Ratnani, Hermin (2017) Pengaruh Penambahan Α-Tocopherol Dalam Pengencer Terhadap Kualitas Spermatozoa, Reactive Oxygen Species Dan Intensitas Kalsium Intraseluler Setelah Pembekuan Cepat Pada Sapi Madura. Doctor thesis, Universitas Brawijaya.
Abstract
Pembekuan cepat adalah metoda alternatif prosesing semen beku dengan uap nitrogen (equilibrasi pada jarak tertentu diatas permukaan nitrogen cair) selama beberapa menit. Tujuan penelitian adalah mengetahui : 1). Jarak yang optimal dalam menentukan kualitas spermatozoa terbaik 2). Efek penambahan berbagai dosis α-Tocopherol terhadap kualitas spermatozoa pada suhu ruang (250C), 50 C (suhu dingin) dan -1960C (suhu beku) 4). Kadar MDA (Malonedialdehyde) pada berbagai suhu penyimpanan 5). Gambaran intensitas Ca++intraseluler semen segar dan semen beku. Materi dan Metoda: peneltian menggunakan semen dari 3 ekor sapi Madura, diencerkan dengan pengencer Tris-aminomethan kuning telur 20%, gliserol 7% dan equilibrasi pada suhu 50 C selama 30 menit, pre-freezing selama 5 menit dan selanjutnya dimasukkan kedalam nitrogen cair (suhu -1960C). Penelitian dilakukan dengan berbagai tahap yaitu 1).Tahap pra penelitian yaitu pemeriksaan makroskopis : volume, konsentrasi, bau dan warna; mikroskopis: motilitas individu, viabilitas, abnormalitas, integritas membran 2). Tahap I yaitu pengaruh berbagai jarak equilibrasi diatas permukaan nitrogen cair secara berturut-turut 5 cm (T1), 7 cm (T2) 10 cm (T3), 15 cm (T4) terhadap kualitas spermatozoa (motilitas individu, viabilitas, abnormalitas, integritas membran) post thawing 3) Tahap II yaitu : Pengaruh penambahan berbagai dosis α-Tocopherol 0,5 mM (P1), 1 mM (P2) dan 1,5 mM (P3) dalam pengencer Trisaminomethan kuning telur terhadap kualitas spermatozoa setelah pengenceran pada suhu kamar (250C), suhu dingin (50C) dan suhu beku (-1960C). 4). Tahap III yaitu pengaruh penambahan berbagai dosis α-Tocopherol dalam pengencer Tris-aminomethan kuning telur terhadap kadar MDA. 5). Intensitas Ca++intraseluer dianalisa dengan CLSM (Confocal Laser Scanning Microscope). Hasil penelitian 1).Tahap I: Pengaruh berbagai jarak equilibrasi uap nitrogen terhadap kualitas spermatozoa diperoleh hasil terbaik pada T1 yaitu pada jarak equilibrasi 5 cm: motilitas (34±5,16)%, viabilitas (61,9±3,02)%, abnormalitas (19,9± 1,85)%, integritas membran (54,1±4,36)%. 2).Tahap II: Pengaruh penambahan berbagai dosis α-Tocopherol terhadap kualitas spermatozoa setelah pengenceran pada suhu kamar, suhu dingin, suhu beku. P2 berbeda signifikan (P≤0,05) dengan P0; P1; P3, sedangkan antar tiga kelompok perlakuan tersebut tidak berbeda signifikan (P≥0), dari beberapa tahap suhu penyimpanan, hasil terbaik diperoleh pada kelompok perlakuan P2 yaitu pada dosis 1 mM a). Setelah Pengenceran pada suhu kamar : motilitas individu (68±4,22)%, viabilitas (87,65±1,11)%, abnormalitas (13,65±1,83)%, integritas membran (77,20±1,78)%, b). Suhu dingin : hasil terbaik motilitas individu (61±2,11)%, viabilitas (73,63±4,88)%, abnormalitas (14,55±2,33)%, integritas membran P2 (67±2,04) c). Suhu beku : 1. Pada jarak equilibrasi diatas permukaan nitrogen cair 5 cm kualitas terbaik adalah P2 masing-masing dengan motilitas (42±4,22)%, viabilitas (68,25±2,44), abnormalitas (6,08±1,16)%, integritas membran (60,5±2,02)%. P2 berbeda signifikan (P≤0,05) dengan P0, P1, P3, sedangan antara 3 kelompok tersebut tidak