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1.
An homologous radioimmunoassay for brown trout vitellogenin (VTG) was developed. Intact VTG, isolated from juvenile brown trout by selective precipitation and anion exchange chromatography was labelled with Na125I, with 1,3,4,6-tetrachloro-3,6-diphenylglycoluril (Iodogen) as the oxidizing agent. Incorporation of Na125I into VTG was higher than 75% and there was little degradation of the labelled protein. Labelled VTG eluted at the same position as unlabelled, purified brown trout VTG when analyzed by gel filtration on Sepharose 6B. Antisera with high titers, i.e. 1250 000, against brown trout VTG were raised in rabbits. The sensitivity of the assay was 5 ng VTG/ml and the standard curve was linear between 10 and 100 ng VTG/ml. Plasma from maturing female brown trout, as well as estradiol-treated and untreated juvenile brown trout diluted parallel to the standard curve, while plasma from maturing female rainbow trout and estradiol-treated arctic charr diluted non-parallel to the standard curve for brown trout VTG. Purified rainbow trout VTG and plasma from maturing female rainbow trout diluted parallel to each other, but with lower sensitivity than for brown trout VTG. Determinations of protein-bound phosphorus in the plasma of estradiol-treated juvenile brown trout correlated well with the RIA determinations of VTG. Repeated freezing and thawing of plasma samples yielded up to a hundred-fold increase in the apparent VTG level, while storage of a plasma sample for one year at –20°C did not affect the VTG level as measured by RIA.  相似文献   

2.
Triploid female fish show impaired gametogenesis and are unable to produce viable offspring. The reproductive physiology of artificially-induced triploid female salmonids has been well described up until the time of first sexual maturation in diploids, but few reports exist for older triploids. This study reports the influence of triploidy on growth, ovarian development and reproductive endocrinology among three age classes of female brook trout (Salvelinus fontinalis) in comparison to sibling diploids. Triploids were larger than diploids for most of the study period, but the difference was statistically significant only during maturation and spawning of 2+ diploids. Plasma estradiol-17 (E2), testosterone (T) and vitellogenin (VTG) levels in triploids were generally lower than in diploids, and VTG was the only parameter to show seasonal fluctuations resembling those of diploids. Triploids showed significantly lower GSI and total oocyte number than diploids of similar age, and only half of all triploids sacrificed during the study (n=56) had developing oocytes in their ovaries. At age 3+, 13 of 19 triploid females had oocytes at various stages of development, including perinucleolar, yolk vesicle and yolk globule stages. In addition, three of these fish had collectively produced 72 mature stage oocytes. Thus, whereas diploid brook trout can produce mature oocytes as two-year-olds, triploids cannot do so until four years of age, with the number of mature oocytes being greatly reduced.  相似文献   

3.
Circulating levels of the egg yolk precursor protein, vitellogenin (VTG), can be used as a biochemical indicator of maturation in female fish. Here we report on purification and partial characterization of VTG from a temperate marine serranid, the gag(Mycteroperca microlepis). Development of a competitive, enzyme-linked immunosorbent assay (ELISA) for gag VTG (gVTG) is also described. The gVTG was purified by DEAE-agarose anion exchange chromatography from a pooled plasma sample collected from several juvenile gag after they were injected with 17estradiol. The protein appeared as a major band of Mr183000 after SDS-PAGE ± Western blotting using either a specific rabbit antiserum to gVTG or a universal monoclonal antibody for vertebrate VTGs. Amino acid composition analysis and N-terminal peptide sequencing verified that gVTG is similar in primary structure to VTG from several other teleost species. The purified gVTG and its specific antiserum were used to develop a sensitive, competitive, antibody-capture ELISA for quantifying the protein in blood plasma from maturing females. VTG levels in maturing female gag were highly correlated with oocyte growth and circulating testosterone and 17-estradiol levels, whereas VTG was non-detectable in juveniles, immature females or males. Two size-based maturity schedules for female gag were constructed, one utilizing detection of VTG in their circulation as a marker of maturity and the other relying on histological evidence that their ovaries were in vitellogenic or later stages of maturation. The two schedules were virtually identical. The gVTG ELISA was also used to detect VTG in blood plasma from mature Nassau grouper (Epinephelus striatus) and red hind (E. guttatus). As with gag, the assay was completely reliable for discriminating between reproductively mature females versus males from these two grouper species.  相似文献   

