Effects of ultra-violet irradiation on sperm motility and diploid gynogenesis induction in large yellow croaker (<Emphasis Type="Italic">Pseudosciaena crocea</Emphasis>) undergoing cold shock

Large yellow croaker, Pseudosciaena crocea, exhibit sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. We have established a protocol to produce all-female croaker P. crocea through induction of meiotic gynogenesis with homologous sperm. The first set of experiments investigated the ultra-violet (UV) irradiation on sperm motility and duration of sperm activity to determine the optimal UV dosage for genetic inactivation of sperm, yet retaining adequate motility for activation of eggs. Milt from several males was diluted 1:100 with Ringer’s solution and UV irradiated with doses ranging from 0–150 J cm⁻². The results indicated that motility and duration of activity generally decreased with increased UV doses. At UV doses greater than 105 J cm⁻², after fertilization, motility was <10% and fertilization rates were significantly lower. Highest hatching rate was obtained at 75 J cm⁻². A second set of experiments was carried out to determine appropriate conditions of cold shock for retention of the 2nd polar body in P. crocea eggs after fertilization with UV-inactivated sperm by altering the timing, temperature and duration of shock. At 20°C, shock applied at 3 min after fertilization resulted in higher survival rate of larvae at 6 h after hatching. Results of different combinations of three shock temperatures (2°C, 3°C or 4°C) and five shock durations (4 min, 8 min, 12 min, 16 min or 20 min) at 3 min after fertilization demonstrated that shocks of 12 min gave highest production of diploid gynogens. Statistical analysis revealed that maximum production of diploid gynogens (44.55 ± 2.99%) were obtained at 3°C. The results of this study indicate that the use of UV-irradiated homologous sperm for activation of P. crocea eggs and cold shock for polar body retention is an effective method for producing gynogenetic offspring.     

Effects of slow release gonadotropin releasing hormone analog on milt characteristics and plasma levels of gonadal steroids in greenback flounder, Rhombosolea tapirina

Spermiated male greenback flounder (Rhombosolea tapirina) were implanted with gonadotropin releasing hormone analog (GnRHa) pellets at different dosages to examine their effects on milt characteristics and plasma levels of testosterone (T), 11-ketotestosterone (11KT) and 17,20β-dihydroxy-4-pregnen-3-one (17,20βP). Milt and blood samples were collected for up to 28 or 42 days post-implantation (p.i.) in two separate experiments using fish that were slightly or moderately spermiated, respectively. In both experiments, fish treated with GnRHa pellets showed a consistent and significant increase in milt volume relative to controls, and the increase of milt volume was more rapid than the increase in numbers of spermatozoa. Spermatocrit (Sct) was significantly lower in GnRHa-treated fish than in controls. Plasma levels of T and 11KT were elevated at 7 and 14 days p.i. in slightly spermiated fish treated with GnRHa and elevated plasma T and 11KT levels were accompanied by increased milt volume early in the spermiation period. In contrast, there was no significant difference in plasma T levels between GnRHa-treated and control fish in fish that were moderately spermiated at the time of implant. Treatment with GnRHa tended to result in lower plasma levels of 11KT and higher levels of 17,20βP relative to controls, and there was a positive relationship between the elevation of plasma 17,20βP and the increase in milt volume in response to implantation of GnRHa pellet. Slow release GnRHa administration had no effect on sperm activity or pH and osmolality of seminal plasma in either experiment.     

Successful Hybridization of Acipenser Species Using Cryopreserved Sperm

Successful hybridization of sterlet (Acipenser ruthenus) × Siberian sturgeon (Acipenser baeri), sterlet × Russian sturgeon (Acipenser gueldenstaedti) and sterlet × European sturgeon (Acipenser sturio) was carried out for the first time using cryopreserved semen of sturgeon males and sterlet × sterlet crosses as controls. Sperm of all three species was diluted with a cryodiluent composed of 23.4 mM sucrose, 0.25 mM KCl, 30 mM Tris (pH 8.0) and 10% methanol. The samples were frozen in plastic straws in the vapor of liquid nitrogen at the height of 3 cm above the level of nitrogen for 3 min. Following thawing approximately 3000 sterlet eggs were fertilized with six straws of frozen-thawed sperm. The hatching rate with sterlet sperm was 30.6% while the hatching rate of A. ruthenus × A. baeri, A. ruthenus × A. gueldenstaedti and A. ruthenus × A. sturio hybrids was 50, 17.4 and 34%, respectively. Morphometric markers as well as random amplified polymorphic DNA (RAPD) assay was used to verify interspecific hybridization.     

鼠尾藻生殖托和气囊的形态结构观察

鼠尾藻的生殖托气囊的构造和精、卵子的形态与放散是研究鼠尾藻有性繁殖过程的基础内容之一。本文以青岛太平角沿岸潮间带的性成熟野生鼠尾藻为对象,采用活体切片、组织切片以及显微照相等方法对鼠尾藻生殖托的形态、内部结构以及挂卵现象进行了观察,并对精子、卵子和气囊的形态做了观察描述,特别是观察到了鼠尾藻精子的鞭毛、雌性生殖窝的结构、气囊的花纹与构造等重要现象。     

精子载体转基因的研究进展

精子载体法培养转基因动物是目前操作最简便，成本低廉且危险隐患相对较低的一种转基因方法。近10年来的研究结果表明，精子载体法可得到转基因阳性动物，并且引起了更多科研人员的关注。     

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.