berbeda signifikan (P≥0,05) 2. Pada jarak equilibrasi diatas permukaan nitrogen cair 7 cm; hasil terbaik : motilitas P2 (38±2,58)%, viabilitas P2 (58,8±2,62)%; abnormalitas terendah P2 x (16,20±1,75)% dan integritas membran P3 (51,45 ±2,31)%. Kualitas spermatozoa yang diperoleh pada jarak equilibrasi diatas permukaan nitrogen cair 5 cm lebih baik dibanding 7 cm. Tahap III: Pengaruh penambahan berbagai dosis α-Tocopherol terhadap kadar MDA pada berbagai suhu, hasil terbaik adalah pada perlakuan α-Tocopherol dosis 1 mM (P2), terjadi penurunan kadar MDA secara berturut-turut pada suhu beku, suhu dingin, suhu ruang dari 190,25±4,24 ng/ml menjadi 175,25±2,32 ng/ml, 171,5±5,62 ng/ml menjadi 137,75±2,97 ng/ml, 161,5±4,32 menjadi 130,25±6,91 ng/ml, penambahan α-Tocopherol dosis 1 mM menghasilkan penurunan kadar MDA pada berbagai suhu penyimpanan dan kadar MDA paling rendah adalah pada suhu ruang. Tahap IV : Rata-rata intensitas Ca++ intraseluler spermatozoa semen segar 196,195 ± 42,11 au dan semen beku (setelah pembekuan cepat) 305,39 ± 88,44 au; intensitas Ca++intraseluler semen segar pada bagian kepala dan dari kepala sampai ekor relatif tinggi; sedangkan untuk semen beku pada bagian kepala masih relatif tinggi, namun dari kepala sampai dengan ekor tampak semakin menurun. Kesimpulan: Penambahan α-Tocopherol dalam pengencer Tris-aminomethan kuning telur berpengaruh terhadap kualitas spermatozoa, reactive oxygen species dan intensitas Ca++intraseleluler setelah pembekuan cepat pada sapi Madura. Penambahan α-Tocopherol yang terbaik adalah dosis 1 mM.
English Abstract
Quick Freezing was an alternative method in frozen semen processing with liquid nitrogen (equilibration on certain range above the surface of liquid nitrogen) for a few minute. The purpose of this research was 1). An optimal range to determine the best spermatozoa quality 2). Effect of α-Tocopherol Supplementation on the sperm quality during room temperate (250C), 50C (before freezing), and -1960C (after freezing) 4). MDA (Malonedialdehyde) level at various temperate of storage 5). Calcium intracellular intensity of fresh and frozen semen. Materials and method : this research use 3 Madurans bull cattle semen, diluted with 20% eggyolk tris-aminomethan, 7% gliserol, and equilibrated on 50C for 30 minutes, 5 minutes prefreezing and plunged into liquid nitrogen (-1960C). This research was done in consecutively step, as follows 1). Macroscopic examination : volume, concentration, odor, and colour; Microscopic examination : individual motility, mass motility, viability, abnormality and membrane integrity 2). The effects of various distance equilibration above liquid nitrogen surface, respectively 5 cm (T1), 7 cm (T2) 10 cm (T3), 15 cm (T4) to sperma quality post thawing. 3) The effect of suplementation various dosage of α-Tocopherol respectively 0.5 mM (P1), 1 mM (P2) , 1.5 mM (P3) in egg yolk Tris-aminomethan extender to sperm quality after ditution at room temperate, before freezing and after freezing. 3). The effects supplementation of various dosages α-Tocopherol in egg yolk Tris-aminomethan extender to MDA level. 