Efficacy of H2O2 inactivated bovine virus diarrhoea virus (BVDV) type 1 vaccine in mice.

BACKGROUND: Bovine viral diarrhea (BVD) is an acute febrile infectious disease caused by the bovine viral diarrhea virus (BVDV), which has brought huge economic losses to the world's cattle industry. At present, commercial inactivated BVDV vaccines may cause some adverse reactions during use. This study aims to develop a safer and more efficient inactivated BVDV vaccine. METHODS: Here, we described the generation and preclinical efficacy of a hydrogen peroxide (H2O2) inactivated BVDV type 1 vaccine in mice, and administered it separately with commercial vaccine (formaldehyde inactivated) in mice to study its efficacy. RESULTS: The BVDV type 1 IgG, IFN- γ, IL-4 and neutralizing antibody in the serum of the H2O2 inactivated vaccine group can be maintained in mice for 70 days. The IgG level reached its maximum value of 0.67 on the 42nd day, significantly higher than the commercial formaldehyde inactivated BVDV type 1 vaccine. IFN- γ and IL-4 reached their maximum values on the 28th day after immunization, at 123.16 pg/ml and 143.80 pg/ml, respectively, slightly higher than commercial vaccines, but the effect was not significant. At the same time the BVDV-1 neutralizing antibody titer reached a maximum of 12 Nu on the 42nd day post vaccination. CONCLUSIONS: The H2O2 inactivated BVDV vaccine has good safety and immunogenicity, which provides a potential solution for the further development of an efficient and safe BVDV vaccine.

Isolation and functional analysis of acid-producing bacteria from bovine rumen.

Ruminants such as cattle rely mainly on microbes in the rumen to digest cellulose and hemicellulose from forage, and the digestion products are mainly absorbed and utilized by the host in the form of short chain fatty acids (SCFAs). This study aimed to isolate acid-producing strains from the cattle rumen and investigate their functions. A total of 980 strains of acid-producing bacteria were isolated from cattle rumen contents using a medium supplemented with bromocresol green. Combined with the test of acid production ability and 16S rRNA amplicon sequencing technology, five strains were selected based on their ability to produce relatively high levels of acid, including Bacillus pumillus, Enterococcus hirae, Enterococcus faecium, and Bacillus subtilis. Sheep were treated by gavage with a mixed bacterial suspension. The results showed that mixed bacteria significantly increased the body weight gain and feed conversion rate of sheep. To investigate the function of acid-producing bacteria in sheep, we used 16S rDNA sequencing technology to analyze the rumen microbes of sheep. We found that mixed bacteria changed the composition and abundance of sheep rumen bacteria. Among them, the abundance of Bacteroidota, Actinobacteriota, Acidobacteriota, and Proteobacteria was significantly increased, and the abundance of Firmicutes was significantly decreased, indicating that the changes in gut microbiota changed the function of the sheep rumen. The acid-producing bacteria isolated in this study can effectively promote the growth of ruminants, such as cattle and sheep, and can be used as additives to improve breeding efficiency, which lays a foundation for subsequent research on probiotics.

Expanded catalogue of metagenome-assembled genomes reveals resistome characteristics and athletic performance-associated microbes in horse.

BACKGROUND: As a domesticated species vital to humans, horses are raised worldwide as a source of mechanical energy for sports, leisure, food production, and transportation. The gut microbiota plays an important role in the health, diseases, athletic performance, and behaviour of horses. RESULTS: Here, using approximately 2.2 Tb of metagenomic sequencing data from gut samples from 242 horses, including 110 samples from the caecum and 132 samples from the rectum (faeces), we assembled 4142 microbial metagenome-assembled genomes (MAG), 4015 (96.93%) of which appear to correspond to new species. From long-read data, we successfully assembled 13 circular whole-chromosome bacterial genomes representing novel species. The MAG contained over 313,568 predicted carbohydrate-active enzymes (CAZy), over 59.77% of which had low similarity match in CAZy public databases. High abundance and diversity of antibiotic resistance genes (ARG) were identified in the MAG, likely showing the wide use of antibiotics in the management of horse. The abundances of at least 36 MAG (e.g. MAG belonging to Lachnospiraceae, Oscillospiraceae, and Ruminococcus) were higher in racehorses than in nonracehorses. These MAG enriched in racehorses contained every gene in a major pathway for producing acetate and butyrate by fibre fermentation, presenting potential for greater amount of short-chain fatty acids available to fuel athletic performance. CONCLUSION: Overall, we assembled 4142 MAG from short- and long-read sequence data in the horse gut. Our dataset represents an exhaustive microbial genome catalogue for the horse gut microbiome and provides a valuable resource for discovery of performance-enhancing microbes and studies of horse gut microbiome. Video Abstract.

