三种人类精液冷冻程序的比较性研究

<正>冷冻方法直接影响到精子的复苏率,也是能否达到技术规范要求的重要指标之一。我们对精液的冷冻存储方法进行了研究分析,分别采用传统的缓慢冷冻储存法(缓冻法)、Planer程序降温仪 (PLANER-KRYO10 Ⅲ,Planer,英国)随机冷冻程序(Planer 随机程序)和Planer改进程序降温仪冷冻程序(Planer改进程序)3种方法行精液冷冻存     

冷冻前平衡处理对人精子冻融后相关参数的影响

目的:研究冻前平衡处理对冷冻精子复苏率的影响,优化一步熏蒸法,以寻找人类精子冷冻的最优方法。方法:使用50例自愿供精者的正常精液标本,同1例供精者的精液一式3份,分别采用不平衡直接冷冻法、室温平衡10 min后冷冻与4℃平衡10 min后冷冻的方法进行处理。复苏后用计算机辅助精液分析(CASA)系统分析3组标本冷冻前后精子运动参数的变化,并检测各组标本精子的畸形率和存活率。结果:4℃平衡组的前向运动精子复苏率[(61.88±16.94)%]显著高于未平衡组[(48.61±16.44)%]和室温平衡组[(49.41±13.77)%](P<0.05),而室温平衡组与未平衡组前向运动精子复苏率则无显著性差异。3组标本经冷冻复苏后其精子畸形率和存活率无显著性差异。结论:冻前4℃平衡10 min的冷冻前处理方法操作简单,实验条件易控,对提高精子复苏率有重要意义。     

溶脲脲原体感染者的精液冷冻保存观察

近几年来 ,由于溶脲脲原体 (UU)在人群中较高的感染率以及对生殖的影响已受到人们的广泛关注。为了研究精液感染 UU后的冷冻生物学表现 ,进行了本实验。一、材料和方法1.UU检测　自制 UU液体培养基 ,以尿素为 UU分解底物[1] 。将 0 .1～ 0 .2 ml精液滴入 2 ml UU液体培基中 ,36 .5℃培养 72 h。2 .精液标本　来源于不育门诊患者 ,液氮冷冻低温保存待测。3.实验一 :精液 UU培养阳性者的 UU培基冷冻后的表现 ,将 5例精液培养阳性者的UU培基置液氮冻存 ,分别于冻存后 1、2、4、8周取出融化 ,取 0 .2 ml滴入新的 2管 UU培基中进行 UU检…     

液氮蒸汽法冷冻精液前不同平衡条件对复苏效果的影响

目的研究精液冷冻前不同的平衡温度与时长对精子复苏率的影响,寻找最佳的液氮熏蒸法冷冻前的平衡条件。方法首先选取20例志愿者精液,每份精液分为4份,在室温下(20℃～25℃),分别采用不平衡直接冷冻法、平衡5min、10min、15min后冷冻,复苏后比较各组复苏率。然后再选取20例志愿者精液,每份精液分为4份,在低温下(4℃～6℃),不平衡直接冷冻、平衡5min、10min、20min后冷冻,复苏后比较各组复苏率。最后再选取20例志愿者精液,每份精液分为3份,比较不平衡直接冷冻法、常温下最佳平衡时长与低温下最佳时长3组的复苏率。结果室温下,平衡5min组复苏率为(66.6±8.0)%,显著高于其他组复苏率[对照组、平衡10min组和15min组分别为(63.5±6.3)%、(63.9±7.1)%、(61.8±7.2)%](P0.05)。低温条件下,平衡10min组复苏率为(66.0±3.2)%,显著高于其他组复苏率[对照组、平衡5min组和20min组分别为(62.7±3.6)%、(63.5±4.1)%、(63.1±4.0)%](P0.05)。室温平衡5min与低温平衡10min两组复苏率[(67.5±8.1)%vs.(68.3±6.4)%]无显著性差异(P0.05)。结论液氮熏蒸法冷冻精液前在室温平衡5min,简单方便,而且可获得较高的精子复苏率。     

