山羊精子介导转染外源DNA的研究   总被引：4，自引：0，他引：4  

赵永聚  郑双艳 《西南大学学报(自然科学版)》2007,29(6):46-50

目的：建立山羊精子介导转染外源DNA的方法,并探索精液离心与否与添加异种精清对转染效率的影响.方法：将采集的山羊精液经过离心处理与标记的线形化pEGFP-N1质粒共育转染,分别用免疫组化的方法检测转染效率、PCR和Southern blotting检测转染外源DNA在精子的结合部位和结合率;同时将实验分为3组,分别为鲜精直接转染、离心后转染和离心山羊精液添加猪精清转染,用免疫组化方法检测转染外源DNA效率.结果：精子核后帽区是结合转染外源DNA的主要部位;3种处理的阳性转染率分别为（26.19±3.25）%、（62.04±7.87）%和（17.04±9.34）%;DNaseⅠ消化后的阳性转染率分别为（11.50±5.05）%、（34.00±5.87）%和（10.00±4.24）%.离心组与未离心组和添加猪精清组差异极显著（p〈0.01）.结论：山羊精子具有自发性结合外源DNA的能力;羊精清和异种精清明显降低精子结合外源基因的效率.  相似文献   

校正的BO液培养法对牛精子体外获能的影响     

阎志春  张天佑  梁裕利  王祖昆 《华南农业大学学报》1991,(Z1)

本试验校正了利用肝素和咖啡因使牛精子体外获能的传统的BO液配方及培养方法;利用去透明带仓鼠卵的穿透试验和顶体的台盼兰——姬姆萨双重染色技术检查了该方法对获能的影响,发现校正的BO液培养法缩短了预培养时间,增强了获能效果。  相似文献   

Study on Cryopreservative Effect of Three Penetrating Cryoprotectants with Three Kinds of Extenders on Rabbit Spermatozoa     

ZHANGGui－xue  LIXiao－juan 《东北农业大学学报(英文版)》2001,8(1):30-36

Glycerol,dimethyl suffixod(DMSO) and acetamide were adopted to verify the cryoprotective ablity of penetrating cryoprotectants with three kinds of extenders for rabbit sperm.Three extenders (A,B and C) and three level of the final concentrations of glycerol and DMSO(2%,3%and 4%),and acetamide 2%,4% and 5% were used with Extender A,4% glycerol had better cryoprotective effect for rabbit sperm motility and 5% acetamide was better with Extender C.4% acetamide was better in being combined with Extender A for sperm acrosome integrity ratio,5% acetamide was better with Extender B and 2% acetamide was better with Extende C. With extender A,there was noe difference among three penetrating cryotectants for plasma membrane integrity.With Extender B,3% glycerol was better than 4% acetamide for plasma membrane integrity,and 3% glycerol with Extender C was better than 2%-4% DMSO and 2% acetamide.  相似文献   

不同糖类对蓝狐精子常温及冷冻保存特性的影响     

李新红邹兴淮  朱淑文华修国 《上海交通大学学报(农业科学版)》2004,22(4):334-338

人工采取6只优质芬兰雄性蓝狐精液,利用含有不同性质糖成分的Tris-卵黄-柠檬酸钠-甘油稀释液稀释后,常温保存及制成细管冻精,荧光免疫方法检测蓝狐鲜精液常温保存及冷冻复苏后冻融精子的质量,目的在于比较不同性质糖在蓝狐精子常温及低温冷冻保存中的作用。结果表明,常温保存时,果糖、木糖及海藻糖试验组精子活力、质膜完整率及顶体完整率较高,与对照组差异显著(P<0.05)或极显著(P<0.01);超低温冷冻保存时,果糖、海藻糖稀释液有利于提高冻融精子质量,活力(44.6%、43.7%)与对照组(35.6%)差异显著(P<0.05),质膜完整率(50.6%、51.2%)、顶体完整率(53.2%、54.1%)与对照组间(39.6%、43.5%)差异极显著(P<0.01),而其他糖分试验组与无糖对照组差别不显著(P>0.05)。结果提示:果糖、木糖、海藻糖亦可作为蓝狐精液常温保存的稀释液成分,而果糖、海藻糖为蓝狐精液超低温冷冻保存较理想的保护剂  相似文献   

