Genomic structure analysis and construction of DNA fingerprint for four sheep populations.

The Yongdeng Qishan sheep (QS) is a sheep population found locally in China. To gain in-depth knowledge of its population characteristics, three control groups were chosen, comprising the Lanzhou fat-tailed sheep (LFT), TAN sheep (TAN), and Minxian black fur sheep (MBF), inhabiting the nearby environments. This study genotyped a total of 120 individuals from four sheep populations: QS, LFT, TAN, and MBF. Using Specific-Locus Amplified Fragment Sequencing, we conducted genetic diversity, population structure, and selective sweep analysis, and constructed the fingerprint of each population. In total, there were 782 535 single nucleotide polymorphism (SNP) variations identified, with most being situated within regions that are intergenic or intronic. The genetic diversity analysis revealed that the QS population exhibited lower genetic diversity compared to the other three populations. Consistent results were obtained from the principal component, phylogenetic tree, and population structure analysis, indicating significant genetic differences between QS and the other three populations. However, a certain degree of differentiation was observed within the QS population. The linkage disequilibrium (LD) patterns among the four populations showed clear distinctions, with the QS group demonstrating the most rapid LD decline. Kinship analysis supported the findings of population structure, dividing the 90 QS individuals into two subgroups consisting of 23 and 67 individuals. Selective sweep analysis identified a range of genes associated with reproduction, immunity, and adaptation to high-altitude hypoxia. These genes hold potential as candidate genes for marker-assisted selection breeding. Additionally, a total of 86 523 runs of homozygosity (ROHs) were detected, showing non-uniform distribution across chromosomes, with chromosome 1 having the highest coverage percentage and chromosome 26 the lowest. In the high-frequency ROH islands, 79 candidate genes were associated with biological processes such as reproduction and fat digestion and absorption. Furthermore, a DNA fingerprint was constructed for the four populations using 349 highly polymorphic SNPs. In summary, our research delves into the genetic diversity and population structure of QS population. The construction of DNA fingerprint profiles for each population can provide valuable references for the identification of sheep breeds both domestically and internationally.

Effects of adding Allium mongolicum Regel powder and yeast cultures to diet on rumen microbial flora of Tibetan sheep (Ovis aries).

The purpose of this experiment was to study the effect of Allium mongolicum Regel powder (AMR) and yeast cultures (YC) on rumen microbial diversity in Tibetan sheep in different Ecological niches. A total of 40 male Tibetan lambs with an initial weight of 18.56 ± 1.49 kg (6 months old) were selected and divided into four groups (10 sheep/pen; n = 10). In the Control Group, each animal was grazed for 8 h per day, in Group I, each animal was supplemented with 200 g of concentrate per day, in Group II, each animal was supplemented with 200 g of concentrate and 10 g of AMR per day, in Group III, each animal was supplemented with 200 g of concentrate and 20 g of YC per day. The experiment lasted 82 days and consisted of a 7-day per-feeding period and a 75-day formal period. The results indicated that at the phylum level, the abundance of Bacteroidota and Verrucomimicrobiota in L-Group II and L-Group III was increased, while the abundance of Proteobacteria was decreased in the LA (Liquid-Associated) groups. The proportion of F/B in S-Group II and S-Group III was increased compared to S-Group I and S-CON in the SA (Soild-Associated) group. At the genus level, the abundance of uncultured_rumen_bacterium and Eubacterium_ruminantium_group in L-Group II and L-Group III was increased. Furthermore, while the abundance of Rikenellaceae_RC9_gut_group was decreased in the LA, the abundance of Prevotella and Eubacterium_ruminantium_group was increased in the S-Group II and S-Group III compared to S-Group I and S-CON. The abundance of probable_genus_10 was the highest in S-Group II in the SA group. After the addition of YC and AMR, there was an increase in rumen microbial abundance, which was found to be beneficial for the stability of rumen flora and had a positive impact on rumen health.

