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。     

史氏鲟精子超低温冷冻保存研究

比较了史氏鲟精子在3种不同配比浓度稀释液的保存效果。试验结果表明,配方Ⅲ作为稀释液,8%甲醇作为抗冻剂,二步法超低温(-196℃)冷冻保存,5h后取出,38℃水浴解冻取得最好的冻后激活率,解冻后激活率为(52.3±3.5)%。解冻精子分别采用井水和激活液D(10mmol/L Tris+10mmol/L NaCl+25mmol/L Glu,pH 8.0)激活,进行人工授精。结果显示配方Ⅲ冻精采用激活液D激活授精获得最高受精率为68.56%,最高孵化率为52.91%。本次试验表明,1～2mmol/L范围内,低浓度K+比高浓度K+对史氏鲟精子保存有利;52～82mosmol/kg范围内,高渗稀释液有利于史氏鲟精子的保存;且激活授精方法是影响冻精受精率和孵化率的关键因素之一。     

日本黄姑鱼精子生理特性及超低温冷冻保存研究

对日本黄姑鱼精子部分生理特性及其超低温冷冻保存技术进行了研究。结果表明,日本黄姑鱼精液pH值为7.0~7.5,精子密度为(11.23±2.78)×109/m l,精子寿命(229.33±17.16)s。在盐度为30~35,pH为7.5~8.5时,精子的活力最高,分别达到(88.33±2.89)%和(88±4.33)%。精子在室温(25℃)条件下,可存活24 h;在低温(4℃)条件下可以存活36 h。以D液作为稀释液,20%的乙二醇作为抗冻剂,利用快速降温法对精子进行超低温冷冻保存,38℃水浴快速解冻,解冻后的精子获得最高的活力为(47±5.78)%。     

超低温冷冻保存中华鲟F_1精子授精方法研究

利用超低温冷冻保存中华鲟子一代(F_1)的精液,研究4种不同激活液、2种不同的授精方式及5种不同冻精授精量对授精效果的影响。结果表明,冷冻保存365d的中华鲟F_1冻精,用养殖水激活授精效果优于4种激活液,获得(15.04±0.58)%的受精率和(14.6±0.59)%的孵化率。相同条件下,试验1、试验2和试验3的湿法受精率和孵化率分别为(18.3±1.45)%、(13.57±0.76)%、(18.65±0.42)%和(16.82±2.01)%、(11.03±0.98)%、(15.87±1.25)%,湿法授精效果均优于干法授精。用10mL中华鲟卵与不同冻精授精量进行湿法授精,精子密度3.82×109个/mL,稀释比1∶2,冻精活力(35.45±5.26)%,冻精用量0.1~0.6mL,随着冻精用量的加大,受精率和孵化率呈现上升趋势,0.6mL时,受精率和孵化率达最高,分别为(16.43±1.57)%和(14.58±1.35)%,但随着冻精用量的继续加大,其受精率和孵化率随之下降。     

棘头梅童鱼精子超低温冷冻保存研究

为保护和利用棘头梅童鱼种质资源,以常用无机盐及葡萄糖配制的5种溶液(依次编号A、B、C、D、E)作为稀释液,不同体积分数的DMSO作为抗冻剂,采用2 mL冻存管和两步降温的方式,对棘头梅童鱼精子的超低温冷冻保存技术进行了研究,并利用人工养殖黄姑鱼的成熟卵子对冻存3年的棘头梅童鱼冷冻精子的授精能力进行检验。结果表明,以E溶液为稀释液、10%DMSO为抗冻剂、两步降温方式冷冻保存的棘头梅童鱼精子在37℃水浴解冻后复活率较高,为(76.67±10.41)%^82.33±4.62%;以上述方法冻存3年的棘头梅童鱼冷冻精子与人工养殖黄姑鱼的成熟卵子杂交,受精率达到(20.26±4.12)%。     

