Integrative proteomic and phosphoproteomic analysis in the female goat hypothalamus to study the onset of puberty.

BACKGROUND: Puberty marks the end of childhood and achieve sexual maturation and fertility. The role of hypothalamic proteins in regulating puberty onset is unclear. We performed a comprehensive differential proteomics and phosphoproteomics analysis in prepubertal and pubertal goats to determine the roles of hypothalamic proteins and phosphoproteins during the onset of puberty. RESULTS: We used peptide and posttranslational modifications peptide quantification and statistical analyses, and identified 69 differentially expressed proteins from 5,057 proteins and 576 differentially expressed phosphopeptides from 1574 phosphorylated proteins. Combined proteomic and phosphoproteomics, 759 correlated proteins were identified, of which 5 were differentially expressed only at the protein level, and 201 were only differentially expressed at the phosphoprotein level. Pathway enrichment analyses revealed that the majority of correlated proteins were associated with glycolysis/gluconeogenesis, Fc gamma R-mediated phagocytosis, focal adhesion, GABAergic synapse, and Rap1 signaling pathway. These pathways are related to cell proliferation, neurocyte migration, and promoting the release of gonadotropin-releasing hormone in the hypothalamus. CTNNB1 occupied important locations in the protein-protein interaction network and is involved in focal adhesion. CONCLUSION: The results demonstrate that the proteins differentially expression only at the protein level or only differentially expressed at the phosphoprotein level and their related signalling pathways are crucial in regulating puberty in goats. These differentially expressed proteins and phosphorylated proteins may constitute the proteomic backgrounds between the two different stages.

Metabolomics Analysis of Sodium Salicylate Improving the Preservation Quality of Ram Sperm.

The aim of this study was to investigate the effects of sodium salicylate (SS) on the preservation and metabolic regulation of sheep sperm. Under 4 °C low-temperature conditions, SS (at 10 µM, 20 µM, 30 µM, and 50 µM) was added to the semen diluent to detect sperm motility, plasma membrane, and acrosome integrity. Based on the selected optimal concentration of SS (20 µM), the effects of 20 µM of SS on sperms' antioxidant capacity and mitochondrial membrane potential (MMP) were evaluated, and metabolomics analysis was conducted. The results showed that on the 20th day of low-temperature storage, the sperm motility of the 20 µM SS group was 62.80%, and the activities of catalase (CAT) and superoxide dismutase (SOD) were significantly higher than those of the control group (p < 0.01). The content of Ca2+, reactive oxygen species (ROS), and malondialdehyde (MDA) were significantly lower than those of the control group (p < 0.01), and the total antioxidant capacity (T-AOC) was significantly higher than that of the control group (p < 0.05); mitochondrial activity and the total cholesterol (TC) content were significantly higher than those in the control group (p < 0.01). An ultrastructural examination showed that in the SS group, the sperm plasma membrane and acrosome were intact, the fibrous sheath and axoneme morphology of the outer dense fibers were normal, and the mitochondria were arranged neatly. In the control group, there was significant swelling of the sperm plasma membrane, rupture of the acrosome, and vacuolization of mitochondria. Using metabolomics analysis, 20 of the most significant differential metabolic markers were screened, mainly involving 6 metabolic pathways, with the amino acid biosynthesis pathway being the most abundant. In summary, 20 µM of SS significantly improved the preservation quality of sheep sperm under low-temperature conditions of 4 °C.

Quantitative proteomics analysis to assess protein expression levels in the ovaries of pubescent goats.