Prickly Ash Seeds improve immunity of Hu sheep by changing the diversity and structure of gut microbiota.

Prickly Ash Seeds (PAS), as a traditional Chinese medicinal herb, have pharmacological effects such as anti-asthma, anti-thrombotic, and anti-bacterial, but their impact on gut microbiota is still unclear. This study used a full-length 16 s rRNA gene sequencing technique to determine the effect of adding PAS to the diet on the structure and distribution of gut microbiota in Hu sheep. All lambs were randomly divided into two groups, the CK group was fed with a basal ration, and the LZS group was given a basal diet with 3% of PAS added to the ration. The levels of inflammatory factors (IL-10, IL-1ß, and TNF-α) in intestinal tissues were measured by enzyme-linked immunosorbent assay (ELISA) for Hu sheep in the CK and LZS group. The results indicate that PAS can increase the diversity and richness of gut microbiota, and can affect the community composition of gut microbiota. LEfSe analysis revealed that Verrucomicrobiota, Kiritimatiella, WCHB 41, and uncultured_rumen_bacterium were significantly enriched in the LZS group. KEGG pathway analysis found that LZS was significantly higher than the CK group in the Excretory system, Folding, sorting and degradation, and Immune system pathways (p < 0.05). The results of ELISA assay showed that the level of IL-10 was significantly higher in the LZS group than in the CK group (p < 0.05), and the levels of TNF-α and IL-1ß were significantly higher in the CK group than in the LZS group (p < 0.05). LEfSe analysis revealed that the dominant flora in the large intestine segment changed from Bacteroidota and Gammaproteobacteria to Akkermansiaceae and Verrucomicrobiae after PAS addition to Hu sheep lambs; the dominant flora in the small intestine segment changed from Lactobacillales and Aeriscardovia to Kiritimatiellae and WCHB1 41. In conclusion, the addition of PAS to sheep diets can increase the number and types of beneficial bacteria in the intestinal tract, improve lamb immunity, and reduce intestinal inflammation. It provides new insights into healthy sheep production.

Identification and functional characterization of developmental-stage-dependent piRNAs in Tibetan sheep testes.

The core function of the testes is to produce sperms, which is the prerequisite for maintaining male fertility. PIWI-interacting RNAs (piRNAs) are a class of non-coding small RNAs that are mainly enriched in the reproductive organ and play a key role in germ cell development and spermatogenesis. However, the expression and function of piRNAs in the testes of Tibetan sheep, a domestic animal endemic to the Tibetan Plateau, remain unknown. In this study, we evaluated the sequence structure, expression profile, and potential function of piRNAs in testicular tissues from Tibetan sheep at different developmental stages (3 months, 1 year, and 3 years of age, respectively) by small RNA sequencing. Of the identified piRNAs, the sequence lengths of 24-26 nt and 29 nt dominate. Most piRNA sequences begin with uracil and have a distinct ping-pong structure which mainly distributes in exons, repeat regions, introns, and other unannotated regions of the genome. The piRNAs in the repeat region are primarily derived from the retrotransposons: long terminal repeats, long interspersed nuclear elements, and short interspersed elements. These piRNAs constitute 2,568 piRNA clusters, which mainly distribute on chromosomes 1, 2, 3, 5, 11, 13, 14, and 24, and of these clusters, a total of 529 piRNA clusters were differentially expressed in at least two age groups. Most of the piRNAs were expressed in a low abundance in the testes of developing Tibetan sheep. A total of 41,552 and 2,529 differential piRNAs were identified in testes from 3 months vs. 1 year, and 1 year vs. 3 years, respectively, presenting significantly increased abundance for most piRNAs in 1 year and 3 years compared with 3 months. The functional evaluation of the target genes showed that the differential piRNAs are mainly involved in regulating gene expression, transcription, protein modification, and cell development during spermatogenesis and testicular development. In conclusion, this study focused on the sequence structure and expression characteristics of piRNAs in the testis of Tibetan sheep and provided new insights into the functional mechanism of piRNAs in testicular development and spermatogenesis of sheep.

