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1.
探讨了不同甘油浓度(3%、5%、7%、11%)和不同平衡时间(30、60、90、120min)对食蟹猴(Macaca fascicularis)精液冷冻效果的影响,以建立和优化食蟹猴精液冷冻的程序。参照TTE稀释液成分组成改良型TTE,冷冻前和解冻后均检测精子的活力、畸形率、质膜完整性、顶体完整率。结果显示,平衡时间为30min时精子的冷冻解冻后活力、复苏率均高于平衡时间90min和120min组,差异显著(P<0.05),比60min组稍好;甘油浓度为3%、5%组的精子冷冻解冻后活力及复苏率均高于甘油浓度11%组,差异显著(P<0.05),比7%组好;不同甘油浓度各组间以及不同平衡时间各组间畸形率、质膜完整性、顶体完整率差异不显著(P<0.05)。由此得出如下结论,在食蟹猴精液冷冻中,在改良TTE中加入3%~5%的甘油且平衡30min可以获得较好效果,精子冻后活率和复苏率达到45%和62%。  相似文献   

2.
不同渗透压的稀释液对猕猴精子低温冷冻保存的影响   总被引:3,自引:0,他引:3  
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。  相似文献   

3.
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。  相似文献   

4.
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).  相似文献   

5.
2008年3月1日至4月27日和2009年3月3日至5月1日,在陕西省珍稀野生动物抢救饲养研究中心对处于繁殖期内的4只雄性秦岭大熊猫(Ailuropoda melanoleuca qinlingensis)精液进行了细管冻精实验。比较组成不同的4种稀释液:葡萄糖-果糖-柠檬酸三钠-卵黄-甘油-双抗(稀释液1)、葡萄糖-蔗糖-柠檬酸三钠-卵黄-甘油-双抗(稀释液2)、葡萄糖-柠檬酸三钠-卵黄-甘油-双抗(稀释液3)和美国进口的TEST(加入3.5%甘油),以及直接降温平衡法(方法 1)与逐级降温平衡法(方法 2)2种冷冻保存操作方法,对秦岭大熊猫精液进行细管冷冻保存后精子活力和顶体完整率的影响。结果表明:稀释液1的精子活力为46.25%±11.67%,顶体完整率为80.75%±7.89%,TEST的精子活力为48.75%±8.54%,顶体完整率为84.50%±7.59%,两者的精子活力和顶体完整率均无明显差异(P0.05),但是都明显高于稀释液2(P﹤0.01)和稀释液3(P﹤0.01);采用方法 1冷冻保存秦岭大熊猫精液,解冻后精子的活力和顶体完整率分别为45.67%±10.54%和81.37%±8.42%,都显著高于方法 2(P﹤0.01);方法 1解冻后畸形率为23.50%±3.51%,明显低于方法 2(P﹤0.01)。经比较确定,方法 1(用稀释液1)是一种较好的细管冷冻保存秦岭大熊猫精液的方法。  相似文献   

6.
人工采取8只优质芬兰雄性蓝狐的精液,分别利用2%、4%、6%和8%甘油浓度的卵黄-Tris-果糖-柠檬酸钠稀释液进行稀释,制成细管冻精。在冻融后0、O.5、2、4、6h检测4种浓度组的精子运动度、质膜完整率、顶体完整率;并利用透射电镜观察冻融前后精子的超微结构变化。冻融后0h,4%甘油浓度组冻融精子的运动度、质膜完整率、顶体的完整率均最高(分别为41.8%、43.6%、48.4%),2%浓度组最低(分别为24.5%、27.6%、31.7%);随着检测时间延长,2%与4%组的精子特性差异显著,但2%、6%、8%3个组间差异不显著;6h时各组间精子的运动度均不超过10%,最高质膜完整率和顶体完整率分别为11.8%、12.7%。说明蓝狐精液稀释剂中甘油的适宜浓度应为4%,冻融后精子的活力维持时间较短。蓝狐精子冻融过程中质膜极易发生膨胀或断裂、顶体囊泡化或溃散,而质膜和顶体丢失现象较少。  相似文献   

