小鼠H-Y单克隆抗体ELISA检测方法的建立   总被引：2，自引：0，他引：2  

杭苏琴  刘为敏  牛树理  冯紫云 《中国兽医学报》2004,24(6):547-549

以纯系 BAL B/ c雄性小鼠脾细胞腹腔注射免疫同系雌性小鼠 9次 ,获得的抗血清经雌、雄鼠脾细胞吸收后用于精子细胞毒性试验 ,测得 H- Y抗血清效价为 1/ 16 0。选取免疫应答最好的雌鼠脾细胞与 SP2 / 0骨髓瘤细胞融合 ,用精子细胞毒性方法筛选效价较高的细胞株制备腹水 ,建立优化的 EL ISA反应条件。优化后的 EL ISA反应条件为 :抗原4℃过夜 ,加 H- Y抗血清 37℃反应 12 0 m in,加 HRP- Ig G 37℃反应 30 min,加 TMB 2 5℃ 30 min。优化后的 EL ISA与常规 EL ISA比较 ,可以明显降低阴性吸光值 ,检测灵敏度从 1/ 32 0上升为 1/ 12 80  相似文献   

Deep Freezing of Boar Semen Packaged in Plastic Bags and Straws     

C.O. Bwanga  S. Einarsson  H. Rodriguez-Martinez 《Reproduction in domestic animals》1991,26(3):117-125

For practical reasons, a large volume (i.e. 5 ml) of frozen boar semen per insemination dose is desirable, but successful freezing has not been achieved, since optimal cooling rates have not yet been established. Post-thaw motility and the acrosome intep'ty of semen from four boars frozen with a programmable freezin machine, in mini-(0.25 ml), maxi-(5 ml) plastic straws and in 10 × 5 cm PVC- or Teflon FEP-plastic bags (0.35 – 0.12 mm thick, 5 ml) was studied. The freezing of the semen was monitored using thermocouples placed in the straws and the bags. The freezing curve started from +5°C, at a rate of −3°C/min, to – 6°C, it was held for 1 min at −6°C, and was followed by further drop to −100°C at a rate of −20°C/min, with subsequent storage in LN₂. The bags had a much shorter freezing point plnteau, compared to the maxi-straws. Post-thaw sperm motility was significantly higher when semen was frozen in mini-straws or in bags than in maxi-straws. The freezing procedure did not cause major acrosomal damages, significantly more normal apical ridges being present in the bags and mini-straws than in the maxi-straws. This in vitro evaluation indicates that the freezing method employed is satisfactory for freezing large volumes of boar semen into plastic bags .  相似文献   

影响精液冷冻和人工授精效果的因素     

吴结革  茆达干 《动物医学进展》2003,24(5):48-50

通过对精液冷冻保存的细胞反应原理的阐述 ,指出冷冻保护剂甘油对精液保存具有利弊效应 ,提出精子膜脂组成的差异使得不同品种的精子对冷冻损伤的易感性不同。雌性生殖道解剖结构的品种差异 ,精子形态 ,精子运行机制的细微差异 ,人工授精时间及精子的运行能力 ,采精方式等因素对精液冷冻保存和人工授精的成功有决定性作用。研究精子质膜的生物学特性可解决低活力精子的问题 ,然而这并不能解决冷冻后精子质量的个体差异。对精细胞基因组的研究可以找出这些个体的遗传差异。因此 ,冷冻精子和精原细胞 (用于细胞外注射 )的差异已经成为完整基因组问题。  相似文献   

杏种子与种胚的超低温保存研究   总被引：3，自引：0，他引：3  

李云  李嘉瑞 《种子》1995,(6):14-16

含水量为６．９％带有内果皮的种子，以－５Ｃｍｉｎ＾－１速率降至－８０℃，放入ＬＮ２，在室温缓慢解冻，成活率可达９１．５％。去掉内果皮含水量为７．１％的种仁，以上述方式保存，成活率也可达到８８．２％。含水量是超低温保存杏种子最重要的因素。研究表明：冰冻保护剂ＤＮＳＯ对杏种子的超低温保存影响不大。含水量为４．８％￣８．１％去掉子叶的种胚超低温保存后ＴＴＣ染色成活率为８２．８％，并能在ＭＳ培养基上发育为  相似文献   

