Pengaruh Penambahan Serum dan atau DNase dalam Medium Disosiasi terhadap Jumlah dan Viabilitas Spermatogonia Ikan Gurame (Osphronemus gouramy Lac.)
The Influence of Serum and or DNase in Dissociation Medium on the Number and Viability of Giant Gouramy (Osphronemus gouramy Lac.)
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Date
2011Author
Syahrani, Nurida Dessalma
Djuwita, Ita
Andriani, Irma
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Dissociation is the first step to obtain a sufficient number of spermatogonia as donor cells for transplantation. Dissociation techniques is perfomed by mechanical and enzymatic methods to accelerate the separation of cells and tissue. This study was aimed to determine the effect of serum and or DNase in the dissociation medium on the number and viability of spermatogonia. Testes were taken from giant gouramy (500-800 gram body of weight) with gonad weight 20 mg. Dissociation was performed using four different media that were PBS containing: (1) 0.5% trypsin, (2) 0.5% trypsin+5% serum, (3) 0.5% trypsin+10 IU/μL DNase, and (4) 0.5% trypsin+5% serum+10 IU/μL DNase, with total volume of 500 μL. The number of spermatogonia was calculated every hour during five hours dissociation using hemocytometer, and the percentage of viability were calculated based on Trypan Blue stainning method. Quantitive datas were analyzed using General Linier Model analysis (GLM) and continued by Duncan’s test if there was a significant difference. The addition of serum in the dissociation medium did not significantly affect on the number of spermatogonia, but significantly increased the viability percentage of spermatogonia. The addition of DNase or combination of serum and DNase could produce the highest number and percentage viability of spermatogonia, the number of viable spermatogonia significantly increased after three hours of DNase addition and two hours for the combination of serum and DNase. Disosiasi adalah langkah awal yang diperlukan untuk mendapatkan jumlah spermatogonia yang cukup sebagai sel donor untuk transplantasi. Teknik disosiasi dilakukan dengan metode mekanik dan enzimatik untuk mempercepat pemisahan sel dan jaringan. Penelitian ini bertujuan untuk mengetahui pengaruh penambahan serum dan atau DNase pada medium disosiasi terhadap jumlah dan viabilitas spermatogonia. Testes diambil dari ikan gurame yang berukuran 500 sampai 800 gram/ekor dengan bobot gonad 20 mg. Disosiasi dilakukan menggunakan empat medium yang berbeda yaitu menggunakan PBS yang mengandung: (1) tripsin 0.5% (2) tripsin 0.5% + serum 5%, (3) tripsin 0.5% + DNase 10 IU/μL, dan (4) tripsin 0.5% + serum 5% + DNase 10 IU/μL, dengan volume total 500 μl. Jumlah spermatogonia dihitung setiap jam selama lima jam disosiasi dengan menggunakan hemositometer, dan viabilitas sel diidentifikasi menggunakan pewarna Trypan Blue (TB). Data kuantitatif yang diperoleh dianalisis menggunakan metode analisis General Linier Model (GLM) dan uji lanjut Duncan’s jika terdapat perbedaan nyata. Penambahan serum dalam medium disosiasi tidak menunjukkan pengaruh yang nyata terhadap jumlah rata-rata spermatogonia, namun meningkatkan secara nyata persentase viabilitas spermatogonia. Penambahan DNase maupun kombinasi serum dan DNase mampu menghasilkan rata-rata jumlah spermatogonia dan persentase viabilitas spermatogonia paling tinggi selama disosiasi, dengan jumlah spermatogonia hidup yang meningkat secara nyata setelah disosiasi selama tiga jam untuk penambahan DNase dan dua jam untuk kombinasi serum dan DNase