补肾填精方对电磁辐射所致小鼠睾丸组织SOD活性和MDA含量的研究

目的:探讨移动电话的电磁辐射对雄鼠生殖功能的影响及补肾填精方的预防作用。方法:用移动电话的模拟辐射源对各组小鼠(空白组除外)进行全身辐射,辐射频率为900MHz,每天2h,连续35d。按平均功率密度分为4个组,即空白组(0uW/cm2)、低辐射组(33uW/cm2)、高辐射组(181uW/cm2)和补肾组(181uW/cm2)。辐射期间,补肾组每天给予0.15mL/10g体重的补肾填精方浓缩液(含生药量0.9g/mL)灌胃,其他各组给予等量生理盐水灌胃。35d后取双侧睾丸和附睾称重,检测睾丸组织超氧化歧化酶(SOD)活性和丙二醛(MDA)水平。结果:各组间的睾丸、附睾及其脏器系数无显著性差异(P0.05);高辐射组睾丸组织SOD活性(13.98±1.64)u/mgprot明显下降、MDA水平(1.26±0.11)nmol/mgprot明显上升(P0.05)。结论:移动电话的电磁辐射可以降低睾丸组织SOD活性并升高睾丸组织MDA水平,补肾填精方对其病理损伤有着一定的预防作用。     

巴戟天根两种提取物对微波损伤大鼠生精功能的影响

目的:探讨巴戟天根不同浓度提取物对微波损伤的雄性SD大鼠生精功能的影响。方法:40只健康雄性SD大鼠先分为正常对照组和辐射组,辐射后再将辐射组分为辐射模型组,巴戟天根水提物治疗组和巴戟天根醇提物治疗组,每组10只。辐射组应用微波信号发生器(900 HZ 1.0 W),功率密度为218μm/cm2,12 h/d,持续辐射2周。治疗组在辐射后分别给予巴戟天根水提物和巴戟天根醇提物20 g/(kg.d)持续灌胃2周。观察各组大鼠生长发育,扑捉潜伏期(CIP)和扑捉次数(CT),睾丸和附睾指数及精子形态学的差异,精子浓度、精子畸形率以及血清睾酮的浓度。结果:与正常对照组[(269.50±36.07)g]相比,辐射模型组[(254.77±20.38)g]大鼠体重稍降低,CIP延长及CT减少(P<0.05),精子浓度[(87.717±12.365)×106/ml]降低及精子畸形率[(0.126±0.100)×106/ml]明显升高(P<0.05);睾丸和附睾出现不同程度的病理损伤改变,睾丸指数降低,血清睾酮水平无明显变化。两治疗组较正常对照组体重显著下降(P<0.05),血清睾酮的水平显著升高,且与辐射模型组相比CT增加、CIP缩短、精子浓度显著升高、畸形率显著下降、血清睾酮的水平升高(P<0.05)。治疗组睾丸的病理性损伤显著修复,附睾管内除见大量精子外,还可见大量脱落的细胞。结论:巴戟天根的水提取物和醇提取物均可促进微波辐射损伤的生殖器官的修复以及精子的生成。     

男性不育患者精子DNA完整性与精浆氧化应激水平的关系及其对体外受精的影响

目的:探讨男性不育患者精子DNA完整性与精液常规参数、精浆氧化应激水平的关系,以及对体外受精(IVF)结局的影响。方法:采用精子染色质扩散法(SCD)检测433个IVF周期中精子DNA损伤性,根据精子DNA碎片指数(DFI)的不同,分为低DFI组(DFI30%)、高DFI组(DFI≥30%),比较2组精子浓度、前向运动精子百分率、快速前向运动精子百分率、精浆丙二醛(MDA)、总抗氧化能力(TAC)水平及受精率、卵裂率、优胚率的差异。结果:高DFI组与低DFI组比较,前向运动精子百分率及快速前向运动精子百分率显著降低[(29.2±16.8)%vs(48.6±16.7)%、(9.4±6.6)%vs(19.0±9.1)%,P0.01],高DFI组精子浓度与低DFI组相比差异无显著性[(52.3±32.4)×106/ml vs(51.4±30.9)×106/ml,P0.05];高DFI组精浆中的MDA含量明显高于低DFI组[(2.28±0.26)nmol/L vs(0.95±0.18)nmol/L,P0.01],高DFI组精浆中的TAC含量明显低于低DFI组[(10.2±3.5)U/L vs(33.2±7.9)U/L,P0.01];高DFI组IVF受精率明显低于低DFI组[(58.9±30.0)%vs(77.2±25.0)%,P0.01],两组间卵裂率[(70.7±35.6)%vs(80.4±15.6]、优胚率[(40.4±31.3)%vs(41.7±29.4)]比较差异均无显著性(P均0.05)。结论:精子DNA损伤与氧化应激有关,同时精子DNA损伤可降低IVF受精率,影响IVF结局。     

