Korelasi Ekspresi HSP70, Kualitas Spermatozoa Post-thawing, dan Fertilitas Sapi Bali Berdasarkan Kelompok Umur
Abstract
Sapi Bali merupakan sapi asli Indonesia yang memiliki berbagai keunggulan, seperti performa yang baik, kualitas karkas yang bagus, mudah beradaptasi, mampu mencerna pakan berkualitas rendah, dan performa reproduksi yang baik. Strategi pemuliaan untuk meningkatkan populasinya salah satunya adalah melalui penerapan teknologi reproduksi berbantuan inseminasi buatan (IB) dengan menggunakan semen beku. Pemanfaatan semen beku dalam penerapan IB mempunyai beberarapa kendala, diantaranya adalah penurunan kualitas spermatozoa oleh pengaruh proses kriopreservasi dan pencairan kembali semen. Selain itu, penuaan merupakan salah satu faktor yang menyebabkan penurunan kualitas semen seperti hilangnya protein-protein (makromolekul) spermatozoa dan berimplikasi pada penurunan fertilitas. Makromolekul spermatozoa seperti heat shock protein 70 (HSP70) memiliki peran yang penting untuk menjaga homeostasis protein dari pengaruh perubahan suhu dan stres oksidatif. Kehadiran protein ini banyak dikaji hubungannya dengan fertilitas. Oleh karena itu, diperlukan kajian untuk mengidentifikasi ekspresi gen dan protein HSP70 serta hubungannya dengan kualitas spermatozoa post-thawing dan fertilitas lapangan sapi Bali.
Penelitian ini menggunakan 6 ekor pejantan sapi Bali yang dikelompokkan berdasarkan umur (3 ekor berumur 9 tahun dan 3 ekor berumur 12 tahun). Computer-assisted semen analysis (CASA) digunakan untuk mengamati motilitas dan parameter kinetik spermatozoa. Abnormalitas morfologi spermatozoa dievaluasi menggunakan pewarnaan Diff-Quick. Pewarnaan fluorescence isothiocyanate-conjugated peanut agglutinin (FITC-PNA) digunakan untuk mengamati integritas akrosom spermatozoa dan pewarnaan acridine-orange (AO) untuk mengamati fragmentasi DNA. Identifikasi ekspresi gen HSP70 pada spermatozoa post-thawing dilakukan menggunakan qRT-PCR dan identifikasi protein HSP70 menggunakan ELISA. Tingkat fertilitas lapangan diperoleh dari nilai conception rate (CR) yang dihitung dari data layanan IB dan pemeriksaan kebuntingan tahun 2019-2021 provinsi Bali yang tersedia di data besar iSIKHNAS. Data dianalisis menggunakan Microsoft Excel 365 dan Microsoft Access 2013 untuk mengetahui tingkat fertilitas pejantan. Perbedaan kualitas spermatozoa post-thawing, ekspresi HSP70, dan fertilitas antar-kelompok umur (9 tahun dan 12 tahun) dianalisis menggunakan t-test independent. Hubungan ekspresi HSP70, kualitas spermatozoa post-thawing, dan fertilitas dianalisis menggunakan Pearson correlation. Data tersebut diolah menggunakan program SPSS versi 25.0.
Hasil penelitian menunjukkan bahwa terdapat perbedaan kualitas spermatozoa post-thawing antara pejantan kelompok umur 9 tahun dan 12 tahun. Motilitas total (63,971,12% vs 57,232,01%), motilitas progresif (58,171,44% vs 51,712,34%), non-motile sperm (36,001,11% vs 42,781,99%), abnormalitas morfologi (kelainan bagian kepala: 0,490,09% vs 0,740,09; kelaian bagian leher dan midpiece: 0,780,15% vs 2,400,67%; kelainan bagian ekor: 7,950,36% vs 9,490,56%), dan integritas akrosom spermatozoa (86,680,72% vs 82,571,02%)
dari pejantan umur 9 tahun secara signifikan terdapat perbedaan dengan pejantan umur 12 tahun, sedangkan pada parameter kinetik dan fragmentasi DNA tidak terdapat perbedaan yang signifikan antara kedua kelompok umur. Teridentifikasi ekspresi gen dan protein HSP70 pada spermatozoa post-thawing sapi jantan Bali, meskipun tidak berbeda signifikan (P>0,05) antara kedua kelompok umur. Tingkat fertilitas lapangan dari semua pejantan tergolong baik, namun kelompok pejantan umur 9 tahun (75,941,09%) dan kelompok pejantan 12 tahun (74,200,39%) tidak terdapat perbedaan yang signifikan (P>0,05). Secara statistik ekspresi gen HSP70 berkorelasi dengan hiperaktif spermatozoa (r = 0,815; P<0,05) dan integritas akrosom spermatozoa (r = 0,882; P<0,05), sedangkan protein HSP70 berkorelasi dengan integritas akrosom spermatozoa (r = 0,860; P<0,05). Selain itu, ekspresi gen HSP70 memiliki hubungan erat dengan fertilitas (r = 0,935; P<0,01). Hubungan yang erat juga terlihat antara hiperaktif (r = 0,927; P<0,01) dan integritas akrosom spermatozoa (r = 0,820; P<0,05) dengan tingkat fertilitas. Oleh sebab itu, ekspresi HSP70 dapat dikaji lebih lanjut sebagai kandidat biomarka fertilitas jantan pada sapi