Impacts of Cryopreservation on Semen Quality and Sperm Protein Profiles of Pesisir Bulls
Abstract
This study aimed to evaluate the impacts of cryopreservation on semen quality and sperm protein profile in Pesisir bulls. Semen samples were collected from three bulls and analyzed in fresh and post-thaw conditions. The sperm motility and kinematic variables were assessed using Computer-Assisted Sperm Analysis (CASA), while sperm viability and plasma membrane integrity (PMI) were evaluated through eosin-nigrosin staining and the hypoosmotic swelling test (HOST), respectively. Subsequently, the total protein concentration (PC) and profile were examined using SDS-PAGE. The results showed that there was a significant decrease in semen quality after thawing, with sperm motility reducing from 81.10% to 70.22%, viability reducing fom 87.47% to 77.27%, and PMI reducing from 85.09% to 71.32% (p<0.05). Kinematic variables such as velocity, straightness, and beat cross frequency also decreased significantly. Protein analysis showed a reduction in total concentration from 1.78 mg/mL to 1.19 mg/mL and alterations in protein band distribution, with the loss of specific high- and low-molecular weight after freezing. These results suggested that cryopreservation negatively impacts semen quality and sperm protein integrity, potentially impairing fertility. Moreover, further studies were recommended to optimize cryopreservation protocols and mitigate adverse effects.
Full text article
References
Afriani, T., Jaswandi, Rastosari, A., Razzak, M. C. A., & Wahyudi, D. (2022). Addition of tomato juice as additive in diluent of egg yolk citrate on the quality of Pesisir cattle semen. Journal of Animal Health and Production, 11(1), 62–67. https://doi.org/10.17582/journal.jahp/2023/11.1.62.67
Agung, P. P., Saputra, F., Zein, M. S. A., Wulandari, A. S., Putra, W. P. B., Said, S., & Jakaria. (2019). Genetic diversity of Indonesian cattle breeds based on microsatellite markers. Asian-Australasian Journal of Animal Sciences, 32(4), 467–476. https://doi.org/10.5713/ajas.18.0283
Ali, M. A., Qin, Z., Dou, S., Huang, A., Wang, Y., Yuan, X., Zhang, Y., Ni, Q., Azmat, R., & Zeng, C. (2023). Cryopreservation induces acetylation of metabolism-related proteins in boar sperm. International Journal of Molecular Sciences, 24(13), 10983. https://doi.org/10.3390/ijms241310983
Alkhawagah, A. R., Ricci, A., Banchi, P., Martino, N. A., Poletto, M. L., Donato, G. G., Nervo, T., & Vincenti, L. (2022). Effect of epidermal growth factor (EGF) on cryopreserved Piedmontese bull semen characteristics. Animals, 12(22), 3179. https://doi.org/10.3390/ani12223179
Arifiantini, R. I. (2012). Teknik koleksi dan evaluasi semen pada hewan. Institut Pertanian Bogor.
