Chromatin and morphometry alterations of bovine sperm head after Percoll (TM) density gradient centrifugation

The aim of this work was to assess the morphometry and chromatin integrity of bovine sperm head after a three layers discontinuous Percoll (TM) density gradient centrifugation. Frozen semen doses were obtained from six bulls of different breeds, including three taurine and three Zebu animals. Three ejaculates per bull were evaluated. The semen doses were thawed and two smears were made from each sample before (control) and after the Percoll (TM) centrifugation (Percoll (TM) group). The smears were stained with toluidine blue and grayscale digital images were captured and processed in Scilab environment software. It was observed that chromatin heterogeneity was reduced (P<0.05) and chromatin decondensation was increased (P<0.05) after the Percollm treatment utilized. In addition, it was observed that sperm head length was higher (P<0.05) and the side symmetry was lower (P<0.05) in centrifuged sperm cells. When analyzed separately by subspecies, it was observed that the decrease (P<0.05) in chromatin heterogeneity and the increase (P<0.05) in chromatin decondensation occurred in Zebu sperm heads. In addition, the length and the width:length ratio of sperm heads was affected by Percoll (TM) centrifugation in Zebu semen. In conclusion, the three layers discontinuous Percoll (TM) centrifugation increased the chromatin decondensation and the morphometric alterations of frozen-thawed bovine semen. However, the real implication of these findings in fertility rates of centrifuged sperm must be investigated. (C) 2011 Elsevier B.V. All rights reserved.

Effects of discontinuous Percoll gradient centrifugation on the quality of bovine spermatozoa evaluated with computer-assisted semen analysis and fluorescent probes association

The objective of this study was to evaluate the quality of bovine frozen-thawed sperm cells after Percoll gradient centrifugation. Frozen semen doses were obtained from six bulls of different breeds, including three taurine and three Zebu animals. Four ejaculates per bull were evaluated before and after discontinuous Percoll gradient centrifugation. Sperm motility was assessed by computer-assisted semen analysis and the integrity of the plasma and acrosomal membranes, as well as mitochondrial function, were evaluated using a combination of fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide. The procedure of Percoll gradient centrifugation increased the percentage of total and progressive sperm motility, beat frequency, rectilinear motility, linearity and rapidly moving cells. In addition, the percentage of cells with intact plasma membrane and mitochondrial membrane potential was increased in post-centrifugation samples. However, the percentage of sperm cells with intact acrosomal membrane was markedly reduced. The method used selected the motile cells with intact plasma membrane and higher mitochondrial functionality in frozen-thawed bull semen, but processing, centrifugation and/or the Percoll medium caused damage to the acrosomal membrane.

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

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.

Habitat use, reproductive traits and social interactions in a stream-dweller treefrog endemic to the Brazilian Cerrado

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Egg traits and physiological neonatal chick parameters from broiler breeder at different ages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)