移动电话辐射对雄性大鼠精液质量的影响

目的 探讨移动电话电磁辐射对成年雄性大鼠精液质量影响.方法 根据移动电话辐射,将30只SD雄性大鼠随机分为A组,B组与C组,每组10只,A组和B组分别每天辐射2h和4h,C组未辐射,饲养30d后处死,对3组精子密度、精子活动率、精子形态进行观察,分析3组观测指标是否有显著差异.结果 C组精子密度、精子活动率分别为(54.18±2.93)×106/ml、(69.63±2.62)％,A组精子密度、精子活动率分别为(50.80±2.39)×106/ml、(66.40±2.72)％,A组与C组比较,差异有统计学意义(P＜0.05);B组精子密度、精子活动率分别为(40.45±2.46)×106/ml、(63.90±2.84)％,B组与C组比较,差异也有统计学意义(P＜0.01).精子正常形态比例A组、B组和C组分别为(69.90±4.95)％、(69.27±4.29)％和(71.82±4.12)％,A组、B组分别与C组比较,差异无统计学意义(P＞0.05).结论 移动电话辐射可引起雄性大鼠精子密度下降、精子活动率减少,且与辐射时间呈正相关；精子正常形态比例与辐射无关.     

不育患者精子DNA链损伤类型的研究及临床意义

目的:观察精子DNA单、双链损伤(SSB、DSB)在男性不育中的特征,探讨DSB与男性不育的关系,为男性不育的诊疗提供新的观察指标与思路。方法:选择男性不育患者60例以及同期因女方因素不孕前来检查的生育力评估正常的健康男性30例作为对照组,分析两组精子DNA损伤及精液主要参数的差异。精子浓度及活力采用计算机辅助精子分析系统,精子存活率分析采用低渗膨胀试验,精子形态采用Diff-Quik染色法,精子DNA损伤采用双尾彗星实验。结果:双尾彗星实验检测精子DNA完整性共有9种彗星模型。不育患者精子DNA损伤指数(DFI)为(33.8±13.1)%,单链损伤指数(SSB-DFI)为(19.2±11.4)%、SSB占所有损伤精子的比率(SSB-DFI/DFI)为(56.8±32.4)%,双链损伤指数(DSB-DFI)为(23.9±13.4)%、DSB占所有损伤精子的比率(DSB-DFI/DFI)为(70.8±19.5)%;与对照组比较[分别为(16.3±7.9)%、(14.9±7.6)%、(91.4±27.8)%、(6.1±2.7)%、(37.4±11.3)%)],SSB-DFI无显著性差异(P0.05),DSB-DFI、DFI和DSB-DFI/DFI显著高于对照组(P均0.01),SSB-DFI/DFI显著降低于对照组(P0.01)。绘制ROC曲线,DSB-DFI/DFI、DSB-DFI及DFI诊断男性不育最佳截断值分别为39.5%、15.85%和18.65%,ROCAUC、敏感度和特异性分别为(0.969,98.3%,90.0%)、(0.912,86.7%,80.0%)、(0.861,90.0%,70.0%)。不育组精子SSB-DFI及SSB-DFI/DFI与精液常规参数均无相关关系(P均0.05),DFI与前向运动精子百分率、精子存活率及正常形态精子百分率呈负相关(P0.05或P0.01),与精子浓度无相关关系(P0.05),精子DSB-DFI及DSB-DFI/DFI与精子浓度、精子存活率、前向运动精子百分率及正常形态精子百分率均呈负相关(P0.05或P0.01)。结论:影响男性不育的DAN损伤因素可能是DSB,而与SSB相关性不大,精子DNA链的损伤类型对男性生殖能力的评估有较大的参考价值。     

