Acta Univ. Agric. Silvic. Mendelianae Brun. 2007, 55(2), 65-70 | DOI: 10.11118/actaun200755020065

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

Zuzana Rečková1,2, Marie Machatková1, Roman Rybář1, Ladislav Máchal2
1 Oddělení genetiky a reprodukce, Výzkumný ústav veterinárního lékařství, Hudcova 70, 621 00 Brno, Česká republika
2 Ústav chovu a šlechtění zvířat, Mendelova zemědělská a lesnická univerzita, Zemědělská 1, 613 00 Brno, Česká republika

Cílem práce bylo standardizovat metodu hodnocení integrity chromatinu u separované populace bovinních spermií a monitorovat změny, ke kterým dochází v průběhu jejich kapacitace stimulované heparinem.
V experimentu bylo použito zmrazené sperma 11 mladých býků českého strakatého plemene s definovanou plodností v in vitro systému (od 12,9 % do 25,8 % embryí) i v inseminaci (od 60,2 % do 66,4 % březosti). Bovinní sperma bylo separováno pomocí Tyrodova média (SP-Talp) na gradientu Percollu a resuspendováno ve fertilizačním médiu (IVF-Talp). Spermie byly inkubovány při laboratorní teplotě v koncentraci 25 × 106 v cm3 po dobu šesti hodin jak v médiu doplněném o heparin (H+), tak i bez heparinu (H-). Vzorky byly odebrány ihned po rozmrazení spermatu (PS), po separaci spermií (P0), jejich, tříhodinové (P3) a šestihodinové (P6) inkubaci. Získané vzorky byly vyšetřeny pomocí průtokové cytometrie, metodou popsanou Evensonem et al., (2002). U každého vzorku bylo celkem hodnoceno 10 tisíc spermií ve dvou měřeních. Podíl spermií s nedetekovatelným DNA fragmentačním indexem (non-DFI sperm), tj. spermií s nepoškozenou strukturou chromatinu byl stanoven pomocí SCSA-soft softwaru. Získané hodnoty byly statisticky zpracovány v programu STATISTICA pomocí Wilcoxnova testu.
Změny integrity chromatinu spermií v průběhu jejich separace a kapacitace byly u sledovaných býků značně individuální. Separace motilních spermií signifikantně zvýšila průměrný podíl non-DFI spermií u všech sledovaných býků, průměrně od 94,2 % do 96,4 % (P ≤ 0,01). Zatímco u většiny býků zůstal průměrný podíl non-DFI spermií během jejich inkubace (H-) téměř konstantní (průměr, P0 - 96,4 %, P3 - 95,6 %, P6 - 95,5 %), tak v průběhu kapacitace (H+) se průměrný podíl non-DFI spermií postupně snižoval (průměr, P0 - 96,4 %, P3 - 95,2 %, P6 - 94,2 %). Zjištěné rozdíly byly statisticky průkazné (P0 vs. P3H+a P0 vs. P6H+, P ≤ 0,05). Statisticky významný rozdíl (P ≤ 0,05) v průměrném podílu non-DFI spermií byl také nalezen mezi kapacitovanými (P6H+) a inkubovanými spermiemi (P6H-).
Z výsledků naší studie je možné vyvodit následující závěry. Separace motilních spermií na gradientu Percollu zvýšila podíl spermií s neporušenou strukturou chromatinu. Inkubace spermií vyvolala mírné poškození integrity chromatinu spermií, které bylo u kapacitovaných spermií potencováno účinkem heparinu. V průběhu procesu kapacitace zůstal podíl spermií s neporušenou strukturou chromatinu relativně vysoký a lze tedy předpokládat, že byl dostatečný pro potenciální fertilizaci oocytů.

býci, spermie, integrita chromatinu, průtoková cytometrie

Monitoring of chromatin integrity changes in the population of motile bovine sperm capacitated in vitro

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.

Keywords: bulls, spermatozoa, chromatin integrity, flow cytometry
Grants and funding:

This study was supported by Grants MZE 0002716201 and 1B44018 of the Ministry of Agriculture of the Czech Republic and by project of MSMT - MSM 6215648905.

Received: February 5, 2007; Published: November 27, 2014  Show citation

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Rečková, Z., Machatková, M., Rybář, R., & Máchal, L. (2007). Monitoring of chromatin integrity changes in the population of motile bovine sperm capacitated in vitro. Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis, 55(2), 65-70. doi: 10.11118/actaun200755020065
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