RINGKASAN Tujuan dari penelitian ini adalah untuk mempelajari aktivasi dari oosit mamalia M-II arSEt hasil IVM untuk partenogenSEis dan stimulasi aktivasi oosit terfertilisasi (IVF) setelah dilakukan mikroinjeksi Ekstrak Spermatozoa (SE) dalam sitoplasma oosit. SE diisolasi dari ejakulat semen nanalia (kambing lokal). Fokus penelitian adalah mempelajari prosSE dan kejadian-kejadian selama aktivasi oosit (partenogenSEis) yaitu corticul granule exoxytosis (CGE), meiotic rSEumption, pronucleus formation (PN), cleavage dan perkembangan lanjut dari sel. Sementara untuk sel oosit terfertilisasi (IVF) mempelajari tingkat cleavage dan pertumbuhan sel embrio mamalia Metode yang digunakan adalah eksperimental laboratorium. Oosit mamalia mature (metafase II) diperoleh dengan melakukan in vitro maturation (IVM) oosit yang diisolasi dari anthral folikel ovaroum dari rumah potong hewan (RPH) kambing. IVM dilakukan dalam medium dasar TCM199 + 10 % FBS + 10 ul FSH + 25 ul LH + gentamycin dalam inkubator C02 5 % selama 24 jam, dan diseleksi M-II mature oosit iii aktivasi. berdasarkan ekstruksi polar bodi I. (PB-I), sampai digunakan untuk perlakuan Mikroinjeksi SEktrak Spermatozoa (SE) dilakukan dengan mikromanipulator-inverted mikroskop dengan mikropipet ukuran standart. Pada perkembangan injeksi SE dilakukan dengan membuat slit pada zona pellucida SE dimasukkan dalam medium dan selanjutnya oosit IVM dengan slit di paparkan dalam medium. Preparasi SEktrak Spermatozoa dilakukan dengan metode preparasi SE kuda dan babi yang telah dimodifikasi. SE yang dihasilkan disimpan pada temperatur – 80 oC sampai digunakan. Konsentrasi protein dalam SE ditentukan berdasarkan konsentrasi awal dalam ejakulat yaitu 10 x 108 spermatozoa/ml medium isolasi nukleus. Percobaan mikroinjeksi SE dilakukan pada aktivasi oosit partenogenSEis dan oosit terfertilisasi (IVF). Injeksi SE dilakukan langsung dalam sitoplasma oosit. Sebagai kontrol adalah induksi aktivasi menggunakan bahan kimia kombinasi Ca-ionophore A-23187 dan 6-DMAP. Variabel yang diamati adalah persentase oosit yang teraktivasi berdasarkan pada Cleavage rate untuk partenogenesis dan oosit IVM yang kemudian digunakan untuk IVF. Untuk konfirmasi adanya aktivasi dilakukan analisis intensitas calsium (Ca++) dengan menggunakan Fluo-3 menggunakan Confocal Laser Scanning Microscope. Hasil penelitian menunjukkan bahwa SE setelah dikarakterisasi menghasilkan 11 macam protein dengan berat molekul yang berbeda, termasuk di dalamnya diketemukan protein 100 kD yang diduga merupakan protein spSEifik berperanan untuk aktivasi oosit. Ada perbedaan antara perlakuan pada intensitas kalsium dan penyebarannya. Penambahan SE pada perlakuan 1. kontrol (M-II parthenogenetik oosit), SE 2.5 ug/ml , 3. 7 % ethanol + 2.5 ug/ml, 4. SE 100 kD dan IVM-IFV oosit. Penambahan SE pada medium aktivasi cenderung menghasilkan kenaikan cleavage rate dan hasilnya lebih tinggi jika dibandingkan dengan hanya penambahan SE spSEifik 100 kD. Hasil ini berlawanan dengan hasil yang diperkirakan sekaligus berarti bahwa SE lebih efektif jika dibandingkan penggunaan protein spSEifik 100 kD. KSEimpulan penelitian adalah bahwa suplementasi SE pada medium Metafase II (IVM) dapat meningkatkan terjadinya aktivasi sel berdasarkan pada adanya kenaikan intensitas calsium ++ dan tingkat cleavage sel. Dengan demikian maka SE mempunyai potensi sebagai kandidat untuk
dikembangkan lebih lanjut sebagai bahan suplemen atau bahan pengganti agen aktivasi kimia. Pada akhirnya diharapkan akan dapat dikembangan sistem kultur sel embrio baru dengan memanfaatkan SE dalam IVM-IVF untuk meningkatkan produksi embrio. Kata Kunci: Ekstrak Spermatozoa, Aktivasi, Calsium, Embrio
SUMMARY The aim of this rSEearch is to improving the culture system IVM-IVF for embryo production parthenogenically using sperm extract suplementation. Sperms extract (SE) was suplemented into chemical activation agent then transfected into M-II oocyte after performing slit of zona pellucida using micromanipulation equipment. The intracellular Ca+2 intensity and the spread of fluorSEcent intentisy (fluo-3) in the ooplasma was invSEtigated by analysis imaging using confocal laser scanning microscope (CLSM). This SE was characterised before and its indicate the 11 kinds of protein weight molecular included the 100 kD. Dilution of SE in chemical activation agent caused a different profile of calsium intensity after both line seriSE and histogram analysis. The rSEult showed that the intensity of Ca+2 is diffefrent among treatments: (1).control (IVM, M-II oocyte), (2). SE 2.5 ug/ml (3) 7 % Ethanol + SE 2.5 ug/ml and (4). SE of 100 kDa protein weight. After washing and cultured in vitro of thSEe oocyte during 24 to 48 hours, activation using SE of 2.5 ug/ml rSEulted in about 36.33 % of 251 oocyte was reached cleavage compare to activation using 100 kDa protein of SE of 2 % of 240 oocytSE. The SE is relatively better than spSEific protein of SE of 100 kDa for their role as activation effect to goat oocytSE. .
Key words: Activation , Sperm Extract, Calcium, Embryo
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