Donkeys are an important domesticated animal, providing labor, meat, milk, and medicinal materials for humans. However, the donkey population is continuously declining and even at risk of extinction. The application of modern animal production technology, such as oocyte in vitro maturation, is a promising method to improve the donkey population. In this study, we explore the gene expression patterns of donkey germinal vesicle (GV) and in vitro matured metaphase II (MII) oocytes using single cell RNA-seq of the candidate genes along with the regulatory mechanisms that affect donkey oocyte maturation. We identified a total of 24,164 oocyte genes of which 9073 were significant differentially expressed in the GV and MII oocytes. Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that these genes were associated with the meiotic cell cycle, mitochondrion activity, and N-glycan biosynthesis, which might be the key genes and regulatory mechanisms affecting the maturation of donkey oocytes. Our study provides considerable understanding regarding the maturation of donkey oocytes and serves as a theoretical basis for improving the development of donkey oocytes, which could ultimately benefit the expansion of the donkey population and conservation of biodiversity and genetic resources.

The selection and evaluation of high-quality embryos are the key factors affecting in vitro embryo development and pregnancy outcome. The timing of first embryonic cleavage has been considered a positive indicator of the in vitro developmental potential of embryos, while the underlying molecular mechanism is still not fully understood. In this study, the embryos generated by parthenogenetic activation (PA) or in vitro fertilization (IVF) were monitored and recorded every 2 h and divided into two groups (early cleavage or late cleavage) based on the cleavage rate and blastocyst formation data. RNA sequencing was used to analyze the gene expression pattern of the embryos. We identified 667 and 71 different expression genes (DEGs) in early cleavage and late cleavage porcine PA and IVF embryos, respectively. Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the DEGs are mainly enriched in pathways concerning the proteasome, DNA repair, cell cycle arrest, autophagy, and apoptosis, suggesting that severe endoplasmic reticulum stress (ERS) and DNA damage may be the key factors that led to the low development potential of late cleavage embryos. This study provides a theoretical basis for the following application and offers important information about the understanding of the timely manner of porcine embryo development.