4.
Turbot and Atlantic halibut are highly valued fish species. However,very little is known about fillet shelf-life characteristics associated withboth species. Thus, fillet -tocopherol content and proximate compositionof wild turbot (1.5 kg) and Atlantic halibut (1.1 kg)caught off the south coast of Ireland and the north-west coast of Iceland,respectively, were investigated. In addition, the susceptibility of fillets, storedunder retail conditions, to lipid oxidation and colour change was studied.Proximate composition analysis showed that turbot had significantly highermoisture (P < 0.001) and lower protein (P < 0.001) contents compared toAtlantic halibut. Atlantic halibut incorporated significantly higher (P <0.001) levels of -tocopherol into fillets than turbot. Over 14 days ofstorage on ice, fillets from Atlantic halibut exhibited significantly lower (P =0.020) levels of lipid oxidation than those of turbot. However, malondialdehyde(MDA) concentrations were generally very low, never exceeding 0.6 gg–1 fillet. Turbot maintained a significantly higher (P< 0.001) pH over the storage period. The lightness (L* values) offillets from both species increased over 14 days of storage, but wassignificantly higher (P < 0.001) in Atlantic halibut than in turbot. Turbotdeveloped a relatively intense yellow colour during storage (decrease in hueangle and increase in b* values), whereas this was not the case forAtlantic halibut. The results of this study demonstrate that fillets of wildAtlantic halibut stored on ice, were less prone to lipid oxidation anddiscolouration than those of wild turbot. However, quality changes in turbotwere very small showing that both fish have tremendous shelf-life capacities interms of lipid oxidation. These findings are considered in the context of knownmaterial for farmed fish.  相似文献   

5.
The objectives of this study were to isolate and characterize vitellogenin from plasma of estradiol-treated protandrous black porgy,Acanthopagrus schlegeli. Vitellogenin concentrations in plasma measured by a validated enzyme-linked immunosorbent assay (ELISA) were also compared. Two-year-old black porgy (n=20) were fed with estradiol-17 (4 mg kg–1 of feed). Plasma was collected for purification of vitellogenin. Two forms of vitellogenin were found in plasma after chromatography on Sepharose CL-2B column and hydroxylapatite, and polyacrylamide gel electrophoresis. Black porgy vitellogenins are phospho-lipo-glycoproteins based on their chemical staining properties. The apparent molecular weights of the two forms of vitellogenin were 636 kDa and 321 kDa, respectively. The amino acid composition of purified vitellogenin was also analyzed after acid hydrolysis. The presence of immunoreactive vitellogenin was identified in the plasma and mucus extract from control and estradiol-induced females on the basis of Western blotting. Serial dilution of the plasma and mucus extract taken from estradiol-induced black porgy showed reactivity to an antiserum against lipovitellin in the ELISA, whereas mucus extract and plasma from male fish did not. Significantly higher concentrations of plasma vitellogenin were detected in estradiol-stimulated black porgy than in the control males.  相似文献   

6.
Extremely low levels of the maturation inducing steroid (MIS) 17,20-dihydroxy-4-pregnen-3-one (17,20-DHP) were found in the ooplasm and ovarian follicle membranes of Atlantic salmonSalmo salar ouananiche, a finding that is at variance with the elevated blood levels of the steroid. The uptake of MIS at physiological concentrations into brook trout follicles occurred by passive diffusion. Uptake of the steroid into the ovarian follicle membrane, consisting of zona radiata and the attached follicle cells, deviated from linearity in a double reciprocal plot. These results suggest that 17,20-DHP is binding to a receptor-like protein in the ovarian follicle or the zona radiata membrane surrounding the oocyte, and extend our previous demonstration of 17,20-DHP receptor-like activity in the zona radiata membrane of the late stage brook trout oocytes. An oocyte cytoplasmic receptor gave subunits on SDS PAGE that were similar to the membrane and cytosol receptors previously described.  相似文献   

