麻醉对小鼠的排卵数量、排极率、存活率及孤雌激活率的影响

实验探讨了麻醉(即氯胺酮)对小鼠卵母细胞成熟和激活的影响。选择达到体成熟的昆明种小鼠实施氯胺酮的三期麻醉剂量,观察小鼠超排卵母细胞数量、排极、存活率及乙醇孤雌激活率的变化。结果发现,氯胺酮可明显减少小鼠超排卵母细胞的数目,而且卵母细胞也出现一定数量的畸形;但氯胺酮对极体释放率影响不显著。随着培养时间的延长,对照组和麻醉组的卵母细胞在培养8h后,均有一定的死亡率,尤其是氯胺酮组的存活率表现出显著降低(与对照组相比)。另外,氯胺酮对孤雌激活的影响也是显著的。     

影响猪卵巢卵母细胞体外成熟的若干因素

介绍了以屠宰场废弃的猪卵巢为材料,通过系统技术途径得到猪早期胚胎的基本方法。新鲜的猪卵巢采集后,25～35℃条件下保存,在3h内进行卵泡卵母细胞与卵丘细胞复合体(COCs)采集与分级,对COCs中卵母细胞体外成熟(IVM)、体外受精(IVF)及其受精卵体外发育(IVD)等关键技术环节进行了系统地探索,重点探讨了不同发育阶段(直径大小)卵泡卵母细胞、不同培养系统与添加成分以及不同培养时间等关键因素对猪卵母细胞IVM的影响,并对其IVF卵裂能力进行了比较与评价。实验结果表明,采用mTCM199作为基础成熟培养系统液,并添加ф5～8mm卵泡液(pFF)和雌二醇(E2),ф2～8mm卵巢卵泡COCs经IVM40h可得到较高的成熟率(66.7%)和IVF卵裂率(29.2%)。本研究初步建立了猪2-4-细胞早期胚胎的体外批量生产技术,可望进一步改进和完善,应用于养猪业科研与生产实践。     

暗纹东方鲀排卵后授精时间与受精率的关系

研究了暗纹东方鲀排卵后授精时间与受精率的关系。对 3龄人工养殖的暗纹东方鲀采用多次注射外源激素的方法, 诱导其卵母细胞成熟并排卵。分别在不同时刻对排卵后的卵母细胞进行人工授精, 在原肠中期检查受精率。结果表明, 随着排卵后授精时间的延长, 受精率逐渐下降, 18h时受精率降为 0。排卵后 0、2、4h时授精的受精率, 分别为 83. 75%、78 .75%和 75. 5%, 无显著差异 (P>0 .05), 而其它不同授精时间之间的平均受精率均具有显著性差异 (P<0. 05)。对于不同时刻的平均受精率与排卵后授精时间两者关系进行拟合, 得出它们之间的关系为y= 0 .0352x3 -0 .9877x2 +1 .7976x+81. 953 (R2 =0 .996 ), 相关系数较高, 说明应用 3阶函数曲线能很好地表达两者之间的关系。     

虹鳟排卵前后血清中性类固醇激素浓度变化的研究

对虹鳟(Salmo gairdneri)排卵前后血清17β-雌二醇(17β-E_2),睾酮(T)和17α-羟-20β-双氢孕酮(17α20βP)的含量进行了测定。17β-E_2从排卵前15天的18.1ng/ml急骤下降到排卵前3—6天的2ng/ml,并继续下降,至排卵时仅为0.9ng/ml。睾酮在虹鳟雌鱼血清中的含量很高,血清浓度从排卵前9天的峰值水平143.3ng/ml缓慢下降,排卵时为24.3ng/ml。17α20βP的血清浓度变化明显,排卵前15天,该激素浓度接近于基线水平或甚至低得不能被检测,排卵前9天开始迅速上升,到排卵前3天达到峰值350.6ng/ml,排卵时为302.2ng/ml。本研究进一步证实了17α20βP在鲑鳟鱼类卵母细胞最后成熟过程中的生理作用,是一种诱发卵母细胞成熟的类固醇激素。临排卵前,血清17β-E_2浓度的下降,可能调节了17α20βP大量分泌的时间;17α20βP的大量分泌是卵母细胞达到最后成熟和排卵不可缺少的一环。注射合成的大麻哈鱼(Oncorhynchus keta)促性腺激素释放激素(s-GnRH)及其类似物(s-GnRH-A),诱发虹鳟血清类固醇激素17β-E_2,睾酮和17α20βP的变化趋势与自然排卵时的变化相类似。经注射药物诱发排卵的鱼,较对照组提早一周排卵,而且排卵较同步和集中。     

