Massively Parallel CRISPR-Cas9 Knockout Screening in Sheep Granulosa Cells for FSH Response Genes.

Follicle-stimulating hormone (FSH) regulates ovarian follicle development through specific gene expression programs. Granulosa cells (GCs) are somatic cells surrounding the oocytes, secreting gonadotropins to regulate ovulation and promote follicular development. By analyzing the effects of different doses of FSH on the proliferation of GCs, we found that adding 10 ng/mL of FSH, as the optimal concentration, could promote the growth of GCs. Furthermore, we have successfully constructed the first CRISPR-Cas9 knockout library targeting the genes on chromosomes 2 and 3 and the X chromosomes of the sheep massively parallel coding gene, as well as an ovarian GCs knockout cell library. For the first time, we have exposed the knockout cell library to a concentration of 10 ng/mL FSH to explore the underlying mechanisms. Through this screening, we have identified 836 positive-negative screening genes that are responsive to FSH, thereby revealing the regulatory mechanisms and screening the functionality of candidate genes. Next, RNA-Seq of control (0 ng/mL), low (10 ng/mL), and high (100 ng/mL) doses of FSH revealed 1708 differentially expressed genes, and combined with 836 genes, we obtained 129 FSH dose-dependent genes with extremely significant differences. This enables us to delve deeper into investigating and identifying the mechanisms by which FSH regulates GCs. More generally, we have discovered new regulatory factors and identified reproductivity-associated major effectors. These findings provide novel research directions for further studies on sheep reproduction.

First detection of Candidatus Rickettsia tarasevichiae in Hyalomma marginatum ticks.

Ticks are important vectors of zoonotic diseases and play a major role in the circulation and transmission of many rickettsial species. The aim of this study was to investigate the carriage of Candidatus Rickettsia tarasevichiae (CRT) in a total of 1168 ticks collected in Inner Mongolia to elucidate the potential public health risk of this pathogen, provide a basis for infectious disease prevention, control and prediction and contribute diagnostic ideas for clinical diseases that present with fever in populations exposed to ticks. A total of four tick species, Haemaphysalis concinna (n = 21), Dermacentor nuttalli (n = 122), Hyalomma marginatum (n = 148), and Ixodes persulcatus (n = 877), were collected at nine sampling sites in Inner Mongolia, China, and identified by morphological and molecular biological methods. Reverse transcription PCR targeting the 16S ribosomal RNA (rrs), gltA, groEL, ompB and Sca4 genes was used to detect CRT DNA. Sequencing was used for pathogen species confirmation. The molecular epidemiological analysis showed that three species of ticks were infected with CRT, and the overall positive rate was as high as 42%. The positive rate of I. persulcatus collected in Hinggan League city was up to 96%, and that of I. persulcatus collected in Hulun Buir city was 50%. The pool positive rates of D. nuttalli and H. marginatum collected in Bayan Nur city and H. concinna collected in Hulun Buir city were 0%, 28% and 40%, respectively. This study revealed the high prevalence of CRT infection in ticks from Inner Mongolia and the first confirmation of CRT detected in H. marginatum in China. The wide host range and high infection rate in Inner Mongolia may dramatically increase the exposure of CRT to humans and other vertebrates. The role of H. marginatum in the transmission of rickettsiosis and its potential risk to public health should be further considered.

Microbial diversity of ticks and a novel typhus group Rickettsia species (Rickettsiales bacterium Ac37b) in Inner Mongolia, China.

Ticks can carry multiple pathogens, and Inner Mongolia's animal husbandry provides excellent environmental conditions for ticks. This study characterized the microbiome of ticks from different geographical locations in Inner Mongolia; 905 Dermacentor nuttalli and 36 Ixodes persulcatus were collected from sheep in three main pasture areas and from bushes within the forested area. Mixed DNA samples were prepared from three specimens from each region and tick species. Microbial diversity was analyzed by 16S rRNA sequencing, and α and ß diversity were determined. The predominant bacterial genera were Rickettsia (54.60%), including Rickettsiales bacterium Ac37b (19.33%) and other Rickettsia (35.27%), Arsenophonus (11.21%), Candidatus Lariskella (10.84%), and Acinetobacter (7.17%). Rickettsia bellii was identified in I. persulcatus, while Rickettsiales bacterium Ac37b was found in D. nuttalli from Ordos and Chifeng. Potential Rickettsia and Anaplasma coinfections were observed in the Ordos region. Tick microbial diversity analysis in Inner Mongolia suggests that sheep at the sampling sites were exposed to multiple pathogens.