The testis in mammals plays an indispensable role in male fertility, in which the development and function are strictly regulated by a variety of non-coding small RNAs. PIWI-interacting RNAs (piRNAs) are the most abundant non-coding small RNAs in mammalian testes, playing an irreplaceable role in testicular development and spermatogenesis. To characterize the piRNA expression and investigate their potential biological function in developmental Tibetan sheep testes, we carried out RNA sequencing. Our results revealed that the length distribution of the identified piRNAs is mostly 24­26 nt and 29 nt, exhibiting a preference for uracil at their 5' end and significant ping-pong structure. Most piRNAs were differentially expressed in Tibetan sheep testes in a development-age-dependent manner, and the vast majority of them were upregulated in postpubertal and adult testes. Based on the functional annotation of piRNA target genes, they were mainly implicated in the regulation of gene expression, transcription, protein modification and development during testicular development and spermatogenesis.

Identification and Functional Assignment of Genes Implicated in Sperm Maturation of Tibetan Sheep.

While traveling through the epididymis, immature sheep spermatozoa undergo a sequence of processes that ultimately give them the capacity to swim and fertilize an egg. Different gene expression patterns may be found in the epididymal caput, corpus, and cauda, conferring variant or unique biological roles during epididymis development and sperm maturation. To search for candidate genes associated with ovine sperm maturation and assess their possible modulating mechanisms, we characterized gene expression in each epididymal segment derived from pre- and post-pubertal Tibetan sheep by RNA sequencing. Compared with pre-puberty, 7730 (3724 upregulated and 4006 downregulated), 7516 (3909 upregulated and 3607 downregulated), and 7586 (4115 elevated and 3471 downregulated) genes were found to be differentially expressed in the post-pubertal caput, corpus, and cauda epididymis, respectively, and real-time quantitative PCR verified the validity of the gathered expression patterns. Based on their functional annotations, most differential genes were assigned to the biological processes and pathways associated with cellular proliferation, differentiation, immune response, or metabolic activities. As for the post-pubertal epididymis, 2801, 197, and 186 genes were specifically expressed in the caput, corpus, and cauda, respectively. Functional annotation revealed that they were mainly enriched to various distinct biological processes associated with reproduction (including the caput binding of sperm to the zona pellucida; fertilization in the caput and corpus; and meiosis in the caput and cauda) and development (such as cell differentiation and developmental maturation in the caput; cell proliferation and metabolism in the corpus; and regulation of tube size and cell division/cell cycle in the cauda). Additionally, we focused on the identification of genes implicated in immunity and sperm maturation, and subsequent functional enrichment analysis revealed that immune-related genes mainly participated in the biological processes or pathways associated with the immune barrier (such as JAM3 and ITGA4/6/9) and immunosuppression (such as TGFB2, TGFBR1, TGFBR2, and SMAD3), thus protecting auto-immunogenic spermatozoa. Additionally, sperm maturation was mostly controlled by genes linked with cellular processes, including cell growth, proliferation, division, migration, morphogenesis, and junction. Altogether, these results suggest that most genes were differentially expressed in developmental epididymal regions to contribute to microenvironment development and sperm maturation. These findings help us better understand the epididymal biology, including sperm maturation pathways and functional differences between the epididymal regions in Tibetan sheep and other sheep breeds.

The regulatory role of BMP4 in testicular Sertoli cells of Tibetan sheep.

