中国对虾（Penaeus chinensis）精子做载体将生长激素基因导入受精卵的研究

本实验采用卡介苗注射器将羊生长激素基因溶液注射到亲虾纳精囊中，以精子为载体，携带基因导入对虾受精卵，而达到转基因对虾的目的。经ＰＣＲ检测表明：１．经ＳＴＥ（ｐＨ８．０）第一次洗涤精子的洗涤液呈阳性信号；第２－４次的洗涤液呈阴性；２．多次洗涤后的精子抽提的ＤＮＡ呈阳性；３．检测 １０个样品１００尾仔虾中有一个样品呈阳性结果，转基因比率在 １％以上。同时斑点杂交结果也印证了上述结果。     

Pregnancy in the Caspian Miniature Horse Using Frozen Semen Cryopreserved with the EquiPRO CryoGuard Freeze Medium and Customized Freezing Protocols

The primary goal of this project was to establish a protocol to freeze the sperm of the Caspian miniature horse in an attempt to start an intensive artificial insemination program to effectively increase the population of this breed, which has been listed as “Critical Rare Breed” by the American Livestock Breed Conservancy and is in danger of extinction. Commercially available equine freezing medium (EquiPRO CyoGuard Complete egg-yolk extender) was used for the initial setup of two different freeze protocols: slow and fast. The fast-freeze protocol had slightly better postthaw results and was used for a fertility demonstration. Five mares of proven fertility, aged 3 to 12 years, were used in the fertility trials, two of which resulted in pregnancy. This is the first report of pregnancy in the Caspian miniature horse using frozen semen, and the results seem to be a promising start to an extensive program to help this endangered breed, although further research on freezing protocols and conditions for this process are necessary to further improve the survival of semen and pregnancy rate.     

Sexing of Dog Sperm by Fluorescence In Situ Hybridization

Effective preselection of sex has been accomplished in several species of livestock and also in humans using the flow cytometric sperm sorting method. A guaranteed high sorting accuracy is a key prerequisite for the widespread use of sperm sexing. The standard validation method is flow cytometric remeasurement of the DNA content of the sexed sperm. Since this method relies on the same instrument that produced the original sperm separation, it is not truly independent. Therefore, to be able to specifically produce either male or female offspring in the dog, we developed a method of direct visualization of sex chromosomes in a single sperm using fluorescence in situ hybridization (FISH) as a validation method. Denaturation of canine spermatozoa by immersion in 1 M NaOH for 4 min yielded consistent hybridization results with over 97% hybridization efficiency and a good preservation of sperm morphology. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X- and Y-chromosome-bearing spermatozoa in any of the three dogs. In addition, the mean purities of flow-sorted sex chromosomes in spermatozoa of the three dogs were 90.8% for the X chromosome fraction and 89.6% for the Y chromosome fraction. This sorting was evaluated by using the dual color FISH protocol. Therefore, our results demonstrated that the FISH protocol worked reliably for both unsorted and sexed sperm samples.     

钝吻黄盖鲽精子冷冻保存及生理特性分析

钝吻黄盖鲽(Pseudopleuronectes yokohamae)是鲆鲽鱼类中重要的天然捕捞和养殖对象,但由于生态环境变化、人工捕捞过度等因素,导致其种质资源数量降低,进行精子冷冻保存技术研究,对其种质资源保存具有重要意义.本研究以性成熟的雄性钝吻黄盖鲽为实验材料,对精子稀释液种类及成分、抗冻剂种类、激活精子海水盐度和冷冻精液授精实验等进行筛选,实验数据利用SPSS 19.0软件进行单因素方差分析和Student-Newman-Keuls分析.结果表明,利用MFs-3(8 g/L NaCl+0.65 g/L KCl+15 g/L Glucose)稀释液分别与20％1,2丙二醇(1,2-propylene glycol,PG)和20％乙二醇(ethylene glycol,EG)抗冻剂作为精子抗冻保存液时,冷冻效果最好,其中PG组对应的精子活力、快速运动时间和寿命分别为(95.26±0.39)％、(46.00±1.00)s和(124.33±4.04)s,EG组则为(95.15±0.41)％、(45.67±0.58)s和(124.00±3.00)s.利用盐度为10～50的人工配制的海水激活解冻后的精子,发现当盐度为30时,精子活力高达(95.07±0.69)％,与对照组相比不存在显著性差异.将PG组和EG组冷冻保存的精子解冻后分别与其卵进行授精实验,PG组受精率和孵化率为(80.08±0.68)％和(77.44±1.76)％,EG组则为(80.17±0.45)％和(77.92±1.33)％,与鲜精相比无显著性差异.利用计算机辅助精子分析系统(computer-aided sperm analysis,CASA)测定MFs-3+20％ PG和MFs-3+20％ EG冷冻保存的钝吻黄盖鲽精子的各项运动参数,结果显示,二者的曲线运动速度(curvilinear velocity,VCL)、直线运动速度(straight line velocity,VSL)、平均鞭打频率(beat cross frequency,BCF)、运动的直线性(linearity,LIN)和运动的前向性(straightness,STR)的数值差异性不显著.本研究利用MFs-3+20％ PG和MFs-3+20％ EG成功冷冻了钝吻黄盖鲽的精子,为钝吻黄盖鲽精子冷冻保存技术、人工杂交育种繁殖及种质资源库的建立奠定了基础.     