BACKGROUND: Changes in the abundance of ovarian proteins play a key role in the regulation of reproduction. However, to date, no studies have investigated such changes in pubescent goats. Herein we applied isobaric tags for relative and absolute quantitation (iTRAQ) and liquid chromatography-tandem mass spectrometry to analyze the expression levels of ovarian proteins in pre-pubertal (n = 3) and pubertal (n = 3) goats. RESULTS: Overall, 7,550 proteins were recognized; 301 (176 up- and 125 downregulated) were identified as differentially abundant proteins (DAPs). Five DAPs were randomly selected for expression level validation by Western blotting; the results of Western blotting and iTRAQ analysis were consistent. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that DAPs were enriched in olfactory transduction, glutathione metabolism, and calcium signaling pathways. Besides, gene ontology functional enrichment analysis revealed that several DAPs enriched in biological processes were associated with cellular process, biological regulation, metabolic process, and response to stimulus. Protein-protein interaction network showed that proteins interacting with CDK1, HSPA1A, and UCK2 were the most abundant. CONCLUSIONS: We identified 301 DAPs, which were enriched in olfactory transduction, glutathione metabolism, and calcium signaling pathways, suggesting the involvement of these processes in the onset of puberty. Further studies are warranted to more comprehensively explore the function of the identified DAPs and aforementioned signaling pathways to gain novel, deeper insights into the mechanisms underlying the onset of puberty.

Morphological changes and functional circRNAs screening of rabbit skeletal muscle development.

BACKGROUND: The temporal expression pattern of circular RNAs (circRNAs) across developmental stages is essential for skeletal muscle growth and functional analysis. However, there are few analyses on the potential functions of circRNAs in rabbit skeletal muscle development. RESULTS: Initially, the paraffin sections showed extremely significant differences in the diameter, number, area and density of skeletal muscle fibers of the fetus, child, adult rabbit hind legs (P < 0.01). Then, RNA-seq libraries of these three stages were constructed. A total of 481 differentially expressed circRNAs (DE-circRNAs) and 5,658 differentially expressed genes (DEGs) were identified. Subsequently, DE-circRNAs, whose host genes were DEGs or non-DEGs, were analyzed by GO respectively. In the fetus vs. child group, up-regulated DE-circRNAs (whose host genes were DEGs) were related to muscle fiber structure, and down-regulated ones were related to mitosis. The up-regulated DE-circRNAs (whose host genes were non-DEGs) were involved in enzyme activity, methylation and glycosylation, and the down-regulated ones were involved in mitosis and catabolism. In the fetus vs. adult group, the up-regulated DE-circRNAs (whose host genes were DEGs) were related to skeletal muscle basic structure, and the down-regulated ones were also associated with cell proliferation. But the up-regulated DE-circRNAs (whose host genes were non-DEGs) were connected with regulation of histone ubiquitination, chromatin and organelles. The down-regulated DE-circRNAs were connected with the catabolism processes. In addition, novel_circ_0022663 and novel_circ_0005489, which might have coding potential, and novel_circ_0004210 and novel_circ_0001669, which might have miRNA sponge capability, were screened out. CONCLUSIONS: In this study, hind leg muscles of fetus, child and adult rabbits were collected for paraffin section and RNA-seq to observe the structural changes of skeletal muscle and obtain circRNA expression profiles at different stages. These data provided a catalog of circRNAs related to muscle development in New Zealand rabbits, allowing us to better understand the functional transitions in mammalian muscle development.

Dynamic changes of miRNAs in skeletal muscle development at New Zealand rabbits.

BACKGROUND: miRNA is one of the crucial roles in the complex and dynamic network that regulates the development of skeletal muscle. The landscape of skeletal muscle miRNAs from fetus to adult in New Zealand rabbits has not been revealed yet. RESULTS: In this study, nine RNA-seq libraries of fetus, child and adult rabbits' leg muscles were constructed. A total of 278 differentially expressed miRNAs (DEmiRNAs) were identified. In the fetus vs. child group, the main functional enrichments were involved in membrane and transport. Pathway enriched terms of up-regulated DEmiRNAs were connected with the differentiation and hypertrophy of skeletal muscle, and down-regulated ones were related to muscle structure and metabolic capacity. In the child vs. adult group, functions were associated to positioning and transportation, and pathways were relevant to ECM, muscle structure and hypertrophy. Finally, ocu-miR-185-3p and ocu-miR-370-3p, which had the most target genes, were identified as hub-miRNAs in these two groups. CONCLUSIONS: In short, we summarized the highly expressed and uniquely expressed DEmiRNAs of fetus, child and adult rabbits' leg muscles. Besides, the potential functional changes of miRNAs in two consecutive stages have been explored. Among them, the ocu-miR-185-3p and ocu-miR-370-3p with the most target genes were selected as hub-miRNAs. These data improved the understanding of the regulatory molecules of meat rabbit development, and provided a novel perspective for molecular breeding of meat rabbits.

