Establishment and functional characterization of immortalized rabbit dermal papilla cell lines.

Hair follicle (HF) undergo periodic growth and development in mammals, which regulated by dermal papilla cells (DPCs) are reported to play an important role in HF morphogenesis and development. However, primary DPCs have low proliferative activity, age quickly, and fresh cell isolation is both time-consuming and laborious. In this study, we introduced the SV40 large T antigen (SV40T) into dissociated early passage rabbit vibrissae DPCs with lentiviral vectors and established seven immortalized DPC lines (R-1, R-2, R-3, R-4, R-5, R-6 and R-7). These cell lines displayed early passage morphology and high alkaline phosphatase activity. RT-PCR and immunofluorescence staining showed that all the immortalized cell lines expressed the DPC markers (α-SMA, IGF1, ALPL, FGF2, BMP2 and TGFß2), but α-SMA was only expressed well in R-3, R-4, and R-7. Furthermore, it was found that R-7 was the only line to survive beyond 50 passages. Compared to melanoma cells, R-7 did not undergo malignant transformation. Karyotyping and cell growth viability analysis illustrated that the R-7 cell line preserved the basic characteristics of primary DPCs. The R-7 DPCs established have potential application for future hair research. The study provides the theoretical basis in the cell research of HF growth and development.

Dermal PapillaCell-Derived Exosomes Regulate Hair Follicle Stem Cell Proliferation via LEF1.

Hair follicle (HF) growth and development are controlled by various cell types, including hair follicle stem cells (HFSCs) and dermal papilla cells (DPCs). Exosomes are nanostructures that participate in many biological processes. Accumulating evidence indicates that DPC-derived exosomes (DPC-Exos) mediate HFSC proliferation and differentiation during the cyclical growth of hair follicles. In this study, we found that DPC-Exos increase ki67 expression and CCK8 cell viability readouts in HFSCs but reduce annexin staining of apoptotic cells. RNA sequencing of DPC-Exos-treated HFSCs identified 3702 significantly differentially expressed genes (DEGs), including BMP4, LEF1, IGF1R, TGFß3, TGFα, and KRT17. These DEGs were enriched in HF growth- and development-related pathways. We further verified the function of LEF1 and showed that overexpression of LEF1 increased the expression of HF development-related genes and proteins, enhanced HFSC proliferation, and reduced HFSC apoptosis, while knockdown of LEF1 reversed these effects. DPC-Exos could also rescue the siRNA-LEF1 effect in HFSCs. In conclusion, this study demonstrates that DPC-Exos mediated cell-to-cell communication can regulate HFSCs proliferation by stimulating LEF1 and provide novel insights into HF growth and development regulatory mechanisms.

DNA Methylation Mediates lncRNA2919 Regulation of Hair Follicle Regeneration.

Hair follicles (HFs) are organs that periodically regenerate during the growth and development of mammals. Long non-coding RNAs (lncRNAs) are non-coding RNAs with crucial roles in many biological processes. Our previous study identified that lncRNA2919 is highly expressed in catagen during the HF cycle. In this study, the in vivo rabbit model was established using intradermal injection of adenovirus-mediated lncRNA2919. The results showed that lncRNA2919 decreased HF depth and density and contributed to HF regrowth, thereby indicating that lncRNA2919 plays a negative role in HF regeneration. Moreover, methylation levels of the lncRNA2919 promoter at different HF cycle stages were detected through bisulfite sequencing. The key CpG site that negatively correlates with lncRNA2919 expression during the HF cycle was identified. 5-Aza-dc-induced demethylation upregulated lncRNA2919 expression, and the core promoter region of lncRNA2919 was verified on the basis of luciferase activity. Furthermore, we found that DNA methylation could prevent the binding of EGR1 to the lncRNA2919 promoter region, thereby affecting the transcriptional expression of lncRNA2919. Collectively, DNA methylation inhibits the transcriptional expression of lncRNA2919, which plays a vital role in the HF cycle and HF regrowth. These findings contribute to the basic theory of epigenetics in HF biology and provide references for further research in HF disease treatment and animal wool production.

Characterization of HTATIP2 and its role during hair follicle cycles in Angora rabbit.