不同稀释液对香港牡蛎精子冷冻保存效果的研究

分别以生理盐水、柠檬酸钠-甘氨酸液、海水、KCl-NaCl溶液、Hank’s液、D-Hank’s液为稀释液,不同浓度二甲基亚砜(dimethylsulfoxide,DMSO)及海藻糖为抗冻剂,研究超低温冷冻保存香港牡蛎(Crassostrea hongkongensis)精子方法及不同浓度含氨海水对复苏精子的激活效果,以筛选出最适宜精子冷冻保存的稀释液和抗冻剂及其浓度。结果显示,柠檬酸钠-甘氨酸液为稀释液复苏后精子成活率最高,为(82.90%±0.19%);其次是生理盐水(77.57%±0.49%)、D-Hank’s液(72.22%±0.36%)、Hank’s液(70.00%±0.28%)。氨海水终浓度为0.0313%时,复苏后精子活力最强,柠檬酸钠-甘氨酸溶液为稀释液精子活力为(61.25%±3.06%),生理盐水为稀释液精子活力为(68.70%±0.74%)。以生理盐水为稀释液、DMSO浓度为14%冷冻保存时,复苏精子存活率最高,含海藻糖精子存活率为(86.91%±3.14%),不含海藻糖精子存活率为(81.81%±3.68%),以柠檬酸钠-甘氨酸液为稀释液、DMSO浓度为12%时,复苏后精子存活率最高,含海藻糖的为(79.62%±1.95%),不含海藻糖的为(70.58%±2.41%)。     

超低温冷冻对俄罗斯鲟精子顶体酶活性及DNA损伤的影响

选取6 ind健康的雄性俄罗斯鲟(Acipenser gueldenstaedti),经人工催产后获得成熟的精子,研究超低温冷冻(-196℃)对俄罗斯鲟精子顶体酶活性及DNA损伤影响。结果显示:俄罗斯鲟鲜精中顶体酶的平均活性为(36.18±2.54)μIU·10-6,经过超低温冷冻后,精子顶体酶活性显著降低,添加抗冻保护液精子中顶体酶活性降至(21.55±0.79)μIU·10-6,未添加抗冻保护液精子中顶体酶活性降至(9.58±1.08)μIU·10-6,且三者间有显著性差异(P0.05)。单细胞凝胶电泳结果表明,俄罗斯鲟鲜精彗星率为(37.33±7.77)%,添加抗冻剂后冻精的彗星率为(63.67±5.13)%,未添加抗冻剂直接冷冻彗星率高达(86.00±3.61)%,三者间有显著差异(P0.05)。用CASP分析软件分析测量彗星拖尾长度(L tail)、彗星尾部DNA的相对含量(Tail DNA)、尾动量(TM)、Olive尾动量(OTM)等各项表征DNA损伤的指标,发现冻精组的各项指标均显著高于鲜精组,未添加抗冻剂直接冷冻组又高于添加抗冻剂组,3组间有显著性差异(P0.05)。本研究结果表明:超低温冷冻能导致精子顶体酶活性下降和DNA损伤,抗冻剂对精子具有保护作用。     

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。     

石鲽、牙鲆精子冷冻保存研究及其在人工杂交中的应用

对石鲽(Kareius bicoloratus )和牙鲆(Paralichthys olivaceus)精子冷冻保存技术进行了研究,筛选到一个优良的稀释液MPRS,用MPRS冷冻保存石鲽、牙鲆精子,冻后成活率在70%以上。在石鲽冻精和牙鲆卵的杂交实验中,杂交组受精率是 28 4%±4 55(n=3),孵化率是 42 7%±7 35(n=3),胚胎发育正常;5 000尾杂交鱼苗有102尾成功度过变态期。在牙鲆冻精和大菱鲆卵的杂交实验中,杂交组的受精率只有2 7%,部分受精卵虽能孵化出膜,但仔鱼全为畸形。这两个杂交实验,特别是石鲽冻精和牙鲆卵的杂交实验,证实了冷冻保存的精子完全可以应用在杂交育种工作中。     

云纹石斑鱼精子冷冻保存

以云纹石斑鱼精液为实验材料,对精子稀释液、抗冻剂种类和适宜浓度、冷冻保存液进行了筛选。结果表明,利用9g/L NaCl、10g/L KHCO3和10%小牛血清配制而成的稀释液EM1-2适宜于云纹石斑鱼精子冷冻保存,以2ml冷冻管为精子容器,在60L液氮生物保存罐中冷冻保存精子,冷冻解冻精子活力可达56.67%±5.77%,要优于TS-2、ES1-3和其他EM系列稀释液冷冻保存精子活力。利用EM1-2为基础液对抗冻保护剂进行筛选,结果显示,10%~20%的二甲基亚砜(DMSO)和1-2-丙二醇(PG)冷冻保存后精子活力无显著差异(P0.05),其中15%的DMSO和10%PG冷冻保存精子效果最优,解冻后精子活力分别可达54.52%±7.81%和57.24%±3.69%。利用冷冻保存1年的精液与云纹石斑鱼卵进行受精,受精率和孵化率均达到80%以上,与新鲜精子无显著性差异(P0.05)。本研究表明,利用EM1-2配制15%的DMSO或10%的PG可用于冷冻保存云纹石斑鱼精液。在此基础上,建立了精子冷冻库,保存精子130ml,为人工繁育和杂交育种提供了丰富的精子源。     

