平衡时间、冷冻方法及解冻程序对马精液冷冻效果的影响

精液平衡、冷冻及解冻是冻精制作过程中三个必不可少的环节,对精液冷冻效果起着决定性作用。在马(Equus caballus)精子冷冻中针对这三个过程的研究较少,为进一步优化马精液冷冻方法,提高精液冻后质量,本研究比较不同平衡时间、冷冻方法及解冻程序对冻融后精子运动参数、质膜完整性及线粒体膜电势的影响。平衡120 min、180 min和240 min组冻融后精液活力及质膜完整性明显高于平衡0 min、45 min、90 min及8 h平衡组;距离液氮面2 cm和4 cm高度熏蒸冷冻获得了与程序冷冻仪冷冻法相似的冷冻效果;采用高温瞬时解冻法(75℃7 s和46℃20 s)比常规方法(37℃30 s)获得了更高的冻后精液活力(P0.05)。综合上述结果,在马精液冷冻过程中综合采用120~240 min平衡,距离液氮面2~4 cm熏蒸法和高温瞬时解冻法(75℃7s和46℃20 s)可获得更好的精液冷冻效果。     

淫羊藿多糖对弱精症精子冷冻保存效果的影响

常用的冷冻保护剂对于弱精症精子冻融效果欠佳,本实验通过在人精子冷冻保护液中添加淫羊藿多糖(EPS),研究其对冻融过程中精子活力及精子功能的影响。选择弱精症精液15例,液化后的精液样本分别与甘油-卵黄-柠檬酸盐(GEYC)冷冻保护液或含有EPS冷冻保护液混匀冷冻。检测其精子的活力、存活率以及精子形态、丙二醛(MDA)以及活性氧(ROS)的含量,精子核碎裂指数(DFI),精子顶体反应率(AR),并通过透射电镜观察精子微观结构的变化。添加EPS后精子MDA和ROS水平明显降低,含有3 mg/mL EPS的冷冻组抗氧化性明显优于其他组(P0.05);含有EPS的冷冻组复苏后的存活率以及精子头部正常形态率都明显高于未添加组,但是两组间的精子前向运动PR无显著差异;此外,添加EPS的冷冻组精子DFI下降显著,AR明显升高;电镜观察精子头部显微结构显示,添加EPS组的精子在质膜以及顶体膜完整性上明显优于未添加组。结果提示,在精液冷冻保护液中添加EPS可降低精子活性氧的水平,保护精子顶体结构和功能,从而改善解冻后精子顶体功能和精子核的完整性。     

圈养黑熊精液的冷冻保存

将采自23 头成年圈养黑熊的精液,分别用3 种稀释液(Ⅰ:Tris - 乳- 果- 卵;Ⅱ:柠- 葡- 蔗- 卵;Ⅲ:Tris - 柠- 果- 葡- 卵)稀释并在4℃ 下保存,通过检测精液在不同稀释液稀释条件下的保存时间,筛选出最适稀释液用于精液的冷冻保存;从精液解冻后精子的活率、活力、畸形率、顶体完整率4 个指标,分别从3种冷冻保护剂(甘油3%、3.5% 、4% )、两种冷冻方法(两步冷冻法和自动冷冻法)两个方面进行了比较试验。结果表明:精子活力在0.3 以上时,稀释液Ⅲ保存时间为175.42 ± 3.04 h,显著高于稀释液Ⅰ和稀释液Ⅱ(P ﹤0.01),稀释液Ⅱ保存时间也明显高于稀释液Ⅰ (P ﹤0.01);含3.5% 甘油浓度的稀释液解冻后精子活率(41.75 ± 3.46% )、活力(32.63 ±5.27% )和顶体完整率(85.62 ± 4.58% )显著高于其他两组(P ﹤0.01),并且精子畸形率(29.32 ± 8.22% )明显低于其他两组(35.95 ± 8.04% ,36.07 ±7.72% )(P ﹤0.01);采用自动冷冻法冷冻保存圈养黑熊精液,解冻后精子活率、活力和顶体完整率分别为41.75 ±3.46% 、32.63 ± 5.27%和85.62 ±4.58% ,都明显高于两步冷冻法(P ﹤0.01);解冻后畸形率为29.32 ± 8.22% ,明显低于两步冷冻法(P ﹤0.01)。     

