Cryopreservation of fluorescence activated cell sorted boar spermatozoa based on extracellular ubiquitinationák A., Krylov V. (2016): Cryopreservation of fluorescence activated cell sorted boar spermatozoa based on extracellular ubiquitination. Czech J. Anim. Sci., 61: 310-316.
download PDF
The present study is focused on the methodology of fluorescence activated cell sorting (FACS) of spermatozoa stained by the antibody against extracellular surface marker ubiquitin (eUb) and subsequent protocol for their long term storage in liquid nitrogen (LN). High level of spermatozoa surface ubiquitination has been previously discussed as a negative quality marker. From a general point of view, any other outer membrane antigen would be compatible with our approach. Regarding our experimental design we found that only those insemination doses with at least 40% of motile spermatozoa after freezing and thawing (F/T) in the egg-yolk medium with lactose are suitable for the subsequent antibody staining and FACS. The sorting rate was sufficient for the preparation of up to 20 spermatozoa aliquots for intracytoplasmic sperm injections (ICSI). Two significantly different groups with good freezability were prepared and stored in LN (0.73% contamination of spermatozoa with high eUb level in non-ubiquitinated group and reversely 6.65% spermatozoa without eUb in highly ubiquitinated group). Sperm viability after FACS varied from 11 to 28% regardless of the used media (P = 0.15). Required viability of F/T sorted spermatozoa was obtained by using Solusem® extender as a load and collection medium. In this case 12% of viable spermatozoa with progressive motility in low eUb level group and 7% in high eUb level group (P < 0.05) were detected. Our approach allows obtaining sufficient number of viable spermatozoa for subsequent artificial fertilization by ICSI. This procedure could be used for a wide variety of spermatozoa sorting based on different surface markers.
Alkmin Diego V., Parrilla Inmaculada, Tarantini Tatiana, Parlapan Laura, del Olmo David, Vazquez Juan M., Martinez Emilio A., Roca Jordi (2014): Intra- and interboar variability in flow cytometric sperm sex sorting. Theriogenology, 82, 501-508
Balao da Silva C.M., Ortega Ferrusola C., Morillo Rodriguez A., Gallardo Bolaños J.M., Plaza Dávila M., Morrell J.M., Rodriguez Martínez H., Tapia J.A., Aparicio I.M., Peña F.J. (2013): Sex sorting increases the permeability of the membrane of stallion spermatozoa. Animal Reproduction Science, 138, 241-251
Barbas J. P., Mascarenhas R. D. (2009): Cryopreservation of domestic animal sperm cells. Cell and Tissue Banking, 10, 49-62
Ciereszko A., Ottobre J.S., Glogowski J. (2000): Effects of season and breed on sperm acrosin activity and semen quality of boars. Animal Reproduction Science, 64, 89-96
del Olmo David, Parrilla Inmaculada, Sanchez-Osorio Jonatan, Gomis Jesus, Angel Miguel A., Tarantini Tatiana, Gil Maria A., Cuello Cristina, Vazquez Jose L., Roca Jordi, Vaquez Juan M., Martinez Emilio A. (2014): Successful laparoscopic insemination with a very low number of flow cytometrically sorted boar sperm in field conditions. Theriogenology, 81, 315-320
Eskandari-Shahraki M., Tavalaee M., Deemeh M. R., Jelodar Gh. A., Nasr-Esfahani M. H. (2013): Proper ubiquitination effect on the fertilisation outcome post-ICSI. Andrologia, 45, 204-210
Garner Duane L. (2006): Flow cytometric sexing of mammalian sperm. Theriogenology, 65, 943-957
Gil M.A., Roca J., Cremades T., Hernandez M., Vazquez J.M., Rodriguez-Martinez H., Martinez E.A. (2005): Does multivariate analysis of post-thaw sperm characteristics accurately estimate in vitro fertility of boar individual ejaculates? Theriogenology, 64, 305–316.
