ISSN 1392-2130. VETERINARIJA IR ZOOTECHNIKA (Vet Med Zoot). T. 73 (95). 2016 84 EFFECTS OF EXPOSURE TO X-RAY AT AIRPORT SECURITY CHECKPOINTS ON MEMBRANE INTEGRITY OF CHILLED CANINE SEMEN Simona Sakalauskaitė 1 , Jakov Šengaut 3 , Neringa Sutkevičienė 2 , Henrikas Žilinskas 2 , Arūnas Rutkauskas 2 , Vita Riškevičienė 1 1 Department of Veterinary Pathobiology 2 Large Animal Clinic, Veterinary Academy, Lithuanian University of Health Sciences, Tilžės 18, LT-47181, Kaunas, Lithuania; e-mail: simona.sakalauskaite@lsmuni.lt 3 Private Small Animal Clinic ‘Jakovo veterinarijos centras’, Gerosios Vilties 1, LT-03147, Vilnius, Lithuania Abstract. This study was conducted to investigate effects of X-irradiation on motility and viability of canine spermatozoids in chilled semen samples over the storage period of 5 days. Diluted semen samples from 10 different dogs were divided into 2 aliquots, and 1 group of aliquots was exposed to X-irradiation by transporting it through airport security X-ray machine (HI-SCAN 100100 T). Evaluation of total and progressive spermatozoids motility was done by Sperm Class Analyzer (SCA) (Spain, 2011). To detect spermatozoa with a biochemically active plasma membrane, a hypo-osmotic swelling test was used. Assessment of spermatozoa with a structurally intact plasma membrane was done using SYBR-14/PI (Molecular Probes) fluorescent staining. Our study showed that the total and progressive motility (P > 0.05) and the percentage of canine spermatozoa with a functional (P ≤ 0.05, P ≤ 0.01) and intact (P > 0.05) membrane over the storage period of 5 days were lower in the samples exposed to X-irradiation compared with the control group. Keywords: canine semen, X-ray, plasma membrane integrity Introduction. Insemination with chilled semen is commonly used in dog breeding. In the recent years, it has become usual to transport chilled canine semen internationally to prevent inbreeding and to improve genetic variety (Kmenta et al., 2011). Nowadays, it is common to use air transportation for biological material, including semen (Hendricks et al., 2010). Canine semen, as every other cargo, is exposed to X-irradiation at airport security checkpoints. It is well known that exposure to X- irradiation can cause double and single DNA strands breaks. In one study, the motility of frozen domestic feline spermatozoa decreased after exposure to X-ray. These results show that exposure to X-irradiation at airport security checkpoints may have a negative impact on semen and other important biological material’s quality parameters (Gloor et al., 2006). However, there is no information whether exposure to X-ray at airports has any negative effects on motility and viability of spermatozoa of chilled canine semen. Although there are several quality parameters of canine semen, one of the most important is integrity of the spermatozoal membrane. The plasma membrane is essential for sperm survival in a female reproductive tract, capacitation and fertilisation (Mocé & Graham, 2008; Santos et al., 2011). These processes depend on the integrity of the spermatozoal plasma membrane, which can be assessed by the hypo-osmotic swelling test (HOST) (Goericke–Pesch & Failing, 2013) and fluorescent staining. The HOST method is based on the principle that under hypo-osmotic conditions biochemically active spermatozoa with a functionally intact membrane absorb water and swell increasing in volume until equilibrium is reached (Jeyendrean et al., 1984; Cabrita et al., 1999; Amorim et al., 2009). This test is used to assess sperm membrane functional integrity (Kargei et al., 2014). Spermatozoal plasma membrane structural integrity is assessed using amphipathic probes with the acyl group, which can penetrate intact membranes (Donoghue et al., 1995). The aim of our study was to investigate effects of exposure to X-ray at airport checkpoints on motility and viability of chilled canine spermatozoa over the storage period of 5 days. Methods and materials. Semen samples were collected from 10 different healthy dogs. The dogs were 2 to 5 years old. The semen was collected by digital manipulation into plastic tubes separating 3 fractions of the ejaculates. Only the sperm rich fraction was used in this study. Each ejaculate was divided into 2 equal aliquots. Both aliquots were diluted with CaniPlus Chill 10 extender (Minitüb, Germany). One group of aliquots was transported through airport security X-ray machine (HI-SCAN 100100 T) one time. Volume of ejaculate, concentration and morphology of spermatozoa and subjective motility of fresh canine semen were assessed using conventional semen evaluation methods (Januškauskas, 2010). Only the samples with normal sperm concentration and low spermatozoa morphological abnormalities were used in the study. The extended semen samples were kept at 4 o C and examined every day over the storage period of 5 days. The total and the progressive motility parameters of the extended semen samples were determined using the computer-assisted sperm analysis (CASA) system Sperm Class Analyzer (SCA V.5.1.) (Microptic. S. L., Spain). The motility of spermatozoa was examined at 37 o C under phase-contrast microscope Olympus BH2 with a pre- warmed 37°C stage (Olympus Optical Co., Ltd., Japan) using a 100X magnification. A 5-µL aliquot was placed on a pre-warmed 37 o C microscope slide, covered with a