Sperm Swimming Movement Characteristics And Cryopreservation For Pinctada Fucata Martensii And Chlamys Nobilis

The germplasm resources of marine bivalve have degenerated continually in the last decades,due to the environment destruction and artificial selection.It is urgent to rejuvenate and preserve the germplasm of bivalve species.And the germplasm bank construction for marine organism is especially meaningful for China.The present study has examined the characteristics of sperm swimming movement in the post-activation,major factor trigging sperm activation for the pearl oyster Pinctada fucata martensii,short-term sperm preserving at lower temperature and sperm cryopreservation for the P.fucata martensii and noble scallop Chlamys nobilis.The present study aims to offer evidence and technical support to genetic breeding,germplasm conservation and optimization of artificial breeding technology for P.fucata martensii and C.nobilis,promoting biological germplasm bank establishment and marine biological resources utilization in China.1.Sperm from P.fucata martensii was a sprint racer,while C.nobilis was a marathon racer.Sperm form P.fucata martensii can not be activated by fresh seawater,but by seawater containing ammonia(NH3-SW).After activated by 2 mM NH3-SW,P.fucata martensii sperm in FA(full activation)lasts for 5.5 min,with 89%total motility(TM),60%rapid sperm(RAP),141 ?m/s curvilinear velocity(VCL),115 ?m/s straight-line velocity(VSL),127?m/s average path velocity(VAP),2.0 ?m head displacement(ALH)and 12 Hz beat-cross frequency(BCF).C.nobilis sperm were activated by fresh seawater,and sperm in FA lasts for 22 min,with 84%TM,58%RAP,168 ?m/s VCL,129 ?m/s VSL,148?m/s VAP,1.5 ?m ALH and 12 Hz BCF.2.Ammonia ion,rather than pH or salinity,in seawater was the major factor in triggering P.fucata martensii sperm motility.Seawater containing 2 mM or 3 mM NH3 was suggested for triggering P.fucata martensii sperm motility.After activated,sperm moved into FA in in 30 seconds,with 90%TM,143 ?m/s VCL,110 ?m/s VSL,130 ?m/s VAP,and 10 Hz BCF.3.Ca-free Hanks' balanced salt solution(Ca-free HBSS)is more suitable for short-term sperm preserving,used as basic diluent extender and storage medium,for the ability of sperm motility inhibiton.The P.fucata martensii sperm motility guarantee period under4? was within 24 h,with 70%TM,126 ?m/s VCL,106 ?m/s VSL,125 ?m/s VAP,1.76 ?m ALH and 9 Hz BCF after activated by 2 mM NH3-SW.The C.nobilis sperm C.nobilis sperm was within 8 h,with 83%TM,150 ?m/s VCL,123 ?m/s VSL,134 ?m/s VAP,1.74 ?m ALH and 11 Hz BCF after activated by fresh seawater.The motility of refrigerated sperm within the motility guarantee period can be used as fresh sperm.4.A programmable freezing method was developed for P.fucata martensii sperm cryopreservation.The procedure was the following:sperm with initial TM>65%after 2 mM NH3-SW activated were selected for further cryopreservation.Ca-free HBSS was used as the extender,and 10%-15%propylene glycol(PG)or 15%ethylene glycol(EG)were used as cryoprotectant agents,with three minutes equilibration on crushed ice.Then sperm were frozen at a rate of 10?/min from 0? to-50?,and maintained at-50?for 1 min before being plunged into liquid nitrogen(LN).By this programmable freezing method,the TM of P.fucata martensii post-thaw sperm was higher than 65%;the VCL,VSL,VAP were higher than 50 ?m/s,20 ?m/s,40 ?m/s respectively;the ALH was higher than 1.11 ?m;the BCF was higher than 4.38 Hz.Furthermore,fertilization rate was higher than 35%.5.A basic programmable freezing method for C.nobilis sperm cryopreservation was developed.The procedure was the following:sperm with initial TM>70%after fresh seawater activated were selected for further cryopreservation.Ca-free HBSS was used as the extender,and 5%-10%dimethyl sulfoxide(DMSO)were used as cryoprotectant agents,with two minutes equilibration on crushed ice.Then sperm were frozen at a rate of 10?/min from 0? to-80? and maintained at-80? for 2 min before being plunged into liquid nitrogen(LN).By this programmable freezing method,the TM of C.nobilis post-thaw sperm was higher than 30%;the VCL,VSL,VAP were higher than 90?m/s,16 ?m/s,18 ?m/s respectively;the ALH was higher than 1.11 ?m.Furthermore,fertilization rate was higher than 26%.