Development of a non-programmable sperm cryopreservation technique in pearl oyster Pinctada fucata martensii and investigation of transcriptomic profile of post-thaw sperm by RNA-seq

The deterioration of germplasm quality and genetic diversity has been considered as the key reason threatening the sustainable development of pearl oyster ( Pinctada fucata martensii ) industry in China. This study has developed a non-programmable sperm cryopreservation technique to provide a reliable strategy for its germplasm preservation to address this issue and investigated the changes in transcriptomic profiles of post-thaw sperm by RNA-seq. The factors important to the development of this cryopreservation technique were assessed, including cryoprotectant agent [dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, glycine, glucose and polyvinylpyrrolidone (PVP)], rack height, thawing temperature and sperm to egg ratio at fertilization. The post-thaw sperm fertilization rate of 40.67 ± 4.04 % was produced when sperm were cryopreserved with 6 % DMSO at 6 cm rack height, thawed at 40 °C temperature and fertilized at a sperm to egg ratio of 10000:1. This rate was further improved to approximately 50 % by adding 0.1 % PVP into 6 % DMSO with 50 % lower sperm to egg ratio. The addition of PVP also improved acrosome integrity, motility and activities of catalase and superoxide dismutase of post-thaw sperm. The transcriptomic profiles showed 207 differentially expressed transcripts between fresh and cryopreserved sperm. The enrichment network analysis of the top 10 most significantly altered Gene Ontology terms from biological process, cellular components and molecular function revealed that the increased abundance transcripts were mainly related to the cellular movement, chromosome segregation and cell division, while the decreased abundance transcripts were mainly related to the immunity. In conclusion, a non-programmable sperm cryopreservation technique has been established to preserve the germplasm in farmed P. fucata martensii in China. The differential transcriptomic profiles between fresh and post-thaw sperm could provide useful information to further improve sperm cryopreservation technique which could also benefit the breeding program and germplasm preservation in both pearl oyster and other related bivalve aquaculture industry.