The aim of the study is to assess the influence of candidate genes encoding aquaporins (AQPs): AQP3, AQP7 and AQP11, associated with indicators of bull semen quality, for their further use as transcriptional biomarkers. Methods. Using quantitative reverse transcription polymerase chain reaction (RT-qPCR), we assessed the expression of selected genes in native and frozen-thawed sperm of 7 Holstein bulls and analyzed the correlations between the expression level of the studied genes with indicators of sperm quality that are significant for survival and fertilization. The following biochemical parameters of native and deconserved bull spermatozoa were assessed: motility, cell morphology, membrane integrity, viability, mitochondrial membrane potential, level of generation of reactive oxygen species (ROS). The scientific novelty of the study lies in the fact that for the first time in our country the relationship between the expression level of the AQP3, AQP7 and AQP11 genes and the quality of sperm of Holstein bulls was assessed. Results. The AQP11 gene can be recommended as a reliable transcriptional biomarker, since it had a high positive correlation with the content of living (0.821, p = 0.0145), normal (0.750, p = 0.0384) cells, and a negative correlation with the content of defective (–0.679, p = 0.0735), dead cells (–0.821, p = 0.0145) and ROS content (-0.821 p=0.0145) in frozen-thawed and native sperm. The AQP7 gene transcript of frozen-thawed sperm had an average negative correlation with indicators of dead sperm content (–0.727, p = 0.0545) and acrosome defects (–0.667, p = 0.0735) at a level close to significant. The AQP3 gene transcript had a significant positive correlation with the content of dead cells (0.786, p = 0.0251) in frozen-thawed sperm and a negative correlation with the content of defective, dead cells and ROS content in frozen-thawed and native sperm.
spermatozoa, bulls, fertility, semen quality, cryopreservation, media, RNA, transcripts, cryoresistance biomarkers, mitochondria, aquaporins
1. Grötter L. G., Cattaneo L., Marini P. E., Kjelland M. E., Ferré L. B. Recent advances in bovine sperm cryopreservation techniques with a focus on sperm post-thaw quality optimization. Reproduction in Domestic Animals. 2019; 54 (4): 30681204. DOI:https://doi.org/10.1111/rda.13409.
2. Khan M. Z., Sathanawongs A., Zhang Y. Impact of cryopreservation on spermatozoa freeze-thawed traits and relevance OMICS to assess sperm cryo-tolerance in farm animals. Frontiers in Veterinary Science. 2021; 8: 33718466. DOI:https://doi.org/10.3389/fvets.2021.609180. EDN: https://elibrary.ru/SDYESZ
3. Aliakbari F., Eshghifar N., Mirfakhraie R., Pourghorban P., Azizi F. Coding and non-coding RNAs, as male fertility and infertility biomarkers. International Journal of Fertility & Sterility. 2021; 15 (3): 34155862. DOI:https://doi.org/10.22074/IJFS.2021.134602.
4. Selvaraju S., Ramya L., Parthipan S., Swathi D., Binsila B.K. Deciphering the complexity of sperm transcriptome reveals genes governing functional membrane and acrosome integrities potentially influence fertility. Cell and Tissue Research. 2021; 385 (1): 33783607. DOI:https://doi.org/10.1007/s00441-021-03443-6. EDN: https://elibrary.ru/PKRVOV
5. Qin Z., Wang W., Ali M. A., Wang Y., Zhang Y. Transcriptome-wide m6A profiling reveals mRNA post-transcriptional modification of boar sperm during cryopreservation. BMC Genomics. 2021; 22 (1): 34344298. DOI:https://doi.org/10.1186/s12864-021-07904-8. EDN: https://elibrary.ru/IKLHCF
6. Shangguan A., Zhou H., Sun W., Ding R., Li X. Cryopreservation Induces Alterations of miRNA and mRNA Fragment Profiles of Bull Sperm. Frontiers in Genetics. 2020; 11: 32431726. DOI:https://doi.org/10.3389/fgene.2020.00419.
7. Kadivar A., Shams Esfandabadi N., Dehghani Nazhvani E., Shirazi A., Ahmadi E. Effects of cryopreservation on stallion sperm protamine messenger RNAs. Reproduction in Domestic Animals. 2020; 55 (3): 31885108. DOI:https://doi.org/10.1111/rda.13615.
8. Faraji S., Rashki Ghaleno L., Sharafi M., Hezavehei M., Totonchi M., et al. Gene Expression Alteration of Sperm-Associated Antigens in Human Cryopreserved Sperm. Biopreservation and Biobanking. 2021; 19 (6): 34009011. DOI:https://doi.org/10.1089/bio.2020.0165. EDN: https://elibrary.ru/SAASKZ
9. Ran M. X., Zhou Y. M., Liang K., Wang W. C., Zhang Y. Comparative analysis of microRNA and mRNA profiles of sperm with different freeze tolerance capacities in boar (Sus scrofa) and giant panda (Ailuropoda melanoleuca). Biomolecules. 2019; 9 (9): 31480517. DOI:https://doi.org/10.3390/biom9090432.
