超低温冻存时间对史氏鲟精子活力的影响

为研究超低温冻存时间对史氏鲟精子活力的影响,使用史氏鲟冻精与鳇鱼卵子进行了杂交授精试验。结果显示:经超低温冷冻后的精液,与对照组的鲜精(126.7±9.4 s)比较,其寿命显著下降(冻存5 h的为78.7±4.11 s,336 d的为72.0±4.32 s,702 d的为57.3±2.05 s),且授精率和受精卵的孵化率受到显著影响。冻存5 h的精液,其授精率和受精卵孵化率分别为(73.31±15.27)%和(55.68±9.81)%,与鲜精比较差异不显著(P>0.05),而冻存时间为336 d[授精率(59.20±2.39)%,受精卵孵化率(53.08±1.23)%]和702 d[授精率(25.29±8.26)%,受精卵孵化率(21.38±9.91)%]的精液则差异显著。结果表明,超低温冷冻对史氏鲟精子造成了一定程度的损伤,长时间的超低温冻存会显著降低精子的活力,影响授精率和受精卵的孵化率。     

条纹锯精液超低温冷冻保存研究

为建立条纹锯精液超低温冷冻保存方法,实验采用计算机辅助精子分析系统（CASA）分析了采用6种抗冻保护剂（GLY[甘油]、DMSO[二甲基亚砜]、PG[丙二醇]、EG[乙二醇]、METH[甲醇]、DMA[二甲基乙酰胺]）在4种浓度（5%、10%、15%、20%,v/v）下对条纹锯精液的冷冻保存效果。结果发现,以HBSS为稀释液,采用程序降温仪分步降温冷冻保存条纹锯精液,37℃水浴解冻后的精子中,15% PG 作为抗冻保护剂的精子运动率最高,达到（93.1±0.9）%,与鲜精差异不显著（P>0.05）,15% PG 作为抗冻保护剂的精子水浴解冻后精子的运动速度最高,平均直线速度、平均曲线速度、平均路径速度分别达到了（88.3±0.3）μm/s、（76.2±0.5） μm/s、（86.7±0.7） μm/s,与鲜精差异不显著（P>0.05）。在不同种类及不同浓度抗冻保护剂保护下,15% PG 作为抗冻保护剂的精子解冻后 1 min内运动率变化与鲜精差异不显著（P>0.05）。研究表明,15% PG为条纹锯最佳抗冻保护剂,可用于条纹锯精液的超低温冷冻保存。     

计算机辅助分析冷冻前后褐牙鲆精子运动特征

观察了褐牙鲆(Paralichthys olivaceus)精子在室温和低温下的活力与寿命,并应用计算机辅助精子分析系统(CASA)对超低温冷冻前后褐牙鲆精子的运动特征进行了分析,结果表明:褐牙鲆精子在室温(25℃)下,可存活4 d,在低温(4℃)下可存活7 d;鲜精的活力为(87. 74±5. 47)%,解冻后,精子的最高活性为(84. 00±3. 67)%;激活0. 5 min时,冻精与鲜精的运动精子占总精子数的百分率(MOT)无显著性差异(P>0. 05),但精子平均曲线运动速度(VCL)、平均直线运动速度(VSL)、平均路径运动速度(VAP)和精子运动路线的曲折程度(LIN)都有显著性差异(P <0. 05);激活4min和10min时,冻精与鲜精的MOT、VCL、VSL、VAP和LIN间都有显著性差异(P <0. 05)。鲜精激活0. 5 min后,直线运动、曲线运动、左右摆动和不运动的精子数目占总精子数的百分比分别为(24. 49±3. 87)%、(48. 53±4. 55)%、(24. 72±2. 86)%和(2. 27±1. 22)%;冻精激活0. 5min后,直线运动、曲线运动、左右摆动和不运动的精子数目占总精子数的百分比分别为(18. 58±1. 33)%、(35. 67±3. 00)%、(35. 24±2. 67)%和(10. 51±1. 33)%。随着激活时间的延长,褐牙鲆鲜精和冻精的运动状态均发生了改变,直线运动和曲线运动的精子数目逐渐减少,而不运动和左右摆动的精子数目逐渐增加。     

