黑鲷精子的超低温冻存及DNA损伤的SCGE检测

以0.5 mL的麦细管为冻存管和DMSO为抗冻剂进行超低温冷冻黑鲷精子,对冻精核DNA的损伤情况进行单细胞凝胶电泳（SCGE）检测,其结果表明,以Cortland溶液为稀释液,5%、10%、15%及20%DMSO为抗冻剂的超低温冻存的黑鲷精子活力、受精率与鲜精无显著差异。其中以10%DMSO为抗冻剂的冻存效果最佳,冻精的激活率、运动时间、寿命及受精率分别达（92.91±1.25）%、（39.90±2.70）min、（53.82±2.84）min及（89.35±1.99）%;而以25%及30%DMSO为抗冻剂时,冻精活力及受精率显著下降。SCGE检测结果显示,DMSO浓度为5%、10%、15%及20%时,黑鲷冻精与鲜精的彗星率及损伤系数差异不显著;DMSO浓度为25%及30%时,冻精与鲜精的彗星率及损伤系数差异显著;冻精的彗星率与抗冻剂DMSO浓度成正相关。黑鲷鲜精及冻精核的DNA损伤主要为轻度和中度损伤,重度损伤比例较低,完全损伤仅存在于25%及30%DMSO为抗冻剂的冻精中,且比例低。分析认为,较高浓度的DMSO是引起冻精核DNA损伤的主要原因。     

环境因子及超低温冻存对黄姑鱼精子活力的影响

通过测定精子的激活率、运动时间及寿命研究了环境因子变化对黄姑鱼精子活力的影响及超低温冻存后黄姑鱼精子的活力。结果表明,黄姑鱼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 8.0～8.5、盐度25条件下,精子激活率达(85.33±2.52)%,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300～500 mmol·L^-1、600 mmol·L^-1、800～1000 mmol·L^-1及900mmol·L^-1;精子在缺少HCO₃^-的人工海水中未能被激活;精子在无Ca²⁺或无Mg²⁺的人工海水中激活率均大于80%,但运动时间及寿命均有所缩短。以Cortland及HBSS溶液为稀释液、10%EG为抗冻剂冻存黄姑鱼精子,冻精激活率>80%,运动时间均超过200s。     

兰州鲇精液超低温冷冻保存技术研究及细胞损伤检测

为保护兰州鲇(Silurus lanzhouensis Chen)种质资源, 促进新品种选育, 文章开展了兰州鲇精液超低温冻存抗冻剂筛选及程序保存技术研究, 并通过精子解冻激活率、核DNA检测、扫描电镜和透射电镜检测技术对冻存效果和冻存损伤情况进行了评测。结果表明: 兰州鲇精液以10% DMSO为抗冻剂, 4℃平衡20min, –80℃平衡30min后立即置于液氮超低温保存, 并于40℃水浴解冻时精子冻存效果较佳, 冻精激活率达(75.56±3.91)%。SCGE检测DNA损伤结果显示超低温冻存10d、20d及30d兰州鲇精液的彗星率和损伤系数无显著差异。扫描电镜检测显示兰州鲇精子明显分头部, 中段及尾部3部分, 属于单鞭毛型, 无顶体, 无侧鳍, 中心粒相互垂直呈“T”形, 鞭毛为典型的“9+2”微管结构。冻存结构损伤主要表现为膜损伤, 细胞质膜与染色质膜发生破损、折皱、囊泡化或整体脱落, 细胞核膜与质膜空隙加大, 核发生变形, 核质疏散, 线粒体结构弥散, 线粒体内容物外流, 中心粒复合体易位, 鞭毛外膜变形脱离, 中段位置断裂, 微管结构基本完好。研究筛选的超低温冷冻保存技术可长期有效保存兰州鲇精液, 为兰州鲇种质资源保护及今后精液超低温冷冻保存技术改良提供理论依据和技术基础。     

鮸鱼精子的生理特性及超低温冻存

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HCO3-的人工海水均能使鮸鱼精子激活,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。     

