Abstract
Experiments were conducted to find the optimal condition for freezing boar spermatozoa packaged in 0.5ml plastic straws using polystyrene foam container and liquid nitrogen (LN2). Spermatozoa from six fertile boars (two Large White, two Durock and two Landrace boars) were suspended in BF5 diluent containing 2.5% (v/v) glycerol and packaged in 0.5ml plastic straws at 5°C. Straws were frozen at 4 to 20cm above LN2 surface. The straws were first placed on a hard rubber rack and then transferred into the freezing container at the height of 4, 10, 15 and 20cm from the surface of LN2, or were placed directly on a steel rack (4cm) which was previously cooled in the container. After 20min, straws were plunged into LN2. Cooling rates of these samples at the temperature zone of -15 to -30°C ranged from 3.5 to 90.5°C/min. Frozen semen was thawed by incubating straws in a water bath at 50°C for 8sec. The terminal temperature after the thawing process was 27°C, and warming rate from -196 to 27°C was 1672°C/min. Frozen-thawed semen was diluted 1:10 with BTS at 30°C, and progressive motility and acrosomal integrity were evaluated during 6hrs incubation at 37°C. Motility rate was highest in semen that was frozen at 4cm above LN2 using a rubber rack. The proportion of spermatozoa with normal acrosome was best maintained in semen frozen at 4cm above LN2 using a steel or rubber rack. These results indicate that the cooling rates of 35 to 90°C/min at the temperature zone of -15 to -30°C are optimal for freezing boar semen packaged in 0.5ml plastic straws. After addition of glycerol, diluted semen was incubated for up to 6hrs at 5°C (glycerol equilibration) and then frozen at 4cm above LN2 using a rubber rack. The glycerol equilibration times had little influences on the viability of spermatozoa after thawing. The simple freezing method developed in this study would be applicable when semen is used for surgical insemination or in vitro insemination.