不同渗透压的稀释液对猕猴精子低温冷冻保存的影响

以稀释液TTE(382mOsm/kg)为对照,研究了5种渗透压(688、389、329、166、43mOsm/kg)的TEST稀释液(TEST、mTEST1、mTEST2、mTEST3、mTEST4)在冷冻过程中对猕猴精子功能的影响。精液一步稀释于含甘油的防冻液中,甘油的终浓度为5%(v/v)。在冷冻前后分别检测精子的运动度和质膜完整性,后者用Hoechst33342和碘化丙锭双色标记流式细胞术分析。结果表明:冷冻之前,与鲜精相比,用TEST和mTEST4稀释的精子运动度和质膜完整性显著降低(P<0·001),其余组中除mTEST2稀释的精子质膜完整性显著降低(P<0·05)外,精子运动度无差异;冷冻复苏后,TTE、mTEST3和mTEST1冻存精子的运动度和质膜完整性最高,其次是mTEST2,TEST和mTEST4冷冻效果最差(P<0·05)。提示等渗、适当高渗或低渗的稀释液适合猕猴精子的冷冻保存;对精子产生高渗毒害作用是导致猕猴精子用TEST冷冻存活率低的主要原因。     

一种简单实用的猪颗粒冻精制作技术

本实验以年龄在2.5岁左右的长白种公猪的精液为材料,在比较了常用的Ⅰ号、Ⅱ号、Ⅲ号猪精液冷冻稀释液和Ⅰ号、Ⅱ号解冻液及冷冻———解冻程序对猪精液的冷冻效果后,依据解冻后精子的活力、质膜完整性等指标,发现:1、Ⅱ号冷冻稀释液的稀释效果(精子活力42.5±5.2精子弯尾率44.7±3.5)和Ⅱ号解冻液的解冻效果(精子活力43.8±2.6精子弯尾率36.2±4.3)明显较好;2、根据制作经验总结,发现了一种与以往资料上介绍的完全不同的猪精液冷冻颗粒制作方法,从而筛选出了一种简单实用的猪颗粒冻精制作技术。     

大熊猫冷冻精液解冻速度和解冻液中添加Pentyoxyfilline对其解冻后活力的影响

建立大熊猫的精子库,进行远距离圈养大熊猫种群间的人工授精和遗传物质的转运,维持遗传多样性,是目前大熊猫遗传管理的优先方法。要成为最有效的工具,精子库保存的精子解冻后的活力必须很好。本文对大熊猫冷冻精液的解冻速度和解冻液中添加化学激活剂Pentyoxyfilline(PF)后的精子活力进行了试验。试验用的精液采自11只成年大熊猫,精液冷冻速度为每分钟-40℃～-100℃。试验Ⅰ:将冷冻精液放入3种不同温度的水浴中解冻:(1)22℃(慢速解冻);(2)37℃(中速解冻)(3)50℃(快速解冻)。将冷冻前精子活力(78 1±2 9%)和解冻后的平均精子活力进行比较,快速解冻后的精子活力(57 5±5 4%)显著地降低(P<0 05),而中速解冻的精子活力(67 5±3 1%)和慢速解冻的精子活力(73 33±2 1%)与冷冻前的活力接近。试验Ⅱ:使用中速解冻方法解冻精液后,分别加入最终浓度为0mM、1mM、5mM和10mM的PF,然后分别保温15min和24h。在PF(0mM、1mM、5mM和10mM)中分别孵育15min的解冻精子活力,运动状态,活率和顶体正常率在试验期的90min内都很相似(P>0 05)。在1mMPF中孵育24h的精子活力没有变化(P>0 05)。在5mM和10mMPF中孵育过的精子活力(5mM:24 0±4 7%;10mM19 5±3 6%)比没有加PF的对照组的精子活力(38 3±5 2%)显著地低(P<0 05)。而且,在10mM     

