Kesimpulan Umum
Berdasarkan penelitian yang telah dilakukan dapat disimpulkan bahwa kombinasi medium TCM-199 sebagai medium maturasi dan CR1aa sebagai medium untuk fertilisasi dan kultur embrio in vitro merupakan kombinasi medium yang dapat digunakan dalam proses produksi embrio domba in vitro secara optimal. Status reproduksi ovarium mempengaruhi jumlah oosit dengan kualitas baik yang terkoleksi dan tingkat pematangan inti oosit domba in vitro. Tingkat fertilisasi dan pembelahan embrio yang berasal dari oosit kualitas baik yang dimatangkan in vitro tidak dipengaruhi oleh status reproduksi dari pasangan ovarium yang digunakan.
Saran
Penelitian dapat dilanjutkan dengan melakukan pembekuan embrio untuk tujuan stok bank genetik dan transfer embrio untuk menguji kualitas embrio yang telah dihasilkan secara in vitro. Disamping itu perlu dilakukan kajian lebih lanjut mengenai hubungan status reproduksi ovarium dengan level hormonal.
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Lampiran 1. Komposisi medium koleksi oosit (modified Phosphate Buffered Saline, m-PBS)
Komponen Jumlah
d-PBS (Gibco, Grand Island, NY) 0.96 g Penisilin-streptomycin (100 IU/ml) 1000 µl
Fetal Bovine Serum (5 %) 5 ml
Lampiran 2. Komposisi medium TCM-199 untuk pematangan oosit in vitro 1. Larutan stok: 100 ml Komponen Jumlah TCM-199 0.95 g NaHCO3 0.2201 g Penicilin-streptomycin (100 IU/ml) 1000 µl 2. Medium pematangan oosit in vitro: 10 ml
Komponen Jumlah
Larutan stok TCM-199 9.5 ml
Fetal Bovine Serum (10 %) 1 ml
FSH (0.01 µg/ml) 10 µl
LH (10 µg/ml) 100 µl
E2 (1 µg/ml) 10 µl
Stok FSH (Antrin®, Denka Pharmaceutical Co., Kawasaki, Japan) Dosis: 0.01 IU/ml ∞ 0.01 mg/ml
Untuk membuat stok, maka 10 IU FSH dilarutkan dengan 1 ml solvent
(pelarut)
Stok LH (Chorulon®, Intervet International B.V., Boxmer, Holland) Dosis: 10 IU/ml
Untuk membuat stok, maka 1500 IU Chorulon dilarutkan dalam 1.5 ml
solvent (pelarut) Stok Estradiol (E2) Dosis: 1 µg/ml
10 mg E2 dilarutkan dengan 10 ml Etanol (EtOH), sehingga untuk 1 ml medium memerlukan 1 µl stok E2.
Lampiran 3. Komposisi medium Charles Rosenkrans-1 (CR1) 1. Larutan stok (10x): 100 ml Komponen Jumlah NaCl 0.6703 g L (+) Lactic acid 0.0546 g KCl 0.0231 g NaHCO3 0.2201 g Sodium Pyruvat 0.0044 g L-Glutamine 0.0146 g Phenol red 1 mg Milli Q water 100 ml
2. Medium pematangan oosit in vitro: 10 ml
Komponen Jumlah
Larutan stok CR1aa 9.5 ml
Fetal Bovine Serum (10 %) 1 ml Penicilin-streptomycin (100 IU/ml) 100 µl MEM Non Esensial Amino Acid 100x 100 µl
MEM BME Amino Acid 50x 200 µl
FSH (0.01 µg/ml) 10 µl
LH (10 µg/ml) 100 µl
3. Medium kapasitasi (CR1aa-kafein): 20 ml
Komponen Jumlah
Larutan stok CR1aa (ml) 20 ml
MEM Non Esensial Amino Acid 100x 200 µl
MEM BME Amino Acid 50x 400 µl
Caffeine benzoate 0.0194 g
Penicillin-streptomycin (100 IU/ml) 200 µl 4. Medium fertilisasi (CR1aa-kafein-heparin): 10 ml
Komponen Jumlah
Larutan stok CR1aa (ml) 10 ml
MEM Non Esensial Amino Acid 100x 100 µl
MEM BME Amino Acid 50x 200 µl