Calcium-45 uptake as a measurement of freeze-thaw injury to chicken spermatozoa in the presence or absence of cryopreservatives

Abstract

Liquid scintillation analysis of Ca 45 uptake from chicken seminal plasma was used to evaluate pre- and postfreeze injury in the absence or presence of cryopreservatives (DMSO, glycerol, sucrose, and glucose). Three freezing methods evaluated were: 1) direct immersion in liquid nitrogen (fast), 2) controlled freezing 1 °C per min (slow), and 3) 1 °C per min to −6 °C followed by 85 °C per min to −196 °C (slow-fast). Freezing, slow or fast, significantly increased Ca 45 uptake in the absence of any cryopreservative. A glycerol level of 12% was necessary to obtain any reduction in freeze-thaw damage with slow or fast freezing. However, a slow-fast combination resulted in a protective effect at lower glycerol levels. DMSO was completely unsuccessful as a cryopreservative with high or low cooling velocities. Neither glycerol nor DMSO affected Ca 45 transport before freezing. Glucose and sucrose stimulated Ca 45 uptake before freezing, making it difficult to evaluate freeze damage. Glucose increased the amount of damage to slow frozen spermatozoa. However, it gave some protection at the highest level (0.08 g) to fast frozen semen. Sucrose levels at or above 0.08 g reduced the amount of freeze-thaw damage from fast freezing. A higher level (0.16 g) was needed to prevent significantly the injury of slow freezing. The liquid scintillation method using Ca 45 appears to be a reliable method for evaluating freeze-thaw injury to spermatozoa.