7.
A group of Atlantic salmon (Salmo salar) was followed through their first year of maturation and spawning. At monthly intervals, starting with juvenile fish in December, 5–7 fish of each sex were killed, and liver and plasma were sampled. The last sampling point was of spawning fish in November a year later. Variables in the cytochrome P450 (P450) system were studied in hepatic microsomes, and estradiol 17 was measured in the plasma of females to assess the maturational status. The P450 1A1-mediated 7-ethoxyresorufin O-deethylase (EROD) started at high levels in winter, but decreased to non-detectable activities in pre-spawning females. Decreases, but not to the same extent, were also observed during this period in total cytochrome P450, cytochrome b5, NADPH-cytochrome P450 reductase, and in the content of two immunochemically determined P450 isozymes. At the same time, LSI levels increased in maturing females (starting in July), and GSI levels increased in both sexes (starting in May). Sex specific differences were observed in pre-spawning fish in September and October, with levels of total P450, b5, NADPH-cytochrome P450 reductase, EROD and P450 isozymes significantly lower in females. At the same time, plasma estradiol-17 levels reached peak values in females. The results point to the important role of sex steroids such as estradiol-17 as major factors in the regulation of final sexual maturation. However, this study also indicates that there may be estradiol-17 independent events of equal importance in the early stages of gonadal maturation that may involve the P450 system. The changes observed in the P450 system (as a major drug and steroid metabolizing system) of Atlantic salmon during sexual maturation may be of importance both in the endogenous transduction of hormonal signals, and as a pharmacological basis for designing therapeutic treatment of diseases in the aquaculture industry.Parts of this work were presented at the 5th International Symposium on Responses of Marine Organisms to Pollutants, April 1989 in Plymouth, United Kingdom (Larsen and Goks\/oyr 1989).  相似文献   

8.
The egg yolk precursor, vitellogenin (VTG), was purified from blood plasma of striped bass by chromatography on hydroxylapatite or DEAE-agarose. The fish were first implanted with estradiol-17β (E2), which induced vitellogenesis. A rabbit antiserum (a-FSPP) raised against plasma from mature female striped bass, and then adsorbed with mature male plasma, was used to detect female-specific plasma protein (FSPP) in the chromatography fractions. Striped bass VTG (s-VTG) was collected from the peak fraction that was induced by E2, reacted with a-FSPP, and contained all detectable phosphoprotein. It appeared as a single band (Mr ≂ 170,000) in SDS-PAGE or Western blots using a-FSPP, and as a pair of closely-spaced phospholipoprotein bands in native gradient-PAGE, suggesting that there is more than one circulating form of s-VTG. The relationship of s-VTG to the yolk proteins was verified using a-FSPP. The antiserum reacted with the main peak from gel filtration of saline ovary extracts, and it specifically immunostained the two main bands in Western blots of the extracts and the yolk granules of mature oocytes. The amino acid composition of s-VTG was similar to that of VTG from other fish and Xenopus. A radial immunodiffusion assay for s-VTG was developed using a-FSPP and purified s-VTG as standard. The s-VTG was not detected in blood plasma of males, immature females, or regressed adult females, but plasma s-VTG levels were highly correlated with plasma E2 and testosterone levels, and oocyte growth, in maturing females. The results indicate that the maturational status of female striped bass can be identified by s-VTG immunoassay.  相似文献   

9.
The major components of the cytochrome P450 (P450) system in liver microsomes of Atlantic salmon were studied using spectrophotometric, catalytic and immunochemical techniques. In juvenile fish sampled during the winter season, high basal activities of 7-ethoxyresorufin O-deethylase (EROD) were found. The Km for 7-ethoxyresorufin was 0.4 µM, and Vmax 1.23 nmol/min/mg protein in juvenile fish. In mature fish sampled from the same group of fish in December, EROD activity was barely detectable (20–30 pmol/min/mg protein). Treatment with the P450 1A1 inducer -naphthoflavone (BNF) resulted in almost 2-fold induction of total P450, and 30–40-fold induction of EROD activity in immature fish. A similar fold increase was seen in mature fish. The differences in EROD activity between untreated and BNF-treated fish, was accompanied by similar differences in a P450 1A1 cross-reacting protein (Mr=58,000 D) in immunochemical studies using rabbit anti-cod P450 1A1 IgG. However, judging from these studies, the levels of P450 1A1-protein in mature salmon far exceeded those accounted for by the measured EROD activity in comparison to immature fish (both before and after BNF-treatment), indicating inhibiting effects of sex steroids on the measured activity. This effect was not seen on 7-ethoxycoumarin O-deethylase activity. A long-term storage experiment indicated that Atlantic salmon liver microsomes can be stored for 2 years at –80°C in 20% glycerol without losing more than 20–40% of its catalytic activity.Parts of this work were presented at the 5th International Symposium on Responses of Marine Organisms to Pollutants, April 1989 in Plymouth, United Kingdom (Larsen and Goksøyr 1989).  相似文献   