Analysis of the eighth intron polymorphism of NR6A1 gene in sheep and its correlation with lumbar spine number.

For revealing molecular markers related to the traits of multiple lumbar vertebrae in sheep, we analyze the relationship between NR6A1 gene polymorphism and lumbar vertebrae number traits in Xinjiang Kazakh sheep. Lumbar muscle tissues were collected from 6-lumbar spine (L6) Kazak sheep and 7-lumbar spine (L7) Kazak sheep and the intron-8 of NR6A1 gene was amplified by PCR. The SNP locus was detected by the PCR-SSCP method. One-Way ANOVA and an Independent Chi-square Test is adopted to analyze the genotype association with lumbar spine number variation. There were two SNP loci in the intron-8 of the NR6A1 gene: IVS8-188 and IVS8-281. One-Way ANOVA and Independent Chi-square Test indicated a significant association between IVS8-281 and lumbar spine number. The SNP locus of NR6A1 gene intron 8 (IVS8-281G > A) could play a certain role in the variation of lumbar spine number in Xinjiang Kazakh sheep and demonstrates potential to accelerate the sheep breeding of selection process.

CRAMdb: a comprehensive database for composition and roles of microbiome in animals.

CRAMdb (a database for composition and roles of animal microbiome) is a comprehensive resource of curated and consistently annotated metagenomes for non-human animals. It focuses on the composition and roles of the microbiome in various animal species. The main goal of the CRAMdb is to facilitate the reuse of animal metagenomic data, and enable cross-host and cross-phenotype comparisons. To this end, we consistently annotated microbiomes (including 16S, 18S, ITS and metagenomics sequencing data) of 516 animals from 475 projects spanning 43 phenotype pairs to construct the database that is equipped with 9430 bacteria, 278 archaea, 2216 fungi and 458 viruses. CRAMdb provides two main contents: microbiome composition data, illustrating the landscape of the microbiota (bacteria, archaea, fungi, and viruses) in various animal species, and microbiome association data, revealing the relationships between the microbiota and various phenotypes across different animal species. More importantly, users can quickly compare the composition of the microbiota of interest cross-host or body site and the associated taxa that differ between phenotype pairs cross-host or cross-phenotype. CRAMdb is freely available at (http://www.ehbio.com/CRAMdb).

A database of animal metagenomes.

With the rapid development of high-throughput sequencing technology, the amount of metagenomic data (including both 16S and whole-genome sequencing data) in public repositories is increasing exponentially. However, owing to the large and decentralized nature of the data, it is still difficult for users to mine, compare, and analyze the data. The animal metagenome database (AnimalMetagenome DB) integrates metagenomic sequencing data with host information, making it easier for users to find data of interest. The AnimalMetagenome DB is designed to contain all public metagenomic data from animals, and the data are divided into domestic and wild animal categories. Users can browse, search, and download animal metagenomic data of interest based on different attributes of the metadata such as animal species, sample site, study purpose, and DNA extraction method. The AnimalMetagenome DB version 1.0 includes metadata for 82,097 metagenomes from 4 domestic animals (pigs, bovines, horses, and sheep) and 540 wild animals. These metagenomes cover 15 years of experiments, 73 countries, 1,044 studies, 63,214 amplicon sequencing data, and 10,672 whole genome sequencing data. All data in the database are hosted and available in figshare https://doi.org/10.6084/m9.figshare.19728619 .

ADDAGMA: A database for domestic animal gut microbiome atlas.

