Expression of endogenous and exogenous growth hormone (GH) messenger (m) RNA in a GH-transgenic tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

We have previously produced transgenic fish from crosses between a wild-type female tilapia (Oreochromis niloticus) and a G1 transgenic male. This line of growth-enhanced tilapia carries a single copy of a chinook salmon (s) growth hormone (GH) gene spliced to an ocean pout antifreeze promoter (OPAFPcsGH) co-ligated to a carp -actin/lacZ reporter gene construct, integrated into the tilapia genome. Because little is known about the expression sites of transgenes, we have characterised the gene expression patterns of sGH and tilapia (t)GH in transgenic tilapia using a newly established real-time PCR to measure the absolute mRNA amounts of both hormones. The sGH gene, which was expected to be expressed mainly in liver, was also found to be expressed in other organs, such as gills, heart, brain, skeletal muscle, kidney, spleen, intestine and testes. However, in pituitary no sGH mRNA but only tGH mRNA was found. Tilapia GH mRNA in wild-type pituitary amounted to 226 ± 30 pg/g total RNA but in transgenics only to 187 ± 43 pg/g total RNA. Liver exhibited the highest level of sGH mRNA (8.3 ± 2.5 pg/g total RNA) but the extrahepatic sites expressed considerable amounts of sGH mRNA ranging from 4.1 ± 2.0 pg/g total RNA in gills to 0.2 ± 0.08 pg/g total RNA in kidney. The widespread expression of the sGH gene is assumed to be due to the tissue specificity of the type III AFP gene promoter. It is assumed that our transgenic experiments, which in contrast to some other approaches caused no obvious organ abnormalities, mimick the GH expression during ontogeny. Because sGH mRNA is expressed both in liver and in extrahepatic sites it may not only promote secretion and release of liver-derived (endocrine) IGF-I leading to an overall growth enhancement but also stimulate IGF-I expression within the different organs in a paracrine/autocrine manner and, thus, further promote organ growth.     

Interactions between Nile tilapia (Oreochromis niloticus) and cladocerans in ponds (Khartoum,Sudan)

Juvenile Nile tilapia (Oreochromis niloticus) are omnivorous, and the question asked in this study is how they affect on their environment? Do they mainly act as predators on the cladoceran zooplankton or do they compete with the cladocerans for phytoplankton? This problem was studied in three ponds with and three ponds without small tilapia (3–5 cm). The fish growth rate, the succession of plankton species and the changes in abiotic conditions, were monitored over a period of 67 days. The fish biomass was kept low and the mean was approximately constant (12.6 g m^?2) during the experiment. Phosphate was added to avoid phytoplankton nutrient limitation. Although the diet of Nile tilapia contained both phytoplankton and zooplankton, the fish affected the ecosystem in a similar way as zooplanktivorous fish. The fish ponds got more phytoplankton due to increase of Chlorophyta. Effects on the other phytoplankton groups Euglenophyta, Bacillariophyta, Cryptophyta and Cyanophyta could not be registered. The ponds without fish had higher densities of Daphnia lumholtzi and D. barbata. The other Cladocerans seemed less influenced by fish presence. The relative fish growth rate was most positively correlated with the density of Daphnia lumholtzi, Diaphanosmoa excisum and Bosmina longirostris. Tilapia seemes to have two feeding modes: (1) preying on large zooplankton and (2) unselective filtration of small planktonic organisms such as phytoplankton. In our experiment the first feeding mode affected the ecosystem more than the second.     

Effect of melatonin on GnIH precursor gene expression in Nile tilapia,Oreochromis niloticus

Sexual maturation and gonadal development of fish is greatly influenced by photic information, an external environmental factor, and melatonin mediates this information to regulate gonadotropin (GTH) secretion and gonadal activation. The relationship between gonadotropin inhibiting hormone (GnIH) and melatonin in fish, however, has not been studied to date. Here, the GnIH expression pattern and daily change of melatonin levels were compared to each other in mature tilapia (body length 16.1 ± 0.2 cm, body weight 77.7 ± 3.43 g), and the effect of melatonin injection on GnIH gene expression was investigated. GnIH gene expression increased at night when the secretion of melatonin increased, whereas gene expression decreased during the day when melatonin secretion decreased. Injecting tilapia intraperitoneally with melatonin increased GnIH gene expression and decreased the expression of gonadotropin releasing hormone (GnRH) and GTH. Furthermore, the injection decreased the 11-KT concentration in male tilapia. These results indicate that melatonin is likely to suppress the hypothalamus-pituitary-gonad (HPG) axis via the action of GnIH in this species.     

