| The sea cucumber Apostichopus japonicus belongs to the phylum Echinodermata and is naturally distributed in the northern West Pacific Ocean.It is one of the important marine aquaculture species in China with high economic and nutritional value.However,in recent years,due to artificial factors such as overfishing and habitat destruction,as well as natural factors such as extreme environments,the wild population of sea cucumbers in natural sea areas in China has sharply decreased,leading to the depletion of natural resources.The wild sea cucumber has been evaluated as an endangered(EN)species on the IUCN Red List.The breeding of sea cucumbers has also encountered problems such as serious degradation of breeding resources,poor stress resistance,frequent disease outbreaks,low survival rate,long breeding cycle,and slow growth rate,which have resulted in a decline in the quality of commercial sea cucumbers and caused serious economic losses.Therefore,the protection and preservation of A.japonicus genetic resources is an urgent issue.The development of cryopreservation protocols is important for protecting germplasm resources and solving the problem of germplasm degradation.Currently,there is limited research on the cryopreservation of A.japonicus sperm,and no unified and comprehensive cryopreservation technology has been established,let alone the commercial and industrial application of A.japonicus cryopreserved sperm.In this study,based on precision instruments such as a programmable freezer and a computer-assisted sperm analysis system,key parameters during the cryopreservation process were systematically and comprehensively screened.We established a cryopreservation technology suitable for A.japonicus sperm,and a combination of low-temperature short-term storage and cryopreservation technology with wider application scope for sea cucumber sperm.The effects of the cryopreservation process on sea cucumber sperm were elucidated from multiple aspects,including morphology,physiology,metabolic function,and transcriptomics.We also established a regression equation for predicting the freezing resistance of A.japonicus sperm based on sea cucumber trait parameters.The main research results are as follows:1.Establishment of cryopreservation technology for A.japonicus spermIn this study,key parameters that may affect the quality of sea cucumber sperm after cryopreservation,such as the type of diluent,type and concentration of cryoprotectant,cooling rate,and thawing rate,were comprehensively screened to establish cryopreservation technology for A.japonicus sperm.The steps for cryopreservation of A.japonicus sperm were summarized.The technology can be summarized as follows:(1)Fresh A.japonicus semen was collected using an anatomical filtration method and stored at 4°C for backup.A small amount of fresh semen was taken for activation with an activator,and the sea cucumber fresh semen with a sperm motility rate of 85%or higher was used for cryopreservation of sperm.(2)Filter-sterilized natural seawater(NSW)was used as a diluent,dimethyl sulfoxide(DMSO)was used as a penetrating cryoprotectant,and glucose(Glu)was used as a non-penetrating cryoprotectant.A freezing protection solution containing 12.5%DMSO and0.1 mol/L Glu was prepared and stored at 4°C for backup.(3)The fresh A.japonicus semen and the freezing protection solution were mixed at a dilution ratio of 1:5,and immediately placed in a programmable cooling instrument for programmed cooling.(4)The cooling program was set to equilibrate at 0°C for 5 min,then cooled to-80℃at a cooling rate of 10℃/min,equilibrated at-80℃for 5 min,and finally cooled to-180℃at a cooling rate of 20℃/min.After equilibrating for 5 min,the sample was removed and stored in liquid nitrogen(-196℃)for long-term storage.(5)When testing the frozen sperm motility,the cryopreserved tube was removed from the liquid nitrogen and thawed in a 20℃water bath,gently shaken to ensure uniform temperature,and removed immediately when only a small amount of solid remained in the tube(approximately 1 min).The sample was further shaken in the air until completely melted.(6)An appropriate amount of freshly thawed frozen semen was taken,activated with an activator,and the sperm motility was detected using a CASA system.2.Optimization of the cryopreservation technique for A.japonicus sperm in production applicationsIn this study,through screening experiments using different cryopreservation devices and volumes,it was found that these factors had no significant impact on the viability of sea cucumber cryopreserved sperm.By exploring the effects of different cryoprotectant formulations on the fertilization and hatching rates of sea cucumber sperm,it was found that the established optimal cryopreservation technique for A.japonicus sperm can obtain frozen sperm with a post-thaw motility of more than 65%,a fertilization rate(in the blastocyst stage)of nearly 80%,and a hatching rate(in the early auricularia larva stage)of more than 65%,which has certain value for production applications.Through screening experiments,a low-temperature preservation technique for A.japonicus sperm was established for the first time,and a combined technique of short-term low-temperature preservation and long-term cryopreservation was also established.This technique can maintain the motility of A.japonicus sperm at more than 70%within 4 days and more than 50%within 10 days.Additionally,the cryopreservation of A.japonicus sperm for 4 days can obtain cryopreserved sperm with post-thaw motility of more than 35%.3.The impact of cryopreservation process on A.japonicus spermIn this study,the effects of cryopreservation process on A.japonicus sperm were examined from multiple perspectives,including morphology,physiology,metabolic function,and transcriptomics.The research found that the process of cryopreservation could significantly reduce the physiological activity indicators of A.japonicus sperm,such as sperm motility,semen density,and sperm movement speed,and the reduction of physiological activity characteristics of different individuals was different.The process of cryopreservation could also cause the ultrastructure of A.japonicus sperm cells to undergo disruption in the middle and tail of the head,as well as damage to the nucleus and irregular shape,increasing the abnormality rate of sperm in semen.Moreover,the process could significantly increase the activity of all enzymes(including total ATPase,succinate dehydrogenase(SDH),lactate dehydrogenase(LDH),superoxide dismutase(SOD),catalase(CAT))detected in the cryopreserved semen seminal plasma of A.japonicus,and increase the activity of SOD,SDH,the total ATPase in the cryopreserved semen sperm of A.japonicus,and changing the metabolic process of sperm.In addition,The cryopreservation process resulted in significant downregulation of 452 genes and upregulation of 162 genes.The differentially expressed genes were mainly enriched in related pathways such as environmental stress,metabolic inhibition,and damage repair,including the"Hippo signaling pathway","retrograde endogenous cannabinoid signaling pathway","repair of double strand breaks through homologous recombination",and"repair of double strand breaks through non homologous end connections",This indicates that the cryopreservation process causes a certain degree of damage to the structure and DNA integrity of sperm cells,thereby affecting their cell growth and fertilization ability,and inducing sperm to actively initiate repair mechanisms...4.Study on the correlation between the morphological characteristics of A.japonicus and the cryopreservation resistance of its spermatozoaIn this study,in order to predict the cryopreservation resistance of A.japonicus spermatozoa based on their morphological characteristics,a correlation analysis was conducted between multiple morphological indicators and the sperm vitality of cryopreserved A.japonicus spermatozoa.The results showed that there was no significant correlation between the superficial morphological indicators of A.japonicus,such as total weight,body wall weight,and gonad weight,and the cryopreservation resistance of their spermatozoa.However,there were significant positive correlations between the cryopreservation resistance of A.japonicus spermatozoa and the relative content of arachidonic acid in the fresh seminal plasma,and there were significant negative correlation between the cryopreservation resistance of A.japonicus spermatozoa and the enzymatic activities of LDH and SOD in the seminal plasma and spermatozoa.Regression equations were established for the enzymatic activities of LDH and SOD in the seminal plasma and spermatozoa,respectively,and the cryopreserved sperm vitality of A.japonicus,which was calculated as cryopreserved sperm vitality(%)=110.093+(-0.367)*LDH enzymatic activity in seminal plasma+(-0.109)*SOD enzymatic activity in seminal plasma,with an R~2 value of 0.724. 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