提高精子转染外源DNA效率的山羊精液冷冻方法

目的筛选一种既提高精子转染外源DNA效率,又保持解冻后精子活力的山羊精液冷冻—解冻方法。方法应用正交设计L9(34),因素分别为稀释液种类、稀释比例、降温时间和解冻液,每个因素选择3个水平,检测和比较解冻后精子转染外源DNA效率和精子活力。结果所选冷冻—解冻各因素对精液转染效率影响不显著[F(8,18)=1.032,P=0.449];平衡时间对冷冻—解冻活力影响极显著[F(2,24)=9.972,P=0.001],平衡1h极显著小于平衡2 h和4 h的精液活力(P=0.003,P=0.000),以平衡4 h最好。用筛选的冷冻—解冻方法处理精液,解冻后精子的活力极明显降低(P=0.002);生存指数降低,GOT释放量增加,菌落数减少,与鲜精相比差异显著(P=0.018;P=0.016;P=0.018);精子畸形率增加,顶体完整率降低,与鲜精相比差异不显著(P=0.494;P=0.084)。结论优化了提高精子转染外源DNA效率的山羊精液冷冻—解冻方法。     

滇南小耳猪精液的直接冷冻保存

目的建立滇南小耳猪精液冷冻保存方法,加速云南省特有小型猪种的小型化选育。方法利用脉冲电刺激模式诱导公猪输精管自助收缩排精。利用直接冷冻新技术（DFM）研究不同冷冻方案对精子的运动度、精子顶体完整性和体内授精胚胎发育能力的影响。结果在直接冷冻方法中,3%甘油防冻剂的作用下,60 s植冰时间和1.5 mm/s的冷冻降温参数对精子运动度保护良好,精子运动复苏率达到61.7%。但是,3%乙二醇虽然与甘油一样对精子的顶体完整性都有很好的保护作用,对精子运动度保护能力较差。此外,3%甘油、60 s植冰、1.5mm/s冷冻速度的直接冷冻的冻精解冻,移植到超数排卵的母猪子宫颈口实施人工授精,获得卵的受精和胚胎发育潜能尚可。结论玻璃管直接冷冻可以完成滇南小耳猪精子的冷冻保存。     

几种冷冻稀释液与单胺类防冻剂对中缅树鼩精子冷冻存活率的影响

该文实验精子采自昆明地区经笼养驯化的树鼩(Tupaia belangeri),检测其冷冻前后运动度、顶体完整率以及检测部分冷冻精子的受精能力。实验一:选用8种已报道的冷冻稀释液TTE、TCG、TCF、TTG、BWW、BTS、DM、SR稀释鲜精,并添加0.4mol/LDMSO,4℃预冷平衡2h后,TTE、DM和SR稀释液的精子的运动度与鲜精无差别(P>0.05),其余处理组均显著下降(P<0.05)。冷冻复苏后,各组的运动度显著低于预冷平衡处理后的运动度(P<0.05);DM组的复苏运动度显著高于其他稀释液组(P<0.05),BWW组最低(P<0.05)。对于顶体完整率,与鲜精相比,4℃平衡2h后,TTE和DM组精子的顶体完整率显著高于BWW、BTS和SR组(P<0.05)。冷冻复苏后,DM组精子的顶体完整率显著高于其它(除了TTE)冷冻组(P<0.05)。实验二:在DM稀释液基础上分别添加4种浓度0.2、0.4、0.8和1.2mol/L的二甲基甲酰胺(DF)、甲酰胺(F)、二甲乙酰胺(DA)和乙酰胺(A)以及0.4mol/LDMSO,经过预冷平衡处理后,与鲜精相比,各防冻剂组的精子运动度没有下降(P>0.05);冷冻解冻后,各冷冻组精子的运动度显著低于预冷平衡处理后的精子运动度(P<0.05);0.8mol/LDF和0.4mol/LDMSO组精子的运动度显著高于其它冷冻组(P<0.05)。对于顶体完整率,预冷平衡处理后各高浓度组的比率显著下降;冷冻复苏后,0.4mol/LF和0.4mol/LDF组精子的顶体完整率相对较高。实验三,人工授精实验中,DM+0.8mol/LDF冷冻精子的受精率为16.7%,DM+0.4mol/LDMSO的受精率为50.0%。以上实验结果提示,含卵黄的非离子冷冻稀释液对树鼩精子冷冻保护效果好,但单胺类防冻剂的防冻效果还需进一步深入研究。     

