| dc.description.abstract | Penilaian fertilitas calon pejantan di Indonesia berdasarkan teknik Breeding Soundness Examination (BSE). Teknik BSE mudah dilakukan, dapat diulangi dan berkorelasi dengan kesuburan pejantan. Pengujian BSE meliputi tiga bagian, yaitu pengamatan fisik (pengamatan genetalia eksternal dan internal melalui eksplorasi rektal), pengukuran lingkar skrotum, tingkah laku seksual (libido), dan analisis kualitas semen. Metode evaluasi semen merupakan metode yang paling umum digunakan untuk penilaian pejantan. Namun, penilaian fertilitas pejantan berdasarkan kualitas semen saja belum dapat menggambarkan fertilitas yang sesungguhnya. Penelitian proteomic dibutuhkan sebagai kajian dalam memprediksi fertilitas pejantan. Beberapa laporan terbaru menunjukkan efektivitas komponen protein plasma semen dapat bersifat menguntungkan atau merugikan bagi fertilitas maupun kualitas semen.
Tujuan penelitian ini untuk melakukan kajian penilaian fertilitas pejantan melalui penentuan protein penanda fertilitas. Tahapan penelitian yang dilakukan mencakup: 1) karakterisasi pejantan sapi simental berdasarkan hubungan antara konsentrasi testosteron, adiponektin, dan kualitas semen; 2) profil protein plasma semen berdasarkan berat molekul dan hubungannya dengan kualitas semen segar pejantan sapi simental; 3) protein plasma semen yang berkaitan dengan fertilitas dan kualitas semen segar pejantan sapi simental.
Sampel plasma semen diambil dari enam ekor sapi simental milik Balai Inseminasi Buatan (BIB) Ungaran dan enam ekor BIB Lembang dengan kisaran umur 3-8 tahun. Kedua belas ekor tersebut dibagi menjadi kelompok pejantan dengan motilitas spermatozoa ≥70% (normal fresh semen) dan motilitas spermatozoa <70% (poor fresh semen). Pengelompokan berdasarkan data sekunder dari kedua BIB tahun 2018-2019. Kelompok normal fresh semen (NFS) dari BIB Ungaran (NFSU), NFS dari BIB Lembang (NFSL), poor fresh semen (PFS) dari BIB Ungaran (PFSU) dan PFS dari Lembang (PFSL). Setiap kelompok masing-masing tiga ekor sapi. Analisis proteomic menggunakan sampel yang berasal dari BIB Lembang (NFSL dan PFSL).
Hasil analisis menunjukkan adanya perbedaan signifikan (p<0,05) antara volume semen dan konsentrasi spermatozoa pada sapi NFSU, PFSU, dan NFSL dengan kelompok sapi PFSL. Sampel NFSU (6,10 ml dan 1455,02×106 /ml), PFSU (6,10 dan 1313,98×106 /ml), serta NFSL (6,73 ml dan 1301,74×106 /ml) lebih tinggi dibandingkan dengan kelompok sapi PFSL (4,37 ml dan 731,35×106 /ml). Rata-rata konsentrasi testosteron pada pejantan sapi simental NFSU, PFSU, NFSL, dan PFSL berturut-turut sebanyak 35,36 ng/ml, 30,42 ng/ml, 34,94 ng/ml, dan 33,06 ng/ml. Konsentrasi adiponektin dalam penelitian ini menunjukkan rata-rata sebesar 5,52 µg/ml (NFSU), 7,23 µg/ml (PFSU), 5,95 µg/ml (NFSL), dan 6,86 µg/ml (PFSL).
Pola sekresi hormon testosteron dan adiponektin pada semua pejantan (NFSU, PFSU, NFSL, dan PFSL) tidak menunjukkan perbedaan konsentrasi yang signifikan (p<0,05). Parameter evaluasi fertilitas untuk seleksi calon pejantan sapi simental berdasarkan evaluasi semen, pengukuran testosteron, dan adiponektin belum dapat menggambarkan kapasitas fertilitas sapi pejantan yang sebenarnya, sehingga perlu ditambahkan dengan penilaian pejantan secara molekular, salah satunya melalui identifikasi protein plasma semen yang berhubungan langsung dengan fertilitas.
