Background: Yak (Bos grunniens) is the most important domestic animal for people living at high altitudes. Yak ordinarily feed by grazing, and this behavior impacts the accuracy of the pedigree record because it is difficult to control mating in grazing yak. This study aimed to evaluate the pedigree system and individual identification in polled yak. Methods: A total of 71 microsatellite loci were selected from the literature, mostly from the studies on cattle. A total of 35 microsatellite loci generated excellent PCR results and were evaluated for the parentage testing and individual identification of 236 unrelated polled yaks. A total of 17 of these 35 microsatellite loci had polymorphic information content (PIC) values greater than 0.5, and these loci were in Hardy–Weinberg equilibrium without linkage disequilibrium. Results: Using multiplex PCR, capillary electrophoresis, and genotyping, very high exclusion probabilities were obtained for the combined core set of 17 loci. The exclusion probability (PE) for one candidate parent when the genotype of the other parent is not known was 0.99718116. PE for one candidate parent when the genotype of the other parent is known was 0.99997381. PE for a known candidate parent pair was 0.99999998. The combined PEI (PE for identity of two unrelated individuals) and PESI (PE for identity of two siblings) were >0.99999999 and 0.99999899, respectively. These findings indicated that the combination of 17 microsatellite markers could be useful for efficient and reliable parentage testing and individual identification in polled yak. Discussion: Many microsatellite loci have been investigated for cattle paternity testing. Nevertheless, these loci cannot be directly applied to yak identification because the two bovid species have different genomic sequences and organization. A total of 17 loci were selected from 71 microsatellite loci based on efficient amplification, unambiguous genotyping, and high PIC values for polled yaks, and were suitable for parentage analysis in polled yak populations.

Yak reproductive characteristics have received extensive attention, though the molecular regulation mechanism of its ovarian activity remains to be explored. Therefore, this study initially conducted a comparative analysis of yak ovarian activities in anestrus, estrus, and pregnancy regarding their morphology and histology, followed by implementing RNA sequencing (RNA-seq) technology to detect the overall gene expression and biological mechanism in different reproductive stages. H&E staining showed that there were more growing follicles and mature follicles in ovarian tissue sections during estrus than ovarian tissues during non-estrus. The RNA-seq analysis of yak ovary tissues in three periods showed that DEGs related to follicular development and hormone metabolism were screened in the three comparison groups, such as COL1A2, NR4A1, THBS2, PTGS2, SCARB1, STAR, and WNT2B. Bioinformatics analysis showed that these DEGs are involved in ion binding, cell development, metabolic processes, enriched in ECM–receptor interactions, steroid biosynthesis, together with aldosterone generation/discharge and Wnt/PI3K-Akt signaling pathways. In addition, we speculate alternate splice development events to have important role/s in regulating ovarian functional genomic expression profiles. These results provide essential knowledge aimed at scrutinizing pivotal biomarkers for yak ovarian activity, together with paving the way for enhancing researchers’ focus on improving yak reproductive performance.

The yak (Bos grunniens) is subjected to nutritional deficiency during the whole winter grazing season; deciphering the adipose metabolism and energy homeostasis under cold and nutrients stress conditions could be a novel way to understand the specific mechanism of energy metabolism. Circular RNAs (circRNAs) have elucidated that they play a key role in many biological events, but the regulatory function of adipose development remains mostly unknown. Therefore, the expression pattern of circRNAs were identified for the first time during yak adipocyte differentiation to gain insight into their potential functional involvement in bovine adipogenesis. We detected 7203 circRNA candidates, most of them contained at least two exons, and multiple circRNA isoforms could be generated from one parental gene. Analysis of differential expression circRNAs displayed that 136 circRNAs were differentially expressed at day 12 (Ad) after adipocyte differentiation, compared with the control at day 0 (Pread 0), while 7 circRNAs were detected on day 2. Sanger sequencing validated that six circRNAs had head-to-tail junction, and quantitative real-time PCR (qPCR) results revealed that the expression patterns of ten circRNAs were consistent with their expression levels from RNA-sequencing (RNA-seq) data. We further predicted the networks of circRNA-miRNA-gene based on miRNAs sponging by circRNAs, in which genes were participated in the adipocyte differentiation-related signaling pathways. After that, we constructed several adipocyte differentiation-related ceRNAs and revealed six circRNAs (novel_circ_0009127, novel_circ_0000628, novel_circ_0011513, novel_circ_0010775, novel_circ_0006981 and novel_circ_0001494) were related to adipogenesis. Furthermore, we analyzed the homology among yak, human and mouse circRNAs and found that 3536 yak circRNAs were homologous to human and mouse circRNAs. In conclusion, these findings provide a solid basis for the investigation of yak adipocyte differentiation-related circRNAs and serve as a great reference to study the energy metabolism of high-altitude animals.

Spermatogenesis is a complex process that involves proliferation and differentiation of diploid male germ cells into haploid flagellated sperm and requires intricate interactions between testicular somatic cells and germ cells. The cellular heterogeneity of this process presents a challenge in analyzing the different cell types at various developmental stages. Single-cell RNA sequencing (scRNA-seq) provides a useful tool for exploring cellular heterogeneity. In this study, we performed a comprehensive and unbiased single-cell transcriptomic study of spermatogenesis in sexually mature 4-year-old yak using 10× Genomics scRNA-seq. Our scRNA-seq analysis identified six somatic cell types and various germ cells, including spermatogonial stem cells, spermatogonia, early-spermatocytes, late-spermatocytes, and spermatids in yak testis. Pseudo-timing analysis showed that Leydig and myoid cells originated from common progenitor cells in yaks. Moreover, functional enrichment analysis demonstrated that the top expressed genes in yak testicular somatic cells were significantly enriched in the cAMP signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, and ECM receptor interactions. Throughout the spermatogenesis process, genes related to spermatogenesis, cell differentiation, DNA binding, and ATP binding were expressed. Using immunohistochemical techniques, we identified candidate marker genes for spermatogonial stem cells and Sertoli cells. Our research provides new insights into yak spermatogenesis and the development of various types of cells in the testis, and presents more reliable marker proteins for in vitro culture and identification of yak spermatogonial stem cells in the later stage.

In order to investigate the composition and differences in volatile organic compounds (VOCs) in yak and cattle-yak meat and determine the key metabolites and metabolic pathways related to flavor formation. In this study, the VOCs and non-volatile metabolites in Longissimus dorsi muscle of two groups of samples were detected and analyzed by gas chromatography–ion migration spectrometry (GC-IMS) and gas chromatography–mass spectrometry (GC-MS). The results showed that 31 VOCs were identified by GC-IMS, including 5 alcohols, 5 ketones, 5 esters, 3 aldehydes, 2 furans, 2 hydrocarbons, 1 amine, 1 acid, 1 thiazole, 1 pyrazine, and 5 others. Most of them were alcohols, ketones, esters, and aldehydes. A total of 75 non-volatile metabolites with significant differences were obtained by GC-MS screening, among which amino acid contents such as serine, glycine, phenylalanine, and aspartic acid were significantly up-regulated in cattle-yak, and glutamic acid and tyrosine were significantly up-regulated in yak. The non-volatile differential metabolites in the two groups were significantly enriched in the metabolic pathways of arginine biosynthesis and oxidative phosphorylation. By combining GC-IMS and GC-MS, this study comprehensively and intuitively reflected the differences in VOCs between yak and cattle-yak meat, and clarified the metabolomic reasons for the differences in VOCs, so as to provide a theoretical basis for meat quality improvement.