冷冻—解冻猪精子的超微结构观察

自从50年代初Polge发现甘油有卓越的防冻作用以后,动物精液冷冻技术发展很快。现在,奶牛和羊冷冻精液已广泛应用于人工授精和体外受精中,效果与鲜精接近,而猪冷冻精液在人工授精后虽也能产仔,但妊娠率与每窝产仔数均下降(Johnson等,1981);体外受精率可达85％以上,但精子浓度需用5—6×10_7精子/ml,比鲜精用量提高25—100倍(Wang等,1991)。顶体形态正常精子的减少是造成冷冻精液受精率低的重要原因之一(Pursel,1979),用光镜不能清楚地观察顶体的形态变化,但运用电镜技术对猪冷冻-解冻精子的顶体形态的超微结构研究报道不多(Courtens等,1985;Hashizume,1990)。针对猪冷冻—解冻精子体内/体外受精能力降低的问题,我们用透射电镜对冷冻的猪精子解冻0 h及4 h后精子顶体与头部质膜的超微结构变化进行了观察。     

Cryopreservation of yellowfin seabream (Acanthopagrus latus) spermatozoa (Teleost, Perciformes, Sparidae)

The effects of both osmolality and cation in the initiation of sperm motility were examined in yellowfin seabream, Acanthopagrus latus. Various factors involved in the cryopreservation of yellowfin seabream spermatozoa on motility are discussed. Extender containing only glucose proved to be a suitable medium for freezing yellowfin seabream spermatozoa to -196 degrees C. Glycerol seems to have a direct osmotic effect on yellowfin seabream sperm cells, and it induced sperm motility before freezing and during thawing. However, this exhausted the energy needed for sperm motility for fertilization. Dimethyl sulfoxide (DMSO) proved superior to ethylene glycerol, propylene glycerol, glycerol and methanol as a cryoprotectant. Prolonged equilibration time had a detrimental effect on both prefreezing and post-thawing sperm motility. The estimated optimum freezing rate was in the range of -20 to -154 degrees C/min. More frozen-thawed than fresh spermatozoa are required to achieve comparable fertilization rates.     

Cryopreservation of spermatozoa from wild-born Namibian cheetahs (Acinonyx jubatus) and influence of glycerol on cryosurvival

Sperm cryopreservation is a valuable tool for the genetic management of ex situ populations. This study was conducted to assess: (1) semen characteristics of wild-born cheetahs; and (2) the impact of three types of glycerol influence (duration of exposure, temperature, and method of addition) on sperm cryosensitivity. To evaluate the impact of duration of glycerol exposure, spermatozoa were incubated in Test Yolk Buffer (TYB) with 4% glycerol at ambient temperature (approximately 22 degrees C) for 15 vs. 60 min before cryopreservation. To evaluate the influence of temperature and method of glycerol addition, spermatozoa were resuspended at ambient temperature either in TYB with 0% glycerol followed by addition of 8% glycerol (1:1 v/v; at ambient temperature vs. 5 degrees C) or directly in TYB with 4% glycerol. All samples were cryopreserved in straws over liquid nitrogen vapor and evaluated for sperm motility and acrosomal integrity after thawing. Semen samples (n = 23; n = 13 males) contained a high proportion (78%) of pleiomorphic spermatozoa. Ejaculates also contained a high proportion of acrosome-intact (86%) and motile spermatozoa (78%). Immediately after thawing, a significant proportion of spermatozoa retained intact acrosomes (range, 48-67%) and motility (range, 40-49%). After thawing, incubation in glycerol for 60 min at ambient temperature before freezing decreased (p < 0.05) sperm motility and acrosomal integrity at one time-point each (pre-centrifugation and post-centrifugation, respectively). However, method or temperature of glycerol addition had no (p > 0.05) impact on sperm cryosurvival. In summary, (1) wild-born cheetahs produce high proportions of pleiomorphic spermatozoa but with a high proportion of intact acrosomes; and (2) resuspension in 4% glycerol, followed by exposure for up to 60 min at ambient temperature, had minimal effect on sperm motility and acrosomal integrity after cryopreservation. Results indicate the feasibility of cryopreserving cheetah spermatozoa under field conditions, providing a user-friendly method to capture and store gametes to enhance genetic management.     