Effects of season and dietary ascorbic acid on some biochemical characteristics of rainbow trout (Oncorhynchus mykiss) semen     

Andrzej Ciereszko  Li Liu  Dr. Konrad Dabrowski 《Fish physiology and biochemistry》1996,15(1):1-10

During the reproductive season, rainbow trout spermatozoa are stored in the sperm ducts for several months. There is no sperm production at this time since spermatogenesis is completed before spawning. To leam more about characteristics of semen during such a long storage, we analyzed changes in protein concentrations, anti-proteinase activity in seminal plasma and sperm aspartate aminotransferase activity during an extended reproductive period during which fish were fed diets supplemented with various ascorbic acid concentrations. Seminal plasma protein concentration and anti-proteinase activity declined toward the end of the reproductive season. These phenomena may be related to oncoming proteolytic events leading to degradation of the sperm. Protein concentrations and anti-proteinase activities were strongly correlated within groups of different ascorbic acid supplementations and several sampling dates (r=0.6–0.9 in most cases, p<0.05). Ascorbic acid deficiency resulted in a decrease in both parameter levels as compared to levels in groups with vitamin C supplement (p<0.08). Deficiency also resulted in lower stimulation of aspartate aminotransferase by an exogenous pyridoxal 5-phosphate in comparison to fish fed vitamin C-supplemented diets (p<0.05). These results support earlier studies suggesting a protective role of ascorbic acid toward maintaining sperm quality.  相似文献   

中间球海胆精子的超低温保存技术   总被引：1，自引：2，他引：1  

王笑月  周遵春  李华林  王丽梅 《水产科学》2002,21(3):1-3

20 0 0年 10～ 11月 ,对大连龙王塘海区成熟的中间球海胆 (Strongylocentrotusintermedius)精子进行超低温保存研究。结果表明 :不同的抗冻保护剂 ,不同的体积分数 ,不同的冷冻方式 ,精子冻存的效果不同。以自然海水为基础溶液 ,以 12 %的二甲亚砜或 9%的甘油作为抗冻保护剂 ,稀释精液至 8× 10 7～ 11× 10 7个 /ml,距液氮表面 10cm处预冷 8min ,再浸入液氮中保存 ,解冻时直接移出液氮于 17～ 18℃自来水流水解冻 ,精子存活率分别是 95 % ,90 % ;受精率分别为 94 0 % ,86 5 %。  相似文献   

Biochemical Composition of Seminal Plasma and Annual Variations in Semen Characteristics of Jundiá <Emphasis Type="Italic">Rhamdia quelen</Emphasis> (Quoy and Gaimard,Pimelodidae)     

Adriana?BorgesEmail author  Daniele?Rodrigues?Siqueira  Daiane?Follmann?Jurinitz  Rebeca?Zanini  Fernanda?do?Amaral  Marcelo?Lacerda?Grillo  Eneder?Rosana?Oberst  Guillermo?Federico?Wassermann 《Fish physiology and biochemistry》2005,31(1):45-53

This study investigated the composition of milt of the South American silver catfish (Rhamdia quelen) or jundiá. The semen was taken from jundiá in different periods during the four seasons. The biochemical composition of seminal fluid and the characteristics of sperm were analyzed. The semen quantity which can be extracted per fish in one day was 0.95 ± 0.08 ml during spring (maximum) and 0.24 ± 0.03 ml during winter (minimum). Sperm density (spermatocrit) showed higher values in the spring (75.1 ± 1.3%) decreasing slightly afterwards and reaching 63.0 ± 2.4% to 65.0 ± 2.2% in the fall and winter. Immediately after water dilution, 90–100% of the spermatozoa presented vigorous straightforward motility that remained for at least 20 s. The total duration of the motility was 47.9 ± 1.3 s in the spring and 38.6 ± 0.6 s in the other seasons (P < 0.05). This pattern of motility is maintained for more than 2 h after storage of the milt at room temperature. The pH from 5 to 10 of the water dilution does not influence the sperm motility. The mean seminal pH and osmolality values were 8.7 ± 0.07 and 274.8 ± 11.2 (mOsm/kg), respectively. The ion concentration was: Na 153.7 ± 2.4, K 10.7 ± 2.4, Cl 139.4 ± 2.1, Ca 4.2 ± 0.2, Mg 0.9 ± 0.05, P 0.9 ± 0.08 (mEq/l). The total protein was 0.6 ± 0.05 mg/dl and cholesterol concentration was 13.9 ± 0.9 mg/dl.  相似文献   

