microRNA transcriptome analysis of granulosa cells predicts that the Notch and insulin pathways affect follicular development in chickens.

MicroRNAs (miRNAs) have been documented to play critical roles in chicken reproduction. Granulosa cell (GC) development of the follicle is closely related to hierarchical follicle ordering, making it an important factor in determining laying performance. Thus, it is meaningful to mine follicular development-related miRNAs. To identify regulatory miRNAs and the biological mechanisms by which they control follicular development, we conducted small RNA sequencing of GCs isolated from prehierarchical follicles named small yellow follicle (SYFG), the smallest hierarchical follicle (F6G), and the largest hierarchical follicle (F1G). A total of 99, 196, and 110 differentially expressed miRNAs (DEMs) were identified in SYFG.vs.F6G, SYFG.vs.F1G, and F6G.vs.F1G, respectively. Of these, 22 miRNAs, including miR-223, miR-103a, miR-449c-3p, and miR-203a, were ubiquitously identified as DEMs in three stages. Target gene prediction suggested that these miRNAs are associated with the MAPK, TGF-ß, and Wnt signaling pathways, which are all associated with follicular development. The Notch and insulin signaling pathways were commonly enriched in all three comparisons. RT-qPCR analysis further indicated that the expression levels of PSEN2, which encodes an essential factor regulating Notch and insulin signaling, was significantly changed in SYFG, F6G, and F1G. The current study provides basic data and offers a new foundation for further exploration of the roles of miRNAs in follicular development in chickens.

The Effects of 1-Deoxynojirimycin from Mulberry on Oxidative Stress and Inflammation in Laying Hens and the Direct Effects on Intestine Epithelium Cells In Vitro.

The intestine is highly vulnerable to various factors and has been proposed as a promising determinant for poultry health. Phytogenic or plant-derived feed additives can be used to help improve intestinal health. In this study, we aimed to investigate the effects of DNJ on the antioxidative parameters, including malondialdehyde (MDA), total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and inflammatory cytokines (IL-6, IL-1ß, and TNF-α), in plasma and intestinal tissues using layers supplemented with or without the DNJ extract of mulberry leaves (DNJ-E) via the ELISA method. A total of 192 healthy Hy-Line Brown layers, aged 47 weeks old, were used to conduct a 56-day study. All hens were randomly separated into four groups as follows: a basal diet containing 0 mg/kg DNJ-E(CON), 50 mg/kg, 100 mg/kg, and 150 mg/kg DNJ-E. Furthermore, the potential mechanism by which DNJ influences intestinal function was also investigated in in vitro cultured intestinal epithelium cells (IEC) with quantification methods including the use of a cell counting kit-8 (CCK8), ELISA, qRT-PCR, and ROS detection. The results showed that CAT in plasma significantly increased following 50 mg/kg DNJ-E supplementation. Moreover, 50 mg/kg DNJ-E supplementation was associated with increases in T-SOD in the jejunum and ileum. However, there was no significant difference in inflammatory cytokines between groups in in vivo experiments. Subsequent in vitro IEC studies revealed that cell viability increased significantly following 5 µM and 10 µM DNJ treatments while decreasing significantly following 20 µM DNJ treatment. Antioxidative parameters improved at 5 µM and 10 µM DNJ concentrations. However, there were no ameliorative effects on antioxidant parameters observed under 20 µM DNJ treatment. The expression levels of Nrf2 mRNA increased significantly under DNJ treatment. DNJ treatment was associated with significant changes in the expression of genes of inflammatory cytokines. In conclusion, our study revealed that DNJ could improve oxidative stress and inflammation responses in the chicken intestine. These findings provide a theoretical reference for the development of functional feed additives that regulate intestinal health and lay the foundation for systematically revealing the mechanism of DNJ.

Effects of quercetin on granulosa cells from prehierarchical follicles by modulating MAPK signaling pathway in chicken.