Trend analysis of the role of circular RNA in goat skeletal muscle development.

BACKGROUND: Circular RNA (circRNA) is produced during the splicing of mRNA (in addition to linear splicing) and is part of the gene regulatory network. The temporal expression patterns the different developmental stages were inseparable from these molecules' function. RESULTS: Skeletal muscles of Anhui white goat (AWG) across seven fetal to postnatal development stages were sequenced and 21 RNA sequencing libraries were constructed. We thereby identified 9090 circRNAs and analyzed their molecular properties, temporal expression patterns, and potential functions at the different stages. CircRNAs showed complexities and diversity of formation as the same host gene produces multiple isoforms of these nucleic acids with different expression profiles. The differential expression of 2881 circRNAs (DECs, P < 0.05) was identified and four were randomly selected and validated by qPCR. Moreover, 1118 DECs under strict selected (SDECs, |log2FC| > 2 and P-adj value < 0.01) showed 4 expression trends (Clusters 0, 19, 16 and 18). Cluster 0 molecules had increasing expression at all stages with effects on muscle through metabolism, regulation of enzyme activity, and biosynthesis. Cluster 16 circRNAs had high expression in the early and late stages and are involved in "Wnt signaling pathway", "AMPK signaling pathway" and others. Cluster 18 molecules were mainly expressed at F120 and participate in "cytoskeletal protein binding", "Notch signaling pathway" and so on. Cluster 19 circRNAs were down-regulated at all stages and related to muscle structure and development. Lastly, the SDECs divided the period of skeletal muscle development into three transitional stages: stage 1 (F45 to F90), which related to muscle satellite cell proliferation and muscle fiber structure; stage 2 (F90 to B1), in which the attachment of the cytoplasmic surface to the actin cytoskeleton initiates; and stage 3, which involved the "cGMP-PKG signaling pathway". Moreover, the paraffin sections messages also validated that there are three transitional stages of skeletal muscle development. CONCLUSION: Our current study provides a catalog of goat muscle-related circRNAs that can stratify skeletal muscle development fetus 45 days to newborn 90 days into three developmental stages. These findings better our understanding of functional transitions during mammalian muscle development.

Transcription profiles of oocytes during maturation and embryos during preimplantation development in vivo in the goat.

RNA sequencing performed on goat matured oocytes and preimplantation embryos generated invivo enabled us to define the transcriptome for goat preimplantation embryo development. The largest proportion of changes in gene expression in goat was found at the 16-cell stage, not as previously defined at the 8-cell stage, and is later than in other mammalian species. In all, 6482 genes were identified to be significantly differentially expressed across all consecutive developmental stage comparisons, and the important signalling pathways involved in each development transition were determined. In addition, we identified genes that appear to be transcribed only at a specific stage of development. Using weighted gene coexpression network analysis, we found nine stage-specific modules of coexpressed genes that represent the corresponding stage of development. Furthermore, we identified conserved key members (or hub genes) of the goat transcriptional networks. Their association with other embryo genes suggests that they may have important regulatory roles in embryo development. Our cross-mammalian species transcriptomic comparisons demonstrate both conserved and goat-specific features of preimplantation development.

The expression and regulation of miR-1 in goat skeletal muscle and satellite cell during muscle growth and development.

