饲料中花生四烯酸对发育前期大菱鲆亲鱼性类固醇激素合成的影响

为研究饲料中不同水平的花生四烯酸(arachidonic acid,ARA)对发育前期的大菱鲆亲鱼性类固醇激素合成量及合成过程的影响,配制3种等氮等脂(脂肪含量13%)的实验饲料,分别含有不同梯度水平的花生四烯酸:0.72%(不添加花生四烯酸精制油的对照组,C),5.63%(添加低水平ARA的处理组,ARA-L)及15.03%(添加高水平ARA的处理组,ARA-H)(各数值均为占总脂肪酸的比例)。每组饲料投喂3个实验桶,每桶放25尾3龄大菱鲆亲鱼(雌雄比例约为1:1)。养殖实验在室内流水系统内进行,每天饱食投喂2次,养殖周期为5个月。养殖结束后,分别取性腺发育前期的雌雄鱼测血清雌二醇和睾酮的含量并检测性腺中性类固醇激素合成相关蛋白质的基因表达量。结果显示:与对照组相比,饲料中高水平的花生四烯酸显著降低了雌鱼血清中雌二醇的含量,而雄鱼血清中睾酮的含量在低水平花生四烯酸处理组显著降低。在卵巢中,饲料中高水平的花生四烯酸显著降低了促卵泡激素受体mRNA的表达量,但是饲料中低水平的花生四烯酸显著提高了17α羟化酶的mRNA表达量。在精巢中,饲料中低水平的花生四烯酸显著降低了固醇合成急性调节蛋白以及17α羟化酶的mRNA表达量,但显著升高了芳香化酶的mRNA表达量。饲料中花生四烯酸提高了性腺、肝脏和肌肉组织中花生四烯酸的含量,但降低了二十碳五烯酸(EPA)的含量,卵巢中的花生四烯酸累积量高于精巢。综上所述,饲料中添加一定量的花生四烯酸抑制了发育前期大菱鲆亲鱼雌二醇和睾酮的合成,在卵巢中,这种抑制作用可能是通过抑制促卵泡激素受体的表达来实现,而在精巢中,可能是通过抑制固醇合成急性调节蛋白以及17α羟化酶来实现。     

Effect of waterborne zinc exposure on lipid deposition and metabolism in hepatopancreas and muscle of grass carp <Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis>

The aim of the present study was to explore the effect of waterborne zinc (control, 0.85, 2.20, 3.10 mg/l, respectively) exposure on lipid deposition and metabolism in the hepatopancreas and muscle of grass carp Ctenopharyngodon idella. The lipid content, Zn accumulation, and the activities and expression levels of several enzymes involved in lipid metabolism were determined in hepatopancreas and muscle. Waterborne Zn exposure reduced growth performance and increased Zn accumulation in both tested tissues. In hepatopancreas, Zn exposure increased lipid content, the activities of lipogenic enzymes, such as 6PGD, G6PD, ME, ICDH and FAS, as well as the mRNA expression level of G6PD, 6PGD, ICDH, FAS and SREBP-1. But the activity of CPT I and the mRNA expression of HSL, CPT Iα1a, CPT Iα2a and PPARα were down-regulated by Zn exposure. In contrast, in muscle, waterborne Zn exposure decreased lipid deposition, activities of 6GPD, ICDH and ME, as well as the mRNA expression level of G6PD, ICDH, ME, FAS and SREBP-1. However, the activity of CPT I as well as the mRNA expression level of PPARα, HSL, CPT Iα2a, CPT Iα1b and CPT Iβ were up-regulated by Zn exposure. Our results indicate that waterborne Zn increases lipid content by up-regulating lipogenesis and down-regulating lipolysis in hepatopancreas. But, in muscle, waterborne Zn reduces lipid accumulation by up-regulating lipolysis and down-regulating lipogenesis. Differential patterns of lipid deposition, enzymatic activities and genes’ expression indicate the tissue-specific regulatory mechanism in fish.     

Gene expression responses to restricted feeding and extracted soybean meal in Atlantic salmon (Salmo salar L.)