10.
Two gonadotropic glycoproteins (PmGTH I and II) were purified by ion-exchange chromatography, gel filtration and preparative SDS-PAGE, from pituitaries of red seabream, a marine teleost which has an asynchronous-type ovary and spawns almost daily during the spawning season. The glycoproteins were composed of distinct subunits and the molecular weights were estimated to be 32 and 38 kDa for PmGTH I and PmGTH II, respectively. Both PmGTH I and II were active in two homologous bioassays: in vitro oocyte maturation and/or in vitro estradiol-17 production assays. These two GTHs were distinct in electrostatic properties, molecular weight, stability and yields from pituitaries during the spawning season. These properties suggest that PmGTH I and II correspond to salmon GTH I and II, respectively.A homologous radioimmunoassay with which to measure PmGTH II was developed using a rabbit antiserum against the subunit of PmGTH II and intact PmGTH II as standards and radioactive competitors. Competition curves for red seabream plasma and pituitary extract were parallel to the standard curve, while PmGTH I had low cross-reactivity (3.1 %) with the antibody. This specific RIA system showed an in vivo LHRHa induced GTH surge in the plasma of female red seabream.  相似文献   

11.
Plasma estradiol-17 (E2), testosterone (T), 17,20-dihydroxy-4-pregnen-3-one (DHP) and 17,20,21-tri-hydroxy-4-pregnen-3-one (20-S) levels were measured by radioimmunoassay (RIA) in white perch (Morone americana) and white bass (M. chrysops) that were induced to undergo final oocyte maturation (FOM) with human chorionic gonadotropin (hCG). Plasma DHP levels increased in females of both species in association with oocyte germinal vesicle migration (GVM) and germinal vesicle breakdown (GVBD) and decreased thereafter. Plasma 20-S levels also increased with oocyte GVM in white bass, but were several-fold lower than DHP levels. Circulating E2 and T levels were greatest during GVM and GVBD in both species and decreased to low levels during oocyte hydration and ovulation. Follicles from white perch and white bass which received a priming injection of hCG in vivo, produced both DHP and 20-S in vitro after exposure to hCG and their oocytes underwent GVBD. Ovarian incubates from unprimed fish of either species produced only E2 and T and their oocytes did not complete GVBD. Oocytes from unprimed bass, but not perch, matured when follicles were exposed to hCG in vitro. Both trilostane and cycloheximide blocked in vitro production of DHP and 20-S and oocyte GVBD by white perch follices. DHP and 20-S were equipotent inducers of FOM in the GVBD bioassay. None of several other structurally-related steroids tested were effective within a physiological range of concentrations. These results indicate a role for DHP and 20-S in the control of FOM in white perch and white bass.  相似文献   

12.
Vitellogenin was purified from the serum of 17‐β estradiol (E2)‐induced juvenile Catla catla using a simple two‐step purification procedure i.e. selective chemical precipitation followed by gel filtration chromatography. Purified protein migrated as single band in a native gradient PAGE which indicated the purity of the sample. The molecular weight of the native catla vitellogenin (~440 kDa) was determined using gel filtration chromatography. In SDS‐PAGE under reducing conditions catla vitellogenin dissociated into three major sub units at 115 kDa, 102 kDa and 73 kDa along with a few faint bands. Confirmation of purified protein as catla vitellogenin was supported by multiple physiological evidences, e.g. absence in male as well as juvenile sera and presence in matured female fish, ability to be synthesized upon estradiol injection in immature fish and certain unique biochemical properties like high molecular weight, phospholipoglycoprotein nature of the molecule. Western blot analysis showed that polyclonal antibody raised against purified protein detected vitellogenin in the sera of catla and in a few species selected from Cyprinidae family. These antisera were used to detect vitellogenin in liver tissue of hormone‐induced catla using immunohistochemistry and its applicability in other immunoassays is discussed.  相似文献   