不同保存方式对嗅鞘细胞活性的影响

[目的]探索嗅鞘细胞最佳保存方式.[方法]20例对数生长期OECs,随机分为5组,分别加入10%DMSO、5%DMSOfi%HES、5%DMSO,冰箱降温或程控降温仪降温并液氮保存,定期复苏,通过观察细胞形态、MTT 比色法、台盼蓝染色法检测细胞活性.[结果]同种降温方式下,5%DMSO-6%HES组细胞活性优于另两组,差异显著;同种低温保护剂下,冰箱降温与程控降温仪降温并液氮保存相比,差异不显著;同种保存方式下低温保存半年,各组回收率差异不显著.复苏标本不洗涤,室温下放置不同时间,细胞活性均下降,其中10%DMSO组下降最大.[结论]推荐5%DMSO-6%HES作为OECs冻存低温保护剂;小样本保存应选用冰箱降温液氮保存方式,大样本保存可选用程控降温仪降温或冰箱降温,并液氮保存方式;OECs低温保存半年仍具有较好的细胞活性.     

超低温液氮冷冻技术冻存复苏黑素细胞移植治疗大面积白癜风

目的:研究应用超低温液氮冷冻技术冻存复苏黑素细胞的生物学活性及分析自体黑素细胞移植治疗大面积白癜风的疗效。方法:负压吸疱获取患者正常表皮片,Hu16培养基体外培养黑素细胞,应用超低温液氮冷冻技术冻存复苏黑素细胞,检测黑素细胞的分裂时间(DOT)、黑素含量(M)、黑素制造量(MP)和树突数。受皮区应用超脉冲二氧化碳激光磨削去表皮后,以黑素细胞密度6～10×104个/cm2进行移植。所有患者均至少跟踪观察疗效6个月。结果:11例患者黑素细胞培养至第二代进行超低温液氮冷冻,15例患者黑素细胞培养至第三代进行冷冻,所有的26例患者冻存6、12和24个月复苏黑素细胞的生物学活性指标DOT、M、MP和树突数均与未冻存的细胞无明显差异。未冻存、冻存6、12和24个月的黑素细胞移植治疗的痊愈率为57.69%、61.54%、57.69%和53.85%,有效率为84.61%、88.46%、80.77%和80.77%。冻存6、12和24个月的黑素细胞移植的痊愈率和有效率与未冻存的黑素细胞移植的痊愈率和有效率均无显著性差异。结论:超低温液氮冷冻技术能很好的储存白癜风患者体外培养的黑素细胞,应用冻存复苏的黑素细胞自体移植治疗大面积白癜风皮损疗效确切。     

超低温液氮冷冻技术冻存复苏黑素细胞移植治疗大面积白癜风

目的:研究应用超低温液氮冷冻技术冻存复苏黑素细胞的生物学活性及分析自体黑素细胞移植治疗大面积白癜风的疗效。方法:负压吸疱获取患者正常表皮片,Hu16培养基体外培养黑素细胞,应用超低温液氮冷冻技术冻存复苏黑素细胞,检测黑素细胞的分裂时间(DOT)、黑素含量(M)、黑素制造量(MP)和树突数。受皮区应用超脉冲二氧化碳激光磨削去表皮后,以黑素细胞密度6～10×104个/cm2进行移植。所有患者均至少跟踪观察疗效6个月。结果:11例患者黑素细胞培养至第二代进行超低温液氮冷冻,15例患者黑素细胞培养至第三代进行冷冻,所有的26例患者冻存6、12和24个月复苏黑素细胞的生物学活性指标DOT、M、MP和树突数均与未冻存的细胞无明显差异。未冻存、冻存6、12和24个月的黑素细胞移植治疗的痊愈率为57.69%、61.54%、57.69%和53.85%,有效率为84.61%、88.46%、80.77%和80.77%。冻存6、12和24个月的黑素细胞移植的痊愈率和有效率与未冻存的黑素细胞移植的痊愈率和有效率均无显著性差异。结论:超低温液氮冷冻技术能很好的储存白癜风患者体外培养的黑素细胞,应用冻存复苏的黑素细胞自体移植治疗大面积白癜风皮损疗效确切。     

