Differentially Expressed MicroRNAs Between Fresh And Cryopreserved Spermatozoa In Giant Pansa(Ailuropoda Melanoleuca)

Natural mating and artificial insemination are usually used in captive breeding of giant panda.However,the artificial insemination of giant pandas often uses fresh semen,and artificial insemination with frozen semen is rare.The lower farrowing rate associated with freeze-thawing semen leads to the poor utilization efficiency of giant panda frozen semen.The reason is a lack of understanding of the mechanisms that cause cryodamage and cryoinjury to giant panda sperm during cryopreservation.Sperm cryopreservation plays an important role in the conservation and artificial breeding of giant panda genetic resources.So far,the cryoinjury mechanism of giant panda sperm has not yet been reported.Previous studies have shown that there are a large number of RNA molecules in sperm,in which miRNA plays an important regulatory role in spermatogenesis and maturation,and miRNA in sperm can affect the development of early embryos and offspring traits.Therefore,high-throughput sequencing technology was used to reveal and analysis the differential expression of miRNAs between giant panda fresh and cryopreserved sperm and their functions in signaling pathway.The results are as follows:（1）16,980,071 raw reads were obtained from the small RNA Library of giant panda fresh sperm,and 19,571,331 raw reads were obtained from cryopreserved sperm.Among them,the proportion of clean reads in fresh sperm was 80.32%,and cryopreserved sperm accounted for 73.94%.（2）A total of 899 miRNAs in giant panda fresh and cryopreserved sperm were detected.284 miRNAs were significantly differential expressed between giant panda fresh and cryopreserved sperm,195 miRNAs of which were up-regulated and 89 miRNAs were down regulated.（3）The results of KEGG with target genes of all differentially expressed miRNAs showed that 328 genes enriched in the olfactory pathway（Olfactory transduction）.The enriched genes in Neuroactive ligand-receptor interaction,Protein processing in endoplasmic reticulum and Axon guidance were the 87,69 and 69,respectively.（4）In the p53 and PI3K-Akt signaling pathways associated with sperm apoptosis,there are 15 mRNAs in the PI3K-Akt pathway,7 of them are novel genes.Among them,ANGPT4,SGK2,Panda_newGene₁97067 and Panda_newGene₅45068 were significantly expressed between giant panda fresh and cryopreserved sperm.However,only SESN2 was expressed in P53 pathway.