Kusumawati, Enike Dwi (2017) Fertilitas Spermatozoa Sapi Peranakan Ongole Setelah Sexing Dengan Menggunakan Metode Yang Berbeda. Doctor thesis, Universitas Brawijaya.
Abstract
Sapi Peranakan Ongole (PO) yang merupakan salah satu sapi lokal Indonesia dengan jumlah populasi yang mengalami penurunan. Peningkatan mutu genetik dan produktifitas sapi PO dapat dilakukan dengan penerapan Inseminasi Buatan (IB). Teknologi IB tersebut dapat ditingkatkan nilainya dengan menggunakan program anak yang dihasilkan mempunyai jenis kelamin sesuai harapan yaitu dengan sexing spermatozoa. Tujuan dari penelitian ini adalah untuk mengetahui fertilitas serta perubahan kualitas dan struktur spermatozoa setelah proses sexing dengan menggunakan metode sentrifugasi gradien densitas percoll (SGDP) dan sedimentasi putih telur (SPT) pada sapi PO. Penelitian ini menggunakan metode eksperimental meliputi penelitian tahap 1 dan 2. Penelitian Tahap 1 untuk mengetahui kualitas spermatozoa semen hasil sexing pada semen cair dan semen beku menggunakan sampel semen sapi PO di Loka Penelitian sapi Potong Grati Pasuruan. Penelitian Tahap 2 dilakukan untuk menguji fertilitas dengan cara Inseminasi Buatan (IB) yang dilakukan di PT Widodo Makmur Perkasa, Pasir Tengah, Cianjur, Jawa Barat. Proses sexing, pengenceran, pendinginan, pembekuan, uji kualitas spermatozoa dilakukan di Loka Penelitian Sapi Potong, Grati, Pasuruan. Analisa superoxide dismutase (SOD) dan malondialdehide (MDA) dilakukan di laboratorium Faal Fakultas Kedokteran, Universitas Brawijaya Malang. Analisis fluorescein isothiocyanate-peanut aglutination (FITC-PNA) dilakukan di Jurusan Biologi, Universitas Brawijaya Malang. Analisis scanning electron microscope (SEM) dilakukan di Laboratorium Sentral, Universitas Negeri Malang. Penghitungan ukuran kepala spermatozoa dilakukan di Laboratorium Terpadu, Fakultas Peternakan, Universitas Kanjuruhan Malang. IB semen sexing dilakukan di PT Widodo Makmur Perkasa, Pasir Tengah, Cianjur, Jawa Barat. Data dianalisa menggunakan analisis varian. Apabila terdapat perbedaan yang nyata atau sangat nyata maka dilakukan pengujian selanjutnya menggunakan Uji Jarak Berganda Duncan. Rancangan penelitian yang digunakan untuk: kualitas spermatozoa semen beku setelah sexing, post thawing, FITC-PNA, SOD dan MDA menggunakan Rancangan Acak Kelompok. Kualitas Semen cair menggunakan Rancangan Acak Lengkap Faktorial. Ukuran kepala spermatozoa dan keberhasilan IB menggunakan chi square. Sampel semen yang digunakan dievaluasi secara makroskopis dan mikroskopis dengan syarat motilitas massa ≥2+, motilitas individu dan viabilitas ≥70%, abnormalitas ≤20%, dan konsentrasi ≥1000 x 106. Penelitian Tahap 1 menggunakan Rancangan Acak Kelompok menggunakan perlakuan semen tanpa sexing, sexing SGDP lapisan atas dan bawah, sexing SPT lapisan atas dan bawah dengan ulangan masing-masing 10 kali. Motilitas progresif diamati dengan mikroskop cahaya pada perbesaran 400x. Viabilitas dan abnormalitas spermatozoa diamati pada preparat apusan dibawah mikroskop cahaya dengan pembesaran 400x. Spermatozoa yang menyerap warna dinyatakan mati dan yang mengalami kerusakan di kepala atau ekor dinyatakan abnormal. Reaksi akrosom spermatozoa dianalisa dengan FITC-PNA. Selain itu juga dianalisa xi kadar SOD dan MDA. Analisa SEM untuk melihat kerusakan ultra struktur spermatozoa. Penelitian Tahap 2 dilakukan untuk menguji fertilitas dengan cara IB menggunakan 135 ekor sapi dengan semen cair hasil sexing dengan perlakuan semen tanpa sexing, sexing SGDP dan SPT masing-masing 45 ekor sapi setiap perlakuan. Angka non return rate (NRR) diamati dalam periode 20-30 hari dan angka kebuntingan atau conception rate (CR) diamati dengan palpasi per rektal (PKB) setelah 90 hari sejak IB. Sampel semen segar yang digunakan dalam penelitian sesuai dengan standar yang digunakan peneliti sebelumnya volume 4,4±2,18 ml, motilitas individu 70±0%, viabiltas 95,12±0,98%, abnormalitas 1,02±0,15%, konsentrasi 1.758±137,66 juta/ml, kadar SOD dan MDA 44,1±19,6 U/ml dan 227,25±32,29 ng/ml, tudung akrosom utuh 98,85±0,74%. Hasil evaluasi menunjukkan bahwa kualitas spermatozoa sexing SPT lapisan atas lebih baik dibandingkan metode sexing SGDP. Tetapi hasil fertilitas menunjukkan bahwa metode sexing SGDP lebih baik dibandingkan sexing SPT. Hasil penelitian tahap 1 menunjukkan bahwa rata-rata persentase motilitas spermatozoa terbesar sampai terkecil saat post thawing berturut-turut 44,2% (tanpa sexing), 34,35% (sexing SPT lapisan atas), 31,45% (sexing SGDP lapisan bawah), 31,2 % (sexing SPT lapisan bawah), 27,45% (sexing SGDP lapisan atas). Viabilitas spermatozoa hasil sexing, before freezing dan post thawing menunjukkan penurunan. Terlihat