Comparative Analysis Of Transcriptomes Between The Fresh And Vitrified-warmed Crossbred Embryos Of The Yak

The crosses derived from femal yaks(Bos grunniens)fertilized with dairy bulls(Bos taurus)are termed Pian Niu.Their milk yield is much higher than the purebred yak.The development of embryo transfer is still in the early stage.Embryo vitrification is an important procedure of embryo transfer,but the effects of vitrification on gene expression of Pian Niu embryo has not been fully understood.Therefore,we used in vitro fertilization(IVF)to produce Pian Niu(yak oocyte × cattle spermatozoa)in this study.Total RNA were extracted from fresh blastocysts and frozen-thawed blastocysts of Pian Niu.The global mRNA amplifications wereperformed using the Smart-seq2 method,and then RNA library was constructed and sequenced.Transcriptome data annotation and bioinformatics analysis were carried out to explore the difference of frozen-thawed and fresh blastocystsblastocysts of Pian Niu.The results were shown as the followings.(1)In vitro production of Pian Niu embryos.The oocytes were treated as three groups: Group A with no residual granulosa cell(GC)on oocytes before fertilization,Group B with residue two or three layers GC,and Group C reserved all GCs on oocytes.The results showed that the blastocyst rates were 13.88%,24.97% and 23.26% for Group A,B and C,respectively.Group A significantly differed with Group B and C(P<0.05),but there was no difference between Group Band C.Both the cleavage and blastocyst rateswere lower whenyak oocytes were co-incubated with Jersey sperm for 12 h than for 18 h and 24 h(P<0.05).(2)Comparativeanalysis of transcriptomes between the fresh blastocysts and frozen-thawed blastocysts.We obtained 51,099,116 and 54,192,358 clean reads from fresh blastocysts and frozen-thawed blastocysts of Pian Niu by RNA-seq,respectively.All results showed that the two libraries were successfully constructed.A total of 39,435,615 and 38,621,872 reads were mapped to referenced genome for fresh blastocysts and frozen-thawed blastocysts of Pian Niu,respectively.A total of 11,196 differentially expressed genes(DEGs)meeting the criteria of q<0.05 and |log2ratio|≥1,in which 7,570 were upregulated,and 3,626 were downregulated in the fresh blastocysts vs.frozen-thawed blastocysts.GO enrichment analysis of DEGs showed that they were enriched molecular function,cellular component,and cellular process.The KEGG enrichment analysis showed that there were 318 pathways from frozen-thawed blastocysts and fresh blastocysts,of which 14 pathways were significantly enriched.Fresh blastocysts and frozen-thawed blastocysts of Pian Niu had 49,016 and 64,352 alternative splicing,respectively.The two largest proportion forms were TSS and TTS.The number of SNP sites selected from fresh blastocysts and frozen-thawed blastocysts were116,681 and 224,750,respectively.This is,as far as we known,the first report to explore the difference of fresh blastocysts and frozen-thawed blastocysts of Pian Niuusing high-throughput sequencing,which might serve as a key resource for for improving embryo vitrification technology.Our study also providedvaluable information to study gene structure of Pian Niu,and discover new genes related to vitrification damage of embryos.