Proteomic analysis identifies potential markers in small white and small yellow follicle development in chickens.

Extensive knowledge of follicular development is imperative for improving egg production in chickens. The functional role of follicles to produce oocytes (eggs) is well recognised; however, specific markers associated with follicle development have been poorly explored. Therefore, a tandem mass tag based proteomic technique was used to identify the status of the proteome of small white follicles (1-4mm) and small yellow follicles (6-8mm). Analysis of differentially expressed proteins (DEP, Fold Change>1.2, P -value<0.05) demonstrated a total of 92 proteins (n =92), of which 35 (n =35) were upregulated and 57 were downregulated. DEP were further used for gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathways. The GO analysis found that DEP were mainly associated with the RNA metabolic process, cellular component organisation, peptide biosynthetic process and protein folding, thereby suggesting a key role in the follicle development process. Kyoto Encyclopedia of Genes and Genomes enrichment pathway analysis of the DEP substantiated the findings of GO analysis and described that DEP are involved in regulation of the cytoskeleton, carbon metabolism and amino acid biosynthesis. The validation of proteomic data through real-time quantitative polymerase chain reaction suggested HSPA8, HSPA2, SOD1 and FKPB3 as potential markers of small white and small yellow follicle development. This study demonstrates an understanding of proteome dynamics and represents the most comprehensive information on the entire Guangxi Ma chicken follicular proteome.

Spatiotemporal single-cell RNA sequencing reveals the role of steroid hormone pathway during chicken primordial follicle formation.

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

Effects of low nitrogen on seedling growth, photosynthetic characteristics and antioxidant system of rice varieties with different nitrogen efficiencies.

Nitrogen plays a significant role in influencing various physiological processes in plants, thereby impacting their ability to withstand abiotic stresses. This study used hydroponics to compare the effects of three nitrogen supply levels (1N, 1/2N and 1/4N) on the antioxidant capacity of rice varieties JJ88 (nitrogen efficient) and XN999 (nitrogen inefficient) with different nitrogen use efficiencies. The results show that compared with the XN999 variety, the JJ88 variety has stronger adaptability to low-nitrogen conditions, which is mainly reflected in the relatively small decrease in dry weight and net photosynthetic rate (Pn); In the early stage of low-nitrogen treatment (0-7 d), the [Formula: see text] production rate, hydrogen peroxide (H2O2) and malondialdehyde (MDA) content of JJ88 variety increased relatively slightly, but the superoxide dismutase (SOD), peroxide The activity of enzyme (POD) and catalase (CAT) increased significantly; After low-nitrogen treatment, the ASA-GSH cycle enzyme activity of JJ88 variety was relatively high, and the dehydroascorbate reductase (DHAR) activity after 14 days of low-nitrogen treatment was higher than that of 1N treatment; The content of reduced ascorbic acid (ASA) in non-enzymatic antioxidants was lower than that of 1N treatment after 14 days of low nitrogen treatment; The contents of oxidized dehydroascorbic acid (DHA) and carotenoids (Car) were higher than those of 1N treatment after 21d and 14d of low nitrogen treatment respectively; The contents of reduced glutathione (GSH), oxidized glutathione (GSSG) and proline (Pro) showed a larger upward trend during the entire low-nitrogen treatment period. In summary, the JJ88 rice variety has a strong ability to regulate oxidative stress and osmotic damage under low nitrogen conditions. It can slow down plant damage by regulating antioxidant enzyme activity and antioxidant content. This provides a basis for achieving nitrogen reduction and efficiency improvement in rice and the breeding of nitrogen-efficient varieties.

Optimization of the base editor BE4max in chicken somatic cells.

Advanced animal reproductive and breeding biotechnology has made it possible to alter traits or create new genetic resources by the direct knock-in or knock-out of target genes. Base editing technology can achieve single-base mutations without double-stranded DNA breaks, and is a promising tool for use in the genetic modification and breeding of livestock. However, the application of base editors (BEs) in chicken has not been optimized. We evaluated the efficacy of BE4max in chicken somatic cells (DF-1). The key element of BE4max, cytosine deaminase (APOBEC), was optimized for chicken. The base editing efficiency of the optimized chBE4max editor, compared with the original BE4max editor, was improved by 10.4% ± 4.6. By inhibiting the expression of the uracil DNA glycosylase-related gene methyl binding domain protein 4 (MBD4) by siRNA in chicken DF-1 cells, the editing efficiency was enhanced by 4.43% ± 1.4 compared to the control. These results suggest that this editor may have applications in poultry breeding studies.

Proteomic Analysis Identifies Potential Markers for Chicken Primary Follicle Development.

