Development of an enzyme-linked immunosorbent assay (ELISA) for vitellogenin measurement in greenback flounder Rhombosolea tapirina

Vitellogenin (Vtg) was purified from the plasma of 17-estradiol (E₂)-injected male greenback flounder,Rhombosolea tapirina. The molecular weight of the native Vtg was estimated by gel filtration as 540 kD. SDS-PAGE and Western blotting analyses indicated that this protein consisted of three bands with molecular weights of 155, 104, 79 kD, respectively. A polyclonal antibody against the highest molecular weight band of putative Vtg was generated in sheep and an indirect antibody-capture competitive enzyme-linked immunosorbent assay (ELISA) was developed. The assay was validatedfor plasma Vtg measurement in greenback flounder. Serial dilutions of plasma from vitellogenic females parallelled the standard Vtg curve, whereas no cross-reaction was observed with the plasma of males in the ELISA. The Vtg ELISA was used to assess the induction of Vtg by E₂ in vivo in males. The induction of Vtg in greenback flounder showed a time- and dose-dependent response as in other species. In E₂-treated fish, detectable levels of Vtg were first found at 48 h, and reached a peak at 96 h post-injection. Plasma levels of Vtg increased as the E2 dose increased with a threshold of 0.1 mg kg^–1.     

Diel feeding rhythms of greenback flounder Rhombosolea tapirina (Günther 1862): the role of light–dark cycles and food deprivation

The role of light–dark (LD) cycles and food deprivation in the regulation of diel feeding rhythms in greenback flounder Rhombosolea tapirina (Günther 1862) was examined. A computerized monitoring system was developed to record uneaten pellets, and food intake was estimated as the difference between pellets presented and those uneaten. Three groups of five fish each (75–281 g) were exposed sequentially to the LD cycles: LD 12:12, LD 12:12 (scotophase advanced by 9 h), LD 6:18, DD, LD 6:6:6:6, LL and LD 12:12 (return to the initial cycle). Illumination was 120 lux at the water surface (complete darkness at scotophase), and water temperature was 15 ± 1 °C. Fish fed mainly during the light hours under LD 12:12 and resynchronized to the scotophase-advanced LD 12:12 regime after about 12 h. Feeding started at the onset of light and extended to the early scotophase under LD 6:18. A circadian feeding rhythm was detected in fish under DD and LD 6:6:6:6 regimes. Under the LL regime, two groups of fish displayed arrhythmic feeding patterns and did not resynchronize to LD 12:12 for at least 13 days. In contrast, the third group of fish exhibited a circadian feeding rhythm under the LL regime and immediately re-entrained to LD 12:12. Fish were deprived of food for a single period of 26–57 h once feeding patterns had been established under LD 12:12, LD 12 : 12 (scotophase advanced by 9 h) and DD regimes, and feed delivery was resumed during the scotophase or subjective night; the timing of feeding rhythms was shifted by the reintroduction of feed, but progressively resynchronized to the LD cycles. The results suggest that the greenback flounder is a diurnal feeder, that a LD 12:12 cycle is a potent environmental cue to entrain circadian feeding rhythms, and that a biological clock is involved in the timing of feeding.     

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.     

Effect of Luteinising Hormone-Releasing Hormone Analogue and Human Chorionic Gonadotropin on Ovulation, Plasma and Ovarian Levels of Gonadal Steroids in Greenback Flounder Rhombosolea tapirina

Female greenback flounder Rhombosolea tapirina were treated with either des Gly¹⁰ [D-Ala⁶] LHRH ethylamide (LHRH-a) at 50 or 100 pg/kg intraperitoneal injection (ipi), LHRH-a cholesterol pellet (LHRH-a pellet) 100 μg/kg implanted intraperitoneally, or human chorionic gonadotropin (hCG) 1,000 IU/kg ipi. Treatment with hCG, LHRH-a (50 μg/kg) ipi and LHRH-a pellet increased the total number of ovulations above control levels. LHRH-a (100 μg/kg) ipi was not consistently more effective than the control and both LHRH-a (50 μg/kg) ipi and LHRH-a pellet induced more ovulations than LHRH-a (100 μg/kg) ipi. Of those fish that ovulated, most ovulated more than twice, and most ovulations occurred at daily intervals. Oocyte diameters increased and oocyte stage advanced significantly in response to all exogenous hormone treatments. This was accompanied by increases in plasma and ovarian levels of 17β-estradiol (E₂, and in most cases, plasma and ovarian levels of testosterone (T). Plasma and ovarian levels of 17,20β-dihydroxy-4-pregnen-3-one (17,20βP) were not consistently elevated in association with reproductive events.     