Bali. Bali cattle are native Indonesian cattle that have various advantages, such as good performance, good carcass quality, adaptability, ability to digest low-quality feed, and good reproductive performance. One of the breeding strategies to increase the population is through assisted reproductive technology artificial insemination (AI) using frozen semen. The use of frozen semen in the application of AI faces various challenges, including a decrease in spermatozoa quality due to the influence of cryopreservation and the thawing of semen. In addition, aging is one of the factors that cause a decrease in semen quality such as loss of spermatozoa protein (macromolecules) and has implications for decreased fertility. Spermatozoa macromolecules such as heat shock protein 70 (HSP70) have an important role in maintaining protein homeostasis from the effects of temperature changes and oxidative stress. The presence of this protein has been widely studied in relation to fertility. Predicting male fertility is very important in the breeding industry. Through AI services, semen from a superior bull can fertilize many eggs. Therefore, studies are needed to identify the HSP70 gene and protein expression and its relationship with the quality of post-thawing spermatozoa and the field fertility of Bali bulls.
This study used 6 bulls of Bali cattle which were grouped by age (3 bulls aged (9 years and 3 bulls aged 12 years). Computer-assisted semen analysis (CASA) was used to observe spermatozoa's motility and kinetic parameters. Spermatozoa morphological abnormalities were evaluated using Diff-Quick staining. Fluorescence isothiocyanate-conjugated peanut agglutinin (FITC-PNA) staining was used to observe the integrity of the spermatozoa acrosome and acridine-orange (AO) staining to observe DNA fragmentation. Identification of HSP70 gene expression in post-thawing spermatozoa was carried out using qRT-PCR and HSP70 protein identification using ELISA. The field fertility rate was obtained from the conception rate (CR) value which was calculated from data on AI services and pregnancy examinations for the 2019-2021 Bali province, which is available in the iSIKHNAS big data. Data were analyzed using Microsoft Excel 365 and Microsoft Access 2013 to determine the level of bull fertility. Differences in post-thawing spermatozoa quality, HSP70 expression, and fertility between age groups (9 years and 12 years) were analyzed using independent t-tests. The relationship between HSP70 expression, quality of post-thawing spermatozoa, and fertility was analyzed using the Pearson correlation. The data were processed using SPSS version 25.0.
The results showed that there were differences in the quality of post-thawing spermatozoa between males in the age group 9 years and 12 years. The total motility (63,971,12% vs 57,232,01%), progressive motility (58,171,44% vs 51,712,34%), non-motile sperm (36,001,11% vs 42,781,99%), morphological abnormalities (head defects: 0,490,09% vs 0,740,09; neck and midpiece defects: 0,780,15% vs 2,400,67%; tail defects: 7,950,36% vs 9,490,56%), and
acrosome integrity 86,680,72% vs 82,571,02%) of bulls aged 9 years were significantly different from those of bulls aged 12 years, whereas there were no significant differences in kinetic parameters and DNA fragmentation of spermatozoa between both groups. The HSP70 gene and protein expression was identified in spermatozoa of post-thawing Bali bulls, although there was no significant difference (P>0.05) between both groups. The field fertility rate of all bulls was good, with the group of bulls aged 9 years 75.941.09% and the group of males 12 years 74.200.39%, but there was no significant difference (P>0.05) between both groups. Statistically, HSP70 gene expression correlated with hyperactive spermatozoa (r = 0.815; P>0.05) and spermatozoa acrosome integrity (r = 0.882; P>0.05), while HSP70 protein correlated with spermatozoa acrosome integrity (r = 0.860; P>0.05). In addition, HSP70 gene expression has a close correlation with fertility (r = 0.935; P<0.01). A strong correlation was also seen between hyperactive spermatozoa (r = 0.927; P<0.01) and spermatozoa acrosome integrity (r = 0.820; P<0.05) with fertility. Therefore, HSP70 expression can be studied further as a candidate biomarker of bull fertility in Bali cattle.
Collections
- MT - Veterinary Science [899]