13.
A dose-response effect was observed on the plasma concentration of vitellogenin (ALPP, alkali-labile protein P) over a 33 day time-course with repetitive estradiol-17 treatment of European eel (Anguilla anguilla). The plasma estradiol-17 concentration in the treated groups was not proportional to the doses injected. In the groups treated with a medium (0.5 mg kg BW-1) or high dose (5 mg kg BW-1) of estradiol there was a significant increase in the plasma total cholesterol concentration during the 33 day time course. The most marked dose-response effect of estradiol was that of plasma total lipids. The distribution of cholesterol between the different lipoprotein classes was dose-related: high density lipoproteins (HDL) predominated in the controls and in the low and medium dose groups, while low density lipoproteins (LDL) predominated over HDL in the group given the high estradiol dose. Plasma HDL concentration was markedly decreased in the high dose treated group; this finding is interesting since HDL plays an important role in the uptake of cholesterol from the extrahepatic tissue.  相似文献   

14.
In vivo induction of vitellogenin (VTG) in response to the administration of 17-estradiol (E2) was achieved and the protein was isolated by gel filtration column chromatography of plasma samples. Adult female trout were injected with the vitellogenic fraction every ten days from July to November and levels were measured by RIA from September to December. The results showed a significant decrease (p<0.01) in plasma E2 levels in injected females compared with the controls. In December, after finishing the treatment, the plasma E2 concentration increased, in injected females to reach a level similar to that of control females at vitellogenesis. The in vitro study showed that in early vitellogenic oocyte (from September) the presence of the vitellogenic fraction in the incubation medium causes a significant decrease (p<0.01) in the synthesis of E2 by the oocytes. These data suggest that the concentration of the VTG into the oocyte can alter VTG production by the liver, moderating the production of E2 by the ovary.  相似文献   

15.
Triploid fish have under-developed gonads due to altered reproductive endocrinology. Triploids of Indian catfish (H. fossilis) showed significantly reduced plasma levels of gonadotropin (GtH-II), testosterone (T) and estradiol-17 (E2) than that of diploids throughout the year, except for the resting phase, irrespective of sex. Plasma levels of GtH-II were significantly different (p<0.001) between diploid and triploid fish during preparatory, prespawning and spawning phase. The plasma testosterone contents in triploids were significantly less (p<0.001) than that of diploids, except during the resting phase. Triploid females showed very low titres of estradiol-17 (<1 ng ml–1) throughout the annual reproductive cycle in contrast to highly fluctuating levels in diploid females. Thus, this study for the first time provides information on reduced levels of GtH-II and sex steroids in plasma of male triploid fish and additional information on species-specific alteration of sex hormones in female triploids.  相似文献   

16.
Blood samples were collected from captive Pacific halibut, Hippoglossus stenolepis , at intervals of about six weeks from early December 1986 to late November 1987. Concentrations of plasma androgen and estradiol-17β were determined by radioimmunoassay. The plasma concentrations of steroid were highest during autumn and winter in halibut that matured during late winter. The concentrations of steroids in samples collected in December were above 2 ng/mL (estradiol) or 1 ng/mL (androgen) in maturing females and below 0.5 ng/mL for both steroids in non-maturing females. The levels of steroids decreased rapidly about one month before spawning. In a mature male, androgen began to rise in August and November, and reached a peak of 7 ng/mL in early December. One month before spawning, the androgen concentration fell to 0.16 ng/mL. Estradiol concentrations were detectable in the male and varied little during the year. In immature fish, neither androgen nor estradiol changed significantly throughout the year. These results suggest that the concentrations of estradiol or androgen measured in blood samples taken during December may be used to determine the sex and state of maturation of Pacific halibut.  相似文献   

17.
Effect of nonylphenol (NP) products from different companies on in vitro vitellogenin (VTG) synthesis was compared using tilapia ( Oreochromis mossambicus ) hepatocytes cultures. Addition of NP at a concentration of 10−3 M to the medium caused death of hepatocytes (NP-A and NP-B) and delay of monolayer formation (NP-C). No cell death was observed at a concentration of 10−4 M (NP-A, -B and -C) but cell adhesion was slower than control. These results suggest that high concentration of NP is toxic against tilapia hepatocytes. When effects of estradiol-17β (E2, 10−7 M) and NP (10−4 M) on in vitro VTG synthesis were examined, addition of E2 and NP-A and NP-B to the media resulted in elevation of VTG. NP-C did not induce VTG in the medium. Co-treatment of NP-B and tamoxifen, a non-steroidal anti-estrogen, reduced VTG synthesis. These results suggest that NP has estrogenic potential in primary cultures of tilapia hepatocytes and acts through binding to the estrogen receptor, and that there is a difference in the induction level of VTG among different NP products.  相似文献   