Hair follicle (HF) growth and cycling is a complex biological process that occurs in most mammals. As HF growth and cycling directly impacts rabbit wool yield, it is important to better understand the potential regulation pattern of HF development. Our previous study demonstrated that HTATIP2 may participate in regulating rabbit HF cycles, but the molecular mechanism of HTATIP2 remained unclear. In this study, the coding sequence of the HTATIP2 gene in Angora rabbit was cloned. The length of the coding region sequence was 840 bp, which could code 279 amino acids, and exhibited high homology in different mammals. Bioinformatics analyses indicated that the HTATIP2 protein is stable, hydrophilic, located around the cytoplasm, and has a putative signal peptide. Moreover, we verified that HTATIP2 is highly expressed during catagen and telogen of the HF cycle. The overexpression vector was constructed and siRNAs were designed. Overexpression and knockdown of HTATIP2 appeared to regulate JAK-STAT pathway genes, such as BCL2, CCND1, c-Myc, and STAT2. It is therefore likely that HTATIP2 promotes cell apoptosis and inhibits cell proliferation. Our results indicate that HTATIP2 is highly expressed during catagen and telogen and may play an important role in JAK-STAT signaling. This study provides a theoretical foundation for investigating HTATIP2 in biological processes.

miR-218-5p regulates skin and hair follicle development through Wnt/ß-catenin signaling pathway by targeting SFRP2.

The hair follicle is a complex biological system involved in a dynamic process governed by gene regulation. MicroRNAs play a critical role in the regulation of gene expression. We demonstrated that the expression of miR-218-5p and SFRP2 showed the opposite relationship in catagen and telogen phases and that miR-218-5p promoted the growth of hair shafts. The luciferase reporter assays confirmed that SFRP2 is the direct target of miR-218-5p. The expression of miR-218-5p may decrease the expression of SFRP2, which activates the Wnt signaling pathway, including the regulation of downstream genes and ß-catenin/T-cell-specific factor transcriptional activity. Moreover, miR-218-5p enhanced apoptosis, but inhibition of miR-218-5p decreased apoptosis and inhibited RAB-9 cell proliferation. In this study, we show that miR-218-5p positively regulates the Wnt signaling pathway by targeting SFRP2 and acts as a dynamic governor during skin and hair follicle development.

Correction to: Morphological Characterization and Gene Expression Patterns for Melanin Pigmentation in Rex Rabbit.

The original article has been published with an incorrect grant number in the Funding section.

Morphological Characterization and Gene Expression Patterns for Melanin Pigmentation in Rex Rabbit.

Animal melanin has an important role in the formation of animal fur and skin, which is determined by its quantities, character, and distribution. To identify the effect of melanin on the formation of multi-colored Rex rabbits (Black, Chinchilla, Beaver, Protein cyan, Protein yellow, White), the structure of hair follicles and melanin content in multi-colored Rex rabbit skins were observed by Hematoxylin and Eosin (H&E) staining and melanin staining, respectively. The melanin granules were primarily found in the epidermis and hair follicle roots. The melanin content of skin was measured by extracting melanin from skin tissue. The results demonstrated that the melanin content was the highest in the skin of black Rex rabbit. Additionally, we measured the mRNA and protein expression levels of melanin-related key genes (MITF and TYR) in the skin of different hair color by quantitative real-time PCR and Wes assay, respectively. The results revealed that the mRNA expression levels in the skin of black Rex rabbit was highly expressed when as compared with other Rex rabbit skin (P < 0.01), and they were the lowest in the skin of white Rex rabbit. Finally, correlation analysis was conducted between melanin content and the expression levels of mRNA and protein. The results indicated a significant correlation between melanin content and the mRNA expression of MITF (P < 0.05), but it was not correlated with the mRNA expression of TYR (P > 0.05). In summary, melanin deposition has important economic value, and the coat color of fur-bearing animals is partly determined by the melanin-related genes.

Slc7a11 Modulated by POU2F1 is Involved in Pigmentation in Rabbit.

