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

Abstract

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.

Isolate® and Optiprep® minigradients as alternatives for sperm selection in bovine in vitro embryo production

“…In contrast, Cesari et al (2006) reported that Percoll † significantly decreased the proportion of sperm with an intact acrosome. The inconsistent results may be due to diverse concentrations and volumes of the Percoll † gradients used in various studies (Oliveira et al 2010). Nevertheless, we believe that this is more related to the gradient concentrations, because in our study, there was no significant difference for various volumes, i.e., 4.0, 1.2 and 0.8 mL for P, MO, and MP and MI, respectively.…”

“…The difference in DNA mass between X and Y chromosome-bearing sperm causes a difference in weight, and density may promote their separation by density gradients (Parrilla et al 2004;Machado et al 2009). It is well known that X and Y sperm have differences in size and DNA content, and consequently a…”

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

“…Several authors are in agreement that the percentage of sperm with intact plasma membrane is improved after Percoll treatment of frozen-thawed bull semen [4,12,14,16]. However, the results are contradictory regarding acrosomal membrane integrity [2,4,12,14,16,22]. …”

Comparison of embryo developmental rates in Nguni, Bonsmara and Boran beef cattle breeds following in vitro fertilization and artificial insemination

“…Percoll gradient subjected semen was generously supplied by Embryo Plus (Embryo Plus, Brits, South Africa). This percoll gradient and centrifugation were used to separate live and motile sperm cells from the immotile and dead sperm cells and thus enrich the live and highly motile sperm population (Oliveira et al, 2012). To analyse for semen quality, a drop of about 10 µl semen was placed on a microscopic slide and covered with a microscopic cover slip.…”

“…It has been previously demonstrated that in frozen/thawed semen about 50% of sperm cells are rendered immotile following thawing and the fertilizing ability of such semen is significantly lower leading to decreased fertilization rates (Watson, 2000). However, advances in semen cryopreservation technologies, such as, the use of percoll gradient and centrifugation, promises to improve postthaw semen qualities and insemination rates as shown by improved fertilizing rates during AI and IVF (Oliveira et al, 2012). Others have argued that reactive oxygen species are higher in such semen resulting in poor 2-4 cell and 8-cells embryonic developmental stages and poor blastocyst inner cell mass formations (Oliveira et al, 2012;Arias et al, 2017).…”