Quercetin (Que), widely found in a huge variety of plants, plays important roles in ovarian function. However, to data, there have been no reports about Que regulating granulosa cells (GCs) in prehierarchical follicles in chicken. Herein, GCs from follicles diameter from 4 to 8 mm in chicken were treated by Que in vitro culture to investigate how Que exerts its effect on follicular development. GCs treated by Que in concentrations of 10, 100, and 1,000 ng/mL were tested for cell proliferation and progesterone secretion. Eight cDNA libraries were constructed from GCs (4 samples per group) to explore transcriptome expression changes. The role of the MAPK/ERK signaling pathway was validated in this process. Treatment with 100 and 1,000 ng/mL levels of Que significantly promoted cell proliferation and progesterone secretion (P < 0.05). RNA-seq analysis data showed that 402 and 263 differentially expressed genes (DEGs) were up- and down-regulated, respectively. Functional enrichment analysis that the pathways related to follicular development included biosynthesis of amino acids, MAPK signaling pathway, and calcium signaling pathway. Notably, the function exerted in GCs of the different levels of Que was associated with the suppression of the MAPK pathway. In conclusion, our results proved that low levels of Que could promote MAPK signaling pathway, but high levels of Que inhibit MAPK signaling pathway in GCs from the prehierarchical follicles, promote cell proliferation and progesterone secretion, and benefit follicle selection.

A cypovirus encoded microRNA negatively regulates the NF-κB pathway to enhance viral multiplication in Silkworm, Bombyx mori.

MicroRNAs (miRNAs) are small non-coding RNAs that function as novel gene expression regulators at the post-transcriptional level. Not with standing that the biogenesis and function of miRNAs are well-understood in eukaryotes, little is known about RNA virus-encoded miRNAs. Bombyx mori cypovirus (BmCPV) is a double-stranded RNA virus with a segmented genome that causes cytoplasmic polyhedrosis disease in silkworm larvae. To date, the interaction between BmCPV and silkworm remains largely unclear. 22 candidate BmCPV-encoded miRNAs were identified in this study through small RNA sequencing, stem-loop RT-PCR and qRT-PCR. Then, generation and function analyses were conducted on one of the candidate miRNAs, BmCPV-miR-1, in the BmN cells and the silkworm larvae by RNA interference, quantitative PCR, dual-luciferase assay. Our results revealed that BmCPV-miR-1 was encoded by BmCPV genome RNA rather than the degraded fragments of the viral genome. Its generation depended on Dicer-1 and might also be correlated with Dicer-2, Argonaute-1 and Argonaute-2. Moreover, BmCPV-miR-1 could suppress the expression of the target gene, B. mori inhibitor of nuclear factor kappa-B kinase subunit beta (BmIKKß), via binding to the target mRNA 3'-untranslated region, which fine-tuned the host NF-κB signaling pathway and consequently enhanced viral replication. Our results provide new evidence supporting the hypothesis that RNA viruses could generate miRNAs to modulate antiviral host defense.

Transcriptome of miRNA during inhibition of Bombyx mori nuclear polyhedrosis virus by geldanamycin in BmN cells.

Bombyx mori nuclear polyhedrosis virus (BmNPV) is one of several viruses that cause great harm to the sericulture industry, and its pathogenic mechanism is still being explored. Geldanamycin (GA), a kind of HSP90 inhibitor, has been verified to suppress BmNPV proliferation. However, the molecular mechanism by which GA inhibits BmNPV is unclear. MicroRNAs (miRNAs) have been shown to play a key role in regulating virus proliferation and host-pathogen interactions. In this study, BmN cells infected with BmNPV were treated by GA and DMSO for 72 h, respectively, then transcriptome analysis of miRNA was performed from the GA group and the control group. As a result, a total of 29 miRNAs were differentially expressed (DE), with 13 upregulated and 16 downregulated. Using bioinformatics analysis, it was found that the target genes of DEmiRNAs were involved in ubiquitin-mediated proteolysis, phagosome, proteasome, endocytosis pathways, and so on. Six DEmiRNAs were verified by quantitative reverse-transcription polymerase chain reaction. DElong noncoding RNA (DElncRNA)-DEmiRNA-messenger RNA (mRNA) regulatory networks involved in apoptosis and immune pathways were constructed in GA-treated BmN cells, which included 12 DEmiRNA, 132 DElncRNA, and 69 mRNAs. This regulatory network enriched the functional role of miRNA in the BmNPV-silkworm interactions and improved our understanding of the molecular mechanism of HSP90 inhibitors on BmNPV proliferation.