RHO/ROCK信号通路在精子抗冷冻损伤中的作用

目的:探讨RHO/ROCK信号通路在人精子抗冷冻损伤中的作用,为高效精液冷冻保护剂的研制提供理论依据。方法:选取健康精液25份,每份精液分为新鲜组、对照组与RHO通路抑制剂组(抑制剂组)。检测冷冻前后各组精液精子活力、精子存活率、精子膜完整率、正常形态精子百分率、精子DNA碎片指数(DFI)、精子顶体酶活性及精子线粒体膜电位变化;免疫荧光染色检测RHOa/ROCK蛋白在精子中的表达。结果:抑制剂组精液冷冻复苏后精子活力[(57.50±6.83)%vs(51.20±7.70)%,P=0.002]、精子存活率[(60.24±5.53)%vs(52.87±5.07)%,P=0.001]、精子膜完整率[(67.10±4.43)%vs(59.78±5.56)%,P=0.001]、正常形态精子百分率[(7.46±1.28)%vs(4.83±1.11)%,P=0.001]、精子DFI[(18.87±4.07)%vs(27.64±6.64)%,P=0.001]、精子线粒体膜电位(63.11±2.97 vs 56.30±4.28,P=0.001)指标均明显优于对照组;抑制剂组冷冻后精子顶体酶活性与对照组差异无统计学意义(98.30±11.33 vs 97.65±9.31,P0.05)。免疫荧光染色显示RHOa/ROCK蛋白在精子头、颈部广泛表达。结论:RHO/ROCK信号通路在精子冷冻损伤中具有一定作用,抑制其通路活性可明显提高精子抗冷冻损伤的能力。     

加味大黄蟅虫颗粒对精索静脉曲张模型大鼠附睾结构改变的干预作用

目的:探讨实验性精索静脉曲张大鼠附睾管精子发育成熟的改变及加味大黄蟅虫颗粒对附睾精子成熟的影响。方法:60只SD雄性大鼠随机分为模型组、假手术组、迈之灵组及加味大黄蟅虫颗粒低、中、高剂量组,每组10只。除假手术组外,各组按Turner方法制作左侧精索静脉曲张大鼠模型。造模完成后,假手术组及模型组大鼠均给予生理盐水;迈之灵组给予迈之灵片54 mg/kg体重药量溶于生理盐水中;加味大黄蟅虫颗粒低、中、高剂量组分别予以含生药0.6、1.2、2.4 g/ml加味大黄蟅虫颗粒水冲剂。各组皆按1 ml/100 g体重灌胃,每日1次,连续8周。给药8周后全部处死各组实验大鼠,取出左侧附睾,检测附睾精子质量及局部显微超微结构的改变,并行组间对比。结果:模型组大鼠附睾管局部上皮细胞退化变性,部分主细胞及基细胞空泡变性,管腔内见各级未成熟精子细胞。与模型组[(12.77±6.25)%]比较,加味大黄蟅虫颗粒中[(32.23±6.55)%]、高[(39.39±8.53)%]剂量组和迈之灵组[(31.10±7.77)%]大鼠附睾精子活率升高(P0.01)。与迈之灵组比较,加味大黄蟅虫颗粒高剂量组附睾精子活率[(31.10±7.77)%vs(39.39±8.53)%]及浓度[(10.69±4.92)×10~6/ml vs(36.37±7.09)×10~6/ml]明显增高(P0.05)。结论:实验性精索静脉曲张大鼠附睾管局部细胞凋亡明显、间质水肿、微血管扩张,加味大黄蟅虫颗粒能提高附睾培育精子成熟的稳定性,为精子的发生发育创造有利的条件。     

通精灵对实验性精索静脉曲张大鼠精子DNA完整性与睾丸组织氧化应激的影响

目的:探讨通精灵对实验性精索精索静脉曲张大鼠精子DNA完整性与睾丸组织氧化应激的影响。方法:雄性Wistar大鼠75只随机分为假手术组、模型组、高剂量组、中剂量组、低剂量组,每组15只。参照文献制备大鼠精索静脉曲张模型,"夹尾激怒法"建立合并中医肝气郁结证模型。造模完成后4周开始给药,持续8周。观察大鼠的一般情况,精子染色质结构分析法(SCSA)检测附睾精子DFI。化学比色法测睾丸过氧化氢(H_2O_2)含量、过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性。结果:与假手术组相比较,模型组大鼠附睾精子DFI[(8.36±2.49)%]显著升高(P0.01),睾丸组织H_2O_2含量[(431.22±97.01)mmol/g prot]显著升高(P0.01),CAT[(10.53±2.69)U/ml]、SOD[(87.30±25.33)U/ml]活性显著升高(P0.01)。与模型组相比较,通精灵各组附睾精子DFI显著降低,睾丸组织H_2O_2的含量显著降低。结论:精索静脉曲张模型大鼠附睾精子DNA完整性降低,睾丸组织呈氧化应激状态,精子DNA完整性与氧化应激有相关性,通精灵可能通过增加CAT、SOD活性,降低H_2O_2含量减轻了睾丸组织氧化应激状态,从而提高精子DNA完整性。     

