PERAN ALBUMIN TERHADAP KUALITAS DAN INTEGRITAS MEMBRAN SPERMATOZOA SAPI PERANAKAN ONGOLE SELAMA SIMPAN DINGIN DAN BEKU DALAM PENGENCER CAUDA EPIDYDYMAL PLASMA (CEP-2)

ACHADIAH RACHMAWATI, Prof. Ir. Ismaya, M.Sc, Ph.D; Prof. Ir. Budi Prasetyo Widyobroto, DESS., DEA, IPU, ASEAN Eng.; Dr. Ir. Sigit Bintara, M.Si, IPM, ASEAN Eng.; Prof. Dr. Ir. Trinil Susilawati, MS, IPU., ASEAN Eng.

2020 | Disertasi | DOKTOR ILMU PETERNAKAN

Berbagai upaya dilakukan untuk meningkatkan kualitas semen cair maupun beku dalam rangka penyebarluasan bibit unggul melalui IB, diantaranya dengan menggunakan pengencer yang mampu mempertahankan kualitas semen sapi Peranakan Ongole (PO) selama penyimpanan dingin dan beku. Pengencer cauda epidydymal plasma-2 (CEP-2) merupakan salah satu pengencer yang berkembang saat ini terbukti lebih unggul dalam menjaga motilitas dan integritas membran spermatozoa dibandingkan dengan pengencer berbasis Tris. Kandungan bovine serum albumin (BSA) dalam CEP-2 sebagai extracellular cryoprotectant mendukung fungsi kuning telur dan menstimulasi motilitas spermatozoa selama simpan dingin dan beku. Bovine serum albumin, sebagai sumber albumin, lebih efektif daripada substansi makromolekul lain dalam mempertahankan motilitas tinggi dalam media buatan dan melindungi spermatozoa dari beberapa efek merusak dari pengenceran. Penelitian ini bertujuan untuk menentukan level BSA terbaik dalam Pengencer CEP-2 terhadap kualitas semen selama penyimpanan dingin dan beku; mengetahui pola kapasitasi dan integritas membran spermatozoa sapi PO dengan level BSA yang berbeda selama simpan dingin dan beku dan hasil aplikasi IB menggunakan semen cair dan beku dengan level BSA yang terbaik dalam Pengencer CEP-2. Penelitian dilaksanakan di Laboratorium Reproduksi Ternak, Fakultas Peternakan, Universitas Brawijaya, Malang (pembuatan Pengencer CEP-2); Loka Penelitian Sapi Potong, Kecamatan Grati, Kabupaten Pasuruan, Propinsi Jawa Timur (prosesing semen cair dan beku) dan Laboratorium Biologi, Matematika; Ilmu Pengetahuan Alam, Universitas Brawijaya, Malang (pengamatan pola kapasitasi spermatozoa hasil simpan dingin dan beku) dan Kecamatan Jabung, Pakis, Tumpang, Poncokusumo, Singosari dan Lawang, Kabupaten Malang, Propinsi Jawa Timur (aplikasi IB). Materi yang digunakan adalah semen Sapi PO yang ditampung satu pekan sekali dengan vagina buatan, Pengencer CEP-2 + 10% kuning telur + level BSA yang berbeda (0; 0,2; 0,4; 0,6; 0,8 dan 1%), pewarna eosin negrosin, larutan hypo-osmotic swelling test (HOST), pewarna Chlortetracycline (CTC) dan pakan Sapi PO jantan (Rumput Gajah, Indigofera spicata, jerami dan konsentrat). Metode yang digunakan adalah percobaan laboratorium (penelitian tahap satu sampai tiga) dan aplikasi IB (penelitian tahap empat). Variabel pengamatan adalah (1) Motilitas individu, viabilitas dan abnormalitas spermatozoa selama simpan dingin (5oC); (2) Motilitas individu, viabilitas dan abnormalitas spermatozoa selama prosesing semen beku; (3) Jumlah spermatozoa belum kapasitasi, kapasitasi, reaksi akrosom dan integritas membran selama simpan dingin dan beku dan (4) Efisiensi reproduksi hasil IB, meliputi: nilai non return rate (NRR), conception