TINGKAT FERTILISASI DAN CLEAVAGE MENGGUNAKAN SPERMA SAPI HASIL PRESERVASI LEUCOCEPHALA TANNIN IN VITRO

EVA ARIYANTI, drh. Agung Budiyanto, MP., Ph.D ; drh. Dwi Liliek Kusindarta, MP., Ph.D

2013 | Tesis | S2 Sain Veteriner

Penelitian ini bertujuan untuk mengetahui kemampuan fertilisasi dan cleavage sperma hasil preservasi Leucocephala tannin (LT) secara in vitro, serta menemukan waktu inkubasi spermatozoa-oosit dan konsentrasi sperma yang optimal. Keberhasilan spermatozoa dalam memfertilisasi oosit sampai terjadinya cleavage menjadi faktor penting untuk terbentuknya blastosit dan implantasi. Sperma yang digunakan pada penelitian ini adalah sperma LT yang telah disimpan selama 7 hari. Sperma dicuci pada media Bracket and Oliphant's (BO) dan disentrifugasi 1500 rpm, 5 menit. Pengenceran sperma menggunakan media BO, untuk mendapatkan konsentrasi 1 X 106; 2,5 X 106 dan 5 X 106 sel/ml. Oosit yang telah dimaturasi selama 24 jam diinkubasi dengan sperma LT selama 3, 5 dan 7 jam pada suhu 38,5 °C, 5% CO2, kelembaban udara 95%. Setelah inkubasi oosit dicuci dan dipindahkan ke media kultur. Pengamatan tingkat fertilisasi berdasarkan terbentuknya polimorfonuklear (PMN) 10 jam setelah inseminasi. Perkembangan embrio diamati setelah 72 jam inkubasi. Pembentukan polimorfonuklear (PMN) diamati dengan pewarnaan aceto-orcein. Penelitian ini dilakukan dalam 3 tahap, penelitian I : uji waktu inkubasi spermatozoa-oosit 3, 5 dan 7 jam (5 X 106 sel/ml), penelitian II : uji konsentrasi sperma 1 X 106; 2,5 X 106 dan 5 X 106 sel/ml (5 jam), penelitian III : uji kemampuan fertilisasi spermatozoa LT dan kontrol sperma beku (5 X106 sel/ml dan 5 jam). Data hasil pengamatan penelitian I dan II dianalisis menggunakan analisis of varians (Anova) one way, sedangkan data hasil penelitian III menggunakan student t-test, terhadap terhadap tingkat fertilisasi dan cleavage. Hasil penelitian menunjukkan semakin panjang waktu inkubasi spermatozoa-oosit 3, 5 dan 7 jam, jumlah oosit yang terfertilisasi (2 PN) dan tingkat cleavage 8 sel semakin tinggi. Waktu inkubasi spermatozoa-oosit yang optimal adalah 5 jam. Semakin tinggi konsentrasi sperma, tingkat fertilisasi dan cleavage semakin tinggi. Konsentrasi sperma yang optimal adalah 5 X 106. Tidak ada perbedaan antara sperma LT dan sperma beku dalam tingkat fertilisasi dan cleavage. Sperma LT mampu memfertilisasi oosit secara in vitro, sehingga sperma LT dapat digunakan untuk fertilisasi.

The aim of this study were to determine the in vitro fertilization and cleavage rate ability of bull sperm preserved by Leucocephala tannin (LT), devine of the optimal ability of incubation time and sperm concentration. The optimal ability of spermatozoa in fertilizing the oocyte to the cleavage was important factors for blastocyst formation and implantation. Sperm used in this study was LT sperm that has been stored for 7 days. Sperm had washed in the Bracket and Oliphant's medium (BO) and centrifuged at 1500 rpm, 5 minutes. Sperm diluted using BO medium, to obtain the concentration of 1 X 106; 2.5 X 106 and 5 X 106 cells/ml. Oocytes had been matured for 24 hours were incubated with sperm LT for 3, 5 and 7 hours at 38.5 °C, 5% CO2, 95% on air humidity. After incubation, oocytes were washed and transferred to the culture medium. Observation of the fertilization rate based on polymorphonuclear (PMN) formation 10 hours post insemination. Embryonic development was observed after 72 hours incubation. PMN formation observed by aceto-orcein staining. These experimentals have done in 3 series. Experiment I: bovine oocyte matured were incubated with LT sperm for 3, 5 and 7 hours (5 X 106 cells/ml). Experiment II: bovine oocyte matured were incubated with 3 sperm concentration 1 X 106; 2.5 X 106 and 5 X 106 cells/ml for 5 hours. Experiment III: LT sperm fertilization ability and control frozen sperm (5 X 106 cells/ml and 5 hours). Experiment I and II were analyzed using analysis of variance (ANOVA) one way, experiment III analyzed using student t-test, on fertilization rate and cleavage. Increasing the time of incubation and sperm concentration had higher effect in fertilization and cleavage rate. The optimal of the time incubation and sperm concentration were 5 hours and 5 X 106 repectively. LT sperm had ability as good as frozen sperm.

Kata Kunci : sperma, Leucocephala tannin, fertilisasi in vitro, tingkat fertilisasi, cleavage.