ULTRASTRUCTURAL AND ANNEXIN V-DETECTED DAMAGES IN RAM FROZEN-THAWED SEMEN

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Abstracts Special issue on Animal Physiology 2013, vol. 2, Abstracts special issue
pages: 10
Article type: Biotechnology of Biotechnology
Abstract: 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.
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