Effect of extender composition and freezing rate on post-thaw motility and fertility of Arctic char, Salvelinus alpinus (L.), spermatozoa

The effects of extender composition and freezing rate on motility and fertility of frozen‐thawed Arctic char, Salvelinus alpinus, spermatozoa were investigated. Three freezing rates, two semen diluents and three cryoprotectants were tested. Semen frozen in 0.3 mol L^?1 glucose diluent with 10% methanol as a cryoprotectant or in a diluent described by Lahnsteiner with 10%N,N‐dimethylacetamide (DMA) resulted in the highest sperm motility. Fertility was the highest for semen frozen in a glucose–methanol extender but was not significantly different than that for semen frozen in Lahnsteiner's diluent with 10% DMA. Dimethyl sulphoxide (DMSO) at 10% was a relatively ineffective cryoprotectant with either semen diluent. Semen frozen at 6 cm above the surface of liquid nitrogen resulted in a higher post‐thaw sperm motility and fertility than semen frozen at 5 cm. The addition of 7% fresh egg yolk to glucose diluent containing methanol or DMSO did not improve the fertility of frozen‐thawed spermatozoa. However, the addition of 7% fresh egg yolk to glucose–DMA extender significantly improved the fertilization percentages of frozen‐thawed spermatozoa. In conclusion, dilution of semen 1:3 in 0.3 mol L^?1 glucose with 10% methanol and freezing 6 cm above the surface of liquid nitrogen (freezing rate of 40±8°C min^?1, mean±SD from ?5 to ?55°C) is a promising protocol for cryopreservation of Arctic char semen.     

超低温冷冻对西伯利亚鲟精子形态结构损伤的观察

为改进西伯利亚鲟精子超低温冷冻保存技术,探讨精子损伤机制,应用扫描和透射电子显微镜,观察了西伯利亚鲟鲜精与冷冻后精子的形态结构.结果显示,经过冷冻保存后精子的形态发生了很大变化.精子顶体长、精子头中部宽、头中部宽与前部宽比值及中段宽与鲜精相比显著增加(P<0.05);中段长度、后外侧延伸物长度比鲜精显著变短(P<0.05).精子经过冷冻后有30.5%的精子在形态、结构上受到不同程度损伤,受损精子显微结构表现为顶体后外侧延伸物与核糅合,顶体内容物丢失;核膜囊泡化、核膜断裂,核内出现空泡;线粒体内嵴弥散,线粒体脱落;鞭毛外膜松弛与鞭毛脱离等.部分受损伤精子出现顶体丢失,中段脱落,鞭毛自中段基部断裂的现象.精子损伤主要集中在膜系统,中心粒等微管系统基本完好.     

Changes in the flagellum morphology of intact and frozen/thawed Siberian sturgeon Acipenser baerii (Brandt) sperm during motility

Morphological investigations on the changes in flagellar beating was carried out on native (taken from the milt) and thawed sperm of the Siberian sturgeon Acipenser baerii (Brandt). Immediately after activation, the pattern of flagellar wave formation and distribution was the same in native and thawed sperm but, after 27–42 s, depending on the samples, the thawed flagella showed asymmetric and poorly developed waves. The swimming trajectories recorded during 1‐s exposure were much shorter in thawed than in native sperm after 26–28 s motility. In native sperm, the flagella remained in the same axis as the head during the entire motility course, while the head of thawed sperm showed a right angle after 47 s. It is concluded that the freezing/thawing procedure induces some alteration in the dynamics of flagellar beating in many sperm, but these sperm still show progressive displacement. Therefore, the change in morphology of the flagellum during motion is a parameter that should be taken into account in the evaluation of the impact of various treatments on sperm motility.     