Follicles' development in chicken imparts a major impact on egg production. To enhance the egg-laying efficiency, comprehensive knowledge of different phases of follicular development is a prerequisite. Therefore, we used the tandem mass tag (TMT) based proteomic approach to find the genes involved in the primary follicular development of chicken. The primary follicles were divided into two groups-small primary follicles (81-150 µm) and developed primary follicles (300-500 µm). Differential expression analysis (fold change > 1.2, p-value < 0.05) revealed a total of 70 differentially expressed proteins (DEPs), of which 38 were upregulated and 32 were downregulated. Gene ontology (GO) enrichment analysis disclosed that DEPs were intricate with cellular protein localization, the establishment of protein localization, and nucleoside phosphate-binding activities. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway indicated the involvement of DEPs in different metabolic pathways such as glycolysis, pyruvate metabolism, galactose metabolism, and fructose and mannose metabolism. The current proteomic analysis suggested suitable markers such as Anxa2, Pdia3, and Capzb, which may serve as a potential role for primary follicle development. The present study provides the first insight into the proteome dynamics of primary follicle development and would play a potential role for further studies in chicken to improve egg productivity.

Genome-wide analysis of the ovodefensin gene family: Monophyletic origin, independent gene duplication and presence of different selection patterns.

Ovodefensins (OvoDs) represent a group of cysteine-rich host defense peptides that are abundant in the egg white. Recent studies have found that ovodefensins are specific to birds and reptiles. However, the entire repertoire and evolutionary relationships of this gene family have not been thoroughly elucidated to date. Following our cross-species and genome-wide computational study, a total of 94 ovodefensin genes with multiple novel cysteine sequence motifs were identified from 22 phylogenetically divergent species. Phylogenetic analysis suggests that a large number of OvoDs evolved by gene duplication after species divergence. Furthermore, the OvoD genes in each species trend to be clustered densely in a syntenic region flanked by the XKR6 and MTMR9 genes, indicating that they are of monophyletic origin and appear to have emerged via independent gene duplication events in snakes, turtles, crocodiles, birds and the green lizard. Furthermore, positive selection sites are located primarily in the mature peptide region of the turtle, lizard and snake OvoD genes. Moreover, the duplicate OvoDAs in birds seem to be maintained in almost identical sequences and functions by strong purifying selection. Genome-wide identification and analyses of the OvoD gene family may greatly improve our understanding of the potential evolutionary relationship scenario of the OvoD gene family. Continued sequence mining and functional studies of OvoDs will be helpful in shedding light on the relationships between OvoDs and other defensin-related gene families.

Transcriptome analysis reveals differentially expressed genes and pathways for oviduct development and defense in prelaying and laying hens.

PROBLEM: The oviduct plays an indispensable role in the formation of eggs, especially the magnum and uterus. The identification of oviduct development in different stages will help to target candidate genes and pathways in regulation of albumen and eggshell formation, as well as defense mechanism in oviduct and egg. METHODS: To identify the function differences and the molecular defense mechanism of the oviduct and egg, we performed transcriptome sequencing analysis of the magnum and uterus in 120-d-old and 300-d-old Lohmann layers, three birds in each group. RESULTS: With fold changes (log2 ratio) ≥ 2 and false discovery rate (FDR) < 0.01, RNA-Seq revealed 1,040 genes expressed differentially in the magnum and 595 genes in the uterus. By combining GO enrichment and KEGG pathway analysis, it served to show that gene activities of the magnum and uterus in prelaying chickens were more likely to concentrate on growth and development, and after egg-laying, they were mainly inclined to enhance the substances transmembrane transport and secretion activities. We further characterized 1579 new genes, while only 803 of them were functionally annotated. A complex mixture of proteins related to defense was measured in this study. A subset of avian ß-defensins (AvBDs) and ovodefensins (OvoDs), that is, AvBD12, AvBD11, AvBD10, OvoDA1, OvoDB1, OvoDA2, OvoDA3, and OvoDBß, was detected to express in the magnum of laying hens at high levels. CONCLUSION: Collectively, the identification and functional analysis of these differentially expressed genes (DEGs), as well as specific expression of avian defensins, may contribute to understand the development and defense mechanisms of oviduct and eggs.

Molecular Cloning and Expression Analysis of Interleukin-8 and -10 in Yellow Catfish and in Response to Bacterial Pathogen Infection.

The yellow catfish (Pelteobagrus fulvidraco) is an important economic freshwater aquaculture species in Asia. However, little is known about its immune response to bacterial pathogen infection. Here, two cytokines, the proinflammatory cytokine interleukin-8 (IL-8) and the anti-inflammatory cytokine interleukin-10 (IL-10), were identified and characterized in the yellow catfish for the first time. We found that the full length of the IL-8 cDNA was 784 bp and contained an open reading frame (ORF) of 336 bp, while the IL-10 gene was 973 bp in length with a 549 bp of ORF. In addition, both the IL-8 and the IL-10 had similar tissue-specific expression patterns. They were more abundant in the spleen and lowest expressed in the liver. Furthermore, IL-10 but not IL-8 was significantly upregulated in the intestine of yellow catfish by feed supplementation of Clostridium butyricum (CB). More importantly, the expression levels of intestinal IL-10 and IL-8 were up- and downregulated by pathogen Aeromonas punctata stimuli with the presence of CB, respectively. Collectively, these results suggest that IL-10 and IL-8 mediate important roles in the immunity of yellow catfish, and feed supplementation of CB may able to reduce the intestinal inflammation caused by bacteria infections through regulating the expression of IL-10 and IL-8.