rate (CR) dan service per conception (S/C) menggunakan semen beku kontrol, semen cair dan semen beku dengan level BSA terbaik. Analisis statistik dilakukan menggunakan software R Studio versi 3.6.1 dengan tingkat signifikasi 5%. Desain penelitian menggunakan metode ANOVA repeated measured atau time series yang dilanjutkan dengan uji Tukey, apabila data memenuhi kaidah normalitas dan homogenitas. Jika asumsi tidak dipenuhi, maka uji perbandingan dianalisis dengan metode Kruskal Walls yang diikuti dengan uji Wilcoxon (penelitian tahap satu sampai tiga). Analisa diskriptif dan chi square untuk penelitian tahap empat. Hasil penelitian menunjukkan bahwa (1) Level BSA 0,6% mampu mempertahankan motilitas tertinggi pada semen cair (46+2,11%) dan beku (42+2,58%); level BSA 0,2% mampu mempertahankan viabilitas tertinggi pada semen cair (89,36+2,65%) dan level BSA 0,8% pada semen beku (82,50+6,41%); level BSA 1% mampu mampu menekan jumlah spermatozoa abnormalitas pada semen cair (5,00+1,80%) dan level BSA 0,6% pada semen beku (5,37+1,66%); dan total spermatozoa motil setelah delapan hari penyimpanan dingin pada level BSA 0,6% (127,33+4,70 juta/ml) dan level BSA 0,6% setelah simpan beku (119,26+8,46 juta/ml); (2) Level BSA 0,4% mampu mempertahankan integritas membran spermatozoa selama simpan dingin (78,41+4,64%) dan beku (84,30+2,56%); level BSA 0,6% mampu menekan jumlah spermatozoa terkapasitasi selama simpan dingin (23,62+4,90%) dan level BSA 0,8% mampu menekan jumlah spermatozoa terkapasitasi selama simpan beku (21,38+1,73%) dan (3) Efisiensi reproduksi sapi induk yang di inseminasi semen beku kontrol, yaitu nilai NRR2 (94,73%) dan NRR3 (89,40%), S/C (1,29) dan CR (89,47%) lebih tinggi dibandingkan dengan nilai NRR2 (84,21%) dan NRR3 (78,94%), S/C (1,6) dan CR (78,94%) induk sapi yang diiseminasi semen cair dengan level BSA 0,6% dan nilai NRR1 (85%), NRR2 (84,21%) dan NRR3 (73,68%), S/C (1,31) dan CR (84,21%) betina yang di inseminasi semen beku dengan level BSA 0,6%. Kesimpulan penelitian adalah penambahan BSA dalam pengencer CEP-2 meningkatkan kualitas semen cair dan beku yang sesuai dengan SNI. Motilitas semen cair dan beku pada level BSA 0,6%; viabilitas tertinggi semen cair pada level BSA 0,2% dan level BSA 0,8% pada semen beku; abnormalitas spermatozoa terendah semen cair pada level BSA 1% dan 0,6% pada semen beku; dan total spermatozoa motil tertinggi semen cair dan beku pada level BSA 0,6%. Spermatozoa terkapasitasi terendah semen cair pada level BSA 0,6% dan beku pada level BSA 0,8%. Integritas membran spermatozoa tertinggi semen cair dan beku pada level BSA 0,4%. Efisiensi reproduksi menggunakan semen cair dan beku menggunakan level BSA 0,6% dikategorikan baik (nilainya di atas 60% untuk NRR dan CR serta di bawah dua untuk S/C). Saran dari penelitian ini adalah level BSA 0,6% direkomendasikan sebagai extracellular cryoprotectant dalam Pengencer CEP-2 untuk produksi semen cair dan beku dan aplikasi IB guna mengukur fertilitas serta keberhasilan perkawinan. Peternak lebih memperhatikan deteksi birahi dan segera melaporkan ke inseminator, memperhatikan manejemen pemeliharaan, terutama lingkungan kandang dan pakan.

