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

Abstract

The 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 Ϯ SD ϭ 113 Ϯ 35 g, n ϭ 10), diluted in Hanks' balanced salt solution (HBSS; osmolality ϭ 300 mosmols/kg), and stored at 4ЊC. 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 Ϯ SD) declined from 90 Ϯ 7% initially to 26 Ϯ 11% after 6 d of storage at 4ЊC. Motility of sperm from crushed testes declined from 80 Ϯ 8% at collection to 28 Ϯ 10% at 1 d of storage. By day 5, motility of sperm from crushed testes was 9 Ϯ 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 15min intervals for 60 min. Initially, motility was 81 Ϯ 11% for the control sperm. At 60 min, motility was 0% for DMA, 30 Ϯ 24% for DMSO, 78 Ϯ 10% for MeOH, and unchanged for the control. For samples frozen in a controlled-rate freezer (CRF), post-thaw motility was 26 Ϯ 10% for samples with 10% DMSO and 51 Ϯ 18% for

Calcium‐induced quiescence of sperm motility in the bluegill (Lepomis macrochirus)

“…Ca 21 -induced quiescence in bluegill sperm motility dilution of sperm into a hypoosmotic medium, tap water, was associated with the onset of motility. These results are consistent with the findings of Bates et al (2005), which demonstrated that the sperm of bluegill are quiescent in 265 mOsm/kg Hank's SIS but become motile in media of lower osmotic concentration. That sucrose inhibited motility similar to NaCl indicates that osmolality plays a primary role in maintaining sperm quiescent.…”

“…This study is unique, however, in demonstrating that relatively low physiological levels of Ca 21 are inhibitory to bluegill sperm motility and contribute to the maintainence of quiescence of sperm in seminal plasma. Although the studies of Bates et al (2005) suggest that bluegill sperm are maintained quiescent by high osmolality, the SIS used in those studies contained 1.3 mmol/L Ca 21 , a level found inhibitory in conjunction with osmolality in this study. That Ca 21 is inhibitory to sperm motility is consistent with the findings of Okuno and Morisawa ('89), which showed motility of demembranated salmonid sperm is inhibited by 40.1 mmol/L Ca 21 .…”

“…Only recently have studies been conducted on the sperm of the Centrarchidae (Leach and Montgomerie, 2000;Neff et al, 2003;Burness et al, 2004Burness et al, , 2005Casselman and Montgomerie, 2004). Research by Bates et al (2005) demonstrated that bluegill sperm remain immotile at high osmolality and become activated on dilution with freshwater. This study demonstrates that in conjunction with osmolality Ca 21 inhibits initiation of bluegill sperm motility at levels relevant to those of seminal plasma.…”

Methanol and Egg Yolk as Cryoprotectants for Atlantic Salmon Spermatozoa

“…Both chemicals function as intracellular cryoprotectants and are believed to achieve their cryoprotective abilities by decreasing the water content within the cells (McGann 1978). However, DMSO is generally more toxic to fish sperm than methanol at similar concentrations (Tiersch et al 2004a(Tiersch et al , 2004bBates et al 2005).…”

Fertilization by refrigerator stored sperm of the Indian major carp,Labeo calbasu(Hamilton, 1822)

“…Methanol permeates the sperm cell quickly to maintain the osmotic balance with the extracellular environment. It could be less toxic to fish sperm than dimethyl sulphoxide at similar concentrations (Tiersch, Wayman, Skapura, Neidig & Grier 2004;Bates, Williams, Lang & Tiersch 2005). Methanol has widespread use in cryopreservation application, in addition to its use in refrigerator storage to prolong the viability of sperm.…”