Production of inbred larvae through self‐fertilization using oocytes and cryopreserved sperm from the same individuals after sex reversal in eastern oyster Crassostrea virginica          下载免费PDF全文

Huiping Yang  Yan Wang  Ximing Guo  Terrence R Tiersch 《Aquaculture Research》2015,46(9):2153-2165

The eastern oyster Crassostrea virginica can change sex which makes self‐fertilization possible if sperm can be cryopreserved. In this study, small (~1 year old) and large (~2–3 years old) oysters were biopsied for sperm collection. Survival of the biopsied oysters after 1 year was 50% for small oysters and 17% for large oysters. Oocytes were collected from sex‐reversed females, and self‐fertilized with cryopreserved sperm. Of the 24 cryopreserved samples, 14 individuals had ≤1% fertility when crossed with oocytes from unrelated females, indicating that the cryopreserved sperm had reduced fertility. The other 10 individuals had a fertility of 39 ± 25% when crossed with oocytes from unrelated females (non‐selfing), but showed a significantly lower success of self‐fertilization (12 ± 16%) (P = 0.008), while aliquots of the same oocytes had a fertilization of 83 ± 11% when crossing with fresh sperm. Larvae were produced at day 3 in the self‐fertilized families (12–94% of the fertilized oocytes), and survived to eyed‐larvae stage at days 11–14. Genotyping with 9 microsatellite markers confirmed that the larvae resulted from self‐fertilization in four families. This study demonstrated the feasibility of creating self‐fertilized inbred lines of oysters by use of non‐lethal sperm collection and cryopreservation.  相似文献   

人工养殖西伯利亚鲟精子超低温冷冻保存研究   总被引：7，自引：6，他引：7  

刘鹏  庄平  章龙珍  王斌  闫文罡 《海洋渔业》2007,29(2):120-127

研究了人工养殖西伯利亚鲟精子的生物学特征及超低温冷冻保存方法。西伯利亚鲟的产精量为113.67±39.86 ml,精子密度为(6.49±3.10)×108/ml,精子活力为(85.4±9.5)%,精子寿命为353±23 s。精子密度与精子快速运动时间、精子寿命之间均存在线性相关,用方程分别表示为:y=1.0384x+1.5089(R2=0.7325);y=2.9069x+74.289(R2=0.6967)。结果表明精子密度可作为一项精子质量评价的标准。通过比较西伯利亚鲟精子在不同稀释液、不同抗冻剂和抗冻剂浓度、降温速率、解冻温度下的保存效果,结果表明:配方2作为稀释液,18%甲醇作为抗冻剂,二步法超低温(-196℃)冷冻保存精子,40℃水浴解冻取得最好的冻后活力,解冻后活力为(51.8±5.8)%。西伯利亚鲟授精的最佳精卵比为106∶1。在此精卵比下用冻精授精分别得到了(72.3±3)%的受精率和(52.9±4.1)%的孵化率,其中受精率与鲜精没有显著性差异,孵化率与鲜精有显著差异(P<0.05)。  相似文献   

栉孔扇贝异精雌核发育四倍体早期胚胎发育的细胞学荧光显微观察     

迟长凤  杨爱国  王清印  刘志鸿  周丽青 《中国水产科学》2007,14(2):175-182

用1500μW/cm2的紫外线照射长牡蛎(Crassostrea gigas)精子60 s以进行灭活处理,并使之与栉孔扇贝(Chlamys farreri)卵子受精,在卵子受精后排出第一极体前用6-DMAP(50 mg/L)处理受精卵,持续处理35 min,抑制第一极体和第二极体的排放,诱导异精雌核发育四倍体。采用二脒基苯基吲哚(DAPI)荧光染色显微观察法,对灭活的长牡蛎精子诱导的栉孔扇贝雌核发育四倍体早期胚胎发育过程进行细胞学观察。结果表明:经紫外线灭活过的长牡蛎精子进入栉孔扇贝卵子后发生轻微膨胀;在第一次卵裂中期,精核形成一致密的染色质小体(DCB),位于两组分开的母本染色体之间,不参与核分裂;第一次卵裂结束时DCB滞留于两卵裂球的分裂沟上或进入其中一分裂球中;第二次卵裂过程中,DCB的去向与第一次卵裂时基本一致。6-DMAP处理有效地抑制了第一极体和第二极体的排出,从而使雌核四倍化。对担轮幼虫染色体倍性分析结果表明,通过本方法可以获得6.25%的四倍体幼虫。本研究还对灭活的异源长牡蛎精子诱导栉孔扇贝雌核发育四倍体过程中产生的复杂倍性、核物质分离紊乱及多精附卵现象进行了观察和分析。  相似文献   

