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。     

半滑舌鳎精子冷冻保存

半滑舌鳎精子冷冻保存对于人工繁殖育苗、杂交育种、雌核发育及其性别控制研究具有重要的意义，为此，本文对半滑舌鳎精子冷冻保存方法进行了研究。分别利用2．8mol／L的二甲基亚砜（DMSO）、甘油（Gly）和1，2-丙二醇（PG）冷冻保存该鱼精子。结果显示，DMSO冷冻保存精子的活力较高。利用MPRS＋2．8mol／L DMSO以1：0．5、1：1、1：1．5和1：2的比例稀释并冷冻精子，1：1比例在冻前能够抑制精子的运动，冻后活力可达82．50±3．54％，显著高于其他稀释比例（P〈0．05）。分别利用冷冻保存液A（MPRS＋2．8mol／L DMSO）和B（TS-2＋2．8mol／LDMSO）稀释平衡精子，精子在A中的冻前快速运动时间、寿命分别为37．75±6．45S和145．00±78．98S，与鲜精无显著差异（P〉0．05）。利用以上两种冷冻稀释液冷冻保存精子,精子在A液中的冻后活力和寿命分别可达53．50±6．69％和98．00±13．51s，冷冻效果优于B液（P〈0．05）。冷冻后精子的受精率和孵化率分别为55．00±5．00％和35．00±13．23％，受精率与鲜精无显著性差异（P〉0．05），因此认为MPRS＋2．8mol／L DMSO可用于半滑舌鳎精子的冷冻保存。     

刀鲚精子超低温冷冻保存技术的研究

本文对刀鲚精子的超低温冷冻保存技术进行了研究.以解冻后精子的运动力为参数,分别探讨了不同的稀释液、不同种类不同浓度的保护剂、不同的平衡时间以及不同的稀释比例对刀鲚精子进行超低温冷冻保存的保护效果.结果显示:采用D-15稀释液,将精液和稀释液按1:2稀释,4℃平衡20 rmin,加入10％ DMSO,混匀后液氮面上方6 cm处平衡10 min,接着在液氮面上平衡5 min,最后投入液氮中保存,一周后,液氮蒸气中平衡5min,37℃水浴解冻,活力效果最佳,达70％左右.     

畜禽冷冻精液稀释液的研究进展

畜禽精液冷冻保存是人工授精技术的一个重大发展。在精液冷冻保存中稀释液起关键作用。稀释液通过粘附到精子膜上,保护膜结构,增加膜流动性,调低精子对冷休克的易感性,增加了精子冻存能力及解冻后精子活力。     

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。     

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。     

鲤、鲢、鳙精子低温短期保存研究

本文对鲤(Cygrinus carpio)、鲢(Hypophthalmichthys molitrix)和鳙(Aristichthys nobilis)精液在2—4℃的短期保存进行了实验研究。筛选出了几种较为理想的稀释保护液配方;确定了精液与稀释液的适宜稀释比例为1:1;研究了抗冻剂二甲亚砜(DMSO)对低温保存条件下精于活力的影响,确定了DMSO的适宜添加浓度为6％左右。鲤精在2—4℃保存10天,活力仍高达70％,少数精子存活时间长达12天;鲢和鳙精子在2—4℃保存7天后活力仍高达60％,达到了生产应用的水平。为水产养殖和鱼类育种研究提供了一种简便易行的精液短期保存技术。     

珍珠龙精子超低温保存的初步研究

为研究适合珍珠龙精子的短期超低温保存方法,比较了不同保存液、保护剂、降温方式、解冻方式对珍珠龙(Scleropages jardini)精液超低温保存后精子活力的影响,初步解释了经超低温短期保存后珍珠龙精子活力明显较鲜精低的原因.结果显示:采用Ringer's液作为保存液,以浓度为16％的二甲亚砜作为保护剂,选取五步法超低温冷冻保存精子[精子缓慢降温至-150℃→液氮面上3 cm(约-170℃),停留4min→液氮面(约-190℃),停留1 min→液氮],40℃水浴解冻,可取得最好的冻后活力,解冻后精子的活力为(36.7±4.1)％.     