The Use of Dairy Protocols for Sperm Cryopreservation of Blue Catfish Ictalurus furcatus

The commercial‐scale production of fish by use of artificial (induced) spawning would require reliable, large‐volume sources of sperm. Cryopreservation can be used to preserve and store sperm within commercial and research germplasm repositories, but is limited in its application to aquaculture. Straw volume and cooling chamber size restrict the quantity of sperm that can be frozen, and straws must be filled by hand. In contrast, the dairy industry has refined methods for freezing of bull sperm, including automation of straw filling and the use of large cooling chambers. These methods could be used for commercial‐scale cryopreservation of fish sperm, although application would require testing. To supply sperm in large volumes, bags originally developed for swine semen could be cooled using dairy protocols and used as a container for fish sperm. The current study documented the use of commercial‐scale dairy cryopreservation techniques for the production of hybrids of channel catfish Ictalurus punctatus (female) by blue catfish Ictalurus furcarus. Four cryoprotectants (methanol, dimethyl sulfoxide, dimethyl acetamide, and glycerol) were initially evaluated for use with blue catfish sperm. During May 2000 and March to April 2001, suspensions of blue catfish sperm were cryopreserved with 10% methanol in 0.5‐mL French straws and in commercial swine semen bags (Cochette^* bags, IMV International. Minneapolis, Minnesota, USA). Cryopreservation took place at a dairy breeding cooperative, using technology employed for bull semen. Sperm motility before freezing was 26 ± 18% during Year 1 (2000) and 62 ± 30% during 2001. Sperm were thawed at 40 C and used to fertilize the eggs of channel catfish (yielding hybrids). Motility after thawing for sperm frozen in 0.5‐mL straws was 11 ± 10% during 2000 and 50 ± 24% during 2001. Motility after thawing was 41 ± 17% for sperm frozen in swine semen bags in 5‐mL aliquots and 43 ± 10% for sperm frozen in 10‐mL aliquots. Neurulation of eggs fertilized with thawed sperm from straws was 83 ± 13% during 2000 and 54 ± 27% during 2001. Neurulation was 57 ± 24% using sperm frozen in swine semen bags in 5‐mL aliquots and 55 ± 10% using sperm frozen in 10‐mL aliquots. There was no correlation between sperm motility before freezing (in 0.5‐mL straws) and after thawing during 2000 (r= 0.52) or during 2001 (r= 0.49). In addition, there was no correlation between initial motility and neurulation of channel catfish eggs fertilized using thawed sperm during 2000 (r= 0.14) or during 2001 (r= 0.29). Sperm of blue catfish can thus be cryopreserved at a commercial scale using dairy protocols and can be made available for the production of hybrid catfish when viable eggs are available.     

Cryopreservation of scaly carp (Cyprinus carpio) sperm: effect of different cryoprotectant concentrations on post-thaw motility, fertilization and hatching success of embryos

The aim of the present study was to determine the effect of various cryoprotectants on post-thaw sperm quality and fertilizing capacity of cryopreserved scaly carp (Cyprinus carpio) semen. The present study focused on freezing of scaly carp sperm utilizing a practical and inexpensive protocol for aquaculture. Semen was diluted with Kurokura’s extender composing 3.6 g/l NaCl, 10 g/l KCl, 0.22 g/l CaCl₂, 0.08 g/l MgCl₂ and 0.2 g/l NaHCO₃. The extender contained three different cryoprotectants (DMSO, DMA and egg yolk) at ratios of 5, 10 and 15 %. Semen was placed into 0.25-ml straws and exposed to liquid nitrogen vapor (?120 °C) using an insulated box with an adjustable tray for 10 min and then plunged into liquid nitrogen (?196 °C) tank. The thawing process was performed in a water bath at 40 °C for 10 s. The results indicated that type of cryoprotectants and their concentrations are rather effective in scaly carp sperm cryopreservation on post-thaw sperm quality, while they are very important in order to obtain high fertilization rates. The highest fertilization rate was determined as 96.4 ± 0.15 % with 15 % egg yolk, while the highest hatching rate was determined as 99.3 ± 0.80 with 15 % DMA. In conclusion, the applied cryopreservation method for scaly carp sperm is suitable to fertilize high amounts of eggs.     

Cryopreservation of common carp sperm

Experiments were carried out to investigate the effect of five extenders (sucrose, glucose, fructose, KCl and a saline carp sperm extender) and two cryoprotectants (dimethyl-sulfoxide (DMSO) and methanol) on the cryopreservation of common carp sperm. Freezing of sperm using glucose extender and methanol as cryoprotectant resulted in the highest post-thaw motility, fertilization as well as hatching rates (63 ± 9%, 74 ± 15% and 67 ± 17% vs. 87 ± 5%, 84 ± 14% and 69 ± 14% using fresh sperm, respectively). In general, sugar-based extenders combined with methanol as cryoprotectant yielded higher motility, fertilization and hatching rates than ionic extenders in combination with DMSO. The jelly-like agglutination observed after thawing in samples frozen with sugar-based extenders did not reduce fertilization and hatching rates. Frozen–thawed sperm samples were able to successfully fertilize 10 g (8000) eggs.     

