Evaluasi Kerusakan DNA Spermatozoa Semen Cair Babi Menggunakan Pewarnaan Toluidine Blue
Evaluation of DNA Fragmentation of Boar Liquid Semen Spermatozoa using Toluidine Blue Staining
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Hermilinda Parera(1*)
Program Studi Kesehatan Hewan, Jurusan Peternakan, Politeknik Pertanian Negeri Kupang
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Victor Lenda(2)
Program Studi Kesehatan Hewan, Jurusan Peternakan, Politeknik Pertanian Negeri Kupang
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Nancy Diana Foeh(3)
Laboratorium Klinik, Reproduksi, Patologi dan Nutrisi, Fakultas Kedokteran dan Kedokteran Hewan, Universitas Nusa Cendana, Kupang
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Muhammad Mirandy Pratama Sirat(4)
Fakultas Peternakan, Universitas Lampung
(*) Corresponding Author
Keywords:
DNA boar sperm, toluidine blue staining, tris citrate fructose
Abstract
The integrity of the deoxyribonucleic acid (DNA) in sperm plays a crucial role in the fertilization process and embryonic development. This study aimed to evaluate the level of the DNA fragmentation of boar sperm in tris-citrate-fructose extender preserved at temperatures between 16-18°C for 3 days. The level of sperm DNA damage was analysed using toluidine blue (TB) staining. Pig semen collected from 4 male pigs aged 1.3-1.8 years was used as the research sample. Semen collected was conducted twice a week. The experimental method using a Completely Randomized Design with 4 storage duration groups as follows: Group I (0 hours of storage), Group II (24 hours of storage), Group III (48 hours of storage), and Group IV (72 hours of storage), each with 5 replications. The data were analysed using analysis of variance (ANOVA) and, if significant differences were found, Duncan's test was performed. The results of the evaluation of the percentage of DNA damage during the storage process at 16-180C at 0 hours, 24 hours, 48 hours and 72 hours was: 0.33 ± 1.66%, 0.80 ± 0.447%, 1.60 ± 1.14% and 2.80 ± 0.447%, respectively. These results demonstrate that liquid boar semen preserved with tris citrate fructose extender at 16-18 °C for 3 days has spermatozoa DNA damage levels within the normal range, making it suitable for artificial insemination and ensuring optimal AI success.
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Shylesh, T., Harshan, H. M., Wilson, M., Promod, K., Usha, A. P., Sunanda, C., & Unnikrishnan, M. P. (2019). Fresh Semen Characteristics of Large white Yorkshire Boar Semen Selected for Liquid Semen Preservation. International Journal of Current Microbiology and Applied Sciences, 8(09), 1584–1590. https://doi.org/10.20546/ijcmas.2019.809.181.
Agarwal, A., Cho, C. L., Esteves, S. C., & Majzoub, A. (2017). Reactive oxygen species and sperm DNA fragmentation. Translational Andrology and Urology, 6(9), S695–S696. https://doi.org/10.21037/tau.2017.05.40
Agarwal, A., Majzoub, A., Baskaran, S., Selvam, M. K. P., Cho, C. L., Henkel, R., Finelli, R., Leisegang, K., Sengupta, P., Barbarosie, C., Parekh, N., Alves, M. G., Ko, E., Arafa, M., Tadros, N., Ramasamy, R., Kavoussi, P., Ambar, R., Kuchakulla, M., … Shah, R. (2020). Sperm DNA fragmentation: A New Guideline for Clinicians. World Journal of Men’s Health, 38(4), 412–471. https://doi.org/10.5534/WJMH.200128
Akhter, S., Rakha, B., Iqbal, R., & Ansari, M. (2014). Effect of Bovine Serum Albumin on Motility, Plasmalemma, Viability and Chromatin Integrity of Buffalo Bull Spermatozoa. Pakistan Journal of Zoology, 46(1), 115–120.
Alves, I. P., Cancelli, C. H. B., Grassi, T. L. M., Oliveira, P. R. H., Franciscato, D. A., Carreira, J. T., & Koivisto, M. B. de. (2018). Evaluation of sperm head dimensions and chromatin integrity of epididymal sperm from domestic cats using the toluidine blue technique. Animal Reproduction Science, 197, 33–39. https://doi.org/https://doi.org/10.1016/j.anireprosci.2018.08.001
Ausejo, R., Martínez, J. M., Mendoza, N., Bolarin, A., Tejedor, M. T., & Falceto, M. V. (2022). Nuclear DNA Fragmentation in Boar Spermatozoa: Measurement Methods and Reproductive Performance Implications. Frontiers in Veterinary Science, 9. https://doi.org/10.3389/fvets.2022.929858
Bebas, W., & Gorda, W. (2019). Penambahan Bovine Serum Albumin pada Beltsville Thawing Solution Dapat Mempertahankan Kualitas Semen Babi yang Disimpan pada 15°C (Addition of Bovine Serum Albumin to Beltsville thawing Solution Could Maintain Qality of Pig Semen Stored at 15°C ). Jurnal Veteriner, 20(3), 330–336. https://doi.org/10.19087/jveteriner.2019.20.3.330
Erenpreisa, J., Erenpreiss, J., Freivalds, T., Slaidina, M., Krampe, R., Butikova, J., Ivanov, A., & Pjanova, D. (2003). Toluidine Blue Test for Sperm DNA Integrity and Elaboration of Image Cytometry Algorithm. Cytometry. Part A : The Journal of the International Society for Analytical Cytology, 52(1), 19–27. https://doi.org/10.1002/cyto.a.10015
Evenson, D. P. (2016). The Sperm Chromatin Structure Assay (SCSA®) and other sperm DNA fragmentation tests for evaluation of sperm nuclear DNA integrity as related to fertility. Animal Reproduction Science, 169, 56–75. https://doi.org/10.1016/j.anireprosci.2016.01.017
Foeh, N. diana frederika katherina, Arifiantini, R. I., & Yusuf, T. laswardi. (2016). Viabilitas spermatozoa semen beku babi duroc dalam extender BTS menggunakan krioprotektan gliserol. 4, 24–32.