Effect of seminal plasma on the cryopreservation of equine spermatozoa

Seminal plasma is generally removed from equine spermatozoa prior to cryopreservation. Two experiments were designed to determine if adding seminal plasma back to spermatozoa, prior to cryopreservation, would benefit the spermatozoa. Experiment 1 determined if different concentrations of seminal plasma affected post-thaw sperm motility, viability and acrosomal integrity of frozen/thawed stallion spermatozoa. Semen was washed through 15% Percoll to remove seminal plasma and spermatozoa resuspended to 350 x 10(6)sperm/mL in a clear Hepes buffered diluent containing either 0, 5, 10, 20, 40 or 80% seminal plasma for 15 min, prior to being diluted to a final concentration of 50 x 10(6)sperm/mL in a Lactose-EDTA freezing diluent and cryopreserved. Sperm motility was analyzed at 10 and 90 min after thawing, while sperm viability and acrosomal integrity were analyzed 20 min after thawing. Seminal plasma did not affect sperm motility, viability or acrosomal integrity (P>0.05). Experiment 2 tested the main affects of seminal plasma level (5 or 20%), incubation temperature (5 or 20 degrees C) and incubation time (2, 4 or 6 h) prior to cryopreservation. In this experiment, spermatozoa were incubated with 5 or 20% seminal plasma for up to 6h at either 5 or 20 degrees C prior to cryopreservation in a skim milk, egg yolk freezing extender. Samples cooled immediately to 5 degrees C, prior to freezing had higher percentages of progressively motile spermatozoa than treatments incubated at 20 degrees C (31 versus 25%, respectively; P<0.05), when analyzed 10 min after thawing. At 90 min post-thaw, total motility was higher for samples incubated at 5 degrees C (42%) compared to 20 degrees C (35%; P<0.05). In addition, samples containing 5% seminal plasma had higher percentages of total and progressively motile spermatozoa (45 and 15%) than samples exposed to 20% seminal plasma (33 and 9%; P<0.05). In conclusion, although the short-term exposure of sperm to seminal plasma had no significant effect on the motility of cryopreserved equine spermatozoa, prolonged exposure to seminal plasma, prior to cryopreservation, was deleterious.     

Influence of sugar supplementation of the extender on motility, viability and acrosomal integrity of dog spermatozoa during freezing

Influence of different sugars supplemented to the extender on the motility, viability and intact acrosome rates of dog spermatozoa during dilution, equilibration and freezing was studied. The ejaculate was divided into 10 aliquots, which were diluted 1:3 with TRIS-citric acid extender containing 240 mMTRIS, 63 mM citric acid, 8% (v/v) glycerol, 20% (v/v) egg yolk and 70 mM sugar, which was either fructose, galactose, glucose, xylose (monosaccharide), lactose, trehalose, maltose, sucrose (disaccharide) or raffinose (trisaccharide). No sugar was added to the extender in the control group. Extended semen samples were cooled to 5 degrees C over 45 min, packaged in 0.25-mL straws, equilibrated for 2 h at 5 degrees C and frozen in liquid nitrogen vapor. Samples were thawed by placing straws into 37 degrees C water for 30 sec. Motility, viable sperm and intact acrosome rates decreased gradually in all groups after equilibration and consecutively freezing (P<0.001). The type of sugar significantly effected motility, viability and acrosomal integrity during equilibration and freezing (P<0.05). Galactose, lactose, trehalose, maltose and sucrose reduced damaged acrosome percentages in equilibrated samples (P<0.05). Sugar supplementation did not enhance motility and viability during equilibration. The disaccharides, except lactose, reduced post-thaw dead sperm and/or damaged acrosome percentages without promoting post-thaw motility (P<0.01), whereas monosaccharides, especially fructose and xylose, improved motility (P<0.05) along with viability and intact acrosome rates (P<0.05). Trehalose, xylose and fructose significantly increased total active sperm rates (motility x live sperm rate x normal acrosome rate) compared to other sugars (P<0.01) and control (P<0.0001) in frozen thawed samples. Therefore, sugar supplementation of the extender influenced post-equilibration and post-thaw sperm quality, and the type or locality of protective impact of the sugar on dog spermatozoa vary according to type of the sugar.     