环境因子对大黄鱼精子活力的影响   总被引：1，自引：1，他引：1  

朱冬发  成永旭  王春琳  薛良义 《水产科学》2005,24(12):4-6

通过观测精子的激活率、活动时间和寿命研究了几种环境因子的变化对养殖大黄鱼精子活力的影响。试验结果表明:精子平均密度为(1.17±0.09)×1010.ml-1,盐度对大黄鱼精子活力影响较大。当海水盐度适宜(19.61~24.87)时,精子的激活率≥90%,活动时间≥9.65 min,寿命≥13.50 min;在pH=4.0~10.0的海水中,精子都能被正常激活(≥70%),适宜的pH值为7.5~8.0;不同浓度的葡萄糖、NaCl和KCl溶液对精子活力的影响不同,不同浓度的EDTA-2Na溶液均不能激活精子;无Ca2 、Mg2 或HCO3-的人工海水对精子的激活率均高达90%,但精子的活动时间却有较大幅度的缩减。  相似文献   

Cryopreservation effects on the sperm quality of cachama blanca Piaractus brachypomus (Cuvier 1818)     

Juan Antonio Ramirez‐Merlano  Víctor Mauricio Medina‐Robles  Pablo Emilio Cruz‐Casallas 《Aquaculture Research》2011,42(6):738-745

The effects of straws volume, cryoprotectants and thawing temperatures were evaluated on the sperm quality of cachama blanca Piaractus brachypomus (Cuvier), an important Colombian fish species. Sexually mature fish were induced to ovulation or spermiation with a carp pituitary extract. A pool of suitable sperm samples was diluted in glucose, egg yolk, dimethyl sulphoxide (DMSO‐10%), methanol (MET‐10%) or ethylene glycol (ETG‐5%) and packed in 0.5, 2.5 or 5.0 mL straws and frozen in nitrogen vapour. The thawing process was performed in a 35 or an 80 °C water bath. The fertility was evaluated after 6 h post fertilization. The highest motility percentage (33 ± 3%) was observed with sperm cryopreserved with DMSO, packed in 5 mL straws and thawed at 35 °C. The treatments with DMSO and MET packed in 0.5 and 5.0 mL straws and thawed at 35 °C showed the highest fertility (higher than 71%) and the lowest fertility was obtained with MET‐2.5 mL (9 ± 5%). In all the treatments, a significant decrease in the sperm quality was observed at 80 °C. Sperm cryopreserved with DMSO‐10% or MET‐10%, packed in 2.5 or 5.0 mL straws are suitable to achieve acceptable fertilization and to fertilize high amounts of eggs.  相似文献   

Plasma membrane of trout spermatozoa: I. Isolation and partial characterization     

C. Labbé  M. Loir 《Fish physiology and biochemistry》1991,9(4):325-338

The plasma membrane from spermatozoa of rainbow trout was isolated by four techniques: sonication, hypotonic shock, mechanical homogenization after freeze-thawing, and nitrogen cavitation, in combination with continuous sucrose gradient centrifugation. Nitrogen cavitation (900 PSI, 20 min equilibration at 4°C) was the most effective technique.Following nitrogen cavitation, four bands were recovered in the sucrose gradient at densities 1.03, 1.05, 1.09 and 1.15 g/ml. Electron microscopy revealed membrane vesicles of various sizes in bands 1 to 3, while enzyme analysis revealed a 3.9 to 5.5-fold enrichment in 5'-nucleotidase and little contamination by lactate dehydrogenase (cytosol) and succinic dehydrogenase (mitochondria). Lipid analysis of bands 1 and 2 indicated a 6 to 7-fold enrichment in cholesterol and a cholesterol: phospholipid ratio of 0.59–0.70. Seven classes of phospholipids were present in bands 1–3 with no significant differences observed among bands. These data indicate that the vesicles (in bands 1 and 2) obtained after nitrogen cavitation are primarily plasma membranes. Membranes in band 3 appear to be slightly contaminated with nuclear membranes.Most of the plasma membrane proteins were acidic to neutral. The 2 main membrane proteins were 42 and 30 Kilodaltons.  相似文献   

不同精子密度冷冻猪5mL细管精液效果比较     

李林林  张德福  马恒东 《畜禽业》2007,(6):20-21

采集上海白猪精液进行5mL细管冷冻,比较冷冻密度分别为0.5×109个/mL、0.7×109个/mL、1.0×109个/mL的冻后精子质量。结果表明:3种密度冷冻猪精子,解冻后精子质量差异不显著(P﹥0.05)。  相似文献   

