Sephadex filtration as successful alternative to density-gradient centrifugation procedures for ram sperm selection with improved kinetics

Abstract

Density-gradients centrifugation (DGC) and filtration columns (FC) are used to separate deformed or dead sperm, debris, and other cells that may negatively affect the fertilizing capacity of sperm in fresh, chilled and frozen/thawed semen. The present study was conducted to evaluate the suitability of DGC (BoviPure®, Percoll® and Accudenz®) and FC (Sephadex G-15®) sperm selection procedures for fresh-extended and cold-stored ram semen by assessment of post-treatment sperm quality variables. Twenty normospermic ejaculates from ten adult Merino rams were used. Sperm concentration of recovered cells was greater (P < 0.001) after BoviPure treatment than other procedures in both fresh and cold semen. With the Sephadex method, there were more desirable values than with use of DGC procedures in several sperm motility variables measured by using the CASA system. In non-refrigerated semen samples, the percentage of progressive sperm motility (%PSM) after Sephadex filtration was greater (P < 0.05) than after BoviPure treatment; the straightline velocity (VSL) value after Sephadex filtration was greater (P < 0.01) than after Accudenz treatment; the amplitude of lateral head displacement (ALH) after Sephadex and Accudenz treatment was less than non-filtered semen (P < 0.001) and after Percoll (P < 0.01) and BoviPure (P < 0.05) treatments. In cold-stored semen samples, the %PSM after Sephadex filtration was greater than non-filtered (P < 0.05) semen and after BoviPure (P < 0.05), Percoll (P < 0.05) and Accudenz (P < 0.001) treatments. It is concluded that Sephadex column filtration can be used to select ram sperm in non-refrigerated and cooled semen, because percentage progressively motile sperm and some other sperm motility characteristics are greater with use of this techniques as compared with use of DGC methods.

Introduction

Artificial insemination has an important role in sheep breeding but its use is limited because the poor fertility achieved when stored semen is used for vaginal insemination (Gil et al., 2003). The success of this procedure in sheep is limited by the anatomic characteristics of the ewe’s cervix and the short time that ram sperm can be stored as a liquid.

The sperm characteristics and fertilizing capacity of extended ram semen may be improved by applying methods for sperm selection prior to artificial insemination. Dead and non-functional sperm, debris and all dead cells could affect live sperm probably as a production of reactive oxygen species (ROS) that accumulates in deleterious concentrations, which have been reported as inductors of apoptosis (Wang et al., 2003). Sperm selection methods are used with the aim of gaining seminal plasma-free suspensions of predominantly motile sperm with reduced presence of abnormal, immature, and dead spermatozoa, epithelial cells, cell debris, lymphocytes, etc. (Valcárcel et al., 1996; Phillips et al., 2012), potentially allowing for an enhanced fertilizing capacity (Graham and Graham, 1990). Sperm selection methods, however, may have adverse "iatrogenic" effects related to mechanical damage due to centrifugation-resuspension procedures, and the promotion of lipid peroxidation during sperm pelleting due to the close vicinity of leukocytes and damaged sperm that may generate reactive oxygen species (ROS)(Sbracia et al., 1996).

With selective washing techniques based on density-gradient centrifugation (DGC), the sperm are selected according to the density, allowing for the isolation of motile and morphologically normal sperm. Currently, there are several commercial solutions available for sperm separation by DGC´s procedures. BoviPure® is an iso-osmotic salt solution containing colloidal silica particles coated with silane used to select bull sperm for use with artificial reproductive techniques (Samardzija et al., 2006). Silane-coated silica media are have also been proposed to be used in a single layer centrifugation (Martinez-Alborcia et al., 2013). Percoll® is a medium composed of colloidal silica particles (15–30 nm in diameter), coated with polyvinylpyrrolidone, and can be used for sperm selection in several mammalian species (Batista et al., 2011). Accudenz® (formerly called Nycodenz) is a non-ionic tri-iodinated derivative of benzoic acid with three aliphatic hydrophilic side chains of high density (2.1 g/ml) due to the presence of a substituted ring, which is linked to hydrophilic groups to enhance water solubility, and has been used in sperm selection of humans (Gellert-Mortimer et al., 1988; Sbracia et al., 1996), and cheetahs (Crosier et al., 2009), among other species. To our knowledge, no studies have been reported on the use of Accudenz® for selecting ram sperm.

Another technique of sperm separation is filtration through Sephadex columns. Sephadex® is a dextran gel available in different pore (filtration) sizes (G-10 to G-120). The post-thaw quality of sperm recovered after filtration through Sephadex was assessed to be highly acceptable when Sephadex was used for human (Drobnis et al., 1991), bull (Januskauskas et al., 2005; Lee et al., 2009), buffalo (Ahmad et al., 2003), stallion (Sieme et al., 2003), and boar (Bussalleu et al., 2008) semen, and was promising when used for filtering ram sperm (Landa et al., 1980; Valcárcel et al., 1996). In addition, with use of semen from bulls with lesser fertility for insemination that had been filtered through Sephadex there was an improved 60–90 day non-return rate (Graham and Graham, 1990). The mechanism for the trapping of sperm in Sephadex columns remains unclear. The filtration, however, is believed to be based on the fact that non-viable sperm tend to adhere to the Sephadex matrix to a greater extent than motile and seemingly functional sperm, and the latter are able to cross the filtration barrier without modification of functional characteristics (Bussalleu et al., 2008).

For ram semen, a number of sperm selection methods have been tried, including Sephadex filtration (see above), swim-up methods (García-López et al., 1996), sucrose washing, and Millipore filtration (Marti et al., 2006), as well as Percoll density gradient centrifugation (DGC) (Valcárcel et al., 1996). Even though there have been great advances in DGC procedures for sperm selection with different species (Santiago-Moreno et al., 2014, 2016), these procedures include centrifugal forces that may be harmful, especially for ram sperm (García-López et al., 1996). Hence, it was hypothesized that methods that involve little or no centrifugation, such as Sephadex filtration methods, may have advantages as compared with DGC for use in sperm selection in this species. In the present research, the effectiveness of Sephadex G-15® was compared with different DGC procedures (BoviPure®, Percoll®, Accudenz®) in fresh-extended semen and cold-stored ram semen.