We assessed effects of feed restriction and inclusion of 200 g kg^?1 extracted soybean meal in the diet on gene expression in Atlantic salmon using a cDNA microarray (SFA2.0) and real‐time qPCR. The trial lasted for 54 days. Restricted feeding and soybean inclusion reduced the thermal growth coefficient by respectively 51% and 22% compared with fish fed with the fishmeal‐based control diet to satiation. Soybean decreased distal intestinal expression of lysosomal (cathepsins C, D, L, Y and Z) and extracellular proteases while genes involved in responses to cellular stress were up‐regulated. Expression changes of immune genes suggested both pro‐ and anti‐inflammatory regulation. The hepatic responses to soybean and restricted feeding were highly similar, which could be because of negative effects of soybean meal on digestion and nutrient absorption. We observed up‐regulation of ribosomal proteins and down‐regulation of genes involved in lipid and steroid metabolism. Of note, growth reduction in both study groups was associated with coordinated down‐regulation of genes involved in oxidative and cellular stress responses, metabolism of xenobiotics and protein degradation. High expression of stress‐related genes in salmon fed with the control diet suggests that maximum growth rates can be associated with health problems.     

急性铜胁迫对暗纹东方鲀组织铜积累、氧化应激、消化酶、组织病变及脂代谢相关基因表达的影响

通过 96 h 急性毒性实验研究水体铜暴露对暗纹东方鲀(Takifugu fasciatus)生理生化及脂代谢相关基因表达的影响。实验设置对照组、0.1 mg/L 和 0.2 mg/L (96-h LC50) 3 个铜处理组。结果表明,铜在暗纹东方鲀肝、肌肉和全鱼中的积累量随着处理浓度的提高而提高。同等浓度处理下,铜的积累量为肝脏>全鱼>肌肉。随着铜处理浓度的提高,肝脏中丙二醛(MDA)含量及抗氧化酶活性[谷胱甘肽过氧化物酶(GSH-PX),总超氧化物歧化酶(SOD)和过氧化氢酶(CAT)]显著上升。急性铜暴露诱发肝脏血细胞沉积以及血窦扩张的症状。在鳃中,诱发上皮细胞增生,顶部棒状以及产生动脉瘤等症状。急性铜胁迫后,暗纹东方鲀肠道中的淀粉酶活性显著上升,但脂肪酶活性显著下降。在肝脏中,随着处理浓度的提高,淀粉酶、蛋白酶和脂肪酶活性显著降低。在 0.1 mg/L 处理组,脂肪合成相关基因(G6PD、6PGD、LPL、Fas 和 Acc)的表达量最高。但在 0.2 mg/L 处理组中,脂肪分解相关基因(HSL 和 CPT 1)的表达最高。急性铜胁迫后对转运因子 PPARα的影响不显著,但转运因子 PPAR γ的表达量显著上升。本实验表明铜对暗纹东方鲀生理生化指标及脂代谢相关基因的表达均产生显著影响,本研究为暗纹东方鲀养殖过程中铜的合理使用提供有益的指导价值,也为生产中更好地监控重金属污染提供一定的技术指标参考。     

Effects of Acute Handling Stress on Expression of Growth‐Related Genes in Pampus argenteus

In fish, growth and development are mainly regulated by growth hormone/insulin‐like growth factors. Common aquaculture practices subject fish to stress. To investigate the effects of acute stress on growth‐related genes in cultured fish, the expression of growth hormone receptors (ghr1 and ghr2), insulin‐like growth factor binding proteins (igfbp1, igfbp4, and igfbp5), preprosomatostatin I and II (ppss1and ppss2), somatostatin receptors (sstr2 and sstr5), myostatin (mstn1 and mstn2), and glucocorticoid receptors (gr1 and gr2) were examined in Pampus argenteus subjected to handling stress. Plasma growth hormone levels increased significantly and peaked at 12 h and then decreased significantly at 24 h after treatment (P<0.05). Plasma cortisol and glucose concentrations in stressed fish began to increase significantly at 2 and 6 h after treatment, respectively. Real‐time quantitative polymerase chain reaction analysis showed that hepatic ghr2 mRNA levels in liver and muscle decreased sharply in response to the stressor. Igfbp1, 4, and 5 mRNA expressions in muscle also decreased sharply after exposure, while expression of ppss1, sstr2, and mstn2 increased significantly. This study showed that acute handling stress can affect expression of growth‐related genes in P. argenteus. Our findings could be helpful for the further study on response to stress in this species.     

Effects of dietary non‐protein energy source levels on growth performance,body composition and lipid metabolism in herbivorous grass carp (Ctenopharyngodon idella Val.)

