Abstracts Special issue on Animal Physiology 2013, vol. 2, Abstracts special issue
Eliška Špaleková, Alexander Makarevich, Elena Kubovičová, Juraj Pivko
Biotechnology of Biotechnology
Semen cryopreservation and thawing processes may cause damage to sperm membrane structures, which can leads to lower viability and motility of post-thaw sperm. The aim of this study was to examine ultastructural alterations and membrane destabilization in frozen-thawed ram semen. Ultrastructural alterations were detected using transmission electron microscopy (TEM). Analyzed sperm heads were classified into 4 categories: I - sperm with intact plasma membrane and intact acrosome; II - sperm with waved or cracked plasma membrane; III – sperm with swollen or damaged acrosome; IV – sperm with pseudoacrosomal reaction and loss of acrosomal content. Sperm phosphatidylserine translocation (PS, membrane destabilization) was detected by fluorescently labelled Annexin-V-Fluos kit (Roche), and evaluated under a Leica fluorescent microscope. An annexin V-positivity in spermatozoa was localized on acrosomal part of sperm head, mitochondrial segment (MIs), post-acrosomal segment (PAs) and an equatorial segment (EQs). Control groups (CG) were represented by fresh sperm samples. In a fresh semen, about 77% of the sperm cells were distributed into categories I (19%) and II (58%), and 23% of the spermatozoa were classified into categories III (17%) and IV (6%). Oppositely, in frozen-thawed semen more noticeable membrane changes were observed. About 32% of sperm cells were belonging to the category III and 10% to the category IV, whilst only 11% were classified to the category I (intact membranes) and 47% to the category II (waved membranes). The most of the annexin-V positive sperm in CG were labelled in the acrosomal part of the head (26%) and in MIs (31%) or in both areas (28%). None of sperm in CG were marked in EQs. In frozen-thawed sperm annexin-V was localized mainly in the acrosomal (31%) and in PAs (23%) and only 10% of sperm were marked in MIs, whilst 3% of sperm were labelled in the EQs. These results indicate negative effect of frozen and thawing process on sperm membrane ultrastructure and stability mainly in acrosomal and post-acrosomal part of the sperm head.