Solute carrier family 7 member 11 (Slc7a11) is a cystine/glutamate xCT transporter that controls the production of pheomelanin pigment to change fur and skin color in animals. Previous studies have found that skin expression levels of Slc7a11 varied significantly with fur color in Rex rabbits. However, the molecular regulation mechanism of Slc7a11 in pigmentation is unknown. Here, rabbit melanocytes were first isolated and identified. The distribution and expression pattern of Slc7a11 was confirmed in skin from rabbits with different fur colors. Slc7a11 affected the expression of pigmentation related genes and thus affected melanogenesis. Meanwhile, Slc7a11 decreased melanocyte apoptosis, but inhibition of Slc7a11 enhanced apoptosis. Furthermore, the POU2F1 protein was found to bind to the -713 to -703 bp region of Slc7a11 promoter to inhibit its activity in a dual-luciferase reporter and site-directed mutagenesis assay. This study reveals the function of the Slc7a11 in melanogenesis and provides in-depth analysis of the mechanism of fur pigmentation.

Characterization and Establishment of an Immortalized Rabbit Melanocyte Cell Line Using the SV40 Large T Antigen.

Melanocytes (MCs) are specialized cells that synthesize melanin within the melanosome. Cultured MCs are useful in order to study their role in relation to pigmentation. However, MC isolation is laborious and the obtained cells have a limited culture time. In this study, we transformed lentivirus-mediated simian virus 40 Large T (SV40-LT) into primary rabbit melanocytes (Pri RMCs) to establish an immortalized cell line. Morphologically, the immortalized RMCs (Im RMC) were indistinguishable from the Pri RMCs, and dendrites were visible following Dopa staining. No significant differences in cell proliferation or growth between immortalized and primary RMCs were observed. Based on melanocyte-specific markers, the expression of MITF, TYR, and TYRP1 were detected by PCR, immunofluorescence staining, and western blot analysis. Through karyotype, soft agar, and tumorigenesis assays, the immortalized RMCs did not undergo malignant transformation. Our results show that Im RMCs can be used as a tool cell for future MC studies on the pigmentation mechanisms of fur animals.

Multi-omics integration identifies regulatory factors underlying bovine subclinical mastitis.

BACKGROUND: Mastitis caused by multiple factors remains one of the most common and costly disease of the dairy industry. Multi-omics approaches enable the comprehensive investigation of the complex interactions between multiple layers of information to provide a more holistic view of disease pathogenesis. Therefore, this study investigated the genomic and epigenomic signatures and the possible regulatory mechanisms underlying subclinical mastitis by integrating RNA sequencing data (mRNA and lncRNA), small RNA sequencing data (miRNA) and DNA methylation sequencing data of milk somatic cells from 10 healthy cows and 20 cows with naturally occurring subclinical mastitis caused by Staphylococcus aureus or Staphylococcus chromogenes. RESULTS: Functional investigation of the data sets through gene set analysis uncovered 3458 biological process GO terms and 170 KEGG pathways with altered activities during subclinical mastitis, provided further insights into subclinical mastitis and revealed the involvement of multi-omics signatures in the altered immune responses and impaired mammary gland productivity during subclinical mastitis. The abundant genomic and epigenomic signatures with significant alterations related to subclinical mastitis were observed, including 30,846, 2552, 1276 and 57 differential methylation haplotype blocks (dMHBs), differentially expressed genes (DEGs), lncRNAs (DELs) and miRNAs (DEMs), respectively. Next, 5 factors presenting the principal variation of differential multi-omics signatures were identified. The important roles of Factor 1 (DEG, DEM and DEL) and Factor 2 (dMHB and DEM), in the regulation of immune defense and impaired mammary gland functions during subclinical mastitis were revealed. Each of the omics within Factors 1 and 2 explained about 20% of the source of variation in subclinical mastitis. Also, networks of important functional gene sets with the involvement of multi-omics signatures were demonstrated, which contributed to a comprehensive view of the possible regulatory mechanisms underlying subclinical mastitis. Furthermore, multi-omics integration enabled the association of the epigenomic regulatory factors (dMHBs, DELs and DEMs) of altered genes in important pathways, such as 'Staphylococcus aureus infection pathway' and 'natural killer cell mediated cytotoxicity pathway', etc., which provides further insights into mastitis regulatory mechanisms. Moreover, few multi-omics signatures (14 dMHBs, 25 DEGs, 18 DELs and 5 DEMs) were identified as candidate discriminant signatures with capacity of distinguishing subclinical mastitis cows from healthy cows. CONCLUSION: The integration of genomic and epigenomic data by multi-omics approaches in this study provided a better understanding of the molecular mechanisms underlying subclinical mastitis and identified multi-omics candidate discriminant signatures for subclinical mastitis, which may ultimately lead to the development of more effective mastitis control and management strategies.