Seminal plasma composition and its relationship with physical spermatological parameters of Grass carp (Ctenopharyngodon idella) semen: with emphasis on sperm motility     

Yusuf Bozkurt  Fatih Öğretmen  Uğur Erçin  Ümmügülsüm Yιldιz 《Aquaculture Research》2008,39(15):1666-1672

In this research, the mineral and organic composition of the seminal plasma, physical spermatological parameters and their physiological relationships were investigated in grass carp (Ctenopharyngodon idella). The seminal plasma contained 98.14±5.23 mM L^?1 (Na⁺), 380.85±25.95 mM L^?1 (K⁺), 30.25±4.96 mg dL^?1 (Ca²⁺), 19.16±1.70 mEq L^?1 (Mg²⁺), 1.36±0.11 mg dL^?1 glucose, 0.37±0.08 g dL^?1 total protein, 12.02±1.18 mg dL^?1 cholesterol, 14.85±1.50 mg dL^?1 triglyceride and 43.5±9.56 mg dL^?1 urea. The following spermatological parameters were found: sperm volume 14.44±1.16 mL, sperm motility 80.60±1.55%, movement duration 67.68±4.32 s, density 15.43±0.72 × 10⁹ mL^?1, total density 337.43+45.86 × 10⁹ and pH 7.24±0.17. The Na⁺ and Ca²⁺ ions correlated negatively with spermatozoa motility (r=?0.453, P>0.05 and r=?0.192, P>0.05) respectively. The K⁺ ion correlated positively with spermatozoa motility (r=0.545, P>0.05). But a statistically significant correlation was not observed between sperm motility and seminal plasma parameters. The following correlations were observed between mineral and organic components. The Mg²⁺ was positively correlated with glucose and cholesterol (r=0.692, P<0.05 and r=0.680, P<0.05) respectively. A highly significant positive relationship was also found between Mg²⁺ and total protein (r=0.837, P<0.01). On the other hand, a significantly negative relationship was found between Ca²⁺ and triglyceride (r=?0.639, P<0.05). These parameters should be considered when developing procedures for either artificial fertilization or for cryopreservation of grass carp sperm.  相似文献   

稀释液中添加海藻糖对猪冷冻精液质量参数和活性氧的影响   总被引：8，自引：2，他引：8  

金一  陈宠霞  方南洙  金英海 《中国畜牧杂志》2005,41(10):6-8

本试验旨在探讨海藻糖对猪精子冷冻效果以及精子内活性氧(ROS)的含量影响。试验分对照组和4个海藻糖处理组(0.025,0.05,0.1和0.2 mol/L)。精子冷却后,添加0.1 mol/L海藻糖处理组精子活力显著(P<0.05)高于对照组,而精子内和稀释液中各组间ROS发生量都没有显著的差异。精子冷冻解冻后,添加0.05 mol/L海藻糖处理组的精子活力显著(P<0.05)高于对照组;添加0.05 mol/L和0.1 mol/L海藻糖处理组的精子生存率显著(P<0.05)高于对照组;添加0.1 mol/L海藻糖处理组顶体完整的精子百分比显著(P<0.05)高于对照组;4个海藻糖处理组膨胀精子百分比都显著(P<0.05)高于对照组;添加0.025 mol/L海藻糖处理组ROS水平显著(P<0.05)低于对照组。结果表明:海藻糖对精子冷冻保存是有益的,且能抑制精子内ROS的发生,但它的作用机制有待于进一步的研究。  相似文献   

精子受精抗原-1(FA-1) mRNA在绵羊睾丸和附睾中的表达   总被引：1，自引：0，他引：1  

娜仁花  旭日干 《畜牧与兽医》2006,38(11):1-3

本研究通过RT-PCR检测了该基因在绵羊睾丸和附睾中的表达情况。首先,提取绵羊睾丸组织、附睾头、体、尾部组织总RNA,以此为模板,反转录合成cDNA,自行设计引物,PCR扩增出462 bp的目的DNA。然后,将目的片断克隆入T载体,通过菌液PCR和重组质粒酶切,鉴定重组质粒中的目的DNA。再经序列分析鉴定目的片断。同时,以β-actin为内参照物,进行RT-PCR半定量分析,比较精子受精抗原(FA-1)在睾丸、附睾头、体、尾组织中的表达量。结果表明,FA-1在绵羊睾丸和附睾中均表达。  相似文献