Authors: I. V. CHISTIAKOVA, junior researcher of laboratory of developmental biology, T. I. KUZMINA, doctor of biological sciences, professor, the head of laboratory of developmental biology, V. Yu. DENISENKO, doctor of biological sciences, the leading researcher of laboratory of developmental biology, All-Russian Research Institute of Genetics and Breeding of Farm Animals – the branch of the Federal Research Center for Livestock – ARIL named after academician L. K. Ernst (ARRIGBA) (55a Moscow highway, 196601, Saint-Petersburg, Pushkin; phone: 8 (812) 451-71-15)
Keywords: viability, freezing, bovine spermatozoa.
Abstract: Previously, it was shown that pre-incubation of spermatozoa before freezing in the presence of compounds providing capacitation (dbcAMP, theophylline) increases the number of viable cells after thawing [1]. In this study, pre-incubation of spermatozoa before freezing was carried out in the presence of compounds that activating capacitation (caffeine) and do not af- fect this process (PRL and GTP).The evaluation of the sperm viability was carried out with using of fluorescent dye propidium iodide, which allows to divide cells into live and dead by changing of cell color. It was shown that the incubation of intact spermatozoa without subsequent freezing in medium without any additives, or in the presence of caffeine (2 mM), as well as with addition of 10 ng / ml prolactin (PRL) and 10 μM guanosine triphosphate (GTP) did not affect the proportion of living and dead cells. In all variants of the experiments, the number of live spermatozoa of bulls exceeded the number of dead cells about 3 times. In spermatozoa, which were incubated for 4 hours before freezing without the above reagents, the ratio of live / dead
cells was 1:1 after thawing. Pre-incubation of sperm cells before freezing in the presence of caffeine after freezing/thawing provided the increase in the number of viable spermatozoa and the decrease in the number of dead cells. Incubation of sperm for 4 hours before freezing in the presence of PRL and GTP, the freezing and the subsequent thawing the number of living and dead cells did not change. Thus, the incubation of cells with caffeine before cryopreservation providing the rise of capacitated cells leads to the increase in the number of viable spermatozoa after thawing.