Azizah, N., Susilowati, S., Utomo, B., Kusumaningrum, D. A., Kostaman, T., Muttaqin, Z., & Arrazy, A. F. (2023). Seminal plasma protein profiles and testosterone levels as biomarker semen quality of candidate Madura bulls. Journal of Advanced Veterinary and Animal Research, 10(3), 429–436. https://doi.org/10.5455/javar.2023.j696
Baharun, A., Rahmi, A., Handarini, R., Maulana, T., Said, S., Iskandar, H., Darussalam, I., Nalley, W., & Arifiantini, R. I. (2024). Semen quality and frozen semen production in Pasundan bulls: A molecular weight perspective on seminal plasma and spermatozoa protein. Journal of Advanced Veterinary and Animal Research, 10(4), 730–737. https://doi.org/10.5455/javar.2023.j728
Bogle, O. A., Kumar, K., Attardo‐Parrinello, C., Lewis, S. E. M., Estanyol, J. M., Ballescà, J. L., & Oliva, R. (2017). Identification of protein changes in human spermatozoa throughout the cryopreservation process. Andrology, 5(1), 10–22. https://doi.org/10.1111/andr.12279
Byrne, K., Leahy, T., McCulloch, R., Colgrave, M. L., & Holland, M. K. (2012). Comprehensive mapping of the bull sperm surface proteome. Proteomics, 12(23–24), 3559–3579. https://doi.org/10.1002/pmic.201200133
Carreira, J. T., Trevizan, J. T., Carvalho, I. R., Kipper, B., Rodrigues, L. H., Silva, C., Perri, S. H. V., Drevet, J. R., & Koivisto, M. B. (2017). Does sperm quality and DNA integrity differ in cryopreserved semen samples from young, adult, and aged Nellore bulls? Basic and Clinical Andrology, 27, 1–8. https://doi.org/10.1186/s12610-017-0056-9
Castaneda, J. M., Miyata, H., Archambeault, D. R., Satouh, Y., Yu, Z., Ikawa, M., & Matzuk, M. M. (2020). Mouse t-complex protein 11 is important for progressive motility in sperm. Biology of Reproduction, 102(4), 852–862. https://doi.org/10.1093/biolre/ioz226
Chaturvedi, D., Dhami, A. J., & Chaudhari, D. V. (2022). Fortification of tris extender with mifepristone, sericin, and taurine improves velocity and kinematics of fresh and frozen-thawed bovine spermatozoa. Indian Journal of Animal Research, 56(3), 255–262. https://doi.org/10.18805/IJAR.B-4315
Cheema, R. S., Bansal, A. K., Patel, M., & Gandotra, V. K. (2016). Purification and characterization of heparin binding proteins from seminal plasma of cross-bred cattle bulls by affinity chromatography, SDS-PAGE and mass spectrometry. Journal of Proteomics & Enzymology, 5(1). https://doi.org/10.4172/2470-1289.1000127
Costa, B. B. D., Marques, L. S., Lassen, P. G., Rodrigues, R. B., & Streit, D. P. (2019). Cryopreservation‐induced morphological changes in the sperm of South American silver catfish (Rhamdia quelen). Journal of Applied Ichthyology, 35, 1–7. https://doi.org/10.1111/jai.13928
Costa, J., Braga, P. C., Rebelo, I., Oliveira, P. F., & Alves, M. G. (2023). Mitochondria quality control and male fertility. Biology, 12(6), 827. https://doi.org/10.3390/biology12060827
D’Amours, O., Frenette, G., Fortier, M., Leclerc, P., & Sullivan, R. (2010). Proteomic comparison of detergent-extracted sperm proteins from bulls with different fertility indexes. Reproduction, 139(3), 545–556. https://doi.org/10.1530/REP-09-0375
Díaz-Ramos, À., Roig-Borrellas, A., García-Melero, A., & López-Alemany, R. (2012). α-Enolase, a multifunctional protein: Its role on pathophysiological situations. Journal of Biomedicine and Biotechnology, 2012, 1–12. https://doi.org/10.1155/2012/156795