Effects of Intratesticular Injection of 70% Glycerin on Stallions

The removal of endogenous germ cells of recipient stallions is a key step to produce donor germ cell-derived sperm using the germ cell transplantation technique. Six Thoroughbred stallions were divided into a treatment (n = 3) and a control group (n = 3), and 70% glycerin (1, 2, 3-trihydroxypropane, 40 mL per testis) or phosphate-buffered saline, respectively, was locally injected into testes. General semen evaluation, libido, and testicular volume were performed weekly from 3 weeks before to 10 weeks after treatment. The number of round germ cells in the ejaculate was counted using a hemocytometer. The hematoxylin and eosin staining was performed on the cross sections of testicular tissue obtained 11th week of treatment. Plasma testosterone levels in blood collected weekly were measured using a colorimetric competitive enzyme immunoassay kit. The sperm number was significantly lower than that of the control group at 5 and 10 weeks after glycerin injection. No differences in the status of spermatogenesis in the cross sections of seminiferous tubules and testicular volume were found between the two groups. The 70% glycerin-treated stallions had reduced total and progressively motile sperm and exhibited a significantly higher population of round germ cells in the ejaculate. Testosterone levels, testicular volumes, and libido of stallions were not significantly different between the groups. In conclusion, although intratesticular injection of 70% glycerin may have caused disassociation of some germ cells in the seminiferous tubules for several weeks, it did not significantly ablate germ cells in the tubules at 11 week in stallions.     

Cloning of human sperm protein(SP17) and expression in escherichia coli DH5α

AIM: To obtain GST fusion protein of hSP17 gene and construct the recombinant plasmidfor expression in E.coli.METHODS:Total fragment of hS P17 cDNA gene were amplified by RT-PCR,then subcoloned into p GEX-3b to generate recombinant hS P17/pGEX.Right orientation of insert are identified by restricted enzyme digestion.Transform the correct recombinant plasmid into the E.coli DH5a.The expression of fusion proteins hS P17-GST were induced by adding isopropylthiogalactoside(IPTG).RESUL TS and CONCLUSION:The recombinant plasmid hS P17/pGEX-3b could express effectively in E.coli and a high level of fusion protein hsp17-GST with the predicted molecular weight was detected.     

黑白花公牛冻精受精力的检测—穿透仓鼠卵的试验

本试验用SPA法(Sperm Penetration Assay)即精子穿透分析法对8头黑白花公牛冻精的受精力进行检测,以预测各公牛精液受精力的水平。结果:每卵表面平均精子数为3.2～9.5个,每卵内精子数为1.6～3.4个,被穿透的卵子比例即穿透率为34.9～65.7％,具雄原核的卵子比例即原核卵率为16.0～32.4％。这些数值与各公牛冻精授精500多头母牛的效果作对照,8头公牛平均80(70～90)天的不返情率为56％(45.8～71.1％),不返情率与雄原核卵率,穿透率呈高度正相关,相关系数分别为 0.82和 0.92。 另一组试验结果表明:授精时间对穿透率特别是对雄原核形成有很大影响。授精2小时后观察,没有雄原核形成,只有附着于卵子表面的精子;授精4小时后,精子已通过卵黄膜进入卵内,并开始形成雄原核;授精6小时后形成发育良好的雄原核。 这些观察结果有助于利用这项技术在生产上对公牛精液的受精力进行客观评定,为选择后备公牛提供重要的生理指标,特别是对评定冷冻精液的品质具有一定的参考价值。并为精子顶体反应、受精机理、体外受精、遗传学和胚胎学等领域的研究提供新的手段。     

异科精子在蚕卵中的命运

取受精囊里的精液(含异科精子)应用离体人工授精经卵孔入卵;即,蓖麻蚕(?)→多化性家蚕(?)及家蚕(?)→蓖麻蚕(?),并作细胞学观察,表明停滞在第一成熟分裂的卵细胞被远缘精子活化,过后,入卵的精子演变则受卵细胞质所控制.但,尚未见雌雄原核的融合,似系假受精作用.无论怎样,精子中心粒一旦与卵细胞器配合而聚成具功能的中心体,可促使子核岛出现调整和分裂.囊胚的形成,是造胚子的临界期.我们曾分别从对照组(家蚕(?)→家蚕(?),30/160)及科间授精组(蓖麻蚕(?)→家蚕(?),2/754)得到子代.讨论了精-卵相互作用等.     

Effect of iodine supplementation on thyroid and testicular morphology and function in euthyroid rats

Seventy-five male weaner euthyroid rats, randomly divided into three equal groups were used to evaluate the effect of iodine supplementation in the diet on growth and spermatogenesis. From the age of six weeks, the rat groups were fed normal diet containing 0.05 mg iodine/Kg diet (A); normal diet supplemented with 0.5 mg/Kg iodine (B) and normal diet supplemented with 3.0 mg/Kg iodine for a period of 90 days. Thereafter, all three groups were fed the normal diet for another 60 days. Body weight and feed consumption were determined; morphomeric studies of thyroid glands, testes and epididymes were carried out. Spermatogenesis was evaluated with epididymal (ESC) and testicular sperm counts (TSC). Increasing iodine intake significantly (p < 0.05) decreased mean body weight from day 30 of supplementation. Iodine supplementation influenced feed conversion ratio and efficiency in feed utilization in an inconsistent pattern. Supplementation did not significantly (p > 0.05) alter the size of thyroid glands, but increased the mean weights of the testes and epididymes to levels significantly (p < 0.05) higher than values for non-supplemented rats at specific stages of the study, especially at the highest (3 ppm) level of iodine supplementation. However, supplementation resulted generally in lower sperm counts, which was significant (p < 0.05) in the case of the epididymes. The results of the study show that iodine supplementation to weaner, non-iodine deficient euthyroid rats at 3ppm not only retard weight gain but could also reduce fertility by lowering epididymal sperm counts.