A study was conducted to determine the effects of dietary non‐protein energy sources on growth, tissue lipid accumulation and lipid metabolism‐related genes expression of grass carp. Triplicate groups of fish were fed for 9 weeks on four isonitrogenous (300 g kg^?1) experimental diets with four levels of non‐protein energy (6.52 kJ g^?1 control diet, 5.32 kJ g^?1 high‐CEL diet, 8.46 kJ g^?1 high‐CHO diet and 8.53 kJ g^?1 high‐LIP diet respectively). Increasing dietary non‐protein energy source levels did not improve the growth, and the high‐CEL diet reduced the growth of grass carp. The high‐CHO diet tended to induce high hepatosomatic index, with high fat and glycogen content of liver. However, the high‐LIP diet caused the high mesenteric fat index, but did not increase liver fat. The mRNA abundance and activities of hepatic lipogenic enzymes were significantly increased in the high‐CHO diet group, whereas the opposite tendencies were observed in the high‐LIP diet group. Peroxisome proliferator‐actived receptor‐α (PPARα) in liver and PPARγ in mesenteric adipose tissue were up‐regulated in the high‐CEL diet group. Lipoprotein lipase (LPL) gene expression was significantly increased both in liver and mesenteric adipose tissue of fish fed the high‐LIP diet, while the LPL gene expression was up‐regulated in liver but down‐regulated in mesenteric adipose tissue of fish fed the high‐CEL diet. These findings suggest that an increase in dietary non‐protein energy sources alters the genes expression of lipid metabolism and increased lipid deposition.     

Rapid, nongenomic steroid actions initiated at the cell surface: lessons from studies with fish

In addition to the classic mechanism of steroid action mediated by binding to nuclear receptors, there is a growing body of evidence that steroids also exert rapid, nongenomic actions that are initiated at the cell surface by binding to specific steroid membrane receptors. However, the lack of information on the molecular structures of any steroid membrane receptors has impeded development of this alternative model of steroid hormone action. One of the best characterized models of nongenomic steroid action is the progestin induction of oocyte maturation (OM) in fish and amphibians via activation of plasma membrane progestin receptors (mPRs) on oocytes. Investigations of the marked changes in mPR abundance during OM in fishes have provided the first clear evidence of hormonal regulation of steroid membrane receptors and its physiological importance. Recently, a novel gene was discovered in the ovaries of spotted seatrout whose protein has the characteristics of an mPR and the intermediary in the progestin induction of oocyte maturation in this species. The putative mPR is also detected on seatrout sperm by Western blot analysis and is likely the mPR previously characterized in this species that mediates progestin initiation of sperm hyperactivity. Both structural and functional studies suggest the seatrout mPR is a G-protein coupled receptor (GPCR). Subsequently, thirteen structurally-related cDNAs were identified in other vertebrate species, and several of them were also shown to have characteristics of mPRs. The discovery of the molecular structure and likely orientation in the plasma membrane of this new class of steroid membrane receptors provides a plausible mechanistic explanation of how steroids acting at the cell surface can cause rapid intracellular responses. Membrane receptors for estrogens (mER) and androgens (mAR) have also been characterized in teleost gonads. In addition, the first clear evidence for endocrine disruption of a nongenomic steroid action by binding to a membrane steroid receptor was obtained with the seatrout mPR. Thus fish are valuable models for investigating nonclassical steroid actions and their interference by environmental contaminants.     

Effects of dietary tea polyphenols on growth,biochemical and antioxidant responses,fatty acid composition and expression of lipid metabolism related genes of large yellow croaker (Larimichthys crocea)

The study was conducted to investigate the effects of dietary tea polyphenols (TP) on growth performance, biochemical and antioxidants responses, fatty acid composition, and lipid metabolism‐related gene expressions of large yellow croaker (Larimichthys crocea). Four diets were formulated with different levels of TP (0.00%, 0.01%, 0.02% and 0.05%). Results showed that growth performance of L. crocea were not different among dietary treatments. Compared with the control group, fish in 0.02% TP group had lower body and hepatic lipid content and lower total cholesterol content. The minimum content of triglycerides and low‐density lipoprotein‐cholesterol were found in 0.05% TP group. Hepatic n‐6 PUFA and n‐3 PUFA were significantly higher in TP supplementation groups. Malondialdehyde content was lower in TP supplementation groups, and superoxide dismutase activity was higher in 0.01% TP group than the control group. The mRNA expressions of carnitine palmitoyltransferase1, acyl‐CoA oxidase and peroxisome proliferators‐activated receptor α were up‐regulated in 0.01% and 0.02% TP groups, while lipoprotein lipase expression was down‐regulated in TP supplementation groups than the control group. Results suggested that 0.01%–0.02% TP supplementation could reduce the deposition of liver lipid of L. crocea caused by high‐lipid diet, which might be due to the increase in lipid oxidation related gene expressions.     

