Flow cytometry and ultrastructure of cryopreserved red seabream ( Pagrus major) sperm

Abstract

The objectives were to assess motility, fertilizing capacity, structural integrity, and mitochondrial function in fresh versus frozen-thawed (15% DMSO was used as a cryoprotectant) sperm from red seabream ( Pagrus major). Mean (±S.D.) rates of motility, fertilization and hatching of frozen-thawed sperm were 81.0 ± 5.4, 92.8 ± 1.9, and 91.8 ± 5.2%, respectively; for fresh sperm, they were 87.5 ± 7.7, 95.8 ± 2.4, and 93.8 ± 4.2%. Although motility was lower in frozen-thawed versus fresh sperm ( P < 0.05), there was no effect ( P > 0.05) of cryopreservation on fertilization or hatching. Based on scanning and transmission electron microscopy, 77.8 ± 5.6% of fresh sperm had normal morphology, whereas for frozen-thawed sperm, 63.0 ± 7.2% had normal morphology, 20.6 ± 3.1% were slightly damaged (e.g. swelling or rupture of head, mid-piece and tail region as well as mitochondria), and 16.4 ± 4.2% were severely damaged. Sperm were stained with propidium iodide and Rhodamine 123 to assess plasma membrane integrity and mitochondrial function, respectively, and examined with flow cytometry. For fresh sperm, 83.9% had an intact membrane and functional mitochondria, whereas for frozen-thawed sperm, 74.8% had an intact membrane and functional mitochondria, 12.7% had a damaged membrane, 9.9% had nonfunctional mitochondria, and 2.6% had both a damaged membrane and nonfunctional mitochondria. In conclusion, ultrastructure and flow cytometry were valuable for assessment of frozen-thawed sperm quality; cryopreservation damaged the sperm but fertilizing ability was not significantly decreased.