Perkembangan penilaian fertilitas pejantan dilakukan dengan identifikasi dan karakterisasi protein penentu fertilitas spermatozoa maupun kualitas semen menggunakan metode 1D-SDS-PAGE (gel electrophoresis) berdasarkan berat molekul protein. Hasil penelitian ini menunjukkan, masing-masing ekspresi protein dengan BM 134-101 kD, 100-71 kD, dan 70 kD tidak ditemukan pada kelompok sapi dengan motilitas spermatozoa <70% (PFSU dan PFSL). Analisis protein berdasarkan BM menggunakan SDS-PAGE dapat dipertimbangkan sebagai indikator tambahan metode BSE. Konfirmasi profiling protein berdasarkan BM, masih perlu dilakukan analisis proteomics menggunakan metode liquid chromatography mass spectrometry (LC-MS/MS) agar memperoleh protein spesifik yang berhubungan langsung dengan fertilitas maupun kualitas semen sehingga hasilnya lebih optimal.
Teknologi proteomic saat ini digunakan sebagai alat yang penting dalam penilaian semen melalui identifikasi protein sebagai penanda fertilitas spermatozoa. Hasil kajian menunjukkan protein yang terekspresikan dalam plasma semen sapi dengan motilitas spermatozoa >70% (NFSL) yang dijadikan kandidat penanda fertilitas pejantan, yaitu: binder sperm protein (BSP1, BSP3, dan BSP5), spermadhesin 2 (SPADH2), serine protease inhibitor 5 (SERPINA5), epididymal secretory protein E1 (ELSPBP1), cysteine rich secretory protein 1 (CRISP1). Sapi-sapi dengan dengan motilitas spermatozoa <70% (PFSL) ekspresi protein di antaranya berperan dalam protein kinase inhibitor (ACTB dan QSOX1), response to oxidative stress (GPX3), signaling caspase activity (RNASE4), dan growth factor activity (NGF). Protein pada kelompok sapi dengan motilitas spermatozoa >70% (NFSU dan NFSL) mengarah pada mekanisme kapasitasi spermatozoa (heparin binding), transportasi protein, ATP binding, motilitas spermatozoa, spermatogenesis, immune tolerance, dan fertilisasi. Protein sapi PFSL mengarah pada fungsi apoptosis dan antigen.
Hasil kajian proteomics dalam disertasi doktoral ini mampu menjelaskan peran langsung protein plasma semen segar, berdasarkan BM maupun protein spesifik. Peran protein spesifik berkaitan dengan mekanisme fungsional spermatozoa dan fungsi reproduksi. Protein plasma semen sapi NFSL berperan dalam fungsi reproduksi seperti single fertilization, fertilization, heparin binding, sperm capacitation, dan cell surface yang berhubungan dengan parameter kualitas semen sebagai penanda fertilitas pejantan. Penelitian ini memberikan peluang baru, yang dapat dijadikan sebagai parameter tambahan dalam proses seleksi dan reseleksi sapi pejantan di Indonesia. | id |
| dc.description.abstract | The assessment for male fertility in Indonesia is based on the breeding soundness examination (BSE) technique. BSE technique is easy to do, repeatable, and correlate with male fertility. BSE testing includes three parts: physical observation (external and internal genital observations through rectal exploration), scrotal circumference measurement, sexual behavior, and analysis of semen quality. The method for semen evaluation is the most commonly used in the male assessment. However, this method has not been sufficient to describe actual fertility. So that, proteomic research is needed to predicting male fertility. Several recent reports have shown the effectiveness of the components seminal plasma protein to be beneficial or detrimental to both fertility and semen quality.
This study aimed to develop a male fertility assessment method based on determinants of fertility proteins. The stages of the research included: 1) characterization of Simental bulls based on the correlation among testosterone, adiponectin concentration, and semen quality; 2) profile of seminal plasma protein-based on molecular weight and their correlation with fresh semen quality of Simmental bulls; 3) seminal plasma protein related to fertility and fresh semen quality in Simmental bulls.
