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1.
Three forms of female-specific plasma protein were purified from blood plasma of estrogen-treated white perch, characterized, and classified as three distinct vitellogenins (VgA, VgB, and VgC). This study describes the first purification of three classes of native Vg from any vertebrate and sets the stage for discovery of the different functions of each type of Vg.  相似文献   

2.
Three types of vitellogenins (Vgs) namely vitellogenin A (VgA), vitellogenin B (VgB) and vitellogenin C (VgC) have been identified in fishes. The existence of VgA and VgB is reported in the Indian freshwater murrel Channa punctatus. Gene-specific primers were designed using available nucleotide sequences in National Centre for Biotechnology Information (NCBI), for amplification of VgA and VgB cDNA. Differential processing of Vgs is evident in many fishes. Adult male murrel expressed both the VgA and VgB genes when estradiol-17β (E2) is injected in vivo and Vg levels in blood quantified by Enzyme linked immunosorbent assay (ELISA) showed a dose-related response in such treatments. Cultured hepatocytes on treatment with E2, however, expressed only VgB as detected by RT-PCR, suggesting different regulatory mechanism for the VgA and VgB genes.  相似文献   

3.
Three forms of female-specific plasma protein (FSPP 1-3) were purified from blood plasma of estrogen-treated white perch (Morone americana) by combining several types of ion-exchange chromatography including a novel, fast flow, strong anion exchanger (POROS media), followed by gel filtration. Native FSPP 1, FSPP 2 and FSPP 3 had molecular masses of 532 kDa, 532 kDa and 426 kDa, respectively. The apparent mass of purified FSPP 1 and FSPP 2 after SDS-PAGE under reducing conditions was ∼ 180 kDa, while FSPP 3 appeared as a major ∼ 148 kDa band. All of the FSPPs resembled one another with respect to amino acid composition but each appeared to be immunologically distinct. In double immunodiffusion using anti-total FSPP (antiserum raised against vitellogenic female plasma pre-absorbed by male plasma), each FSPP formed one precipitin line that crossed those produced by both others. A rabbit antiserum was raised against each FSPP and absorbed with combinations of the other two FSPPs to ensure specificity. Using the antisera, each FSPP was detected by immuno-electrophoresis in plasma from vitellogenic females or estrogen-treated male or immature fish, but no FSPP was detected in normal male plasma. Endoprotease (Asp-N) digests of the FSPPs were subjected to HPLC separation for N-terminal sequencing and mapping of isolated peptides to published vitellogenin (Vg) sequences. Results of these analyses indicate that white perch FSPP 1, FSPP 2, and FSPP 3 can be classified into three Vg groups identified in previous studies: VgA, VgB, and VgC-like protein, respectively. This is the first report, of which we are aware, on isolation of more than two Vg proteins from any species of vertebrate except the chicken. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

4.
运用建立化学发光免疫(chemiluminescence immunoassay,CLIA)检测法,对梭鱼血清中主要卵黄蛋白原(Vg)类型(B类型;VgB)进行定量测定。梭鱼VgBCLIA法按两步法操作进行,使用纯化的梭鱼VgB制备特异型抗血清(a-VgB)。优化抗体浓度和培育时间等检验条件后,检验范围设定为3.91~500ng/mL。卵黄发生期的梭鱼雌鱼血清和雌激素处理后的梭鱼稚鱼血清稀释曲线与纯化的梭鱼Vg曲线平行,而梭鱼雄鱼稀释血清中几乎不发生相应的免疫学反应。对天津原种场养殖的10尾梭鱼血清VgB水平的定量检测结果表明,在雌鱼(9尾)血清中检测到VgB但个体间差异较大,变化范围为3.0~2700.1μg/mL,并表现出随着卵巢发育而增长的变化趋势。相对而言,成熟梭鱼雄鱼血清VgB水平则极低(2.7μg/mL),表明在其生长环境中不存在雌激素活性。另外,所有个体的组织学观察也未呈现出明显的性腺异常情况。研究为梭鱼主要雌激素诱导生物标志物(VgB)的量化提供了一种新的手段,较以往的检验方法具有更高的灵敏度,有助于建立多元化的水生环境中雌激素活性的检测体系。  相似文献   

