Is the protective effect of egg yolk against osmotic and cryogenic damage on dog spermatozoa dose-dependent?

Abstract

Egg yolk (EY) is conventionally used to reduce sperm cryodamage, however, there has not be evaluation of whether there is a dose-dependent effect with inclusion of EY in semen extender. To enhance the knowledge about the protective effect of EY during cryopreservation of dog semen, a specific study was designed to evaluate the dose-dependent protection of the EY against osmotic and cryogenic damage of dog sperm. In the first experiment, sperm stored in an extender that contained graded EY concentrations (0 %, 5 %, 10 %, and 20 %) were diluted with hypo- or hyper-osmotic solutions (final osmolality of 75, 150, 300, 500, 1000 mOsm/kg). Results from sperm kinetic, membrane integrity (MI), mitochondrial activity, and normal morphology evaluations indicated osmotic stress has especially marked effects on the kinetic capacity of spermatozoa, however, there were no direct effects on mitochondrial activity. In both hypo- and hyper-osmotic conditions, EY had a protective effect regardless of concentration. In the second experiment, semen samples were diluted in extenders at increasing EY concentrations (0 %, 5 %, 10 %, and 20 %) and cryopreserved. Effects on sperm kinetics, membrane and acrosome integrity and mitochondrial membrane potential indicated there was improved sperm viability after thawing when the EY concentration was 5 % and 10 %, and lesser viability when it was 20 %. These results indicate, for the first time, that EY reduces osmotic and cryogenic damage when used at 5 % or 10 % concentrations, and that these concentrations can be used to protect dog spermatozoa more effectively than the conventionally used concentration (20 %).

Introduction

Semen cryopreservation allows for long-term storage of viable and functional spermatozoa (Leroy et al., 2011). This technology has several advantages because semen can be stored for a long period (i.e., years) without losing fertilizing capacity, transported for great distances, or can also be used when the female is in oestrus without having a male in close proximity to mate with the female (Thomassen and Farstad, 2009).

Cryopreservation, however, has detrimental effects on mammalian sperm viability and fertilizing capacity. A reduction in progressive motility (Jones and Stewart, 1979), alterations of membrane permeability and stability (Holt and North, 1986; Watson, 2000), and an increase in the radical species of oxygen (ROS) generation (Alvarez and Storey, 1992; Chatterjee and Gagnon, 2001) have been reported in cryopreserved mammalian spermatozoa as compared with the values for these variables in raw semen. To reduce the effects of temperatures that are used for cryopreservation of spermatozoa, extenders with specific composition were developed. Among the different components of the freezing extender, egg yolk (EY) appears to be a necessary component of extenders if these are to be effective for semen cryopreservation and maintenance of sperm viability after thawing. Results of previous studies indicate the use of EY in the freezing medium reduces cellular damage (Phillips and Lardy, 1940; Pace and Graham, 1974; De Leeuw et al., 1993). Although the protective function of the EY with cryopreservation of sperm is widely recognized, the underlying mechanism of action has not been ascertained. Results of most studies indicate that the protective capacity of EY is related to the content of low density lipoproteins (LDL) (Pace and Graham, 1974; Moussa et al., 2002; Bencharif et al., 2010). It has been proposed that these components adhere to and interact with the sperm membrane (Foulkes, 1977; Graham and Foote, 1987; Manjunath et al., 2002; Bergeron et al., 2004). In some studies the metabolism of spermatozoa, however, is inhibited by some EY components, and this could affect sperm motility (Pace and Graham, 1974; Wall and Foote, 1999).

Traditionally, while EY is added to dog semen extender at a 20 % concentration when there is cryopreservation of sperm (Anderson, 1972; Peña et al., 1998; Silva et al., 2002), there have been few studies in which there has been assessment of whether the cryoprotective effect of EY is dose-dependent. In other males, such as the stallion, EY is effectively used for semen cryopreservation at a 2 % concentration, without a reduction of sperm quality and fertilisation capacity (Pillet et al., 2008).

The damage induced by cryopreservation on spermatozoa is multimodal because in several studies there was a combination of cold shock (Amann and Pickett, 1987), peroxidation (Slaweta et al., 1988), and osmotic stress (Watson, 2000). When the temperature reduction is beyond the freezing point for semen, water forms ice crystals first in the extracellular compartment. This increases the solute concentration in the free uncrystallised water outside the cell, inducing hyperosmotic stress (Sieme et al., 2016). During thawing, however, the ice crystals melt in the free water that enters the plasma membrane, thus sperm undergo hypo-osmotic stress.

Although Foulkes (Foulkes, 1977) suggested that the protective effect of the EY during cryopreservation could contribute to the colloid pressure maintenance of the external medium, few studies have been focused on the functions of EY as a protective compound against the osmotic stress. Even though there is broad understanding of the importance of EY as a semen extender component and consequent wide use of EY for sperm cryopreservation in dogs, there have been surprisingly few studies conducted to clarify the dose-dependent protective effect of this component. Furthermore, a relevant part of the damage during cryopreservation could be attributed to the osmotic stress, but few studies focused on the protective functions of the EY against osmotic stress in dog spermatozoa. Thus, to increase the knowledge of the biology and manipulation of reproduction in dogs, the present study was designed to ascertain, for the first time, the protective effect of EY at different concentrations (0 %, 5 %, 10 %, and 20 %) on dog spermatozoa in different anisosmotic conditions. Furthermore, the aim of the present study was to evaluate the cryoprotective effect of EY, added at the same concentrations (0 %, 5 %, 10 %, and 20 %) to the freezing medium, for dog sperm cryopreservation.