February 15-18, 2009 Washington State Convention Center Seattle ...

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SPERM VITRIFICATION OF A LIVE-BEARING FISH, THE GREEN SWORDTAIL
Xiphophorus helleri
Rafael Cuevas-Uribe*, S.P. Leibo and Terrence R. Tiersch
Aquaculture Research Station
Louisiana State University Agricultural Center
Baton Rouge, LA, 70820 USA
ruribe@agcenter.lsu.edu
Vitrification is an alternative method of cryopreservation where the sample (including the extracellular and intracellular
fractions) forms glass (non-crystalline ice). The probability of successful vitrification depends on interactions of three
major factors: sample viscosity, cooling and warming rates, and sample volume. The first step in protocol development is the
selection of a suitable and least toxic vitrification solution. Sperm vitrification is new in fish and there is little knowledge of
cryoprotectants that vitrify at non-toxic concentrations. The goal of the project was to develop streamlined protocols that could
be integrated into a standardized approach for vitrification of aquatic species germplasm. The specific objectives were to:
1) measure acute toxicity of cryoprotectants at varied concentrations; 2) test various solutions for vitrification; 3) evaluate
different warming methods, and 4) evaluate the ability to fertilize eggs.
Sperm were collected from the green swordtail Xiphophorus helleri by crushing of dissected testis and diluting to a final concentration
of 1 x 10 8 cells per ml using Hanks’ balanced salt solution at 500 mOsmol/kg (HBSS 500). The acute toxicity of
11 cryoprotectants, 50 vitrification solutions, and 5 commercial vitrification solutions were tested. For each cryoprotectant,
concentrations were evaluated ranging from 5 to 50% (Figure 1). In Experiment 2, the glass formation of 44 solutions was
evaluated using 5-mm nichrome loops. After plunging the solutions in liquid nitrogen, glass formation was identified by a transparent
state (crystallization produced a milky appearance; Figure 1). In Experiment 3, four different warming solutions (fresh
water, HBSS 300, HBSS 500, and 7% ethylene glycol + 7% 1,2 propanediol) at two temperatures (24 and 37 °C) were tested.
In Experiment 4, 20 females of X. maculatus were artificially inseminated with two replicates of vitrified sperm from each of
10 males. In addition, five albino females of X. helleri were inseminated with vitrified sperm.
From 226 treatments evaluated for acute toxicity, 44 were selected for further testing, and of these, 21 formed glass. The average
motility of sperm in these vitrifying solutions was low (0-20%; control 60%). The best warming solution was found to be
HBSS 300, and there was no difference between 24 °C and 37 °C. The best post-thaw motility (≤ 10%) was found in 40%
glycerol, and in 20% ethylene glycol + 20% glycerol with an exposure time of ≤ 2 min before cooling. For artificial insemination,
the loop was warmed in 5-µl of HBSS 300 at 24 °C. A 5-µl volume was injected into the females. Vitrification can be
simple, fast, and cheap, does not require expensive equipment and can be performed in the field. This is important for fishes
such as Xiphophorus which are valuable biomedical models, yet many are threatened species in the wild.