Expression of a Novel Piscine Growth Hormone Gene Results in Growth Enhancement in Transgenic Tilapia (Oreochromis Niloticus)

Several lines of transgenic G1 and G2 tilapia fish (Oreochromis niloticus) have been produced following egg injection with gene constructs carrying growth hormone coding sequences of fish origin. Using a construct in which an ocean pout antifreeze promoter drives a chinook salmon growth hormone gene, dramatic growth enhancement has been demonstrated, in which the mean weight of the 7 month old G2 transgenic fish is more than three fold that of their non transgenic siblings. Somewhat surprisingly G1 fish transgenic for a construct consisting of a sockeye salmon metallothionein promoter spliced to a sockeye salmon growth hormone gene exhibited no growth enhancement, although salmon transgenic for this construct do show greatly enhanced growth. The growth enhanced transgenic lines were also strongly positive in a radio-immuno assay for the specific hormone in their serum, whereas the non growth enhanced lines were negative. Attempts to induce expression from the metallo thionein promoter by exposing fish to increased levels of zinc were also unsuccessful.Homozygous transgenic fish have been produced from the ocean pout antifreeze/chinook salmon GH construct and preliminary trials suggest that their growth performance is similar to that of the hemizygous transgenics. No abnormalities were apparent in the growth enhanced fish, although minor changes to skull shape and reduced fertility were noted in some fish. There is also preliminary evidence for improved food conversion ratios when growth enhanced transgenic tilapia are compared to their non-transgenic siblings.The long term objective of this study is to produce lines of tilapia which are both growth enhanced and sterile, so offering improved strains of this important food fish for aquaculture.     

尼罗罗非鱼生长激素及其受体的cDNA克隆与mRNA表达的雌雄差异

尼罗罗非鱼(Oreochromis niloticus)雌雄鱼生长差异明显,为了探讨其原因,本文采用RT-PCR方法克隆了尼罗罗非鱼生长激素(Growthhormone,GH)及其受体(Growth hormone receptor,GHR)的cDNA序列,并应用半定量RT-PCR方法比较了雌、雄尼罗罗非鱼垂体GHmRNA、肝脏GHRmRNA、肌肉GHRmRNA的表达差异。序列分析表明:GH开放阅读框为615bp,共编码204个氨基酸;GHR开放阅读框为1908bp,共编码635个氨基酸。以RT-PCR方法研究了GH、GHR在各组织的分布情况,结果表明:GH仅在垂体中检测到有表达,而GHR在所检测的18种组织中均有表达,其中以肝脏、肌肉、性腺、下丘脑、胸腺表达量较高。以半定量RT-PCR方法进一步比较了雌、雄尼罗罗非鱼垂体GHmRNA、肝脏GHRmRNA、肌肉GHRmRNA的表达量,结果表明:雄鱼垂体GHmRNA和肝脏GHRmRNA的表达量均显著高于雌鱼,肌肉GHRmRNA的表达量则无显著差异,推测垂体GHmRNA和肝脏GHRmRNA表达的雌雄差异是尼罗罗非鱼雌雄生长差异的主要原因之一。     

Growth and energy budget of F2'all-fish' growth hormone gene transgenic common carp

The growth and energy budget for F₂‘all‐fish’ growth hormone gene transgenic common carp Cyprinus carpio of two body sizes were investigated at 29·2° C for 21 days. Specific growth rate, feed intake, feed efficiency, digestibility coefficients of dry matter and protein, gross energy intake (I_E), and the proportion of I_E utilized for heat production (H_E) were significantly higher in the transgenics than in the controls. The proportion of I_E directed to waste products [faecal energy (F_E) and excretory energy loss (Z_E+U_E) where Z_E is through the gills and U_E through the kidney], and the proportion of metabolizable energy (M_E) for recovered energy (R_E) were significantly lower in the transgenics than in the controls. The average energy budget equation of transgenic fish was as follows: 100 I_E= 19·3 F_E+ 6·0 (Z_E+U_E) + 45·2 H_E+ 29·5 R_E or 100 M_E= 60·5 H_E+ 39·5 R_E. The average energy budget equation of the controls was: 100 I_E= 25·2 F_E+ 7·4 (Z_E+U_E) + 35·5 H_E+ 31·9 R_E or 100 M_E= 52·7 H_E+ 47·3 R_E. These findings indicate that the high growth rate of ‘all‐fish’ transgenic common carp relative to their non‐transgenic counterparts was due to their increased feed intake, reduced lose of waste productions and improved feed efficiency. The benefit of the increased energy intake by transgenic fish, however, was diminished by their increased metabolism.     