MicroRNA-1 (miR-1) has been shown to play an important role in muscle growth and development, however, it was mainly discovered in model animals. To explore the function and mechanism of miR-1 in goat, we firstly explored the expression profile of miR-1 in goat tissues and cells. Furthermore, the target gene of miR-1 was predicted, and the relationship between miR-1 and one of its target genes, histone deacetylase 4 (HDAC4), was analyzed through double luciferase reporter assay, real-time PCR, and western blot. It was found that the miR-1 is most abundantly expressed in goat heart and skeletal muscle tissue. Meanwhile, the expression of miR-1 showed an increasing tendency from new-born goats to the 7-month-old goats, and then its expression decreases as the goats mature further. In addition, the expression levels of miR-1 decreased in goat skeletal muscle satellite cells with the algebraic increasing of cells. At last, the results showed that HDAC4 is a target gene of miR-1 in goat, and miR-1 can inhibit the post-transcriptional expression of HDAC4, but had no significant influence on the mRNA level of HDAC4. It was hypothesized that miR-1 promotes muscle development by inhibiting the post-transcriptional expression of HDAC4 in goat.

Local origin or external input: modern horse origin in East Asia.

BACKGROUND: Despite decades of research, the horse domestication scenario in East Asia remains poorly understood. RESULTS: The study identified 16 haplogroups with fine-scale phylogenetic resolution using mitochondrial genomes of 317 horse samples. The time to the most recent common ancestor of the 16 haplogroups ranges from [0.8-3.1] thousand years ago (KYA) to [7.9-27.1] KYA. With combined analyses of the mitochondrial control region for 35 extant Przewalski's horses, 3544 modern and 203 ancient horses across the world, researchers provide evidence for that East Asian prevalent haplogroups Q and R were indigenously domesticated or they were involved in numerous distinct genetic components from wild horses in the southern part of East Asia. These events of haplotypes Q and R occurred during 4.7 to 16.3 KYA and 2.1 to 11.5 KYA, respectively. The diffusion of preponderant European haplogroups L from west to East Asia is consistent with the external gene input. Furthermore, genetic differences were detected between northern East Asia and southern East Asia cohorts by Principal Component Analysis, Analysis of Molecular Variance test, the χ2 test and phylogeographic analyses. CONCLUSIONS: All results suggest a complex picture of horse domestication, as well as geographic pattern in East Asia. Both local origin and external input occurred in East Asia horse populations. And besides, there are at least two different domestication or hybridization centers in East Asia.

Comprehensive Analysis of LncRNA Reveals the Temporal-Specific Module of Goat Skeletal Muscle Development

A series of complex processes regulate muscle development, and lncRNAs play essential roles in the regulation of skeletal myogenesis. Using RNA sequencing, we profiled the lncRNA expression during goat (Capra hircus) skeletal muscle development, which included seven stages across fetal 45 (F45), 65 (F65), 90 (F90), 120 (F120), 135 (F135) days, born for 24 h (B1) and 90 (B90) days. A total of 15,079 lncRNAs were identified in the seven stages, and they were less conservative with other species (human, cow, and mouse). Among them, 547 were differentially expressed, and they divided the seven stages into three functional transition periods. Following weighted gene co-expression network analysis (WGCNA), five lncRNA modules specific for developmental stages were defined as three types: 'Early modules', 'late modules', and 'individual-stage-specific modules'. The enrichment content showed that 'early modules' were related to muscle structure formation, 'late modules' participated in the 'p53 signaling pathway' and other pathways, the F90-highly related module was involved in the 'MAPK signaling pathway', and other pathways. Furthermore, we identified hub-lncRNA in three types of modules, and LNC_011371, LNC_ 007561, and LNC_001728 may play important roles in goat skeletal muscle. These data will facilitate further exploration of skeletal muscle lncRNA functions at different developmental stages in goats.

Filtered reproductive long non-coding RNAs by genome-wide analyses of goat ovary at different estrus periods.