Title: Diversité microbienne des tiques et nouvelle espèce de Rickettsia du groupe du typhus (bactérie Rickettsiales Ac37b) en Mongolie intérieure, Chine. Abstract: Les tiques peuvent être porteuses de plusieurs agents pathogènes et l'élevage en Mongolie intérieure offre d'excellentes conditions environnementales pour les tiques. Cette étude a caractérisé le microbiome des tiques de différentes zones géographiques de Mongolie intérieure; 905 Dermacentor nuttalli et 36 Ixodes persulcatus ont été collectés sur des moutons dans trois principales zones de pâturage et dans des buissons de la zone forestière. Des échantillons d'ADN mixtes ont été préparés à partir de trois spécimens de chaque région et espèce de tique. La diversité microbienne a été analysée par séquençage de l'ARNr 16S et la diversité α et ß a été déterminée. Les genres bactériens prédominants étaient les Rickettsia (54,60 %), dont la bactérie Rickettsiales Ac37b (19,33 %) et d'autres Rickettsia (35,27 %), Arsenophonus (11,21 %), Candidatus Lariskella (10,84 %) et Acinetobacter (7,17 %). Rickettsia bellii a été identifiée chez I. persulcatus, tandis que la bactérie Rickettsiales Ac37b a été trouvée chez D. nuttalli d'Ordos et Chifeng. Des co-infections potentielles à Rickettsia et Anaplasma ont été observées dans la région d'Ordos. L'analyse de la diversité microbienne des tiques en Mongolie intérieure montre que les moutons présents sur les sites d'échantillonnage sont exposés à plusieurs agents pathogènes.

Integrated Multi-Tissue Transcriptome Profiling Characterizes the Genetic Basis and Biomarkers Affecting Reproduction in Sheep (Ovis aries).

The heritability of litter size in sheep is low and controlled by multiple genes, but the research on its related genes is not sufficient. Here, to explore the expression pattern of multi-tissue genes in Chinese native sheep, we selected 10 tissues of the three adult ewes with the highest estimated breeding value in the early study of the prolific Xinggao sheep population. The global gene expression analysis showed that the ovary, uterus, and hypothalamus expressed the most genes. Using the Uniform Manifold Approximation and Projection (UMAP) cluster analysis, these samples were clustered into eight clusters. The functional enrichment analysis showed that the genes expressed in the spleen, uterus, and ovary were significantly enriched in the Ataxia Telangiectasia Mutated Protein (ATM) signaling pathway, and most genes in the liver, spleen, and ovary were enriched in the immune response pathway. Moreover, we focus on the expression genes of the hypothalamic-pituitary-ovarian axis (HPO) and found that 11,016 genes were co-expressed in the three tissues, and different tissues have different functions, but the oxytocin signaling pathway was widely enriched. To further explore the differences in the expression genes (DEGs) of HPO in different sheep breeds, we downloaded the transcriptome data in the public data, and the analysis of DEGs (Xinggao sheep vs. Sunite sheep in Hypothalamus, Xinggao sheep vs. Sunite sheep in Pituitary, and Xinggao sheep vs. Suffolk sheep in Ovary) revealed the neuroactive ligand-receptor interactions. In addition, the gene subsets of the transcription factors (TFs) of DEGs were identified. The results suggest that 51 TF genes and the homeobox TF may play an important role in transcriptional variation across the HPO. Altogether, our study provided the first fundamental resource to investigate the physiological functions and regulation mechanisms in sheep. This important data contributes to improving our understanding of the reproductive biology of sheep and isolating effecting molecular markers that can be used for genetic selection in sheep.

Estimates of Genomic Heritability and the Marker-Derived Gene for Re(Production) Traits in Xinggao Sheep.