18.
A procedure is described for the isolation of intact vitellogenin (c-VTG) from the carp, Cyprinus carpio. VTG was induced in juvenile females using oestradiol-17β and purified from the plasma using a combination of gel-filtration chromatography on Sepharose 6B and ion exchange chromatography on DEAE-cellulose. Purification procedures were conducted at low temperatures (below 9°C) in the presence of the proteolytic enzyme inhibitor aprotinin to prevent degradation. Intact c-VTG had an apparent molecular mass of 390,000 Daltons, but when extracted from plasma in the absence of aprotinin it underwent proteolysis into at least 2 protein fragments (apparent molecular masses of 230,000 and 96,000 Daltons), showing an instability of the native dimer. An amino acid analysis of c-VTG showed that its composition was almost identical to goldfish VTG, a species closely allied to the true carps and also similar to other oviparous vertebrate VTGs. Collectively, these data indicate that using these purification procedures VTG from carp, and probably other teleost species, can be isolated in an intact, highly purified form.  相似文献   

19.
Vitellogenin (VTG) was purified from the plasma of estradiol-17β (E2) treated male brook trout (Salvelinus fontinalis; bt) using gel filtration and anion exchange chromatography. An antiserum to bt-VTG was raised in rabbits, then used to detect bt-VTG by Western blot analysis. Purified bt-VTG and its antibody were then used to develop an antibody-capture, competitive enzyme-linked immunosorbant assay (ELISA). Confirmation of the purified protein as bt-VTG was based on the characteristics of high molecular weight (∼562 kDa), dominant plasma protein in vitellogenic females and induction by exogenous E2 treatment in males. The antiserum recognized a major 184 kDa polypeptide as well as minor bt-VTG degradation products (150–66 kDa) in purified bt-VTG and in plasma from vitellogenic females. Low levels of antibody cross reactivity were shown with plasma from nonvitellogenic females, control rabbit serum, and several other antisera known to not contain VTG. The ELISA had a minimum detection limit of 8 ng ml−1 and intra- and inter-coefficients of variation less than 9% and 15%, respectively. The ELISA demonstrated parallel binding slopes among dilution curves of purified bt-VTG standard and plasma from diploid and triploid brook trout females. Using the ELISA, maximum plasma VTG levels of 93.5±33.6 mg ml−1 were detected in vitellogenic diploid females, whereas only 0.18±0.15 mg ml−1 were detected in triploid females of the same age (n=5 for each). Diploid and triploid males cohabitating with vitellogenic females showed measurable levels of plasma VTG during vitellogenesis in females (i.e., 0.17 and 0.06 mg ml−1, respectively (n=1)). This revised version was published online in August 2006 with corrections to the Cover Date.  相似文献   

20.
Major challenges in culture of Atlantic halibut larvae have been slow growth during the late larval stages and inferior juvenile quality due to pigmentation errors and incomplete eye migration during metamorphosis. The hypothesis of this study was that feeding on‐grown Artemia would alleviate these problems. Artemia were grown for 3–4 days on Origreen or Origo. The growth and nutrient composition of Artemia nauplii and on‐grown Artemia were analysed, and both Artemia types were fed to Atlantic halibut larvae, on‐grown Artemia from 15 days post‐first feeding (dpff). The body length of Artemia increased with 20%–70% in response to on‐growing. In all experiments, protein, free amino acids and the ratio of phospholipid to total lipid increased, while lipid and glycogen decreased. The fatty acid composition improved in some cases and not in others. The micronutrient profiles were not negatively affected in on‐grown Artemia. All these changes are thought to be beneficial for marine fish larvae. The final weight of Atlantic halibut postlarvae was similar, and 90% of the juveniles had complete eye migration in both groups. It is concluded that the present version of Artemia nauplii probably covers the nutrient requirements of Atlantic halibut larvae.  相似文献   

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