BACKGROUND: The goat is an important farm animal. Reproduction is an important process of goat farming. The ovary is the most important reproductive organ for goats. In recent years, an increasing number of long non-coding RNAs (lncRNAs) have been implicated in the regulation of mammal reproduction. However, there are few studies on the function of lncRNAs in reproduction, particularly lncRNAs in the ovary. RESULTS: The sequencing of goat ovaries generated 1,122,014,112 clean reads, and 4926 lncRNAs and 1454 TUCPs (transcripts of uncertain coding potential) were identified for further analysis by using the coding potential analysis software, CNCI, CPC and Pfam-sca. There were 115 /22 differential lncRNAs /TUCPs transcripts between the ovaries of the luteal phase and the follicular phase. We predicted the related genes of lncRNA /TUCP based on co-expression and co-localization methods. In total, 2584 /904 genes were predicted by co-expression, and 326/73 genes were predicted by co-localization. The functions of these genes were further analyzed with GO and KEGG analysis. The results showed that lncRNAs /TUCPs, which are highly expressed in goat ovaries in the luteal phase, are mainly associated with the synthesis of progesterone, and we filtered the lncRNAs /TUCPs, such as XR_001918177.1 and TUCP_001362, which may regulate the synthesis of progesterone; lncRNAs /TUCPs, which are highly expressed in goat ovaries in the follicular phase, are mainly associated with oogenesis and the maturation of oocytes, and we filtered the lncRNAs /TUCPs that may regulate the oogenesis and maturation of oocyte, such as XR_001917388.1 and TUCP_000849. CONCLUSION: The present study provided the genome expression profile of lncRNAs /TUCPs in goat ovaries at different estrus periods and filtered the potential lncRNAs /TUCPs associated with goat reproduction. These results are helpful to further study the molecular mechanisms of goat reproduction.

RNA-seq analysis of lncRNA-controlled developmental gene expression during puberty in goat & rat.

BACKGROUND: Puberty is a pivotal stage in female animal development, and marks the onset of reproductive capability. However, little is known about the function of lncRNAs (long noncoding RNAs) in puberty. Therefore, RNA-seq analysis were performed between goats and rats to clarify the roles of lncRNAs and mRNAs in the onset of puberty. RESULTS: In the present study, the length of lncRNAs, the length of the open reading frame and the exon count were compared between the two species. Furthermore, functional annotation analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis of lncRNAs target genes and differentially expressed mRNA demonstrated the significantly enriched terms, such as AMPK signaling pathway, oxytocin signaling pathway, insulin secretion as well as pheromone receptor activity, and some other signaling pathways which were involved in the regulation of female puberty. Moreover, our results of siRNA interference in vitro showed the candidate lncRNA XLOC_446331 may play a crucial role in regulating female puberty. CONCLUSION: In conclusion, the RNA-seq analysis between goat and rat provide novel candidate regulators for genetic and molecular studies on female puberty.

The interaction between DNA methylation and long non-coding RNA during the onset of puberty in goats.

Epigenetics plays an important role in controlling female puberty. Both DNA methylation and long non-coding RNAs (lncRNA) regulate the initiation of puberty by affecting the expression of genes related to puberty. While recent studies have indicated that DNA methylation of lncRNA represses the expression of lncRNA, its role in regulating puberty remains unclear. To explore the mechanism between DNA methylation and lncRNAs during puberty onset, we performed whole-genome bisulphite sequencing (WGBS) and RNA-sequencing (RNA-seq). We found that DNA methylation was inversely correlated to gene expression levels during puberty. Methylation levels gradually decreased near the transcription initiation site and were present at high levels in the exon, intron and 3' untranslated regions. In the promoter, lncRNA expression was negatively related to DNA methylation. We reported hypermethylation in the gene body and downstream of the lncRNA compared with upstream regions. In GO and KEGG analyses, we found enriched target genes of lncRNA, XLOC_960044 and XLOC_767346. During puberty, methylation of these genes increased while expression decreased. Our study indicates that DNA methylation of the promoter is negatively correlated with lncRNA during puberty onset, and methylation regulates the initiation of puberty via lncRNA, which provides new insight into the epigenetic mechanism of puberty onset.

Screening and evaluating of long noncoding RNAs in the puberty of goats.