Evaluation of growth performance and lipid metabolism in zebrafish fed fructooligosaccharide using RNA sequencing

This study evaluated the effects of fructooligosaccharide (FOS) on growth performance and lipid metabolism in zebrafish (Danio rerio) by RNA sequencing. A total of 240 healthy zebrafish were randomly distributed into two groups. The control group was fed a basal diet, and the treatment group was fed a basal diet supplemented with 0.4% FOS. The results showed that there was no significant difference in growth performance (p > 0.05). The lipid content, total cholesterol, triglyceride, free fatty acid and low‐density lipoprotein were significantly lower (p < 0.05) in the liver of fish fed the 0.4% FOS diet than those of the fish fed the control diet, while the fish fed the 0.4% FOS diet had significantly higher (p < 0.05) high‐density lipoproteins than those of the control fish. Malic enzyme and fatty acid synthetase activities were significantly reduced by adding FOS. KEGG pathway analysis showed that the two pathways of steroid hormone biosynthesis and steroid biosynthesis were significantly enriched (p < 0.05). The profile of genes in these two pathways was affected by FOS in zebrafish. The results of the two pathways suggested new mechanisms underlying the lipid metabolism mechanism of FOS.     

Effect of dietary iron (Fe) levels on growth performance,hepatic lipid metabolism and antioxidant responses in juvenile yellow catfish Pelteobagrus fulvidraco

This study was conducted to determine effects of dietary Fe levels on growth performance, hepatic lipid metabolism and antioxidant response for juvenile yellow catfish Pelteobagrus fulvidraco. Yellow catfish were fed six isonitrogenous and isolipidic diets containing Fe levels of 16.20, 34.80, 54.50, 76.44, 100.42 and 118.25 mg/kg for 8 weeks. Weight gain (WG) and specific growth rate (SGR) increased with dietary Fe levels from 16.20 to 54.50 mg/kg diet and then plateaued over the level. Feed conversion rate (FCR) was highest and protein efficiency rate (PER) was lowest for fish fed the lowest Fe levels of diet. Fe contents in whole body and liver increased with increasing dietary Fe levels. Hepatic lipid content was lowest, but mRNA levels of carnitine palmitoyltransferase (CPT‐1) and peroxisome proliferator‐activated receptor α (PPARα) were highest for fish fed 54.50 mg Fe/kg diet. Fish fed adequate dietary Fe levels reduced hepatic malondialdehyde (MDA) level and increased activities of antioxidant enzymes Superoxide dismutase (SOD), Catalase (CAT) and GS. Based on the broken‐line regression analysis of WG against dietary Fe levels, optimal dietary Fe requirement for yellow catfish was 55.73 mg Fe/kg diets. Fe‐induced changes in MDA levels and antioxidant enzymatic activities paralleled with the change in hepatic lipid content, suggesting the potential relationship between oxidative stress and hepatic lipid accumulation in yellow catfish.     

三丁基锡对褐菖鲉胚胎卵黄囊吸收的影响

鱼类胚胎期的营养对各种器官的发育至关重要,环境条件对营养吸收的影响会增加畸形率和死亡率。研究三丁基锡(tributyltin,TBT)对鱼类胚胎期营养吸收的影响有助于深入认识TBT对鱼类胚胎发育的毒性。本实验以海洋经济鱼类褐菖鲉(Sebastiscus marmoratus)为对象,研究环境水平TBT(0.01、0.1、1、10 ng.L-1)对胚胎卵黄囊吸收的影响。通过油红O染色,发现TBT暴露引起褐菖鲉胚胎血管系统中脂类染色信号减少,而卵黄中的脂类染色正常,说明TBT可能影响了胚胎正常血液循环,从而影响了正常的营养运输。另外,TBT对Na+,K+-ATP酶、碱性磷酸酶活性的抑制以及对甲状腺激素受体α和糖皮质激素受体基因表达的影响可能和其对卵黄囊的吸收抑制有关。TBT对鱼类胚胎卵黄吸收的抑制将影响鱼类的生长和发育,增加畸形率和死亡率,最终导致鱼类种群数量减少。     