Fayyaz, M. H., Andrabi, S. M. H., Haider, M. S., Khalique, M. A., & Shah, S. A. H. (2022). Kisspeptin-10 in cryodiluent improves the post-thaw quality of Nili-Ravi buffalo (Bubalus bubalis) bull spermatozoa. Andrologia, 54(10), e14564. https://doi.org/10.1111/and.14564
Fu, X., Xu, B., Jiang, J., Du, X., Yu, X., Yan, Y., Li, S., Inglis, B. M., Ma, H., Wang, H., Pei, X., & Si, W. (2020). Effects of cryopreservation and long-term culture on biological characteristics and proteomic profiles of human umbilical cord-derived mesenchymal stem cells. Clinical Proteomics, 17(15), 1–18. https://doi.org/10.1186/s12014-020-09279-6
Fujii, T., Hirayama, H., Fukuda, S., Kageyama, S., Naito, A., Yoshino, H., Moriyasu, S., Yamazaki, T., Sakamoto, K., Hayakawa, H., Takahashi, K., Takahashi, Y., & Sawai, K. (2018). Expression and localization of aquaporins 3 and 7 in bull spermatozoa and their relevance to sperm motility after cryopreservation. Journal of Reproduction and Development, 64(4), 327–335. https://doi.org/10.1262/jrd.2017-166
Harayama, H., Nishijima, K., Murase, T., Sakase, M., & Fukushima, M. (2010). Relationship of protein tyrosine phosphorylation state with tolerance to frozen storage and the potential to undergo cyclic AMP-dependent hyperactivation in the spermatozoa of Japanese Black bulls. Molecular Reproduction and Development, 77(10), 910–921. https://doi.org/10.1002/mrd.21233
Harima, R., Hara, K., & Tanemura, K. (2025). TCTEX1D2 is essential for sperm flagellum formation in mice. Scientific Reports, 15(1), 2413. https://doi.org/10.1038/s41598-024-83424-1
Hartatik, T., Priyadi, D. A., Panjono, P., Bintara, S., Ismaya, I., Budisatria, I. G. S., Widyobroto, B. P., & Agus, A. (2019). Association of IGFBP-3 gene polymorphism g. 3.930 G>A with birth size and birth weight in crossbred beef cattle. Journal of the Indonesian Tropical Animal Agriculture, 44(4), 356–363. https://doi.org/10.14710/jitaa.44.4.356-363
Hendri, Jaswandi, Indriastuti, R., & Ananda. (2024). Sperm kinematics of Pesisir bull thawed at different temperatures and times. Buletin Peternakan, 48(4), 233–241. https://doi.org/10.21059/buletinpeternak.v48i4.96459
Hinsch, K.-D., De Pinto, V., Aires, V. A., Schneider, X., Messina, A., & Hinsch, E. (2004). Voltage-dependent anion-selective channels VDAC2 and VDAC3 are abundant proteins in bovine outer dense fibers, a cytoskeletal component of the sperm flagellum. Journal of Biological Chemistry, 279(15), 15281–15288. https://doi.org/10.1074/jbc.M313433200
Huang, Z., Danshina, P. V., Mohr, K., Qu, W., Goodson, S. G., O’Connell, T. M., & O’Brien, D. A. (2017). Sperm function, protein phosphorylation, and metabolism differ in mice lacking successive sperm-specific glycolytic enzymes. Biology of Reproduction, 97(4), 586–597. https://doi.org/10.1093/biolre/iox103
Isnaini, N., Reksadinata, E. K. D., Andri, F., Harsi, T., & Irfan, I. Z. (2022). Effects of bull age on the fresh and frozen semen quality of Aceh cattle. Journal of Animal Health and Production, 10(4). https://doi.org/10.17582/journal.jahp/2022/10.4.475.478
Jaswandi, Prastowo, S., Widyastuti, R., & Ananda. (2024). Quality and protein profiles in local Indonesian ram sperm before and after cryopreservation. International Journal of Veterinary Science, 13(6), 841–852. https://doi.org/10.47278/journal.ijvs/2024.175