Sex steroids concentrations in Russian sturgeon (Acipenser guel denstaedti Br.) serum and coelomic fluid at final oocyte maturation

Sex steroid levels in the coelomic fluid (CF) were lower than in the blood serum (BS) both in spring form (SF) and winter form (WF) of the female Russian sturgeon at the time of ovulation. CF hormone profile may be of relevance for maintaining the viability of ovulated eggs.     

饲料卵磷脂对施氏鲟血清卵黄蛋白原、卵径及性类固醇激素水平的影响

采用不同大豆卵磷脂添加量[0、1%、2%、4%(w/w饲料)]的4种人工配合饲料(分别简称SLD、SL1、SL2和SL4)投喂5+龄施氏鲟(Acipenser schrenckii)后备亲鱼14个月,通过比较各饲料组实验鱼的血清卵黄蛋白原(Vg)、卵径和性类同醇激素水平,探讨大豆卵磷脂对施氏鲟性腺发育的影响机理,并为今后深入研究鲟亲鱼磷脂营养代谢提供基础数据.结果表明,饲料中大豆卵磷脂含量对施氏鲟的卵径发育无显著影响,对血清中Vg、睾酮(T)和雌二醇(E2)的浓度影响显著.投喂量为2%组的血清E2质量浓度(147.91 pg·ml-1)显著高于0%(113.72pg·ml-1)和1%(129.63pg·mL-1)组(P<0.05),而4%组的血清E2质量浓度为139.68 pg-mL-1介于二者之间.随着投喂时间和投喂浓度的增长,施氏鲟后备亲鱼的血清T含量浓度降低,而血清Vg浓度随日粮卵磷脂的添加水平和时间增加而升高.结果提示:①施氏鲟卵黄发生期后备亲鱼饲料卵磷脂的适宜添加量为2%,不足或过量对后备亲鱼的生殖机能均有不利影响;②通过影响血清T的产生从而影响施氏鲟的生殖可能是饲料中卵磷脂在施氏鲟生殖机制中的作用之一;③在施氏鲟后备亲鱼强化培育中可通过适当添加卵磷脂的量,从而实现性腺的正常发育,进而改善性腺发育状况、提高施氏鲟的繁殖性能.     

Seasonal levels of serum vitellogenin and oocyte growth in the channel catfish Ictalurus punctatus

Seasonal levels of serum vitellogenin (VG) were measured in adult and juvenile (10–30 months old) female channel catfish using an enzyme-linked immunosorbent assay (ELISA). In adults sampled during a 2-year period, the highest mean (±s.e.) VG level was 30.21 ± 5.36 mg/ml in May before spawning and declined to 3.79 ± 0.89 mg/ml in June after the fish had spawned. Oocyte diameter increased from September to November, remained the same until February, then increased again until maximum mean diameter of 2356 ± 49 μm was measured in May. Vitellogenin was detected in the serum of juvenile fish that were 12 months old, but yolk granules were not visible in hematoxylin and eosin stained sections of oocytes until the fish were 18 months old. Mean serum VG levels of juvenile fish were lower than the adults, and no VG peak was observed in the juvenile fish. Vitellogenin was present in the serum throughout the year in adult fish and in juveniles older than 12 months.     

漠斑牙鲆胚胎及仔稚鱼发育的形态学观察

对漠斑牙鲆(Paralichthys lethostigma)胚胎及胚后发育进行观察,描述各发育时期的发育时序和形态特征.在水温18.5±0.5℃,盐度32～33的条件下,受精卵历时49 h脱膜而出,孵化后仔鱼在水温20.5±0.5℃时,4日龄开口摄食,26日龄进入变态期,45日龄左右完成变态成为幼鱼.漠斑牙鲆的卵子为浮性卵,油球一个.刚孵化仔鱼的消化道细直,3日龄肛门开通,4日龄卵黄囊消耗殆尽,开口摄食.在原肠期胚体下包约1/2时,在视囊和胚盘上出现点状星状的黑色素细胞.孵化后体两侧菊花状黑色素均匀分布;进入变态期后,体两侧的幼体色素逐渐收缩消失,同时在有眼侧出现个体较小的圆形成体色素细胞.在早期发育过程中,伴随着冠状鳍条的发生、伸长和缩短吸收.无鳔器官的发生.     