Two Putative Cypovirus-Encoded miRNAs Co-regulate the Host Gene of GTP-Binding Nuclear Protein Ran and Facilitate Virus Replication.

microRNA (miRNA) plays important roles in regulating various biological processes, including host-pathogen interaction. Recent studies have demonstrated that virus-encoded miRNAs can manipulate host gene expression to ensure viral effective multiplication. Bombyx mori cypovirus (BmCPV), a double-stranded RNA virus with a segmented genome, is one of the important pathogens for the economically important insect silkworm. Our present study indicated that two putative miRNAs encoded by BmCPV could promote viral replication by inhibiting the gene expression of B. mori GTP-binding nuclear protein Ran (BmRan), an essential component of the exportin-5-mediated nucleocytoplasmic transport of small RNAs. BmCPV-miR-1 and BmCPV-miR-3 are two of the BmCPV-encoded miRNAs identified in our previous studies. BmRan is a common target gene of them with binding sites all located in the 3'-untranslated region (3'-UTR) of its mRNA. The expression levels of the two miRNAs in the midgut of larvae infected with BmCPV gradually increased with the advance of infection, while the expression of the target gene BmRan decreased gradually. The miRNAs and the recombinant target gene consisting of reporter gene mCherry and 3'-UTR of BmRan mRNA were expressed in HEK293T cells for validating the interaction between the miRNAs and the target gene. qRT-PCR results revealed that BmCPV-miR-1 and BmCPV-miR-3 negatively regulate target gene expression not only separately but also cooperatively by binding to the 3'-UTR of BmRan mRNA. By transfecting miRNA mimics into BmN cells and injecting the mimics into the body of silkworm larvae, it was indicated that both BmCPV-miR-1 and BmCPV-miR-3 could repress the expression of BmRan in BmN cells and in the silkworm, and the cooperative action of the two miRNAs could enhance the repression of BmRan expression. Furthermore, the repression of BmRan could facilitate the replication of BmCPV genomic RNAs. It is speculated that BmCPV-miR-1 and BmCPV-miR-3 might reduce the generation of host miRNAs by inhibiting expression of BmRan, thus creating a favorable intracellular environment for virus replication. Our results are helpful to better understand the pathogenic mechanism of BmCPV to the silkworm, and provide insights into one of the evasion strategies used by viruses to counter the host defense for their effective multiplication.

Effects of Energy-Restricted Feeding during Rearing on the Performance, Uniformity, and Development of Rugao Layer Breeders at the Initiation of the Laying Period.

The aim of this study was to assess the effects of energy-restricted feeding during rearing on the performance, uniformity, and development of layer breeders at the initiation of the laying period. A total of 2400 8-week-old Rugao layer breeders were randomly assigned to one of five groups (480 pullets per group) with eight replicates and were fed one of five diets that were nutritionally equal with the exception of apparent metabolizable energy corrected for nitrogen (AMEn) content (2850, 2750, 2650, 2550, and 2450 kcal AMEn/kg) from 8 to 18 weeks of age. The daily amount of feed was restricted to the absolute quantity of the diet consumed by laying hens fed 2850 kcal AMEn per kg diet ad libitum (control). From 18 to 21 weeks of age, all hens were fed a basal diet ad libitum. The body weight (BW) of the laying pullets decreased linearly with increasing energy restriction (p < 0.001) but recovered within 3 weeks of ad libitum feeding (p = 0.290). A gradual increase in the degree of energy restriction resulted in a gradual decrease in average daily weight gain (ADG) and a gradual increase in the feed conversion ratio (FCR) and energy conversion ratio (ECR) from 8 to 18 weeks of age (p < 0.001, p < 0.001, p = 0.008). In contrast, the ADG and ADFI (p < 0.001, p < 0.001) gradually increased, while the FCR and ECR (p < 0.001, p < 0.001) gradually improved from 18 to 21 weeks of age. From 8 to 21 weeks of age, ECR improved (p = 0.005) with an increasing degree of energy restriction. The energy-restricted feeding for 6 weeks to the end of the trial improved BW uniformity (p < 0.05). The relative length and circumference of tarsus (p < 0.001, p < 0.001), and the relative weights and lengths of the small intestine, duodenum, jejunum, ileum, and caeca increased linearly (p < 0.001, p = 0.012, p < 0.007, p = 0.012, p = 0.040; p < 0.001, p = 0.003, p = 0.032, p = 0.029, p = 0.040) with increasing energy restriction at 18 weeks of age. After switching to ad libitum feeding for 3 weeks, the relative weights and lengths of the small intestine, duodenum, and jejunum of laying pullets increased linearly with increasing energy restriction (p < 0.001, p = 0.016, p = 0.011; p = 0.009, p = 0.028, p = 0.032). In conclusion, moderate energy restriction (85.97%, 2450 vs. 2850 kcal AMEn/kg) from 8 to 18 weeks of age and switching to ad libitum feeding from 18 to 21 weeks of age can be used to improve BW uniformity and stimulate the development of the duodenum and jejunum of native layer breeders at the initiation of the laying period without compromising BW.

