Abstract: 【Objective】The objectives of this study were to investigate the transcriptome differences and identify function, classification and metabolic pathways of the differentially expressed genes (DEG) at different developmental stages of yak embryos derived from in vitro fertilization (IVF), which are necessary to better understand the mechanism that regulates embryonic development and provide theoretical basis for improving in vitro embryo production in the yak (Bos grunniens). 【Method】Total RNA was extracted from IVF derived yak embryos at 2-cell, 4-cell, 8-cell, morula and blastocyst stages and amplified via the Smart-seq2 method, and the constructed RNA libraries were sequenced using the HiSeq^TM2500 high-throughput sequencing method. 【Result】After IVF, the average cleavage rates and blastocyst rates were 69.3% and 26.2%, respectively. A total of 47 355 570 to 50 855 888 clean reads were obtained from 2-cell, 4-cell, 8-cell, morula and blastocyst stages, respectively, of which, 85.65% to 90.02% were covered in the yak reference genome. In total, the number of transcripts mapped to yak genome was highest for 8-cell (14 893) and lowest for blastocysts (9 827). The transcripts mainly had five patterns of alternative splice, of which, the two largest proportions were transcription start site (TSS) and transcription terminal site (TTS). The SNP numbers of the five stages of yak embryonic transcripts were 116 601, 234 131, 196 420, 70 841 and 94 840, respectively. A total of 1 221, 1 116, 142 and 564 transcripts were first detected at the 4-cell, 8-cell, morula and blastocyst stages, respectively. As embryo development proceeded, maternally derived transcripts such as BMP15, KIT, GDF9, STAT3, ZP3 and ZP4 etc.were decreased, whereas embryonic transcripts such as SARS,IL18, ACO2, TXN2, ATP5B, PCGF4, UBE3A, MAPK13, SNURF and JUP etc. were increased at specific stages. When |log₂ratio| ≥1 and Q-value＜0.05 were set as thresholds for identifying DEGs, a total of 6 922, 7 601, 8 071 and 10 555 DEGs were identified from 2-cell vs. 4 cell, 4-cell vs. 8-cell, 8-cell vs. morula, and morula vs. blastocyst, respectively. The GO distributions of the DEGs were classified into three categories: biological processes (BP), cellular components (CC), molecular functions (MF) with a total of 62 subcategories of two successive stages. KEGG enrichment analysis of DEGs showed that DEGs of 2-cell vs. 4-cell participated in 308 pathways, and significantly enriched in 11 pathways such as spliceosome, RNA transport and ubiquitin mediated proteolysis etc. DEGs of 4-cell vs. 8-cell participated in 310 pathways, and significantly enriched in 9 pathways such as olfactory transduction, neuroactive ligand-receptor interaction and nucleotide excision repair etc. DEGs of 8-cell vs. morula participated in 316 pathways, and significantly enriched in 10 pathways such as olfactory transduction, ubiquitin mediated proteolysis and neuroactive ligand-receptor interaction etc. DEGs of morula vs. blastocyst participated in 315 pathways, significantly enriched in 2 pathways i.e., spliceosome and RNA transport.【Conclusion】This is the first study for analyzing the transcriptomes of IVF derived yak-embryos at different stages using high-throughput sequencing. A number of DEGs and their function, classification and metabolic pathways were discovered, which enriched transcriptome information for yak embryos. In addition, the results provided a foundation and reference to uncover the mechanism that regulates embryonic development and improves in vitro embryo production of the yak species. 

Key words: yak, in vitro fertilization, embryo, transcriptome, RNA-Seq