title: 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.
authors.affiliation: Center of Biotechnology R&D, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China.
authors.forename: Q H
authors.initials: QH
authors.lastname: Liu
authors.forename: J
authors.initials: J
authors.lastname: Li
authors.forename: S C
authors.initials: SC
authors.lastname: Zhang
authors.forename: Z Z
authors.initials: ZZ
authors.lastname: Xiao
authors.forename: F H
authors.initials: FH
authors.lastname: Ding
authors.forename: D D
authors.initials: DD
authors.lastname: Yu
authors.forename: X Z
authors.initials: XZ
authors.lastname: Xu
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000379:methods*; D005260:Female; D005307:Fertilization in Vitro; D005434:Flow Cytometry; D005453:Fluorescence; D008297:Male; D008854:Microscopy, Electron; D021541:Sea Bream; D013075:Sperm Capacitation; D013081:Sperm Motility; D013094:Spermatozoa / Q000166:cytology* / Q000648:ultrastructure*; D013194:Staining and Labeling