Xinggao sheep are a breed of Chinese domestic sheep that are adapted to the extremely cold climatic features of the Hinggan League in China. The economically vital reproductive trait of ewes (litter size, LS) and productive traits of lambs (birth weight, BWT; weaning weight, WWT; and average daily gain, ADG) are expressed in females and later in life after most of the selection decisions have been made. This study estimated the genetic parameters for four traits to explore the genetic mechanisms underlying the variation, and we performed genome-wide association study (GWAS) tests on a small sample size to identify novel marker trait associations (MTAs) associated with prolificacy and growth. We detected two suggestive significant single-nucleotide polymorphisms (SNPs) associated with LS and eight significant SNPs for BWT, WWT, and ADG. These candidate loci and genes also provide valuable information for further fine-mapping of QTLs and improvement of reproductive and productive traits in sheep.

Clearance of bacteria from lymph nodes in sheep immunized with Brucella suis S2 vaccine is associated with M1 macrophage activation.

Ovine brucellosis is a global zoonotic disease of sheep caused by Brucella melitensis, which inflicts a significant burden on human and animal health. Brucella suis strain S2 (B. suis S2) is a smooth live attenuated vaccine for the prevention of ovine brucellosis in China. However, no previous studies have assessed the immunogenicity of B. suis S2 vaccine after oral immunization in sheep. Here, we attempted to evaluate the ovine immune response over the course of B. suis S2 immunization and to identify in vivo predictors for vaccine development. Body temperature, serum Brucella antibodies, serum cytokines (IL-12p70 and interferon [IFN]-γ), and bacterial load in the mandibular lymph nodes (LN), superficial cervical LN, superficial inguinal LN, and spleen were investigated to determine the safety and efficacy of the vaccine. The abnormal body temperature of sheep occurred within 8 days post-infection (dpi). Brucella suis S2 persisted for a short time (< 21 dpi) in the mandibular LN. The highest level of IL-12p70 was observed at 9 dpi, whereas serum IFN-γ levels peaked at 12 dpi. Transcriptome analysis and quantitative reverse transcription PCR were performed to determine gene expression profiles in the mandibular LN of sheep. Antigen processing and presentation pathway was the dominant pathway related to the dataset. Our studies suggest that the immune response in ovine LN resembled type 1 immunity with the secretion of IL-12p70 and IFN-γ after B.suis S2 immunization and the vaccine may eliminate Brucella via stimulation of M1 macrophages through the course of Th cells.

Effects of dietary oat supplementation on carcass traits, muscle metabolites, amino acid profiles, and its association with meat quality of Small-tail Han sheep.

Oat supplementation of the ruminant diet can improve growth performance and meat quality traits, but the role of muscle metabolites has not been evaluated. This study aimed to establish whether oat grass supplementation (OS) of Small-tail Han sheep improved growth performance and muscle tissue metabolites that are associated with better meat quality and flavor. After 90-day, OS fed sheep had higher live-weight and carcass-weight, and lower carcass fat. Muscle metabolomics analysis showed that OS fed sheep had higher levels of taurine, l-carnitine, inosine-5'-monophospgate, cholic acid, and taurocholic acid, which are primarily involved in taurine and hypotaurine metabolism, purine metabolism, and bile acid biosynthesis and secretion, decreased fat accumulation and they promote functional or flavor metabolites. OS also increased muscle levels of amino acids that are attributed to better quality and flavorsome mutton. These findings provided further evidence for supplementing sheep with oat grass to improve growth performance and meat quality.

Design and characterization of a high-resolution multiple-SNP capture array by target sequencing for sheep.

The efficiency of molecular breeding largely depends on inexpensive genotyping arrays. In this study, we aimed to develop an ovine high-resolution multiple-single-nucleotide polymorphism (SNP) capture array, based on genotyping by target sequencing (GBTS) system with capture-in-solution (liquid chip) technology. All the markers were from 40K captured regions, including genes located within selective sweep regions, breed-specific regions, quantitative trait loci (QTL), and the potential functional SNPs on the sheep genome. The results showed that a total of 210K high-quality SNPs were identified in the 40K regions, indicating a high average capture ratio (99.7%) for the target genomic regions. Using genotyped data (n = 317) from liquid chip technology, we further performed genome-wide association studies (GWAS) to detect the genetic loci affecting sheep hair types and teat number. A single significant association signal for hair types was identified on 6.7-7.1 Mb of chromosome 25. The IRF2BP2 gene (chr25: 7,067,974-7,071,785), which is located within this genomic region, has been previously known to be involved in hair/wool traits in sheep. The results further showed a new candidate region around 26.4 Mb of chromosome 13, between the ARHGAP21 and KIAA1217 genes, that was significantly related to teat number in sheep. The haplotype patterns of this region also showed differences in animals with 2, 3, or 4 teats. Advances in using the high-accuracy and low-cost liquid chip are expected to accelerate sheep genomic and breeding studies in the coming years.