Relationship between transmembrane potential and activation of motility in rainbow trout (Salmo gairdneri)     

Andrew P. Blaber  F. Ross Hallett 《Fish physiology and biochemistry》1988,5(1):21-30

A hypothesis is developed that activation of motility in rainbow trout spermatozoa is a result of membrane hyperpolarization. This hypothesis was developed to explain experimental observations of a relationship between membrane potential and motility as revealed by the use of voltage sensitive fluorescent dyes. The results lead to the following conclusions: a) Transmembrane potential hyperpolarizes with decreasing KCl concentration in 100 mM NaCl. b) Transmembrane potential hyperpolarizes with decreasing NaCl concentration. c) NaCl is three time less effective in changing transmembrane potential and two orders of magnitude less effective in inhibiting activation of motility than KCl. d) Chloride ions have little effect on transmembrane potential or motility. e) Increases in osmotic pressure with the non-ionic molecule sucrose increased the amount of KCl required to inhibit activation. f) The major effect of Na⁺ on K⁺ inhibition may be osmotic.It is suggested that while sperm cells are in the seminal plasma in the reproductive tract of the male rainbow trout their transmembrane potential is maintained above an activation threshold, probably through Na/K pumps which are found in almost all animal cells. Since K⁺ is the most important ion in determining the transmembrane potential, hyperpolarization of the plasma membrane below an activation threshold occurs when the sperm cells are diluted, during spawning, into the low K⁺ environment of freshwater.  相似文献   

The development of a sperm cryopreservation protocol for winter flounder Pseudopleuronectes americanus (Walbaum): evaluation of cryoprotectants and diluents   总被引：1，自引：0，他引：1  

R M Rideout  M K Litvak  & E A Trippel 《Aquaculture Research》2003,34(8):653-659

Three cryoprotectants (dimethyl sulphoxide, propylene glycol and glycerol) and two diluents (sucrose based and saline based) were mixed (9 parts diluent–1 part cryoprotectant) factorially to produce six extenders that were tested to develop an effective sperm cryopreservation protocol for winter flounder Pseudopleuronectes americanus (Walbaum). Sperm were diluted 1:3 with each extender and frozen by flotation on liquid nitrogen before being submerged and stored for 30 days. Sperm left unfrozen in each extender for 20 min showed no toxic effects on motility. Extenders containing propylene glycol (PG) as cryoprotectant yielded higher post‐thaw sperm motilities than those containing dimethyl sulphoxide (DMSO) or glycerol. The sucrose‐based diluent performed better than the saline‐based diluent when DMSO was used as cryoprotectant, but there were no differences in post‐thaw motility between diluents for the other cryoprotectants. Activating sperm with ovarian fluid and sea water instead of sea water alone had no effect on post‐thaw motility. In fertilization trials, no differences were observed between any of the extenders and fresh milt when milt, eggs and sea water were left in contact for 1 h. When sperm were forced to compete for eggs by reducing contact time to 20 s, fertilization results followed those of sperm motility rates. Percentage hatch and morphology of larvae at hatching did not differ for eggs fertilized by cryopreserved and fresh sperm. This study represents the first reported successful attempt at cryopreserving winter flounder sperm and should improve gamete and broodstock management protocols for this species.  相似文献   

The presence of uric acid, an antioxidantive substance, in fish seminal plasma     

A. Ciereszko  K. Dabrowski  D. Kucharczyk  S. Dobosz  K. Goryczko  J. Glogowski 《Fish physiology and biochemistry》1999,21(4):313-315

High concentrations of uric acid in seminal plasma of a range of teleost fish species are reported for the first time. Concentrations of urate amounted to 223.4; 121.9–130.0, 355.9, 735.6, 124.0, 192.7 and 148.0 M for rainbow trout (Oncorhynchus mykiss), yellow perch (Perca flavescens), muskellunge (Esox masquinongy), Northern pike (Esox lucius), carp koi (Cyprinus carpio), bream (Abramis brama), and tench (Tinca Tinca), respectively. Uric acid is an important anti-oxidant, therefore, it has a potential role in protection of fish spermatozoa against oxidative damage.  相似文献