7.
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。  相似文献   

8.
树鼩作为一种新型的、接近灵长类的实验动物,在医学生物学上的应用受到越来越多的重视。精子的结构特性研究及冷冻后结构的完整性分析是精子生物学的主要内容,也有助于树鼩的实验室快速繁殖。该研究采用人工饲养的中缅树鼩(Tupaia belangeri chinensis),结果显示其睾丸占总体重的(1.05±0.07)%,总体积为(1.12±0.10)mL。附睾尾及输精管精子总量估计在2.2×107~8.8×107,其运动度和顶体完整率分别为(68.8±3.9)%和(90.0±2.1)%。利用扫描电子显微镜和透射电子显微镜对树鼩附睾精子的超微结构进行的观察和分析显示精子头部呈圆形或卵圆形;头部长度、宽度平均分别为6.65和5.82μm;精子尾部中段、主段、尾段和精子总长度平均分别为13.39、52.35、65.74和73.05μm;尾部中段的线粒体螺旋数量为48个,其轴丝结构为典型的"9+9+2"结构。冷冻解冻后的精子主要表现在顶体与质膜不完整、精子断裂、尾部扭曲和膨大。上述结果提示树鼩精子与其他哺乳动物精子的结构特征相似,但是精子大小和线粒体螺旋数目有明显的差别,且超微结构改变仍是冷冻精子运动和受精能力下降的主要原因。  相似文献   

9.
选用5只年龄为3~4岁的波尔山羊公羊研究在稀释液中添加海藻糖对山羊精子功能和膜完整性的影响。山羊精子分别用含6.6 mmol/L、13.2 mmol/L、19.8 mmol/L、26.4 mmol/L、39.6 mmol/L、52.9 mmol/L、66.1mmol/L、79.3 mmol/L的不同海藻糖的Tris-柠檬酸-葡糖糖(TCG)稀释液(卵黄:18%;甘油:6%)稀释和冷冻。结果表明:39.6 mmol/L、52.9 mmol/L、66.1 mmol/L、79.3 mmol/L组降温后的精子活率显著(P<0.05)降低;52.9 mmol/L、66.1 mmol/L、79.3 mmol/L组降温后的精子畸形率和39.6 mmol/L组降温后的膨胀精子率显著(P<0.05)提高。26.4 mmol/L组和39.6 mmol/L组冻融后的精子活率显著(P<0.05)高于对照组;66.1mmol/L和79.3 mmol/L组冻融后的精子活率、畸形率分别显著(P<0.05)低于和高于对照组。19.8 mmol/L、26.4 mmol/L、39.6 mmol/L组冻融后精子获能率显著(P<0.05)低于对照组。39.6 mmol/L组冻融后顶体完整率和膨胀精子率显著(P<0.05)高于对照组,而66.1 mmol/L组和79.3 mmol/L组显著(P<0.05)低于对照组。39.6 mmol/L组的受胎率显著(P<0.05)高于对照组,而66.1mmol/L组和79.3 mmol/L组的受胎率显著(P<0.05)低于对照组。结果表明,在含18%的卵黄(v/v)、6%甘油(v/v)的TCG稀释液中,添加适宜浓度(26.4mmol/L和39.6 mmol/L)海藻糖,可显著提高山羊精子功能和膜的完整性。  相似文献   