bahwa rata-rata persentase viabilitas pada saat post thawing berurutan dari yang terbesar ke yang terkecil yaitu 91,68% (semen tanpa sexing), 82,82% (sexing SPT lapisan atas), 81,43% (sexing SGDP lapisan bawah), 77,29% (sexing SPT lapisan bawah), 76,11% (sexing SGDP lapisan atas). Abnormalitas hasil sexing, before freezing dan post thawing menunjukkan peningkatan. Persentase abnormalitas spermatozoa hasil sexing, before freezing dan post thawing dengan metode SPT bagian bawah (spermatozoa Y) lebih tinggi dibandingkan lapisan atas (spermatozoa X). Sedangkan metode SGDP lapisan atas (spermatozoa Y) lebih tinggi dibandingkan lapisan bawah (spermatozoa X). Semen tanpa sexing tetap menunjukkan persentase abnormalitas yang lebih rendah dibandingkan semen sexing. Abnormalitas tersebut masih tergolong rendah karena masih kurang dari 10%. Nilai SOD semakin menurun setelah sexing dan post thawing, sedangkan MDA semakin meningkat. Peningkatan kadar MDA tertinggi pada SGDP lapisan bawah yaitu sebesar 624,25 ng/ml pada waktu post thawing, sedangkan terendah pada sexing SPT lapisan atas sebesar 480,00±107,56 ng/ml. Hasil analisa FITC PNA yang menunjukkan bahwa jumlah spermatozoa yang mengalami reaksi akrosom lebih banyak terjadi pada sexing SPT 6,32% dibandingkan SGDP (4,52%). Hasil SEM menunjukkan bahwa dampak sexing sangat merusak membran spermatozoa. Hasil Penelitian tahap 2 menunjukkan bahwa hasil pemeriksaan kebuntingan pada umur 3 bulan didapatkan 21 ekor sapi bunting dari 45 ekor sapi yang di IB dengan semen non sexing (kontrol), 14 ekor sapi bunting dari 45 ekor sapi yang di IB dengan semen sexing sedimentasi putih telur, 18 ekor sapi bunting dari 45 ekor sapi yang di IB dengan semen sexing gradien densitas percoll. Hasil analisa data dengan uji Chi-Square menunjukkan bahwa nilai CR menggunakan semen beku hasil sexing sedimentasi putih telur dan sentrifugasi gradien densitas percoll serta tanpa sexing berbeda sangat nyata (P<0,01). Nilai CR masih lebih tinggi semen tanpa sexing (46,67%) kemudian diikuti sexing SGDP (40%) dan sexing SPT (31,11%). Kualitas (motilitas, viabilitas, abnormalitas) dan struktur spermatozoa setelah proses sexing dan post thawing menggunakan metode sedimentasi putih xii telur menunjukkan hasil yang lebih baik dibandingkan metode sentrifugasi gradien densitas percoll, tetapi tingkat spermatozoa yang sedang mengalami reaksi akrosom dan telah mengalami reaksi akrosom lebih tinggi sehingga nilai conception rate lebih rendah dibandingkan sexing menggunakan metode sentrifugasi gradien densitas percoll. Fertilitas spermatozoa menunjukkan hasil sexing SGDP lebih baik dibandingkan sexing SPT berdasarkan hasil IB semen cair dengan analisa CR
English Abstract
Improvement of genetic quality and productivity of Filial Ongole Bulls (PO) can be done with the application of Artificial Insemination (AI). The AI technology can be scaled up by using a calf program that has sex according to expectation by sexed sperm. The process of sexed can lead to stress on sexually transmitted sperm cells that often result in membrane damage and decreased quality of sperm. The purpose of this study was to determine the fertility and changes in the quality and structure of sperm after the process of sexed by using the centrifugation method of percoll density gradient and egg white sedimentation in PO Bulls. This research uses experimental method including research step 1 and 2. Step 1 to find out the quality of sperm result of sexed on liquid and frozen sperm using PO bulls sample at Beef Cattle Research Station, Pasuruan, Indonesia. The sperm samples used were evaluated macroscopically and microscopically on the condition of mass motility ≥2+, individual motility and viability ≥70%, ≤20% abnormalities, and concentrations ≥1000x106/ml. Step 1 study using Randomized Block Design using sperm treatment without sexed, sexed Percoll Density Gradient Centrifugation (SGDP) of upper and under layer, sexed Egg Sedimentation (SPT) of upper and under layer with 10 times repetition. Progressive motility is observed with a light microscope at 400× magnification. The sperm viability and abnormalities were observed in smear preparations under a light microscope with 400× magnification. Sperm that absorb the color are declared dead and those damaged in the head or tail are declared abnormal. The acrosome reaction of sperm was analyzed by fluorescein isothiocyanatepeanut aglutination (FITC-PNA) conducted at the Biology Laboratory, Faculty of Mathematics and Natural Sciences, Brawijaya University, Malang according to the operational standard of local procedure. Analysis of superoxide dismutase (SOD) and malondialdehide (MDA) levels was