长牡蛎精子超低温冷冻后超微结构损伤研究

采用程序降温仪分步降温冷冻保存长牡蛎（Crassostrea gigas）精液,并用扫描电镜、透射电镜研究了精子的超微结构损伤。超低温冷冻保存后长牡蛎精子的运动率、受精率及孵化率与鲜精无显著差异。鲜精中84.5%的精子形态结构正常,冻精中73%的精子形态结构正常。形态结构正常的精子表现为顶体、质膜、线粒体与鞭毛结构完整、染色质形状规则,顶体、线粒体及中心粒结构正常,鞭毛形态完整、微管结构清晰;形态结构异常的精子表现为顶体脱落、解体,精子头部质膜膨胀、破裂、染色质肿胀、破裂、解体,线粒体移位、脱落、膨胀,嵴退化或消失,鞭毛弯折、断裂,微管解聚。结果显示,以10% DMSO为抗冻保护剂,HBSS溶液为稀释液,1:4的稀释比例,添加海藻糖,采用分步降温法冷冻保存,对长牡蛎精子具有较好的抗冻保护作用,合适的冻存方法可以有效的保护太平洋牡蛎精子冷冻过程中结构损伤。研究有助于长牡蛎种质资源的收集保存及应用。     

软鳍新光唇鱼精子的超低温冷冻保存

2011年,对软鳍新光唇鱼(Neolissochilus benasi)进行了精子超低温冷冻保存研究。以解冻后的精子活力为指标,采用稀释液D-15,设计不同的抗冻剂种类和浓度,以及不同的实验条件(包括冷冻体积、4℃平衡时间和解冻温度等)探索软鳍新光唇鱼精子的超低温冷冻保存方法。筛选出了适合软鳍新光唇鱼精子超低温冷冻保存的两种抗冻保护剂及其浓度分别为10%MeOH和15%EG,确定了精液与稀释液的最适稀释比例为1:7、4℃平衡时间区间为10～60min、冷冻体积为60μL,以及复苏方法为37℃水浴快速解冻30s。当鲜精活力为(62.33±2.05)%,综合以上最佳实验条件进行保存,解冻后精子的最高活力为(29.67±0.47)%,但效果不理想,不能达到广泛生产运用水平;产生这一结果,可能与异地保育物种的饲养管理有关。因此,在亲鱼培育管理中要最大限度地降低捕获诱发的压力,尽量提供适合的养殖条件。在珍稀鱼类异地保育时,繁殖用雄鱼的培育与雌鱼同等重要,是获得大量高质量仔鱼的关键。     

黄鳝精液-80℃超低温冷冻保存试验

采用-80℃超低温冷冻方法对黄鳝精液冷冻保存技术进行了研究.获得如下结果:黄鳝精子在冻存前不需低温平衡过程;10%DMSO作为抗冻保护剂效果最好,以200 μL离心管为冻存容器,保存168 h,精子相对活力可达79%;以细管为冻存容器,精子相对活力可达88%.此结果为黄鳝精子冷冻保存库的建立提供了实验依据.     

滇南小耳猪精液的直接冷冻保存

目的建立滇南小耳猪精液冷冻保存方法,加速云南省特有小型猪种的小型化选育。方法利用脉冲电刺激模式诱导公猪输精管自助收缩排精。利用直接冷冻新技术（DFM）研究不同冷冻方案对精子的运动度、精子顶体完整性和体内授精胚胎发育能力的影响。结果在直接冷冻方法中,3%甘油防冻剂的作用下,60 s植冰时间和1.5 mm/s的冷冻降温参数对精子运动度保护良好,精子运动复苏率达到61.7%。但是,3%乙二醇虽然与甘油一样对精子的顶体完整性都有很好的保护作用,对精子运动度保护能力较差。此外,3%甘油、60 s植冰、1.5mm/s冷冻速度的直接冷冻的冻精解冻,移植到超数排卵的母猪子宫颈口实施人工授精,获得卵的受精和胚胎发育潜能尚可。结论玻璃管直接冷冻可以完成滇南小耳猪精子的冷冻保存。     