10.
长牡蛎精子超低温冷冻后超微结构损伤研究   总被引:1,自引:0,他引:1  
采用程序降温仪分步降温冷冻保存长牡蛎(Crassostrea gigas)精液,并用扫描电镜、透射电镜研究了精子的超微结构损伤。超低温冷冻保存后长牡蛎精子的运动率、受精率及孵化率与鲜精无显著差异。鲜精中84.5%的精子形态结构正常,冻精中73%的精子形态结构正常。形态结构正常的精子表现为顶体、质膜、线粒体与鞭毛结构完整、染色质形状规则,顶体、线粒体及中心粒结构正常,鞭毛形态完整、微管结构清晰;形态结构异常的精子表现为顶体脱落、解体,精子头部质膜膨胀、破裂、染色质肿胀、破裂、解体,线粒体移位、脱落、膨胀,嵴退化或消失,鞭毛弯折、断裂,微管解聚。结果显示,以10% DMSO为抗冻保护剂,HBSS溶液为稀释液,1:4的稀释比例,添加海藻糖,采用分步降温法冷冻保存,对长牡蛎精子具有较好的抗冻保护作用,合适的冻存方法可以有效的保护太平洋牡蛎精子冷冻过程中结构损伤。研究有助于长牡蛎种质资源的收集保存及应用。  相似文献   

11.
Ping S  Wang F  Zhang Y  Wu C  Tang W  Luo Y  Yang S 《Theriogenology》2011,76(1):39-46
Cryopreservation of sperm from tree shrews, which are considered primitive primates, would enhance genetic management and breeding programs. Epididymal sperm were surgically harvested from male tree shrews, cryopreserved in two Tes-Tris-based cryodiluents, and used in four experiments. In Experiment 1, there were no significant differences in motility and acrosome integrity among five concentrations of egg yolk in TTE after cooling to 4 °C. However, sperm frozen in TTE containing 20% egg yolk at −172 °C/min had better (P < 0.05) post-thaw motility and acrosome integrity. In Experiment 2, sperm held for 10 min prior to storage in liquid nitrogen had greater motility than those held for 5 or 15 min (P < 0.05), but acrosome integrity was not different (P > 0.05) among treatments. In Experiment 3, sperm frozen in TTE diluent had higher (P < 0.05) motility and acrosome integrity than those in TEST diluent. In Experiment 4, there were no differences (P > 0.05) in the fertilization rate of oocytes and the proportion of tree shrews yielding fertilized oocytes, following AI with fresh versus frozen sperm. In conclusion, tree shrew epididymal sperm were successfully cryopreserved, as assessed by post-thaw motility, acrosome integrity, and fertilizing ability.  相似文献   

12.
Ejaculated spermatozoa from cynomolgus monkeys and rhesus monkeys were frozen in straws with six different extenders (TTE, DM, mDM, LG-DM, G-DM, and TCG) containing glycerol. Sperm motility and head membrane and acrosomal integrity were evaluated after freezing and thawing, and the cryoprotective effects were compared among the extenders and the two species studied. The results showed that sperm motility and motility recovery with the six extenders were comparable for the cynomolgus and rhesus monkeys. There was no significant difference in sperm motility and head membrane integrity among the six extenders in either the cynomolgus or rhesus monkeys (P>0.05). However, a slightly but statistically lower percentage of acrosomal integrity was found with TCG in both species compared to the other extenders (P<0.05). These findings demonstrate that TTE, DM, mDM, LG-DM, G-DM, and TCG are equally suitable extenders for the cryopreservation of spermatozoa from cynomolgus and rhesus monkeys.  相似文献   

13.
Singh MP  Sinha AK  Singh BK 《Theriogenology》1995,43(6):1047-1053
Semen samples were obtained from 12 bucks (3 Beetal, 3 Black Bengal and 6 Beetal x Black Bengal) and 10 different extenders were constituted with varying concentrations of glycerol, DMSO, glycerol + DMSO and glycerol + lactose as the sperm cryoprotective agents. After the collection of semen samples, they were assessed for quality, diluted in different extenders after removal of seminal plasma, packaged in ministraws and frozen after equilibration (5'C) for 5 h. The samples were evaluated immediately after equilibration and again 24 h after freezing for progressive motility, percentage of live spermatozoa and acrosome, head and tail abnormalities. Both motility and the percentage of live spermatozoa were most affected by extenders containing only DMSO and these values improved in glycerol + DMSO extenders as the concentration of glycerol was increased while DMSO was decreased. However, these values were significantly higher in extenders containing glycerol + lactose as the cryoprotective agents, and were found to increase with increased concentration of lactose, being highest in TYGL (180). Acrosomal and tail abnormalities tended to increase between post equilibration and post thawing stage, and were higher in extenders containing the higher levels of DMSO. Significantly (P < 0.01) lower percentages of abnormalities were recorded in the glycerol + lactose extenders. The fertility results showed nonsignificant effect of extenders on the conception rate of does.  相似文献   