重金属对鱼类早期发育的生态毒理效应

鱼类早期生活阶段对外界环境极为敏感,即使低浓度的污染物暴露也可能对其胚胎发育、仔鱼生长存活造成损害,导致种群数量和质量的降低。研究污染物对鱼类早期生活阶段的毒理效应和机制是揭示污染物对生物种群和水生生态系统潜在风险的有效途径。重金属对鱼类的影响在精、卵发生时即可显现。例如,抑制卵(精)母细胞成熟和精子活力降低,使受精率降低;影响受精卵吸水膨胀过程,改变胚胎发育速率、引起胚胎畸形、影响胚胎心率、改变孵化时间、降低孵化率;影响初孵仔鱼体长、造成仔鱼畸形、影响卵黄囊吸收和胚后仔鱼发育生长、摄食行为等。重金属对鱼类早期发育阶段的毒性效应主要取决于金属种类、暴露浓度和受试鱼种及其发育阶段。此外,外界环境条件等因素也会对重金属的毒性产生显著影响。本研究综合分析国内外关于重金属污染物对鱼类精卵、胚胎、仔鱼等早期发育过程生态毒理效应的研究进展,并结合相关报道阐述了其毒性机制。     

锯缘青蟹卵巢发育的组织学观察

根据锯缘青蟹卵巢的外部形态和内部组织学特征,卵巢发育可以划分为六个时期:未发育期、发育早期、发育期、将成熟期、成熟期和排卵后期。在发育早期,多数卵母细胞胞质中有多个嗜酸性液泡,这种结构在其他十足类卵母细胞中较少见。少数个体在发育早期发生卵母细胞全面退化,这种现象可能是缺乏交配或饥饿造成的。     

牛、羊卵母细胞孤雌激活的研究

本实验主要比较了离子霉素、电脉冲2种方法激活牛、羊体外成熟卵母细胞的效率。2种激活方法中,牛胚胎卵裂率无显著差异(90.61%VS94.40%,P>0.05),而离子霉素激活胚胎的囊胚发育率极显著高于电激活方法(12.3%VS2.4%,P<0.01)。2种方法对羊的胚胎的研究中,羊胚胎卵裂率无显著差异(72.4%VS77.4%,P>0.05)。但是离子霉素激活胚胎的囊胚发育率显著高于电激活方法(3.67%VS10.4%,P<0.05)。本实验中还比较了用化学激活(离子霉素)激活牛体外成熟卵母细胞后,用SoFaa体系培养,换液与不换液对孤雌激活胚胎体外发育的影响。结果表明:在第四天不换液的胚胎卵裂率和囊胚率极显著高于换液的胚胎(11.64%VS3.49%,P<0.01)。     

中华绒螯蟹卵黄发生期卵母细胞和卵泡细胞超微结构观察

通过透射电镜技术观察了中华绒螯蟹第二次卵巢发育过程中卵巢的超微结构变化.结果表明:(1)中华绒螯蟹第二次卵巢发育过程中卵黄发生期可分为初期和后期;(2)卵黄发生初期(雌蟹第一次排卵后的16 d内),卵黄生成以卵母细胞内源性合成为主,此时卵母细胞胞质中存在大量内质网囊泡、高尔基体和线粒体,这些细胞器参与胞内卵黄物质的合成.内源性合成后期,卵母细胞膜形态多样,呈现触手状、波浪状和断裂状,为外源合成期做准备.此期卵泡细胞还未向卵母细胞靠近,两类细胞间存在着由淋巴细胞吐出的絮状物;(3)卵黄发生后期,首先为卵泡细胞与卵母细胞的结合阶段(排卵后16～21 d),此后,卵泡细胞胞质中含有大量内质网囊泡、卵黄颗粒和脂滴,卵母细胞与卵泡细胞膜变为链珠状便于物质交换,卵母细胞的卵黄合成能力减少,转由卵泡细胞进行外源性物质吸收和卵黄物质合成(21～36 d);(4)卵黄发生结束后,双层卵膜形成,卵黄体和脂肪滴均匀分布在卵母细胞胞质中.     

