| The King Grouper (Epinephelus lanceolatus) is a kind of marine food fish withgood properties and high commercial value in southeast coastal area of China.However, massive breeding production has been seriously restricted by itsunsynchronized gamete release between female and male broodstock. With thedevelopment of preservation technipues of gametes in recent years,the problem can bewell resolved through sperm storage at4℃and cryopreservation. In this paper, thesuitable conditions of the King Grouper sperm storage at4℃and cryopreservation,plasma membrane integrity before and after preservation checked by eosin-Y stainingand the activities of total adenosine-triphosphate(ATP) enzyme, lactate dehydrogenase(LDH), superoxide dismutase(SOD), glutathione peroxidase(GSH-PX) in fresh andpreserved King Grouper sperm were studied for establishing the methods of the KingGrouper sperm storage at4℃and cryopreservation, revealing effects ofcryopreservation on sperm structures and functions and clarifying the mechanism ofsprem cryo-damage.1.Results of experiments on the King Grouper sperm storage at4℃:When sperm was separately stored in modified Mounib’s medium(MMM),Hanks’ balanced salt solution(HBSS) and150mM NaCl. The activating rate, rapidmoving time and life span reduced with prolongation of storage period. Among thethree extenders,the activating rate, rapid moving time and life span of sperm in MMMwere highest. And the activating rate was over60%after storage for96hours.With the increasing of the sperm dilution ratios from1:1to1:100,the activatingrate,rapid moving time and life span of the King Grouper sperm rose first and thendropped after storage at4℃for24hours. And the highest activating rate was found inthe sperm dilution ratio of1:50, the longest rapid moving time and life span werefound in the dilution ratio of1:20.With the increasing of preservation volumes from200μl to1400μl in a2mlcentrifuge tube, the activating rate,rapid moving time and life span of the KingGrouper sperm rose first and then dropped after storage at4℃for24hours.Thehighest activating rate(89.1%) and the longest rapid moving time(20.2min) and life span(22.3min) were found in the preservation volume of800μl.2.Results of experiments on cryopreservation of the King Grouper sperm:Activating rate, viability,rapid moving time,life span and enzymes activities ofthe King Grouper sperm diluted by MMM were all higher than those diluted by HBSSand150mM NaCl after cryopreservation for7days. While the highest activatingrate,viability,rapid moving time, life span and enzymes(total ATP enzyme, LDH,GSH-PX, SOD) activities were obtained when15%DMSO was used ascryoprotectant,and the values were respectively63.3%,69.91%,15.9min,17.4min,2.0771umolPi/108sperm/hour,8660.14U/L,551.84U/L,277.45U/ml.With the increasing of croprotectant DMSO concentration from5%to20%, theactivating rate, viability,rapid moving time, life span and enzymes(total ATP enzyme,LDH, GSH-PX, SOD) activities of frozen-thawed sperm rose first and then droppedafter cryopreservation for7days. And the highest activating rate, viability, rapidmoving time, life span and enzymes(total ATP enzyme,LDH, GSH-PX,SOD)activities were found in the DMSO concentration of15%.After cryopreservation for7days,activating rate, viability, rapid moving time andlife span of frozen-thawed sperm increased as the cryoprotectant trehaloseconcentration increased from5%to20%, and the highest vualues54.52%,56.97%,8.5min and17.4min, respectively, were found in the concentration of20%.The King Grouper sperm diluted in MMM and15%DMSO were placed atdifferent distances above the liquid nitrogen surface for cryopreservation, and as thedistance increased from3cm to12cm,activating rate,viability, rapid moving time, lifespan of frozen-thawed sperm rose first and then dropped, the highest values(respectively average63.91%,71.72%,16.8min and18.5min) were observed at adistance of6cm, while no significant difference was found in the activity of fourenzymes (total ATP enzyme, LDH, GSH-PX, SOD).The increasing of dilution time from10to100reduced the activating rate, rapidmoving time,life span of frozen-thawed sperm, while there was no significantdifference among dilution times of2,10,20.The increasing of equilibration period from5to60min before freezing loweredthe activating rate,rapid moving time and life span of frozen-thawed sperm, and thehighest activating rate, rapid moving time and life span was obtained at theequilibration period of5min, respectively average64.98%,14.1min and15.13min,which was significantly higher than those without equilibration. While increasing of the period of sperm staying at6cm distance above the liquid nitrogen surface from5to60min did not modify activating rate, rapid moving time and life span offrozen-thawed sperm.Increasing of the thawing temperature from35℃to41℃did not effect theactivating rate, rapid moving time and life span of frozen-thawed sperm, while lowervalues were observed when thawing at43℃.In conclusion, results in this paper indicated that different extenders and spermpreservation volumes have an effect on the King Grouper sperm storage at4℃, anddifferent extenders, cryoprotectants, cryoprotectant concentrations, cooling modes,dilution times and equilibration time have an effect on sperm cryopreservation. |
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