Hansen C., Vermeiden T., Vermeiden J.P.W., Simmet C., Day B.C., Feitsma H. (2006): Comparison of FACSCount AF system, Improved Neubauer hemocytometer, Corning 254 photometer, SpermVision, UltiMate and NucleoCounter SP-100 for determination of sperm concentration of boar semen. Theriogenology, 66, 2188-2194
Hodjat M, Akhondi MA, Al-Hasani S, Mobaraki M, Sadeghi MR (2008): Increased sperm ubiquitination correlates with abnormal chromatin integrity. Reproductive BioMedicine Online, 17, 324-330
Jo Hyun-Tae, Bang Jae-Il, Kim Seong-Su, Choi Byung-Hyun, Jin Jong-In, Kim Heyng-Lyool, Jung In-Suk, Suh Tae-Kwang, Ghanem Nasser, Wang Zhongde, Kong Il-Keun (2014): Production of female bovine embryos with sex-sorted sperm using intracytoplasmic sperm injection: Efficiency and in vitro developmental competence. Theriogenology, 81, 675-682.e1
Knox Robert V., Yantis Brandon M. (2014): The effect of numbers of frozen-thawed boar sperm and addition of prostaglandin F2α at insemination on fertility in pigs. Animal Reproduction Science, 151, 194-200
Kren R., Kikuchi K., Nakai M., Miyano T., Ogushi S., Nagai T., Suzuki S., Fulka J., Fulka Jr. J. (2003): Intracytoplasmic sperm injection in the pig: where is the problem? The Journal of Reproduction and Development, 49, 271–273.
Marchiani S., Tamburrino L., Forti G., Baldi E., Muratori M. (2007): M540 bodies and their impact on flow cytometric analyses of human spermatozoa. Society of Reproduction and Fertility Supplement, 65, 509–514.
Martinez-Alborcia Maria J., Valverde Anthony, Parrilla Inmaculada, Vazquez Juan M., Martinez Emilio A., Roca Jordi, Schlatt Stefan (2012): Detrimental Effects of Non-Functional Spermatozoa on the Freezability of Functional Spermatozoa from Boar Ejaculate. PLoS ONE, 7, e36550-
Medrano A., Holt W. V., Watson P. F. (2009): Controlled freezing studies on boar sperm cryopreservation. Andrologia, 41, 246-250
Muratori M. (2004): Sperm ubiquitination positively correlates to normal morphology in human semen. Human Reproduction, 20, 1035-1043
Nagashima H, Matsunari H, Nakano K, Watanabe M, Umeyama K, Nagaya M (2012): Advancing Pig Cloning Technologies Towards Application in Regenerative Medicine. Reproduction in Domestic Animals, 47, 120-126
Ozanon C. (2005): Clinical adaptation of the sperm ubuquitin tag immunoassay (SUTI): relationship of sperm ubiquitylation with sperm quality in gradient-purified semen samples from 93 men from a general infertility clinic population. Human Reproduction, 20, 2271-2278
Purdy P.H. (2008): Ubiquitination and its influence in boar sperm physiology and cryopreservation. Theriogenology, 70, 818-826
Pursel V. G., Johnson L. A., Schulman L. L. (1972): Interaction of Extender Composition and Incubation Period on Cold Shock Susceptibility of Boar Spermatozoa. Journal of Animal Science, 35, 580-
Sutovsky Peter, Neuber Evelyn, Schatten Gerald (2002): Ubiquitin-dependent sperm quality control mechanism recognizes spermatozoa with DNA defects as revealed by dual ubiquitin-TUNEL assay. Molecular Reproduction and Development, 61, 406-413
Sutovsky Peter, Aarabi Mahmoud, Miranda-Vizuete Antonio, Oko Richard (2015): Negative biomarker based male fertility evaluation: Sperm phenotypes associated with molecular-level anomalies. Asian Journal of Andrology, 17, 554-
Varum Sandra, Bento Carla, Sousa Ana Paula M., Gomes-Santos Carina S.S., Henriques Paula, Almeida-Santos Teresa, Teodósio Cristina, Paiva Artur, Ramalho-Santos João (2007): Characterization of human sperm populations using conventional parameters, surface ubiquitination, and apoptotic markers. Fertility and Sterility, 87, 572-583
Vazquez J.M., Parrilla I., Roca J., Gil M.A., Cuello C., Vazquez J.L., Martínez E.A. (2009): Sex-sorting sperm by flow cytometry in pigs: Issues and perspectives. Theriogenology, 71, 80-88
Watson PF (1995): Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reproduction, Fertility and Development, 7, 871-
White IG (1993): Lipids and calcium uptake of sperm in relation to cold shock and preservation: a review. Reproduction, Fertility and Development, 5, 639-
Yi Y.J, Im G.S, Park C.S (2002): Lactose-egg yolk diluent supplemented with N-acetyl-d-glucosamine affect acrosome morphology and motility of frozen-thawed boar sperm. Animal Reproduction Science, 74, 187-194
download PDF

© 2019 Czech Academy of Agricultural Sciences