BACKGROUND: Long noncoding RNAs (lncRNAs) are involved in regulating animal development, however, their function in the onset of puberty in goats remain largely unexplored. To identify the genes controlling the regulation of puberty in goats, we measured lncRNA and mRNA expression levels from the hypothalamus. RESULTS: We applied RNA sequencing analysis to examine the hypothalamus of pubertal (case; n = 3) and prepubertal (control; n = 3) goats. Our results showed 2943 predicted lncRNAs, including 2012 differentially expressed lncRNAs, which corresponded to 5412 target genes. We also investigated the role of lncRNAs that act cis and trans to the target genes and found a number of lncRNAs involved in the regulation of puberty and reproduction, as well as several pathways related to these processes. For example, oxytocin signaling pathway, sterol biosynthetic process, and pheromone receptor activity signaling pathway were enriched as Kyoto Encyclopedia of Genes and Genomes (KEGG) or gene ontology (GO) analyses showed. CONCLUSION: Our results clearly demonstrate that lncRNAs play an important role in regulating puberty in goats. However, further research is needed to explore the functions of lncRNAs and their predicted targets to provide a detailed expression profile of lncRNAs on goat puberty.

Transcriptome differences between Cry1Ab resistant and susceptible strains of Asian corn borer.

BACKGROUND: Asian corn borer (ACB), Ostrinia furnacalis (Guenée), is the major insect pest of maize in China and countries of East and Southeast Asia, the Pacific and Australasia. ACB can develop strong resistance to the transgenic Bt maize expressing Cry1Ab, the most widely commercialized Bt maize worldwide. However, the molecular basis for the resistance mechanisms of ACB to Cry1Ab remained unclear. Two biological replicates of the transcriptome of Bt susceptible (ACB-BtS) and Cry1Ab resistant (ACB-AbR) strains of ACB were sequenced using Solexa/Illumina RNA-Seq technology to identify Cry1Ab resistance-relevant genes. RESULTS: The numbers of unigenes for two biological replications were 63,032 and 53,710 for ACB-BtS and 57,770 and 54,468 for ACB-AbR. There were 35,723 annotated unigenes from ACB reads found by BLAST searching NCBI non-redundant, NCBI non-redundant nucleotide, Swiss-prot protein, Kyoto Encyclopedia of Genes and Genomes, Cluster of Orthologous Groups of proteins, and Gene Ontology databases. Based on the NOISeq method, 3,793 unigenes were judged to be differentially expressed between ACB-BtS and ACB-AbR. Cry1Ab resistance appeared to be associated with change in the transcription level of enzymes involved in growth regulation, detoxification and metabolic/catabolic process. Among previously described Bt toxin receptors, the differentially expressed unigenes associated with aminopeptidase N and chymotrypsin/trypsin were up-regulated in ACB-AbR. Whereas, other putative Cry receptors, cadherin-like protein, alkaline phosphatase, glycolipid, actin, V-type proton ATPase vatalytic, heat shock protein, were under-transcripted. Finally, GPI-anchor biosynthesis was found to be involved in the significantly enriched pathway, and all genes mapped to the pathway were substantially down-regulated in ACB-AbR. CONCLUSION: To our knowledge, this is the first comparative transcriptome study to discover candidate genes involved in ACB Bt resistance. This study identified differentially expressed unigenes related to general Bt resistance in ACB. The assembled, annotated transcriptomes provides a valuable genomic resource for further understanding of the molecular basis of ACB Bt resistance mechanisms.

Identification and characterization of microRNAs in the ovaries of multiple and uniparous goats (Capra hircus) during follicular phase.

BACKGROUND: Superior kidding rate is an important economic trait in production of meat goat, and ovulation rate is the precondition of kidding rate. MicroRNAs (miRNAs) play critical roles in almost all ovarian biological processes, including folliculogenesis, follicle development, follicle atresia, luteal development and regression. To find out the different ovarian activity and follicle recruitment with miRNA-mediated posttranscriptional regulation, the small RNAs expressed pattern in the ovarian tissues of multiple and uniparous Anhui White goats during follicular phase was analyzed using Solexa sequencing data. RESULTS: 1008 miRNAs co-expressed, 309 and 433 miRNAs specifically expressed in the ovaries of multiple and uniparous goats during follicular phase were identified. The 10 most highly expressed miRNAs in the multiple library were also the highest expressed in the uniparous library, and there were no significantly different between each other. The highest specific expressed miRNA in the multiple library was miR-29c, and the one in the uniparous library was miR-6406. 35 novel miRNAs were predicted in total. GO annotation and KEGG Pathway analyses were implemented on target genes of all miRNA in two libraries. RT-PCR was applied to detect the expression level of 5 randomly selected miRNAs in multiple and uniparous hircine ovaries, and the results were consistent with the Solexa sequencing data. CONCLUSIONS: In the present study, the different expression of miRNAs in the ovaries of multiple and uniparous goats during follicular phase were characterized and investigated using deep sequencing technology. The result will help to further understand the role of miRNAs in kidding rate regulation and also may help to identify miRNAs which could be potentially used to increase hircine ovulation rate and kidding rate in the future.