The anti-Müllerian hormone gene's second exon is associated with the reproductive performance of Jinghai Yellow chickens.

Anti-Müllerian hormone (AMH), a member of the transforming growth factor- ß superfamily, plays important regulatory roles in follicular development and sex differentiation. Although much has been learned about the impact of polymorphisms of AMH on reproduction in animals, the effect on chicken reproduction is not well explored. In this study, the polymorphism of five exons of AMH gene and its effect on the reproductive performance of Jinghai Yellow chickens were studied. Primers for the amplification of AMH exons were designed, and Sanger sequencing was performed. Finally, only the polymorphism in the second exon of the AMH gene was found in the present population. Polymorphisms in the second exon of the AMH gene in 246 Jinghai Yellow hens and their associations with reproductive traits were analyzed. In total, four single nucleotide polymorphism (SNP) mutations were detected in the second exon of the AMH gene: g.1868A > C (AA, aa and Aa); g.1883G > A (BB, bb and Bb); g.1987G > A (CC, cc and Cc); and g.1996A > G (DD, dd and Dd). Only the mutation of g.1996A > G affected the reproductive traits: the age of laying first egg (AFE) of dd genotype was significantly ( p < 0.01 ) earlier than that in the DD and Dd hens. Moreover, the egg number by 300 d old (EN300) of dd individuals was significantly higher than that of DD and Dd individuals ( p < 0.01 ). Thus, we inferred that the dd genotype is the beneficial genotype. Additionally, AFE and EN300 showed significantly better performance in both the H2H2 and H7H7 diplotypes compared with other diplotype individuals ( p < 0.01 ). Thus, the H2H2 and H7H7 genotype had the best combination of AFE and EN300. Our study may allow for molecular marker section in poultry breeding.

Identification of long noncoding RNAs in silkworm larvae infected with Bombyx mori cypovirus.

Bombyx mori cypovirus (BmCPV) is one of the most important pathogens causing severe disease to silkworm. Emerging evidence indicates that long noncoding RNAs (lncRNAs) play importantly regulatory roles in virus infection and host immune response. To better understand the interaction between silkworm, Bombyx mori and BmCPV, we performed a comparative transcriptome analysis on lncRNAs and mRNAs between the virus-infected and noninfected silkworm larvae midgut at two time points postinoculation. A total of 16,753 genes and 1845 candidate lncRNAs were identified, among which 356 messenger RNA (mRNAs) and 41 lncRNAs were differentially expressed (DE). Target gene prediction revealed that most of DEmRNAs (123) were coexpressed with 28 DElncRNAs, suggesting that the expression of mRNA is mainly affected through trans- regulation by BmCPV-induced lncRNAs, and a regulatory network of DElncRNAs and DEmRNAs was then constructed. According to the network, many genes involved in apoptosis, autophagy, and antiviral response, such as ATG3, PDCD6, IBP2, and MFB1, could be targeted by different DElncRNAs, implying the essential roles of these genes and lncRNAs in BmCPV infection. In all, our studies revealed for the first time the alteration of lncRNA expression in BmCPV-infected larvae and its potential influence on BmCPV replication, providing a new perspective for host-cypovirus interaction studies.

