鲑鳟鱼类精子超低温保存研究进展

鱼类精子超低温冷冻保存技术发展至今已有60年的历史,取得了巨大的成就,尤其在鲑科鱼类的精液冷冻保存及应用方面。经过统计发现,国外学者在精子超低温冷冻方面取得的主要突破都集中在鲑鳟鱼类领域。本文综述了鲑鳟鱼类精子超低温保存技术的发展史、相应的关键技术和冷冻保存研究的意义、基本生物学原理以及有关精液冷冻保存的研究现状与进展。     

鱼类精液超低温冷冻保存研究进展

鱼类精液超低温冷冻保存研究是从五十年代开始的,经过三十年的努力已取得很大成就。国外的工作主要是在海水鱼类和鲑科鱼类中开展的,并在某些鱼类,如鳕鱼、虹鳟、鲑鱼,大马哈鱼等的精液冷冻保存中获得成功。不少学者还对精液冷冻的原理,冷冻保存的技术环节如稀释液的配制、抗冻剂的种类及浓度、降温平衡、冷冻速度及保存方法等进行了较为详细的研究。我国在鱼类精液超低温冷冻保存方面的工作开展得比较晚。近年来,广东、广西和新疆等地对草、鲢、鳙、鲮等鱼的精液冷冻保存作了一些     

条纹锯鮨精液超低温冷冻保存研究

为建立条纹锯鮨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)下对条纹锯鮨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鮨精液,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(P0.05),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(P0.05)。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(P0.05)。研究表明,15%PG为条纹锯鮨最佳抗冻保护剂,可用于条纹锯鮨精液的超低温冷冻保存。     

棘皮动物精子超低温冷冻保存技术研究进展

棘皮动物(Echinodermata)是无脊椎动物中进化地位最高等的类群,其海参纲(Holothurioidea)和海胆纲(Echinoidea)的一些物种具有非常高的经济价值和营养价值。由于受到人类活动的影响,很多棘皮动物物种数量和生物多样性遭到了严重破坏,种质资源保存迫在眉睫。超低温冷冻保存是种质资源长期保存的重要方法,具有突破地理隔离、实现远缘杂交、保护种质资源、解决种质退化和保护濒危物种等作用。本文综述了海胆、海参和海星3种主要棘皮动物的精子超低温冷冻保存研究进展,并展开描述了精子收集、稀释剂和抗冻剂制备、平衡、冷却、解冻和质量评价等精子冷冻保存过程的各个步骤,以期为棘皮动物种质资源保存的进一步研究及产业化应用提供参考。     

鱼类精子冷冻保存研究进展

鱼类精子的长期保存对于遗传育种、生物多样性保护、濒危物种保护、渔业可持续发展具有重要意义,引起了人们越来越广泛的关注和重视.低温生物学的不断研究和发展为精子的长期稳定保存提供了理论和技术方面的支持.近年来鱼类精子的冷冻保存在保存原理和技术方面都取得了极大的成就,实现了多种鱼类精子冷冻保存的技术突破,并对冷冻保存的原理、冷冻损伤机制等方面有了更深刻的认识,在冷冻精子的生产应用方面也越来越趋于成熟稳定.本文仅针对鱼类精子冷冻保存的研究现状及发展前景作以阐述,以期对更加深入广泛的研究工作有所帮助.     

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。     

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。     

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)%。     

白鲢冻精后代的生长观察

鱼类精液是鱼类自然繁殖及人工授精的基础，其品质的优劣直接关系到后代的性状。近年来，鱼精冷冻保存作为鱼类选种育种的一项新手段为水产科技界所重视，并取得可喜的进展。实验证明，鱼类精液经超低温(-196℃)冷冻保存之后，解冻复苏的精子具有正常的授精能力且胚胎发育正常，     

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。     

Sperm quality and cryopreservation of Brazilian freshwater fish species: a review

