Abstract
The aim of study was to find out the best concentration and exposure time of calcimycin andionomycin in order to produce parthenogenetic embryos. Female Swiss Webster mice were fisrtlyprimed with Pregnant Mare’s Serum Gonadotropin (PMSG) and Human Chorionic Gonadotropin (hCG)with an interval of 48 hours. Sixteen hours after injection of hCG oocyte was collected by Dulbecco’sPhosphate Buffer Saline (dPBS) as a flushing medium. To separate the eggs from cumulus cells wereused hyaluronidase enzyme. The good quality oocytes were incubated in activation medium that isionomycin or calcimycin with a concentration of 3, 6, or 9 iM and exposure time 1, 4, or 7 minutes. Toyield diploid embryos were used 5 ?g/ml cytochlasin B for four hours at 37°C, 5% CO2. Activatedoocytes characterized by the formation of pronuclei washed three times in Potassium SimplexOptimization Medium (KSOM) and subsequently cultured in the same medium until blastocyst stage.The results showed that oocytes activated at calcimycin, the best results was presented at concentration6 ?M and exposure time four minutes, i.e. activation rate reached 96%, cleavage rate 82% and blastocystrate 28%. On the other hand, oocytes activated in ionomycin, the best results was presented atconcentration 3 ?M and exposure time four minutes, i.e. activation rate reached 82%, cleavage rate64% and blastocyst rate of 4%. It was concluded that the best concentration and exposure timecalcimycin on mice oocytes were 6 ?M for four minutes, whereas ionomycin were 3 ?M for four minutes.
Highlights
The aim of study was to find out the best concentration and exposure time of calcimycin and ionomycin in order to produce parthenogenetic embryos
The good quality oocytes were incubated in activation medium that is ionomycin or calcimycin with a concentration of 3, 6, or 9 ìM and exposure time 1, 4, or 7 minutes
The results showed that oocytes activated at calcimycin, the best results was presented at concentration 6 μM and exposure time four minutes, i.e. activation rate reached 96%, cleavage rate 82% and blastocyst rate 28%
Summary
Superovulasi dan Koleksi Sel Telur Superovulasi dilakukan dengan menyuntik mencit Swiss Webster betina yang berumur 812 minggu dengan pregnant mare’s serum gonadotropin (PMSG; Intervet) 5 IU dan human chorionic gonadotropin (hCG; Intervet) 5 IU pada selang waktu penyuntikkan 48 jam. Kompleks kumulus-sel telur (Gambar 1A) diaspirasi dengan menggunakan pipet Pasteur, dan ditransfer ke dalam medium yang sama yang disuplementasi dengan hyaluronidase (Sigma) 0,03%, di bawah satu menit. Sel telur dicuci tiga kali dalam medium yang sama dan dilakukan seleksi untuk memisahkan sel telur yang layak dan tidak layak. Aktivasi Partenogenesis Sebanyak 900 sel telur yang sudah matang (memiliki polar body I) dan berkualitas baik diacak secara lengkap untuk ditempatkan dalam larutan dPBS berbentuk drop 100 μL yang disuplementasi dengan calcimycin atau ionomycin dengan konsentrasi 3, 6, 9 μM dan lama pemaparan 1, 4, atau 7 menit pada suhu ruang. Kultur Embrio Sel telur yang teraktivasi dan memiliki dua pronukleus (Gambar 2A) dicuci tiga kali dalam drop 20 μL Potassium Simplex Optimation Medium (KSOM) yang disuplementasi dengan fetal bovine serum (FBS) 10%, dan selanjutnya dikultur dalam medium yang sama hingga stadium blastosis. Analisis menggunakan software SPSS 19.0 for windows dan MS office Excell 2007
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