Quercetin (Que) possesses diverse biological activities and has been extensively investigated in various fields, but its impact on rooster reproductive performance and the underlying mechanisms remains poorly understood. The present study investigated the effect of quercetin on reproductive performance of roosters and preliminarily explored its underlying regulatory mechanism. Forty-eight 100-day-old roosters were randomly divided into control group and three quercetin groups (Que_5mg/d, Que_10mg/d, and Que_20mg/d). Daily gavage was conducted continuously for 60 days. Semen quality was evaluated using a sperm analyzer. Then, metabolomics, proteomics, network pharmacology, molecular dynamics simulation, hormone detection, qRT-PCR, and their combination analysis was employed for mechanism validation. The result of semen quality evaluation and testicular tissue morphology observation showed that quercetin can significantly increase the semen collection volume, semen motility (P < 0.05), sperm density was significantly higher in the Que_5mg/d and Que_10mg/d groups than in the control group (P < 0.05), and the diameter of the seminiferous tubules, the height of the seminiferous epithelium of the testes (P < 0.05). Consistently, both testicular metabolomics and hormone detection results indicated that quercetin significantly increased testosterone levels (P < 0.05). Metabolite KEGG enrichment analysis revealed a significant upregulation of the steroid hormone biosynthesis. Proteomics and qRT-PCR assays confirmed that quercetin upregulated the expression of genes such as CYP11A1, CYP17A1, and molecular docking and molecular dynamics simulations further indicate that quercetin has a favorable binding with steroid hormone biosynthesis related protein CYP11A1. These results demonstrates that supplementation with quercetin at a dosage of 10 mg/d can enhances reproductive performance in roosters by targeting steroid hormone biosynthesis-related proteins to promote hormone synthesis.

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.

[目的]鉴定出在蛋壳形成过程中可能参与钙转运的溶质载体(SLC)基因超家族成员,并分析SLC基因在蛋壳形成不同时期的表达特征,为进一步探究SLC基因与蛋壳形成的相关性及揭示钙转运调控机制打下基础.[方法]基于NCBI和Ensembl数据库,检索并整理鸡SLC基因超家族全基因信息,通过ProtParam、Pfam、TBtool、NPS@:SOPMA、Euk-mPLoc 2.0及MEGA 11.0等在线软件进行生物信息学分析,并利用实时荧光定量PCR分析钙转运相关SLC基因在蛋壳形成不同时期的表达特征.[结果]鸡SLC基因超家族包括56个亚家族,分布在包括Z染色体在内的32条染色体上,其编码蛋白主要分布在内质网和细胞膜上,大多数为疏水的碱性蛋白,涵盖81种结构域,主要参与核苷酸、糖类、氨基酸、神经递质、无机离子及有机离子等物质的运输,其中与钙离子(Ca2+)相关的SLC亚家族基因有SLC4、SLC8、SLC9、SLC24和SLC30.SLC8和SLC24亚家族基因在进化分支上的亲缘关系很近,存在直接与Ca2+转运的结构域,主要定位在细胞膜和内质网上.实时荧光定量PCR检测结果表明,控制Ca2+的SLC8和SLC24基因在产蛋鸡子宫部的表达显著高于未产蛋鸡(P<0.05,下同),进一步说明二者确实参与调控Ca2+跨膜运输,但各成员间分工有所不同.[结论]鸡SLC基因超家族编码产物多为疏水的碱性蛋白,含有结合多种物质的结构域,定位于细胞或多种亚细胞结构膜上,表明SLC家族蛋白成员参与多种物质的跨膜转运过程.其中,SLC8和SLC24亚家族基因在蛋壳形成过程中协同调控Ca2+从内质网释放并完成跨膜运输,分泌到输卵管子宫部而参与蛋壳的形成.

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.

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.

Newcastle disease (ND) is an avian respiratory disease caused by the Newcastle disease virus (NDV) and is prevalent worldwide but lacks a reliable treatment to surmount it. The single-chain fragment variable (scFv) antibody is the smallest functional unit in an immunoglobulin retaining antigen-binding activities. Previous studies have demonstrated that by acting against the NDV phosphoprotein, scFv is capable of neutralizing the virus in infected cells. In this study, two anti-NDV scFv expressing cassettes driven by CAG promoter and EF1α promoter, respectively, were constructed using the piggyBac transposon plasmid backbone. The recombinant plasmids were transfected into DF-1 and 293T cells and four stable cell lines CAG-scFv-DF-1, EF1α-scFv-DF-1, CAG-scFv-293T and EF1α-scFv-293T were established after puromycin screening. RT-PCR results confirmed the presence of anti-NDV scFv mRNA in all four cell lines. Western blot results showed that scFv was detected in the culture supernatant of the CAG-scFv-DF-1 and EF1α-scFv-DF-1 lines, indicating that the scFv antibody was secretory after expression in the cells. Cytopathic effect assay showed that cells expressing scFv were more resistant to NDV F48E9 than those of control when the virus titer was not higher than 60×TCID50. Results in this study offer information to the generation of transgenic chickens resistant to NDV infection and for production of neutralizing antibodies against NDV.

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.

