Mitochondrial injury to ram sperm during procedures associated with artificial insemination or frozen storage

The effect of a number of procedures involved in the preparation of ram semen for freezing or artificial insemination on sperm mitochondrial function were examined. Ram sperm mitochondrial function was assessed using the membrane potential probe rhodamine 123 (R123). R123 uptake was increased rather than reduced by incubation of sperm at a high rate of dilution (1: 30), and was unaffected by the addition of 1% serum albumin (BSA). Washing sperm also had no effect on R123 uptake. ptake was reduced in ram sperm exposed to either hypertonic (450 mosmol l−1) or hypotonic (150 mosmol l−1) media (P < 0.05) but was unaffected by varying pH from 5.9 to 8.9. There was no interaction between the effects of osmolality and pH. ondrial injury due to cold shock became apparent when sperm were rapidly cooled below 10°C (P < 0.05). Further damage occurred between 10 and 5°C (P < 0.01). The effects of cold shock were not ameliorated by the addition of 1% BSA, 2 mM butylated hydroxytoluene (BHT) or 50 mM erythritol. fect of freezing on ram sperm R123 uptake was greater than that of cold shock (P < 0.01). R123 uptake after freezing and thawing in sperm treated with 250 mM sorbitol or 250 mM dimethylsulphoxide (DMSO) was greater than that of unprotected sperm, or sperm exposed to 4% glycerol (P < 0.05). lated R123 was released rapidly from frozen ram sperm (P < 0.01) but released more slowly from cold shocked sperm (P < 0.01). This suggests that the two stressors induce different types of mitochondrial injury.