不同冷冻保护剂在猪精液冷冻中的作用分析

本实验采用一定浓度的甘油、EG、DMA、DMSO及其两两组合物作为冷冻保护剂。用含冷冻保护制的稀释液将精液稀释后,常温保存,观察精子活率,比较精子生存指数。结果表明：在常温下对精子毒害作用最大的是DMSO,其次是甘油,EG和DMA对精子毒害作用最小。以一定浓度的10种冷冻保护剂将对精液冷冻后观察解冻活率,发现EG和DMA混合保护剂解冻后精子活率最高。     

The development of a sperm cryopreservation protocol for winter flounder Pseudopleuronectes americanus (Walbaum): evaluation of cryoprotectants and diluents

Three cryoprotectants (dimethyl sulphoxide, propylene glycol and glycerol) and two diluents (sucrose based and saline based) were mixed (9 parts diluent–1 part cryoprotectant) factorially to produce six extenders that were tested to develop an effective sperm cryopreservation protocol for winter flounder Pseudopleuronectes americanus (Walbaum). Sperm were diluted 1:3 with each extender and frozen by flotation on liquid nitrogen before being submerged and stored for 30 days. Sperm left unfrozen in each extender for 20 min showed no toxic effects on motility. Extenders containing propylene glycol (PG) as cryoprotectant yielded higher post‐thaw sperm motilities than those containing dimethyl sulphoxide (DMSO) or glycerol. The sucrose‐based diluent performed better than the saline‐based diluent when DMSO was used as cryoprotectant, but there were no differences in post‐thaw motility between diluents for the other cryoprotectants. Activating sperm with ovarian fluid and sea water instead of sea water alone had no effect on post‐thaw motility. In fertilization trials, no differences were observed between any of the extenders and fresh milt when milt, eggs and sea water were left in contact for 1 h. When sperm were forced to compete for eggs by reducing contact time to 20 s, fertilization results followed those of sperm motility rates. Percentage hatch and morphology of larvae at hatching did not differ for eggs fertilized by cryopreserved and fresh sperm. This study represents the first reported successful attempt at cryopreserving winter flounder sperm and should improve gamete and broodstock management protocols for this species.     

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.     

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.     

Cryopreservation of Arctic charr, Salvelinus alpinus (L.), semen in various extenders and in three sizes of straw

Cryopreservation of Arctic charr Salvelinus alpinus (L.) semen was investigated using three diluents, three cryoprotectants [10% dimethyl sulphoxide (DMSO), 10% dimethylacetamide (DMA) or 20% glycerol] and three sizes of straw. The three diluents and three cryoprotectants were combined, resulting in nine extenders. One part semen was added to three parts extender, and motility was evaluated to assess the toxicity of six of the extenders. Semen in nine extenders was frozen in 0.5‐mL straws using liquid nitrogen vapour. Semen extended in 0.3 m glucose and each of the cryoprotectants was also frozen in 0.5‐mL, 1.7‐mL (flat) or 2.5‐mL straws. The freezing rate in each size of straw was measured. Fertility trials were conducted to determine the post‐thaw viability of the frozen semen. The motility of activated spermatozoa was higher in the DMA and DMSO extenders than in the glycerol extender. For the trial using 0.5‐mL straws, post‐thaw fertility results were higher for all extenders containing DMSO, or 0.3 m glucose and DMA, than for all other combinations of diluent and cryoprotectant. For the straw size comparison, the highest fertility was obtained for the 1.7‐mL straw using either DMSO or DMA and for the 2.5‐mL straw using DMSO. For all cryopreservation trials, fertility was low for extenders containing glycerol.     

