Effect of three different diluent on canine semen submitted to two thawing protocols

Abstract

To verify the effect of three different extenders and two thawing temperatures on frozen-thawed canine sperm characteristics, one ejaculate from nine dogs were separately collected and processed (n=9). Each ejaculate was divided into 3 equal samples and centrifuged. The pellets were re-suspended using: 1st pellet - Glicyne-egg yolk extender (GEY), 2nd pellet - TRIS extender and 3rd pellet - M9 extender and all samples were filled in 0.5ml straws. A total of 84 straws (28 for each protocol) were done. The sperm motility, vigour and plasma membrane integrity from each protocol were immediately evaluated (M1) and the straws were brought to a refrigerator at 5ºC for 60 minutes. After that, a sample from each protocol was warmed up in a water bath 37ºC for 5 min. and sperm motility, vigour and plasma membrane integrity were evaluated (M2). The straws were frozen in liquid nitrogen. One straw from each protocol was thawed at 37ºC for 30 sec and another at 72ºC for 8 sec and the sperm motility; vigour, CASA and plasmatic membrane integrity and acrossomal status using FITC-PNA stain were evaluated (M3). Plasma membrane, sperm motility and vigour were evaluated after a 1-hour incubation at 37ºC (M4). Statistical analysis showed that the higher temperature had positive effect on froze-thawed canine sperm characteristics. The best results were taken when canine semen was frozen in GEY extender and thawed 72ºC for 8 sec.