Large-scale genotyping platforms are valuable tools for animal selection and breeding programs. The bead chip has been widely used in both research and commercial applications for a long time. A highly efficient and economical genotyping platform has been developed recently. In the present study, by combining the advantages of resequencing and bead chips, we developed a high-resolution capture array based on target sequencing with capture-in-solution technology (liquid chip), including updated functional probes according to the latest research. We further evaluated this approach by using 317 individuals and found that 210K single-nucleotide polymorphisms can be accurately genotyped, confirming the ratio of the captured regions compared with the designed rations is around 99.7%. Genome-wide association studies conducted using this chip suggested IRF2BP2 gene may be involved in hair types and ARHGAP21-KIAA1217 locus may be related to teats number. The liquid chip with high accuracy and low cost can be widely used in genome-wide association studies and genome selection, supporting efforts in molecular breeding and genetic improvement of sheep.

Effects of exogenous melatonin on expressional differences of immune-related genes in cashmere goats.

The interplay between melatonin and immune system is well recognized in humans. The true integration of research on cashmere goat is still far from clear, especially for cashmere goat maintained in wool and cashmere growth. In this study, we applied various approaches to identify the complex regulated network between the immune-related genes and transcription factors (TFs) and to explore the relationship between melatonin and gene expression in cashmere goats. In total, 1,599 and 1756 immune-related genes were found in the blood and skin of cashmere goats, respectively, and 24 differentially expressed immune-related GO terms were highly expressed in blood after melatonin implantation. We studied the melatonin-dependent networks between the TFs and immune-related genes in cashmere goat. The 3 major regulatory networks were interconnected through TFs. The TFs, such as PHF5A, REXO4, STRAP, JUNB, GATAD2A, ZNF710, and VDR, were also expressed in the blood and skin tissue of cashmere goat. In addition, most genes in these networks, such as VDR, JUNB, and Trib3, were involved in WNT pathway, which is related to cashmere wool growth regulation. On the network basis, we developed a knockout mouse model to identify the network interaction. We observed that 8 high-sulfur protein genes, 12 keratin (KRT) genes, and 19 keratin associated protein (KRTAP) genes related to the growth of cashmere wool were almost not expressed in Trib3 -/- rat skin. Our results suggested that the expression of genes related to wool and cashmere growth may be regulated by the interaction network between genes affected by melatonin and immune-related genes. In summary, we outlined some particularly promising ways for future research on immune-related genes of cashmere goats and the role of melatonin in wool and cashmere growth.

Identification and genetic diversity analysis of Rickettsia in Dermacentor nuttalli within inner Mongolia, China.

BACKGROUND: The genus Rickettsia contains the lineages spotted fever group (SFG), typhus group (TG), and transitional group (TRG). The spotted fever group Rickettsia (SFGR) is transmitted by ticks. The tick species Dermacentor nuttalli is considered the main vector carrying SFGR in Inner Mongolia. Studying the genetic diversity and population structure of Rickettsia is essential for developing effective control strategies and predicting evolutionary trends of Rickettsia. METHODS: In 2019 we collected 408 D. nuttalli in the Inner Mongolia Autonomous Region, detected the percentage of Rickettsia-positive specimens, and characterized the haplotypes. From the Rickettsia-positive ticks, the gltA and ompA genes were extracted, amplified, and sequenced. RESULTS: Ten haplotypes of the gltA gene and 22 haplotypes of the ompA gene were obtained. The phylogenetic analysis showed that the haplotypes G1-G7 and G9 of the gltA gene cluster with Rickettsia raoultii, while G8 and G10 cluster with Rickettsia sibirica. Haplotypes O1-O15, O18 and O20-O22 of the ompA gene cluster with R. raoultii, while O16 and O19 cluster with R. sibirica. The average haplotype diversity was 0.3 for gltA and 0.7 for ompA. The average nucleotide diversity was greater than 0.05. Neutrality tests were nonsignificant for Tajima's D results and Fu's Fs results. The fixation index values (FST) showed that the degree of genetic differentiation between most sampled populations was small (FST < 0.05), whereas some populations showed a medium (FST > 0.05) or large (FST > 0.15) degree of differentiation. Analysis of molecular variance (AMOVA) revealed that the variation within populations was greater than that between populations. The mismatch analysis of Rickettsia showed double peaks. CONCLUSIONS: We found two Rickettsia spp. (R. raoultii and R. sibirica). The high genetic disparity of Rickettsia allows for easy adaption to different environments. Genetic differentiation between populations is small, and Rickettsia populations do not show a geographically differentiated structure. The high rates of retention and infection of Rickettsia in D. nuttalli together with the animal husbandry exchange in Inner Mongolia gradually led to the harmonization of genetic characteristics of Rickettsia across various regions. Overall, the significant genetic diversity and geographical structure of Rickettsia in D. nuttalli are critical for SFGR control.