Cloning of lipoprotein lipase (LPL) and the effects of dietary lipid levels on LPL expression in GIFT tilapia (Oreochromis niloticus)

Genetically improved farmed tilapia (GIFT) (Oreochromis niloticus) is an important aquaculture species. Lipoprotein lipase (LPL) is considered as a key enzyme in lipid metabolism and deposition. The present study was conducted to investigate the nutritional regulation of LPL in GIFT. We cloned and characterized the LPL gene from GIFT. Finally, we determined the effects of dietary lipid levels and refeeding on hepatic LPL gene expression in GIFT. The LPL gene of GIFT (Oreochromis niloticus) (O.nLPL) was 2,298 bp in length and encoded 515 amino acids. Sequence analysis showed that O.nLPL shared 57.3–87.9 % identity with LPLs from other piscine species. To study LPL expression patterns, juveniles GIFT were fed diets containing 3.7, 7.7 or 16.6 % crude lipid for 90 days and the expression of hepatic O.nLPL was examined using real-time PCR. The abundance of hepatic LPL mRNA increased with increasing dietary lipid. The expression of O.nLPL mRNA in the 16.6 % dietary lipid group was significantly higher than that of the 3.7 % lipid group (P < 0.05). The expression of O.nLPL was increased in GIFT following a 48-h fast and decreased 12 h after refeeding. Hepatic LPL mRNA returned to fasting levels 48 h after refeeding. In summary, high dietary lipid induced expression of liver O.nLPL, and expression of liver LPL is regulated by fasting and refeeding.     

Effects of recombinant vertebrate ancient long opsin on reproduction in goldfish, <Emphasis Type="Italic">Carassius auratus</Emphasis>: profiling green-wavelength light

This study was conducted to identify the possible effect of recombinant vertebrate ancient long (VAL) opsin as a non-visual “photoreceptor” in the deep brain of goldfish, Carassius auratus. In addition, we investigated the effects of green-wavelength light on the predictable reproductive function of VAL-opsin as a green-sensitive pigment in the deep brain. To determine this, we quantified changes in gonadotropin hormone (GTH) [GTHα, follicle stimulating hormone (FSH) and luteinizing hormone (LH)] and estrogen receptor (ER; ERα and ERβ) mRNA expression levels associated with goldfish reproduction as well as changes in plasma FSH, LH, and 17β-estradiol (E₂) activities after injection of recombinant VAL-opsin protein in two concentrations (0.1 or 0.5 μg/g body mass) for 4 weeks (injection once weekly) and examined the possible impact of green-wavelength light (500, 520, and 540 nm) on the function of VAL-opsin. As a result, all parameters associated with reproduction significantly increased with time and light-emitting diode (LED) exposure. Based on these results, we suggested that VAL-opsin in the deep brain is involved in goldfish maturation, and it is possible that green-wavelength light improves the ability of VAL-opsin to promote maturation by increasing VAL-opsin expression.     

Distinct expression of three estrogen receptors in response to bisphenol A and nonylphenol in male Nile tilapias (Oreochromis niloticus)

Environmental estrogens, such as bisphenol A (BisA) and nonylphenol (NP), have been shown to affect the estrogen receptor (ER) expression and induce male reproductive abnormalities. To elucidate molecular mechanisms of action of xenoestrogenic chemicals on the expression of estrogen receptors in the testes of Nile tilapia (Oreochromis niloticus), three full-length cDNAs respectively encoding ntERα, ntERβ1 and ntERβ2 were cloned from testes. The amino acid sequences of ntERα, ntERβ1 and ntERβ2 showed a high degree of similarity to the relevant fish species. Tissue-specific expression study showed that three receptors were highly expressed in pituitary, liver, testis, kidney and intestine tissues. The ntERα, ntERβ1 and ntERβ2 mRNA expressions were significantly higher at the sexual early recrudescing stage than at other recrudesced stages. After being exposed to xenoestrogens from weeks 2 to 4, the ntERα mRNA levels were increased significantly in testes after NP treatment at all sampling times or after 4 weeks of exposure to BPA. The ntERβ1 mRNA levels remained unchanged, while a significant decrease of the ntERβ2 mRNA level was observed in testes after exposure to NP and BPA. The present study demonstrates that the regulation of all three ntER subtypes in testes may act via different molecular mechanisms of exposure to NP and BPA.