Transcriptome Analysis of Long Noncoding RNAs and mRNAs in Granulosa Cells of Jinghai Yellow Chickens Illuminated With Red Light.

Jinghai Yellow chickens are a new indigenous breed with a dual purpose in China, but their egg laying performance is limited. Compared with white light (WL), exposure to red light (RL) can improve the egg laying performance of hens. Herein, to elucidate the molecular mechanism by which RL affects the egg laying performance, RNA sequencing was used to analyze long noncoding RNAs (lncRNAs) and mRNAs from granulosa cells of small yellow follicles from Jinghai Yellow chickens in RL and WL groups. A total of 12,466 lncRNAs were identified among the assembled transcripts, of which 168 lncRNAs were significantly different between the RL and WL groups (101 downregulated and 67 upregulated). Additionally, 1182 differentially expressed mRNAs were identified (958 downregulated and 224 upregulated). Integrated network analysis demonstrated that numerous differential mRNAs were involved in follicular development through steroid hormone synthesis, oocyte meiosis, and the PI3K-Akt signaling pathway. The impact of lncRNAs on cis and trans target mRNAs indicates that some lncRNAs play important roles in follicular development of small yellow follicles. The results provide a starting point for studies aimed at understanding the molecular mechanisms by which monochromatic light affects follicular development and egg production in hens.

Integrative analysis of circRNA/miRNA/mRNA regulatory network reveals the potential immune function of circRNAs in the Bombyx mori fat body.

Bombyx mori nucleopolyhedrosis virus (BmNPV) is one of the greatest threats to sustainable development of the sericulture industry. Circular RNA (circRNA), a type of non-coding RNA, has been shown to play important roles in gene expression regulation, immune response, and diseases. The fat body is a tissue with both metabolic and immune functions. To explore the potential immune function of circRNAs, we analyzed differentially expressed (DE)circRNAs, microRNAs(miRNAs), and mRNAs in the B. mori fat body in response to BmNPV infection using high-throughput RNA sequencing. A total of 77 DEcircRNAs, 32 DEmiRNAs, and 730 DEmRNAs that are associated with BmNPV infection were identified. We constructed a DEcircRNA/DEmiRNA/DEmRNA and DEcircRNA/DEmiRNA/BmNPV gene regulatory network and validated the differential expression of circ_0001432 and its corresponding miRNA (miR-2774c and miR-3406-5p) and mRNA (778467 and 101745232) in the network. Tissue-specific expression of circ_0001432 and its expression at different time points were also examined. KEGG pathway analysis of DEmRNAs, target genes of DEmiRNAs, and host genes of DEcircRNAs in the network showed that these genes were enriched in several metabolic pathways and signaling pathways, which could play important roles in insect immune responses. Our results suggest that circRNA could be involved in immune responses of the B. mori fat body and help in understanding the molecular mechanisms underlying silkworm-pathogen interactions.

1-Deoxynojirimycin from mulberry leaves changes gut digestion and microbiota composition in geese.