猪卵泡卵母细胞体外成熟与冷冻保存   总被引：1，自引：0，他引：1  

王锋  蔡令波  顾熟琴  杭苏琴  牛树理 《中国兽医学报》2004,24(6):605-608

研究了激素、猪卵泡液、不同类型血清对猪卵泡卵母细胞体外成熟的影响 ;比较了卵泡直径小于 2 mm、2～ 5 mm和大于 5 mm的卵母细胞体外成熟能力的差异 ,并对不同发育阶段猪卵母细胞的冷冻保存进行了研究。结果表明 :猪卵母细胞体外培养 4 8h时 ,培养的前 2 4 h培养液中加入激素 ,后 2 4 h去掉激素 ,卵母细胞的 A级成熟率 (5 1.73% )和总成熟率 (83.2 5 % )最高 ,极显著高于前 2 4 h不加激素 ,后 2 4 h添加激素培养的成熟率 (P<0 .0 1) ;也显著高于不含激素的培养液连续培养 4 8h的成熟率 (P<0 .0 5 ) ;但与添加激素连续培养 4 8h的成熟率差异不显著 (P>0 .0 5 )。在体外成熟培养液中 ,添加 10 % (体积分数 ) ECS的成熟率 (72 .86 % )显著高于添加 10 % (体积分数 ) NCS的成熟率(6 2 .2 1% ) (P<0 .0 5 ) ,而添加 10 % p FF则抑制卵母细胞的体外成熟。随着卵泡直径的增大 ,卵母细胞体外成熟能力逐渐增强。采用程序冷冻保存方法 ,成熟卵母细胞的成活率 (35 .5 9% )显著高于培养前 (2 4 .6 4 % )和培养 2 4 h(2 3.36 % ) (P<0 .0 5 )。培养 2 4 h(77.2 2 % )和培养成熟 (72 .81% )的卵母细胞解冻后的形态完整率均显著高于培养前(5 3.2 4 % ) (P<0 .0 5 )  相似文献   

水牛精子在胞质内注射后的早期形态变化   总被引：1，自引：0，他引：1  

孟凡丽  韦精卫  石德顺  卢晟盛 《中国兽医学报》2004,24(6):609-612

应用胞质内精子注射 (intracytoplasmic sperm injection,ICSI)技术探讨了水牛卵母细胞胞质与注射精子间的相互作用以及 ICSI精子的形态变化。ICSI后 13h,5 9.4 %的精子头部已膨大 ,且有 18.8%的精子进入解聚状态。雌雄原核发育不同步 ,雄原核在 ICSI后 16 h和 19h的形成率分别为 3.2 %和 4 0 .0 % ;雌原核在 ICSI后 13h已达到 71.9% ,16 h时提高到 90 .3%。经离子霉素与 6 - DMAP联合激活 ,ICSI后 19h产生的胚胎核型以 2 PN PB1 为主 ,其雌原核形成 (激活 )率为 91.3% ,雄原核形成率为 4 0 .0 % ,5 1.3%为孤雌胚。用 5 mm ol/ L 的 DTT预处理精子 1h,可提高精子的解聚率 (30 .9%比 12 .7% ,P<0 .0 5 ) ,但对雄原核的形成率无显著影响 (33.3%比 32 .7% ,P>0 .0 5 )。结果表明 ,水牛精子 ICSI后的雄原核形成时间晚于卵子雌原核形成时间 ,且其比率低于卵子 ,DTT处理精子能提高其解聚率 ,但对雄原核形成无影响  相似文献   

第十九期鸡胚原始生殖细胞的冷冻保存     

肖小珺  蔡琳琳  秦洁  李碧春 《黑龙江畜牧兽医》2005,(2):7-9

采用Ficoll密度梯度离心,提取第 19期(孵化 72h)性腺中的PGCs,对其应用不同的冷冻保护液和不同的平衡方法进行冷冻保存,并于复苏后进行体外培养。复苏后的PGCS用台盼蓝染色检测其存活率,结果发现:从第 19期性腺中获取的PGCs在同一种冷冻保护液下,采用不同的平衡方法进行冷冻,对PGCs的存活率有显著影响(P<0.05)或极显著影响(P<0.01);平衡方法相同,在不同冷冻保护液之间存在显著(P<0.05)或极显著 (P<0.01)差异。PGCs经体外培养 24h后再进行冷冻保存,复苏后其存活率、体外培养存活时间均极显著(P<0.01)短于分离后直接冷冻的PGCs。  相似文献   

In vitro studies of Norwegian Red bovine semen immobilized and cryopreserved in alginate solid gel network          下载免费PDF全文

AH Alm‐Kristiansen  ER Gaustad  G Bai  FB Standerholen  G Klinkenberg  E Kommisrud  KE Waterhouse 《Reproduction in domestic animals》2018,53(2):365-370