The Effect of Ghrelin on the Maturation of Sheep Oocytes and Early Embryonic Development In Vitro.

In vitro maturation (IVM) of sheep oocytes and early embryonic development are of great scientific importance for the study of reproductive development in sheep. Ghrelin is an important hormone that regulates the secretion of the growth hormone (GH). In this study, different gradients of ghrelin (0, 100, 200, and 300 ng/mL) were added to the IVM system of sheep oocytes to observe their cell morphology, and Hosesth 33342 staining was used to determine the time taken for oocytes to reach different developmental stages. We found 200 ng/mL ghrelin to be the optimal concentration. The RNA-seq analysis showed that many signaling pathways were significantly altered by ghrelin. Cell cycle, Wnt, and oxidative phosphorylation were activated; the P53 was inhibited. These pathways together regulate the maturation of oocytes and early embryonic development in vitro. The effects of the addition of ghrelin were verified by the expression of GLUT1 in early embryonic development. The results suggest that adding ghrelin shortens the duration of the IVM of sheep oocytes and hinders early embryonic development. This study provides new insights into the effects of exogenous ghrelin on sheep oocyte maturation and early embryonic development in vitro.

Downregulation of Three Novel miRNAs in the Lymph Nodes of Sheep Immunized With the Brucella suis Strain 2 Vaccine.

Ovine and caprine brucellosis, both caused by Brucella melitensis, lead to substantial economic losses in the animal industry and health problems in human populations. Brucella suis strain 2 (B.suis S2), as a live attenuated vaccine, is used extensively in China to prevent brucellosis. It has been proven that microRNA (miRNAs) are involved in the immunopathogenesis of brucellosis; however, the miRNA-driven mechanism of immune response to B.suis S2 in vivo remains unknown. To determine which new miRNAs are involved in the host immune response to B.suis S2 and elucidate the function of these miRNAs, we performed a comprehensive analysis of miRNA expression profiles in sheep immunized with B.suis S2 using the high-throughput sequencing approach. The submandibular lymphatic nodes from sheep seropositive for Brucella were collected at 7, 14, 21, 30, 60 and 90 days post-immunization. MiRNA sequencing analysis revealed that 282 differentially expressed miRNAs (|log2 fold-change |>0.5 and p < 0.05) were significantly enriched in the immune pathways, including the NF-kappa B signaling pathway, B cell receptor signaling pathway, p53 signaling pathway and complement and coagulation cascades. Increasing the threshold to |log2 fold change|>1 and p < 0.01 revealed 48 differentially expressed miRNAs, 31 of which were novel miRNAs. Thirteen of these novel miRNAs, which were differentially expressed for at least two time points, were detected via RT-qPCR assays. The novel_229, novel_609, novel_973 and oar-miR-181a assessed by RT-qPCR were detectable and consistent with the expression patterns obtained by miRNA sequencing. Functional analyses of these miRNAs demonstrated that their target genes participated in the immune response pathways, including the innate and adaptive immunity pathways. The immune-related target genes of novel_229 included ENSOARG00000000649 and TMED1, as well as LCN2, PDPK1 and LPO were novel_609 target genes. The immune-related target genes of novel_973 included C6orf58, SPPL3, BPIFB1, ENSOARG00000021083, MPTX1, CCL28, FGB, IDO1, OLR1 and ENSOARG00000020393. The immune-related target genes of oar-miR-181a included ENSOARG00000002722, ARHGEF2, MFAP4 and DOK2. These results will deepen our understanding of the host miRNA-driven defense mechanism in sheep immunized with B.suis S2 vaccine, and provide the valuable information for optimizing vaccines and developing molecular diagnostic targets.

