Abstract
Sperm cryopreservation and artificial insemination are important methods for giant panda breeding and preservation of extant genetic diversity. Lower conception rates limit the use of artificial insemination with frozen-thawed giant panda sperm, due to the lack of understanding of the cryodamaging or cryoinjuring mechanisms in cryopreservation. Long non-coding RNAs (lncRNAs) are involved in regulating spermatogenesis. However, their roles during cryopreservation remain largely unexplored. Therefore, this study aimed to identify differentially expressed lncRNAs and mRNAs associated with cryodamage or freeze tolerance in frozen-thawed sperm through high throughput sequencing. A total of 61.05 Gb clean reads and 22,774 lncRNA transcripts were obtained. From the sequencing results, 1477 significantly up-regulated and 1,396 significantly down-regulated lncRNA transcripts from fresh and frozen-thawed sperm of giant panda were identified. GO and KEGG showed that the significantly dysregulated lncRNAs and mRNAs were mainly involved in regulating responses to cold stress and apoptosis, such as the integral component of membrane, calcium transport, and various signaling pathways including PI3K-Akt, p53 and cAMP. Our work is the first systematic profiling of lncRNA and mRNA in fresh and frozen-thawed giant panda sperm, and provides valuableinsights into the potential mechanism of cryodamage in sperm.
Highlights
The giant panda (Ailuropoda melanoleuca) is an endangered species confined to south-central China
We identified a total of 22,774 Long non-coding RNAs (lncRNAs) (Figure 2a), among which 16,110 of them were lincRNAs including 1086 aTnhteiseqnusaelilfniecRdNtrAasn,s4c3r6ip9tisntwroenreic alnncaRlyNzAeds, aunsidn1g20th9eseCnNseClnI,cRCNPCA-.sIcnanadsdoifttiwona,r3e2. ,3W22e pidroentetiinfi-ecdodaintgottaral nosfcr2ip2,t7s7w4 elrnecaRlNsoAidse(nFtiigfiuerde, w2ah)i,chamcoonntgainwsh1i3c,h18166n,1e1w0 goefnethse(mTabwleesrSe1lainncdRSN2A).s including 1086 antisense lncRNAs, 4369 intronic lncRNAs, and 1209 sense lncRNA (Figure 2b)
We acquired a total of 22,774 predicted lncRNAs and 32,322 mRNAs from giant panda sperms
Summary
The giant panda (Ailuropoda melanoleuca) is an endangered species confined to south-central China. Natural mating and artificial insemination are common approaches for breeding of the giant panda in captivity. The artificial insemination of giant pandas mainly employs fresh sperm, while frozen-thawed sperm is rarely used in this procedure. The lower farrowing rate explains the poor utilization efficiency of giant panda frozen-thawed sperm. Only about 25% (3/11) of giant pandas at the ideal breeding age are mating naturally [1]. It has been reported that more than 30% of frozen-thawed sperm can be utilized for artificial insemination with successful conception and birth of giant panda [3]. 4 out of 7 giant pandas were pregnant after artificial insemination performed by Huang and co-authors, yielding a breeding success rate of 57.1% [4]
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