不同冻存液对长江鲟鳍条细胞冷冻效果的影响

为了探究不同浓度配比冻存液对长江鲟(Acipenser dabryanus)鳍条细胞(Dabry′s sturgeon fin-derived cells,DSFCs)冷冻效果的影响,选择传代培养处于对数生长期的DSFCs,将第8代DSFCs置于液氮(-196℃)中冻存。根据冻存液中培养基(MEM)、胎牛血清(Fetal bovine serum,FBS)及二甲基亚砜(Dimethyl sulfoxide,DMSO)的配比不同,分为8个实验组:①含不同浓度DMSO配比实验组:5%DMSO+45%MEM+50%FBS,10%DMSO+40%MEM+50%FBS,20%DMSO+30%MEM+50%FBS;②含不同浓度FBS配比实验组:10%DMSO+90%FBS+0%MEM,10%DMSO+70%FBS+20%MEM,10%DMSO+50%FBS+40%MEM,10%DMSO+30%FBS+60%MEM,10%DMSO+10%FBS+80%MEM,对各组分采用形态学观察、CCK-8法、流式细胞仪检测细胞凋亡和细胞周期,综合分析不同冻存液组分对DSFCs冻存后活力的影响。结果显示,不同浓度DMSO配比实验组中,DMSO的浓度为20%时,细胞复苏后难以贴壁,存活率下降非常明显,仅为38.3%;而5%和10%DMSO细胞生长状态良好,存活率分别为80.2%和78.7%。不同浓度FBS配比实验组中,90%、30%和10%实验组细胞活力显著高于70%和50%实验组。FBS浓度为30%和10%的冻存组细胞凋亡率显著高于90%的冻存组(8.24%,15.35%vs 3.54%),3组之间相互比较有明显差异(P<0.05);细胞周期结果显示,与FBS浓度为90%的冻存组比较,FBS浓度为30%和10%冻存组G 0/G 1期细胞比例均明显增加(P<0.05),S期和G 2+M期细胞比例均显著降低(P<0.05),FBS浓度30%与10%冻存组比较差异不明显(P>0.05)。研究表明,长江鲟细胞的长期冷冻保存宜采用稍低浓度的DMSO(5%~10%)和高浓度的FBS。     

瓦氏黄颡鱼精子的生理特性及其超低温冷冻保存的初步研究

对瓦氏黄颡鱼(Pelteobagrus vachelli)精子在不同盐度和pH下的精子活力进行观察,同时研究了精子在4种不同稀释液与2种不同浓度抗冻剂组成的保存液中的超低温冷冻保存,并开展了冻精的授精实验。结果表明,瓦氏黄颡鱼精子浓度为(2.035±0.179)×1012cell·mL-1,在盐度为5.8、pH为7.17时,精子的活力都高达95%。以A液作为稀释液、10%甲醇作为抗冻剂时,冷冻保存精子效果最好,解冻后精子活力为(81.7±0.9)%。用解冻后的精子进行人工授精,获得的受精率为(88.4±2.1)%,孵化率为(74.0±0.8)%;而鲜精受精率为(91.0±0.8)%,孵化率(82±1.6)%,冻精与鲜精均无显著性差异。人工授精实验证明了解冻后的精子能正常用于该鱼的人工繁殖。     

