計畫目標: 本計畫想經由量化早期母系補充物質( 如p53及IGF-IR )和分子標定物( molecular marker )的建立, 以幫助早期魚卵品質快速的鑑定.初步探討淡水魚吳郭魚n-3不飽和脂肪酸( n-3polyunsaturated fatty acid, n-3PUFA )對卵質的影響及對p53和IGF-IR的關係, 以建立分子指標模式, 進而推廣到其他養殖魚種.架構( 重要工作項目 ): 本計劃主要初步探討對n-3不飽和脂肪酸( n-3PUFA )缺乏對卵品質所造成的影響, 並從受精率、孵化率、胚胎發育過程來逐步探討對卵品質的鑑定, 從而建立一些外觀的觀測的變化及一些內在的分子指標( molecular marker ).( 1 )吳郭魚n-3PUFA缺乏時, 卵品質的影響: 我們打算利用3-6個月大的吳郭魚, 投餵同脂肪量( isolipidic )但不同脂肪成分的飼料.在飼養初期區別雄、雌分開餵食一段時間, 將實驗分成三組.第一組是對照組雄、雌均餵食正常飼料.第二組是實驗組雄魚餵食正常飼料、雌魚餵食缺乏n-3PUFA的飼料.第三組是實驗組雄、雌魚同時餵食缺乏n-3PUFA的飼料.在餵食一段時間之後再進行交配實驗, 計算其每次產卵數、授精率、孵化率等.( 2 )卵質鑑定分子指標之建立: 在脊椎動物中, 早期母系補充物質的充分與否, 會影響整個胚胎發育過程是否完整.因此IGF-IR及p53均可視為早期卵品質鑑定的分子指標.我們在本計畫中打算利用前一項`吳郭魚n-3PUFA缺乏時, 卵品質的影響'所產生的卵, 利用非放射性核醣核酸&
Abstract:In oviparous fish, maternal supply factors, such as p53suppressor protein and insulin-like growth factor-1receptor, play pivotal roles for embryonic developmental stages.We like to set up methods to quantitate the maternal supply factors ( p53and IGF-IR )and establish molecular marker model for egg quality control in oviparous fish.In this proposal, we like to use the tilapia species as a model to evaluate the role of n-3polyunsaturated fatty acid ( PUFA )on the egg quality and its relationship to the p53and IGF-IR mRNA expression.Experimental approach: The egg quality is determined by maternal supply factors.In this preliminary approach, the functional role of n-3polyunsaturated fatty acid ( PUFA )on the fertilization rate, embryo hatchability will be tested and its association with maternal supply molecular markers, such as p53and IGF-IR, will be evaluated.( 1 ): The influence of n-3PUFA deficiency in tilapia egg quality.In this preliminary approach, we plan to use 3to 6month old of both sex tilapia and all fish will receive separate feeding until mating.The feeding group will simply divided into three groups: ( 1 )the control group of both male and female tilapia feed with normal diet.( 2 )The test group 1of male will receive normal diet but female with isolipidic diet with n-3PUFA deficiency.( 3 )Both male and female will feed with isolipidic diet with n-3PUFA deficiency.After certain feeding period, the mating is carried by putting male and female together, the effect of n-3( PUFA )on the egg quality will be evaluated by the egg phenotypic changes, number of spawning egg, fertilization rate, and embryo hatchability.( 2 ).establishment of molecular maker on egg quality.The types and quantities of maternal supply factors will influence the early embryo development.In vertebrate, the insulin-like growth factor-1receptor ( IGF-IR )and p53protein have been recognized to play important roles on oocyte maturation and final embryo development.We like to establish p53and IGF-IR as egg quality indicator molecules.In this proposal, we plan to use eggs generated from ”Step 1”by using ribonuclease protection assay ( RPA )or semi-competitive PCR to quantitate the p53and IGF-IR mRNA expression and use these molecular marker to compare results from the ”Step 1”.The established molecular marker will be valuable tool to immediately test the quality of fish eggs and to prevent the time wasting during the hatching process.Expected results: The fish egg quality is mainly influenced by maternal supply factors, we can compare the molecular marker system to the effect of control and tested n-3PUFA deficiency group on the egg quality by evaluating egg phenotypic structure, number of egg, fertilization rate, and embryo hatchability.This system allow us to test the fish egg quality by a scientific evaluation system and further understand the egg quality related to maternal supply factors and the diets composition on brood stocks.