14.
In this study we describe acrosome staining and motility characteristics of fresh and cryopreserved sterlet (Acipenser ruthenus L.) spermatozoa using soybean trypsin inhibitor-Alexa conjugate fluorescent staining and computer-aided sperm analysis (CASA), respectively. Methanol or dimethylsulfoxide (DMSO) were used as cryoprotectants. After cryopreservation a decline in sperm motility characteristics occurred, but no differential effect between cryoprotectant was observed. Cryopreservation caused a significant increase in the percentage of spermatozoa with acrosome stained by SBTI-Alexa for samples cryopreserved using DMSO compared to methanol. These data suggest that the low usefulness of DMSO for cryopreservation of sturgeon spermatozoa is related to its harmful specific effect towards the acrosome, probably by causing its precocious triggering, much before any egg contact.  相似文献   

15.
The aim of this study was to evaluate the cryoprotective effect of different freezing extenders against cryopreservation injuries on Iberian boar sperm. The sperm-rich fraction was collected and pooled from six sexually mature Iberian boars, and was frozen in different extenders containing glucose, lactose or fructose as sugar source and including Orvus ES Paste only in the freezing extender-2 (Glucose; Lactose and Fructose) or in both freezing extenders (Glucose2; Lactose2 and Fructose2). During the cryopreservation process, the supernatant was removed after the centrifugation step, then was extended with freezing extender-1 for the equilibration period and with freezing extender-2 immediately before freezing. Post-thaw sperm characteristics, such as plasma membrane integrity (SYBR-14/PI), mitochondrial function (Rhodamine 123) and acrosome integrity (NAR), were monitored. Overall sperm motility and the individual kinematic parameters of motile spermatozoa (assessed by the computer-aided sperm analysis system Sperm Class Analyzer [SCA]) were recorded in the different experimental treatments. Measurements were taken at 30 and 150 min post-thaw. The state of the acrosome after thawing did not show significant differences between the freezing extenders studied. Freezing–thawing caused a significant decrease (P < 0.001) in plasma membrane integrity and in mitochondrial activity in the spermatozoa frozen with Orvus ES Paste in both freezing extenders. Furthermore, spermatozoa frozen with Orvus ES Paste in both freezing extenders exhibited lower (P < 0.05) motility and kinematic parameters than those frozen in the absence of Orvus ES Paste in the first freezing extender. The spermatozoa frozen with the Lactose extender and with Orvus ES Paste only in the second freezing extender showed a better evolution of the motility and kinematic characteristics (P < 0.05) over time. The deterioration in post-thaw sperm motility and kinematic parameters were concurrent with reduced sperm characteristics. It can be suggested that in the Iberian pig, the beneficial effects of Orvus ES Paste during the freezing process of spermatozoa is time dependent. The analysis of different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, determined that the extenders studied in the present experiment affected the quality of frozen-thawed semen in Iberian boar.  相似文献   