The Brazilian freshwater fish diversity is the richest in the world. Only 0.7% of all Brazilian species have had any aspect of their sperm biology addressed up to this date. The majority of the fish species described in this review migrate during the spawning season (a phenomenon known as piracema). Urbanization, pollution, hydroelectric dams and deforestation are some of the causes of stock depletion or even local extinction of some of these species. The knowledge concerning sperm quality and minimum sperm:egg ratio is important to maximize the use of males without reducing hatching rates. Furthermore, sperm cryopreservation and gene banking can guarantee the conservation of genetic diversity and development of adequate breeding programs of native fish species. In this review, we present and evaluate the existing information on Brazilian fish species that have been subject to sperm quality and cryopreservation studies. The following parameters were evaluated: volume of extractable sperm, sperm motility, sperm concentration, freezing media, freezing methods, and post-thaw sperm quality. Although the existing protocols yield relatively high post-thaw motility and fertilization rates, the use of cryopreserved sperm in routine hatchery production is still limited in Brazil.     

Cryopreservation of sperm in marine fish

Since the first work of Blaxter in 1953, fish sperm cryopreservation has been attempted on about 30 marine species. The present paper reviews the techniques used and the results published in these species. Particular attention is paid to the handling procedure of sperm before freezing, the problems of semen ageing and semen contamination with urine. The quality of frozen–thawed semen was evaluated using previously standardized biotests, such as a two‐step motility activation technique adapted for the different species and fertilization assays using a discriminating insemination technique. Most extenders used in marine fish are saline or sugar solutions. From the investigated cryoprotectants, dimethyl sulphoxide (DMSO) generally leads to the best results. Cooling rates range from 8 °C to 99 °C min^?1; the thawing rate is generally high. Compared with freshwater species, a high percentage of spermatozoa survives cryopreservation. Therefore, and because of the simplicity of the techniques, the cryopreservation of marine fish sperm is suited for application in aquaculture.     

Development of cryopreservation for maintaining yellow catfish Pelteobagrus fulvidraco sperm

Yellow catfish (Pelteobagrus fulvidraco) is a candidate freshwater fish for aquaculture in China with its high consumer demand. The aim of this study was to examine the possibility of storage of the sperm of yellow catfish by cryopreservation in liquid nitrogen. Experiments were designed to investigate the effects of the different combinations of three extenders (Ringer extender, Kurokura-1 extender and D-15 extender) and three cryoprotectants (DMSO, Glycerol and Methanol) on the cryopreservation of yellow catfish sperm. Post-thaw sperm motility, fertilization and hatching rate were detected to evaluate the reliability of sperm cryopreservation. The results demonstrated that Ringer extender and 10% methanol was the best combination for protecting the sperm during freezing in liquid nitrogen by a three-step method and thawing in a water bath at 37 °C for 60 s. In this combination for cryopreservation, sperm maintained the highest post-thaw motility (65 ± 5%), fertilization (90.47 ± 3.67%) and hatching rate (88 ± 4%). And more interestingly, the fertilization and hatching rate were similar to those of fresh sperm (97.55 ± 2.74% and 92 ± 5%). Successful sperm cryopreservation techniques for yellow catfish have been developed for hatchery purpose.     

Technologies used in the study of sperm function in cryopreserved fish spermatozoa

Sperm cryopreservation has led to transcendental changes in the reproductive biotechnology of both mammals and fish, and is a basic tool for animal improvement. However, these protocols generate damage to cell structure and physiology, altering sperm function as a result of cryoinjuries during freezing and thawing. This review is a compilation of the techniques developed and standardised for assessing sperm function in cryopreserved fish semen. Recent studies have analysed sperm function objectively, applying cellular and molecular techniques to characterise cryodamage. The Computer Assisted Sperm Analysis system has facilitated the assessment of motility, while electron microscopy (SEM and TEM) and cryo‐microscopy have made it possible to study sperm morphology and ultra‐structure. The effects of cryodamage on nuclear DNA have also been analysed using various methods, including the comet Fluorescence in situ Hybridization test, TUNEL, Sperm Chromatin Structure Assay, specific DNA sequences using RT‐PCR and specific genes by qPCR. The latter technique is used to study the mitochondrial genome (mtDNA), together with some candidate genes which are associated with bioenergy activity and sperm motility. Other parameters assessed are mitochondrial membrane potential and ATP content using high performance liquid chromatography, nuclear magnetic resonance spectrometry and cell respiration. All this information makes it possible to establish study and assessment criteria for cryopreserved fish spermatozoa. This work focuses on the use of technologies to study of quality of fish spermatozoa during cryopreservation.     

