Main Article Content
The main objective of this preliminary study was to improve the ram semen quality by the removal of dead and apoptotic cells from the ejaculates. For this purpose, ram spermatozoa were incubated with the Dead Cell Removal kit and magnetically sorted using two procedures with different sample loading rate (Deplete and Depletes) by a fully automated cell sorter. Fresh semen samples (control) as well as both sorted fractions (negative and positive) were analysed for motility parameters using CASA and for the proportion of dead cells using flow cytometry. As expected, a significant increase (P < 0.05) in the number of dead cells and decrease (P < 0.05) in the spermatozoa motility were observed in the positive fractions when sorted by both procedures. However, the viability of negatively sorted spermatozoa was not improved and their motility was insignificantly decreased. In conclusion, although the presented study demonstrates the possible use of a MACS technique for the elimination of ram spermatozoa with compromised membrane, the chosen sorting procedures seem to be insufficient to obtain high purity of spermatozoa with intact membranes. More sensitive depletion programmes should be tested in further studies.
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
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
Dogan, S., Mason, M. C., Govindaraju, A., Belser, L., Kaya, A., Stokes, J. & Memili, E. (2013). Interrelationships between apoptosis and fertility in bull sperm. Journal of Reproduction and Development, 59(1), 18−26. https://doi.org/10.1262/jrd.2012-068
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
Januskauskas, A., Johannisson, A. & Rodriguez-Martinez, H. (2003). Subtle membrane changes in cryopreserved bull semen in relation with sperm viability, chromatin structure, and field fertility. Theriogenology, 60(4), 743−758. https://doi.org/10.1016/S0093-691X(03) 00050-5
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
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, 298. 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
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
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., 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
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