自制金属冷冻板法冷冻人附睾精子的实验研究

目的:研究一种新型人精子冷冻方法对精子复苏率的影响,以探索人附睾精子、睾丸穿刺精子的最佳冷冻方法。方法:选取76例梗阻性无精子症患者的附睾穿刺(PESA)标本,按照自制金属冷冻板法和传统冷冻法分两组冷冻。采用计算机辅助精液分析系统检测冷冻前、后前向运动精子百分率,并对比两种方法对精子膜功能、DNA碎片指数(DFI)、顶体酶活性和精子畸形百分率的影响。结果:复苏后自制金属冷冻板法和传统冷冻法前向运动精子百分率[(12.0±7.5)%vs(8.0±5.1)%,P0.05]和低渗肿胀精子百分率[(22.0±17.5)%vs(18.0±20.5)%]比较有显著性差异(P0.05),均较冷冻前[(20.7±8.8)%和(30.0±13.5)%]显著下降(P0.05)。自制金属冷冻板法复苏后精子顶体酶活性显著高于传统冷冻法[(75.2±9.5)μIU/10~6精子vs(55.7±8.3)μIU/10~6精子,P0.05],均较冷冻前(120.0±10.5)μIU/10~6精子显著下降(P0.05)。两种方法复苏后畸形精子百分率和DFI无显著差异[(98.7±8.8)%vs(98.5±9.2)%,P0.05]和[(38.2±8.5)%vs(39.5±10.2)%,P0.05],并均较冷冻前[(97.2±9.5)%和(30.8±9.7)%]显著升高(P0.05)。自制金属冷冻板法和传统冷冻法冷冻复苏率[(65.2±12.0)%vs(52.3±18.0)%]有显著性差异(P0.05)。结论:自制金属冷冻板法是一种经济高效、操作简单的精子冷冻方法且能最大限度的节约精子;复苏后可以保证较好的精子复苏率、活动力和顶体酶活性。     

Chk1/2基因表达对精子浓度和活力的影响

目的:研究精子中细胞周期检测点激酶1/2(Chk1和Chk2)基因的表达对精子浓度及活力的影响。方法:将精液样本根据精子浓度和活力(前向运动精子百分率)分为正常对照组、少精子症组、弱精子症组和少弱精子症组,每组20例。分别检测各组精子DNA碎片指数(DFI)、精子存活率,采用RT-PCR和Western印迹方法分别检测各组精子Chk1、Chk2的表达。结果:①4组DFI分别为21.24±6.93、19.67±7.64、21.52±6.92、19.28±11.55,无显著差异(P0.05);4组精子存活率分别为(83.48±9.87)%、(63.86±9.16)%、(50.45±16.99)%、(39.21±15.74%),与正常对照组相比,其余3组均有显著下降(P均0.05);②DFI30%和DFI≤30%两组精子的浓度、活力和精子存活率之间的差异有统计学意义(P0.01);③ RT-PCR检测结果显示,4组Chk1 mRNA相对表达量分别为0.73±0.22、0.62±0.14、1.03±0.39、0.92±0.071,各组间比较差异有显著性(P0.01),其与精子浓度呈正相关(b=80.661,P0.01),与精子活力呈负相关(b=-19.275,P0.01);4组Chk2mRNA相对表达量分别为0.66±0.30、0.27±0.09、0.59±0.19、0.42±0.11,各组间比较差异有显著性(P0.01),其与精子浓度呈负相关(b=-90.809,P0.01),与精子活力呈正相关(b=27.507,P0.01)。④Western印迹结果显示,4组Chk1蛋白相对表达量分别为0.63±0.05、0.42±0.03、1.13±0.08、0.87±0.07,各组间比较差异有显著性(P0.01),其与精子浓度呈正相关(b=55.74,P0.01),与精子活力呈负相关(b=-22.649,P0.01);4组Chk2蛋白相对表达量分别为1.23±0.36、0.37±0.16、0.87±0.08、0.68±0.12,各组间比较差异有显著性(P0.01),其与精子浓度呈负相关(b=-53.001,P0.01),与精子活力呈正相关(b=16.676,P0.01)。结论:Chk1和Chk2在人类精子中均有显著表达,在精子DNA出现损伤后,Chk1表达的增强可能促进了精子的凋亡导致弱精子症,而Chk2表达的增强则可能抑制了精子的生成而导致少精子症。     