Time-course RNA-seq analysis reveals stage-specific and melatonin-triggered gene expression patterns during the hair follicle growth cycle in Capra hircus.

BACKGROUND: Cashmere goat is famous for its high-quality fibers. The growth of cashmere in secondary hair follicles exhibits a seasonal pattern arising from circannual changes in the natural photoperiod. Although several studies have compared and analyzed the differences in gene expression between different hair follicle growth stages, the selection of samples in these studies relies on research experience or morphological evidence. Distinguishing hair follicle growth cycle according to gene expression patterns may help to explore the regulation mechanisms related to cashmere growth and the effect of melatonin from a molecular level more accurately. RESULTS: In this study, we applied RNA-sequencing to the hair follicles of three normal and three melatonin-treated Inner Mongolian cashmere goats sampled every month during a whole hair follicle growth cycle. A total of 3559 and 988 genes were subjected as seasonal changing genes (SCGs) in the control and treated groups, respectively. The SCGs in the normal group were divided into three clusters, and their specific expression patterns help to group the hair follicle growth cycle into anagen, catagen and telogen stages. Some canonical pathways such as Wnt, TGF-beta and Hippo signaling pathways were detected as promoting the hair follicle growth, while Cell adhesion molecules (CAMs), Cytokine-cytokine receptor interaction, Jak-STAT, Fc epsilon RI, NOD-like receptor, Rap1, PI3K-Akt, cAMP, NF-kappa B and many immune-related pathways were detected in the catagen and telogen stages. The PI3K-Akt signaling, ECM-receptor interaction and Focal adhesion were found in the transition stage between telogen to anagen, which may serve as candidate biomarkers for telogen-anagen regeneration. A total of 16 signaling pathways, 145 pathway mRNAs, and 93 lncRNAs were enrolled to construct the pathway-mRNA-lncRNA network, which indicated the function of lncRNAs through interacting with their co-expressed mRNAs. Pairwise comparisons between the control and melatonin-treated groups also indicated 941 monthly differentially expressed genes (monthly DEGs). These monthly DEGs were mainly distributed from April and September, which revealed a potential signal pathway map regulating the anagen stage triggered by melatonin. Enrichment analysis showed that Wnt, Hedgehog, ECM, Chemokines and NF-kappa B signaling pathways may be involved in the regulation of non-quiescence and secondary shedding under the influence of melatonin. CONCLUSIONS: Our study decoded the key regulators of the whole hair follicle growth cycle, laying the foundation for the control of hair follicle growth and improvement of cashmere yield.

Effects of oat (Avena sativa L.) hay diet supplementation on the intestinal microbiome and metabolome of Small-tail Han sheep.

Supplementation of the sheep diet with oats (Avena sativa L.) improves animal growth and meat quality, however effects on intestinal microbes and their metabolites was not clear. This study aimed to establish the effect of dietary oat supplementation on rumen and colonic microbial abundance and explore the relationship with subsequent changes in digesta metabolites. Twenty Small-tail Han sheep were randomly assigned to a diet containing 30 g/100 g of maize straw (Control) or oat hay (Oat). After 90-days on experimental diets, rumen and colon digesta were collected and microbial diversity was determined by 16S rRNA gene Illumina NovaSeq sequencing and metabolomics was conducted using Ultra-high performance liquid chromatography Q-Exactive mass spectrometry (UHPLC-QE-MS). Compared to Control group, oat hay increased the abundance of Bacteroidetes and Fibrobacteres as well as known short-chain fatty acid (SCFA) producers Prevotellaceae, Ruminococcaceae and Fibrobacteraceae in rumen (p < 0.05). In rumen digesta, the Oat group showed had higher levels of (3Z,6Z)-3,6-nonadienal, Limonene-1,2-epoxide, P-tolualdehyde, and Salicylaldehyde compared to Control (p < 0.05) and these metabolites were positively correlated with the abundance of cecal Prevotellaceae NK3B31. In conclusion, supplementation of the sheep diet with oat hay improved desirable microbes and metabolites in the rumen, providing insight into mechanisms whereby meat quality can be improved by oat hay supplementation.