Animal gut microbiomes play important roles in the health, diseases, and production of animal hosts. The volume of animal gut metagenomic data, including both 16S amplicon and metagenomic sequencing data, has been increasing exponentially in recent years, making it increasingly difficult for researchers to query, retrieve, and reanalyze experimental data and explore new hypotheses. We designed a database called the domestic animal gut microbiome atlas (ADDAGMA) to house all publicly available, high-throughput sequencing data for the gut microbiome in domestic animals. ADDAGMA enhances the availability and accessibility of the rapidly growing body of metagenomic data. We annotated microbial and metadata from four domestic animals (cattle, horse, pig, and chicken) from 356 published papers to construct a comprehensive database that is equipped with browse and search functions, enabling users to make customized, complicated, biologically relevant queries. Users can quickly and accurately obtain experimental information on sample types, conditions, and sequencing platforms, and experimental results including microbial relative abundances, microbial taxon-associated host phenotype, and P-values for gut microbes of interest. The current version of ADDAGMA includes 290,422 quantification events (changes in abundance) for 3215 microbial taxa associated with 48 phenotypes. ADDAGMA presently covers gut microbiota sequencing data from pig, cattle, horse, and chicken, but will be expanded to include other domestic animals. ADDAGMA is freely available at (http://addagma.omicsbio.info/).

Whole-genome resequencing to investigate the determinants of the multi-lumbar vertebrae trait in sheep.

Multi-lumbar vertebrae trait is a beneficial mutation that can significantly improve livestock meat production. However, the genetic basis of the multi-lumbar vertebrae in sheep is still unclear. Here, we analysed the number of lumbar vertebrae of Duolang sheep and found three different traits of lumbar vertebrae number. Compared with the normal sheep, the length and weight of animal carcass from the multi-lumbar vertebrae sheep increased by 2.21 cm and 0.78 kg, respectively. We performed high-throughput genome resequencing on multi-lumbar vertebrae (n = 18) and normal (n = 11) Duolang sheep and obtained a total of more than 528.87 GB data. We found that the most significantly selective region were located in the 49.68-49.74 MB of chromosome 4 by selective-sweep analysis. We annotated this region and found that it contains SFRP4 which is known to regulate bone development. We further used the PCR-SSCP technology to detect the single nucleotide polymorphism (SNP) of the putative candidate SFRP4 and found that the two SNPs (rs600370085:C > T and rs415133338: A > G) of this gene were significantly associated with the multi-lumbar vertebrae of Duolang sheep. Our study indicates that the SFRP4 may be a potential major gene that affects the number of lumbar vertebrae in Duolang sheep, and has the potential to be utilized for sheep breeding in the future.

CRISPR-Cas13a-Based Detection for Bovine Viral Diarrhea Virus.

Bovine Viral Diarrhea Virus (BVDV) is the main pathogen of bovine viral diarrhea disease (BVD), which leads to enormous economic losses in the cattle industry. A sensitive and specific detection for BVDV is advantageous to the control of BVDV. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have been used for detecting virus RNA. In this study, the expression and purification of LwCas13a protein was optimized and the RNase activity of LwCas13a in vitro was verified. CRISPR-LwCas13a system could detect BVDV virus and BVDV RNA with high specificity and simplicity. The detection limit of the LwCas13a system was 103 pM, and there were no cross-reactions with HEK293T and MDBK. In summary, a sensitive, specific, and simple nucleic acid detection method based on CRISPR-Cas13a was developed for BVDV. This method provides a new detection strategy for early diagnosis of BVDV.

Modes of genetic adaptations underlying functional innovations in the rumen.