Changes in serum vitellogenin levels and immunohistochemical localization of vitellogenin in hepatic cells during ovarian development in the Japanese eel

ABSTRACT: The present study was designed to quantify changes in serum vitellogenin (VTG) levels and to localize VTG in hepatic cells immunohistochemically during artificial maturation of the Japanese eel Anguilla japonica . Hormone treatment resulted in a gradual rise in serum VTG levels, in which the highest values were attained at the migratory nucleus stage. Hepatocytes responded to hormone treatment by increased immunoreactivity; both staining area and intensity were enhanced with ongoing gonadal development. Moreover, cytoplasmic vacuoles increased in number during artificial maturation, occupying large areas inside hepatocytes. The unusual appearance of hepatocytes may reflect the effect of artificial maturation.     

条斑星鲽精巢年周期发育规律和血液性类固醇激素含量变化

采用组织学方法和放射免疫法(RIA)等技术方法,研究了人工养殖条件下条斑星鲽雄性亲鱼精巢发育规律和性类固醇激素的年周期变化规律。实验结果表明,条斑星鲽精巢中可见5个时相的生殖细胞类型,精巢发育可分为5期。条斑星鲽雄鱼GSI值自10月开始升高,12月达峰值(P≤0.05),之后显著下降并保持较低值至下一次生殖周期开始。HSI值在11～12月保持较高表达值,其他各月份保持相对稳定水平。CF值在5～8月保持较高水平,其他月份保持平稳状态。雄鱼血浆中睾酮(T)水平自9月开始升高并在12月(Ⅴ期精巢)达到峰值,其后显著下降并在其后的月份保持较低水平,而雌二醇(E2)在2月出现峰值,其他月份保持相对稳定水平。     

全雌牙鲆和普通牙鲆胚胎发育与鱼苗生长速度的比较

北戴河中心实验站应用异源真鲷精子,通过紫外线照射、冷休克诱导牙鲆雌核发育和高温诱导伪雄鱼,解决了牙鲆性别控制和全雌苗种规模化生产的关键技术问题,实现了牙鲆全雌苗种的规模化生产.全雌鱼苗比普通鱼苗生长速度快是公认的,但其生长速度差异的确切数据目前还没有报道,2008年我们设计了全雌苗种和普通苗种胚胎发育时间和苗种生长增长的测定试验.因条件限制,本试验仅做了胚胎发育和初孵仔鱼长到鱼苗全长5 cm这一阶段的比较试验.     

施氏鲟不同年龄性腺发育与性类固醇激素浓度关系

采用放射免疫方法测得养殖施氏鲟群体 5个年龄组及亲鱼 (8龄 )血液中睾酮 (Testosterone ,T)、雌二醇 (17β-estradi ol,E2 )的不同含量 ,并与其对应年龄的性腺组织学观察进行分析。结果显示 ,1龄性腺刚刚完成性分化发育初期 ,T( X±SD) =(1.8± 0 .72 4 )nmol/L ,α =0 .0 1;E2 ( X±SD) =(5 5 .8± 2 .879)pmol/L ,α =0 .0 5。 2龄精巢Ⅱ期 ,卵巢I期 ,T( X±SD)=(2 .2± 0 .934)nmol/L ,α =0 .0 1。E2 ( X±SD) =(38.9± 2 .343)pmol/L ,α =0 .0 5。 3～ 4龄鱼卵巢发育在Ⅰ～Ⅱ期 ,精巢Ⅲ-Ⅳ期。 3龄T( X±SD) =(9.6± 1.936 )nmol/L ,α =0 .0 5 ;E2 ( X±SD) =(4 4 .8± 2 .6 0 5 )pmol/L ,α =0 .0 5。4龄鱼T( X±SD)=(2 6 .3± 2 .10 5 )nmol/L ,α=0 .0 5 ;E2 ( X±SD) =(5 5 .3± 3.0 5 3)pmol/L ,α =0 .0 5。 5龄鱼卵巢发育在Ⅲ期左右 ,精巢已发育成熟 ,T( X±SD) =(13.9± 1.6 5 2 )nmol/L ,α=0 .0 5。E2 ( X±SD) =(137.7± 5 .880 ) pmol/L ,α=0 .0 5。雄亲鱼血液中T含量变化在 4 0 .5～ 74 .6nmol/L ,E2 含量变化在 5 6～ 116pmol/L。雌亲鱼血液中T含量变化为 39.7～ 73.2nmol/L ,E2 含量变化在 5 4 4～ 90 4pmol/L     