Development of new semen cryopreservation techniques improving sperm survival and ensuring availability of viable spermatozoa for a prolonged time‐period after AI is promising tools to reduce sensitivity of timing of AI and enhance overall fertility. The SpermVital^® technology utilizes immobilization of bull spermatozoa in a solid network of alginate gel prior to freezing, which will provide a gradual release of spermatozoa after AI. The objective of this study was to compare post‐thaw sperm quality and in vitro sperm survival over time of Norwegian Red bull semen processed by the SpermVital^® (SV) technology, the first commercialized production line of SpermVital^® (C) and by conventional procedure applying Biladyl^® extender (B). Post‐thaw sperm motility was not significantly different between SV, C and B semen (p > .05). However, sperm viability and acrosome intactness were higher for SV than C and B semen (p < .05). Small differences in DNA quality were observed (p < .05). Sperm viability after storage in uterus ex vivo was higher for SV than for C semen (p < .05). Furthermore, sperm survival in vitro over time at physiological temperature was significantly higher for SV semen than C semen as well as B semen during the incubation period of 48 hr (p < .05). In conclusion, the SpermVital^® technology is improved and is more efficient in conserving post‐thaw sperm quality and results in higher sperm viability over time in vitro for SV than for C and B semen.  相似文献   

Disaccharide combinations and the expression of enolase3 and plasma membrane Ca2+ ATPase isoform in sturgeon sperm cryopreservation          下载免费PDF全文

P Li  H Du  XM Qiao  ZG Liu  QW Wei 《Reproduction in domestic animals》2018,53(2):472-483

Acipenser sinensis and Acipenser dabryanus are critically endangered species, so germplasm conservation via cryopreservation of sperm is necessary. Disaccharides can act as membrane‐impermeable cryoprotectants, and enolase3 (ENO3) and plasma membrane Ca²⁺ ATPase isoform (PMCA2) are proteins associated with sperm quality. We considered seven characteristics of sperm quality in cultured brood stock from A. sinensis and A. dabryanus. We tested use of sucrose or trehalose alone and in combination at different concentrations for cryopreservation of A. dabryanus sperm. A low concentration of sucrose plus trehalose (S₁₅T₁₅) was optimal. Mixing of the extender with sucrose, lactose, or trehalose alone or with pairwise mixtures revealed that a mixture of lactose and trehalose (L₁₅T₁₅) gave the best results for both A. sinensis and A. dabryanus. Enolase3 and PMCA2 expression levels were measured in cryopreserved A. sinensis sperm via Western blotting. Relative ENO3 and PMCA2 expression levels were examined, and the relationship between disaccharide composition, sperm quality and protein expression was explored in A. sinensis. The results showed that relative ENO3 and PMCA2 expression levels were the highest at L₁₅T₁₅ in cryopreserved A. sinensis sperm. There were significant positive correlations between ENO3 expression and percentage membrane integrity, and between PMCA2 expression and sperm motility parameters (percentage of motile sperm, curvilinear velocity, straight‐line velocity and average path velocity; p < .05) in cryopreserved A. sinensis sperm. Our results indicate the optimal disaccharide combination and concentrations for cryopreservation of A. sinensis and A. dabryanus sperm and suggest that ENO3 and PMCA2 expression levels could serve as a valuable indicator of sperm quality in A. sinensis.  相似文献   

Effects of five cryoprotectants on proliferation and differentiation‐related gene expression of frozen‐thawed bovine calf testicular tissue          下载免费PDF全文

Hao Li  Yi‐Lin Bian  Nicola Schreurs  Xiao‐Gang Zhang  Sayed Haidar Abbas Raza  Qian Fang  Li‐Qiang Wang  Jian‐Hong Hu 《Reproduction in domestic animals》2018,53(5):1211-1218

The cryopreservation of testicular tissue is a potential method for preserving male fertility. However, the effect of cryopreservation on bovine calf testicular tissue is scarce. This study investigated the effect of different cryoprotectants on bovine calf testicular tissue at the molecular level. Testicular tissue from ten immature bovine calves (6 months) was collected after slaughter and cryopreserved in an extender containing different concentrations of the following five cryopreservation solutions (CP): bovine serum albumin (BSA) with 5% dimethyl sulfoxide (DMSO), trehalose with 5% DMSO, DMSO and glycerol and ethylene glycol (EG). After 7‐day cryopreservation, the expression levels of three spermatogonial stem cell (SSC)‐related genes, octamer‐4 (OCT4), KIT ligand (MGF/SCF) and kit oncogene (C‐KIT), were investigated by quantitative PCR (qPCR). The cell viability was highest for the tissues preserved with 30 mg/ml BSA (77.82% ± 1.22) and 40 mg/ml trehalose (74.23% ± 1.16) compared with other groups (p < 0.05), and the level of expression of the three genes was highest with 30 mg/ml BSA (p < 0.05). Compared with other CPs, the 30 mg/ml BSA and 40 mg/ml trehalose have the better cryopreserve protection. The 30 mg/ml BSA is the most viable media for the cryopreservation of testicular tissue from cattle.  相似文献