High Diversity of Long Terminal Repeat Retrotransposons in Compact Vertebrate Genomes: Insights from Genomes of Tetraodontiformes.

This study aimed to investigate the evolutionary profile (including diversity, activity, and abundance) of retrotransposons (RTNs) with long terminal repeats (LTRs) in ten species of Tetraodontiformes. These species, Arothron firmamentum, Lagocephalus sceleratus, Pao palembangensis, Takifugu bimaculatus, Takifugu flavidus, Takifugu ocellatus, Takifugu rubripes, Tetraodon nigroviridis, Mola mola, and Thamnaconus septentrionalis, are known for having the smallest genomes among vertebrates. Data mining revealed a high diversity and wide distribution of LTR retrotransposons (LTR-RTNs) in these compact vertebrate genomes, with varying abundances among species. A total of 819 full-length LTR-RTN sequences were identified across these genomes, categorized into nine families belonging to four different superfamilies: ERV (Orthoretrovirinae and Epsilon retrovirus), Copia, BEL-PAO, and Gypsy (Gmr, Mag, V-clade, CsRN1, and Barthez). The Gypsy superfamily exhibited the highest diversity. LTR family distribution varied among species, with Takifugu bimaculatus, Takifugu flavidus, Takifugu ocellatus, and Takifugu rubripes having the highest richness of LTR families and sequences. Additionally, evidence of recent invasions was observed in specific tetraodontiform genomes, suggesting potential transposition activity. This study provides insights into the evolution of LTR retrotransposons in Tetraodontiformes, enhancing our understanding of their impact on the structure and evolution of host genomes.

Phylogenetic Relationships among TnpB-Containing Mobile Elements in Six Bacterial Species.

Some families of mobile elements in bacterial genomes encode not only a transposase but also an accessory TnpB gene. This gene has been shown to encode an RNA-guided DNA endonuclease, co-evolving with Y1 transposase and serine recombinase in mobile elements IS605 and IS607. In this paper, we reveal the evolutionary relationships among TnpB-containing mobile elements (TCMEs) in well-assembled genomes of six bacterial species: Bacillus cereus, Clostridioides difficile, Deinococcus radiodurans, Escherichia coli, Helicobacter pylori and Salmonella enterica. In total, 9996 TCMEs were identified in 4594 genomes. They belonged to 39 different insertion sequences (ISs). Based on their genetic structures and sequence identities, the 39 TCMEs were classified into three main groups and six subgroups. According to our phylogenetic analysis, TnpBs include two main branches (TnpB-A and TnpB-B) and two minor branches (TnpB-C and TnpB-D). The key TnpB motifs and the associated Y1 and serine recombinases were highly conserved across species, even though their overall sequence identities were low. Substantial variation was observed for the rate of invasion across bacterial species and strains. Over 80% of the genomes of B. cereus, C. difficile, D. radiodurans and E. coli contained TCMEs; however, only 64% of the genomes of H. pylori and 44% of S. enterica genomes contained TCMEs. IS605 showed the largest rate of invasion in these species, while IS607 and IS1341 had a relatively narrow distribution. Co-invasions of IS605, IS607 and IS1341 elements were observed in various genomes. The largest average copy number was observed for IS605b elements in C. difficile. The average copy numbers of most other TCMEs were smaller than four. Our findings have important implications for understanding the co-evolution of TnpB-containing mobile elements and their biological roles in host genome evolution.

Comparative Analysis and Phylogenetic Insights of Cas14-Homology Proteins in Bacteria and Archaea.

