DEPLETION OF DEAD SPERMATOZOA DID NOT SUFFICIENTLY IMPROVE THE QUALITY OF RAM SEMEN: SHORT COMMUNICATION
Keywords:
ram semen, CASA, MACS, depletion, dead spermatozoa, flow cytometryAbstract
The main objective of this study was to improve the motility and overall quality of spermatozoa from the fresh ram semen samples using more sensitive depletion programmes of AutoMACS Pro Separator to remove dead sperm cells. Briefly, ram spermatozoa, given either at high (108) or low (107) concentrations, were incubated with the Dead Cell Removal kit reagent and magnetically sorted using two very sensitive depletion programmes (Depl05 and Depl025) with different sample loading rates. Fresh unsorted semen samples (control) as well as both sorted fractions (negative and positive) were analysed using computer-assisted sperm assay (CASA) to assess the motility parameters and using flow cytometry to evaluate the proportion of dead cells and overall magnetic-activated cell sorting (MACS) efficiency. We obtained significantly (P < 0.01) lower percentage of dead spermatozoa only after sorting the high concentrated spermatozoa using a Depl025 programme compared to control samples. However, the negative fractions still contained more than 20 % of dead cells irrespective of the sorting programme used. In addition, the motility parameters were significantly improved neither by the used sorting strategy nor by the adjustment of spermatozoa concentration. In conclusion, further optimization of this method is required in order to sufficiently remove dead spermatozoa and to improve the spermatozoa motility.
References
Agarwal, A., Varghese, A. C. & Sharma, R. K. (2009). Markers of oxidative stress and sperm chromatin integrity. In Molecular Endocrinology (pp. 377−402). Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-378-7_24
Baláži, A., Vašíček, J., Svoradová, A., Macháč, M., Jurčík, R., Huba, J., Pavlík, I. & Chrenek, P. (2020). Comparison of three different methods for the analysis of ram sperm concentration. Slovak Journal of Animal Science, 53(2), 53−58.
Bucar, S., Gonçalves, A., Rocha, E., Barros, A., Sousa, M. & Sá, R. (2015). DNA fragmentation in human sperm after magnetic-activated cell sorting. Journal of Assisted Reproduction and Genetics, 32(1), 147−154. https://doi.org/10.1007/s10815-014-0370-5
Delbès, G., Herrero, M. B., Troeung, E. T. & Chan, P. T. K. (2013). The use of complimentary assays to evaluate the enrichment of human sperm quality in asthenoteratozoospermic and teratozoospermic samples processed with Annexin-V magnetic activated cell sorting. Andrology, 1(5), 698−706. https://doi.org/10.1111/j.2047-2927.2013.00106.x
Di Santo, M., Tarozzi, N., Nadalini, M., & Borini, A. (2011). Human sperm cryopreservation: update on techniques, effect on DNA integrity, and implications for ART. Advances in Urology, 2012. https://doi.org/10.1155/2012/854837
Esteves, S. C., Zini, A., Aziz, N., Alvarez, J. G., Sabanegh Jr, E. S., & Agarwal, A. (2012). Critical appraisal of World Health Organization's new reference values for human semen characteristics and effect on diagnosis and treatment of subfertile men. Urology, 79(1), 16−22. https://doi.org/10.1016/j.urology.2011.08.003
Glander, H. J., Schiller, J., Süss, R., Paasch, U., Grunewald, S. & Arnhold, J. (2002). Deterioration of spermatozoal plasma membrane is associated with an increase of sperm lyso-phosphatidylcholines. Andrologia, 34(6), 360−366. https://doi.org/10.1046/j.1439-0272.2002.00508.x
