A two-step cooling procedure was employed to cryopreserve Pseudosciaena crocea sperm,and the sperm ultrastructure after which was observed under transmission electron microscopy.The results show that there were no significant differences between frozen-thawed sperm and fresh sperm by comparing the activation rate,moving time and life-span.Both the fresh sperm and cryopreserved sperm had ultrastructural damages in various degree.The deformation rate of the fresh and cryopreservated sperms were 28.5% and 37%,respectively.The following aspects of cryopreserved sperm with normal morphology was observed:the plasma and nuclear membrane;the sleeve,axoneme and centriole;the mitochondrion obtained integrity and with well-developed cristae.On the contrary,the sperm cryodamages were observed as follows:swelled or disrupted plasma and nuclear membrane;partially damaged nucleus;the swelled,dislocated or disarticulated mitochondrion with degenerated or vanished cristae.The results show that Cortland solutions and 10% DMSO are the best choice for extender and cryoprotecant,which are helpful for improving the frozen-thawed P.crocea sperm quality.

以甘油(Gly)为抗冻剂、0.5 mL麦细管为冻存管,两步降温法超低温冷冻保存大黄鱼精子,用单细胞凝胶电泳(SCGE)法检测冻精核的DNA损伤.结果表明:以Cortland液为稀释溶液,5％～20％ Gly为抗冻剂,超低温冷冻保存大黄鱼精子的效果较佳,冻精活力与鲜精活力无显著差异;Gly浓度为10％,冻精的激活率和运动时间分别达89.93％和8.91min;25％、30％Gly组冻精活力显著下降;SCGE检测显示,Gly浓度5％～20％时,冻精核DNA损伤与鲜精无显著差异,Gly浓度25％及30％时,冻精核DNA损伤与鲜精差异显著;鲜精与冻精的DNA损伤主要表现为轻度和中度损伤,重度损伤比率较低,完全损伤只存在于25％Gly及30％Gly组的冻精中,且比例低.

为了解超低温冻存对大黄鱼精子细胞结构损伤的影响,本研究以Cortland溶液为稀释液,二甲基亚砜(DMSO)及乙二醇(EG)为抗冻剂,0.25 mL的麦细管为冻存管,两步降温法(平放在离液氮面3～4 cm处3～5 min后入液氮中保存)超低温冻存大黄鱼(Pseudosciaena crocea)精子,并用单细胞凝胶电泳(SCGE)技术检测了冻精的DNA损伤,荧光双染色-流式细胞术(FCM)检测了冻精的细胞膜性结构损伤.结果表明,DMSO及EG浓度在5％～20％时,冻精的活力与鲜精相比无显著差异；其中DMSO及EG浓度在10％时,冻精的激活率、运动时间、寿命分别为(87.00±2.45)％、(8.99±0.24) min和(13.11±0.65) min及(87.50±2.52)％、(8.45±0.48)min和(12.84±0.50) min; DMSO及EG浓度在25％和30％时,冻精的活力显著下降.SCGE检测显示,DMSO及EG浓度在5％～20％时,冻精的DNA损伤与鲜精相比差异不显著；DMSO及EG浓度为25％和30％时,冻精的DNA损伤明显加重；冻精的DNA损伤与抗冻剂DMSO及EG的浓度成正相关.FCM检测显示,DMSO及EG浓度在5％～20％时,冻精中线粒体及细胞膜结构保持完整性的精子比例与鲜精相比无显著差异；DMSO及EG浓度在25％和30％时,冻精中的线粒体及细胞膜结构保持完整性的精子比例明显下降.分析认为,较高浓度的DMSO及EG是引起冻精活力下降,DNA、线粒体及细胞膜结构损伤加重的主要原因；为确保大黄鱼冻精的质量,应以10％DMSO或10％EG为抗冻剂.