Genetic diversity analysis of Dermacentor nuttalli within Inner Mongolia, China.

BACKGROUND: Ticks (Arthropoda, Ixodida), after mosquitoes, are the second most prevalent vector of infectious diseases. They are responsible for spreading a multitude of pathogens and threatening the health and welfare of animals and human beings. However, given the history of tick-borne pathogen infections in the Inner Mongolia Autonomous Region of China, surprisingly, neither the genetic diversity nor the spatial distribution of haplotypes within ticks has been studied. METHODS: We characterized the haplotype distribution of Dermacentor nuttalli in four main pastoral areas of the Inner Mongolia Autonomous Region, by sampling 109 individuals (recovered from sheep) in April-August 2019. The 16S rRNA gene, cytochrome c oxidase subunit I (COI), and the internal transcribed spacer 2 region (ITS2) were amplified and sequenced from extracted DNA. RESULTS: Twenty-six haplotypes were identified using 16S rRNA sequences, 57 haplotypes were identified with COI sequences, and 75 haplotypes were identified with ITS2 sequences. Among the three genes, total haplotype diversity was greater than 0.7, while total nucleotide diversity was greater than 0.06. Neutrality tests revealed a significantly negative Tajima's D result, while Fu's Fs was not significantly positive. Fixation index values (FST) indicated that the degree of genetic differentiation among some sampled populations was small, while for others it was moderate. Analysis of molecular variance (AMOVA) revealed that the variation within populations was greater than that among populations. The mismatch analysis of D. nuttalli exhibited double peaks. CONCLUSION: The genetic diversity of D. nuttalli populations in our region can likely adapt to different geographical environments, thereby leading to genetic diversity, and creating genetic differentiation among different populations. However, genetic differentiation is cryptic and does not form a pedigree geographical structure.

Identification of piRNAs in the testes of Sunite and Small-tailed Han sheep.

P-element-induced wimpy testis-interacting RNAs (piRNAs) are small RNAs that are essential for mammalian spermatogenesis and testicular development. Comparative analyses of the molecular mechanisms of spermatogenesis among different organisms are therefore dependent on accurate piRNA characterizations. In this study, we identified piRNAs in the testes of two breeds of Mongolian sheep: the Sunite (SN), which has a low reproductive rate, and Small-tailed Han (STH), which has a high reproductive rate. A thorough understanding of the mechanisms underlying the differences in fecundity between the two breeds might provide insights for the improvement of fertility and reproductive success in these and other sheep breeds. We identified 835 piRNAs and 206 piRNA clusters across the two breeds. Of these, 29 putative piRNAs were expressed in the SN samples only, and 229 putative piRNAs were expressed in the STH samples only. In addition, 206 piRNA clusters were upregulated in STH sheep as compared to the SN sheep. Functional pathway analysis indicated that the genes neighboring the predicted piRNAs were likely associated with spermatogenesis. piRNAs might thus be linked to male fecundity in sheep. Our results increase knowledge of the association between piRNAs and male fertility.

Comparative Transcriptome Analysis Reveals that a Ubiquitin-Mediated Proteolysis Pathway Is Important for Primary and Secondary Hair Follicle Development in Cashmere Goats.

BACKGROUND: The fleece of cashmere goats contains two distinct populations of fibers, a short and fine non-medullated insulating cashmere fiber and a long and coarse medullated guard hair. The former is produced by secondary follicles (SFs) and the later by primary follicles (PFs). Evidence suggests that the induction of PFs and SFs may require different signaling pathways. The regulation of BMP2/4 signaling by noggin and Edar signaling via Downless genes are essential for the induction of SFs and PFs, respectively. However, these differently expressed genes of the signaling pathway cannot directly distinguish between the PFs and SFs. RESULTS: In this study, we selected RNA samples from 11 PFs and 7 SFs that included 145,525 exons. The pathway analysis of 4512 differentially expressed exons revealed that the most statistically significant metabolic pathway was related to the ubiquitin-mediated proteolysis pathway (UMPP) (P<3.32x 10-7). In addition, the 51 exons of the UMPP that were differentially expressed between the different types of hair follicle (HFs) were compared by cluster analysis. This resulted in the PFs and SFs being divided into two classes. The expression level of two selected exons was analyzed by qRT-PCR, and the results indicated that the expression patterns were consistent with the deep sequencing results obtained by RNA-Seq. CONCLUSIONS: Based on the comparative transcriptome analysis of 18 HFs from cashmere goats, a large number of differentially expressed exons were identified using a high-throughput sequencing approach. This study suggests that UMPP activation is a prominent signaling pathway for distinguishing the PFs and SFs of cashmere goats. It is also a meaningful contribution to the theoretical basis of the biological study of the HFs of cashmere goats and other mammals.