The rumen is the hallmark organ of ruminants and hosts a diverse ecosystem of microorganisms that facilitates efficient digestion of plant fibers. We analyzed 897 transcriptomes from three Cetartiodactyla lineages: ruminants, camels and cetaceans, as well as data from ruminant comparative genomics and functional assays to explore the genetic basis of rumen functional innovations. We identified genes with relatively high expression in the rumen, of which many appeared to be recruited from other tissues. These genes show functional enrichment in ketone body metabolism, regulation of microbial community, and epithelium absorption, which are the most prominent biological processes involved in rumen innovations. Several modes of genetic change underlying rumen functional innovations were uncovered, including coding mutations, genes newly evolved, and changes of regulatory elements. We validated that the key ketogenesis rate-limiting gene (HMGCS2) with five ruminant-specific mutations was under positive selection and exhibits higher synthesis activity than those of other mammals. Two newly evolved genes (LYZ1 and DEFB1) are resistant to Gram-positive bacteria and thereby may regulate microbial community equilibrium. Furthermore, we confirmed that the changes of regulatory elements accounted for the majority of rumen gene recruitment. These results greatly improve our understanding of rumen evolution and organ evo-devo in general.

Identification and Profiling of Pituitary microRNAs of Sheep during Anestrus and Estrus Stages.

MicroRNAs (miRNAs) are a class of small non-coding RNAs, molecules of 21 to 25 nucleotides in length, that regulate gene expression by binding to their target mRNA and play a significant role in animal development. The expression and role of miRNAs in regulating sheep estrus, however, remain elusive. Transcriptome analysis is helpful to understand the biological roles of miRNAs in the pituitary gland of sheep. A sheep's pituitary gland has a significant difference between estrus and anestrus states. Here, we investigate the expression profiles of sheep anterior pituitary microRNAs (miRNAs) in two states, estrus and anestrus, using Illumina HiSeq-technology. This study identified a total of 199 miRNAs and 25 differentially expressed miRNAs in the estrus and anestrus pituitary gland in sheep. Reverse transcription quantitative-PCR (RT-qPCR) analysis shows six differentially (p < 0.05) expressed miRNAs, that are miR-143, miR-199a, miR-181a, miR-200a, miR-218, and miR-221 in both estrus and anestrus states. miRNAs containing estrus-related terms and pathways regulation are enriched using enrichment analysis from gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Moreover, we also envisioned a miRNA-mRNA interaction network to understand the function of miRNAs involved in the pituitary gland regulatory network. In conclusion, miRNA expression profiles in sheep pituitary gland in the anestrus and estrus deliver a theoretical basis for the study of pituitary gland biology in sheep.

Analysis of pituitary transcriptomics indicates that lncRNAs are involved in the regulation of sheep estrus.

Seasonal estrus is a key factor limiting animal fertility, and understanding the molecular mechanisms that regulate animal estrus is important for improving animal fertility. The pituitary gland, which is the most important endocrine gland in mammals, plays an important role in regulating the physiological processes such as growth, development, and reproduction of animals. Here, we used RNA-seq technology to study the expression profile of lncRNAs in the anterior pituitary of sheep during estrus and anestrus. In this study, we identified a total of 995 lncRNAs, of which 335 lncRNAs were differentially expressed in two states (including 38 up-regulated and 297 down-regulated lncRNAs). RT-qPCR verified the expression levels of several lncRNAs. Target predictive analysis revealed that these lncRNAs can act in cis or trans and regulate the expression of genes involved in the regulation of sheep estrus. Target gene enrichment analysis of differentially expressed lncRNAs indicates that these lncRNAs can regulate sheep estrus by regulating hormone metabolism and energy metabolism. Through our research, we provide the expression profile of lncRNAs in the pituitary of sheep, which provides a valuable resource for further understanding of the genetic regulation of seasonal estrus in sheep from the perspective of lncRNAs.

Screening and evaluating of long non-coding RNAs in prenatal and postnatal pituitary gland of sheep.

Long non-coding RNAs are transcribed into RNA molecules that are >200 nucleotides in length. However, the expression and function analysis of lncRNAs in the sheep pituitary gland are still lacking. In this study, we identified 1755 lncRNAs (545 annotated lncRNAs and 1210 novel lncRNAs) from RNA-seq data in the pituitary gland of embryonic and adult sheep. A total of 235 lncRNAs were differentially expressed between embryonic and adult group. We verified the presence of some lncRNAs using RT-PCR and DNA sequencing, and identified some differentially expressed lncRNAs using qPCR. We also investigated the role of cis-acting lncRNAs on target genes. GO and KEGG enrichment analysis revealed that the target genes of lncRNAs were involved in the regulation of hormones secretion and some signaling pathways in the sheep pituitary gland. Our study provides comprehensive expression profiles of lncRNAs and valuable resource for understanding their function in the pituitary gland.

