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Background The temperature of testes exposed to hyperthermic conditions can affect livestock reproductive performance. This study aimed to explore the difference in semen quality, testicular morphological structure, and gene expression profiles of testes of Angora rabbits in spring (no heat stress), summer (intense heat stress), and autumn (no heat stress) seasons. Results Heat stress during summer significantly reduced semen quality and damaged testicular morphology and spermatogenesis, which recovered to normal levels in autumn, although semen quality recovery was notably slow. RNA-Seq analysis showed that the expression levels of 8703 genes changed significantly in summer, but their expression levels in autumn returned to those in spring, which was consistent with the testicular morphology analysis results in different seasons. Enrichment analysis revealed that the DEGs were primarily associated with spermatogenesis, sperm motility, spermatid development, cell death, regulation of apoptotic processes, and responses to external stimuli. The MAPK, Rap1, TNF, Ras, Apoptosis, and Wnt signaling pathways regulated reproduction under heat stress. In addition, minimal variations in testicular morphology and gene expression profiles were observed between autumn and spring. Gene expression pattern analysis showed that genes with high expression in summer mainly participated in the regulation of cell apoptosis, immunity, and response to heat stress, whereas genes with low expression in summer mainly participated in the regulation of spermatogenesis. Conclusions This study investigated the influence of different seasons on the reproductive performance of male Angora rabbits and provided initial insights into the molecular regulatory mechanisms underlying the testicular response to heat stress during summer.

Colostrum intake is a crucial determinant of survival in newborn rabbits. Neonates rely entirely on passive immunity transfer from their mothers while suckling colostrum. The goal of this study was to explore the protein differences of rabbit milk during different lactation periods. Our findings showed that the daily milk yield exhibited an increasing trend from the 2nd to the 21st day of lactation. A data-independent acquisition proteomics approach identified a total of 2011 proteins. Significantly, different abundances were found for 525 proteins in the colostrum and the mature milk samples. Eleven differentially abundant proteins (DAPs) were examined using parallel reaction monitoring, which verified the reliability of the proteomic data. Gene Ontology analysis revealed that these DAPs were primarily associated with glycosyltransferase activity, macromolecule transmembrane transporter activity, and regulation of acute inflammatory response. The dominant metabolic pathways of the DAPs involve the complement and coagulation cascades. A protein–protein interaction analysis identified apolipoprotein B, apolipoprotein A1, triose phosphate isomerase 1, and albumin as the hub proteins responsible for distinguishing differences between biological properties in rabbit colostrum and mature milk. These findings enhance our comprehension of the rabbit milk proteome, particularly in expanding our knowledge regarding the requirements of neonatal rabbits.

Wool fiber diameter (WFD) is an important index of wool traits and the main determinant of wool quality and value. However, the genetic determinants of fiber diameter have not yet been fully elucidated. Here, coarse and fine wool of Wan strain Angora rabbits and their hair follicle traits were characterized. The results indicated significant differences in the diameters of wool fibers and their hair follicles. The RNA sequencing (RNA-Seq) technique was used to identify differences in gene expression in hair follicles between coarse and fine wool. In total, 2574 differentially expressed genes (DEGs) were found between the two hair follicle groups. Transcription factors, keratin-associated protein (KAP) and keratin (KRT) families, and ECM-related genes may control the structure of fine fibers in rabbits. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that skin development, epidermal cell and keratinocyte differentiation, epithelium development, and Notch and ribosome signaling pathways were significantly enriched, respectively. GSEA further filtered six important pathways and related core genes. PPI analysis also mined functional DEGs associated with hair structure, including LEF1, FZD3, SMAD3, ITGB6, and BMP4. Our findings provide valuable information for researching the molecular mechanisms regulating wool fiber and could facilitate enhanced selection of super-fine wool rabbits through gene-assisted selection in the future.

Copy number variations (CNVs), which include duplications and deletions of DNA segments, are significant structural variants that play crucial roles in the genetics of complex traits in livestock. High-throughput sequencing technologies enable the systematic identification of structural variants across genomes. However, CNV-based analyses of whole-genome sequencing data in rabbits remain largely unexplored. Herein, we characterized genome-wide CNVs of two rabbit breeds, Jiuyishan rabbit (JY) and Hyplus rabbit (HP), using whole-genome resequencing to elucidate their genetic characteristics and selection signatures. In total, 5,599 CNV regions (CNVRs) were identified between JY and HP, covering 0.98% of the reference genome. To identify selection signatures, we employed variance stabilizing transformation (Vst) values, selecting the top 1% of CNVRs with the highest Vst values, resulting in 56 CNVRs. These CNVRs harbored 27 genes. Functional analyses indicated that these genes were associated with important traits such as growth ( HOMER1 , NOS1AP , PDE4B , LEPROT ) and reproduction ( FRAS1 , CFAP43 , TM9SF2 , and CTNND2 ). This study aims to enhance our understanding of CNVs and selection signals in rabbits, provide insights into the genetic differences between Chinese indigenous breeds and Western commercial lines, and offer valuable resources for investigating the genetic basis of complex traits.