A morphological classification proposal for curimba (Prochilodus lineatus) sperm damages after cryopreservation

The present study evaluated some cryoprotectants and concentrations for curimba (Prochilodus lineatus) semen preservation from a simple methodology of analysis of sperm morphology. Semen of nine males was diluted (1:4) into eight cryosolutions, all of them containing 5% of BTS^? Minitüb, still comprising methanol or dimethyl sulphoxide (DMSO) at four concentrations: 5%, 7.5%, 10% and 12.5%. Morphological sperm analysis was performed on an optical microscope at × 1000 magnification. Damages on the head, midpiece and tail rest (the main and terminal portions) were investigated and classified as primary or secondary, according to the assumed influence on fertilization. There was a linear reduction in the total damages with an increase in the cryoprotectant concentration. Samples with DMSO showed the lowest percentage of fractured tails and tail stumps. The cryopreservation protocol retained the fertilizing potential of the sperm after freezing and both methanol 5% and DMSO 7.5% conferred adequate protection to the curimba sperm cells. Sperm morphology, as pointed out here, must be incorporated into routine fish sperm (cryopreserved or not) analysis once sperm defects influence fertilization directly. To the best of our knowledge, this was the first report on curimba sperm damages after cryopreservation by optical microscopy. The classification model presented in this study can be adequate to tackle fish sperm damages.     

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.     

Factors Affecting Cryopreservation Efficiency and Enzyme Activity in Northern Pike,Esox lucius,Sperm

Abstract

Two freezing techniques (straws and pellets), three cryo-protectants (DMSO, glycerol, and DMA) in four concentrations, and several extenders were tested to determine their suitability for cryopreservation of northern pike, Esox iucius, sperm. Activity of aspar-tate aminotransferase (AspAT) and acid phosphatase (AcP) in cryo-preserved milt was determined. Fertilization ability of cryopreserved milt was affected by the freezing technique, by type and concentration of cryoprotectant, as well as by the kind of extender used. These factors also influenced AspAT and AcP activity assayed in cryopreserved sperm. Extender containing 0.6 M sucrose + 15% DMSO + 10% egg yolk was most suitable for cryopreservation of pike sperm in pellet form (90.5% of eyed eggs, as compared to control group, which was 89.1%).     

Cryopreservation of summer flounder,Paralichthys dentatus L., sperm

The summer flounder, Paralichthys dentatus L., is a high‐value species and considerable research has been conducted to determine practices conducive for its culture. As milt can be limited in this species, experiments were conducted to develop a practical sperm cryopreservation protocol for hatchery use. Two dilution ratios (1:2 and 1:4; sperm:extender), 2 diluents (saline and sucrose‐based), 2 cryoprotectants (10% DMSO and 12% glycerol) and 3 freezing rates (?5, ?10 and ?15°C min^?1) were evaluated using differential staining to assess post‐thaw sperm survival. Seven combinations of the factors examined reduced post‐thaw viability by less than 30%. The average viability of sperm from fresh, pooled flounder milt (67.2 ± 2.9%) was not different from that of thawed milt diluted 1:4 with sucrose diluent (10% DMSO) frozen at ?5°C min^?1 (38.4 ± 7.7%) and fertilization and hatch success were not different in trials using fresh or thawed, cryopreserved sperm. From these experiments a practical sperm cryopreservation method was developed, but further refinement of the freezing protocol is necessary to optimize results.