二甲双胍对不同日龄小鼠卵母细胞体外培养的影响研究

通过研究二甲双胍对不同日龄小鼠卵母细胞体外培养促进作用,明确二甲双胍作为体外培养促进剂的可行性,为二甲双胍在其他哺乳动物卵母细胞体外培养中的应用研究奠定基础。试验研究二甲双胍对卵母细胞体外成熟和体外受精的影响。结果表明卵母细胞成熟率、卵裂率及囊胚率均无明显变化,二甲双胍能够促进卵巢卵泡发育,增加卵母细胞数量。二甲双胍一定程度上对小鼠卵母细胞体外培养具有促进作用。     

渗透胁迫对中华绒螯蟹胚胎生化组成及消化酶活性的影响

研究了高渗与低渗胁迫对离体培养的中华绒螯蟹(Eriocheir sinensis)不同发育期胚胎中生化成分含量及消化酶活性的影响。结果显示:从原肠期发育到出膜前期,对照组(盐度15)胚胎中蛋白含量降低(P<0.05)、胰蛋白酶与胃蛋白酶活性升高(P<0.05),脂类含量(P>0.05)和脂肪酶活性(P<0.05)降低,碳水化合物含量和淀粉酶活性均在眼点期升高(P<0.05)而在出膜前又降低(P<0.05);低渗胁迫引起胚胎蛋白含量降低(P<0.05),而高渗胁迫导致胚胎蛋白含量升高(P<0.05);高渗胁迫对胚胎脂类含量无显著影响(P>0.05),而低渗胁迫引起出膜前期胚胎中脂类含量显著降低(P<0.05);高、低渗胁迫对胚胎碳水化合物含量均无显著影响(P>0.05);除高、低渗胁迫均显著降低眼点期胚胎淀粉酶活性(P<0.05)而引起出膜前期胚胎胰蛋白酶活性略降低(P>0.05)外,低渗胁迫导致不同发育时期胚胎消化酶活性升高,而高渗胁迫导致酶活性降低;除脂肪酶外,渗透胁迫对原肠期胚胎消化酶活性的影响均具有显著性(P<0.05)。研究结果说明,蛋白质和脂类是中华绒螯蟹胚胎发育过程中重要的能源物质与结构物质,渗透胁迫通过改变中华绒螯蟹胚胎消化酶的活性进而影响胚胎对卵黄物质的分解与利用,最终影响胚胎的发育。本实验结果提示,河口盐度过高或过低均可能对中华绒螯蟹早期胚胎的发育及后期幼体的质量产生潜在的不利影响。     

A new classification of pre‐ovulatory oocyte maturation stages in pikeperch,Sander lucioperca (L.), and its application during artificial reproduction

This study aimed at improving the reproduction effectiveness and synchronization of ovulation in the pikeperch, Sander lucioperca (L.), during induced spawning, which is one of the main bottlenecks in the aquaculture of this species. For this purpose, a new categorization of maturation stages in pre‐ovulatory oocytes was applied. It is generally based on two morphological indicators: germinal vesicle migration or its breakdown (GVBD) and different oil droplet coalescence rates. This categorization covered seven stages (from I to VII) – from the end of vitellogenesis to ovulation. The categorization was verified by controlled reproduction with the use of hormonal stimulation (500 IU of hCG per kg of female body weight) and low spawning temperature (12 °C), which extended the latency time. In addition, some morphological indicators (pseudo‐gonadosomatic index, Fulton's condition coefficient) of females were calculated in order to determine their usability in determining the maturation stage. However, these indicators proved to be ineffective for this purpose, further highlighting the need to determine the maturational stages in pre‐ovulatory oocytes to synchronize ovulation in pikeperch. During the experiment, ovulation seemed to be synchronized among the experimental treatments. Statistical differences were found in terms of latency time between experimental groups at different maturity stages (II – 78–98 h; III – 57–78 h; IV – 48–58 h; V – 32–49 h; VI – 5–30 h) according to the proposed classification. This classification and the results presented in the study significantly improved the synchronization of ovulation, which may positively affect the effectiveness of pikeperch production under controlled conditions.     