Rapid visual detection of FecB gene expression in sheep.

Sheep play an important role in agricultural production and people's lives, and fecundity is one of the most important economic traits of sheep for sheep breeders. The Booroola fecundity (FecB) gene has a certain correlation with litter size in sheep. Therefore, this study aims to detect FecB expression quickly, accurately and visually. Here, we used the nucleic acid dye SYBR Green I to detect FecB with the amplification refractory mutation system (ARMS), which can efficiently, rapidly, economically and visually detect FecB expression in sheep. After ARMS polymerase chain reaction (PCR), SYBR Green I was directly added to the ARMS products, and whether the sheep carried FecB was judged by directly observing the color change in the PCR tube. Homozygous (BB) or heterozygous (B+) samples with FecB mutation were bright green, while wild type (++) samples without FecB were orange yellow. This study suggested that this method has 100% accuracy and 0.5 ng/µL sensitivity. To our knowledge, this is the first report that shows the integration of the ARMS with SYBR Green I to detect FecB expression in sheep visually.

Circ-HIPK3 plays an active role in regulating myoblast differentiation.

The development of skeletal muscle is a complex biological process involving a variety of regulatory pathways. A deeper understanding of the developmental mechanisms of skeletal muscle is of great significance for the treatment of muscular lesions. In recent years, studies have shown that circRNAs, as a new class of post-transcriptional regulators, play an important regulatory role in a variety of biological processes, but their function in skeletal muscle development remains unclear. Here, we used C2C12 myoblasts to study circ-HIPK3, which has significant differences in the developmental stages of skeletal muscle and is highly expressed. We found that the expression level of circ-HIPK3 was significantly up-regulated (p < .05) with the persistence of C2C12 cell differentiation. Combining the results of bioinformatics analysis and dual luciferase reporter experiments, we found that circ-HIPK3 is a sponge of miR-124 and miR-379 that regulate muscle differentiation. Our study shows that circ-HIPK3 can promote the differentiation of C2C12 myoblasts, providing a scientific basis for further research on the development of skeletal muscle at the level of circRNAs.

Whole-Genome Resequencing Reveals Loci Associated With Thoracic Vertebrae Number in Sheep.

The number of vertebrae, especially thoracic vertebrae, is an important economic trait that may influence carcass length and meat production in animals. However, the genetic basis of vertebrae number in sheep is still poorly understood. To detect the candidate genes, 400 increased number of thoracic vertebrae (T14L6) and 200 normal (T13L6) Kazakh sheep were collected. We generated and sequenced 60 pools of genomic DNA (each pool prepared by mixing genomic DNA from 10 sheep with the same thoracic traits), with an average depth of coverage of 25.65×. We identified a total of 42,075,402 SNPs and 11 putatively selected genomic regions, including the VRTN gene and the HoxA gene family that regulate vertebral development. The most prominent areas of selective elimination were located in a region of chromosome 7, including VRTN, which regulates spinal development and morphology. Further investigation indicated that the expression level of the VRTN gene during fetal development was significantly higher in sheep with more thoracic vertebrae than in those with a normal number of thoracic vertebrae. A genome-wide comparison between sheep with increased and normal numbers of thoracic vertebrae showed that the VRTN gene is the major selection locus for the number of thoracic vertebrae in sheep and has the potential to be utilized in sheep breeding in the future.

Association analysis of polymorphism in the NR6A1 gene with the lumbar vertebrae number traits in sheep.