Grunewald, S. & Paasch, U. (2013). Sperm selection for ICSI using annexin V. In Spermatogenesis (pp. 257−262). Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-038-0_23
Grunewald, S., Paasch, U. & Glander, H. J. (2001). Enrichment of non–apoptotic human spermatozoa after cryopreservation by immunomagnetic cell sorting. Cell and Tissue Banking, 2(3), 127−133. https://doi.org/10.1023/A:1020188913551
Merino-Ruiz, M., Morales-Martínez, F. A., Navar-Vizcarra, E., Valdés-Martínez, O. H., Sordia-Hernández, L. H., Saldívar-Rodríguez, D. & Vidal-Gutiérrez, O. (2019). The elimination of apoptotic sperm in IVF procedures and its effect on pregnancy rate. JBRA Assisted Reproduction, 23(2), 112. https://doi.org/10.5935/1518-0557.20190007
Mrkun, J., Dolenšek, T., Knific, T., Pišlar, A., Kosec, M., Kos, J. & Zrimšek, P. (2014). Elimination of apoptotic boar spermatozoa using magnetic activated cell sorting. Acta Veterinaria Brno, 83(1), 13−18. https://doi.org/10.2754/avb201483010013
Niemann, H., Kuhla, B., & Flachowsky, G. (2011). Perspectives for feed-efficient animal production. Journal of Animal Science, 89(12), 4344−4363. https://doi.org/10.2527/jas.2011-4235
Oehninger, S., Duru, N. K., Srisombut, C., & Morshedi, M. (2000). Assessment of sperm cryodamage and strategies to improve outcome. Molecular and Cellular Endocrinology, 169(1−2), 3−10. https://doi.org/10.1016/s0303-7207(00)00343-9
Oseguera-López, I., Ruiz-Díaz, S., Ramos-Ibeas, P. & Pérez-Cerezales, S. (2019). Novel techniques of sperm selection for improving IVF and ICSI outcomes. Frontiers in Cell and Developmental Biology, 7. https://doi.org/10.3389/fcell.2019.00298
Paasch, U., Grunewald, S., Fitzl, G. & Glander, H. J. (2003). Deterioration of plasma membrane is associated with activated caspases in human spermatozoa. Journal of Andrology, 24(2), 246−252. https://doi.org/10.1002/j.1939-4640.2003.tb02669.x
Roca, J., Martinez-Alborcia, M. J., Gil, M. A., Parrilla, I., & Martinez, E. A. (2013). Dead spermatozoa in raw semen samples impair in vitro fertilization outcomes of frozen-thawed spermatozoa. Fertility and Sterility, 100(3), 875−881. https://doi.org/10.1016/j.fertnstert.2013.05.020
Said, T., Agarwal, A., Grunewald, S., Rasch, M., Baumann, T., Kriegel, C. & Paasch, U. (2006). Selection of nonapoptotic spermatozoa as a new tool for enhancing assisted reproduction outcomes: an in vitro model. Biology of Reproduction, 74(3), 530−537. https://doi.org/10.1095/biolreprod.105.046607
Tournaye, H. (2012). Male factor infertility and ART. Asian Journal of Andrology, 14(1), 103. https://doi.org/10.1038/aja.2011.65
Vašíček J., Baláži A., Kuželová L. & Chrenek P. (2014b). Comparison of two commercial kits for the elimination of rabbit spermatozoa with damaged membrane via MACS technique. In Animal Physiology 2014 (pp. 277−283), Prušánky, 2014. Brno: Mendel University in Brno. ISBN 978-80-7375-971-1
Vasicek, J., Pivko, J. & Chrenek, P. (2014a). Reproductive performance of New Zealand White rabbits after depletion of apoptotic spermatozoa. Folia Biologica, 62(2), 109−117. https://doi.org/10.3409/fb62_2.109
Vašíček, J., Svoradová, A., Baláži, A., Jurčík, R., Macháč, M., & Chrenek, P. (2020). Use of dead cell removal kit for the improvement of ram semen quality. Slovak Journal of Animal Science, 53(04), 205−208.
Vendrell, X., Ferrer, M., García-Mengual, E., Munoz, P., Trivino, J. C., Calatayud, C. & Ruiz-Jorro, M. (2014). Correlation between aneuploidy, apoptotic markers and DNA fragmentation in spermatozoa from normozoospermic patients. Reproductive Biomedicine Online, 28(4), 492−502. https://doi.org/10.1016/j.rbmo.2013.12.001