The use of luteinizing hormone releasing hormone analogue for ovulation induction in black sea bass (Centropristis striata)

Interest in commercial production of black sea bass has increased in recent years, but reliable spawning methods remain problematic. The objective of this study was to evaluate the effects of oocyte size and luteinizing hormone releasing hormone analogue (LHRHa) dosage and delivery systems on ovulatory success for in vitro fertilization. Vitellogenic females with maximum oocyte diameters of 400–625 μm were implanted with a 95% cholesterol–5% cellulose pellet containing 50 μg of LHRHa. Fish with maximum oocyte diameters < 450 μm failed to ovulate. In contrast, 90% of fish with 500 μm oocytes spawned within 36 h and 40% of this group ovulated a second time. All of the females containing oocytes > 550 μm ovulated. In a second experiment, females with uniformly vitellogenic oocytes (> 500 μm) and implanted with 50 μg LHRHa ovulated substantial numbers of eggs (45,000–192,000 eggs/kg body weight (BW), but fertility was consistently low (0–15%). In a third experiment, 19 of 39 females receiving implants containing 6.3–23.6 μg LHRHa/kg BW during the spawning season ovulated, but fecundity (17,000–339,000 eggs/kg) and fertilization (0–98%) were highly variable. When fish were grouped by developmental index, calculated as the number of oocytes with diameters > 400 μm/total number of oocytes measured, there were no statistical differences among groups with respect to the number of spawns, fecundity or fertilization success. In a fourth experiment, 11 of 13 females with a clutch of fully vitellogenic oocytes that were injected with 20 or 100 μg/kg BW LHRHa ovulated between 1 and 2 times on consecutive days. Five of seven given an implant containing 12.5 μg LHRHa ovulated one or more times. Fish implanted with shams or injected with vehicle alone did not ovulate in any of the experiments. No differences were found in the number of spawns, fecundity or fertilization success from fish receiving different doses of injected or implanted LHRHa. Incubation of pooled eggs produced 155,000 larvae (60% hatch) and 95,000 one-gram juveniles. These results demonstrate that injected or implanted LHRHa is effective for inducing ovulation in black sea bass with maximum oocyte diameters > 500 μm.     

Reproductive parameters of the chub mackerel <Emphasis Type="Italic">Scomber japonicus</Emphasis> estimated from human chorionic gonadotropin-induced final oocyte maturation and ovulation in captivity

ABSTRACT:   Final oocyte maturation and ovulation of captive chub mackerel Scomber japonicus with fully yolk-accumulated oocytes were induced by a single injection of human chorionic gonadotropin. Reproductive parameters, including spawning frequency and batch fecundity, which are required to estimate spawning biomass in pelagic fish by the daily egg production method, were analyzed. Germinal vesicle migration (GVM) occurred at 18–24 h post-injection, and the hydration and ovulation of oocytes were completed at 30 and 36 h post-injection, respectively. The results of the maturation process suggest that fish with GVM-stage ovaries captured in the daytime from the field are capable of spawning on the night following their capture. The oocytes used in the oocyte size-frequency distribution method for batch fecundity estimates should be at late GVM and more advanced stages. The results of sequential artificial insemination showed that the quality of ovulated eggs held in the ovarian lumen rapidly deteriorated as time progressed after ovulation. This indicates that the fertilization window for the ovulated eggs of chub mackerel lasts only a few hours, and spawning behavior should be performed within a few hours after ovulation in the wild population.     

The role of prostaglandins in the control of ovulation in yellow perch,Perca flavescens