Accurate sperm morphology assessment predicts sperm function

Sperm morphology has been associated with in vitro as well as in vivo fertilisation. The study aimed to evaluate the possible relation between the percentage of spermatozoa with normal morphology and the following sperm functional assays: (i) zona-induced acrosome reaction (ZIAR); (ii) DNA integrity; (iii) chromatin condensation; (iv) sperm apoptosis; and (v) fertilisation rates. Regression analysis was employed to calculate the association between morphology and different functional tests. Normal sperm morphology correlated significantly with the percentages of live acrosome-reacted spermatozoa in the ZIAR (r = 0.518; P < 0.0001; n = 92), DNA integrity (r = -0.515; P = 0.0018; n = 34), CMA(3) -positive spermatozoa (r = -0.745; P < 0.0001; n = 92), sperm apoptosis (r = -0.395; P = 0.0206; n = 34) and necrosis (r = -0.545; P = 0.0009; n = 34). Negative correlations existed between for the acrosome reaction, and DNA integrity, while negative associations were recorded with the percentages of CMA(3) -positive spermatozoa, apoptotic and necrotic spermatozoa. Sperm morphology is related to sperm dysfunction such as poor chromatin condensation, acrosome reaction and DNA integrity. Negative and significant correlations existed between normal sperm morphology and chromatin condensation, the percentage of spermatozoa with abnormal DNA and spermatozoa with apoptotic activity. The authors do not regard sperm morphology as the only test for the diagnosis of male fertility, but sperm morphology can serve as a valuable indicator of underlying dysfunction.     

附睾、睾丸精子卵浆内单精子注射治疗阻塞性无精症引起不育

为评价附睾或睾丸精子卵浆内单精子注射（ＩＣＳＩ）治疗阻塞性无精症引起不育的疗效，对３１例无精子男性不育患者配偶进行超排卵治疗３７个周期，获卵当日从患者附睾取精，其中２４例获得活动精子，７例失败，改用钳取睾丸曲细精管从中分离精子以供ＩＣＳＩ。结果：共获卵４５３个，附睾、睾丸精子ＩＣＳＩ受精率分别为５５７％、６１７％，平均每周期移植胚胎３７个，总妊娠率每周期２９７％。结论：只要获得活动精子ＩＣＳＩ，阻塞性无精症患者也有机会生育     

Computerized sperm motility and application of sperm cryopreservation

An objective method for measuring sperm motion characteristics was developed on an Intellect 100 Quantel Image Analysis System suitable for various image cytometric applications. It provided overall analysis of percent motility (% MS) as well as individual and mean measurements of motion characteristics, including vigor characteristics such as curvilinear velocity (Vc), straight line velocity (Vsl), and trajectory pattern characteristics, that is, progressiveness ratio (PR) and amplitude of lateral head displacement (Alh). Evaluation of the method for reproducibility and accuracy showed reliable measurements of these parameters measured on a minimum of 70 motile sperm, sufficient to describe adequately the sperm population. A study was performed comparing motion characteristics of 30 semen samples falling in a normal range before and after cryopreservation in cryoprotector medium (CM). A mean motility rate of recovery (MRR) of 45% was obtained. Only sperm count and concentration in motile forms among initial semen variables correlated weakly with MRR. Velocity recovery rate (VRR) approached 1 with a marked variability among ejaculates. Distribution profile of Vc was highly modified by freezing in CM: spermatozoa that were initially fast and progressive were the most resistant to cryoaggression. PR and Alh values were little affected by freezing in CM. The tolerance of various samples from a given patient was highly variable for % MS and Alh and less variable for Vc and PR. This illustrates the difficulty in predicting the effect of freezing on motility characteristics and, therefore, of extrapolating from semen variables the ability of frozen-thawed samples to fertilize.     