performed at the Faal Laboratory, Faculty of Medicine, Brawijaya University, Malang. Scanning electron microscope (SEM) analysis to see the ultra-structural damage of sperm was done at Central Laboratory of Malang State University. Step 2 was conducted to test fertility by AI using 135 head of cows with liquid sperm resulted from sexed with sperm treatment without sexed, sexed SGDP and SPT each 45 cows each treatment. Non-return rate (NRR) is observed within a period of 20-30 days and the pregnancy rate (CR) is observed with per rectal palpation (PKB) after 90 days from AI. Data were analyzed using variance analysis. If there is a real or very real difference then the next test is done using Duncan Multiple Range Test. The research design used for: frozen sperm quality after sexed, post thawing, FITC-PNA, SOD and MDA using Randomized Block Design. Head size and success of AI (NRR and CR) using chi square. Quality of liquid sperm using Completely Randomized Design Factorial. Fresh sperm samples used in the study were in accordance with the standard used by previous researchers volume 4.4±2.18 ml, individual motility 70±0%, viability 95.12±0.98%, abnormality 1.02±0.15%, concentration 1,758±137.66 million/ml, SOD and MDA content 44.1±19.6 U/ml and xiv 227.25±32.29 ng/ml, 98.85±0.74% whole acrosome hood. The results of the evaluation showed that the quality of upper layer SPT sexed sperm was better than the SGDP sexed method. But fertility results show that SGDP sexed method is better than SPT sexed. The mean percentage of post thawing sperm motility from the largest to the smallest were on without sexed (44.2%), sexed of upper layer SPT (34.35%), sexed of under layer SGDP (31.45%), sexed of under layer SPT (31.2%), sexed of upper layer SGDP (27.45%). The viability of sperm after sexing, before freezing and post-thawing showed a decrease. It can be seen that the average percentage of viability during post thawing sequence from the largest to the smallest is sperm without sexed (91.68%), sexed upper layer SPT (82.82%), sexing under layer SGDP (81.43%), sexed under layer SPT (77.29%), sexed SGDP upper layer (76.11%). Abnormalities of sexing results, before freezing and post thawing showed improvement. Percentage of sperm abnormalities result of sexing, before freezing and post thawing with under layer SPT method (Y sperm) higher than upper layer (X sperm). While the upper layer SGDP method (Y sperm) is higher than the bottom layer (X sperm). Sperm without sexing still shows a lower percentage of abnormality than sexed sperm. The abnormality is still relatively low because it is still less than 10%. The value of SOD decreases after sexing and post thawing, while MDA is increasing. The highest increase of MDA levels in the under layer SGDP was 624.25 ng/ml at post thawing time, while the lowest on upper layer SPT was 480.00±107.56 ng/ml. SEM results show that the impact of sexing is very damaging to the membrane of spermatozoa. The result of pregnancy examination at the age of 3 months found 21 head of pregnant cows from 45 cows in AI with non-sexing sperm (control), 14 head of pregnant cows from 45 cows in the AI with sperm sexed SPT, 18 head of pregnant cows from 45 cows in AI with sperm sexed SGDP. Result of data analysis with Chi-Square test showed that CR value using frozen semen result of sexed SPT and SGDP and without sexed were very significant (P<0.01). CR value is still higher sperm without sexed (46.67%) followed by sexed SGDP (40%) and sexed SPT (31.11%). This is related to the result of FITC-PNA analysis which shows that the number of sperm that experienced acrosome reaction is more common in sexed SPT of 6.32% than SGDP (4.52%). Quality (motility, viability, abnormality) and sperm structure after sexed process and post thawing using the egg white sedimentation method showed better results than percoll density gradient centrifugation method, but the sperm level undergoing acrosome reaction and had undergone higher acrosome reaction so that the conception rate was lower than sexing using percoll density gradient centrifugation method. Fertility of sperm showed better sexed SGDP outcomes than sexed SPT based on AI sperm results with CR analysis.
| Item Type: | Thesis (Doctor) |
|---|---|
| Identification Number: | DIS/636.208 2/KUS/f/2017/061709039 |
| Uncontrolled Keywords: | BULLS - BREEDING, BEEF CATTLE - REPRODUCTION, CATTLE - REPRODUCTION, BULLS - REPRODUCTION, CATTLE - SPERMATOZOA |
| 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: | 04 Apr 2018 02:08 |
| Last Modified: | 08 Dec 2020 01:33 |
| URI: | http://repository.ub.ac.id/id/eprint/9144 |
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