Preliminary Assessment of Refrigerated and Frozen Storage of Sperm of the Coppernose Bluegill

Abstract

AbstractThe coppernose bluegill (CBG) Lepomis macrochirus purpurescens is a bluegill subspecies that is native to Florida. The CBGs and their hybrids are of interest as potential food fish. Our objectives were to (1) develop an osmolality activation curve for CBG sperm, (2) determine suitable cryoprotectants for the cryopreservation of CBG sperm, and (3) demonstrate the ability of cryopreserved CBG sperm to fertilize eggs. Sperm were stripped from mature CBGs (mean ± SD = 113 ± 35 g, n = 10), diluted in Hanks' balanced salt solution (HBSS; osmolality = 300 mosmols/kg), and stored at 4°C. An activation curve was generated by exposing the sperm to solutions prepared by serial dilution of HBSS with de‐ionized water. The sperm remained inactive at osmolalities above 265 mosmols/kg. Dilution below 115 mosmols/kg was required for complete activation. The motility of sperm stored in these solutions was estimated at 24‐h intervals. Stripped sperm motility (mean ± SD) declined from 90 ± 7% initially to 26 ± 11% after 6 d of storage at 4°C. Motility of sperm from crushed testes declined from 80 ± 8% at collection to 28 ± 10% at 1 d of storage. By day 5, motility of sperm from crushed testes was 9 ± 9%; no motility was observed at 6 d. Sperm were exposed to 10% concentrations of the cryoprotectants dimethyl acetamide (DMA), dimethyl sulfoxide (DMSO), and methanol (MeOH), and motility was evaluated at 15‐min intervals for 60 min. Initially, motility was 81 ± 11% for the control sperm. At 60 min, motility was 0% for DMA, 30 ± 24% for DMSO, 78 ± 10% for MeOH, and unchanged for the control. For samples frozen in a controlled‐rate freezer (CRF), post‐thaw motility was 26 ± 10% for samples with 10% DMSO and 51 ± 18% for samples with 10% MeOH. For samples frozen by means of dairy industry technology (DIT), post‐thaw motility was 46 ± 21% for samples with 10% DMSO and 56 ± 13% for samples with 10% MeOH. Overall, samples frozen in 10% MeOH had significantly higher post‐thaw motility (P = 0.043) than did those frozen in 10% DMSO. However, post‐thaw motility did not differ between samples frozen by use of the CRF and DIT (P = 0.13). For sperm frozen in the CRF, fertilization was 15% for samples with 10% DMSO and 50% for samples with 10% MeOH. For sperm frozen by means of DIT, fertilization was 50% for samples with 10% DMSO and 75% for samples with 10% MeOH.