Jumeau, F., Sigala, J., Dossou-Gbete, F., Frimat, K., Barbotin, A. L., Buée, L., Béhal, H., Sergeant, N., & Mitchell, V. (2018). A-kinase anchor protein 4 precursor (pro-AKAP4) in human spermatozoa. Andrology, 6(6), 854–859. https://doi.org/10.1111/andr.12524
Kadirvel, G., Periasamy, S., & Kumar, S. (2012). Effect of cryopreservation on apoptotic-like events and its relationship with cryocapacitation of buffalo (Bubalus bubalis) sperm. Reproduction in Domestic Animals, 47(1), 143–150. https://doi.org/10.1111/j.1439-0531.2011.01818.x
Liu, Y., Jiang, M., Li, C., Yang, P., Sun, H., Tao, D., Zhang, S., & Ma, Y. (2011). Human t-complex protein 11 (TCP11), a testis-specific gene product, is a potential determinant of the sperm morphology. The Tohoku Journal of Experimental Medicine, 224(2), 111–117. https://doi.org/10.1620/tjem.224.111
Luconi, M., Carloni, V., Marra, F., Ferruzzi, P., Forti, G., & Baldi, E. (2004). Increased phosphorylation of AKAP by inhibition of phosphatidylinositol 3-kinase enhances human sperm motility through tail recruitment of protein kinase A. Journal of Cell Science, 117(7), 1235–1246. https://doi.org/10.1242/jcs.00931
Ma, L., Jung, D.-J., Jung, E.-J., Lee, W.-J., Hwang, J.-M., Bae, J.-W., Kim, D.-H., Yi, J. K., Lee, S. M., Ha, J. J., & Kwon, W.-S. (2021). Assessment of cryopreserved sperm functions of Korean native brindled cattle (Chikso) from different region research centers of Korea. Journal of Animal Reproduction and Biotechnology, 36(2), 106–115. https://doi.org/10.12750/JARB.36.2.106
Mahmoud, K. Gh., Sakr, A. M., Ibrahim, S. R., Sosa, A. S., Hasanain, M. H., & Nawito, M. F. (2021). GnRHR gene polymorphism and its correlation with semen quality in buffalo bulls (Bubalus bubalis). Iraqi Journal of Veterinary Sciences, 35(2), 381–386. https://doi.org/10.33899/ijvs.2020.126886.1407
Margaryan, H., Dorosh, A., Capkova, J., Manaskova-Postlerova, P., Philimonenko, A., Hozak, P., & Peknicova, J. (2015). Characterization and possible function of glyceraldehyde-3-phosphate dehydrogenase-spermatogenic protein GAPDHS in mammalian sperm. Reproductive Biology and Endocrinology, 13, 1–9. https://doi.org/10.1186/s12958-015-0008-1
Marques, J. C. C., Cezar, A. R. R., do Nascimento, A. D., da Silva, J. P., Batista, A. M., Guerra, M. M. P., & Câmara, D. R. (2023). Relationship between Na/K-ATPase in thawed sperm and fertility of Angus bulls. Animal Reproduction, 20(4), e20220066. https://doi.org/10.1590/1984-3143-ar2022-0066
Masrizal, Udin, Z., Hendri, Afriani, T., Jaswandi, & Ananda. (2025). Optimizing equilibration time for enhanced post-thaw quality of Pesisir bull frozen semen. International Journal of Veterinary Science, 14(2), 281–288. https://doi.org/10.47278/journal.ijvs/2024.239
Morrell, J. M., Valeanu, A. S., Lundeheim, N., & Johannisson, A. (2018). Sperm quality in frozen beef and dairy bull semen. Acta Veterinaria Scandinavica, 60(41), 1–10. https://doi.org/10.1186/s13028-018-0396-2
Mostek, A., Słowińska, M., Judycka, S., Karol, H., Ciereszko, A., & Dietrich, M. A. (2018). Identification of oxidatively modified proteins due to cryopreservation of carp semen. Journal of Animal Science, 96(4), 1453–1465. https://doi.org/10.1093/jas/sky063
Nagy, Á., Polichronopoulos, T., Gáspárdy, A., Solti, L., & Cseh, S. (2015). Correlation between bull fertility and sperm cell velocity parameters generated by computer-assisted semen analysis. Acta Veterinaria Hungarica, 63(3), 370–381. https://doi.org/10.1556/004.2015.035