Type-V-F Cas12f proteins, also known as Cas14, have drawn significant interest within the diverse CRISPR-Cas nucleases due to their compact size. This study involves analyzing and comparing Cas14-homology proteins in prokaryotic genomes through mining, sequence comparisons, a phylogenetic analysis, and an array/repeat analysis. In our analysis, we identified and mined a total of 93 Cas14-homology proteins that ranged in size from 344 aa to 843 aa. The majority of the Cas14-homology proteins discovered in this analysis were found within the Firmicutes group, which contained 37 species, representing 42% of all the Cas14-homology proteins identified. In archaea, the DPANN group had the highest number of species containing Cas14-homology proteins, a total of three species. The phylogenetic analysis results demonstrate the division of Cas14-homology proteins into three clades: Cas14-A, Cas14-B, and Cas14-U. Extensive similarity was observed at the C-terminal end (CTD) through a domain comparison of the three clades, suggesting a potentially shared mechanism of action due to the presence of cutting domains in that region. Additionally, a sequence similarity analysis of all the identified Cas14 sequences indicated a low level of similarity (18%) between the protein variants. The analysis of repeats/arrays in the extended nucleotide sequences of the identified Cas14-homology proteins highlighted that 44 out of the total mined proteins possessed CRISPR-associated repeats, with 20 of them being specific to Cas14. Our study contributes to the increased understanding of Cas14 proteins across prokaryotic genomes. These homologous proteins have the potential for future applications in the mining and engineering of Cas14 proteins.

PiggyBac Transposon Mining in the Small Genomes of Animals.

TEs, including DNA transposons, are major contributors of genome expansions, and have played a very significant role in shaping the evolution of animal genomes, due to their capacity to jump from one genomic position to the other. In this study, we investigated the evolution landscapes of PB transposons, including their distribution, diversity, activity and structure organization in 79 species of small (compact) genomes of animals comprising both vertebrate and invertebrates. Overall, 212 PB transposon types were detected from almost half (37) of the total number of the small genome species (79) investigated. The detected PB transposon types, which were unevenly distributed in various genera and phyla, have been classified into seven distinct clades or families with good bootstrap support (>80%). The PB transposon types that were identified have a length ranging from 1.23 kb to 9.51 kb. They encode transposases of approximately ≥500 amino acids in length, and possess terminal inverted repeats (TIRs) ranging from 4 bp to 24 bp. Though some of the transposon types have long TIRs (528 bp), they still maintain the consistent and reliable 4 bp target site duplication (TSD) of TTAA. However, PiggyBac-2_Cvir transposon originating from the Crassostrea virginica species exhibits a unique TSD of TATG. The TIRs of the transposons in all the seven families display high divergence, with a highly conserved 5' end motif. The core transposase domains (DDD) were better conserved among the seven different families compared to the other protein domains, which were less prevalent in the vertebrate genome. The divergent evolution dynamics analysis also indicated that the majority of the PB transposon types identified in this study are either relatively young or old, with some being active. Additionally, numerous invasions of PB transposons were found in the genomes of both vertebrate and invertebrate animals. The data reveals that the PB superfamily is widely distributed in these species. PB transposons exhibit high diversity and activity in the small genomes of animals, and might play a crucial role in shaping the evolution of these small genomes of animals.

Exosomal miRNA-181a-5p from the cells of the hair follicle dermal papilla promotes the hair follicle growth and development via the Wnt/ß-catenin signaling pathway.

Exosomal miRNAs are verified critical biomarkers, which participate in several biological processes. The growth and development of the hair follicle (HF) are typically controlled by the exosomal miRNAs via cell-to-cell communication. This study identified a high expression of miR-181a-5p in the low-passage DPC-Exos (exosomes derived from dermal papilla cell), revealing the transportation patterns of the DPC-Exos-derived miR-181a-5p entering the HFSC (hair follicle stem cell). The exosomal miR-181a-5p activates the Wnt/ß-catenin signaling pathway by targeting the Wnt inhibitor WIF1 and thereby regulates the proteins and genes related to HF growth and development. Moreover, the exosomal miR-181a-5p was found to suppress the HFSC apoptosis but promoted the HFSC proliferation. The in vitro culture of the HF organ revealed that the exosomal miR-181a-5p possesses a positive role in hair growth. Collectively, the exosomal miR-181a-5p affects the HF growth and development through the Wnt/ß-catenin signaling pathway. The exosomal miR-181a-5p might, therefore, act as the novel biomarker and therapeutic target for treating hair-related diseases and wool production in mammals.

lncRNA2919 Suppresses Rabbit Dermal Papilla Cell Proliferation via trans-Regulatory Actions.