Rapid freezing without cooling equilibration in canine sperm

The aim of this study was to develop a rapid method of canine semen freezing without cooling equilibration using treatment with different cryoprotectant agents (CPAs) and freezing in liquid nitrogen (LN(2)) vapor in a 0.5-mL straw via modifying vitrification. Ejaculates from eight beagle dogs were frozen with different CPAs (CPA-free, 5% glycerol, 5% ethylene glycol, and 10% ethylene glycol) and freezing times (direct plunging into LN(2) or freezing for 1, 2, 3, or 10 min in LN(2) vapor before plunging into LN(2)). Frozen-thawed sperm were evaluated for motility, viability, normal morphology, and plasma- and acrosome-membrane integrities. The 5% glycerol treatment resulted in improved sperm motility, plasma-membrane integrity and acrosome-membrane integrity (P<0.05). Freezing in LN(2) vapor showed improved sperm motility, viability, and plasma membrane integrity (P<0.05), and freezing for more than 2 min in LN(2) vapor increased acrosome-membrane integrity compared with direct plunging into LN(2) (P<0.05). The direct plunging into LN(2) showed no motile sperm. However, freezing for more than 2 min in LN(2) vapor increased the total abnormalities compared to direct plunging into LN(2) (P<0.05). In conclusion, use of 5% glycerol and freezing in LN(2) vapor were essential for the rapid freezing of canine sperm without cooling equilibration. In particular, holding for 2 min in LN(2) vapor was sufficient to yield successful rapid freezing. This rapid freezing method is simple and effective in canine sperm and would be helpful to offer information for trial of vitrification in large volumes of canine sperm.     

Comparative semen cryopreservation in ferrets (Mustela putorius furo) and pregnancies after laparoscopic intrauterine insemination with frozen-thawed spermatozoa

A study was conducted to determine an optimum technique for semen cryopreservation and the biological competence of frozen-thawed ferret spermatozoa. Fifty-two fresh electroejaculates from 4 males were evaluated for sperm percentage motility, forward progressive motility, motility index (SMI) and acrosomal integrity. To determine the optimum temperature for maintaining sperm motility in vitro and the influence of glycerol on sperm motility, seminal aliquants were diluted in TEST diluent (containing either 0 or 4% glycerol) and maintained at 25 degrees or 37 degrees C. For cryopreservation, semen was diluted in each of 3 cryodiluents (TEST, PDV, BF5F), cooled for 30 min at 5 degrees C and pelleted on solid CO2 or frozen in 0.25 ml straws (20 degrees C/min to -100 degrees C). Following thawing, SMI and acrosomal integrity were determined. Ten females with maximum vulval swelling were given 90 i.u. human chorionic gonadotrophin and laparoscopically inseminated in utero with spermatozoa previously frozen using the optimum diluent and freeze-thaw method. The maintenance temperature of 25 degrees C was superior (P less than 0.05) to 37 degrees C for sustaining sperm motility, and glycerol did not influence (P greater than 0.05) motility for up to 11 h of culture. After thawing, motile spermatozoa were recovered in all treatment groups, but sperm motility and normal acrosomal ratings were highest using the PDV diluent, the pelleting method and thawing at 37 degrees C (P less than 0.05). Seven of the 10 ferrets (70%) inseminated with spermatozoa frozen by this approach became pregnant and produced 31 kits (mean litter size 4.4; range 1-9 kits).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of egg yolk during the freezing step of cryopreservation on the viability of goat spermatozoa