5.
Two forms of vitellogenin (Vg: Vg1 and Vg2) were purified from the plasma of estradiol-17β (E2)-treated Indian walking catfish, Clarias batrachus, by gel filtration and adsorption chromatography. Native Vg1 and Vg2 had apparent molecular masses of 375 and 450 kDa, respectively, and both Vgs resolved into two similar major bands (95 and 67 kDa) in SDS-PAGE under reducing condition. Polyclonal antisera raised against each form of Vg were absorbed with a combination of hypophysectomized male catfish serum proteins and alternate Vg to ensure specificity. Immunological analyses verified the presence of Vg1 and Vg2 in the plasma of female catfish. Homologous ELISAs were developed for Vg1 and Vg2 using their respective harvested antisera, which exhibited the detection limit of 100 ng ml?1 for Vg1 and 40 ng ml?1 for Vg2, and low level of cross-reactivity (not parallel to the standard) was found with alternate Vg in each assay. Treatment of male catfish with E2 induced both Vgs showing a proportionate ratio of Vg1 to Vg2 at 5.6:1. Plasma concentrations of both Vgs measured by ELISAs at different reproductive phases of field collected female catfish increased in accordance with the ovarian development, keeping the proportionate ratio of Vg1 to Vg2 at about 2:1 in fish undergoing vitellogenesis during prespawning period and 1:20 during spawning period, suggesting that Vg1 may be the major Vg to contribute in yolk formation, whereas Vg2, besides its role in yolk formation, may facilitate other physiological functions. The present study, thus, demonstrates the occurrence of two unequally synthesized Vgs in the catfish.  相似文献   

6.
A large size (400 kDa) non-collagenous protein was detected as a major component in the extract, with neutral salt solution, from the dermis of sea cucumber Apostichopus armata. On SDS-PAGE analysis, the 400 K component shifted to a lower molecular weight component (about 200 K) by reduction with 2-mercaptoethanol, and they were both reactive for periodic acid-Schiff (PAS) reaction staining. From these results, this protein was suggested to be a glycoprotein consisting of disulfide-bonded two subunits with almost equal molecular weight (200 K). In addition to relatively high contents (>100/1,000 residues) of aspartic and glutamic acids, cysteine was also detected (6.1/1,000 residues) in amino acid analyses of this protein partially purified by anion-exchange column chromatography. These combined results suggest the structural similarity of the 400 K component to fibronectins from other vertebrate and invertebrate animals.  相似文献   

7.
ABSTRACT:   Lipovitellin (Lv), the major yolk protein derived from vitellogenin (Vg), was purified from vitellogenic ovaries of Pacific saury Cololabis saira using hydroxylapatite column chromatography followed by gel filtration. The apparent native mass of purified Lv was approximately 420 kDa, while the tertiary structure of Lv revealed by sodium dodecylsulfate–polyacrylamide gel electrophoresis was typical of teleost Lvs, consisting of a heavy chain (∼99 kDa) and a light chain (∼34 kDa). Western blot analysis using rabbit antiserum raised against Pacific saury Lv revealed a specific reaction with a polypeptide (∼194 kDa) that is present in serum from female Pacific saury but not in male serum, suggesting the approximately 194-kDa polypeptide to be the Vg monomer. This study describes the first step toward the development of specific immunoassays for Pacific saury Vg, which will be an effective tool for monitoring the reproductive development of this species.  相似文献   

8.
We sought to provide a useful indicator of the presence of endocrine-disrupting contaminants along the marine coast of the South Pacific using Chilean flounder (Paralichthys adspersus). In light of the lack of information on vitellogenin for this species, we induced, purified, and identified the plasma vitellogenin of Chilean flounder inhabiting the Chilean coast. Vitellogenin (Vg) from Chilean flounder was purified by size exclusion and ion-exchange chromatography using plasma from juvenile males induced by injecting 17β-estradiol. The Vg was detected by SDS–PAGE and Western blot analyses using an antibody against turbot (Scophthalmus maximus) vitellogenin. These analyses revealed a protein band of 205 kDa and three minor bands of 120, 90, and 68 kDa. These proteins were identified as Vg by means of mass spectrometry (LCQ Duo ESI-IT-MS), matching sequences of tryptic peptides to known sequences for several other fish species. The matches showed the presence of vitellogenin (VgI, VgII, Vg A and Vg B) in Chilean flounder, similar to species such as mummichog (Fundulus heteroclitus), Japanese medaka (Oryzias latipes), and white perch (Morone americana). These results are discussed in terms of identifying Vg in Paralichthys adspersus with the antibody to turbot Vg. Moreover, we compare the molecular size of Vg from Chilean flounder (large) with that of other flatfish species. Finally, we discuss the potential use of this molecule as a biomarker for the presence of xeno-estrogenic compounds along the Chilean coastline.  相似文献   