Identification and characterization of the myeloid differentiation factor 88 gene in yellow catfish.

Myeloid differentiation factor 88 (MyD88) is an important adapter protein of the innate immune system, but it has never before been reported in yellow catfish (Pelteobagrus fulvidraco). In this study, we cloned and characterized the yellow catfish MyD88 gene. The gene was 1230 bp in length and contained an 876-bp open reading frame which encodes a polypeptide of 291 amino acid residues. The theoretical molecular mass and isoelectric point of this polypeptide were 33.4341 kDa and 5.17, respectively. Furthermore, bioinformatic and phylogenetic analyses grouped yellow catfish MyD88 with MyD88 of other fish. We found that the deduced amino acid sequence showed that the conserved N-terminal death domain and the C-terminal typical Toll/interleukin-1 receptor domain were very similar to those of other fish. Moreover, reverse transcription PCR showed that yellow catfish MyD88 is ubiquitously expressed in all tissues examined, with highest expression levels observed in the spleen and lowest levels in the intestine. Importantly, MyD88 was shown to be significantly up-regulated in the intestines after 30-day dietary supplement of Clostridium butyricum. Collectively, these results indicate that yellow catfish MyD88 has a conserved structure and is probably an important component of innate immunity in yellow catfish. This study is the first to identify and characterize MyD88 in yellow catfish, thereby providing a reference for further research into the yellow catfish innate immune system.

Molecular characterization of a novel ovodefensin gene in chickens.

Host defense peptides (HDPs) represent a large group of diverse small peptides that play important roles in host defense and disease resistance. In vertebrates, one of the main types of HDPs belong to defensins, which are less than 100 amino acid residues and characterized by a highly conserved motif of cysteine residues. Recently, a subfamily of defensins, namely ovodefensins (OvoDs), has been identified in birds and reptiles. However, both their family members and evolutionary relationships remain unclear. In the present study, we cloned and characterized a novel gene namely OvoDBß in chickens. Our results showed that the full length of chicken OvoDBß mRNA contains 344 bp nucleotides and encodes a 61-amino acid protein. We further revealed that the mRNA of OvoDBß is abundant in the oviduct of laying hens but absent in many other tissues. Additionally, sequences comparison and analyses suggested that OvoDBß is orthologous to the gene previously known as zebra finch OvoDB1, albeit it might exhibit specific structures. Furthermore, both OvoDBα and OvoDBß were existent in the genome of each bird, implying that two types of OvoDBs sharing same cysteine motif have already emerged before the species divergence. More importantly, recombinant OvoDBß mature peptide exerted antibacterial activity against Escherischia coli (CICC23657 strain) in vitro. These results collectively indicated that the putative sequence, namely chicken OvoDBß, is a function gene with potential antimicrobial property. Discovery and function characterization of novel HDP genes may help us develop novel antimicrobial agents in the future.

Molecular cloning and expression analysis of Foxp3 from Nile tilapia.

Foxp3 is a crucial transcription factor for the development and function of CD4(+)CD25(+) regulatory T cells (Tr) which play a key role in preventing autoimmune diseases and maintaining maternal-fetal tolerance in mammals. However, the knowledge of Foxp3 and its regulation in teleosts is limited. In the present study, the Foxp3 cDNA was cloned from Nile tilapia and characterized. The full length cDNA of Nile tilapia (nt)Foxp3 contained a 5'UTR of 104 bp, a 3'UTR of 239 bp and an open reading frame of 1134 bp. The putative ntFoxp3 contained a C2H2 zinc finger domain, a winged-helix/fork head domain and a leucine zipper-like domain with a consensus of V-x(6)-L-x(6)-L-x(6)-L, which are typical motifs of a Foxp3. The highest mRNA expression of ntFoxp3 was observed in the spleen, with moderate expression in the head kidney, intestine, kidney, liver and brain. Stimulation of peripheral blood mononuclear cells with PHA and LPS led to a significant increase of ntFoxp3 mRNA expression at 6 and 24h, respectively. In contrast, stimulation with PMA had no effect during the sampling period. After injecting 1×10(7) cells of live Aeromonas hydrophila into adult female Nile tilapias, the mRNA expression of ntFoxp3 significantly increased in the gill and intestine at 6h, while unchanged in the spleen. In all the tissues examined, a clearly up-regulation of ntFoxp3 expression was detected at 24h after the second challenge. These results suggest that ntFoxp3 might be involved into lymphocyte activation and immune responses as reported in mammals. Meanwhile, the correlation between the expression profile of ntFoxp3 and serum estrogen (17-beta-estradiol, E2) concentration was investigated by real-time PCR and enzyme immunoassay. The results showed that E2 could regulate the expression of ntFoxp3 in the intestine and kidney but not in the spleen. This work will help future investigations into Tr cells and extend our understanding of interactions between sex hormones and immune related molecules in teleosts.