71 THE EFFECT OF CRUDE GLYCEROL ON SELECTED RABBIT SPERMATOZOA MOTILITY PARAMETERS IN VITRO Dušan Paál 1 , Tomáš Slanina 2 , František Strejček 1 , Miroslav Perniš 1 , Martin Morovič 3 , Peter Massányi 2 1 Department of Botany and Genetics, Constantine the Philosopher University inNitra, Nábrežie mládeže 91, 949 74 Nitra, Slovakia, dusan.paal@ukf.sk, fstrejcek@ukf.sk 2 Department of Animal Physiology, Slovak University of Agriculture inNitra, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia, xslanina@is.uniag.sk, peter.massanyi@uniag.sk 3 Department of Zoology and Anthropology, Constantine the Philosopher University inNitra, Nábrežie mládeže 91, 949 74 Nitra, Slovakia, mmorovic@ukf.sk Abstract Glycerol is widely used as permeating cryoprotectant, sugar substitute and animal dietary enhancer. There are several types of glycerol differing in relation to industrial processing and thus its further application. Due to high energy level, crude glycerol presents a promising alternative to energy-rich feed ingredients traditionally used in rabbit diets. Depending on the concentration, glycerol can exert toxic effects on spermatozoa plasma membranes. This study was designed to assess the effect of different crude glycerol concentrations (5%, 10%, 15%, and 20%) on New Zealand white rabbit spermatozoa in vitro. In total 5 motility parameters - total motility (MOT), progressive motility (PRO), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) - proven to be associated with fertilisation rate were evaluatedin defined time intervals (0.5h, 1h, 2h, 3h, 24h) after the application of glycerol. The addition of 5% glycerol to the semen slightly decreased the MO parameter. However, 10%, 15% and 20% glycerol caused noticeable drop of MOT in all time intervals. Similar effect was observed in PRO, where 5% glycerol did not markedly attenuate VCL. On the contrary, 10%, 15% and 20% glycerol radically reduced VCL values. BCF values at 5% glycerol were slightly lower compared to control. The other glycerol concentrations exerted a toxic effect by considerable decrease of BCF. The most toxic effect of glycerol on spermatozoa were recorded just at 10%, 15% and 20% glycerol in all time intervals. Measurement of ALH returned results analogous to the previous motility parameters. Keywords Spermatozoa. Motility. Glycerol. Rabbit. Toxicity. INTRODUCTION Glycerol is a chemical compound widely used due to its cryoprotective properties in deep freezing of male gametes (Mazur et al., 2008, Samper and Morris, 1998, Woods et al., 2004) and in food industry as a sugar substitute. Moreover, crude glycerol frequently utilized as a supplement in diets for farmed animals (Hansen et al., 2009, Harzia et al., 2013). Many studies have found glycerol to be a suitable feed ingredient for cattle, pigs, poultry and rabbits. While rabbit digestive organs allow for inclusion of several agro-industrial by- products in feed (Mateos and Vidal, 1996), a number of studies have focused on finding alternative nutrition sources such as glycerol. There are a few types of glycerol differing in their relation to the degree of industrial processing. Crude glycerol compared to semi-purified glycerol, features by high content of fatty acids accompanied by sodium and potassium residues. Due to its high energy level crude glycerol presents a promising alternative to energy-rich dietary items traditionally used in rabbit diets (Retore et al., 2012). However, depending on the concentration, glycerol can exert chemical and osmotic toxicities on cell plasma membranes (Hammerstedt al., 1990, Garcia et al., 2012) including spermatozoa. Thereby it comes to considerable reduction of the total quality of semen, which is frequently used in artificial insemination (AI). AI is thought to be one of the most advanced approaches in animal breeding. It requires semen of acceptable characteristics and sufficient motility (Brun et al., 2002; Ducci et al., 2002). Methods such as computer-assisted semen analysis (CASA) have been, and are still, widely used for determining a broad range of