Objective: The Wannan Black pig is a typical Chinese indigenous, disease-resistant pig breed with high fertility, and a crude-feed tolerance that has been bred by artificial selection in the south of Anhui province for a long time. However, genome variation, genetic relationships with other pig breeds, and domestication, remain poorly understood. Here, we focus on elucidating the genetic characteristics of the Wannan Black pig and identifying selection signatures during domestication and breeding. Methods: We identified the whole-genome variation in the Wannan Black pig and performed population admixture analyses to determine genetic relationships with other domesticated pig breeds and wild boars. Then, we identified the selection signatures between the Wannan Black pig and Asian wild boars in 100-kb windows sliding in 10 kb steps by using two approaches: the fixation index (FST) and π ratios. Results: Resequencing the Wannan Black pig genome yielded 501.52 G of raw data. After calling single-nucleotide variants (SNVs) and insertions/deletions (InDels), we identified 21,316,754 SNVs and 5,067,206 InDels (2,898,582 inserts and 2,168,624 deletions). Additionally, we found genes associated with growth, immunity, and digestive functions. Conclusion: Our findings help in explaining the unique genetic and phenotypic characteristics of Wannan Black pigs, which in turn can be informative for future breeding programs of Wannan Black pigs. KCI Citation Count: 8

Objective: Considering the physiological and clinical importance of leptin receptor (LEPR) in regulating obesity and the fact that porcine LEPR expression is not known to be controlled by lncRNAs and miRNAs, we aim to characterize this gene as a potential target of SSC-miR-323 and the lncRNA TCONS_00010987. Methods: Bioinformatics analyses revealed that lncRNA TCONS_00010987 and LEPR have SSC-miR-323-binding sites and that LEPR might be a target of lncRNA TCONS_00010987 based on cis prediction. Wild-type and mutant TCONS_00010987-target sequence fragments and wild-type and mutant LEPR 3′-UTR fragments were generated and cloned into pmiR-RB-REPORTTM-Control vectors to construct respective recombinant plasmids. HEK293T cells were co-transfected with the SSC-miR-323 mimics or a negative control with constructs harboring the corresponding binding sites and relative luciferase activities were determined. Tissue expression patterns of lncRNA TCONS_00010987, SSC-miR-323, and LEPR in Anqing six-end-white (AQ, the obese breed) and Large White (LW, the lean breed) pigs were detected by real-time quantitative polymerase chain reaction; backfat expression of LEPR protein was detected by western blotting. Results: Target gene fragments were successfully cloned, and the four recombinant vectors were constructed. Compared to the negative control, SSC-miR-323 mimics significantly inhibited luciferase activity from the wild-type TCONS_00010987-target sequence and wild-type LEPR-3′-UTR (p<0.01 for both) but not from the mutant TCONS_00010987-target sequence and mutant LEPR-3′-UTR (p>0.05 for both). Backfat expression levels of TCONS_ 00010987 and LEPR in AQ pigs were significantly higher than those in LW pigs (p<0.01), whereas levels of SSC-miR-323 in AQ pigs were significantly lower than those in LW pigs (p<0.05). LEPR protein levels in the backfat tissues of AQ pigs were markedly higher than those in LW pigs (p<0.01). Conclusion: LEPR is a potential target of SSC-miR-323, and TCONS_00010987 might act as a sponge for SSC-miR-323 to regulate LEPR expression. KCI Citation Count: 2

ObjectiveMutations in low-density lipoprotein receptor (LDLR), which encodes a critical protein for cholesterol homeostasis and lipid metabolism in mammals, are involved in cardiometabolic diseases, such as familial hypercholesterolemia in pigs. Whereas microRNAs (miRNAs) can control LDLR regulation, their involvement in circulating cholesterol and lipid levels with respect to cardiometabolic diseases in pigs is unclear. We aimed to identify and analyze LDLR as a potential target gene of SSC-miR-20a.MethodsBioinformatic analysis predicted that porcine LDLR is a target of SSC-miR-20a. Wild-type and mutant LDLR 3′-untranslated region (UTR) fragments were generated by polymerase chain reaction (PCR) and cloned into the pGL3-Control vector to construct pGL3 Control LDLR wild-3′-UTR and pGL3 Control LDLR mutant-3′-UTR recombinant plasmids, respectively. An miR-20a expression plasmid was constructed by inserting the porcine pre-miR-20a-coding sequence between the HindIII and BamHI sites in pMR-mCherry, and constructs were confirmed by sequencing. HEK293T cells were co-transfected with the miR-20a expression or pMR-mCherry control plasmids and constructs harboring the corresponding 3′-UTR, and relative luciferase activity was determined. The relative expression levels of miR-20a and LDLR mRNA and their correlation in terms of expression levels in porcine liver tissue were analyzed using reverse-transcription quantitative PCR.ResultsGel electrophoresis and sequencing showed that target gene fragments were successfully cloned, and the three recombinant vectors were successfully constructed. Compared to pMR-mCherry, the miR-20a expression vector significantly inhibited wild-type LDLR-3′-UTR-driven (p<0.01), but not mutant LDLR-3′-UTR-driven (p>0.05), luciferase reporter activity. Further, miR-20a and LDLR were expressed at relatively high levels in porcine liver tissues. Pearson correlation analysis revealed that porcine liver miR-20a and LDLR levels were significantly negatively correlated (r = −0.656, p<0.05).ConclusionLDLR is a potential target of miR-20a, which might directly bind the LDLR 3′-UTR to post-transcriptionally inhibit expression. These results have implications in understanding the pathogenesis and progression of porcine cardiovascular diseases.