Growth efficiency in transgenic tilapia (Oreochromis sp.) carrying a single copy of an homologous cDNA growth hormone

Growth hormone (GH) has been shown to have a profound impact on fish physiology and metabolism. However, detailed studies in transgenic fish have not been conducted. We have characterized the food conversion efficiency, protein profile, and biochemical correlates of growth rate in transgenic tilapia expressing the tilapia GH cDNA under the control of human cytomegalovirus regulatory sequences. Transgenic tilapia exhibited about 3.6-fold less food consumption than nontransgenic controls (P < 0.001). The food conversion efficiency was significantly (P < 0.05) higher (290%) in transgenic tilapia (2.3 +/- 0.4) than in the control group (0.8 +/- 0.2). Efficiency of growth, synthesis retention, anabolic stimulation, and average protein synthesis were higher in transgenic than in nontransgenic tilapia. Distinctive metabolic differences were found in transgenic juvenile tilapia. We had found differences in hepatic glucose, and in agreement with previous results we observed differences in the level of enzymatic activities in target organs. We conclude that GH-transgenic juvenile tilapia show altered physiological and metabolic conditions and are biologically more efficient.     

Effect of water temperature,feeding frequency,and protein percent in the diet on water quality,growth and behavior of Nile tilapia Oreochromis niloticus (Linnaeus, 1758)

This study evaluated how water temperature (26, 28, and 30°C), number of meals per day (one or two meals), and protein percent in diet (20, 25 and 30%) impact growth performance, biometric indices, and feeding behavior of Nile tilapia, Oreochromis niloticus. Fish were randomly allocated into 18 equal replicate groups. Higher final body weight was observed in fish reared at 30°C and fed one meal per day containing 30% crude protein. Better weight gain, weight gain %, feed conversion ratio, specific growth rate, and condition factor were recorded in fish reared at 26°C and fed one meal per day containing 30% protein. The best length weight relationship was obtained in fish reared at 26°C and fed one meal per day containing 30% crude protein. Shorter feeding duration and duration of appetite inhibition latency were recorded in fish reared at 30°C, fed one meal per day, and given a diet containing 30% protein. The highest proactivity was recorded in fish reared at 30°C, received one meal per day, and with 25% crude protein in their diet. Conclusively, rearing Nile tilapia at 26–30°C with a lower feeding frequency (one meal/day) and a 30% crude protein diet achieved better performance and feeding behavior.     

First data on rabbit dung as a water fertilizer in tropical fish culture and its effect on the growth of Oreochromis niloticus (Teleostei, Cichlidae)

The influence of rabbit dung on water quality, plankton occurrence and Nile tilapia growth (Oreochromis niloticus) was studied. pH and total alkalinity increased, dissolved oxygen concentration decreased but not below the critical level whilst the nitrogen parameters were not affected in a significant way. An important increase of plankton was recorded and was reflected in a decrease of transparency: the phytoplankton density was 2.7 times that of the controls. Tilapia growth rate in the manure treatments was 5.1 times that of the control group; no significant difference in survival rates was observed.     

Identification of a sex-determining region in Nile tilapia (Oreochromis niloticus) using bulked segregant analysis

Sex determination in the Nile tilapia (Oreochromis niloticus) is thought to be an XX-XY (male heterogametic) system controlled by a major gene. We searched for DNA markers linked to this major locus using bulked segregant analysis. Ten microsatellite markers belonging to linkage group 8 were found to be linked to phenotypic sex. The putative Y-chromosome alleles correctly predict the sex of 95% of male and female individuals in two families. Our results suggest a major sex-determining locus within a few centimorgans of markers UNH995 and UNH104. A third family from the same population showed no evidence for linkage of this region with phenotypic sex, indicating that additional genetic and/or environmental factors regulate sex determination in some families. These markers have immediate utility for studying the strength of different Y chromosome alleles, and for identifying broodstock carrying one or more copies of the Y haplotype.     