Cryopreservation of silver barb Puntius gonionotus (Bleeker) spermatozoa: effect of extender composition,cryoprotective agents and freezing rate on their postthawing fertilization ability

The effects of extender composition, cryoprotectant concentration and freezing and thawing on the fertilization efficiency of cryopreserved spermatozoa of Puntius gonionotus were evaluated. Computer‐aided motility analysis of semen was conducted to check the suitability of spermatozoa for cryopreservation after mixing with different extenders and cryoprotective agents (CPAs). Extender‐4 with an osmolality 260 mOsmol kg⁻¹and pH 7.6 was used for the cryopreservation study. Among the CPAs, dimethyl sulphoxide (DMSO) was least toxic and more than 60% fertilization was achieved when used at 1.4 M at 0 °C for 10 and 30 min, whereas the toxicity of all CPAs to spermatozoa was evident when tested at 30 °C. Semen frozen at −16 °C min⁻¹ with 1.4 M DMSO showed 70% fertilization, which was significantly higher (P<0.05) than other freezing rates. Samples thawed at 35 °C water showed a fertilization rate comparable with that of fresh semen. Computer‐assisted semen analysis of fresh and frozen semen after thawing showed variations in different types of motility in spermatozoa and in their class. There was no significant difference in motility before or after cryopreservation; however, significant differences could be observed in the average path velocity (VAP), straight line velocity (VSL) and curve linear velocity (VCL). Semen of silver barb could be cryopreserved with extender‐4 by addition of 1.4 M DMSO to a final cryopreservation medium (MED 2) cooled at a rate of −16 °C min⁻¹, stored in liquid nitrogen (−196 °C) and utilized after thawing at 35±2 °C.     

An Efficient Methodology for Cryopreservation of Spermatozoa of Red Seabream, Pagrus major, with 2-mL Cryovials

The aim of this study was to optimize the cryopreservation protocols for the sperm of red seabream, Pagrus major. The 2‐mL cryovials and programmable freezer were employed for cryopreservation. Six extenders, six cryoprotectants in various concentrations ranging from 6 to 20% (v/v), four cooling rates, and three thawing temperatures were evaluated by postthaw sperm motility and fertility. The ratio of sperm to egg for postthaw sperm fertilization trials was experimentally standardized and was optimal at 500:1. The best motility of postthaw sperm (79.4 ± 4.7% to 88.6 ± 8.0%), fertilization rates (89.6 ± 2.9 to 95.6 ± 1.9%), and hatching rates (85.3 ± 5.1% to 91.4 ± 4.3%) were achieved when Cortland extender, dimethyl sulfoxide (15, 18, and 20%) or ethylene glycol (9, 12%) as cryoprotectants, 20 C/min as the cooling rate, and 40 C as the thawing temperature were employed. Moreover, the results on embryonic development were not significantly different between cryopreserved sperm and fresh sperm during incubation process. In conclusion, these methods of cryopreservation of red seabream sperm are suitable for routine aquaculture application and preservation of genetic resources.     

Cryopreservation of Atlantic cod (Gadus morhua) sperm in large‐volume straws: applications for commercial production and gene banking

In our study, we used a full factorial analysis of variance design to examine the effects of diluent [Mounib's sucrose‐based diluent+hen's egg yolk (EY) and Hanks' balanced salt solution (HBSS)+EY], freezing rate (?2.5, ?5.0 and ?7.5 °C min^?1) and thawing rate (2.5, 5.0 and 7.5 °C min^?1) on motility and velocity of Atlantic cod sperm cryopreserved in 2.5 mL cryogenic straws. We found that post‐thaw sperm performance was strongly influenced by the presence of higher‐order interactions of the factors we tested. For all models broken down by diluent, the 2.5 °C min^?1 thawing rate had the lowest sperm motility recovery index. Mounib's sucrose‐based diluent+EY had the highest motility recovery index at all thawing rates. Mean per cent motility for fresh sperm (87.7±2.9%) was not significantly different than of sperm cryopreserved using Mounib's sucrose‐based diluent+EY, frozen at ?2.5 °C min^?1 and thawed at 5.0 °C min^?1 (77.1±2.9%). For Mounib's sucrose‐based diluent+EY, velocity was significantly higher with sperm thawed at 7.5 °C min^?1, than sperm thawed at 2.5 °C min^?1, while thawing rate had no effect for HBSS+EY. Our findings have implications for cod mariculture and aiding in conservation efforts for a dominant marine fish species.