Title page for ETD etd-0903102-205825

Type of Document Master's Thesis
Author Whaley, C. Brian
Author's Email Address cwhale2@lsu.edu
URN etd-0903102-205825
Title Hybridization of Lepomid Sunfishes by Use of Cryopreservation
Degree Master of Science (M.S.)
Department Forestry, Wildlife, and Fisheries
Advisory Committee
Advisor Name Title
Terrence R. Tiersch Committee Chair
Mark C. Bates Committee Member
Robert C. Reigh Committee Member
Robert P. Romaire Committee Member
  • sperm preservation
  • sunfish reproduction
  • hybridization
Date of Defense 2002-07-31
Availability unrestricted
The goal of this project was to integrate cryopreservation into the hybridization of sunfishes. The first objective was to evaluate the conditioning of sunfishes to spawn in cages in warmwater ponds. Bluegill and green sunfish were stocked in cages in ponds heated to 27 C by geothermal water. Fish in heated ponds did not spawn on artificial nests in cages. Fish were also brought into the laboratory for artificial spawning. There were 22 spawns yielding 47 ± 19% (mean ± SD) fertilization.

The second objective was to develop methods for the refrigerated and frozen storage of sperm of bluegill Lepomis macrochirus and green sunfish Lepomis cyanellus. Sperm were stored in Hanks’ balanced salt solution prepared at 300 mOsmol/kg, and activated with solutions prepared at 80 mOsmol/kg. Sperm stored at 4 C maintained motility for as long as10 d, but the optimum use of sperm was on the day it is collected. Cryopreserved sperm should be thawed in a 40 C water bath for 7 s. The motility of cryopreserved sperm lasted for less than 1 min. Cryopreserved sperm of bluegill and green sunfish were able to fertilize green sunfish eggs.

The third objective was to improve methods of cryopreservation of sperm of bluegill and coppernose bluegill Lepomis macrochirus purpurescens to produce hybrids with green sunfish. Sperm were exposed to five concentrations of five cryoprotectants and motility was estimated at 10 min intervals for 30 min. There were concentration-dependent effects on motility over time. One concentration of each cryoprotectant was used for cryopreservation. Sperm cryopreserved with methanol had the highest post-thaw motility while sperm cryopreserved with propylene glycol yielded the highest fertilization. Sperm of bluegill and coppernose bluegill were cryopreserved and used to fertilize eggs of the same green sunfish. There were no significant differences between the subspecies for motility before freezing (P = 0.4704), post-thaw motility (P = 0.5841), fertilizing ability (P = 0.1351) and hatch (P = 0.1133).

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