转大麻哈鱼生长激素基因鲤生态安全性检测与分析

评价了转大麻哈鱼（Oncorhynchus Suckley）生长激素基因鲤生态安全性问题及研究转基因鱼对天然野生鲤群体遗传污染程度。通过RAPD和SSLP方法,用265个RAPD标记和35对鲤的微卫星标记对受杂交鲤污染的哈尔滨江段黑龙江鲤群体、未受污染的抚远江段黑龙江鲤群体及模拟转基因鲤占普通鲤群体的1%和10%比例获得繁殖子代等实验群体的DNA样本进行全基因组扫描统计分析得出结论,即转基因鲤占普通鲤群体1%时对普通群体的基因污染程度是微乎其微的,远远低于杂交鲤对野生群体基因污染,转基因鲤占普通鲤群体10%时对普通鲤遗传背景的影响稍有升高,但仍然远远低于杂交鲤对野生群体基因污染程度。总之,在现有的检测技术条件及有效的监控条件下,与杂交鲤相比转基因鲤对鲤野生群体的遗传背景的影响是微弱的,而外来种和杂交种则对生态环境有严重威胁。     

Evaluation of an antisense RNA transgene for inhibiting growth hormone gene expression in transgenic rats

We compared the levels of growth hormone (GH) mRNA in the pituitary, plasma GH concentration, and altered phenotype in rats heterozygous and homozygous for an antisense RNA transgene targeted to the rat GH gene, with those in nontransgenic rats. We initially investigated whether the transgene promoter, which is connected to four copies of a thyroid hormone response element (TRE) that increases promoter activity, affected in vivo transgene expression in the pituitary of the transgenic rats. Plasma GH concentration correlated negatively with T, injection in surgically thyroidectomized heterozygous transgenic rats. There was a reduction of about ?35–40% in GH mRNA levels in the pituitary of homozygous animals compared with those in non-transgenic rats. Plasma GH concentration was significantly ?25–32 and ?29–41% lower in heterozygous and homozygous transgenic rats, respectively, compared with that in nontransgenic animals. Furthermore, the growth rates in homozygous transgenic rats were reduced by ?72–81 and ?51–70% compared with those of their heterozygous and nontransgenic littermates, respectively. The results of these studies suggested that the biological effect of GH in vivo is modulated dose-dependently by the antisense RNA transgene. The rat GH gene can therefore be targeted by antisense RNA produced from a transgene, as reflected in the protein and RNA levels. © 1995 Wiley-Liss, Inc.     

Effect of bisphenol A toxicity on growth performance,biochemical variables,and oxidative stress biomarkers of Nile tilapia,Oreochromis niloticus (L.)

Bisphenol A (BPA) is one of the industrial chemical compound which is used in the production of polycarbonate plastics and epoxy resins. BPA is used throughout the world and it could enter the aquatic ecosystems causing serious problems. To evaluate the potential effects of BPA toxicity on Nile tilapia, Oreochromis niloticus (L.) performance, its lethal concentration (LC₅₀) was determined and it was 13.13 µg/L. After that, fish (33.9 ± 0.55 g/fish) were exposed to 0.0, 1.64, or 3.28 µg/L of BPA for 6 weeks after which growth performance, biochemical variables, and oxidative defense system were assessed. The results showed that fish growth and feed intake were significantly reduced as BPA levels increased with no significant difference in fish survival. Total protein, albumin, globulin, and acetylcholine esterase decreased significantly; meanwhile, aspartate transferase, alanine transferase, alkaline phosphatase, uric acid, and creatinine increased significantly with exposure to BPA in a dose dependent manner. Furthermore, malondialdehyde value and the activities of superoxide dismutase and catalase increased significantly; while glutathione peroxidase and glutathione S‐transferase decreased significantly as BPA levels increased. In conclusion, BPA exposure in aquatic environment deteriorated fish performance and health causing liver and kidney dysfunction. Thus, fish exerted oxidative defense enzymes as a protection tool against BPA toxicity.     

