MONITORING Of ChROMATIN INTEGRITY ChANGES IN ThE POPULATION Of MOTILE BOVINE SPERM CAPACITATED IN VITRO

REČKOVá, Z., MACHATKOVá, M., RyBář, R., MáCHAL, L.: Monitoring of chromatin integrity changes in the population of motile bovine sperm capacitated in vitro. Acta univ. agric. et silvic. Mendel. Brun., 2007, LV, No. 2, pp. 65–70 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. bulls, spermatozoa, chromatin integrity, flow cytometry 66 Z. Rečková, M, Machatková, R. Rybář, L. Máchal A sufficient number of viable and motile spermatozoa with intact acrosome and undamaged chromatin structure are needed to be able to penetrate and fertilize oocytes in in vitro conditions. Different separation techniques can be used to obtain populations of viable and motile spermatozoa from frozen-thawed bull sperm. The most commonly used separation method for frozen-thawed sperm separation is the Percoll gradient centrifugation (Galli et al., 2003). Major advantage of motile sperm separation by Percoll gradient compared to other methods is a relatively high yield of spermatozoa (Parrish et al., 1995). Sperm separation also increases the proportion of viable spermatozoa with intact acrosome (Alomar et al., 2006), however, their chromatin structure can be disrupted by the separation process. Sperm of some bulls are highly sensitive to such a treatment, therefore more careful methods are needed for separation, as are the swim-up method or centrifugation on Sephadex column. further, it was confirmed that increased levels of reactive oxygen groups can occur during centrifugation with a subsequent risk of sperm chromatin damage (Aitken and Clarkson, 1988; Zalata et al., 1995). Successful oocyte fertilization necessitates a certain portion of spermatozoa to undergo an acrosomal reaction in a corresponding time. Acrosome reaction of spermatozoa can be stimulated by supplementation of the fertilization media with different capacitation agents, most frequently heparin (Pereira et al., 2000; Mendes et al., 2003). Both separation and capacitation of spermatozoa can induce specific damage of the chromatin structure (Silva and Gadella, 2006). Boe-Hansen et al. (2003) suppose that lower fertility of some bulls under in vitro conditions could be due to chromatin integrity changes during preparation of spermatozoa for oocyte fertilization or at their capacitation. fatehi et al (2006) found that bovine sperm with damaged chromatin are able of fertilization, however, their embryos show impaired development which is in correlation with the level of sperm chromatin damage. Assessment of sperm DNA integrity and other qualitative and quantitative characteristics as are viability, acrosome intactness and mitochondrial activity can be done by flow cytometry (Graham et al., 1990; Watson et al., 2002; Nagy et al., 2004; Gillan et al., 2005). Sperm Chromatin Structure Assay described by Evenson et al. (2002) is a suitable method for the assessment of bovine sperm chromatin integrity. Monitoring of chromatin changes in the population of spermatozoa capacitated under standard conditions could result in more precise prediction of the fertilization ability in individual bulls. Therefore it is of importance to find out whether chromatin integrity changes occur during separation and capacitation of spermatozoa and to elucidate impact of those changes on efficiency of in vitro fertilization in individual bulls. The objective of our study was to standardize of the method of chromatin integrity assessment in the population of motile spermatozoa, and monitoring of changes occurring during their separation and capacitation. MATERIALS AND METhODS