Previous studies have shown that 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P) can induce both germinal vesicle breakdown and ovulationin vitro of yellow perch (Perca flavescens) oocytes. The stimulation of ovulation can be blocked by indomethacin and restored by the subsequent addition of several primary prostaglandins (Goetz and Theofan 1979). In the present investigation, medium levels of prostaglandin F (PGF) and E (PGE) were measured by radioimmunoassay duringin vitro 17α,20β-P-induced ovulation of perch oocytes. PGF levels increased significantly (compared to controls) from 30 to 36h of incubation. Hourly samples taken through the time of ovulation revealed that the increase in PGF was very closely correlated to the time of ovulation though it did not preceed it. Cortisol, testosterone, estradiol-17β, 17α,20α-dihydroxy-4-pregnen-3-one and 17α-hydorxyprogesterone did not increase PGF levels by 48h of incubation, however, several other progestational steroids including 20β-dihydroprogesterone (20β-P) and progesterone did. 17α,20β-P, 20β-P and progesterone also stimulated an increase in PGF in spontaneously ovulating oocytes (in which all oocytes ovulated including controls), indicating that the increase in PGF was not merely a result of the physical process of ovulation but was related to the presence of the steroid. Based on work supported by the National Science Foundation under grant DCB-8517718 and DCB-8718178.     

Egg collection from hatchery-reared broodstock of spotted halibut Verasper variegatus treated with LHRH analog

The objective of this study is to establish a method for acquiring large quantities of high-quality eggs for artificial fertilization from hatchery-reared broodstock of spotted halibut Verasper variegatus. We estimated the optimum conditions for implantation of luteinizing hormone-releasing hormone analog (LHRHa) cholesterol pellet (LHRHa-CP) to induce ovulation from two experiments which focused on the dose and the timing combined with monitoring of oocyte development by ovarian cannulation. From the results of cannulation, vitellogenesis of hatchery-reared broodstock was suggested to occur normally, although final oocyte maturation was not initiated in the first clutch of oocytes. In late December, oocytes in the most advanced clutch still underwent vitellogenesis, having diameter of about 0.82?mm. LHRHa-CP implantation during this period had no remarkable effects, except for administration at high dose (100???g/kg). In contrast, in mid January, when oocyte diameter reached about 0.95?mm, ovulation occurred in most individuals, even at low dose (20???g/kg). In mid February atretic oocytes became remarkable and LHRHa-CP implantation showed much lower performance in terms of egg quality. The diameter of growing oocytes converged to about 0.95?mm, which was that of fully grown postvitellogenic oocytes. Thus, oocyte diameter is suggested to be an effective indicator to estimate the timing of LHRHa-CP implantation.     

Involvement of sex steroids in final stages of oogenesis in Eurasian perch, Perca fluviatilis

Significant plasma 17,20β-dihydroxy-4-pregnen-3-one peaks were measured for the first time in female Eurasian perch, Perca fluviatilis, during the pre-ovulatory period, reaching 3.5 ng ml^?1, but was not synchronized with final maturation and ovulation stages.     

Controlled reproduction in Anguilla anguilla (L.): comparison between spontaneous spawning and stripping‐insemination approaches

This study aimed to compare the fertility of eggs between artificially matured female silver eels that spawned spontaneously and those that were spawned by manual stripping. The effects of the two methods of spawning on ovulation and fertilization rate were also investigated. For this purpose, 18 wild female European eels captured in Bonello lagoon (North Adriatic Sea) were carp pituitary extract‐injected to undergo sexual maturation and ovulation; a final injection of 17,20β‐dihydroxy‐4‐pregnen‐3‐one (DHP) was administered when at least 30% of the oocytes were fully transparent. After the DHP‐injection, nine eels were transferred to a new closed recirculating aquaculture system, where they were housed with spermiating males (sex ratio 4/1) to allow spontaneous spawning (SPT‐group); the remaining nine eels were transferred to a 250 L tank and ovulation was checked at four‐hourly intervals by manual stripping (STR‐group). The number of eggs per female in the SPT‐group was significantly greater than that in the STR‐group. Furthermore, fertilization rates in the SPT‐group were notably higher than those observed in the STR‐group. Significantly, the best performances were obtained among eels in which at least 50% of oocytes were fully transparent at the time DHP was administered. We conclude that the fertility of eggs from spontaneously spawning eels is superior to that of eggs acquired by strip‐spawning and artificial fertilization.     

