Kriopreservasi embrio kambing peranakan etawah hasil produksi in vitro menggunakan metode vitrifikasi cryoloop

WAHYUNINGSIH, Ir. Diah Tri Widayati, MP., Ph.D

2009 | Tesis | S2 Ilmu Peternakan

Penelitian ini dilakukan untuk mengetahui metode kriopreservasi yang efektif dengan vitrifikasi cryoloop pada embrio kambing Peranakan Etawahhasil fertilisasi in vitro. Ovarium diambil dari Rumah Potong Hewan (RPH), dibawa ke laboratorium dalam medium NaCl fisiologis 0,9%, 31 – 34ºC. Oosit diaspirasi dari folikel yang berukuran 3,1 – 6 mm dengan siring 5 ml, jarum 23 G yang berisi Dulbecco’ Phosphate Bufferred Saline (D-PBS). Pencarian oosit dilakukan di bawah mikroskop stereo, oosit dimaturasi dengan medium G-IVF 50 μl dalam inkubator CO2 pada suhu 39oC, kadar CO2 5%, kelembaban 95% selama 24 jam. Oosit yang matur (masak) difertilisasi dengan semen beku kambing PE yang sebelumnya telah dicuci dengan 50 μl sperm rinse, kemudian disentrifus dengan kecepatan 1800 rpm (485 G) selama 15 menit, supernatan diambil dan diulangi sebanyak 3 kali. Endapan spermatozoa ditambahkan dengan medium ferlilisasi G-IVF 50 μl, kemudian dibuat tetes spermatozoa dan diinkubasi selama 2,5 jam untuk kapasitasi. Fertilisasi dilakukan dalam inkubator seperti pada proses maturasi selama 5 jam. Oosit yang telah difertilisasi dipindahkan ke dalam 50 μl tetes G–1 dan dikultur, perkembangan embrio diamati setiap 24 jam. Pembekuan embrio dilakukan dengan vitrifikasi cryoloop pada pembelahan 2 dan 4 sel. Embrio dicuci dalam medium DPBS sebanyak 3 kali kemudian masuk dalam tetes 20 μl solution I selama 2 menit, solution II 20 μl selama 30 detik. Cryoloop dimasukkan ke dalam solution II untuk membuat film yang tipis. Embrio dibiarkan pada film dan diuapkan di atas nitogen cair sampai membeku. Cryoloop disimpan dalam nitrogen cair. Embrio dihangatkan kembali dengan memasukkan cryoloop ke dalam tetes 20 μl solution out I selama 2 menit, 20 μl solution out II selama 3 menit, dan dalam tetes medium basal (G – MOB) selama 5 menit dan medium kultur G – 1 selama 24 jam dan dilakukan pengamatan. Beberapa kualitas embrio yang didapatkan sebelum vitrifikasi beragam dengan persentase kualitas A, B, dan C secara berurutan adalah 28,57; 57,14; dan 14,29%. Sedangkan kualitas embrio setelah vitrifikasi masing – masing grade B, C, dan D secara berurutan adalah 14,29; 28,57; dan 57,14%. Analisis data menggunakan Student-t test menunjukkan perbedaan yang nyata (P≤0,05) antara kualitas sebelum dan sesudah vitrifikasi. Berdasarkan hasil penelitian disimpulkan bahwa ditinjau dari kepraktisan pengerjaan, kebutuhan medium, dan waktu, metode vitrifikasi cryoloop dipandang cukup efektif.

A research was conducted to evaluate the cryopreservation of in vitro Etawah Cross Bred embryo production using cryoloop vitrification (CLV) method. Ovaries were collected from slaughterhouse as soon as the goat was slaughtered and transported to the laboratory in NaCl 0.9% at 31-34ºC. Oocyte was aspirated from follicles with diameter 3,1 – 6 mm using 5 ml disposable syringe with 23 G needle which had filled 1 ml Dulbecco’ Phosphate Bufferred Saline (D-PBS). Oocytes were searched using stereo microscope and washed three times using D-PBS. In vitro maturation was done by placing oocytes in to drop of 50 μl covered by mineral oil, then incubated under 39oC, 5% CO2, 95% humidity for 24 hours. Frozen semen were thawed in warm water (35ºC) then placed into centrifuge tube, added 0.5 ml sperm rinse, then centrifuged 1800 rpm (485 G) for 15 min, 3 times, supernatant was removed and added with 0.5 ml G-IVF, sperm concentration was 12.5 x 106/ml and 70% motility. Spermatozoa drop (50 μl) was prepared in disposable culture dish that covered with mineral oil and incubated under 39oC, 5% CO2, 95% humidity for 2.5 hours for capacitation. The in vitro mature oocytes were transffered into sperm drop then incubated under 39oC, 5% CO2, 95% humidity for 5 hours. The fertilized oocytes were washed third using D-PBS then cultured into 50 μl drop G – 1, then incubated on 39oC, 5% CO2, 95% humidity. Embryo development was monitored and examined every 24 hours. Two cell embryos were vitrified in solution I (7.5% DMSO, 7.5% EG in 10 ml PBS) for 2 min and then transferred onto a film of CLV solution II (15% DMSO, 15% EG, 10 mg/ml ficoll, and 0.65 M sucrose 2.22 gr in 10 ml PBS) for 30 sec suspended in the cryoloop. The cryoloop was plunged in to the liquid nitrogen. The embryos were thawed with incubation in solution out I (0.25 M sucrose in 10 ml PBS) for 2 min, in solution out II (0.125 M sucrose in 10 ml PBS) for 3 min, and in basal medium (G – MOB) for 5 min, and then cultured in G1 for 24 hr. The cleavage rate occured in 48 hr after culture, there were 2 and 4 stage. The grade percentage of embryos were 28.57; 57.14; and 14.29% respectively. Data analyzed using Student-t test showed there were differences (P≤0.05) between embryos before and after vitrification. It can be concluded that cryoloop vitrification was efective refer to practical processing, medium, and time.

Kata Kunci : Oosit kambing,Fertilisasi in vitro,Pembelahan,Vitrifikasi cryoloop,Thawing, Does oocytes, In vitro fertilization, Clevage, Cryoloop vitrification, Thawing, Embryo quality)