain for DLY, while the increase for JW was only 4.26%. A 16S rRNA sequencing analysis revealed that the roughage diet increased the relative abundance of beneficial bacteria, such as Lactobacillus and Clostridium, while reducing the relative abundance of some potential pathogens, thus improving the gut microbiota environment. After being fed with the roughage diet, the abundance of bacterial genera, such as Treponema, Terrisporobacter, Coprococcus, and Ruminococcaceae, which aid in the digestion and utilization of dietary fiber, were significantly higher in Jinwu compared to DLY, indicating that these bacterial genera confer Jinwu with a higher tolerance to roughage than DLY.

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.

Effects of Different Photoperiods on the Transcriptome of the Ovary and Small White Follicles in Zhedong White Geese.

Photoperiod can regulate the broodiness of geese and thus increase their egg-laying rate. The laying performance of geese is mainly determined by ovary and follicle development. To understand the effect of photoperiod on the ovary and small white follicles, sixteen 220-day-old healthy female Zhedong white geese were randomly divided into two groups for long photoperiods (15L:9D) and short photoperiods (9L:15D). The geese were euthanized after two months of feeding, and their ovaries and follicles were collected for transcriptome sequencing. RNA-seq analysis identified 187 and 448 differentially expressed genes in ovaries and small white follicles of different photoperiod groups, respectively. A long photoperiod promotes high expression of SPP1, C6, MZB1, GP1BA, and FCGBP genes in the ovaries, and increases the expression of SPP1, ANGPTL5, ALPL, ZP1, and CHRNA4 genes in small white follicles. Functional enrichment analysis showed that photoperiod could affect respiratory system development, smooth muscle cell proliferation in ovaries, and extracellular matrix-related function in small white follicles. WGCNA revealed 31 gene modules, of which 2 were significantly associated with ovarian weight and 17 with the number of small white follicles. Our results provide a better understanding of the molecular regulation in the photoperiod affecting goose reproduction.

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.

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.

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.