Various attempts are made to improve the quality of liquid and frozen semen in the context of disseminating superior germs through IB, including by using diluents that were able to maintain the quality of semen of Ongole Cattle Breeds (PO) during cold and frozen storage. Cauda epidydymal plasma-2 (CEP-2) diluent is one of the growing diluents that is currently proven to be superior in maintaining motility and integrity of sperm membranes compared to Tris-based. The content of Bovine serum albumin (BSA) in CEP-2 as an extracellular cryoprotectant supports the function of egg yolk and stimulates the motility of spermatozoa during cold and frozen storage. Bovine serum albumin, as a source of albumin, is more effective than other macromolecular substances in maintaining high motility in artificial media and protecting spermatozoa from some of the damaging effects of dilution. This study aims to determine the best BSA level in CEP-2 for the quality of semen during cold and freezing storage; know the capacitation pattern and membrane integrity of Ongole grade bull sperm with different BSA levels during cold and frozen storage and the results of AI applications using liquid and frozen semen with the best BSA levels in CEP-2. The study was conducted at the Animal Reproduction Laboratory, Faculty of Animal Science, University of Brawijaya, Malang (making of CEP-2 diluents); Beef Cattle Research Station, Grati District, Pasuruan Regency, East Java Province (liquid and frozen semen processing) and Biology Laboratory, Mathematics and Natural Sciences, University of Brawijaya, Malang (observing the pattern of sperm capacitations resulting from liquid and frozen storage) and the Districts of Jabung, Pakis, Tumpang, Poncokusumo, Singosari and Lawang, Malang Regency, East Java Province (IB application). The materials were PO semen which was collected once a week with artificial vagina, CEP-2 diluent + 10% egg yolk + different BSA levels (0; 0.2; 0.4; 0.6; 0.6 and 0.8 %), negrosin eosin staining, Hypo-Osmotic Swelling Test (HOST) solution, Chlortetracycline (CTC) staining and feed for bulls (Elephant grass, Indigofera spicata, straw and concentrate). The method used was a laboratory experiment (research stages one to three) and AI application (research stage four). Observation variables were (1) individual motility, viability and abnormal sperm during chilling storage (5oC); (2) Individual motility, viability and abnormality of sperm during the processing of frozen semen; (3) The number of sperm uncapacitated, capacitated, acrosomal reaction and membrane integrity during chilling and frozen storage and (4) Reproductive efficiency of AI results, including: non return rate (NRR), conception rate (CR) and service per conception (S/C) using control frozen semen, liquid and frozen semen with the best BSA level. Statistical analysis was performed using R Studio software version 3.6.1 with a significance level of 5%. The study design used the ANOVA repeated measured or time series method followed by the Tukey Test, if the data met the norms of normality and homogeneity. If the assumptions were not met, the comparison test was analyzed by the Kruskal Walls Method followed by the Wilcoxon Test. The results showed that (1) BSA level 0.6% was able to maintain the highest motility in liquid and frozen semen and BSA level 0.8% was able to maintain the highest viability in liquid and frozen semen and BSA level 1% was able to suppress the number of sperm abnormalities in liquid and frozen semen; (2) BSA level 0,4% is able to maintain the integrity of spermatozoa membrane during chilling and freezing storage and BSA level 0,6% was able to suppress the number of sperm that are capitalized during cold storage and BSA level 0.8% able to reduce the amount of sperm during frozen storage and (3) the reproductive efficiency of the cows inseminated by frozen semen, namely NRR2 (94.73%) and NRR3 (89.40%), S/C (1.29 times) and CR (89.47%) were higher than with NRR2 (84.21%) and NRR3 (78.94%), S/C (1.6) and CR (78.94%) inseminated by BSA level 0.6% and NRR1 value (85%), NRR2 (84.21%) and NRR3 (73.68%), S/C (1.31 times) and CR (84.21%) inseminated by frozen semen with 0.6% BSA level. Reproductive efficiency using liquid and frozen semen using BSA levels was categorized as good, because the values are above 60% for NRR and CR and two times for S/C. Based on the description and discussion that has been presented in the previous chapter, it can be concluded, as follows: The addition of BSA in CEP-2 diluents improving the quality of liquid and frozen semen in accordance with INS. Motility of liquid and frozen semen at BSA level 0.6%; highest viability of liquid cement at BSA level of 0.2% and BSA level of 0.8% in frozen semen; lowest sperm abnormalities in liquid semen at BSA level 1% and 0.6% in frozen semen; and the highest total motile sperm are liquid and frozen semen at BSA level 0.6%. The lowest sperm are capacitated by liquid semen at BSA level 0.6% and frozen at BSA level 0.8%. The highest integrity of sperm membrane is liquid and frozen semen at BSA level 0.4%. Reproductive efficiency using liquid and frozen semen using a BSA level of 0.6% is well categorized (values above 60% for NRR and CR and under two for S/C). Suggestions from this study are the 0.6% BSA level recommended as extracellular cryoprotectant in CEP-2 diluents for the production of liquid and frozen semen and AI applications to measure fertility and marital success. Farmers pay more attention to the detection of estrus and immediately report to the inseminator, pay attention to maintenance management, especially the environment of the stall and feed.

Kata Kunci : Semen, Cryoprotectant, Kapasitasi, Reaksi Akrosom, Efisiensi Reproduksi