This study was aimed to investigate whether 1-deoxynojirimycin (DNJ) affects the digestion system of young geese and assess whether mulberry leaf, which contains this substance, has disadvantages that compromise its value as poultry feed. One hundred and twenty-eight 12-day-old male Wanxi white geese were randomly assigned into 4 treatment groups. The control group was fed an ordinary diet without DNJ. The other groups namely L-DNJ, M-DNJ, and H-DNJ had their basic diets supplemented with 0.05 mg/g, 0.1 mg/g, and 0.15 mg/g DNJ, respectively. The geese were fed for 6 wk, and the apparent digestibility test was conducted in the last week. Intestinal parameters, digestive organs, and enzymes were determined. 16S rRNA gene sequencing was conducted for cecal flora composition. The results revealed that DNJ decreased body and liver weight and increased feed conversion ratio in comparison with the control (P < 0.05); however, it did not influence the weight and length of the intestine or the pancreas weight. The utilization of organic matter, metabolizable energy, ether extract, acid detergent fiber, and calcium in feed were reduced in the M-DNJ and L-DNJ groups compared with those in the control (P < 0.05); however, the utilization of crude protein was increased in all DNJ-treated groups (P < 0.01). In the H-DNJ group, the usage of soluble phosphorus was also increased (P < 0.05). High-dose DNJ increased the activity of trypsin in the pancreas but reduced those of amylase (P < 0.05) and lipase (P > 0.05) in the pancreas and duodenum. The intestinal villi were short, even impaired, in DNJ-treated groups. High-throughput sequencing data revealed that DNJ supplement reduced the α-diversity indices of the cecal microbiota. The principal component analysis further suggested a difference in community structure between the DNJ treatment groups and control. High-dose DNJ increased the relative abundance of Bacteroides, Escherichia-Shigella, and Butyricicoccus but reduced that of unclassified Ruminococcaceae compared with the control (P < 0.05). In conclusion, changes in the digestive system caused by DNJ seriously affected the metabolism of nutrients in geese and reduced their growth performance. Attention should be paid to the adverse effects of DNJ when using mulberry leaves as poultry feed.

Comprehensive analysis of lncRNA-mRNA regulatory network in BmNPV infected cells treated with Hsp90 inhibitor.

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the main pathogens that seriously affect the sustainable development of sericulture industry. Inhibition of Hsp90 by Hsp90 inhibitor, geldanamycin (GA) significantly suppresses BmNPV proliferation in Bombyx mori, while the functional mechanism is not clear. LncRNA has been widely reported to play an important role in immune responses and host-virus interactions in mammalian. However, related research has been rarely reported on silkworm. In this study, firstly, we confirmed the decrease of BmNPV ORF75 protein in the BmNPV-infected BmN cells treated with GA. Next, by using a genome-wide transcriptome analysis, we compared the lncRNA and mRNA expression profiles in BmNPV infected BmN cells treated with or without GA and identified a total of 282 differentially expressed lncRNAs (DElncRNAs) and 523 DEmRNAs. KEGG pathway analysis revealed DEmRNA were mainly involved in ubiquitin mediated proteolysis, spliceosome, RNA transport and oxidative phosphorylation. Further, we selected 27 immune-related DEmRNAs, which displayed the similar changes of expression patterns on both protein level and transcript level to construct DElncRNA-DEmRNA network. In addition, based on the DElncRNA-bmo-miR-278-3p-BmHSC70 regulatory network, we explored the potential function of several lncRNAs as sponges to inhibit the regulatory effect of bmo-278-3p on Bombyx mori heat shock protein cognate 70 (BmHSC70). Our finding suggests that lncRNAs play a role in the regulation of BmNPV proliferation by Hsp90.

Expression profile analysis of circular RNAs in BmN cells (Bombyx mori) upon BmNPV infection.

The disease caused by Bombyx mori nucleopolyhedrovirus (BmNPV) has always been difficult to control, resulting in tremendous economic losses in the sericulture industry. Although much has been learned about the impact of noncoding RNAs on pathogenesis, the role of circular RNA (circRNA) in insect immunity remains unclear. To explore circRNA regulation involved in BmNPV infection, we used transcriptome analysis of BmN cells with or without BmNPV infection to generate circRNA data set. A total of 444 novel circRNAs were identified in BmN cells, with 198 pervasively distributed both in the control group and BmNPV-infection group. The host genes were enriched inMAPK signaling pathway, dorso-ventral axis formation, and ECM-receptor interaction, which were required for the normal larval growth. A total of 75 circRNAs were differentially expressed (DE) on BmNPV infection. Six downregulated circRNAs were validated by Sanger sequencing and qRT-PCR. DEcircRNA-miRNA-DEmRNA network was constructed based on the six validated circRNAs. Pathway analysis indicated that the predicted target genes were mainly enriched in the metabolic pathway and immune-related signaling pathway. Our results may provide a basis for further studies on circRNA function in BmN cells challenged by BmNPV infection and offer an insight into the molecular mechanism on silkworm-virus interaction.