Comprehensive analyses of 435 goat transcriptomes provides insight into male reproduction.

A systematic analysis of genes related to reproduction is crucial for obtaining a comprehensive understanding of the molecular mechanisms that underlie male reproductive traits in mammals. Here, we utilized 435 goat transcriptome datasets to unveil the testicular tissue-specific genes (TSGs), allele-specific expression (ASE) genes and their uncharacterized transcriptional features related to male goat reproduction. Results showed a total of 1790 TSGs were identified in goat testis, which was the most among all tissues. GO enrichment analyses suggested that testicular TSGs were mainly involved in spermatogenesis, multicellular organism development, spermatid development, and flagellated sperm motility. Subsequently, a total of 95 highly conserved TSGs (HCTSGs), 508 middle conserved TSGs (MCTSGs) and 42 no conserved TSGs (NCTSGs) were identified in goat testis. GO enrichment analyses suggested that the HCTSGs and MCTSGs has a more important association with male reproduction than NCTSGs. Additionally, we identified 644 ASE genes, including 88 tissue-specific ASE (TS-ASE) genes (e.g., FSIP2, TDRD9). GO enrichment analyses indicated that both ASE genes and TS-ASE genes were associated with goat male reproduction. Overall, this study revealed an extensive gene set involved in the regulation of male goat reproduction and their dynamic transcription patterns. Data reported here provide valuable insights for a further improvement of the economic benefits of goats as well as future treatments for male infertility.

Transcriptome analysis revealed differences in gene expression in sheep muscle tissue at different developmental stages.

BACKGROUND: The analysis of differentially expressed genes in muscle tissues of sheep at different ages is helpful to analyze the gene expression trends during muscle development. In this study, the longissimus dorsi muscle of pure breeding Hu sheep (H), Suffolk sheep and Hu sheep hybrid F1 generation (SH) and East Friesian and Hu sheep hybrid sheep (EHH) three strains of sheep born 2 days (B2) and 8 months (M8) was used as the research object, and transcriptome sequencing technology was used to identify the differentially expressed genes of sheep longissimus dorsi muscle in these two stages. Subsequently, GO and KEGG enrichment analysis were performed on the differential genes. Nine differentially expressed genes were randomly selected and their expression levels were verified by qRT-PCR. RESULTS: The results showed that 842, 1301 and 1137 differentially expressed genes were identified in H group, SH group and EHH group, respectively. Among them, 191 differential genes were enriched in these three strains, including pre-folding protein subunit 6 (PFDN6), DnaJ heat shock protein family member A4 (DNAJA4), myosin heavy chain 8 (MYH8) and so on. GO and KEGG enrichment analysis was performed on 191 differentially expressed genes shared by the three strains to determine common biological pathways. The results showed that the differentially expressed genes were significantly enriched in ribosomes, unfolded protein binding, FoxO signaling pathway, glycolysis / glycogen generation and glutathione signaling pathway that regulate muscle protein synthesis and energy metabolism. The results of qRT-PCR were consistent with transcriptome sequencing, which proved that the sequencing results were reliable. CONCLUSIONS: Overall, this study revealed the important genes and signaling pathways related to sheep skeletal muscle development, and the result laid a foundation for further understanding the mechanism of sheep skeletal muscle development.

Multi-Omics Analysis Reveals the Regulatory Mechanism of Probiotics on the Growth Performance of Fattening Sheep.