Stimulation of spermiation and induction of ovulation in pike (Esox lucius)

Mature northern pike were given various hormonal treatments in March or April in order to stimulate spermiation or to induce ovulation. In males the total amount of sperm collected after treatment increased, in comparison with saline-injected males, by 3–11 times with partially purified salmon gonadotropin (PPSG-activity: half of the highly purified s-GTH; injected at doses of between 5 and 100 μg/kg body weight); 3–6 times with crude carp pituitary extract (0.5–3 mg/kg body weight); and 3–7 times with fresh pike pituitaries (14 and 1.2 mg wet weight/kg body weight). The sperm obtained after hormonal treatment was of good quality. Intracardiac injection of superactive LRH analogue had no effect. In females, PPSG induced 90 and 100% ovulation at the doses of 50 and 25 μg/kg body weight. Dried salmon pituitaries (2.5 mg/kg, equivalent to 50 μg of PPSG) gave 25% ovulation; at 10 mg/kg, 25% complete ovulation was again recorded, but in addition 70% of the females showed oocyte maturation and partial ovulation. Similarly, dried carp pituitary (3 mg/kg) induced only oocyte maturation but no ovulation. The oocytes obtained after hormonal treatment were in general fertile. Intraperitoneal injection of LRH in an emulsified form induced neither oocyte maturation nor ovulation. The lack of effect of LRH analogue is discussed and shows that the use of this compound as a substitute for pituitary preparation is not very promising.     

Spawning induction of pejerrey Odontesthes bonariensis in captivity using sustained‐release gonadotropin releasing hormone agonist implants

The aim of this study was to induce and synchronize spawning of pejerrey Odontesthes bonariensis (Valenciennes, 1835), using gonadotropin releasing hormone agonist (GnRHa) implants. In the first experiment, the ovarian condition was assessed by ovarian biopsies and the measurement of the genital pore width (GPW). Females having the leading clutch of oocytes with a diameter of around 800–900 μm and a GPW between 4.5 and 5.5 mm were treated with GnRHa implants. Eighty per cent of females spawned between 2 and 9 days after treatment, 12 days earlier than 20% of the fish in the control group that presented signs of spawning activity. In order to avoid any possible ovarian injury and/or stress by the catheterization procedure, in a second experiment, females were selected only by visual inspection of the abdomen and GPW measurement. As in experiment 1, 80% of females spawned between 2 and 8 days after treatment, 8 days earlier than 30% of the fish that spawned in the control group. In both experiments, fertilization and hatching success were similar between control and GnRHa‐treated groups. These results clearly demonstrated that GnRHa implantation can advance and synchronize ovulation and spawning in pejerrey without affecting egg quality.     

Preliminary Observations on the Reproductive Physiology of Female Orangemouth Corvina in Captivity

Seasonal changes In oocyte growth and plasma estradiol, testosterone, thyroid hormones and vitellogenin levels were monitored in three captive adult female orangemouth corvina Cynoscion xanthulus subjected to a condensed (8 mo) seasonal cycle of photoperiod and water temperature. Plasma concentrations of estradiol and testosterone began to rise when temperature increased to 28 C and photoperiod to 15 h light (midsummer conditions). This was accompanied by elevated circulating levels of vitellogenin and the appearance of vitellogenic oocytes (diameter > 100 μm). Estradiol concentrations and mean oocyte size increased concurrently during late summer conditions and were maximum during fall conditions, approximately 8 wk after the beginning of ovarian recrudescence. In contrast, plasma levels of thyroid hormones did not show any distinct seasonal changes. Gonadally recrudesced females contained several size classes of vitellogenic oocytes. Injection of these fish with a luteinizing hormone-releasing hormone analog (LHRHa) caused maturation and spawning of the largest oocytes (mean diameter of follicles × 2500 μm). Another group of vitellogenic oocytes had been recruited into this size class by 2 wk after the end of spawning, which suggests that this species is capable of repeated spawning during the reproductive season. Injection of LHRHa resulted in increased plasma levels of gonadotropin and repeated spawning over several days. LHRHa-induced final oocyte maturation, ovulation and spawning were preceded by increases in plasma levels of two teleostean maturation-inducing steroids, 17α,20β, 21-trihydroxy-4-pregnen-3-one (20β-S) and 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P₄). The results provide preliminary evidence that oocyte growth is stimulated in orangemouth corvina subjected to an abbreviated seasonal cycle in captivity under midsummer photoperiod and temperature conditions and is associated with seasonal increases in plasma estradiol concentrations and vitellogenin production. However, the relative importance of 20β-S versus 17α, 20β-P₄ in the control of final oocyte maturation in this species could not be determined from the results of the present study.