The temperature of testes exposed to hyperthermic conditions can affect livestock reproductive performance. This study aimed to explore the difference in semen quality, testicular morphological structure, and gene expression profiles of testes of Angora rabbits in spring (no heat stress), summer (intense heat stress), and autumn (no heat stress) seasons. Heat stress during summer significantly reduced semen quality and damaged testicular morphology and spermatogenesis, which recovered to normal levels in autumn, although semen quality recovery was notably slow. RNA-Seq analysis showed that the expression levels of 8703 genes changed significantly in summer, but their expression levels in autumn returned to those in spring, which was consistent with the testicular morphology analysis results in different seasons. Enrichment analysis revealed that the DEGs were primarily associated with spermatogenesis, sperm motility, spermatid development, cell death, regulation of apoptotic processes, and responses to external stimuli. The MAPK, Rap1, TNF, Ras, Apoptosis, and Wnt signaling pathways regulated reproduction under heat stress. In addition, minimal variations in testicular morphology and gene expression profiles were observed between autumn and spring. Gene expression pattern analysis showed that genes with high expression in summer mainly participated in the regulation of cell apoptosis, immunity, and response to heat stress, whereas genes with low expression in summer mainly participated in the regulation of spermatogenesis. This study investigated the influence of different seasons on the reproductive performance of male Angora rabbits and provided initial insights into the molecular regulatory mechanisms underlying the testicular response to heat stress during summer.

ABSTRACT The present work aimed to explore the molecular mechanisms and candidate genes associated with fat metabolism in Anqingliubai (obese) and Yorkshire (lean) pigs. The transcriptome profiling of backfat between Anqingliubai and Yorkshire pigs was carried out by RNA-sequencing technology. The sum of clean reads were 288.3 and 365.3 million which was obtained from the RNA sequencing data in the Anqingliubai and Yorkshire pigs, respectively. Most reads were located in exonic region, while less reads were located in intergenic and intronic regions. There were 2601 upregulated genes, but 284 downregulated genes in Yorkshire pigs compared with those in Anqingliubai pigs. The top 10 most significant Gene Ontology (GO) terms included catalytic activity, binding, cell, cytoplasm, positive regulation of multicellular organismal process, biological regulation, cellular process, etc. There were 54 significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including cytokine-cytokine receptor interaction, biosynthesis of unsaturated fatty acids, fatty acid metabolism, regulation of lipolysis in adipocytes, glycerolipid metabolism, etc. The results of differentially expressed genes from sequence were highly reliable by qRT-PCR confirmation. The present work will help understanding of the different mechanisms involved in fat deposition between lean and obese pigs.

Fiber diameter is an important characteristic that determines the quality and economic value of rabbit wool. This study aimed to investigate the genetic determinants of wool fiber diameter through an integration analysis using transcriptomic and proteomic datasets from hair follicles of coarse and fine wool from Angora rabbits. Using a 4D label-free technique, we identified 423 differentially expressed proteins (DEPs) in hair follicles of coarse and fine wool in Angora rabbits. Eighteen DEPs were examined using parallel reaction monitoring, which verified the reliability of our proteomic data. Functional enrichment analysis revealed that a set of biological processes and signaling pathways related to wool growth and hair diameter were strongly enriched by DEPs with fold changes greater than two, such as keratinocyte differentiation, skin development, epidermal and epithelial cell differentiation, epidermis and epithelium development, keratinization, and estrogen signaling pathway. Association analysis and protein–protein interaction network analysis further showed that the keratin (KRT) family members, including KRT77, KRT82, KRT72, KRT32, and KRT10, as well as CASP14 and CDSN, might be key factors contributing to differences in fiber diameter. Our results identified DEPs in hair follicles of coarse and fine wool and promoted understanding of the molecular mechanisms underlying wool fiber diameter variation among Angora rabbits.