In this study,DMSO was used as cryoprotectants,and HBSS solution was used as extender.Two-step cooling for cryopreservation of Nibea albiflora spermatozoa in 0.25 mL straws.DNA damage,mitochondria and membrane damage of N.albiflora sperm in response to the cryopreservation process by SCGE and FCM were also investigated.The results demonstrated that there were no significant differences between fresh sperm and frozen-thawed sperm which was diluted with 5%-20% DMSO in vitality.The activation rate,moving time and life-span of frozen-thawed sperm were 85.25% ±3.95%,(3.23±0.27) min and(3.83±0.33) min when 10% DMSO was used as cryoprotectant.However,a significant drop in sperm vitality was observed when DMSO concentration was increased to 25% and 30%.The SCGE showed that there were no significant differences between fresh sperm and frozen-thawed sperm which was diluted with 5%-15% DMSO in DNA fragments,but DNA fragments differed significantly with fresh sperm when DMSO concentration rose to 20%,25%,30%.In fact,there was a positive correlation between comet rate of frozen-thawed sperm and concentration of DMSO in protocol.Also,we found that the integrity of mitochondria and membrane of frozen-thawed sperm obtained high proportion when using 5%-20% DMSO as cryoprotectant,and there were no significant differences between fresh sperm and frozen-thawed sperm.But,the mitochondrial and membrane integrity had a significant drop when using 25% and 30% DMSO as cryoprotectants.So we concluded that toxicity of cryoprotectant was the main factor of DNA damage,mitochondrial membrane damage in frozen-thawed sperm.

通过测定精子的激活率、运动时间及寿命,研究了鮸鱼精子的生理特性,以0.5mL麦细管为冻存管、两步降温法超低温冻存鮸鱼精子.结果表明,鮸鱼精子激活与运动的适宜盐度为20～30、适宜pH值为5.5～9.0,适宜的KCl、NaCl、CaCl2溶液浓度分别为(500～600)mmoL/L、(400-500)mmol/L、(300～400)mmol/L,适宜的葡萄糖溶液浓度为(800～900)mmol/L.无Ca2+、Mg2+及HCO-3的人工海水均能使娩鱼精子激活,但运动时间及寿命有所下降.以Cortland溶液为稀释液,10%Gly、15%Gly、5%DMSO、10%DMSO、15%DMSO、10%EG、10%PG、15%PG及20%PG为抗冻剂,超低温冻存鮸鱼精子15d后,冻精的活力与鲜精相比无显著差异,其中,以10%Gly为抗冻剂冻存精子的效果最好,冻精的激活率、运动时间及寿命分别达(86.38±1.63)%、(8.24±1.37)min及(10.21±0.42)min.

The effects of pH value,salinity,ions and glucose solutions on the sperm vitality of Boleophthalmus pectinirostris are investigated through determination of activation ratio,moving duration and life-span of sperm. The results indicate that the activation ratio of sperm exceeds(81.67±7.21)%in the solutions with salinity ranging from 10 to 25.The highest activation ratio,the longest moving duration and life-span of sperm are found to be(94.33±4.04)%,(87.67±8.02)min and(115.33±9.24)min,respectively,in the case that the sperm is activated in the suitable solutions with salinity 20.Also,in the solutions of pH value 6.0~9.0,the activation ratio, the longest moving duration and life-span of sperm are all greater than(80.33±4.51)%,(63±4)min and(80±4.36) min respectively.The vitality reaches its maximum in the solutions of pH 8.The sperm has a greater activation ratio in the solutions with ions concentrations of NaCl,KCl,MgCl2,CaCl2,NaH2PO being 150,300,300,100, 100 mmol·L- 1,respectively.

因名贵经济鱼类育种或种质资源保存的需要,镜子超低温冻存及冻精质量评价的研究已越来越引起研究者的重视.在海产鱼类中,已见对大黄鱼(Pseudosciaenacrocea)[1,2]、黑(鱼周)(Sparus mactinirostris)[3,4]、大弹涂鱼(Boleophthalmus pectinirostris)[4]、中华乌塘(酆)(Bostrichthys sinensis)[5]、花鲈(Lateolabrax japonicus)[6]、大菱鲆(Scophthalmus maximus)[7]、细须石首鱼(Micropogonias undulatus)[8]等种类精子冷冻保存的研究报道.

We investigated the effects of environmental factors and cryopreservation on the vitality of sperm in Nibea albiflora.The results indicated that the suitable salinity for sperm activation and movement was 25-35 while pH value 7.5-8.5.The highest activation rate,the longest moving time and life-span of sperm were(87.00±5.54)%,(336±14.02) s,(405.33±12.22) s,respectively when the sperm was activated in the solutions(salinity25,pH 8.0-8.5).Also the suitable concentration of KCl,NaCl,MgCl_2,glucose solutions for sperm activation was 300-500mmol·L~(-1),600mmol·L~(-1),800-1000 mmol·L~(-1) and 900 mmol·L~(-1),respectively.The sperm could not be activated by HCO_3~_-free artificial seawater and more than 80%of sperms were activated by Ca~(2+)-free,Mg~(2+)- free artificial seawater while the moving time and life-span of sperm was shortened significantly.The activation rate and moving time of cryopreservated sperm exceeded 80%and 200s when Cortland and HBSS solutions were used as extender and 10%EG as cryoprotectant.