精子冻存技术及上游法精子处理技术对精子DNA完整性的影响

目的通过对冷冻前后及精子处理前后精子DNA完整性的比较,探讨冷冻技术、冷冻时间及上游法精子处理技术对精子DNA完整性的影响。方法（1）精液常规检测正常的患者30例,手淫法取精,精液液化混匀后分4份,分别用于精子染色质扩散（SCD）实验检测精子DNA完整性、上游法处理精液、以及两组冻存实验（不加保护剂直接冻存的为冻存1组;添加蛋黄葡萄糖保护剂冻存的为冻存2组）;（2）上游法处理后的精液一部分用于检测精子动力和形态,一部分用于精子DNA完整性检测;（3）冻存1组分别于冻存第7天和第90天解冻,SCD实验检测精子DNA完整性;（4）冻存2组分别于第7天和第90天解冻,0．1ml用于精子DNA完整性检测,剩余精液应用上游法处理,检测上游处理前后精子DNA的完整性。结果（1）冻存1组中,冻存90d后解冻的精子DNA损伤率[（25．6土7．3）％]显著高于冻存7d者[（22．4±7．4）％]（P〈0．05）,且均显著高于新鲜精液的精子DNA损伤率[（20．6±7．3）％]（P〈O．05）;冻存2组中,冻存90d后精子DNA损伤率显著高于冻存7d者[（25．9±7．2）％VS．（23．6土7．8）％]（P〈O．05）,且均显著高于新鲜精液（P〈0．05）;而冻存7d和90d后,两个冻存组间比较,精子DNA损伤率均无显著差异（P〉0．05）。（2）新鲜精液经上游法处理后,精子DNA损伤率由处理前的（20．6±7．3）％降为（6．4±2．5）％（P〈O．05）;冻存2组中精液冻存7d和90d后复苏上游法处理后,精子DNA损伤率较未经上游法处理者均显著降低[分别为（9．38±2．8）％VS．（23．6±7．8）％和（9．7±2．6）％VS．（25．9±7．2）％]（P％0．05）。结论冻存对精子DNA有损伤,冻存时间对于精子DNA完整性有影响。不添加保护剂直接冻存和添加保护剂对精子DNA完整性的影响无显著差异。不论是新鲜精液还是冻存复苏精液,上游法处理并不会增加精子DNA的损伤,且有利于筛选出具有更好DNA完整性的精子。     

深低温冷冻保存气管的实验研究

目的　寻找最适于冷冻气管的冷冻保护剂。方法　选择目前常用的几种冷冻保护剂 ,分别冷冻犬气管碎片。经程控降温 ,液氮冻储不同时间后复温 ,通过病理学、电镜以及酶组织化学检查 ,对其活性进行比较。结果　以 10 %二甲基亚砜 0 1mol L海藻糖作为冷冻保护剂冷冻的气管 ,复温后其活性明显高于其它冷冻保护剂 ,且冻储 8个月时气管的活性与冻储 1小时相比仅有微小变化。结论　程控降温 ,液氮冻储可以长期保存气管 ,10 %二甲基亚砜 0 1mol L海藻糖是目前冷冻气管的最佳冷冻保护剂配方。     

小牛血清及维生素对小鼠生精上皮细胞冻存的影响

目的 :探讨维生素C(VC)、维生素E(VE)及小牛血清 (NBS)对冻存小鼠生精上皮细胞的影响。　方法 :在DMEM培养液 10 %二甲基亚砜 (DMSO)的基础上 ,分别添加 0～ 2 0 %NBS ,以及在DMEM培养液 10 %DMSO 10 %NBS的基础上 ,分别添加 15 0 μg/mlVC及 5 0 μg/mlVE,冷冻保存 7d龄小鼠生精上皮细胞 ,37℃水浴复苏 ,锥虫蓝染色检查细胞复苏率。　结果 :冻存液中分别含 0、5 %、10 %及 2 0 %NBS时 ,细胞复苏率分别为 83.4 %、84 .7%、85 .7%及 83.6 % ,各复苏率之间差异无显著性 (P >0 .0 5 )。冻存液中分别含 15 0 μg/mlVC及 5 0 μg/mlVE时 ,细胞复苏率分别为 88.0 %及 82 .9% ,其复苏率与未加维生素的冻存液组 (85 .7% )相比差异无显著性 (P >0 .0 5 )。　结论 :在冻存液中添加不同浓度的NBS及添加抗氧化剂VC或VE,对小鼠生精上皮单细胞冻存没有明显的保护作用 ,不能显著提高细胞复苏率     