Nibali, S. C., Battiato, G., Pappalardo, X. G., & De Pinto, V. (2024). Voltage-dependent anion channels in male reproductive cells: Players in healthy fertility? Biomolecules, 14(10), 1290. https://doi.org/10.3390/biom14101290
Oliveira, L. Z., de Arruda, R. P., de Andrade, A. F. C., Celeghini, E. C. C., dos Santos, R. M., Beletti, M. E., Peres, R. F. G., Oliveira, C. S., & de Lima, V. F. M. H. (2012). Assessment of field fertility and several in vitro sperm characteristics following the use of different Angus sires in a timed-AI program with suckled Nelore cows. Livestock Science, 146(1), 38–46. https://doi.org/10.1016/j.livsci.2012.02.018
Özbek, M., Hitit, M., Kaya, A., Jousan, F. D., & Memili, E. (2021). Sperm functional genome associated with bull fertility. Frontiers in Veterinary Science, 8, 610888. https://doi.org/10.3389/fvets.2021.610888
Perez-Patiño, C., Barranco, I., Li, J., Padilla, L., Martinez, E. A., Rodriguez-Martinez, H., Roca, J., & Parrilla, I. (2019). Cryopreservation differentially alters the proteome of epididymal and ejaculated pig spermatozoa. International Journal of Molecular Sciences, 20(7), 1791. https://doi.org/10.3390/ijms20071791
Peris-Frau, P., Martín-Maestro, A., Iniesta-Cuerda, M., Sánchez-Ajofrín, I., Cesari, A., Garde, J. J., Villar, M., & Soler, A. J. (2020). Cryopreservation of ram sperm alters the dynamic changes associated with in vitro capacitation. Theriogenology, 145, 100–108. https://doi.org/10.1016/j.theriogenology.2020.01.046
Prieto-Martínez, N., Morató, R., Muiño, R., Hidalgo, C. O., Rodríguez-Gil, J. E., Bonet, S., & Yeste, M. (2017). Aquaglyceroporins 3 and 7 in bull spermatozoa: Identification, localisation and their relationship with sperm cryotolerance. Reproduction, Fertility and Development, 29(6), 1249–1259. https://doi.org/10.1071/RD16077
Putra, D., Sumadi, S., Kanazawa, T., & Hartatik, T. (2016). Identification of growth hormone gene polymorphism for beef cattle in Pesisir Selatan district, West Sumatra, Indonesia. Biodiversitas Journal of Biological Diversity, 17(2), 246–251. https://doi.org/10.13057/biodiv/d170246
Rajamanickam, G. D., Kastelic, J. P., & Thundathil, J. C. (2017). Content of testis-specific isoform of Na/K-ATPase (ATP1A4) is increased during bovine sperm capacitation through translation in mitochondrial ribosomes. Cell and Tissue Research, 368(1), 187–200. https://doi.org/10.1007/s00441-016-2514-7
Rana, R. S., Slathia, I., & Rana, R. S. (2020). Study the morphometry of fresh and cryopreserved Murrah bull spermatozoa. Advances in Zoology and Botany, 8(4), 279–290. https://doi.org/10.13189/azb.2020.080401
Rosyada, Z. N. A., Pardede, B. P., Kaiin, E. M., Tumbelaka, L. I. T. A., Solihin, D. D., Purwantara, B., & Ulum, M. F. (2023). Identification of heat shock protein70-2 and protamine-1 mRNA, proteins, and analyses of their association with fertility using frozen-thawed sperm in Madura bulls. Animal Bioscience, 36(12), 1796–1805. https://doi.org/10.5713/ab.23.0142
Ryu, D.-Y., Song, W.-H., Pang, W.-K., Yoon, S.-J., Rahman, M. S., & Pang, M.-G. (2019). Freezability biomarkers in bull epididymal spermatozoa. Scientific Reports, 9(1), 12797. https://doi.org/10.1038/s41598-019-49378-5
Saranholi, D. A. C., Paula, R. R. de, Pytilak, E., Afonso, F., Canela, L. F., Almeida, A. B. M. de, Hidalgo, M. M. T., Martins, M. I. M., Blaschi, W., & Barreiros, T. R. R. (2021). Comparison of seminal characteristics of Aberdeen Angus, Holstein and Nelore bulls before and after cryopreservation. Research, Society and Development, 10(16), e408101623382. https://doi.org/10.33448/rsd-v10i16.23382
