A quantifiable biochemical marker for cold shock damage to porcine sperm

Abstract

s / Theriogenology 578 10% FBS, 10% DMSO, and 0.07 M sucrose; (B) DMEM with 10% FBS and 10% DMSO; and (C) FBS with 10% DMSO. The vials were held at 80 8C for 24 h, and then transferred to liquid nitrogen ( 196 8C). After 3 weeks, cells were thawed in a water bath at 38 8C for 2 min. After adding 2 mL of DMEM with 10% FBS, cells were centrifuged and resuspended to 0.5 mL in DMEM with 1% BSA. Cell viability was assessed by Trypan blue exclusion immediately after isolation (L0), before adding the freezing medium (L1), and after freezing and thawing (L2). Survival rate (S) was defined as L2/L1 100. The effect of treatment on L2 or S was evaluated using one-way ANOVA. Cell viability after isolation, culture, and freezing and thawing (L0, L1 and L2) was compared using ANOVA for repeated measurements (SAS Institute Inc., Cary, NC). There was no effect of treatment on L2 or S. Overall mean values ( S.D.) of L0, L1 and L2 were 99.1 1.9, 78.5 14.9 and 67.8 20.5%, respectively (P < 0.05). Overall survival rate was 86.4 21.5%. We concluded that the three freezing media tested were equally suited for freezing equine testicular cells. Enzymatic digestion yielded a cell suspension with excellent viability, which decreased after incubation and cryopreservation. Freezing equine testicular cells immediately after isolation may result in better postthaw viability. However, functional assays are still required to positively identify SSCs among testicular cells, and to determine the ability of this cell population to survive freezing and thawing.