Temperature-induced sperm nuclear vacuolisation is dependent on sperm preparation

The influence of temperature during incubation on the degree of sperm nuclear vacuolisation was assessed by two different experiments. In a first experiment, motile spermatozoa from 24 patients were prepared by the swim-up technique and incubated either at room temperature or at 37 °C for up to 4 h. The presence of sperm nuclear vacuoles was determined by contrast-enhanced high magnification microscopy. No statistically significant difference was found in the degree of sperm nuclear vacuoles in both groups (RT: 45.6 ± 17.6%; 37 °C: 48.4 ± 17.0%) following 4 h of incubation. In a second experiment, spermatozoa from six patients were either prepared by swim-up or washed and incubated at 37 °C. After 4 h of incubation, a significant increase in sperm nuclear vacuolisation was found in washed sperm (from 51.5 ± 15.4% to 68.6 ± 9.0%; P < 0.05) but not in swim-up sperm (from 51.5 ± 15.4% to 48.2 ± 17.1%; n.s.). Our data show that the mode of sperm preparation does influence sperm nuclear vacuolisation at 37 °C (Experiment II). However, sperm nuclear vacuolisation is unaffected by temperature in motile sperm after preparation and isolation by swim-up.     

Efficacy of sperm wash media in improving sperm motility

Spermatozoa from 15 fertile men were washed with Ham's F10 and incubated with two commercially available sperm nutrient media for 2, 4, 6, and 24 h. Both sperm capacitation medium (Irvine Scientific Co., Santa Ana, CA) and Pro-ception (Milex Products, Inc., Chicago, IL) proved to be capable of improving sperm motion characteristics. These media may be used for incubating sperm for intrauterine insemination or for in vitro fertilization.     

Effects of altered epididymal sperm transit time on sperm quality

The epididymal sperm transit time seems to have an important role in the process of sperm maturation, and it seems that alterations to the transit can harm the process. The aim of the present work was to evaluate the influence of altered sperm transit time through the epididymis on sperm parameters and fertility of rats, as well as the role of testosterone in the alterations. Sprague–Dawley adult male rats were randomly assigned to four different groups and were treated for 12 days: (i) 10 μg/rat/day DES, to accelerate the transit; (ii) 6.25 mg/kg/day guanethidine sulphate, to delay the transit; (iii) same treatment as group 1, plus androgen supplementation; (iv) control animals received the vehicles. Guanethidine treatment delayed the sperm transit time through the epididymal cauda, provoking increased sperm reserves in this region. Animals exposed to DES showed an acceleration of sperm transit time in the epididymis, and consequently decreased sperm density in both epididymal regions, the caput-corpus and cauda, and diminished sperm motility. In both cases sperm production was not altered. Testosterone supplementation was able to restore the transit time to values close to normality, as they were higher than in the control rats. The same occurred in relation to sperm motility. Rats exposed to DES presented lower fertility after in utero artificial insemination using sperm collected from the proximal cauda epididymis. Therefore, it was concluded that the acceleration of rat sperm transit time appeared to harm normal sperm maturation, thus decreasing sperm quality and fertility capacity, in an androgen-dependent way.     

Assessing sperm function

This article reviews basic semen tests and new fertility tests that are providing great insights to the rapidly developing understanding of male infertility. Finally, promising new tests under development are mentioned with their potential clinical applications.     

Canine sperm vitrification with sucrose: effect on sperm function

The ability of sucrose to protect spermatozoa against mitochondrial damage, artificial acrosome reaction and DNA fragmentation during ultra‐rapid cryopreservation in canine sperm was investigated. Swim‐up selected spermatozoa of second‐fraction semen were vitrified with different concentrations of sucrose (0.1, 0.25 and 0.4 m ) in proportion 1 : 1 v/v with HTF–BSA 1%. From each group, 30‐μl suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by submerging the spheres in HTF with 1% BSA at 37 °C. The number of progressively motile spermatozoa was significantly higher in the sucrose 0.25 m + HTF–BSA 1% (42.5 ± 2.3%, P < 0.01) than in HTF only (1.66 ± 0.3%). The same combination of sucrose 0.25 m + HTF–BSA 1% (42.7 ± 1.5%) had a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P < 0.05) and decreased the DNA fragmentation (2.8 ± 0.5%) as compared with HTF only (1.93 ± 0.6% and 5.6 ± 0.6% respectively). With respect to acrosome‐reacted spermatozoa, no significant difference was found between the groups investigated (P > 0.05). It is concluded that sucrose, a nonpermeable cryoprotectant, can effectively preserve important physiological parameters such as mitochondrial membrane potential and DNA integrity during ultra‐rapid cryopreservation.