养殖密度对四种鲟鱼生长的影响

经过56d对五种不同养殖密度下鲟鱼生长情况的观察，发现其生长曲线基本符合逻辑斯谛方程。通过计算，得出在试验条件下鲟鱼潜在的最大增长率为0．0124。     

超低温保存对罗氏沼虾胚胎几种酶活性的影响

研究了超低温冷冻保存(-196℃)对罗氏沼虾胚胎内总三磷酸腺苷酶(ATPase)、肌酸激酶(CK)、琥珀酸脱氢酶(SDH)、乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)等酶活性的影响。运用试剂盒分别测定了冷冻前后罗氏沼虾胚胎内酶活性的变化。结果表明,经过超低温冷冻保存后,胚胎内7种酶的活性均显著下降(P0.05),其中总ATPase、CK、LDH和SDH的活性分别从冻前的1.322±0.162 U/mg prot、0.404±0.015 U/mg prot、352.225±23.214 U/gprot和2.067±0.139 U/mg prot下降到0.087±0.003 U/mg prot、0.010±0.002 U/mg prot、5.890±0.658 U/gprot和0.552±0.138 U/mg prot;SOD、CAT和GSH-Px的活性分别从冻前的19.217±0.677 U/mg prot、3.587±0.233 U/mg prot和7.626±1.106 U/(min.mg)下降至3.579±0.234 U/mg prot、1.773±0.227 U/mg prot和1.524±0.096 U/(min.mg);MDA的活性从1.015±0.038 n mol/mg prot上升到20.937±0.320 n mol/mgprot,超低温冷冻对罗氏沼虾胚胎酶活性有显著性影响。     

长期超低温冷冻保存对鞍带石斑鱼精子超微结构及酶活性的影响

【目的】本文旨在探究长期超低温冷冻保存中鞍带石斑鱼精子质膜、活力、超微结构及酶活性的变化,为阐明影响鞍带石斑鱼精子冷冻保存质量的相关机制提供理论依据。【方法】采集2022年鞍带石斑鱼鲜精及储存时间分别为23、49、61个月冷冻保存精液,用伊红-苯胺黑染色方法检测精子质膜完整性;用计算机辅助精子分析仪（CASA）检测精子运动参数;测量精浆和精子中琥珀酸脱氢酶（SDH）、过氧化氢酶（CAT）、谷胱甘肽还原酶（GR）、总超氧化物歧化酶（T-SOD）、谷胱甘肽过氧化物酶（GSH-PX）和肌酸激酶（CK）共六种酶活性的变化及三磷酸腺苷（ATP）浓度变化;用扫描电镜和透射电镜观察鲜精和冻精超微结构。【结果】伊红-苯胺黑染色检测结果发现,鲜精质膜完整性最高为83.43±2.73 %,经过超低温冷冻后,精子质膜完整性显著降低（P<0.05）,且随着冷冻保存时间的延长而逐渐降低。CASA结果显示鲜精活力最高为90.47±3.34 %,经过超低温冷冻后精子活力显著降低（P<0.05）,但长期保存23-61个月精子活力无显著性差异,精子活力保持在63.95±3.66 %-68.58±2.73 %,具有稳定的活力,且鲜精与冻精之间精子平均直线运动速度（VSL）、平均曲线运动速度（VCL）和平均路径速度（VAP）均没有显著差异（P>0.05）。精子超微结构显示,鲜精形态结构正常、线粒体排列结构规则、形态大小正常。经过超低温冷冻保存后,精子形态结构损伤明显,表现为精子头部质膜破损、细胞质外漏、细胞核膜破损、尾部鞭毛断裂或脱落等损伤;鞍带石斑鱼精浆和精子超低温冷冻前后六种酶活性的变化及ATP含量结果显示,经过超低温冷冻后,精子内SOD、GSH-PX和CAT三种酶及ATP含量均有显著性降低（P<0.05）。精浆中酶活力升高,除GR和CAT外,其余酶活性均差异显著（P<0.05）。【结论】长期超低温冷冻对鞍带石斑鱼精浆和精子酶活性、精子活力及精子超微结构均具有较显著影响,【意义】研究结果为鱼类精子冷冻损伤机理研究积累了丰富的数据,为鱼类精子长期冷冻保存提供了技术参考和评价指标。     

Evaluation of fertilizing capacity of cryopreserved rainbow trout sperm

ABSTRACT:   Experimental insemination was performed using artificially produced low-motility sperm. A mathematical model was applied to the results of the insemination in order to clarify the relationship between sperm motility, the density of sperm and the fertilization rate of eggs. In the model, the probability of fertilization by individual spermatozoa was a function of sperm density in the insemination solution. The results showed that the probability of fertilization clearly decreased with increased sperm density, and the maximum possible fertilizing rate by increasing the sperm density was constrained by the proportion of motile sperm (% motility). The model was also applied to the results of insemination tests of cryopreserved sperm in order to evaluate the fertilizing capacity of cryopreserved sperm. It was proven that cryopreserved sperm needed a higher density to obtain the maximum fertilization rate compared with fresh sperm, and it was anticipated that the ratio of the motile inseminated cryopreserved sperm should be more than 5.0% to achieve an egg fertilization rate greater than 90%.     