胭脂鱼(Myxocyprinus asiaficus)精液超低温冷冻保存及酶活性测定

为了找到适合珍稀鱼类胭脂鱼的物种保存方法,本研究比较了不同的稀释液(D-17, D-20, Ringer液和Kurokura-1)以及不同的稀释比例(1∶2, 1∶3, 1∶6)、不同的抗冻剂(二甲亚砜,甘油和甲醇)以及不同的添加浓度(8%, 10%, 12%)对胭脂鱼精子超低温冷冻保存效果,结果显示:稀释液D-17、D-20保存效果显著优于Ringer液、Kurokura-1 (p0.05);D-17稀释液的最佳的稀释倍数为1∶3,D-20稀释液的最适稀释比例为1∶3或1∶6;冷冻保存107 d后,D-20 (1∶6)配方的激活率最高,达(81.67±2.89)%;抗冻剂二甲亚砜保存效果显著优于甘油和甲醇(p0.05),二甲亚砜的最适添加浓度为8%。同时测定了添加D-17 (1∶3)抗冻液、Ringer液(1∶3)抗冻液和未添加抗冻液冻存样本精浆中酶活力,结果显示未添加抗冻液精浆中ATPase、SDH、LDH、CK、GOT活性均高于添加抗冻液的样品;添加抗冻液D-17 (1∶3)的样品精浆中ATPase、SDH、CK、GOT的含量低于冻存相同天数的Ringer液(1∶3)样品;精浆中ATPase、SDH、LDH、CK、GOT含量随冻存时间的延长呈上升趋势。本研究为保护淡水珍稀鱼类胭脂鱼的种质资源提供了理论基础。     

四种渗透性防冻剂在猕猴精子低温冷冻保存中对精子功能状态的影响

以冷冻精子的复苏运动度、荧光染料Hoechst 3 3 2 5 8检测的细胞膜完整率、异硫氰酸荧光素标记的花生凝集素 (FITC PNA)检测的顶体完整率作为精子功能状态的指标 ,对甘油、二甲亚砜、乙二醇和丙二醇 4种常用渗透性防冻剂在猕猴精子冷冻保存过程中的作用进行了比较。结果表明 :冷冻保存精子的复苏运动度 ,甘油 ( 4 7 3± 5 7% )和乙二醇 ( 4 4 8± 6 7% ) >二甲亚砜 ( 2 2 9± 0 9% ) >丙二醇 ( 0± 0 % ) ;细胞膜完整率 ,甘油 ( 5 4 8± 3 2 % )和乙二醇 ( 5 4 0± 6 7% ) >二甲亚砜 ( 3 7 5± 7 0 % ) >丙二醇 ( 2 8 3± 6 5 % ) ;顶体完整率 ,甘油 ( 82 2± 2 4 % )和乙二醇 ( 82 4± 2 4 % ) >二甲亚砜 ( 6 8 7± 5 7% )和丙二醇 ( 72 3±3 5 % ) (P <0 0 5 )。结果提示 :二甲亚砜和丙二醇 ,尤其是丙二醇并不适合猕猴精子的冷冻保存 ;而乙二醇具有和甘油相似的保护作用 ,是一种极具潜力的猕猴精子冷冻保存的渗透性防冻剂。     

Semen cryopreservation of yellow croaker <Emphasis Type="Italic">Larimichthys polyactis</Emphasis>

The effects of various extenders and cryoprotectants on movable spermatozoa ratio (MSR), spermatozoa velocity (SV) and duration of spermatozoa motility (DSM) of post-thawed spermatozoa were examined. The MSR, SV and DSM of post-thawed sperm in artificial seminal plasma (ASP) extender were higher than those in marine fish Ringer’s solution (MFRS) extender (P < 0.01) and was not significantly different from that of fresh sperm. No significant differences were observed in the motility parameters between fresh spermatozoa and frozen-thawed spermatozoa cryopreserved with ASP extender supplement 10% EG (ethylene glycol) cryoprotectant. Using the above method, yellow croaker semen was cryopreserved with extender ASP and 10% EG. As a result, at the spermatozoa/egg ratio of 100,000:1, the fertilization rate and hatching rate of the frozen-thawed spermatozoa cryopreserved for 1 week or 1 year in liquid nitrogen (45.7 ± 3.2% and 27.2 ± 5.0% or 37.5 ± 4.4% and 27.2 ± 5.0%) were similar to that of fresh spermatozoa (51.0 ± 3.1% and 36.7 ± 2.2%). There was a small alternation of shape in cryopreserved spermatozoa compared with fresh spermatozoa. In conclusion, the optimal conditions for yellow croaker semen cryopreservation are ASP extender supplement 10% EG cryoprotectant. This is the first report on semen cryopreservation of yellow croaker Larimichthys polyactis.     