Hair follicles (HFs) are complex organs that grow cyclically during mammals' growth and development. Long non-coding RNAs (lncRNAs) cannot be translated into proteins and play crucial roles in many biological processes. In our previous study, candidate lncRNAs associated with HF cyclic regeneration were screened, and we identified that the novel lncRNA, lncRNA2919, was significantly expressed during catagen. Here, we identified that lncRNA2919 has no coding potentiality and is highly expressed in the cell nucleus, and downregulates HF growth and development-related genes, inhibits cell proliferation, and promotes cell apoptosis in rabbit dermal papilla cells. lncRNA2919 recruits STAT1 to form a compound. As a key transcription factor, STAT1 regulates the transcriptional expression of KRTAP11-1. Our study revealed that lncRNA2919 is involved in HF cyclic regeneration through the trans-regulatory lncRNA2919-STAT1-KRTAP11-1 axis. This study elucidates the mechanism through which lncRNA2919 regulates HF growth and development and the role of lncRNA2919 as a new therapeutic target in animal wool production and human hair-related disease treatment.

CDK1 promotes the proliferation of melanocytes in Rex rabbits.

BACKGROUND: The fur color constitutes one of the most important economic characteristics of fur animals and is determined by the content of melanin. A previous study has shown that the cyclin-dependent kinase 1 (CDK1) is a member of the protein kinase family, involved in forming the color of the fur in Rex rabbits. However, its effect on the melanocytes remains unclear. OBJECTIVE: This study aimed to provide evidence for the role of CDK1 in melanogenesis. METHODS: This study measured the expression of CDK1 in Rex rabbit skins of six coat colors using qRT-PCR. The CDK1-mediated regulation of the pigmentation-related genes and cyclin-dependent kinases were analyzed. The melanin content, proliferation, and apoptosis of the melanocytes were analyzed using the NaOH, CCK8, and Annexin V-FITC methods. RESULTS: The CDK1 expression in the skin of the rex rabbits with different coat colors was found to be regular, and the expression level was found to be the highest in the skin of the black rex rabbits (P < 0.05). The overexpression/knockdown of CDK1 was found to significantly increase/decrease the melanin content in the melanocytes (P < 0.01). Besides, CDK1 was found to significantly promote the proliferation of the melanocyte and inhibit apoptosis (P < 0.01). Furthermore, the overexpression of CDK1 was found to significantly affect the expression of the other melanin-related genes like TYR, PMEL, DCT, as well as the mRNA expression of the cyclin-dependent kinases CDK4, CDK6, CDK8, CCNB1. CONCLUSIONS: The results indicated that CDK1 can serve as a key gene regulating melanogenesis, melanocyte proliferation, and apoptosis, providing a new theoretical basis for studying the mechanism by which the different colors of the fur evolve in mammals.

Characterization and functional analysis of SMAD2 regulation in hair follicle cycle in Angora rabbits.

Hair follicle (HF) development is characterized by periodic growth cycles regulated by numerous factors. We previously showed that SMAD2 might be involved in the HF growth cycle in Angora rabbits. However, its extra role in the HF growth and development remains obscure. In this study, we cloned the complete coding sequence (CDS) of the Angora rabbit SMAD2 gene. Within SMAD2 CDS, we identified the open reading frame (ORF) had a length of 1314 bp and encoding 437 amino acids. Bioinformatics analyses revealed that the SMAD2 protein is unstable and hydrophilic, and predominatelylocalizesin the cell nucleus. We identified that SMAD2 expression was elevated in the telogen phase of the during HF cycle. The knockdown and overexpression of SMAD2 could regulate HF growth and development related genes, such as WNT2, FGF2, and LEF1.Furthermore, SMAD2 may upregulate TGF-ß signaling pathway-related genes, including TFDP1, E2F4, and RBL1. In conclusion, our results indicate that SMAD2 plays a vital role in HF development by regulating the TGF-ß signaling pathway.