Transcriptomic Analysis of circRNAs and mRNAs Reveals a Complex Regulatory Network That Participate in Follicular Development in Chickens.

Follicular development plays a key role in poultry reproduction, affecting clutch traits and thus egg production. Follicular growth is determined by granulosa cells (GCs), theca cells (TCs), and oocyte at the transcription, translation, and secretion levels. With the development of bioinformatic and experimental techniques, non-coding RNAs have been shown to participate in many life events. In this study, we investigated the transcriptomes of GCs and TCs in three different physiological stages: small yellow follicle (SYF), smallest hierarchical follicle (F6), and largest hierarchical follicle (F1) stages. A differential expression (DE) analysis, weighted gene co-expression network analysis (WGCNA), and bioinformatic analyses were performed. A total of 18,016 novel circular RNAs (circRNAs) were detected in GCs and TCs, 8127 of which were abundantly expressed in both cell types. and more circRNAs were differentially expressed between GCs and TCs than mRNAs. Enrichment analysis showed that the DE transcripts were mainly involved in cell growth, proliferation, differentiation, and apoptosis. In the WGCNA analysis, we identified six specific modules that were related to the different cell types in different stages of development. A series of central hub genes, including MAPK1, CITED4, SOD2, STC1, MOS, GDF9, MDH1, CAPN2, and novel_circ0004730, were incorporated into a Cytoscape network. Notably, using both DE analysis and WGCNA, ESR1 was identified as a key gene during follicular development. Our results provide valuable information on the circRNAs involved in follicle development and identify potential genes for further research to determine their roles in the regulation of different biological processes during follicle growth.

Transcriptome Analysis of circRNA and mRNA in Theca Cells during Follicular Development in Chickens.

Development of ovarian follicles requires interactions between granulosa cells, theca cells, and oocytes. Multiple transcription levels are involved but information about the role of noncoding RNAs, especially circular RNAs (circRNAs), is lacking. Here, we used RNA sequencing to profile circRNAs and mRNAs in theca cells from three types of follicle: small yellow follicles (SYF), the smallest hierarchical follicles (F6), and the largest hierarchical follicles (F1). Using bioinformatics analysis, we identified a total of 14,502 circRNAs in all theca cells, with 5622 widely distributed in all stages of development. Differential expression analysis suggested that some genes display differential isoforms during follicular development. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed enrichment of both differentially expressed circRNAs and mRNAs in pathways associated with reproduction, including the TGF-b signaling pathway, oocyte meiosis, and vascular smooth muscle contraction. Our study provides the first visual information about circRNAs and mRNAs in theca cells during follicle development in chickens and adds to the growing body of knowledge about theca cells.

Analysis of lncRNA-mediated gene regulatory network of Bombyx mori in response to BmNPV infection.

Bombyx mori nucleopolyhedrosis virus (BmNPV) has always been a great challenge to the development and stability of the sericulture industry. LncRNAs have been reported to play important roles in gene expression regulation, development and immune response but the roles of lncRNAs in BmNPV infection and silkworm-BmNPV interaction are not clear. We used a genome-wide transcriptome analysis to identify the lncRNAs in Bombyx mori cells (BmN cells) and analyzed the differentially expressed lncRNAs, microRNAs and protein-coding genes in silkworm cells with or without BmNPV infection. A total of 13,159 candidate lncRNAs were identified in the BmN cells, among which 4450 lncRNAs were differentially expressed (DE) with 2837 up-regulated and 1613 down-regulated. In addition, 66 differentially expressed miRNAs (DEmiRNAs) and 7448 differentially expressed mRNAs (DEmRNAs) were identified, and DElncRNA-DEmiRNA-DEmRNA regulatory network was constructed. Gene expression was variable in 4973 of predicted lncRNA cis target genes in BmNPV infected cells. KEGG pathway analysis indicated that the target genes of DElncRNAs are enriched in ubiquitin mediated proteolysis, endocytosis and lysosome pathways. qRT-PCR validated the differential expression of several lncRNAs and miRNAs. Our results suggested that DElncRNAs participate in host response to BmNPV infection via interactions with their target genes and miRNAs. Our results will help us to improve our understanding of lncRNA-mediated regulatory roles in BmNPV infection and provide new insights into silkworm-pathogen interactions.

