Abstract
Freezing process in ram spermatozoa caused damage of the plasma membrane and acrosome thatlead to the decrease of spermatozoa fertility. Research was conducted to evaluate acrosomal damageduring freezing process using lectin histochemical technique. Semen was collected twice a week usingartificial vagina from 1-2 years old Garut ram. Immediately after collection, characteristic of semenquality was evaluated then diluted with Niwa and Sasaki Freezing (NSF) medium. Semen was loadedinto 0.25 mL mini straws and equilibrated at 4oC for two hours. Straws were then frozen and stored inliquid nitrogen. Evaluation of sperm characteristic (motility, viability and plasma membrane integrity)and acrosomal integrity were done during the freezing process. Detection of acrosomal integrity was observedusing Fluorescens isothiocyanate (FITC) and Avidin-Biotin-Complex (ABC) staining methods. Data ofcharacteristic spermatozoa and acrosomal integrity were analyzed using ANOVA. Result of the experimentsshowed that the percentage of motility, viability and plasma membrane integrity of spermatozoa before freezing (83 ± 2.7%; 88.8 ± 2.6%; 88.2 ± 3.7%) were significantly decreased (P<0.05) after equilibration (71± 4.2%; 84.2 ± 5.0%; 76.2 ± 1.3%) and after thawing (40 ± 3.5%; 61.08 ± 3.3%; 51.2 ± 10.4%). The percentageof intact acrosomal spermatozoa using FITC and ABC methods during freezing process were 93.63 ±2.73%; 88.04 ± 3.2% and 81.73 ± 4.77% VS 94.54 ± 0.26%; 88.17 ± 0.38% and 79.38 ± 2.06%, respectively.In conclusion, the characteristic of spermatozoa were significantly decrease (P<0.05) during freezing process.Furthermore, the integrity of acrosome spermatozoa can be well analyzed during freezing process by usinglectin histochemical staining methods.
Highlights
Akrosom memegang peranan penting dalam proses fertilisasi
Freezing process in ram spermatozoa caused damage of the plasma membrane and acrosome that lead to the decrease of spermatozoa fertility
After collection, characteristic of semen quality was evaluated diluted with Niwa and Sasaki Freezing (NSF) medium
Summary
Koleksi Semen Semen segar dikoleksi dari domba garut jantan dewasa kelamin yang berumur antara 1-2 tahun dengan bobot badan berkisar antara 25-30 kg. Pakan hijauan dan konsentrat sebagai sumber pakan pokok bagi domba, sedangkan air minum diberikan secara ad libitum. Kriopreservasi Spermatozoa Semen segar yang sudah dievaluasi karakteristiknya diencerkan dengan medium Niwa dan Sasaki Freezing (NSF) untuk kemudian dibekukan (Kikuchi et al, 1999; Karja et al, 2006). Semen yang mempunyai karakteristik baik dengan persentase motilitas minimal 70% digunakan dalam proses pembekuan. Penambahan bahan pengencer dilakukan dengan metode two step freezing yaitu dimulai dengan penambahan medium NSF I kemudian dilakukan ekuilibrasi pada suhu 4°C selama dua jam sebelum ditambahkan dengan medium NSF II dan diekuilibrasi kembali pada suhu yang sama selama lima menit. Semen dikemas ke dalam straw berukuran 0,25 mL (I.V.M., France) kemudian diletakan pada styrofoam plate dalam uap nitrogen cair berjarak sekitar 4 cm dari permukaan nitrogen cair selama 20 menit kemudian segera dimasukan dalam kontainer nitrogen cair dengan suhu -196oC untuk penyimpanan
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