Evaluation of DNA damage in Dicentrarchus labrax sperm following cryopreservation

In this paper, DNA laddering analysis and single-cell gel electrophoresis (SCGE) or Comet assay, were used to detect DNA damage in response to a cryopreservation process in sea bass spermatozoa. The results obtained demonstrate that the cryopreservation protocol used to cryopreserve the sea bass sperm cause significantly damage at DNA level. In fact, the degree of DNA damage in frozen-thawed sperm (%DNAT=38.2+/-11.2, MT=498.9+/-166.4, n=3) was different (P<0.01) from that measured in fresh sperm (%DNAT=32.7+/-11.1, MT=375.2+/-190.7, n=3). Data here reported also demonstrated the fundamental role played by cryoprotectants (BSA and Me2SO) in reducing fish sperm DNA fragmentation. Finally, from our results, the ability of SCGE to reveal DNA fragmentation in fish sperm is also confirmed.     

Semen cryopreservation of small abalone (Haliotis diversicolor supertexa)

Methods for cryopreserving spermatozoa and maximizing fertilization rate in Taiwan small abalone, Haliotis diversicolor supertexa, were developed. The gametes (spermatozoa and eggs) of small abalone were viable 3 h post-spawning, with fertilization, and development rate decreasing with time. A minimum of 10(2) cell/ml sperm concentration and a contact time of 2 min between gametes is recommended for artificial insemination of small abalone eggs. Eight cryoprotectants, dimethyl sulfoxide (DMSO), dimethyl acetamide (DMA), ethylene glycol (EG), propylene glycol (PG), butylene glycol (BG), polyethylene glycol, glycerol and methanol, were tested at concentrations between 5 and 25% to evaluate their effect on motility of spermatozoa exposed to cryoprotectant for up to 60 min at 25 degrees C before freezing. The least toxic cryoprotectant, 10% DMSO, was added to artificial seawater (ASW) to formulate the extender for freezing. Semen was diluted 1:1 with the extender, inserted into 1.5 ml microtubes and frozen using a cooling rate between -3.5 and -20 degrees C/min to various transition temperatures (0, -30, -60, -90 and -120 degrees C), followed by transfer and storage in liquid nitrogen (-196 degrees C). The microtubes were thawed from +45 to +145 degrees C/min. Spermatozoa, cooled to -90 degrees C at a cooling rate of -12 or -15 degrees C/min and then immersed in liquid nitrogen, had the best post-thaw motility. Post-thaw sperm motility was markedly reduced compared to fresh sperm. More frozen-thawed spermatozoa are required to achieve fertilization rates comparable to those achieved using fresh spermatozoa.     

DNA fragmentation in chicken spermatozoa during cryopreservation

Semen cryopreservation is fundamental both for the practice of artificial insemination, and for the conservation of genetic resources in cryobanks; nevertheless, there is still not an efficient standard freezing procedure assuring a steady and suitable level of fertility in fowl, and consequently there is no systematic use of frozen semen in the poultry industry. This study examined changes in motility (CASA), cell membrane integrity (Ethidium Bromide (EtBr) exclusion procedure and stress test) and DNA fragmentation (neutral comet assay) in fowl spermatozoa before, during and after cryopreservation and storage at −196 °C. An optimized comet assay for chicken semen was studied and applied to the analyses. Semen collected from 18 Mericanel della Brianza (local Italian breed) male chicken breeders was frozen in pellets and thawed in a water bath at 60 °C. Measurements were performed on fresh semen soon after dilution, after equilibration with 6% dimethylacetamide at 4 °C (processed semen) and after thawing. Sperm DNA damage occurred during cryopreservation of chicken semen and the proportion of spermatozoa with damaged DNA significantly increased from 6.2% in fresh and 6.4% in processed semen to 19.8% in frozen-thawed semen. The proportion of DNA in the comet tail of damaged spermatozoa was also significantly affected by cryopreservation, with an increase found from fresh (26.3%) to frozen-thawed (30.9%) sperm, whereas processed semen (30.1%) didn't show significant differences. The proportion of total membrane damaged spermatozoa (EtBr exclusion procedure) did not increase by 4 °C equilibration time, and greatly and significantly increased by cryopreservation; the values recorded in fresh, processed and frozen semen were 2.9, 5.6, and 66.7% respectively. As regards the proportion of damaged cells in the stress test, all values differed significantly (7.1% fresh semen, 11.7% processed semen, 63.7% frozen semen). Total motility was not affected by equilibration (52.1% fresh semen, 51.9% processed semen), whereas it decreased significantly after cryopreservation (19.8%). These results suggest a low sensitivity of frozen-thawed chicken spermatozoa to DNA fragmentation, therefore it should not be considered as a major cause of sperm injuries during cryopreservation.     