INTRODUCTION: The vertebral number is an economically significant trait, which is associated with body length and carcass traits. Nuclear Receptor Subfamily 6, Group A, Member 1 (NR6A1) is a member of the nuclear receptor superfamily and it plays an important role in the early development of embryos. OBJECTIVES: The NR6A1 gene was considered as an important candidate for influence vertebrae number, while the potential associations between this gene and the number of lumbar vertebrae traits of sheep have not been explored. METHODS: In this study, we detected the genetic variants of NR6A1 gene and analyzed the associations of the polymorphisms with lumbar number traits in 130 Kazakh sheep. We use single-strand conformation polymorphism (SSCP) technique to detect single nucleotide polymorphism (SNP) of NR6A1 gene, and the association of the genotype and lumbar number variation was analyzed by independent Chi-square test. RESULTS: We detect SNP of NR6A1 gene by PCR-SSCP technique, and polymorphisms were only found in the coding region of exon-6 and exon-8 of NR6A1 gene. In order to investigate the connection between the SNP locus and lumbar number traits in sheep, we conducted a Chi-square test for independence for exon-6 and exon-8 of NR6A1 gene, respectively. Association analysis revealed significant associations between the SNP (rs414302710: A >C) in the exon-8 of NR6A1 gene with the number of lumbar vertebrae (P < 0.01). CONCLUSION: Our study indicated that this SNP (rs414302710: A>C) locus of exon-8 of NR6A1 gene in sheep possible influence the number of lumbar vertebrae, which has the potential to be applied in selective breeding of sheep.

Circular RNA circ-FoxO3 Inhibits Myoblast Cells Differentiation.

CircRNA is a type of closed circular non-coding RNA formed by reverse splicing and plays an important role in regulating the growth and development of plants and animals. To investigate the function of circ-FoxO3 in mouse myoblast cells' (C2C12) differentiation and proliferation, we used RT-qPCR to detect the expression level of circ-FoxO3 in mouse myoblast cells at different densities and different differentiation stages, and the specific interference fragment was used to inhibit the expression level of circ-FoxO3 in myoblast cells to observe its effect on myoblast cells proliferation and differentiation. We found that the expression level of circ-FoxO3 in myoblast cells increased with the prolongation of myoblast cells differentiation time, and its expression level decreased with the proliferation of myoblast cells. At the same time, we found that the differentiation ability of the cells was significantly increased (p < 0.05), but the cell proliferation was unchanged (p > 0.05) after inhibiting the expression of circ-FoxO3 in myoblast cells. Combining the results of bioinformatics analysis and the dual luciferase reporter experiment, we found that circ-FoxO3 is a sponge of miR-138-5p, which regulates muscle differentiation. Our study shows that circ-FoxO3 can inhibit the differentiation of C2C12 myoblast cells and lay a scientific foundation for further study of skeletal muscle development at circRNA levels.

Genetic basis of ruminant headgear and rapid antler regeneration.

Ruminants are the only extant mammalian group possessing bony (osseous) headgear. We obtained 221 transcriptomes from bovids and cervids and sequenced three genomes representing the only two pecoran lineages that convergently lack headgear. Comparative analyses reveal that bovid horns and cervid antlers share similar gene expression profiles and a common cellular basis developed from neural crest stem cells. The rapid regenerative properties of antler tissue involve exploitation of oncogenetic pathways, and at the same time some tumor suppressor genes are under strong selection in deer. These results provide insights into the evolutionary origin of ruminant headgear as well as mammalian organ regeneration and oncogenesis.

Comprehensive analysis of circRNAs expression profiles in different periods of MDBK cells infected with bovine viral diarrhea virus.

CircRNAs play an important regulatory role in the regulation of disease. However, we have a limited understanding of the role of circRNAs in the host's complex protective and pathological mechanisms of BVDV infection. Transcriptome analysis of circRNAs in host cells after BVDV infection may allow us to understand the biological functions of circRNAs in the regulation of BVDV infection. Here, we identified a total of 19,118 circRNAs from the MBDK cells (at 12 h, 24 h, and 48 h post-infection) infected with BVDV by using RNA-seq technology. We confirmed several circRNAs using RT-PCR and DNA sequencing, and qRT-PCR analysis was performed to identify several circRNAs expression and circRNAs resistance to RNase R digestion. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis showed that the host genes of differentially expressed circRNAs were involved in the regulation of cell proliferation, apoptosis, cycle and viral infection related signaling pathways. These results indicate that circRNA in host cells plays a broad regulatory role after BVDV infection and provides a valuable resource for studying circRNA biology in host cells after BVDV infection.