J Vet Sci.  2011 Sep;12(3):267-272. 10.4142/jvs.2011.12.3.267.

Transmission electron microscopy for characterization of acrosomal damage after Percoll gradient centrifugation of cryopreserved bovine spermatozoa

• Oliveira L
• Hossepian de Lima VFM
• Levenhagen MA
• dos Santos RM
• Assumpcao
• Jacomini J
• de Andrade AFC
• de Arruda
• Beletti ME

• KMID: 1067399
• DOI: http://doi.org/10.4142/jvs.2011.12.3.267

Abstract

The objective of this study was to characterize acrosomal ultrastructure following discontinuous Percoll gradient centrifugation of cryopreserved bovine sperm. Semen was collected from six bulls of different breeds and three ejaculates per bull were evaluated. Frozen semen samples were thawed and the acrosomal region of sperm cells was evaluated by transmission electron microscopy (TEM) before (n = 18) and after (n = 18) Percoll centrifugation. The evaluation of 20 sperm heads from each of the 36 samples analyzed ensured that a large number of cells were investigated. The data were subjected to analysis of variance at a level of significance of 5%. Percoll centrifugation reduced the percentage of sperm exhibiting normal acrosomes (from 61.77 to 30.24%), reduced the percentage of sperm presenting atypical acrosome reactions (from 28.38 to 4.84%) and increased the percentage of sperm exhibiting damage in the acrosome (from 6.14 to 64.26%). The percentage of sperm with typical acrosome reactions was not significantly different before (3.70%) and after (0.67%) centrifugation. TEM distinguished four different types of acrosomal status and enabled ultrastructural characterization of acrosomal injuries. The percentage of sperm exhibiting normal acrosomes decreased and damage in the acrosome was the most frequent acrosomal injury with the Percoll gradient centrifugation protocol utilized.

Keyword

acrosomal ultrastructure; bovine; Percoll; sperm selection

MeSH Terms

Acrosome/*pathology/ultrastructure
Animals
Cattle/*physiology
Cell Membrane/*pathology/ultrastructure
Cell Separation/veterinary
Centrifugation, Density Gradient/veterinary
Cryopreservation/veterinary
Male
Microscopy, Electron, Transmission/veterinary
Povidone/*adverse effects
Silicon Dioxide/*adverse effects
Spermatozoa/pathology/ultrastructure

Figure

• 

  Fig. 1 Electron micrographs of a sagittal section of a bovine sperm head showing a spermatozoon. (A) Normal acrosome, sperm head presenting an intact acrosomal membrane completely surrounding the acrosomal ground substance. (B) "Physiological" acrosome reaction, sperm head presenting swelling of acrosomal ground substance with vesicles of fused plasma and outer acrosomal membranes. (C) Atypical acrosome reaction, sperm head presenting swelling of acrosomal ground substance dispersed under the swollen outer acrosomal membrane. (D) Damage in the acrosome, sperm head presenting rupture of the acrosomal membrane with swelling of acrosomal ground substance in restricted points. A: ×85,000; B~D: ×50,000.

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