16.
There has been a lack of research into equine sperm vitrification to date, but studies of other species suggest it may have significant potential. To evaluate the impact of various cryoprotectant agents (CPA) and vitrification on equine sperm quality, a controlled study was carried out. A total of 12 ejaculates were subjected to exposure to CPA and vitrification. Sperm was diluted in a range of CPA: fresh, control (BSA), sucrose (0.15M, 0.3M and 0.5M), trehalose (0.15M, 0.3M and 0.5M) and the combination of sucrose and trehalose (M1: 0.15M sucrose+0.5M trehalose; M2: 0.5M sucrose+0.15M trehalose). Sperm motility, viability, acrosome integrity and DNA fragmentation were assessed at the time of CPA exposure and after vitrification. The exposure of spermatozoa to various concentrations of sucrose and/or trehalose significantly reduced sperm motility, with lower concentrations resulting in higher sperm motility. Sperm viability and DNA fragmentation did not vary after exposure to CPA, but acrosome integrity fell significantly when spermatozoa were exposed to CPA with high osmolality. When spermatozoa were vitrified, motility values were significantly higher than those obtained during the exposure. Low concentrations of sucrose (0.15M and 0.3M) and trehalose (0.15M) showed the best progressive sperm motility. The vitrification-warmed procedure significantly reduced sperm viability and acrosome integrity, but DNA did not vary with any of CPA used. Equine sperm vitrification demonstrates a low capacity for preserving sperm motility, and extenders containing trehalose or sucrose at lower concentrations are associated with a better protective effect on sperm motility. After vitrification, acrosome and plasma membranes were severely impaired, while the DNA structure was maintained. Equine spermatozoa partially recover the motility after vitrification, but there is a need for further studies into the preservation of sperm membranes.  相似文献   

17.
The effects of three amino acids (proline, glutamine, and glycine) added to the freezing medium Tes-Tris-egg yolk (TTE) for cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa were studied. This is the first report on the effects of amino acids on nonhuman primate sperm cryopreservation. The addition of 5mM proline, 10mM glutamine, and 10 or 20mM glycine each significantly improved post-thaw sperm motility and membrane and acrosome integrity compared with the control (TTE alone). However, a significant decrease in motility and membrane/acrosome integrity was observed when amino acid concentrations increased to 60mM for proline and glutamine, and 80 mM for glycine. The results suggest that adding a limited amount of amino acids to the freezing media is beneficial for freezing cynomolgus monkey sperm.  相似文献   

18.
Aboagla EM  Maeda T 《Theriogenology》2011,76(3):538-546
Arbutin (4-hydroxyphenyl-glucopyranoside) is a glycosylated hydroquinone present in high concentrations in the leaves of several plants capable of surviving prolonged, extreme dehydration. Two experiments were conducted to determine the effects of arbutin on cryopreservation of goat sperm. In Experiment 1, goat sperm were frozen in extenders with various ratios of Tris-citric acid-glucose (TCG) and arbutin; concentrations of the latter were 0.0 (only TCG), 0.1, 0.2, 0.3, and 0.4 M (only arbutin)]. All extenders had 20% (v/v) egg yolk (EY) and 4% (v/v) glycerol (osmolality = 370 mOsm, pH = 7.0). Sperm motility and acrosome integrity were assessed using CASA, and fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA), respectively. Percentages of motile and progressively motile sperm improved with the addition of arbutin; results were optimal (89.0 and 70.0%, respectively; P < 0.05), with 0.4 M arbutin. Furthermore, arbutin improved (P < 0.05) post-thaw recovery rates for both motility and progressive motility. After incubation for 3 h, motility of frozen-thawed washed sperm improved (70%, P < 0.05) with arbutin in the extender. The percentage of sperm with an intact acrosome peaked (77.2%, P < 0.05) with 0.4 M arbutin in the extender. In Experiment 2, the percentage of cells with merocyanine 540/Yo-Pro staining was higher in sperm treated with arbutin than with TCG (P < 0.05), with the best result (58.0%) with 0.4 M arbutin; therefore, arbutin increased membrane fluidity. In conclusion, substitution of a TCG-EY diluent composition with arbutin improved freezability of goat sperm (apparently due to increased membrane fluidity). Furthermore removal of arbutin by centrifugation after freezing and thawing increased sperm longevity.  相似文献   

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