Four experiments were conducted to investigate the effects of egg yolk during the freezing step of cryopreservation (namely, the process except for the cooling step), on the viability of goat spermatozoa. The effects of egg yolk on sperm motility and acrosome integrity during the freezing step were investigated in Experiment 1. Spermatozoa diluted with Tris-citric acid-glucose (TCG) solution containing 20% (v/v) egg yolk were cooled to 5 degrees C, washed, and then frozen in TCG with egg yolk (TCG-Y), TCG without egg yolk (TGG-NY), 0.370 M trehalose with egg yolk (TH-Y), or trehalose without egg yolk (TH-NY). All extenders contained glycerol. In frozen-thawed spermatozoa, the inclusion of egg yolk in the freezing extenders increased (P<0.05) percentages of motile sperm, progressively motile sperm, and the recovery rate (ratio of post-thaw to pre-freeze values), but decreased (P<0.05) acrosomal integrity. Moreover, extenders with trehalose had better (P<0.05) post-thaw sperm viability. In Experiment 2, the effects of egg yolk on acrosome status before and after freezing were studied. Egg yolk significantly decreased the proportion of intact acrosomes before freezing, leading to fewer (P<0.05) intact acrosomes post-thaw and lower (P<0.05) recovery rates for intact acrosomes. In Experiment 3, including sodium dodecyl sulfate (SDS) in a diluent containing egg yolk tended to preserve the acrosome compared with the egg yolk containing diluent free of SDS, however, spermatozoa had a lower (P<0.05) proportion of intact acrosomes than those in a yolk-free diluent. However, after cooling, spermatozoa were diluted with a glycerolated extender containing egg yolk. Therefore, the objective of Experiment 4 was to explore whether the egg yolk or glycerol was responsible for the reduced intact acrosome percentage. In this experiment, after cooling and washing the spermatozoa were diluted in TCG with glycerol and/or egg yolk. The combination of glycerol and egg yolk in the extender reduced (P<0.05) the proportion of intact acrosomes compared with egg yolk or glycerol alone. In conclusion, the inclusion of egg yolk significantly improved sperm motility, indicating its beneficial effects during the freezing step of cryopreservation; trehalose appeared to synergistically increase its cryoprotective effects. Furthermore, although neither glycerol nor egg yolk per se affected the proportion of intact acrosomes, the combination of the two significantly reduced the proportion of acrosome-intact spermatozoa.     

In vitro evaluation of ram sperm frozen with glycerol, ethylene glycol or acetamide

The aim was to assess the in vitro effect of glycerol, ethylene glycol or acetamide on frozen-thawed ram spermatozoa. Aliquots of each sixteen ejaculates from four rams of the Morada Nova breed were diluted in Tris-egg yolk with glycerol (5%), ethylene glycol (3% or 5%) or acetamide (3% or 5%) and frozen at -196°C. After thawing, progressive sperm motility was greater (P<0.05) in cryopreservation with glycerol 5% and ethylene glycol (3% or 5%) than with acetamide (3% or 5%). Acrosome integrity was greater (P<0.05) with ethylene glycol 5% than acetamide (3% or 5%). The percentage of sperm without oxidative stress was greater (P<0.05) with ethylene glycol (3% or 5%) than with acetamide (3% or 5%). Plasma membrane integrity was greater with glycerol 5% (P<0.05) than with the other cryoprotectants. Thus, it is concluded that glycerol 5% and ethylene glycol 3% or 5% protect ram sperm against the harmful effects of freezing and that glycerol 5% offers greater protection to sperm plasma membrane.     

Cryopreservation of kangaroo spermatozoa using alternative approaches that reduce cytotoxic exposure to glycerol

Alternative techniques for the cryopreservation of kangaroo spermatozoa that reduced or eliminated the need for glycerol were investigated including; (1) freezing spermatozoa with 20% glycerol in pre-packaged 0.25 mL Cassou straws to enable rapid dilution of the glycerol post-thaw, (2) investigating the efficacy of 20% (v/v) dimethyl sulphoxide (DMSO) and dimethylacetamide (DMA—10%, 15% and 20% v/v) as cryoprotectants and (3) vitrification of spermatozoa with or without cryoprotectant (20% v/v glycerol, 20% v/v DMSO and 20% v/v DMA). Immediate in-straw post-thaw dilution of 20% glycerol and cryopreservation of spermatozoa in 20% DMSO produced no significant improvement in post-thaw viability of kangaroo spermatozoa. Spermatozoa frozen in 20% DMA showed post-thaw motility and plasma membrane integrity of 12.7 ± 1.9% and 22.7 ± 5.4%, respectively, while kangaroo spermatozoa frozen by ultra-rapid freezing techniques showed no evidence of post-thaw viability. The use of 10–20% DMA represents a modest but significant improvement in the development of a sperm cryopreservation procedure for kangaroos.