一种适于家蚕细胞激素研究的双荧光素酶报告基因系统的内参质粒的构建

双荧光素酶报告基因系统能够提供灵敏的读数,但该系统需要依赖组成型表达的内参对读数进行归一化。然而,大多数内参并不是在所有条件下都组成型表达。为此,文中建立了一个有效的方法制备适于家蚕细胞双荧光素酶报告基因系统的内参质粒。首先,突变BmV gP78启动子上的激素应答相关元件,获得了在家蚕细胞中稳定表达的组成型启动子BmV gP78M;然后,用BmV gP78M替换pRL-SV40质粒上的SV40启动子和嵌合内含子序列,成功构建了pRL-V gP78M内参质粒;最后,通过细胞转染实验证实pRL-V gP78M内参在家蚕细胞系中稳定表达,并且pRL-V gP78M内参的表达活性不受蜕皮激素、保幼激素及激素相关转录因子的影响。最终,获得了在家蚕细胞中稳定表达且表达量适中的内参质粒pRL-V gP78M。该内参可以有效地作为双荧光素酶报告基因系统的内参质粒用于家蚕细胞系中激素的研究。同时,该内参质粒的构建方法也为构建适于其他物种细胞系的双荧光素酶报告基因系统的内参质粒提供了参考。     

Effect of feeding pineapple waste on growth performance,texture quality and flesh colour of nile tilapia (Oreochromis niloticus) fingerlings

The study aims to evaluate the effects of pineapples waste on the growth, texture quality and flesh colour of Nile tilapia (Oreochromis niloticus) fingerlings. Fingerlings were fed with four different levels of pineapple waste diets throughout 56 days, which contain a control group (Diet 1) and experimental diets that formulated with 10% (Diet 2), 20% (Diet 3) and 30% (Diet 4) of pineapple waste. The experimental diet was formulated with rice bran, fish meal, soybean meal, vitamin and mineral premix, vegetable oil and binder to attain 32% dietary protein. The results revealed that the formulated fish diet with pineapple waste given the optimum weight gain, weight gain percentage, specific growth rate than the control group, where Diet 4 has shown the highest value (p < 0.05). There were no effects of the pineapple waste diet on the texture quality of the fillet, while only red chromaticity (a*) showed a significant difference (p < 0.05). In conclusion, the addition of pineapple waste can improve the growth rate of Nile tilapia, and the supplementation level of the pineapple waste in the diet was 30% of the total feed formulation.     

Germ line transmission and expression of alacZ containing transgene in tilapia (Oreochromis niloticus)

Transgenic tilapia were produced using a carp -actinlacZ gene construct and the transmission of the transgene to F₁ progeny was followed. Of 36 founder fish analysed, 9 (25%) were found to be transgenic in at least one tissue and two of these were found to transmit the transgene to progeny. One male produced only three out of 28 transgene DNA positive progeny (10.7%) but none of these were found to express the transgene. The other male produced 243 out of 1601 (15.2%)lacZ expressing embryos; 40 of these were analysed by Southern blotting and all were found to be positive for the presence of the transgene but revealed two different patterns of transgene arrangement. Three different expression patterns were also observed and presumed to be a result of multiple integration events and resulting position effects.     

Morphological and quantitative evaluation of the ovarian recrudescence in Nile tilapia (Oreochromis niloticus) after spawning in captivity

The Nile tilapia is one of the most important fish species for aquaculture worldwide. Understanding their reproductive biology is essential for improving their aquaculture methods. The morphological and quantitative dynamics of ovarian recrudescence of Oreochromis niloticus was studied for 21 days postspawning. To accomplish this, breeding females were kept in controlled conditions and ovarian samples were collected weekly for histological, ultrastructural and morphometric analyses. Ovarian follicle morphology revealed an intense synthesis activity of the follicular cells, which actively contributed to formation of the zona radiata and oocyte development following spawning. Recently spawned ovaries contained follicles at all developmental stages, but they were predominantly early primary growth (～42%) and full‐grown follicles (～20%). Remnants of spawning, postovulatory follicle complexes represented approximately 5% of the former ovarian follicles immediately after spawning, and less than 1% after 7 days. Atretic follicles accounted for approximately 2% of the follicles studied during the period. The stock of primary growth follicles was stable during ovarian recrudescence, indicating their availability for continuous recruitment. Only the frequency of full‐grown follicles significantly increased in the ovaries during recrudescence, representing approximately 35% of the follicles 21 days postspawning. The diameters of all follicles were significantly different between the periods analyzed. The ovaries' morphological characteristics, the maintenance of young follicles stocks and the gradual and significant increase in the proportion and diameter of full‐grown follicles showed a rapid ovarian recovery and follicular growth of O. niloticus, in 21 days at 29.5°C, necessary for the next spawning. J. Morphol. 275:348–356, 2014. © 2013 Wiley Periodicals, Inc.