Satrio, F. A., Karja, N. W. K., Setiadi, M. A., Kaiin, E. M., Pardede, B. P., & Purwantara, B. (2024). Age-dependent variations in proteomic characteristics of spermatozoa in Simmental bull. Frontiers in Veterinary Science, 11, 1393706. https://doi.org/10.3389/fvets.2024.1393706
Shanmugam, M., & Mahapatra, R. (2019). Pellet method of semen cryopreservation: Effect of cryoprotectants, semen diluents and chicken lines. Brazilian Archives of Biology and Technology, 62(e19180188), 1–10. https://doi.org/10.1590/1678-4324-2019180188
Shoshan-Barmatz, V., Ben-Hail, D., Admoni, L., Krelin, Y., & Tripathi, S. S. (2015). The mitochondrial voltage-dependent anion channel 1 in tumor cells. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1848(10), 2547–2575. https://doi.org/10.1016/j.bbamem.2014.10.040
Somashekar, L., Selvaraju, S., Parthipan, S., & Ravindra, J. P. (2015). Profiling of sperm proteins and association of sperm PDC-109 with bull fertility. Systems Biology in Reproductive Medicine, 61(6), 376–387. https://doi.org/10.3109/19396368.2015.1094837
Sutarno, & Setyawan, A. D. (2016). Review: The diversity of local cattle in Indonesia and the efforts to develop superior indigenous cattle breeds. Biodiversitas Journal of Biological Diversity, 17(1), 275–295. https://doi.org/10.13057/biodiv/d170139
Wahyudi, D., Udin, Z., & Afriani, T. (2023). Analysis of motility characteristic of Pesisir bulls sexed semen with different pre-freezing method based on computer-assisted sperm analyzer (CASA). Buletin Peternakan, 47(3), 136–141. https://doi.org/10.21059/buletinpeternak.v47i3.84331
Wang, P., Wang, Y.-F., Wang, H., Wang, C.-W., Zan, L.-S., Hu, J.-H., Li, Q.-W., Jia, Y.-H., & Ma, G.-J. (2014). HSP90 expression correlation with the freezing resistance of bull sperm. Zygote, 22(2), 239–245. https://doi.org/10.1017/S096719941300004X
Yoon, J.-W., Lee, S.-E., Kim, W.-J., Kim, D.-C., Hyun, C.-H., Lee, S.-J., Park, H.-J., Kim, S.-H., Oh, S.-H., Lee, D.-G., Pyeon, D.-B., Kim, E.-Y., & Park, S.-P. (2022). Evaluation of semen quality of Jeju Black Cattle (JBC) to select bulls optimal for breeding and establish freezing conditions suitable for JBC sperm. Animals, 12(5), 535. https://doi.org/10.3390/ani12050535
Zhang, X. (2018). Effects of different cytoprotectants combination on sperm survival, fertility and embryo development in Amur Sturgeon (Acipenser schrenckii). Animal and Veterinary Sciences, 6(4), 51–57. https://doi.org/10.11648/j.avs.20180604.11
Authors
Authors submitting manuscripts should understand and agree that copyright of manuscripts of the article shall be assigned/transferred to Tropical Animal Science Journal. The statement to release the copyright to Tropical Animal Science Journal is stated in Form A. This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License (CC BY-SA) where Authors and Readers can copy and redistribute the material in any medium or format, as well as remix, transform, and build upon the material for any purpose, but they must give appropriate credit (cite to the article or content), provide a link to the license, and indicate if changes were made. If you remix, transform, or build upon the material, you must distribute your contributions under the same license as the original.
Article Details
How to Cite