Pituitary and plasma AVT content in the flounder (Platichthys flesus)

Radioimmunoassay measurement of pituitary AVT content and plasma AVT concentration indicated comparable levels in fully adapted sea water (SW) and fresh water (FW) flounders. Circulating AVT represented less than 0.1% of the pituitary AVT reserve. The urophysis contained AVT but the total content was only 2 or 3 fold that of circulating AVT. In fish adapted to hypertonic media, there was a close correlation between plasma AVT concentration and plasma Na⁺ concentration or osmolality. The present study examined the effects of acute osmotic challenge, associated with FW to SW transfer, and the influence of extracellular fluid volume status on AVT secretion. Short-term transfer of fish from FW to SW (up to 3 days) did not evoke a clear change in plasma AVT levels, though pituitary content was reduced at 24 h. During the first 3 days after transfer to SW, only small increments in plasma tonicity were apparent. The sensitivity of AVT secretion to osmotic stimuli may only be expressed when plasma osmolality has exceeded a specific threshold, which was probably not reached in these transfer studies. Fish in hypotonic media showed no relationship between plasma osmolality/tonicity and plasma AVT concentration. Acute extracellular fluid volume expansion of SW adapted fish also abolished the normally observed relationship between plasma osmolality and AVT concentration in these hypertonic media fish. This trend indicates that volume status may modulate the sensitivity of AVT secretion to osmotic stimuli as occurs in tetrapods.     

三丁基锡对罗氏沼虾卵黄蛋白原（VTG）表达和性腺发育的影响

将罗氏沼虾成虾浸浴于含三丁基锡（Tributyltin, TBT） (0.1、0.2、0.4mg/L）的水体中,研究TBT对其肝胰脏和性腺中卵黄蛋白原(VTG)表达和性腺发育的影响。结果表明,罗氏沼虾在TBT中浸浴10d,TBT能够诱导雌虾的肝胰腺VTG基因表达,而对卵巢VTG基因的表达则表现为抑制作用;TBT对雄虾的肝胰腺VTG基因表达有抑制作用,而对精巢VTG基因的表达无明显影响。性腺组织切片显示,TBT浸浴能够促进罗氏沼虾精巢的发育,且随浸浴剂量的增加对精巢发育的促进作用逐步增强,但TBT对罗氏沼虾卵巢发育无明显影响。     

Dietary fish oil replacement by soybean oil: Effect on plasma vitellogenin,sex steroids and ovarian steroidogenesis in Chinese strip‐necked turtles (Mauremys sinensis)

In order to investigate the effects of dietary fish oil replacement, the turtles (Mauremys sinensis) were fed four experimental diets for 10 months: FO (100% fish oil), FSO (70% fish oil and 30% soybean oil), SFO (30% fish oil and 70% soybean oil) and SO (100% soybean oil), sampled at pre‐vitellogenesis, vitellogenesis and post‐vitellogenesis. The results showed that plasma gonadotropin‐releasing hormone (GnRH) levels were the highest at pre‐vitellogenesis, which promoted the secretion of gonadotropin and sex steroids. Therefore, plasma luteinizing hormone (LH) and estrogen (E₂) levels were significantly increased at post‐vitellogenesis (p < 0.05), while follicle‐stimulating hormone (FSH) levels increased at vitellogenesis (p < 0.05). The FO and FSO groups had significantly higher GnRH and E₂ levels than the other two groups (p < 0.05). In addition, plasma vitellogenin (Vtg) levels significantly increased at vitellogenesis and post‐vitellogenesis (p < 0.05), which were significantly higher in the groups of FO and FSO than SO (p < 0.05). Moreover, the expression levels of hepatic estrogen receptor α (Erα) mRNA were significantly increased at vitellogenesis and post‐vitellogenesis while ovarian Cyp19α1α mRNA were significantly increased at post‐vitellogenesis (p < 0.05), and both were the lowest in SO. Taken together, the replacement of fish oil with 66.7% soybean oil is feasible.     