9.
Vitellogenin (Vg) of the barfin plaice Liopsetta pinnifasciata was isolated and purified. In native polyacrylamide gel electrophoresis, Vg appeared as one band. After being subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE), Vg fraction produced several polypeptides with molecular masses of 180, 98, 70, 52, 41 and 37 kDa. MALDI–TOF mass spectrometry (MS) of the 180- and 98-kDa Vg polypeptides from the SDS–PAGE gel and de novo sequencing of their four peptide fragments based on MS/MS analysis confirmed that the purified proteins were vitellogenins, which shared high similarity with the Vgs of the barfin flounder Verasper moseri and Atlantic halibut Hippoglossus hippoglossus. The most part of the predicted sequences obtained from the L. pinnifasciata 180-kDa polypeptide has previously been found in the V. moseri vitellogenin type B, the sequences obtained from the 98-kDa polypeptide were found in V. moseri vitellogenin type A, so these findings allow us to propose that L. pinnifasciata has at least two different forms of Vg. Rabbit polyclonal antibodies against Vg were produced, and a quantitative enzyme-linked immunosorbent assay was developed. The concentration of Vg in barfin plaice from the moderately contaminated area of Amursky Bay in the Sea of Japan was detected based on the maturity stage of their gonads. In November 2008, the Vg concentration in the plasma of females with advanced oogenesis varied from 5.295 to 28.367 mg/ml (mean 16.38 ± 6.73 mg/ml, CV = 41.1%); in the plasma of males, the concentration ranged from non-detectable to 0.957 mg/ml (0.29 ± 0.42 mg/ml, CV = 127.9%). In October 2009, the Vg concentration in female plasma was lower than in November 2008 (2.21–13.87 mg/ml). High individual variability of plasma Vg was characteristic for maturing males (CV = 200.3%) and immature females (CV = 255.5%), and there was no significant difference between plasma Vg concentrations in males captured in November 2008 and October 2009 or in maturing males and immature females. Vacuolisation of hepatocytes was more typical for males with low plasma Vg concentrations and females with high plasma Vg concentrations. Necrosis and pyknosis of hepatocyte nuclei were more frequent in males with high Vg concentrations and in females with low plasma Vg concentrations.  相似文献   

10.
11.
In order to identify maturational factors that can serve as indicators of egg quality in artificially matured Japanese eel, Anguilla japonica, maturation-promoting factor (MPF; consists of cdc2 and cyclin B) related proteins and chromosomes were examined. Mitogen-activated protein kinase (MAPK; 44 kDa band) and cdc2 (35 kDa and 34 kDa band) were detected. Chromosomes aligned on a vertical spindle appeared normal; however, chromosomes on a horizontal spindle and dispersed chromosomes seemed unusual.  相似文献   

12.
The optimal conditions for protein hydrolysates preparation from spotted babylon Babylonia areolata exhibiting tyrosinase inhibitory activity and antioxidant activity using alkaline protease (Protease G6) were undiluted Protease G6 (5.8 × 105 DU/g) at 60 min of hydrolysis time and eightfold diluted Protease G6 (7.25 × 104 DU/g) at 240 min of hydrolysis time. These two conditions were fractioned using molecular weight (MW) cutoff values of 10, 5, and 3 kDa membranes, and their anti-melanogenic and antioxidant properties were further analyzed. Among the fractions, the MW < 3 kDa fraction exhibited high levels of inhibitory activity toward the mono- and diphenolase activities of tyrosinase, with IC50 values of 1.758 and 8.995 μg/mL, respectively. Kinetic studies revealed that this fraction behaved as an uncompetitive inhibitor. The results demonstrated that the MW < 3 kDa fraction suppressed melanin synthesis and decreased cellular tyrosinase activity with no cytotoxicity to B16F10 melanoma cells.  相似文献   