Cryopreservation of turbot (Scophthalmus maximus) spermatozoa

The aim of this study was to develop a method for cryopreserving turbot semen and to compare sperm motility characteristics, metabolic status and fertilization capacity of frozenthawed and fresh semen. The best results were obtained when spermatozoa were diluted at a 1:2 ratio with a modified Mounib extender, supplemented with 10% BSA and 10% DMSO. For freezing sperm samples, straws were placed at 6.5 cm above the surface of liquid nitrogen (LN) and plunged in LN. The straws were thawed in water bath at 30 degrees C for 5 sec. Use of this simple method resulted in a 60 to 80% reactivation rate of the thawed spermatozoa. Although the percentage of motile spermatozoa in the frozen-thawed semen samples was significantly lower than in fresh semen, spermatozoa velocity and respiratory rate remained unchanged. The process of cryopreservation significantly decreased intracellular ATP content. The fertilization rate of frozen-thawed spermatozoa was significantly lower than that of fresh spermatozoa, but it increased with sperm concentration.     

Flow cytometry and ultrastructure of cryopreserved red seabream (Pagrus major) sperm

The objectives were to assess motility, fertilizing capacity, structural integrity, and mitochondrial function in fresh versus frozen-thawed (15% DMSO was used as a cryoprotectant) sperm from red seabream (Pagrus major). Mean (+/-S.D.) rates of motility, fertilization and hatching of frozen-thawed sperm were 81.0+/-5.4, 92.8+/-1.9, and 91.8+/-5.2%, respectively; for fresh sperm, they were 87.5+/-7.7, 95.8+/-2.4, and 93.8+/-4.2%. Although motility was lower in frozen-thawed versus fresh sperm (P<0.05), there was no effect (P>0.05) of cryopreservation on fertilization or hatching. Based on scanning and transmission electron microscopy, 77.8+/-5.6% of fresh sperm had normal morphology, whereas for frozen-thawed sperm, 63.0+/-7.2% had normal morphology, 20.6+/-3.1% were slightly damaged (e.g. swelling or rupture of head, mid-piece and tail region as well as mitochondria), and 16.4+/-4.2% were severely damaged. Sperm were stained with propidium iodide and Rhodamine 123 to assess plasma membrane integrity and mitochondrial function, respectively, and examined with flow cytometry. For fresh sperm, 83.9% had an intact membrane and functional mitochondria, whereas for frozen-thawed sperm, 74.8% had an intact membrane and functional mitochondria, 12.7% had a damaged membrane, 9.9% had nonfunctional mitochondria, and 2.6% had both a damaged membrane and nonfunctional mitochondria. In conclusion, ultrastructure and flow cytometry were valuable for assessment of frozen-thawed sperm quality; cryopreservation damaged the sperm but fertilizing ability was not significantly decreased.     

Effects of extender composition,cooling rate,and freezing on the motility of sea bass (Dicentrarchus labrax,L.) spermatozoa after thawing

A successful cryopreservation procedure for sperm must guarantee recovery of the morphological and functional characteristics of the cells following thawing so that preserved semen can to be used comparably with non-preserved semen. The aim of this work was to identify a species-specific freezing protocol for sea bass (Dicentrarchus labrax) spermatozoa by optimising all the stages in the cryopreservation procedure. In the first stage of the experiments, the cryoprotectants and the relative concentrations that had the least toxic effect on motility at room temperature were selected. The capacity of the selected cryoprotectant substances was then assessed in freezing tests as follows: dimethyl sulfoxide (Me(2)SO) 5% and 7%, ethylene glycol (EG) 7% and 10%, propylene glycol (PG) 7% and 10%. The cryoprotectant that gave the best results in this second stage of the experiments was EG 10%, and this was then used for the optimisation of the different stages in the freezing procedure: two different times of adaptation to the cryoprotectant were tested (15min and 6h), as well as the effects of adding an energy substrate (1.25mM sodium pyruvate) to assess its possible use as an energy source. Lastly, using the extender (diluent+Na-pyruvate+EG10%) and the adaptation procedure (6h at 0-2 degrees C) that had given the best results in the preceding stages of the experiments, four cooling rates were tested: 10, 12, 15, 24 degrees C/min. It was shown that the semen that was diluted immediately after collection in extender that contained the cryoprotectant (EG 10%), was equilibrated for 6h at 0-2 degrees C and then cooled at a rate of 15 degrees C/min, showed motility on thawing comparable to that of fresh semen (P=0.045).