七带石斑鱼和云纹石斑鱼幼鱼在封闭循环水条件下的生长特性

观测研究了七带石斑鱼(Epinephelus septemfasciatus)幼鱼和云纹石斑鱼(Epinephelus moara)幼鱼在封闭式循环养殖系统中的生长特性,用SPSS 17.0软件中的Curve Estimation对相关数据进行模型分析与参数估计。结果表明,经过122 d的养殖,七带石斑鱼幼鱼平均体质量由(114.836±25.343)g增加到(213.861±38.604)g,相对增长率为0.707%,全长(TL)与养殖时间(t)的函数关系式为TL=-0.006t3+1.622t+13.954,体质量(W)与体长(BL)的关系式为W=0.436BL2.055;云纹石斑鱼幼鱼平均体质量由(79.620±13.007)g增加到(238.086±46.307)g,相对增长率为1.631%,全长(TL)与养殖时间(t)的函数关系式为TL=-0.013t2+2.008t+11.540,体质量(W)与体长(BL)的关系式为W=0.018BL3.083。两种幼鱼丰满度生长差异不显著(P0.05),都保持在2.2-3.4之间。     

Motility parameters of perch spermatozoa (Perca fluviatilis L.) with cryoprotectors addition

The addition of cryoprotectants during the freezing of semen in liquid nitrogen protects spermatozoa from the negative influence of freezing. Every species needs an appropriate cryoprotectant that has to be experimentally selected. Semen obtained from five perches was diluted with the Kobayashi buffer solution at 1:9 ratio. To determine the influence of cryoprotectants on spermatozoa motility parameters, the same type of buffer solution was applied with the addition of methanol, dimethyl sulfoxide (DMSO) and dimethylacetamide (DMA) using the concentration of 10, 5, 2.5 %, respectively, glycerol (15; 7.5 %), sucrose and trehalose (0.45; 0.225; 0.113 M). After the preparation of such tests, parameters of spermatozoa motility were measured, using the CASA system (Image House CRISMAS Company Ltd.). Among used cryoprotectants, methanol did not cause any effect on the sperm motility parameters. The lowest percentage concentrations of DMA, DMSO, glycerol, sucrose and trehalose did not significantly influence the percentage of motile spermatozoa. Higher concentrations of these compounds considerably lowered all motility parameters. As for glycerol and saccharides, their addition resulted in the lowering of the spermatozoa motility possibly due to a higher viscosity of the solution. However, DMA and DMSO were most probably toxic to perch sperm cells. The obtained results indicate that the best cryoprotectant to be used with perch spermatozoa is methanol.     

Suitable methods for cryopreservation of semen from Atlantic halibut, Hippoglossus hippoglossus L.

Decrease in the quality and quantity of Atlantic halibut, Hippoglossus hippoglossus L., semen towards the end of the reproductive season hampers production of good-quality embryos. Therefore, cryopreservation of spermatozoa is a method showing potential to facilitate controlled reproduction in Atlantic halibut. The present study aimed at establishing the appropriate cryopreservation procedure. We tested 20 extenders composed of four various diluents and five cryoprotectants (DMSO, DMA, methanol, propylene glycol, and glycerol) to determine the best extender. Then, we examined cryopreservation quality using various methods of loading and various volumes of cryopreserved samples. In most of the tested variants, sperm diluted with an extender showed high motility after 24-h incubation despite the high osmotic pressure of the extender. Modified turbot extender (MTE) was the best of the tested diluents, securing the highest post-thaw motility (P < 0.05), and DMSO, DMA, and methanol were the best cryoprotectants (P < 0.05). There was no significant effect of 15-min equilibration of semen in MTE-based extenders prior to freezing on post-thaw motility (P > 0.05). MTE-based extender was chosen as the most suitable. Semen cryopreserved in straws, Eppendorfs or Ziploc bags in volumes ranging from 0.25 to 20 ml showed similar high fertilization ability. Survival of larvae produced with the cryopreserved sperm did not differ from controls produced with freshly collected sperm. Motility 3 h after thawing was high but depended on the type of cryoprotectant and the volume of cryopreserved sperm (P < 0.05). The developed cryopreservation procedure has been applied at our Atlantic halibut breeding station for seed production.     