Whole-body concentrations of cortisol and sex steroids in white sturgeon (Acipenser transmontanus, Richardson 1836) during early development and stress response

In general little is known about hormones and the ontogeny of the stress response in the early developmental stages of chondrostean fishes and in particular of white sturgeon (Acipenser transmontanus, Richardson 1836). In this study, we measured for the first time cortisol and sex steroids (testosterone and estradiol) in eggs, larvae, post-larvae, and fry of white sturgeon by radioimmunoassay (RIA), to elucidate some endocrine aspects of its development. The cortisol, testosterone, and 17β-estradiol of maternal origin found in unfertilized eggs of white sturgeon probably regulate both growth and development of the embryo. Cortisol decreased after fertilization, whereas testosterone and 17β-estradiol did not significantly change. During the late stages of embryo development and immediately after hatching, endogenous production of cortisol and sexual steroids, respectively, occurred. Sex steroids may be physiological inducers of gonad sex differentiation in sturgeon. All steroids showed an increase 10 days post-hatch (dph), near the transition from an endogenous to an exogenous energy source. Cortisol maintained the same basal levels even after metamorphosis, whereas testosterone and 17β-estradiol declined significantly in post-larvae at 35 and 45 days post-hatch. In addition, to evaluate the ontogeny of a functional hypothalamic-pituitary-interrenal (HPI) axis, larvae and fry were submitted to acute stress. The HPI axis did not seem to be functional on the first day post-hatch, but became so from the third day post-hatch onward.     

Relationship between vitamin C and plasma concentrations of testosterone in female rainbow trout,Oncorhynchus mykiss

Two-year old rainbow trout females were fed diets containing 0, 30, 110, 220, 440 and 870 mg kg^-1 ascorbyl-2-monophosphate Mg⁺ salt (groups 1, 2, 3, 4, 5, and 6, respectively) from August until March. At the time of spawning (February–March) blood was sampled and the ovulating females were hand stripped. Estradiol (E₂) and testosterone (T) concentrations in plasma, and ascorbic acid (AA) concentrations in plasma and eggs were determined. The mean plasma concentrations of T were higher in group 4, 5, or 6 than in group 2 or 3 (p < 0.05). Moreover, the average plasma concentration of T in fish fed the diets with AA level below National Research Council (NRC) recommendations (groups 1, 2 and 3) was significantly lower (p<0.01) than the average plasma concentration in fish fed diets with AA level above NRC recommendations (groups 4, 5, and 6). These data are consistent with the hypothesis that AA can influence production of steroids in female rainbow trout.Corresponding author     

Changes in oocyte and blood plasma osmotic components of ayu, Plecoglossus altivelis Temminck & Schlegel during oocyte maturation

Oocyte maturation in teleosts is characterized by a complex series of cytoplasmic and nuclear changes. The purpose of this study is to present sequential changes in the oocyte constituents and regulatory mechanisms involved in the hydration and osmotic regulation of oocytes during final maturation of ayu, Plecoglossus altivelis Temminck & Schlegel. Oocyte volumes and water contents in ayu (P. altivelis) markedly increased from 0.372 mm³ and 60.5% at the mature stage to 0.712 mm³ and 79.4% at the time of oviposition. In the same period, net increases in total free amino acids and electrolytes were noted, and involvements of these constituents in the water uptake by oocytes are discussed. Moderate oocyte hydration in ayu is accompanied by steady increases in Na⁺, but not K⁺, and these observations are in contrast to results for all other marine teleosts reported elsewhere. In the present study, a steady increase in oocyte Na⁺ during maturation until ovulation pre‐dominantly results from diffusional leakage due to concentration gradients, while at the time of oviposition, both active transport and diffusional pathways play important roles in osmotic regulation in ayu oocytes.     

Genetic characterization, morphometrics and gonad development of induced interspecific hybrids between yellowtail flounder, Pleuronectes ferrugineus (Storer) and winter flounder, Pleuronectes americanus (Walbaum)

Viable interspecific hybrids between yellowtail flounder (Pleuronectes ferrugineus, Storer) and winter flounder (Pleuronectes americanus, Walbaum) were produced by artificial insemination of yellowtail flounder eggs with winter flounder sperm. However, mean fertilization rate, hatching success and early survival up to 3 weeks post hatch were significantly lower than those of parental pure cross controls (P < 0.01). Overall, cytogenetic traits (karyological analysis and estimation of cellular DNA contents using flow cytometry) of hybrid flounder were intermediate between the two parental species. Microsatellite assay was used to distinguish the parental genomes in the hybrids; in most cases, one allele was specific to each of the parents. Morphometrics assessed by body proportions indicated that hybrids generally displayed a morphology intermediate between the maternal and paternal species. Interspecific hybrids exhibited abnormal and retarded gonad development in both sexes based on histological analysis of gonads from adult fish. The sterility of the hybrids presents a significant advantage for their use in aquaculture, as potential escapees would not be capable of reproducing in the wild and contaminating natural stocks.