This study aimed to determine the regulatory mechanism of bone morphogenetic protein 4 (BMP4) gene in the testes of Tibetan sheep and its role in the blood-testis barrier (BTB). First, we cloned BMP4 gene for bioinformatics analysis, and detected the mRNA and protein expression levels of BMP4 in the testes of Tibetan sheep pre-puberty (3-mo-old), during sexual maturity (1-yr-old), and in adulthood (3-yr-old) by qRT-PCR and Western blot. In addition, the subcellular localization of BMP4 was analyzed by immunohistochemical staining. Next, BMP4 overexpression and silencing vectors were constructed and transfected into primary Sertoli cells (SCs) to promote and inhibit the proliferation of BMP4, respectively. Then, CCK-8 was used to detect the proliferation effect of SCs. The expression of BMP4 and downstream genes, pathway receptors, tight junction-related proteins, and cell proliferation and apoptosis-related genes in SCs were studied using qRT-PCR and Western blot. The results revealed that the relative expression of BMP4 mRNA and protein in testicular tissues of 1Y group and 3Y group was dramatically higher than that of 3M group (P < 0.01), and BMP4 protein is mainly located in SCs and Leydig cells at different development stages. The CDS region of the Tibetan sheep BMP4 gene was 1,229 bp. CCK-8 results demonstrated that the proliferation rate of BMP4 was significantly increased in the overexpression group (pc-DNA-3.1(+)-BMP4; P < 0.05). In addition, the mRNA and protein expressions of SMAD5, BMPR1A, and BMPR1B and tight junction-related proteins Claudin11, Occludin, and ZO1 were significantly increased (P < 0.05). The mRNA expression of cell proliferation-related gene Bcl2 was significantly enhanced (P < 0.05), and the expression of GDNF was enhanced (P > 0.05). The mRNA expression of apoptosis-related genes Caspase3 and Bax decreased significantly (P < 0.05), while the mRNA expression of cell cycle-related genes CyclinA2 and CDK2 increased significantly (P < 0.05). It is worth noting that the opposite results were observed after transfection with si-BMP4. In summary, what should be clear from the results reported here is that BMP4 affects testicular development by regulating the Sertoli cells and BTB, thereby modulating the spermatogenesis of Tibetan sheep.

The fertility of male Tibetan sheep is mainly affected by testicular development and spermatogenesis. In these processes, Sertoli cells (SCs) play a central role and are regulated by a variety of genes and factors. BMP4 is mainly distributed in Sertoli cells, and its expression level increases with age. Overexpression of the BMP4 gene in Tibetan sheep testis SCs revealed elevated expression of BMP4 protein and its downstream genes SMAD5, pathway receptor proteins BMPR1A and BMPR1B; followed by elevated expression levels of cell proliferation-related genes and decreased expression levels of apoptosis-related genes. Meanwhile, the expression of tight junction proteins was also elevated. These results indicate that BMP4 affects testicular development by regulating the Sertoli cells and blood­testis barrier, thereby affecting the spermatogenesis of Tibetan sheep.

Differential tissue expression of sex steroid-synthesizing enzyme CYP11A1 in male Tibetan sheep (Ovis aries).

Steroid metabolism is a fundament to testicular development and function. The cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) is a key rate-limiting enzyme for catalyzing the conversion of cholesterol to pregnenolone. However, despite its importance, what expression and roles of CYP11A1 possesses and how it regulates the testicular development and spermatogenesis in Tibetan sheep remains largely unknown. Based on this, we evaluated the expression and localization patterns of CYP11A1 in testes and epididymides of Tibetan sheep at three developmental stages (three-month-old, pre-puberty; one-year-old, sexual maturity and three-year-old, adult) by quantitative real-time PCR (qPCR), western blot and immunofluorescence. The results showed that CYP11A1 mRNA and protein were expressed in testes and epididymides throughout the development stages and obviously more intense in one- and three-year-old groups than three-month-old group (except for the caput epididymidis). Immunofluorescence assay showed that the CYP11A1 protein was mainly located in Leydig cells and epididymal epithelial cells. In addition, positive signals of CYP11A1 protein were observed in germ cells, epididymal connective tissue and sperms stored in the epididymal lumen. Collectively, these results suggested that the CYP11A1 gene might be mainly involved in regulating spermatogenesis and androgen synthesis in developmental Tibetan sheep testis and epididymis.