Study on cryogenic preservation of grey mullet sperm

Preliminary techniques of mass propagation of grey mullet were established in Taiwan some years ago. Among the many problems concerned, the cryogenic preservation of grey mullet sperm has been studied since 1971. The aim of this study is not only to ensure the availability of mullet semen anywhere and anytime it is needed, but also to contribute to the international cooperation of cross breeding of grey mullet in the future. Results of the study on cryogenic preservation of grey mullet sperm made during the experimental mass propagation of mullet in the Tungkang Marine Laboratory for the past 3 years are summarized in this paper.The gonadosomatic index (GSI) of male grey mullet migrating near the coast of Tungkang ranged from 4 to 20 during the spawning season. The pH value of the semen was 7.4. Each spermatozoon was composed of a head part measuring 2.3 μ × 1.4 μ and a tail part four to five times as long as the head. There were about 5.3 × 10¹⁰ sperms in each ml of semen. Eosin-nigrosin staining was used for clearer identification. Sperm motility was preserved for up to 23 days in the case of raw semen at 5° C. Cryoprotective agents were needed at the ultra-low temperature (?196° C) of preservation in liquid nitrogen. Feasible procedures of freezing the grey mullet sperm were determined. Fresh semen diluted with cryoprotective agents was dispensed into 0.5 ml straws which were then sealed. These pretreatments prior to cryopreservation had to be done within the correct equilibration time of 1 h or less. Semen in straws was precooled in liquid nitrogen vapor until a temperature of ?80° C was reached. Straws in the canister were then put into liquid nitrogen for long-term preservation. The optimum effect of cryoprotective agents was found with 5–10% glycerine or dimethylsulfoxide (DMSO) at 1:1, 1:5, and 1:10 dilutions. In this condition, both good motility and fertility before freezing and cryoprotection were obtained. So far the best result of frozen thawed mullet sperm was moderate motility and 2.7% fertility of the semen cryopreserved for 1 year and 4 days.     

日本蟳精子超低温冷冻保存技术的研究

以精子存活率作为评价指标,采用两步降温法研究了稀释剂、抗冻保护剂和预冷时间对日本蟳精子超低温冷冻保存效果的影响。结果表明:以采用稀释剂II、15%二甲基亚砜(DMSO)作为抗冻保护剂的保存效果最佳。在液氮中保存24 h后,精子存活率可达83.76%,保存一年后可达73.81%。精液的第一次预冷时间以25 min为宜。     

Cryopreservation of sperm in farmed blacklip abalone (Haliotis rubra Leach, 1814)

This study developed a technique of sperm cryopreservation using liquid nitrogen (LN) vapour in farmed blacklip abalone Haliotis rubra through evaluating the following five key factors: (1) cryoprotectant agent (CPA) toxicity; (2) cooling temperature; (3) thawing temperature; (4) sperm to egg ratio and (5) sugar addition, using sperm motility or fertilization rate as quality assessment indicators. The results demonstrated that 6% dimethyl sulfoxide (DMSO) was the best single CPA for sperm cryopreservation in this species. The highest post‐thaw sperm motility was achieved when sperm were exposed to LN vapour for 10 min at 5.2 cm above the LN surface and thawed at 60°C and recovered at 16°C in seawater baths. Post‐thaw sperm motility was found to be significantly higher when 6% DMSO was used in combination with 1% or 2% glucose than 6% DMSO alone. Further evaluation of fertilization rate between these CPAs showed that 6% DMSO+2% glucose achieved the highest fertilization rate of 70% at a sperm to egg ratio of 10 000:1.