13.
ABSTRACT:   Because blood vitellogenin (Vg) has been considered a biomarker for environmental estrogens, the basal levels of Vg and 17β-estradiol (E2) were determined in male Japanese whiting reared under natural conditions. Serum levels of Vg and E2 were measured and gonadal development was assessed by gonadosomatic index (GSI) and histological observation in 8–10 male fish at monthly intervals throughout the annual reproductive cycle. Serum E2 was <60 pg/mL throughout the study period. In contrast, serum Vg exhibited seasonal changes: serum levels of Vg gradually increased from April to May (mean 63 ± 13 ng/mL and 124 ± 48 ng/mL in April and May, respectively), and then reached a peak value (mean 352 ± 68 ng/mL) in June. Thereafter, serum Vg gradually decreased, reaching undetectable levels (<50 ng/mL) in October. Serum levels of Vg tended to increase in the male fish in which the GSI was >1%. Histological observation revealed that testes in such male fish were in active spermatogenesis and then all of the testes of male fish in which serum Vg decreased to ND levels were regressed. These results suggest that Vg productive potency (sensitivity to estrogens) may increase in the spermatogenic stage, resulting in production of Vg in response to very low levels of natural or xenobiotic estrogens.  相似文献   

14.
The biological activities of catfish LH-like (semi-purified: s200a and purified Qa) and FSH-like (semi-purified: s200b and purified: Qb) were compared in intact and hypophysectomized female catfish, Clarias batrachus, during preparatory and the pre-spawning periods on vitellogenesis and ovarian maintenance, as well as in vitro final maturation of oocytes, germinal vesicle breakdown (GVBD). During preparatory period, in intact catfish, semi-purified FSH-like induced complete vitellogenesis through the production of estradiol-17β (E2) and vitellogenin (Vg) accompanied by the formation of SIII yolky oocytes. On the other hand, semi-purified LH-like had induced the formation of only SII (characterized by the appearance of cortical alveoli in cytoplasm) oocytes, which indicates the initiation of vitellogenesis. In hypophysectomized female catfish, purified LH-like but not FSH-like induced the formation of SII oocytes in the ovaries. Treatment with semi-purified LH- and FSH-like at the dose level of 5 µg/fish/day for 7 days significantly maintained the yolky oocytes in gravid catfish after hypophysectomy with a significant reduction in plasma Vg, but not E2 levels, indicating some unknown GtH-induced factor doing the job. In in vitro oocytes culture, both LH- and FSH-like induced GVBD, but the response was significantly more with LH-like than FSH-like. All these findings revealed that both LH-like and FSH-like have overlapping physiological functions, but their responses differ depending on the physiological status of the catfish.  相似文献   

15.
The aim of this study was to evaluate the addition of artificial substrates in biofloc technology systems that are used for the intensive production of Litopenaeus vannamei. The experiment lasted 35 days. Tanks each with a useful volume of 800 L were filled with water containing bioflocs (25 % of the total volume) and filtered seawater. Three treatments with three replicates each were compared: (1) Ctrl (control), i.e., without the addition of artificial substrate, (2) 200 and (3) 400, with added artificial substrate equivalent to 200 and 400 % of the lateral area of the tanks, respectively. The shrimp with initial weight of 0.40 ± 0.15 g were stocked at a density of 300 shrimp/m2. Feed (Guabi/38 Active, Brazil) was supplied two times per day. The water quality parameters were suitable for the production of L. vannamei. The settleable solids (SS) were higher in the Ctrl treatment (82.1 ± 19 mL L?1) and differed statistically from the 200 and 400 treatments that presented average SS values below 10 mL L?1. The reuse of water with bioflocs accelerated nitrification in the tanks, and no significant differences were shown between tanks. The presence of biofilm did not interfere with ammonium or nitrite levels, but it did serve as a food source that optimized shrimp performance, as shown by higher final weights. More studies are needed to evaluate the circulation and mixing intensity effects of the substrates on water throughout the production cycle.  相似文献   