[Transcriptome Analysis of Rabbit Spleen with Hog Cholera Lapinized Virus Infection Based on High-throughput Sequencing Technology].

In the current study, rabbit spleen was analysed at 12 h,24h,30 h,36h,and 48hpost-infection with hog cholera lapinized virus(C strain)using high-throughput sequencing. The rabbit genome was used as a reference to identify differentially expressed genes(DEGs)at different time points post-infection. The top 10 DEGs were filtered based on significance, and we searched for their biological functions through the Uniprot and NCBI databases. The former three time points have 10co-expressing genes, many of which have a relationship to immunity and inflammation. The latter two time points for the top 10 DEGs are identical, and B2 M,RLA-DR-ALPHA,CD74,and IGJ are involved in the antiviral immune response. GO functional annotation revealed that in biological processes at each time point,except for 24hpost-infection,the immune response has the most terms, followed by metabolism and regulation. According to the KEGG database, the DEGs for 24hpost-infection were enriched for the RIG-I-like receptor signaling pathway and the DEGs for 30hpost-infection were found to have a focal adhesion and ECM-receptor interaction pathway. Moreover, the DEGs for 36 hand 48hpost-infection have seven identical pathways, of which were directly or indirectly related to the antiviral response. These pathways included the proteasome, lysosome, ribosome, chemokine signaling pathways, B cell receptor signaling pathway, antigen processing, and presentation pathway, and the Fc gamma R-mediated phagocytosis pathway.These results provide novel insight into the gene expression in rabbit spleens post-infection with C strain, and provide a theoretical basis for further understanding of the molecular mechanisms by which rabbits adapt to infection with C strain.

[Research Progress in the Core Proteins of the Classical Swine Fever Virus].

The core protein (CP) of the classical swine fever virus (CSFV) is one of its structural proteins. Apart from forming the nucleocapsid to protect internal viral genomic RNA, this protein is involved in transcriptional regulation. Also, during viral infection, the CP is involved in interactions with many host proteins. In this review, we combine study of this protein with its disorders, structural/functional characteristics, as well as its interactions with the non-structural proteins NS3, NS5B and host proteins such as SUMO-1, UBC9, OS9 and IQGAP1. We also summarize the important part played by the CP in CSFV pathogenicity, virulence and replication of genomic RNA. We also provide guidelines for further studies in the CP of the CSFV.

Comparative genomic approach reveals novel conserved microRNAs in Inner Mongolia cashmere goat skin and longissimus dorsi.

MicroRNA (miRNA) is a large class of non-coding RNA which usually acts a fine-tuned regulation in repressing gene expression on transcriptional and post-transcriptional level during ontogenetic development, metabolism, and occurrence of disease. Due to the lateness of goat genome investigation, registered goat microRNAs are little known and the function of it is poorly understood. In this study, we identified 5 novel miRNAs express in cashmere goat skin and longissimus dorsi using comparative genomic approach combined with expression profile analysis. Further qPCR and sequencing validation demonstrated that the novel miRNAs we identified expressed in goat skin and longissimus dorsi, three of them with the highest expression in February and two in October, indicating these novel microRNAs were involved in growth and cessation of goat hair production. Subsequently, potential target genes were explored via base-pairing with complementary sequences miRNA/mRNA interaction searching. WDR12 and CSNK1A1 involved in Notch/Wnt signal transduction pathway were finally identified. Collectively, the approach of comparative genomics combining expression profile analysis is a powerful tool to identify novel miRNA in goat and the 5 identified novel miRNAs(chi-miR-2284n, chi-miR-421*, chi-miR-421, chi-miR-1839 and chi-miR-374; Accession number: JQ002550-JQ002554) play a essential role in goat hair production of cashmere goat, both in entering growth and cessation phase. This study is also a meaningful complement to the present goat miRNA database for further understanding function of miRNAs in regulation of goat cashmere production.