10. Fraser L., Brym P., Pareek C.S., Mogielnicka-Brzozowska M., Paukszto Ł. et al. Transcriptome analysis of boar spermatozoa with different freezability using RNA-Seq. Theriogenology. 2020; 142: 31711689. DOI:https://doi.org/10.1016/j.theriogenology.2019.11.001.
11. Peris-Frau P., Soler A. J., Iniesta-Cuerda M., et al. Sperm Cryodamage in Ruminants: Understanding the Molecular Changes Induced by the Cryopreservation Process to Optimize Sperm Quality. International Journal of Molecular Sciences. 2020; 21 (8): 32316334. DOI:https://doi.org/10.3390/ijms21082781.
12. Kordowitzki P., Kranc W., Bryl R., Kempisty B., Skowronska A., Skowronski M. T. The relevance of Aquaporins for the physiology, pathology, and aging of the female reproductive system in mammals. Cells. 2020; 9 (12): 33271827. DOI:https://doi.org/10.3390/cells9122570.
13. Fujii T., Hirayama H., Fukuda S., Kageyama S., Naito A., Yoshino H., Moriyasu S., Yamazaki T., Sakamoto K., Hayakawa H. Expression and localization of aquaporins 3 and 7 in bull spermatozoa and their relevance to sperm motility after cryopreservation. Journal of Reproduction and Development. 2018; 64: 29798965. DOI:https://doi.org/10.1262/jrd.2017-166.
14. Prieto-Martínez N., Morató R., Muiño R., Hidalgo C. O., Rodríguez-Gil J. E., Bonet S., Yeste M. Aquaglyceroporins 3 and 7 in bull spermatozoa: Identification, localisation and their relationship with sperm cryotolerance. Reproduction, Fertility and Development. 2017; 29: 27221122. DOI:https://doi.org/10.1071/RD16077.
15. Calamita G., Delporte C. Involvement of aquaglyceroporins in energy metabolism in health and disease. Biochimie. 2021; 188: 33689852. DOI:https://doi.org/10.1016/j.biochi.2021.03.001. EDN: https://elibrary.ru/VWSKUO
16. Prieto-Martínez N., Vilagran I., Morató R., Rivera Del Álamo M. M., Rodríguez-Gil J. E., Bonet S., Yeste M. Relationship of aquaporins 3 (AQP3), 7 (AQP7), and 11 (AQP11) with boar sperm resilience to withstand freeze-thawing procedures. Andrology. 2017; 5 (6): 28941027. DOI:https://doi.org/10.1111/andr.12410.
17. Morató R., Prieto-Martínez N., Muiño R., Hidalgo C. O., Rodríguez-Gil J. E., Bonet S., Yeste M. Aquaporin 11 is related to cryotolerance and fertilising ability of frozen-thawed bull spermatozoa. Reproduction, Fertility and Development. 2018; 30: 29365310. DOI:https://doi.org/10.1071/RD17340.
18. Highland H. N., Rishika A. S., Almira S. S., Kanthi P. B. Ficoll-400 density gradient method as an effective sperm preparation technique for assisted reproductive techniques. Journal of Human Reproductive Sciences. 2016; 9 (3): 27803588. DOI:https://doi.org/10.4103/0974-1208.192070. EDN: https://elibrary.ru/YEUFSH
19. Harshitha R., Arunraj D. R. Real-time quantitative PCR: A tool for absolute and relative quantification. Biochemistry and Molecular Biology Education. 2021; 49 (5): 34132460. DOI:https://doi.org/10.1002/bmb.21552. EDN: https://elibrary.ru/CKJOCM
20. Bonilla-Correal S., Noto F., Garcia-Bonavila E., Rodríguez-Gil J. E., Yeste M., Miro J. First evidence for the presence of aquaporins in stallion sperm. Reproduction in Domestic Animals. 2017; 4: 29052325. DOI:https://doi.org/10.1111/rda.13059.
21. Delgado-Bermúdez A., Recuero S., Llavanera M., Mateo-Otero Y., Sandu A., Barranco I., Ribas-Maynou J., Yeste M. Aquaporins are essential to maintain motility and membrane lipid architecture during mammalian sperm capacitation. Frontiers in Cell and Developmental Biology. 2021; 9: 34540822. DOI:https://doi.org/10.3389/fcell.2021.656438. EDN: https://elibrary.ru/CLUUTN