16.
A study was conducted to isolate, partial characterize Asian sea bass (Lates calcarifer) vitellogenin (vtg). Two-year-old juvenile L. calcarifer (n = 10) were given three intraperitoneal injections of 17-β estradiol (E2) at a dose of 2 mg/kg body weight to induce vitellogenesis. Blood was collected 3 days after the last injection, and plasma was purified through gel filtration chromatography. A broad single symmetrical peak consisting of vtg molecule was produced. Protein concentration was 0.059 mg/ml as determined by Bradfrod assay using bovine serum albumin as a standard. The protein appeared as one circulating form in Native PAGE considering the dimeric form of putative vtg with molecular weight of 545 kDa. In SDS-PAGE under reducing conditions, two major bands appeared at 232.86 and 118.80 kDa and minor bands at 100.60, 85.80 and 39.92 kDa, respectively. The purified vtg was used to generate a polyclonal antibody, and the specificity of antibody was assessed by Western blot analysis. Two major bands were immunoreacted, but no cross-reactivity was observed with plasma from non-induced males. The protein was characterized as phosphoglycolipoprotein as it positively stained for the presence of lipid, phosphorus and carbohydrate using Sudan Black B, methyl green and periodic acid/Schiff reagent solution, respectively. The amino acid composition was analyzed by high sensitivity amino acid analysis that showed high percentage of non-polar amino acids (~48 %). The results suggest the potential utilization of vtg as a basis tool to further study about reproductive physiology of this important economical species.  相似文献   

17.
A 10-week feeding experiment was conducted to evaluate the effects of dietary protein to lipid ratios (P/L) on growth, intestinal digestive enzyme activities and body composition in juvenile rice field eel (Monopterus albus) (initial mean body weight of 65.76 ± 1.07 g, mean ± SEM). Nine test diets were formulated in a 3 × 3 factorial design to contain three protein levels (350, 400 and 450 g kg?1) for each of three lipid levels (40, 80 and 120 g kg?1), respectively. Each diet was randomly assigned to triplicate groups of 60 fish per net cage (1.5 × 2.0 × 1.5 m). Results showed the survival was above 96 % and was not affected by dietary treatments. Fish fed the diet with 450 g kg?1 protein and 40 g kg?1 lipid showed the best weight gain (WG) (103.95 %) and feed conversion ratio (1.60) (P < 0.05). WG, protein efficiency ratio and energy retention increased with the increasing in lipid at 350 g kg?1 protein level (P < 0.05). However, WG showed a little decline with increasing dietary lipid when fish fed the diets with 400 and 450 g kg?1 protein level, but no significant difference was observed (P < 0.05). Hepatosomatic index, visceralsomatic index and intestinal lipase activity increased with the increasing of dietary lipid level irrespective protein level. Intestinal trypsin activity increased with the increasing of dietary lipid level when fish fed the diets with 350 g kg?1 protein, but showed converse trend when fish fed the diets with 400 and 450 g kg?1 protein. Serum triglyceride, body lipid and energy were positively correlated with the dietary lipid. Results of the present study showed that the dietary protein/lipid ratio of 450/40 g kg?1 is considered optimum for rice field eel under culture conditions, and the increase in dietary lipid level has no efficient protein-sparing effect when fish fed the diets with 400 and 450 g kg?1 protein level.  相似文献   

18.
A feeding experiment was conducted to evaluate the efficacy of, and determine the requirements for dietary AsA (l-ascorbic acid) using its popular derivative l-ascorbyl-2-monophosphate-Mg (APM) for juvenile Pacific bluefin tuna (PBT). The five test diets (with APM-0, -400, -800, -1200 and -1600) were prepared by adding 0, 400, 800, 1200 and 1600 mg of APM kg diet?1, respectively. These diets, together with chopped sand lance (the control diet), were administered, in duplicate to 0.27 g PBT at 25 days after hatching (300 juveniles per 15 m3 tank). The PBT were fed to apparent satiety 6 times a day for 2 weeks. While final body weight, feed conversion and survival showed no differences among diets APM 400–1600, PBT fed APM-0 indicated lower growth performance together with anorexia, dark pigmentation and ataxia from 5 days and reached 80 % mortality at 12 days after the start of the feeding trial. In addition, liver and brain ascorbic acid (AsA) concentrations increased with increasing dietary APM levels, the values were similar in PBT fed diets with APM-1200 and APM-1600, and liver and brain concentrations of AsA were significantly higher in these fish than in the PBT fed the other diets (P < 0.05). The PBT responded to increasing dietary APM, and the fish fed APM-1200 had the highest weight gain, protein and lipid content. The data indicate that PBT can utilize APM as an AsA source and suggest that dietary supplementation with APM-1200 (454 mg AsA kg?1 diet) would result in satisfactory growth.  相似文献   