捐精者精子冷冻复苏率影响因素分析

目的:探讨季节、血型及精液参数等对捐精者精子冷冻复苏率的影响。方法:回顾性分析陕西省人类精子库捐精者4 088份精液标本,研究季节、血型、禁欲时间、精液量、精子形态、冷冻前精子活力及浓度对精子冷冻复苏率的影响。结果:捐精者精子冷冻复苏率随着精子浓度增高而增加,相关性分析提示精子浓度与冷冻复苏率呈正相关(r=0.247,P0.01)。而精子冷冻前活力和精子冷冻复苏率呈负相关(r=-0.262,P0.01)。禁欲第6天组的精子冷冻复苏率[(70.2±5.4)%]明显高于其他禁欲时间组(P0.01)。精子正常形态率20%组的精子冷冻复苏率[(71.4±5.1)%]要高于其他各组(P0.01)。A型血精子冷冻复苏率明显高于B型血[(69.1±4.8)%vs(69.8±4.7)%,P0.01];季节、精液量与精子冷冻复苏率之间无明显相关性(P0.05)。结论:捐精者的精子浓度、活力、形态及禁欲时间对于预测精子冷冻复苏率有一定的价值,而季节、血型、精液量与捐精者精子冷冻复苏率无明显相关性。     

Kruger strict morphology and post-thaw progressive motility in cryopreserved human spermatozoa

The purpose of this prospective study was to evaluate Kruger strict morphology and conventional semen analysis in predicting cryosurvival and the progressive motility recovery rate of frozen spermatozoa. Our study included 56 semen samples with >10 million spermatozoa per ejaculate. The main outcome measures were conventional semen analysis, strict morphology analysis by the Kruger method, cryosurvival rate and post-thaw sperm motility. A significant reduction in sperm motility after cryopreservation was demonstrated. The freeze-thawing process caused a 66% reduction in rapid progressive motile spermatozoa, a 45% reduction in slow progressive motile spermatozoa and a 2% reduction in nonprogressive motile spermatozoa. The cryosurvival and progressive motility recovery rates were not correlated with parameters of conventional semen analysis, such as sperm concentration, motility, WHO morphology and total motile count, but the progressive motility recovery rate was significantly correlated with the percentage of spermatozoa exhibiting Kruger normal morphology (P = 0.028). The recovery rate of rapidly progressive motility was profoundly decreased compared with slow progressive motility following the frozen-thaw procedure of semen. Kruger strict morphology assessment was a better predictor of the progressive motility recovery rate following the freezing-thaw procedure than parameters of conventional semen analysis.     

Effect of Cryoprotective Media and Dilution Methods on the Preservation of Human Spermatozoa

Human Sperm Preservation Medium (HSPM) containing 15% glycerol, 0.05 M sucrose and 1% glycine had an equal or better capacity to preserve sperm motility and vitality than egg yolk-citrate medium (ECM) containing 15% glycerol. Insemination of patients with semen frozen in HSPM resulted in a higher pregnancy rate than semen frozen in ECM. Glycerol was superior to dimethyl sulphoxide (DMSO) or ethylene glycol (EG) as a cryoprotectant, although the cryoprotective ability of 7.5% glycerol and 7.5% EG did not differ significantly. Neither the addition temperature nor the method of addition of the media containing glycerol, DMSO or EG had any influence on the sperm cryosurvival. Raffinose (0.025 to 0.05 M) or sucrose (0.05 to 0.1 M) improved cryosurvival of spermatozoa. The pH of HSPM had no significant influence on the cryosurvival of spermatozoa but the optimal pH was 6.5 to 7. Ratios of 1:1, 1:2, 1:3, and 1:4 of diluent to semen did not affect sperm cryosurvival.     

Cryopreservation of human semen. Comparison of cryopreservatives, sources of variability, and prediction of post-thaw survival.

Human semen was cryopreserved using Human Sperm Preservation Medium, TEST-Yolk buffer, or glycerol alone. Sperm characteristics for each specimen were measured before and after freezing to determine which cryopreservative resulted in better cryosurvival and recovery of motile sperm. Sperm frozen in Human Sperm Preservation Medium had a significantly better recovery of all semen parameters (motility, velocity, and recovery) than either TEST-Yolk or glycerol alone. Statistical analyses also were done to examine the variability between and within donor semen specimens. Differences between donors, between specimens, and measurements within donors all contributed to variability of sperm characteristics. Specimen-to-specimen variability for a given donor represented 12% to 47% of the total variability, whereas processing and measurement variability represented 12% to 41%. Donors also varied in the ability of their sperm to tolerate freezing. There was a relationship between motile count after dilution with cryopreservative and post-thaw motile count. This relationship allows the prediction of poor-thaw survival before freezing a specimen.     

