The rumen microbiota plays a key role in the utilization of plant materials by ruminants, yet little is known about the key taxa and their genetic functions of the rumen sub-environment involved in the ruminal degradation process. Understanding the differences in the composition and function of ruminal microbiota in the liquid-associated (LA) and solid-associated (SA) systems is needed to further study and regulate rumen function and health. In this study, rumen contents of nine sheep were collected to separate LA and SA systems with elution and centrifugal precipitation. Metagenome sequencing was used to investigate the differences in microbial composition and genetic functions of LA and SA systems, with special emphasis on their degradational potential toward carbohydrates. Results showed that the dominant species composition was similar between the two systems, but SA microorganisms had a higher relative abundance than LA microorganisms in all taxa. The concentration of fiber-degrading bacteria, such as Ruminococcus, Treponema, and Fibrobacter, was higher and Prevotella was lower in the SA vs. LA system. Additionally, SA microorganisms dominated in cellulose degradation, while LA microorganisms were more important in starch utilization based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO)'s functional categories and Carbohydrate-Active Enzymes (CAZymes). In general, SA microorganisms are more abundant and important in metabolic functions than LA, such as carbohydrate and amino acid metabolisms. In summary, the key differential biomarkers between LA and SA systems were Prevotella, Ruminococcus, Treponema, and Fibrobacter. Ruminal microbes degraded carbohydrates synergistically with SA, thus, more focusing on cellulose and hemicellulose, while LA is more important to starch.

Background The detection of selective traits in different populations can not only reveal current mechanisms of artificial selection for breeding, but also provide new insights into phenotypic variation in new varieties and the search for genes associated with important traits. Panou sheep is a cultivated breed of Tibetan sheep in China with stable genetic performance, consistent appearance and fast growth and development after decades of artificial selection and cultivation. Due to long-term adaptation to the high altitude, cold and hypoxic environment in the plateau area, they may have formed a unique gene pool that is different from other Tibetan sheep breeds. To explore the genetic resources of Panou sheep, we used next-generation sequencing technology for the first time to investigate the genome-wide population structure, genetic diversity, and candidate signatures of positive selection in Panou sheep. Results Comparative genomic analysis with the closely related species Oula sheep (a native breed of Tibetan sheep in China) was used to screen the population selection signal of Panou sheep. Principal component analysis and neighbor joining tree showed that Panou sheep and Oula sheep had differences in population differentiation. Furthermore, analyses of population structure, they came from the same ancestor, and when K = 2, the two populations could be distinguished. Panou sheep exhibit genetic diversity comparable to Oula sheep, as shown by observed heterozygosity, expected heterozygosity and runs of homozygosity. Genome-wide scanning using the Fst and π ratio methods revealed a list of potentially selected related genes in Panou sheep compared to Oula sheep, including histone deacetylase 9 (HDAC9), protein tyrosine kinase 2 (PTK2), microphthalmia-related transcription factor (MITF), vesicular amine transporter 1 (VAT1), trichohyalin-like 1 (TCHHL1), amine oxidase, copper containing 3 (AOC3), interferon-inducible protein 35 (IFI35). Conclusions The results suggest that traits related to growth and development and plateau adaptation may be selection targets for the domestication and breeding improvement of Tibetan sheep. This study provides the fundamental footprints for Panou sheep breeding and management.

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 months old), during sexual maturity (1 year old), and in adulthood (3 years 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 1229 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, 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 blood testes barrier, thereby modulating the spermatogenesis of Tibetan sheep.

Testis has an indispensable function in male reproduction of domestic animals. Tibetan sheep (Ovis aries) is a locally adapted breed of sheep raised in the Qinghai-Tibet Plateau, with outsized roles in providing the livelihood for millions of residents. Nevertheless, less is known on how protein expression and their functional roles in developmental testes of such breed limit their use in breeding efforts. In this study, we obtained comprehensive protein profiles from testes of Tibetan sheep at three developmental stages (including pre-puberty, post-puberty, and adulthood) using data-independent acquisition-based proteomic strategy to quantitatively identify the differentially abundant proteins (DAPs) associated with testicular development and function and to unravel the molecular basis of spermatogenesis. A total of 6,221 proteins were differentially expressed in an age-dependent manner. The reliability of the gene expression abundance was corroborated by quantitative PCR and targeted parallel reaction monitoring. These DAPs were significantly enriched to biological processes concerning spermatid development and sperm deformation, mitosis, glycolytic process, cell-cell/extracellular matrix (ECM) junctions, cell proliferation, apoptosis, and migration and to the pathways including, developmental process and sexual reproduction-related (such as VEGF, estrogen, insulin, GnRH, Hippo, PI3K-Akt, mTOR, MAPK, and AMPK), and testicular cell events-related pathways (such as tight/gap/adherens junctions, ECM-receptor interaction, regulation of actin cytoskeleton, glycolysis, cell cycle, and meiosis). Based on these bioinformatics analysis, we constructed four protein–protein interaction network, among which the proteins are involved in mitosis, meiosis, spermiogenesis, and testicular microenvironment, respectively. Altogether, these bioinformatics-based sequencing results suggest that many protein-coding genes were expressed in a development-dependent manner in Tibetan sheep testes to contribute to the testicular cell development and their surrounding microenvironment remodeling at various stages of spermatogenesis. These findings have important implications for further understanding of the mechanisms underlying spermatogenesis in sheep and even other plateau-adapted animals.

Glycolysis in Sertoli cells (SCs) can provide energy substrates for the development of spermatogenic cells. Triose phosphate isomerase 1 (TPI1) is one of the key catalytic enzymes involved in glycolysis. However, the biological function of TPI1 in SCs and its role in glycolytic metabolic pathways are poorly understood. On the basis of a previous research, we isolated primary SCs from Tibetan sheep, and overexpressed TPI1 gene to determine its effect on the proliferation, glycolysis, and apoptosis of SCs. Secondly, we investigated the relationship between TPI1 and miR-1285-3p, and whether miR-1285-3p regulates the proliferation and apoptosis of SCs, and participates in glycolysis by targeting TPI1. Results showed that overexpression of TPI1 increased the proliferation rate and decreased apoptosis of SCs. In addition, overexpression of TPI1 altered glycolysis and metabolism signaling pathways and significantly increased amount of the final product lactic acid. Further analysis showed that miR-1285-3p inhibited TPI1 by directly targeting its 3’untranslated region. Overexpression of miR-1285-3p suppressed the proliferation of SCs, and this effect was partially reversed by restoration of TPI1 expression. In summary, this study shows that the miR-1285-3p/TPI1 axis regulates glycolysis in SCs. These findings add to our understanding on the regulation of spermatogenesis in sheep and other mammals.