Genetic evaluation of eggshell color based on additive and dominance models in laying hens.

OBJECTIVE: Eggshells with a uniform color and intensity are important for egg production because many consumers assess the quality of an egg according to the shell color. In the present study, we evaluated the influence of dominant effects on the variations in eggshell color after 32 weeks in a crossbred population. METHODS: This study was conducted using 7,878 eggshell records from 2,626 hens. Heritability was estimated using a univariate animal model, which included inbreeding coefficients as a fixed effect and animal additive genetic, dominant genetic, and residuals as random effects. Genetic correlations were obtained using a bivariate animal model. The optimal diagnostic criteria identified in this study were: L* value (lightness) using a dominance model, and a* (redness), and b* (yellowness) value using an additive model. RESULTS: The estimated heritabilities were 0.65 for shell lightness, 0.42 for redness, and 0.60 for yellowness. The dominance heritability was 0.23 for lightness. The estimated genetic correlations were 0.61 between lightness and redness, -0.84 between lightness and yellowness, and -0.39 between redness and yellowness. CONCLUSION: These results indicate that dominant genetic effects could help to explain the phenotypic variance in eggshell color, especially based on data from blue-shelled chickens. Considering the dominant genetic variation identified for shell color, this variation should be employed to produce blue eggs for commercial purposes using a planned mating system.

Genome-wide association study provides insights into the genetic architecture of bone size and mass in chickens.

Bone size is an important trait for chickens because of its association with osteoporosis in layers and meat production in broilers. Here, we employed high density genotyping platforms to detect candidate genes for bone traits. Estimates of the narrow heritabilities ranged from 0.37 ± 0.04 for shank length to 0.59 ± 0.04 for tibia length. The dominance heritability was 0.12 ± 0.04 for shank length. Using a linear mixed model approach, we identified a promising locus within NCAPG on chromosome 4, which was associated with tibia length and mass, femur length and area, and shank length. In addition, three other loci were associated with bone size or mass at a Bonferroni-corrected genome-wide significance threshold of 1%. One region on chicken chromosome 1 between 168.38 and 171.82 Mb harbored HTR2A, LPAR6, CAB39L, and TRPC4. A second region that accounted for 2.2% of the phenotypic variance was located around WNT9A on chromosome 2, where allele substitution was predicted to be associated with tibia length. Four candidate genes identified on chromosome 27 comprising SPOP, NGFR, GIP, and HOXB3 were associated with tibia length and mass, femur length and area, and shank length. Genome partitioning analysis indicated that the variance explained by each chromosome was proportional to its length.

Identification of potential genomic regions and candidate genes for egg albumen quality by a genome-wide association study.

Albumen quality is a leading economic trait in the chicken industry. Major studies have paid attention to genetic architecture underlying albumen quality. However, the putative quantitative trait locus (QTL) for this trait is still unclear. In this genome-wide association study, we used an F 2 resource population to study longitudinal albumen quality. Seven single-nucleotide polymorphism (SNP) loci were found to be significantly ( p < 8.43 × 10 - 7 ) related to albumen quality by univariate analysis, while 11 SNPs were significantly ( p < 8.43 × 10 - 7 ) associated with albumen quality by multivariate analysis. A QTL on GGA4 had a pervasive function on albumen quality, including a SNP at the missense of NCAPG, and a SNP at the intergenic region of FGFPB1. It was further found that the putative QTLs at GGA1, GGA2, and GGA7 had the strongest effects on albumen height (AH) at 32 weeks, Haugh units (HU) at 44 weeks, and AH at 55 weeks. Moreover, novel SNPs on GGA5 and GGA3 were associated with AH and HU at 32, 44, and 48 weeks of age. These results confirmed the regions for egg weight that were detected in a previous study and were similar with QTL for albumen quality. These results showed that GGA4 had the strongest effect on albumen quality. Only a few significant loci were detected for most characteristics probably reflecting the attributes of a pleiotropic gene and a minor-polygene in quantitative traits.