季节因素对供精志愿者精液冷冻前后相关参数的影响

目的:本研究旨在了解季节因素对供精志愿者冷冻前后精液参数及前向运动精子冷冻复苏率的影响。方法:6414份精液样本均来自2006年9月～2008年6月在浙江省人类精子库捐精的1135例供精志愿者,年龄22～32岁;按取精时间划分为春、夏、秋、冬季组;对所有精液标本均进行精液常规分析,对其中达到精子库冷冻标准的精液进行分装、冷冻,并进行复苏后的精液常规分析。结果:精液量春季最多[(2.92±1.17)ml],明显高于夏、秋、冬3个季节[(2.71±1.07)、(2.74±1.15)、(2.83±1.15)ml],有显著性差异(P均<0.05);精子密度秋季最高[(105.60±39.76)×106/ml],明显高于春、夏、冬3个季节[(101.18±40.16)×106/ml、(93.54±35.10)×106/ml、(101.29±38.37)×106/ml],有显著性差异(P均<0.05);前向运动精子百分率春季最高[(58.49±10.04)%],明显高于夏、秋、冬3个季节[(57.60±8.97)%、(56.76±9.63)%、(56.60±8.56)%],有显著性差异(P均<0.05);夏季精液的前向运动精子冷冻复苏率最低[(66.98±15.68)%],明显低于春、秋、冬3个季节[(69.04±14.26)%、(69.35±13.42)%、(69.31±15.11)%],有显著性差异(P均<0.05);春季精液冷冻复苏后前向运动精子密度最高[(28.82±11.29)×106/ml],明显高于夏、秋、冬3个季节[(25.57±10.08)×106/ml、(26.97±9.68)×106/ml、(26.21±9.47)×106/ml],有显著性差异(P均<0.05);夏季精液冷冻复苏后前向运动精子百分率最低[(39.75±9.48)%],明显低于春、秋、冬3个季节[(41.87±9.28)%、(41.44±8.45)%、(41.03±9.13)%],有显著性差异(P均<0.05)。结论:季节因素对供精志愿者精液参数及前向运动精子冷冻复苏率有影响,春季精液量多、前向运动精子百分率高、冷冻复苏率高、冷冻复苏后前向运动精子密度高,是捐精的最佳季节。     

Comparative evaluation of fresh and washed human sperm cryopreserved in vapor and liquid phases of liquid nitrogen.

Infectious organisms such as hepatitis B were recently shown to survive in liquid nitrogen. To prevent cross-contamination of semen samples via liquid nitrogen, studies were undertaken to evaluate human sperm survival in the vapor phase of liquid nitrogen at -189 degrees C. The study was conducted in 2 separate experiments. In the first experiment, a total of 30 unwashed, fresh semen samples (15 normozoospermic and 15 oligozoospermic) were evaluated for motility, vitality, and morphology after freeze-thaw survival in vaporous (-189 degrees C) and liquid nitrogen (-196 degrees C; control) phases. Similar evaluations were carried out in a second experiment on 27 samples (15 normozoospermic and 12 oligozoospermic) that were previously washed by the swim-up method. Motile sperm recovery rates were significantly different between liquid and vapor phases (unwashed, normozoospermic: 42.76% +/- 3.23% vs 45.52% +/- 4.44%, P < .05; washed, normozoospermic: 34.44% +/- 4.41% vs 37.58% +/- 3.90%, P < .05; unwashed, oligozoospermic: 16.53% +/- 3.34% vs 18.25% +/- 4.36%, P < .05; washed, oligozoospermic: 10.32% +/- 2.54% vs 12.25% +/- 2.81%, P < .05). Recovery rates for motility were much higher for unwashed samples compared with washed semen samples. In all experiments the recovery of normal and live forms showed no significant differences between the vapor and liquid nitrogen storage phases (P > .05). The results demonstrate that both washed and unwashed human sperm survive satisfactorily with good recovery in the vapor of liquid nitrogen and can be recommended for future storage in medically assisted conception programs.     