Bacitracin Methylene Disalicylate Improves Intestinal Health by Modulating Its Development and Microbiota in Weaned Rabbits.

Intestinal infections are a major cause of morbidity and mortality in humans and agricultural animals, especially newborns and weaned animals. Preventive treatments that help weaned animals maintain homeostasis and balance the hindgut microbial populations are desirable. The present study aimed to explore the impact of bacitracin methylene disalicylate (BMD) on the intestinal health by analyzing the intestinal environment, morphology, expression of peptidoglycan recognition proteins (PGRPs), and flora of weaned rabbits. A total of 300 New Zealand weaned rabbits were randomly divided into the following five treatment groups for a 35-day feed trial: control group (basal diet), bacitracin zinc (BZ) group (50 mg/kg BZ), BMDa group (100 mg/kg BMD), BMDb group (50 mg/kg BMD), and BMDc group (rabbits fed a basal diet supplemented with 25 mg/kg BMD). In each treatment group, 28 rabbits were slaughtered for experimental analysis. The results showed that the supplementation of BMD increased the environmental acidity of the cecum of the weaned rabbits and reduced the ammonia-nitrogen concentration, which was beneficial to the survival of useful bacteria in the intestine. The morphology analysis of the duodenum using hematoxylin and eosin staining revealed that the villus length, villus/crypt ratio, and intestinal wall thickness increased in the BMD group, thereby improving the structure of the duodenum and the absorption capacity of the small intestine. Moreover, real-time polymerase chain reaction test showed that PGRPs (especially PGLYRP-1 and PGLYRP-2) in the intestinal had an antagonistic effect with BMD in the process of inhibiting pathogenic bacteria, resulting in their decreased expression (P < 0.05). Furthermore, through 16S rRNA sequencing in the cecal content, the abundance of the predominant phyla in the BMDa and BZ groups was found to be the closest. The abundance of the genera Lachnospira, Erysipelotrichaceae (p-75-a5), Paraprevotellaceae (YRC22), Mogibacterium, Peptococcaceae (rc4-4), Anaerovibrio, Succinivibrio, and Sphaerochaeta increased in the BMDa and BZ groups (P < 0.05). The relative abundance of Alistipes, Sedimentibacter, and Dorea significantly increased only in the BMDa group (P < 0.05). Conclusively, BMD, as well as microbes, improved the intestinal environment and structure to maintain the intestinal health of weaned rabbits.

Distinct Retrotransposon Evolution Profile in the Genome of Rabbit (Oryctolagus cuniculus).

Although the rabbit genome has already been annotated, it is mobilome remains largely unknown. Here, multiple pipelines were used to de novo mine and annotate the mobilome in rabbit. Four families and 19 subfamilies of LINE1s, two families and nine subfamilies of SINEs, and 12 ERV families were defined in rabbit based on sequence identity, structural organization, and phylogenetic tree. The analysis of insertion age and polymerase chain reaction suggests that a number of families are very young and may remain active, such as L1B, L1D, OcuSINEA, and OcuERV1. RepeatMasker annotation revealed a distinct transposable element landscape within the genome, with approximately two million copies of SINEs, representing the greatest proportion of the genome (19.61%), followed by LINEs (15.44%), and LTRs (4.11%), respectively, considerably different from most other mammal mobilomes except hedgehog and tree shrew, in which LINEs have the highest proportion. Furthermore, a very high rate of insertion polymorphisms (>85%) for the youngest subfamily (OcuSINEA1) was identified by polymerase chain reaction. The majority of retrotransposon insertions overlapped with protein-coding regions (>80%) and lncRNA (90%) genes. Genomic distribution bias was observed for retrotransposons, with those immediately upstream (-1 kb) and downstream (1 kb) of genes significantly depleted. Local GC content in 50-kb widows had significantly negative correlations with LINE (rs=-0.996) and LTR (rs=-0.829) insertions. The current study revealed a distinct mobilome landscape in rabbit, which will assist in the elucidation of the evolution of the genome of lagomorphs, and even other mammals.