Probiotics have been proven to improve the growth performance of livestock and poultry. The aim of this experiment was to investigate the effects of probiotic supplementation on the growth performance; rumen and intestinal microbiota; rumen fluid, serum, and urine metabolism; and rumen epithelial cell transcriptomics of fattening meat sheep. Twelve Hu sheep were selected and randomly divided into two groups. They were fed a basal diet (CON) or a basal diet supplemented with 1.5 × 108 CFU/g probiotics (PRB). The results show that the average daily weight gain, and volatile fatty acid and serum antioxidant capacity concentrations of the PRB group were significantly higher than those of the CON group (p < 0.05). Compared to the CON group, the thickness of the rumen muscle layer in the PRB group was significantly decreased (p < 0.01); the thickness of the duodenal muscle layer in the fattening sheep was significantly reduced; and the length of the duodenal villi, the thickness of the cecal and rectal mucosal muscle layers, and the thickness of the cecal, colon, and rectal mucosal layers (p < 0.05) were significantly increased. At the genus level, the addition of probiotics altered the composition of the rumen and intestinal microbiota, significantly upregulating the relative abundance of Subdivision5_genera_incertae_sedis and Acinetobacter in the rumen microbiota, and significantly downregulating the relative abundance of Butyrivibrio, Saccharofermentans, and Fibrobacter. The relative abundance of faecalicoccus was significantly upregulated in the intestinal microbiota, while the relative abundance of Coprococcus, Porphyromonas, and Anaerobacterium were significantly downregulated (p < 0.05). There were significant differences in the rumen, serum, and urine metabolites between the PRB group and the CON group, with 188, 138, and 104 metabolites (p < 0.05), mainly affecting pathways such as vitamin B2, vitamin B3, vitamin B6, and a series of amino acid metabolisms. The differential genes in the transcriptome sequencing were mainly enriched in protein modification regulation (especially histone modification), immune function regulation, and energy metabolism. Therefore, adding probiotics improved the growth performance of fattening sheep by altering the rumen and intestinal microbiota; the rumen, serum, and urine metabolome; and the transcriptome.

Positive In Vitro Effect of ROCK Pathway Inhibitor Y-27632 on Qualitative Characteristics of Goat Sperm Stored at Low Temperatures.

Y-27632, as a cytoskeleton protector, is commonly used for low-temperature preservation of cells. Goat sperm are prone to damage to the cytoskeleton under low-temperature conditions, leading to a loss of sperm vitality. However, the Y-27632 small molecule has not yet been used in research on low-temperature preservation of goat semen. This study aims to address the issue of low temperature-induced loss of sperm motility in goats by using Y-27632, and explore the regulation of Y-27632 on goat sperm metabolism. At a low temperature of 4 °C, different concentrations of Y-27632 were added to the sperm diluent. The regulation of Y-27632 on the quality of low temperature-preserved goat semen was evaluated by detecting goat sperm motility, antioxidant capacity, mitochondrial activity, cholesterol levels, and metabolomics analysis. The results indicated that 20 µM Y-27632 significantly increased plasma membrane integrity (p < 0.05), and acrosome integrity (p < 0.05) and sperm motility (p < 0.05), increased levels of superoxide dismutase (SOD) and catalase (CAT) (p < 0.01), increased total antioxidant capacity (T-AOC) (p < 0.05), decreased levels of malondialdehyde (MDA) and reactive oxygen species (ROS) (p < 0.01), and significantly increased mitochondrial membrane potential (MMP). The levels of ATP, Ca2+, and TC in sperm increased (p < 0.01). Twenty metabolites with significant differences were identified, with six metabolic pathways having a significant impact, among which the D-glutamic acid and D-glutamine metabolic pathways had the most significant impact. The artificial insemination effect of goat semen treated with 20 µM Y-27632 was not significantly different from that of fresh semen. This study indicates that Y-27632 improves the quality of low-temperature preservation of sperm by protecting the sperm plasma membrane, enhancing sperm antioxidant capacity, regulating D-glutamine and D-glutamate metabolism, and promoting the application of low-temperature preservation of semen in artificial insemination technology.