速冻和缓慢冷冻法对精子运动特征的影响

目的了解冷冻方法对人精子运动特征的影响。方法精液标本进行速冻和缓慢冷冻保存,应用计算机精液分析仪进行精子运动特征分析。结果冷冻复温后精子运动能力与冷冻前精子运动能力比较明显下降(P<0.001,P<0.05);速冻与缓慢冷冻方法保存的精子运动参数相比较差异均无显著性(P>0.05)。结论冷冻保存易导致精子运动能力下降,速冻与缓慢冷冻方法对精子运动参数影响无明显差异。     

Cryopreservation alters the levels of the bull sperm surface protein P25b

Fertility of frozen-thawed bull sperm is reduced by cryopreservation. Freezing-thawing procedures can result in as much as a sevenfold fertility decrease. Sperm mortality and loss of motility do not fully explain the reduced fertility of cryopreserved semen; they may be partially explained by the loss of sperm surface proteins, which are necessary for fertilization. We have previously identified P25b, a sperm surface protein, which is associated with the fertility index of bulls used for artificial insemination. Using Western blotting techniques, we have evaluated P25b levels before and after cryopreservation of bull spermatozoa in extenders based on either egg yolk or milk. Long storage periods (28 days) in liquid nitrogen results in a threefold decrease of P25b levels associated with cryopreserved versus fresh spermatozoa. Over a short storage period (3-7 days), a stable P25b level was observed on spermatozoa cryopreserved in extender containing either egg yolk or milk. A decrease in P25b levels associated with spermatozoa was observed after 5 days of storage in egg yolk extender, whereas a significant decrease was observed after 14 days of sperm storage in milk extender (P < .05). Therefore, the loss of P25b may be responsible, at least in part, for the decrease in fertility following the freezing-thawing procedure of bull semen. Moreover, the cryopreservation extender used may have different effects on the loss of sperm surface proteins after even brief storage periods in liquid nitrogen. Considering that a sperm protein similar to P25b exists in humans (P34H), these results may have significant clinical applications in which frozen semen is used.     

Effect of pre-freezing conditions on the progressive motility recovery rate of human frozen spermatozoa

We evaluated the effects of sperm concentration, progressive motility, sperm morphology, duration of abstinence and collection season on the progressive motility recovery rate of human frozen spermatozoa to identify characteristics that predict the progressive motility recovery rate of human frozen spermatozoa and improve the protocol for sperm collecting in sperm banks. A total of 14 190 semen samples donated at Zhejiang human sperm bank of China between September 2006 and June 2011 were collected from 1624 donors. Semen was evaluated according to WHO standard procedures for sperm concentration. Progressive motility, sperm morphology, ejaculate collection season and abstinence time were recorded. After freezing and thawing, the progressive motility was assessed. Results showed that sperm concentration, progressive motility and normal morphology were significantly associated with the progressive motility recovery rate of human frozen spermatozoa. In addition, the abstinence time and collection season also significantly affected progressive motility recovery rate. Our results indicated that sperm concentration, progressive motility and normal morphology could be valuable in predicting the progressive motility recovery rate of human frozen spermatozoa. As such, progressive motility recovery may be improved by donating semen when abstinent for 3–5 days and during seasons other than summer.     

Influence of cryopreservation on the microelectrophoretic motility (EPM) of human spermatozoa

The net surface charge of spermatozoa represents a characteristic of the plasmamembrane. It can be evaluated indirectly, by the measurement of the sperm electrophoretic motility (EPM). The EPM of washed human spermatozoa was determined in the microelectrophoresis to investigate three problems: (a) intra- and interindividual variation coefficients of EPM of cryopreserved spermatozoa, (b) effects of cryopreservation medium, temperature shock and cryoprotection on the EPM, (c) relationship between the EPM and the cryotolerance of the spermatozoa. The inter- and intrain-dividual variation coefficients of the sperm EPM of 24 cryopreserved semen samples of 6 fertile semen donors amounted to 7% and to 6%, respectively. The cryopreservation did not cause a significant change of sperm EPM in contrast to the temperature shock in liquid nitrogen. Temporary incubation of spermatozoa with egg yolk decreased their EPM. A significant correlation between EPM and the cryotolerance ascertained by the alteration of sperm motile efficiency could be found. After contact with egg yolk spermatozoa achieved a behaviour that corresponded to a better cryotolerance.