Simple method for freezing boar spermatozoa packaged in 0.5 ml plastic straws: effects of freezing conditions and glycerol equilibration times

摘要

Experiments were conducted to find the optimal condition for freezing boar spermatozoa packaged in 0.5 ml plastic straws using polystyrene foam container and liquid nitrogen (LN_2). Spermatozoa from six fertile boars (two Large White, two Durock andtwo Landrace boars) were suspended in BF5 diluent containing 2.5 percent (v/v) glycerol and packaged in 0.5 ml plastic straws at 5 deg C. Straws were frozen at 4 to 20cm above LN_2 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 20 cm from the surface of LN_2, or were placed directly on a steel rack (4cm) which was previously cooled in the container. After 20 min, straws were plunged into LN_2. Cooling rates ofthese samples at the temperature zone of -15 to -30 deg C ranged from 3.5 to 90.5 deg C/min. Frozen semen was thawed by incubating straws in a water bath at 50 deg C for 8 sec. The terminal temperature after the thawing process was 27 deg C, and warmingrate from -196 to 27 deg C was 1672 deg C/min. Frozen-thawed semen was diluted 1 : 10 with BTS at 30DC, and progressive motility and acrosomal integrity were evaluated during 6 hrs incubation at 37 deg C. Motility rate was highest in semen that was frozen at 4cm above LN_2 using a rubber rack. The proportion of spermatozoa with normal acrosome was best maintained in semen frozen at 4 cm above LN_2 using a steel or rubber rack. These results indicate that the cooling rates of 35 to 90 deg C/min at the temperature zone of -15 to -30 deg C are optimal for freezing boar semen packaged in 0.5m/ plastic straws. After addition of glycerol, diluted semen was incubated for up to 6 hrs at 5 deg C (glycerol equilibration) and then frozen at 4 cm above LN_2 usinga 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.