19.
Changes in the levels of plasma vitellogenin (Vg), estradiol (E2) and testosterone (T) were examined following gonadal development induced by carp gonadotropin treatment (cGTH) of freshwater female yellow and silver eels (Anguilla anguilla L.). The animals received injections of cGTH (250 μg kg−1 body weight) or saline vehicle three times a week, for 6 to 8 weeks. No effect of vehicle was observed. Steroidogenic activity of the ovary was stimulated by cGTH treatment as shown by the increase in circulating steroid levels in both stages. However, the responses of T, E2 and Vg differed according to the stage of development of eels. At the yellow stage, the initial steroid plasma levels were undetectable (< 0.01 ng ml−1) before treatment and ovarian steroidogenic activity was slightly stimulated following cGTH treatment; steroid levels reached their highest values after 3 weeks and 6 weeks of treatment for E2 (0.62 ± 0.13 ng ml−1 and T (0.33 ± 0.30 ng ml−1), respectively. The cGTH treatment slightly increased plasma Vg levels (0.2–0.7 μg ml−1 during the experiment compared with the initial values of the group. At the silver stage, the initial steroid levels were detectable (0.7 ng ml−1 for E2 and 0.1 ng ml−1 for T); cGTH treatment did not significantly increase plasma E2 level which remained at initial levels. Nevertheless, plasma T levels dramatically increased from 0.1 to 3 ng ml−1 and peaked after 1 or 2 weeks of cGTH treatment; a rapid increase in plasma Vg levels occurred, reaching its highest value at 5 mg ml−1 after 3 weeks of treatment. Thus, the steroid kinetic profiles in relation to the appearance of Vg in the plasma following cGTH treatment was closely related to androgen levels and there was a strong vitellogenic response induced by chronic cGTH treatment. In order to test if androgens could be implicated in the vitellogenic response, we evaluated the potencies of various androgens (testosterone and 5α-androstane-3β,17β-diol)in vivo andin vitro, associated with E2 to induce the production of Vg.In vitro experiments showed that Vg synthesis was induced by high doses (10−6 to 10−5 M) of androgen in the eel. Tamoxifen totally inhibited the action of androgens suggesting that androgens were acting through binding to the E2 receptor.In vivo, androgens given alone at 50 μg kg−1 3 times a week for 1 months had no significant effect on plasma Vg levels. In addition, E2-androgen cotreatment showed that the presence of androgen did not modify the vitellogenic response induced by E2.  相似文献   

20.
The experiment was conducted to investigate the effects of prepared hairtail protein hydrolysate–Fe2+ (PH–Fe2+) complexes on growth and non-specific immune responses of crayfish (Procambarus clarkii). The diets with five different levels of PH–Fe2+ (200, 400, 600, 800, and 1000 mg/kg) were fed to crayfish for 60 days. The results indicated that the survival rate, weight gain percentage, specific growth rate, and muscle index of crayfish with 400–1000 mg/kg of PH–Fe2+ feeding were significantly higher, 9.94–10.10, 8.55–8.72, 0.24–0.29 %/day, and 2.39–3.05 %, respectively, than the control values after 60 days of feeding. Additionally, after 30 days of feeding, 200–400 mg/kg of PH–Fe2+ showed no significant (P > 0.05) improvement effect on activities of superoxide dismutase (SOD), phenoloxidase (PO), lysozyme (LSZ), and acid phosphatase (ACP) in serum or hepatopancreas of crayfish. However, significant improvement effects were observed in 800–1000 mg/kg groups. After 60 days of feeding, 400–600 mg/kg of PH–Fe2+ significantly (P < 0.05) improved the activity of SOD and LSZ, but did not affect the activity of PO and ACP significantly (P > 0.05). Moreover, the activities of SOD, PO, LSZ, and ACP in serum and hepatopancreas were all significantly enhanced upon treatment with 800–1000 mg/kg of PH–Fe2+. For these reasons, PH–Fe2+ can be recommended as a supplement in crayfish feed to increase the growth and immunity.  相似文献   

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