Monitorování změn integrity chromatinu v populaci motilních bovinních spermií kapacitovaných v podmínkách in vitro

Abstract

The objective of our study was to standardize a method for chromatin integrity assessment in a separated population of bovine sperm and monitor the changes occurring during sperm capacitation stimulated with heparin. Frozen sperm of 11 young bulls of the Czech pied breed with a defined fertility in both in vitro system (from 12.9% to 25.8% embryos) and in insemination (from 60.2% to 66.4% pregnancy) was used in our experiments.Bovine spermatozoa were isolated by Percoll gradient centrifugation from frozen-thawed semen using Tyrode’s medium (SP-Talp) and resuspended in a fertilization medium (IVF-Talp). The spermatozoa were incubated at laboratory temperature at a concentration 25 × 106 per cm3 for 6 h either in IVF-Talp medium with heparin (H+) or without heparin (H–). Samples were obtained immediately after sperm thawing (PS), following motile spermatozoa separation (P0), and their three (P3) and six hour (P6) incubation. The samples were examined by flow cytometry. Two measurements were carried out in each of the samples so that a total of 10 thousand spermatozoa were analysed. Proportion of spermatozoa with undetectable DNA fragmentation index (non-DFI sperm) i.e. spermatozoa with undamaged chromatin structure were determined using SCSA-soft software.Chromatin integrity changes of spermatozoa before and after separation and capacitation differed markedly in individual bulls. Separation of motile spermatozoa increased significantly the mean proportion of non-DFI sperm in tested bulls (from 94.2 to 96.4%, P ≤ 0.01). While in most of the bulls the mean proportion of non-DFI sperm remained nearly constant during incubation (H–) (mean, P0 – 96.4%, P3 – 95.6%, P6 – 95.5%), it gradually decreased during capacitation (H+) (mean, P0 – 96.4%, P3 – 95.2%, P6 – 94.2%). The differences were statistically significant (P0 vs. P3H+, P0 vs. P6H+, P ≤ 0.05). Significant difference (P ≤ 0.05) in the mean proportion on non-DFI sperm was also found between capacitated (P6H+) and incubated (P6H–) spermatozoa.The results of our study suggest the following outcomes. Separation of motile spermatozoa by Percoll gradient increased the proportion of spermatozoa with undamaged chromatin structure. Sperm incubation induced gentle damage of chromatin integrity which was potentiated by heparin in capacitated spermatozoa. The proportion of spermatozoa with undamaged chromatin structure remained relatively high in the course of capacitation, therefore we can assume it to be high enough for a potential oocyte fertilization.