Abstract—Target of this study was the analysis of the impact of crude glycerol on canine spermatozoa motility, morphology, viability, and membrane integrity. Experiments were realized in vitro. In the study, semen from 5 large dog breeds was used. They were typical representatives of large breeds, coming from healthy rearing, regularly vaccinated and integrated to the further breeding. Semen collections were realized at the owners of animals and in the veterinary clinic. Subsequently the experiments were realized at the Department of Animal Physiology of the SUA in Nitra. The spermatozoa motility was evaluated using CASA analyzer (SpermVision TM , Minitub, Germany) at the temperature 5 and 37°C for 5 hours. In the study, 13 motility parameters were evaluated. Generally, crude glycerol has generally negative effect on spermatozoa motility. Morphological analysis was realized using Hancock staining and the preparations were evaluated at magnification 1000x using classification tables of morphologically changed spermatozoa. Data clearly detected the highest number of morphologically changed spermatozoa in the experimental groups (know twisted tails, tail torso and tail coiling). For acrosome alterations swelled acrosomes, removed acrosomes and acrosomes with undulated membrane were detected. In this study also the effect of crude glycerol on spermatozoa membrane integrity were analyzed. The highest crude glycerol concentration significantly affects spermatozoa integrity. Results of this study show that crude glycerol has effect of spermatozoa motility, viability, and membrane integrity. Detected changes are related to crude glycerol concentration, temperature, as well as time of incubation. Keywords—Dog, semen, spermatozoa, acrosome, glycerol, CASA, viability. I. INTRODUCTION LYCEROL (CH 3 H 8 O 3 ), a highly permeable polyhydric alcohol is the cryo-protector most frequently used in semen freezing in different species [1]. However, this cryo- protector exhibits toxic effects on spermatozoa, such as physicochemical alterations that can lead to rupture of the plasma membrane or removal of important membrane proteins, as well as cause acrosome damage, which will be reflected in reduced fertility [2], [3]. Since glycerol has a relative high molecular weight (92 kDa) and does not readily penetrate into the spermatozoa membrane, it causes tremendous osmotic stress during the thawing procedure, P. Massanyi, L. Kichi, T. Slanina, E. Kolesar, N. Lukac, E. Tvrda, A. Kolesarova are with the Department of Animal Physiology, Slovak University of Agriculture in Nitra, Slovak Republic (corresponding author, phone: +421- 37-3414284; e-mail: massanyi@yahoo.com). J. Danko is with Department of Anatomy, Histology and Physiology, University of Veterinary Medicine and Pharmacy in Kosice, Slovak Republic (e-mail: danko@uvlf.sk). R. Stawarz and P. Massanyi are with Institute of Biology, Pedagogical University of Krakow, Poland (e-mail: robert.stawarz@gmail.com). because it is easily removed from the cell membranes [4]. The ideal concentration of glycerol in the extender represents a balance between its toxicant and protecting effects; high concentrations can also affect the fertilizing capacity of the spermatozoa [5]. However, the sensitivity of spermatozoa to glycerol is highly species dependent [6]. The aim of this study was to analyze the influence of different crude glycerol concentrations on canine spermatozoa motility parameters during short in vitro cultivation. II. MATERIAL AND METHODS In this study, semen was obtained from sexually mature male of five large dog breeds: Boxer, Border collie, Rottweiler, Greyhound and Husky. Semen was diluted in a ratio of 1 part of semen and 10 parts of physiological solution (Sodium chloride 0.9% Braun, B. Braun Melsungen AG, Melsungen, Germany) – control sample (GK), or commercial diluent (GR). In the same ratio, the semen was diluted with different concentration of crude glycerol solution: GA: 0.11 ml/ml, GB: 0.05 ml/ml, GC: 0.027 ml/ml, GD: 0.013 ml/ml, GE: 0.0069 ml/ml, GF: 0.0034 ml/ml, GG: 0.0017 ml/ml diluted in the physiological solution. Samples were cultured at 5 and 37°C (T) and recorded at five time periods: 0, 1, 2, 3, and 4 hours. Characteristic of used glycerol – crude glycerol: content at least 80.06%; NaCl content 7.15%; water 8.0%; methanol 0.001%; Cd<0.01 mg/l, Pb<0.1 mg/l, Cu<0.04 mg.l, Mn<0.03 mg/l, Zn 2.5 mg/l, Fe 15 mg/l, Ni 2.5 mg/l, Co 12.5 mg/l and Cr 7.5 mg/l. To evaluate spermatozoa motility parameters Computer Assisted Semen Analyzer (CASA) system – SpermVision program (Minitube, Germany) equipped with a microscope Olympus BX 51 (Olympus, Japan) was used. Each sample was placed into Makler Counting Chamber (depth of 10 μm, Sefi– Medical Instruments, Germany) and then placed in a microscopic field. Using the canine specific set up motility parameters were evaluated (mainly total motile spermatozoa and progressively motile spermatozoa). For morphological analysis, the staining according to Hancock was used. The preparations were evaluated at magnification 1000x. The changes were classified according to the classification tables of morphologically changed forms of spermatozoa. Obtained data were statistically analyzed with the help of the PC program Excel and a statistics package SAS 9.1 (SAS Institute Inc., USA) using Student’s t–test and Scheffe test. Statistical significance was indicated by P values of less than 0.05; 0.01 and 0.001. Crude Glycerol Affects Canine Sperm Motility: Computer Assisted Semen Analysis in vitro P. Massanyi, L. Kichi, T. Slanina, E. Kolesar, J. Danko, N. Lukac, E. Tvrda, R. Stawarz, A. Kolesarova G World Academy of Science, Engineering and Technology International Journal of Animal and Veterinary Sciences Vol:9, No:11, 2015 1151 International Scholarly and Scientific Research & Innovation 9(11) 2015 scholar.waset.org/1307-6892/10002589 International Science Index, Animal and Veterinary Sciences Vol:9, No:11, 2015 waset.org/Publication/10002589