[目的]探索在慢羽鸡成纤维细胞中敲除ev21基因的可行性,净化鸡群内源性逆转录病毒,同时为快速培育缺失ev21基因的慢羽鸡配套系打下基础.[方法]根据ev21基因序列(KY235336)特点,分别在其5'和3'端各设计2个sgRNA,用于构建4种不同sgRNA的打靶质粒,筛选出在5'和3'端打靶效率较高的sgRNA.然后基于CRISPR/Cas9基因编辑技术对ev21基因进行剪切,并通过同源重组方式以红色荧光蛋白(mCherry)的DNA片段(CAG-mCherry)替换ev21基因,实现对慢羽鸡成纤维细胞内源性白血病病毒ev21基因定点敲除.[结果]在慢羽鸡成纤维细胞中能检测到ev21基因,构建的4种sgRNA(sgRNA1～sgRNA4)均能成功插入对应的打靶质粒中,经嘌呤霉素筛选及T7E1酶切检测,发现转染4种不同sgRNA打靶质粒后慢羽鸡成纤维细胞均有不同程度的死亡,其中又以sgRNA1和sgRNA3的基因敲除效率较高.同时针对同源位点左右同源臂构建表达mCherry的供体质粒,以其转染293T细胞12 h后均能表达出mCherry.以sgRNA1和sgRNA3打靶质粒及供体质粒共同转染慢羽鸡成纤维细胞,观察发现成纤维细胞内的mCher-ry持续表达,至转染后第30 d通过流式细胞仪分选收集红色荧光阳性成纤维细胞,并提取其总DNA进行PCR鉴定与基因测序,结果显示红色荧光阳性成纤维细胞中有目的片段(CAG-mCherry)插入,即以插入替换方式能实现对ev21基因的敲除.[结论]基于crispr/cas9基因编辑技术的基因敲除方法能成功敲除慢羽鸡成纤维细胞内源性白血病病毒ev21基因,为培育缺失ev21基因的慢羽鸡品系提供技术支持.

[目的]明确FoxO3转录因子在不同日龄鸡卵巢组织中的表达模式及其具体定位情况,为后续开展家禽卵泡激活及发育调控等相关机理研究提供科学依据.[方法]在GenBank中搜索鸡与其他物种(鸭、鹅、老鼠、猪、牛及人类)的FoxO3氨基酸序列,通过LaserGene分析鸡FoxO3氨基酸序列与其他物种FoxO3氨基酸序列间的亲缘关系及同源差异情况;利用RT-PCR鉴定FoxO3基因是否在鸡卵巢组织中表达,再采用实时荧光定量PCR检测不同发育阶段鸡卵巢组织中FoxO3基因的表达水平,最后以免疫荧光检测FoxO3蛋白在鸡卵巢组织中的定位情况.[结果]鸡与鸭和鹅的FoxO3氨基酸序列相似性分别为92.7％和95.3％,基于FoxO3氨基酸序列相似性构建的系统发育进化树也显示鸡与鸭和鹅的亲缘关系较近,说明FoxO3基因的进化相对较保守.在不同发育阶段的鸡卵巢组织中均能检测到FoxO3基因表达,且FoxO3基因在0日龄鸡卵巢组织中的相对表达量最高,显著高于在其他发育阶段的相对表达量(P<0.05).在0日龄鸡卵巢组织中未检测到FoxO3蛋白,但在21日龄和成年鸡的卵巢组织中均能检测到FoxO3蛋白;在21日龄鸡卵巢组织中FoxO3蛋白主要定位在原始卵泡细胞周围,在卵泡内部没有表达;而在成年鸡卵巢组织中FoxO3蛋白仅定位于大卵泡细胞边缘,小卵泡细胞内并未发现FoxO3蛋白.[结论]由于鸡和哺乳动物的FoxO3氨基酸序列高度同源,且FoxO3蛋白在鸡卵巢组织中的定位与在哺乳动物卵巢组织中的定位相似,说明FoxO3在鸡卵巢组织中发挥着与哺乳动物相似的功能,即在卵泡激活与成熟过程中发挥重要作用,因此通过调控FoxO3能有效提高鸡的繁殖性能.

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.

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 β-defensins (AvBDs) and ovodefensins (OvoDs), that is, AvBD12, AvBD11, AvBD10, OvoDA1, OvoDB1, OvoDA2, OvoDA3, and OvoDBβ, 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.

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.

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 OvoDBβ in chickens. Our results showed that the full length of chicken OvoDBβ mRNA contains 344 bp nucleotides and encodes a 61-amino acid protein. We further revealed that the mRNA of OvoDBβ is abundant in the oviduct of laying hens but absent in many other tissues. Additionally, sequences comparison and analyses suggested that OvoDBβ is orthologous to the gene previously known as zebra finch OvoDB1, albeit it might exhibit specific structures. Furthermore, both OvoDBα and OvoDBβ 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 OvoDBβ mature peptide exerted antibacterial activity against Escherischia coli (CICC23657 strain) in vitro. These results collectively indicated that the putative sequence, namely chicken OvoDBβ, 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.

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.

为探讨不同饲养方式对淘汰蛋鸡生产性能及蛋肉品质的影响,以240只淘汰笼养芦花蛋鸡为试验材料,随机分为两个处理,分别为大棚平养和继续舍内笼养,测定两种饲养方式下淘汰蛋鸡的产蛋性能和蛋品质,以及处理前、处理5周及10周的屠宰性能和肉品质.结果显示:平养产蛋率显著低于笼养,但蛋白高度和哈夫单位显著高于笼养;平养能有效降低淘汰蛋鸡的腹脂重,但其整体屠宰性能较笼养差;平养在肉色和滴水损失指标上好于笼养,但在肌内脂肪、粗水分、肌纤维直径与密度、氨基酸指标上差异不显著.综合分析,平养对淘汰芦花蛋鸡的部分蛋、肉品质指标有一定改善,但笼养生产性能相对更好.