With sexually matured and artificial breeding Acrossocheilus fasciatus as parent fish,this paper adopted the way of artificial insemination to obtain fertilized eggs.Fertilized eggs were incubated in the micro-flow water poor and the process of embryonic development was observed.The result shows that the eggs were 1.84~2.14 mm in diameter,sinkable,sticky and golden yellow.The diameter of the fertilized eggs expanded to 2.32~2.54 mm after hydrating.The process of embryonic development in Acrossocheilus fasciatus underwent the following six stages: blastoderm formation,cleavage,blastula,gastrula,neurula,and organ differentiation.When water temperature reaches(25±1) ℃,42 h 53 min were needed to hatch the embryos.The newly hatched larvae were 6.5±0.2 mm in length.The fore part of yolk sac expanded into a spherical shape and the hind part looked like a bar.In the process of embryonic development,their optic vesicle and olfactory placode,caudal fin and forrmation of lens appeared almost simultaneously.

The effects of pH value,salinity,glucose and ions solutions on the sperm vitality of Csiaenops ocellatus were investigated by measuring the activation ration,moving time and life-span of sperm.The results indicated that the average density of sperm was (1.203±0.22)×10~(10)·ml~(-1) and the suitable salinity for sperm activation and movement was 20-35,while pH value 6.0～8.5.The activation rate,moving time and life-ban of sperm reached to 94.32%,9.14 min and 12.55 min respectively when the sperm was activated in salinity 25.The proper concentration of KCl,NaCl,CaCl_2 solutions for sperm activation and movement was 600～700 mmol·L-1,600 mmol·L~(-1) and 400～600 mmol·L~(-1) respectively,while the proper concentration of glucose solution was 700～900mmol·L~(-1).No significant differences were found in the activation rate when the sperm was activated by the artificial seawater lacking of HCO_3~-,Mg~(2+) or Ca~(2+),but the moving time and survival time of sperm became shorter.

In this paper,the effect of several factors on the vitality of Acrossocheilus fasciatus sperm were investigated through determination of activation rate,moving time and life-span.The results indicated that the suitable pH for sperm activation and movement was 6.5-8.0.When the sperm was in the solutins of pH values 7.0,the activation rate and moving time were(93.33±2.89)%,(30.33±1.53) s,respectedly.The activation rate,the moving time and life-span of sperm were(93.67±2.89)%,(36.00±1.00) s,(44.67±1.53) s,respectively when the sperm was activated in 0.4%g·L-1 NaCl solutions.Mealwhile in 0.7%g·L-1KCl solutions,the activation rate,the longest moving time and life-span of sperm were(93.33 ±2.89)%,(55.33 ±1.53) s,(65.33 ±2.31) s,respectively.But the sperm could not be activated in the solutions that the concentrations of CaCl2 and glucose ranged from 0.1% to 1.0% g·L-1.When the sperm was laid at room temperature 28-30 ℃ and low temperature4 ℃,a significant drop appeared after 3 h atroom temperature.But 5 hours later,no significant differences were found when the sperm was laid at low temperature.In addition,we found that CF-HBSS and 1.0% g·L-1 NaCl could be used for sperm cryopreservation through the filtering of several extenders.

通过测定黄姑鱼精子的激活率、运动时间及寿命研究了不同环境因子对黄姑鱼精子活力的影响。结果表明，黄姑鱼精液中精子浓度为（1.41±0.12）×1010个/ ml；精子激活与运动的适宜盐度为25~35、适宜pH为7.5~8.5。在pH 8.0~8.3、盐度25条件下，精子激活率达（87.00±5.54）%，运动时间及寿命分别为（336±14.02）s及（405.33±12.22）s。精子激活与运动的适宜NaCl、KCl、MgCl2及葡萄糖溶液浓度分别为300~500mmol•L-1、600mmol•L-1、800~1000 mmol•L-1及900 mmol•L-1；精子在CaCl2溶液中有聚集现象出现，且运动时间及寿命明显缩短；精子在缺少HCO3-的人工海水中未能被激活；精子在无Ca2+或Mg2+的人工海水中激活率均超过80%，但运动时间及寿命均有所缩短。