González-Marín, C., Gosálvez, J., & Roy, R. (2012). Types, causes, detection and repair of DNA fragmentation in animal and human sperm cells. International Journal of Molecular Sciences, 13(11), 14026–14052. https://doi.org/10.3390/ijms131114026
Juan, C. A., de la Lastra, J. M. P.,
Plou, F. J., & Pérez-Lebeña, E. (2021). The chemistry of reactive oxygen species (Ros) revisited: Outlining their role in biological macromolecules (dna, lipids and proteins) and induced pathologies. International Journal of Molecular Sciences, 22(9). https://doi.org/10.3390/ijms22094642
Kim, H. S., Kang, M. J., Kim, S. A., Oh, S. K., Kim, H., Ku, S. Y., Kim, S. H., Moon, S. Y., & Choi, Y. M. (2013). The utility of sperm DNA damage assay using toluidine blue and aniline blue staining in routine semen analysis. Clinical and Experimental Reproductive Medicine, 40(1), 23–28. https://doi.org/10.5653/cerm.2013.40.1.23
Kumaresan, A., Das Gupta, M., Datta, T. K., & Morrell, J. M. (2020). Sperm DNA Integrity and Male Fertility in Farm Animals: A Review. Frontiers in Veterinary Science, 7(6), 1–15. https://doi.org/10.3389/fvets.2020.00321
Lewis, S. E., John A, R., Conner, S. J., ConIuliis, G. D., Evenson, D. P., Henkel, R., Giwercman, A., & & Gharagozloo, P. (2013). The impact of sperm DNA damage in assisted conception and beyond: recent advances in diagnosis and treatment. Reproductive Biomedicine Online, 27(4), 325–337. https://doi.org/10.1016/j.rbmo.2013.06.014
Lin, C., & Tsai, S. (2016). The effect of cryopreservation on DNA damage, gene expression and protein abundance in vertebrate. Italian Journal of Animal Science ISSN:, 11(21), 119–122. https://doi.org/10.4081/ijas.2012.e21
Martı´nezmartı´nez-Pastor, F., Mata-Campuzano, M., ´ Lvarez-Rodrı´guezrodrı´guez, M. A., Lvarez, M. A. ´, Anel, L., & De Paz, P. (2010). Probes and Techniques for Sperm Evaluation by Flow Cytometry. 45(2), 67–78. https://doi.org/10.1111/j.1439-0531.2010.01622.x
Monachesi, N. E., Neild, D., & Carretero, M. (2019). Dog sperm DNA: Raw semen evaluation with Toluidine blue stain. Reproduction in Domestic Animals Zuchthygiene, 54(8), 1078–1084. https://doi.org/10.1111/rda.13490
Nasr-Esfahani, M., & Tavalaee, M. (2021). Sperm Chromatin Structure: Toluidine Blue Staining (pp. 156–162). https://doi.org/10.1017/9781108878715.021
Parera, H., & Lenda, V. (2023). Evaluation of Motility, Viability and Abnormality of Boar Spermatozoa in Various Modified Extenders. Jurnal
Peternakan Ilmiah Terpadu, 11(1), 13–33. ttps://jurnal.fp.unila.ac.id/index.php/JIPT/article/view/6508/4577
Priyanto, L., Arifiantini, R. I., Yusuf, T. L., Reproduksi, B., Kebidanan, D., & Hewan, K. (2015). Deteksi Kerusakan DNA Spermatozoa Semen Segar dan Semen Beku Sapi Menggunakan Pewarnaan Toluidine Blue (Detection Of Sperm DNA Damage Infresh and Frozen Semen Using Toluidine Blue Staining). In Jurnal Veteriner Maret (Vol. 16, Issue 1).
Rui, B. R., Angrimani, D. S. R., Bicudo, L. C., Losano, J. D. A., Nichi, M., & Pereira, R. J. G. (2018). A fast, low-cost and efficient method for the diagnosis of sperm DNA fragmentation in several species. Reproduction in Domestic Animals, 53(1), 171–175. https://doi.org/10.1111/rda.13087
Shylesh, T., Harshan, H. M., Wilson, M., Promod, K., Usha, A. P., Sunanda, C., & Unnikrishnan, M. P. (2019). Fresh Semen Characteristics of Large white Yorkshire Boar Semen Selected for Liquid Semen Preservation. International Journal of Current Microbiology and Applied Sciences, 8(09), 1584–1590. https://doi.org/10.20546/ijcmas.2019.809.181.
Published
2024-06-22
How to Cite
Parera, H., Lenda, V., Foeh, N., & Sirat, M. M. (2024). Evaluasi Kerusakan DNA Spermatozoa Semen Cair Babi Menggunakan Pewarnaan Toluidine Blue. JURNAL KAJIAN VETERINER, 12(1), 41-51. https://doi.org/10.35508/jkv.v12i1.14732
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