Cryopreservation of Atlantic salmon Salmo salar sperm: effects on sperm physiology

The objective of this study was to determine the effect of freezing on the function in Atlantic salmon Salmo salar spermatozoa. The semen was frozen in Cortland's medium+1.3M dimethyl sulphoxide+0.3M glucose+2% bovine serum albumin (final concentration) in a ratio of 1:3 (semen:cryoprotectant) as the treatment (T) and fresh semen as the control (F). Straws of 05ml of sperm suspension were frozen in 4cm of N2L. They were thawed in a thermoregulated bath (40 degrees C). After thawing, the percentage of spermatozoa with fragmented DNA [transferase dUTP (deoxyuridine triphosphate) nick-end labelling (TUNEL)], plasma membrane integrity (SYBR-14/PI) and mitochondrial membrane potential (MMit, JC-1) were evaluated by flow cytometry and motility was evaluated by optical microscope under stroboscopic light. The fertilization rates of the control and treatment semen were tested at a sperm density of 15x10(7) spermatozoa oocyte(-1), by observation of the first cleavages after 16h incubation at 10 degrees C. In the cryopreserved semen (T), the mean +/- s.d. DNA fragmentation was 48 +/- 25%; plasma membrane integrity 752 +/- 63%; mitochondrial membrane potential 517 +/- 36%; motility 585 +/- 53%; curved line velocity (V-CL) 612 +/- 174 mu ms(-1); average-path velocity (V-AP) 501 +/- 173 mu ms(-1); straight-line velocity (V-SL) 591 +/- 184 mu ms(-1); fertilization rate 816 +/- 19%. There were significant differences in the plasma membrane integrity, mitochondrial membrane potential, motility, fertilization rate, V-CL, V-AP and V-SL compared with the controls (P<005). Also the mitochondrial membrane potential correlated with motility, fertilization rate, V-CL and V-SL (r=075; r=059; r=077 and r=079, respectively; P<005); and the fertilization rate correlated with V-CL and V-SL (r=059 and r=055, respectively).