NONLETHAL SPERM COLLECTION AND CRYOPRESERVATION IN THE EASTERN OYSTER CRASSOSTREA VIRGINICA

摘要: Cryopreservation can preserve genetic materials in perpetuity and be applied to oyster culture for breeding programs. Protocols exist sperm cryopreservation the eastern Crassostrea virginica, but nonlethal sample collection is needed valuable individuals, such as tetraploids, or specific lines. The goal of this study was develop methods oyster. objectives were (1) evaluate natural spawning a method, (2) anesthesia induce shell opening biopsy, (3) mechanical notching (4) verify combined with biopsy cryopreservation. Five males (of 60 oysters) spawned naturally after 7 h, an average concentration 1.9 ± 1.0310 3 cells/mL (in 2 L seawater). No oysters (n ¼ 30) responded by during 36 h treatment 5% Dead Sea salt (containing 33.3% MgCl2), 22 opened 36-h Epson (MgSO4). Sperm collected had fresh motility 3%-80% postthaw 1%-5%; production 4.53 10 5 2.3310 8 cells per male. Mechanical did not cause mortality 20). After 18-G 20-G needles, survival 80% (16 20 each). 5.42 needle 8) 16± 12% 3± 2%, 1.35310 9) 21± 20% 4%. differences observed between samples biopsied sizes (P $ 0.074). To collection, total 39 sampled obtain males, which averaged 99.48 23.17 g weight, 74.1 6.0 mm height, 60.9 7.2 length. 3.6 2.1 Biopsied showed 23 motility, 13 6% postequilibration (after equilibration 10% DMSO 30-60 min before freezing), 6± 4% motility. Flow cytometry analysis indicated 84± intact plasma membranes sperm, 59± 9% sperm. Fertilization thawed 20± 22% (from 1%-87%). significant dissected (lethal collection) 0.550), 1.000), 0.101), membrane integrity difference fertilization 0.039; samples, 22%; 68 40%). Overall, developed Eastern that individuals