Cryopreservation of Persian sturgeon (Acipenser persicus) sperm: effects of cryoprotectants,antioxidant, membrane stabilizer,equilibration time and dilution ratio on sperm motility and fertility

Three experiments were performed to develop protocols for cryopreservation of Persian sturgeon Acipenser persicus, sperm. In the first experiment, sperm from six males was individually split in three subsamples and cryopreserved using Modified Tsvetkova's extender (mT) supplemented with dimethyl sulfoxide (DMSO), methanol (MeOH), glycerol (Gly) and ethylene glycol (EG) at concentration of 5%, 10%, 15% and 20%. In the second set of experiments, the effects of six equilibration times (0, 5, 10, 20, 40 and 60 min) and dilution ratios (volume sperm: volume extender 1:0.5, 1:1, 1:2, 1:3, 1:5 and 1:10) and the additive advantage of bovine serum albumin (BSA; 0, 2.5, 5 and 10 mg mL^?1) and ascorbic acid (0, 2.5, 5 and 10 U mL^?1), on the post‐thaw survival of sperm (triplicate set of six fish) were evaluated. Then, sperm was diluted in 1:1 mT extender with 10 mg mL^?1 BSA with selected cryoprotectants (15% MeOH and 10% DMSO) for 5 min. After a month of storage in liquid nitrogen, post‐thawed sperm motility; fertilization and hatching rate and viability of derived larvae were measured (Exp.3). Evaluation of cryoprotectants efficiency showed that MeOH 15% and DMSO 10% were suitable for cryopreservation of Persian sturgeon sperm. Gly and EG resulted in very low post‐thaw motility rates even at lowest concentration. No significant difference was observed among the four different equilibration times (0, 5, 10, 20 min) (P > 0.05) although higher equilibration times than 20 min resulted low post‐thaw motility (P < 0.05). The motility of frozen–thawed sperm did not significantly change when dilution ratio was increased from 1:0.5 to 1:3 (P > 0.05). However, higher dilution ratios (1:5 and 1:10) reduced the percentage of motile sperm. Supplementation of the cryoprotectant solution with 10 mg mL^?1 BSA significantly improved post‐thaw motility (P < 0.05), but ascorbic acid did not improve post‐thaw motility (P > 0.05). The results of experiment 3 showed that the highest fertilization (30.2 ± 5.75) and hatching rates (28.2 ± 5.25) were observed when samples were frozen with 15% MeOH (P > 0.05). Our study indicates that the use of mT extender consisting of 10 mg mL^?1 BSA in 15% MeOH diluted with sperm at 1:1 ratio for 5 min can be recommended cryopreservation method for Persian sturgeon sperm.     

不同盐度激活液、K+浓度及保存时间对日本鳗鲡精子活力的影响

在6种不同盐度(34、32、30、28、26和22)激活液、3种不同K+浓度(25 mmol/L、30 mmol/L和35 mmol/L)稀释液和不同保存时间(0 h、24 h、48 h、72 h和96 h)条件下,对日本鳗鲡(Anguilla japonica)精子的活力进行观察和测定.结果表明,激活液盐度为30时精...     

Motility and fertility of cryopreserved spermatozoa of the Japanese sea cucumber Apostichopus japonicus

We developed both a cryopreservation method for Japanese sea cucumber spermatozoa and an artificial fertilization method using post‐thaw spermatozoa. Twenty per cent dimethyl sulfoxide (DMSO), 16% foetal bovine serum, and 64% artificial seawater were suitable cryodiluent, and the diluent was pre‐cooled to 0°C. Semen was diluted with the solution and enclosed in a 250 μl straw, cooled to ?50°C at 10.4 ± 0.4°C/min, and immediately immersed in liquid nitrogen. Although this method showed the highest post‐thaw motility in all the conditions we examined, its post‐thaw motility was still less than approximately 15%. Artificial fertilization was carried out by adding post‐thaw semen with a cryodiluent to the oocytes. The fertilization rate of 200 oocytes/ml seawater increased with the amount of post‐thaw semen from 1 to 5 μl but showed a significant decrease at 25 μl. This decrease was considered to be due to DMSO in the cryodiluent, because the fertilization rate of the fresh semen decreased sharply when the DMSO concentration around the oocytes was 1.0% or more. Further improvement in increasing post‐thaw motility and lowering the cryoprotectant concentration is necessary for commercial‐scale artificial fertilization.     

