Embryo Cryopreservation And Transcriptome Analysis Of Surviving Larvae In Kelp Grouper (Epinephelus Moara)

Kelp grouper(Epinephelus moara)is one of the most economically important aquaculture species in Southeast Asia,and is mainly distributed in the western North Pacific and coastal areas of China.Mariculture of E.moara has increased in recent years due to high demand and increased market price.Overfishing,artificial release of fishery resources,and the degradation of wild germplasm resources have led to the degradation of many wild fish species,reducing their genetic diversity across the world.Cryopreservation of fish embryos is important method to conserve germplasm and genetic diversity to improve fish stocks for aquaculture.However,owing to their large size,syncytial layer,large perivitelline space,and sensitivity to chilling,successful cryopreservation at-196 °C has been more difficult than mammalian embryos.At present,only a few species have yielded surviving embryos after cryopreservation.Therefore,it is important to strengthen basic research on embryo cryopreservation of fish.Our results offer important insights into further improving cryopreservation and the mechanisms by which cryopreservation damages fish embryos.There was four chapters of experimental process and results this dissertation(1)A preliminary study on the effect of vitrification solution on kelp grouper(Epinephelus moara)embryosThis chapter was a pre-experiment for the embryo cryopreservation experiment in the following section.In this part,the effects of vitrification solution PMG3 S and PMG3 T on the survival of embryo and their capacity of cryoprotection were studied.It was concluded that kelp grouper embryos had shown high enough tolerance that could be cryopreserved after vitrification.(2)Optimization of vitrification factors for embryo cryopreservation of kelp grouper(Epinephelus moara)In this part,we optimized vitrification factors to improve the survival and hatching rate of kelp grouper(Epinephelus moara)embryo after cryopreservation.Included in the experiment were further investigating and screening the equilibrium time(25-45min)of embryos in vitrification solution and the influence for embryonic volume,investigating the effects of 11 vitrification solution concentrations(25-50%)on the survival rate of embryos at 4 stages(16S,18 S,22S,TB),the tolerance of embryos at 5 stages(4-6S,16 S,22S,TB,HB)in two effective vitrification solutions(35% PMG3 S and 35% PMG3T),and the pre-chilling temperature(-5°C and 4°C)and time before freezing,cryopreserving 9855 embryos at 10 various stages(from optic capsule stage to pre-hatch stage).The results of the experiment indicate that for kelp grouper,the equilibration time of 30 min had the best protective and dehydration effect on embryo.Tail-bud stage exhibited greater tolerance to vitrification than embryos at other stages.PMG3 S was more suitable for preserving several embryonic development stages than PMG3 T.Pre-chilling treatment can improve the embryonic viability before and after freezing.After optimizing,the average survival,normal development and malformation rates of cryopreserved embryos were 6.32%,2.36% and 3.49%,and the average hatching rate of surviving embryos after cryopreservation was 39.85%.The hatched larvae gradually died at 12 th day after cultivation and the longest lived for 16 days.The research has recorded valuable data for further improving survival and hatching rate of grouper cryopreserved embryo and provided references for further exploring techniques in fish embryo cryopreservation.(3)Cryopreservation of kelp grouper(Epinephelus moara)embryos using nonpermeating cryoprotectantsIn this part,we present a new approach to cryopreserve kelp grouper embryos using non-permeating cryoprotectants(NPC).Various cryopreservation factors(embryo container,cryoprotectant solution,embryonic stage,equilibrium time,solution concentration,and thawing temperature and time)were screened in preliminary experiments.We found that using 1M sucrose to equilibrate for 5,6,and 7 mins and using cryotube II as the container for embryos was most effective for the kelp grouper embryos at the 16 S and TB stages.Thawing was most effective at 24 °C for 30 s.In six of the experiments,at least four groups of embryos survived freezing and thawing.In total,1195 viable embryos were recovered from those four experiments.The survival rate was 12.60%-31.3%,the hatching rate was 5.08%-41.28%,and maximum alive time of the thawed embryos ranged from 70 hours to 9 days.These results are encouraging for future experiments on cryopreservation of kelp grouper embryos using NPCs,and our study provides new cryopreservation conditions for grouper embryos.(4)Transcriptome comparative analysis of larvae hatched from fresh and cryopreserved kelp grouper(Epinephelus moara)embryosCryopreservation of marine fish embryos causes to severe cryogenic damage,and to date,adults have not been reared from embryos that were cryopreserved.In this part,we gave a series of reasonable explanation for these negative phenomena,through comparing the transcriptome of larvae hatched from fresh and cryopreserved embryo of kelp grouper,based on the enrichment analysis of go and KEGG.The results show that dehydration / rehydration or freezing/thawing during cryopreservation may have affected development of the central nervous system including the primordia of the eyes,leading to the expression of multiple genes that directly or indirectly mediate the light response process and genes encoding photoreceptor proteins.There was a decrease in the amount,in which a decrease in the expression of the mtco1 gene would indirectly cause optic neuropathy and visual impairment in mice.The inactivation of cnga3 gene directly leads to the selective loss of cone-mediated photoresponse,which eventually leads to the almost complete absence of cone cells,resulting in abnormal development of lens in some larvae.The expression levels of several genes(glrb,gabrd,cacng3,gabra6)related to the central nervous system's major inhibitory effects have significantly decreased,which will cause sudden abnormal discharges of brain neurons and have alter gene expression in the neuroectoderm.This results in abnormal gene expression in the Wnt signal pathway,Notch signal pathway,and dorsal and ventral axis formation pathways of fry,which leads to the mismigration of the prechordal plate mesoderm,causing the subsequent deformity of the trunk of the larvae.