5). The Ca++intracelllar intensity was analyzed using CLSM (Confocal Laser Scanning Microscope). The result of the research were Step : I).The effects of the various distance of equilibration to sperm quality, that the best sperm quality at 5 cm with motility (34±5.16)%, viability (61.9±3.02)%, abnormality (19.9± 1.85)%, membrane integrity (54.1±4.36)%. II). The effect supplementation of various dosage of α-Tocopherol after dilution at room temperate, before freezing and after freezing there was significantly difference (P≤0,05) between P2 with P0; P1; P3 and between P0, P1, P3 were non significantly difference (P≥0), the best result from several temperate of storage was at suplementation 1 mM (P2) α-Tocopherol a). After dilution at room temperate resulted sperm motility (68±4.22)%, viability (87.65±1.11)%, abnormality (13.65±1.83)%, integritas membran (77.20±1.78)%, b). Before freezing resulted sperm motility (61±2.11)%, viability (73.63±4.88)%, abnormality (14.55±2.33)%, membrane integrity (67±2.04)%; c). After freezing : 1. Equilibration by the distance of 5cm above liquid nitrogen was found the best result at 1 mM (P2) with sperm motility (42±4.22)%, viability (68.25±2.44), abnormality (6.08±1.16)%, membrane integrity (60.5±2.02)%. There was significantly difference (P≤0,05) between P2 with with P0, P1, P3 and between P0, P1, P3 there was non significantly difference (P≥0.05 2. Equilibration by the distance of 7 cm above liquid nitrogen was found the best result : 1 mM (P2) with sperm motility (38±2.58)% and viability (58,8 ± 2,62)%, abnormality (16.20±1.75)%, and 1,5 mM (P3) with membrane integrity (51.45 ±2.31)%); Sperm quality at 5 cm equilibration was better than 7 cm. III).The effects supplementation of α-Tocopherol in egg yolk Tris-aminomethan extender to MDA level x at vaious temperate, the best result was dosage α-Tocopherol 1 mM (P2), there was decreased of MDA level, respectively at room temperate, before freezing, after freezing from 161.5±4.32 to 130.25±6.91 ng/ml, 171.5±5.62 ng/ml to 137.75±297 ng/ml, 190.25±4.24 ng/ml to 175.25±2.32 ng/ml, supplementation of α-Tocopherol with 1mM dosage resulted a decrease MDA level at various degree of storage and the lowest MDA level was at room temperate. IV). The average intensity of calcium intracellular on fresh semen and frozen semen respectively 196.195 ± 42.11 au and 305.39 ± 88.44 au; the Ca++ intracellular intensity of sperm was different between fresh semen and frozen semen on the head part and from the head to the tail, which is on the head part and from the head to the tail in fresh semen consitently high, but in frozen semen on the head part still relatively high and from the head to the tail was decreased. Conclusion : the supplementation of 1 mM α-Tocopherol in semen extender resulted beneficial effect on sperm motility, viability, abnormality, and plasma membrane integrity, ROS and Ca++ intracellular intensity after quick freezing in Madurans bull cattle
| Item Type: | Thesis (Doctor) |
|---|---|
| Identification Number: | DIS/636.082 4/RAT/p/2017/061710214 |
| Uncontrolled Keywords: | CATTLE - SPERMATOZOA, VITAMIN E, INTTRACELLULAR CALCIUM, FROZEN SEMEN |
| Subjects: | 600 Technology (Applied sciences) > 636 Animal husbandry > 636.2 Cattle and related animals > 636.208 2 Cattle and related animals (Breeding) |
| Divisions: | S2/S3 > Doktor Ilmu Ternak, Fakultas Peternakan |
| Depositing User: | Nur Cholis |
| Date Deposited: | 10 Nov 2017 01:23 |
| Last Modified: | 09 Dec 2020 14:43 |
| URI: | http://repository.ub.ac.id/id/eprint/5236 |
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