Fertilization efficiency of cryopreserved sperm from striped catfish, Pangasius hypophthalmus (Sauvage)

The fertilization efficiency of cryopreserved sperm was compared with fresh sperm from striped catfish, Pangasius hypophthalmus . Of the two sets of experiments carried out, the first compared four sperm doses using fresh sperm and fresh eggs. The second experiment compared six concentrations of cryopreserved sperm ranging from 6.94 × 10⁷ to 6.94 × 10¹⁰ to fertilize 100 eggs per batch. Fertilization, hatch and survival rates were compared between cryopreserved and fresh sperm. The highest fertilization rate (53.75±1.62%) was achieved with a sperm dose of 6.94 × 10⁸. Increasing the sperm dose to 3.47 × 10⁹ did not increase the fertilization rate, indicating that the optimum sperm:egg ratio lies between 6.94 × 10⁶ and 3.47 × 10⁷ sperm per egg. Both highest (6.94 × 10¹⁰) and the lowest (6.94 × 10⁷) sperm doses resulted in lower fertilization rates (2.04% and 16.90% respectively). No significant differences were found among four fresh sperm doses compared. Mean hatch and survival rates resulting from fresh and cryopreserved sperm were similar. The experiment shows that while only 1.89 × 10⁶ fresh spermatozoa was required to fertilize a fresh egg, 6.94 × 10⁶ (or 3.67 times more) cryopreserved sperm was required to achieve the same level of fertilization. This provides important information for making decision to cryopreserve sperm for commercial and/or conservation purposes.     

Larval growth of turbot, Scophthalmus maximus (L.) produced with fresh and cryopreserved sperm

The present paper assesses the fertilization and hatching rates, as well as the growth, of larvae obtained from four artificial fertilizations (AF) using fresh and cryopreserved sperm of the turbot Scophthalmus maximus (L.). Larvae growth in both sperm groups, measured in terms of length and weight at culture days 0, 7, 14 and 31, are compared, as well as their growth rates. The two groups' fertilization and hatching rates were not significantly different. Likewise, no significant differences in length and wet weight of 7‐ and 14‐day‐old larvae were found using fresh and cryopreserved sperm; however, significant differences were found in 31‐day‐old larvae, which were more attributable to the variability inherent in larval turbot culture, and to variability in the reproductive specimens used in our study, than to the type of sperm employed. These results indicate that the type of sperm used in artificial fertilization, i.e. fresh or cryopreserved, is not a determining factor, either for fertilization and hatching, or for subsequent larval development. Our results also confirm once again the high quality of cryopreserved turbot sperm, and its usefulness in commercial hatcheries.     

Cryopreservation of sea cucumber Apostichopus japonicus (Selenka) sperm

A simple and convenient method for the cryopreservation of sea cucumber Apostichopus japonicus (Selenka) sperm was tested in the present study. The highest motility (76.7±2.9%) of post‐thawing sperm was obtained in 15% dimethyl sulphoxide (DMSO) with a 1:9 dilution (semen volume to DMSO volume) when 0.5 mL semen–DMSO mixture was frozen at 6 cm above liquid N₂ in a closed styrofoam box. After thawing, sperm cryopreserved in glycerol almost lost motility entirely. Although there was no significant difference in percentage of motile sperm between 15% and 20% DMSO, the duration of sperm motility of 15% DMSO group was longer than that of 20% DMSO group. The motility of post‐thawing sperm enhanced when the dilution ratio of semen increased from 1:1 to 1:9. Morphological changes such as the loss of mitochondria, swollen plasma membrane and broken or rolled‐up tails were observed in post‐thawing sperm using an eosin–nigrosin staining. The fertility of cryopreserved sperm was significantly lower than that of unfrozen sperm. The 10‐fold increase in sperm to egg ratio resulted in double fertility for cryopreserved sperm, and about 70% fertility relative to the control.