Seminal plasma samples came from six Simmental bulls belonging to the Ungaran Artificial Insemination Center (AIC) and six AIC Lembang bulls aged 3-8 years. The twelve bulls were divided into groups with 70% spermatozoa motility (normal fresh semen) and <70% spermatozoa motility (poor fresh semen). The groups were based on secondary data from the two AICs for 2018-2019. Normal fresh cement (NFS) from AIC Ungaran (NFSU), NFS from AIC Lembang (NFSL), poor fresh cement (PFS) from AIC Ungaran (PFSU) and PFS from Lembang (PFSL). Each group has three bulls. Proteomic analysis used samples from AIC Lembang (NFSL and PFSL).
The result shows there were significant differences (p<0.05) between the semen volume and concentration of spermatozoa in bulls with spermatozoa motility >70, both samples from AIC Ungaran (NFSU: 6.10 mL and 1455.02×106 mL-1), and samples from AIC Lembang (NFSL) (5.99 mL and 1035.07×106 mL-1) were higher than the group of bulls whose motility was <70% (PFSL) (4.37 mL and 731.35×106 mL-1). The mean testosterone levels in all Simmental bulls (NFSU, NFSL, and PFSL) were 35.36 ng mL-1, 34.94 ng mL-1, and 33.06 ng mL-1, respectively. The level of adiponectin concentration showed an average of 5.52 µg mL-1 (NFSU), 5.95 µg mL-1 (NFSL), and 6.86 µg mL-1 (PFSL). The analysis showed that the level of testosterone and adiponectin hormones in all bulls (NFSU, NFSL, and PFSL) did not show a significant difference (p>0.05). Fertility evaluation parameters for the Simmental bull’s selection based on semen evaluation, testosterone, and adiponectin measurements have not been able to describe the actual fertility capacity, so it is necessary to add the molecular assessment of bulls, one which is through the identification of seminal plasma proteins that are related to fertility.
The development of bull fertility assessments was conducted by identifying and characterizing protein that determines sperm fertility and semen quality using the 1D-SDS-PAGE (gel electrophoresis) method based on molecular protein weight. The result showed that each protein expression with BM 134-101 kD, 100-71 kD, and 70 kD was not found in the group of bulls with spermatozoa motility <70% (PFSL). Protein analysis based on molecular weight using SDS-PAGE can be considered an additional indicator for the BSE method for bull’s candidate selection. However, confirmation of protein profiling base on BM still needs to be conducted by proteomics analysis using liquid chromatography-mass spectrometry (LC-MS/MS) method to obtain specific proteins that are directly related to fertility and semen quality for the results are more optimal.
Proteomic technology is currently being used as an essential tool in assessing semen through the identified proteins related to sperm fertility. The results showed that proteins expressed explicitly in seminal plasma with normal fresh semen quality (NFSU and NSFL) bulls were used as candidates for male fertility biomarkers, namely: binder sperm protein (BSP1, BSP3, dan BSP5), spermadhesin 2 (SPADH2), a serine protease inhibitor 5 (SERPINA5), epididymal secretory protein E1 (ELSPBP1), cysteine-rich secretory protein 1 (CRISP1). PFSL bulls protein expression includes a role in protein kinase inhibitor (ACTB dan QSOX1), response to oxidative stress (GPX3), signaling caspase activity (RNASE4), and growth factor activity (NGF). NFSU and NFSL proteins lead to the mechanism of spermatozoa capacitation (heparin-binding), protein transport, ATP binding, spermatozoa motility, spermatogenesis, immune tolerance, and fertilization. On the other hand, PFSL bull protein leads to apoptotic and antigen functions. The results in this doctoral dissertation can explain the direct role in fresh semen of seminal plasma of protein-based on molecular weight and specific protein on spermatozoa functional mechanisms and reproductive functions such as single fertilization, fertilization, heparin-binding, sperm capacitation, cell surface, ATP binding, dan calcium-binding associated with semen quality parameters as biomarkers of male fertility. This research provides a new opportunity that can be used as additional parameters in the selection and reselection process for bulls in Indonesia. | id |
