uncertainties, we examined some of the fundamental aspects of tetraploid production, Ž
. including dynamics of development in triploid eggs Eudeline et al., 2000 . Here we
report the results of our investigations into improving production of tetraploids in C. gigas.
2. Materials and methods
2.1. Brood stock and gametes Triploid Pacific oysters used in this study were 2 years old, and produced by blocking
the release of PB 2 with CB. Ploidy was confirmed in all individuals by flow cytometry prior to spawning. Diploid Pacific oysters used in this study were 2 years old and came
Ž .
from a randomly mated population reared in Willapa Bay Washington State . Both Ž
. diploid and triploid brood stock were kept in a warm seawater pond about 238C before
being transferred in 10,000-l conditioning tanks for about 1.5 – 2 months before spawning.
2.2. Preparation of gametes Certified triploids were randomly selected, opened, and sexed under the microscope.
Males and females were separated to prevent any accidental fertilization. All surfaces in contact with broodstock were cleaned with diluted bleach. Gametes were obtained by
strip-spawning. Ž
Eggs from each individual female were stripped without adding water i.e., without .
aid of a spray bottle ; seawater was added only when all females from an experiment Ž
. had been stripped. This ‘‘dry stripping’’ Allen and Bushek, 1992 ensured that all eggs
were exposed to seawater simultaneously, which is important to get a synchronous Ž
. development and a consistent duration of hydration Eudeline et al., 2000 . Eggs were
separated from cellular debris by passing them through an 80-mm Nytex screen. Eggs Ž
. were caught on a 25-mm screen, then resuspended in 1-mm filtered seawater FSW at
258C and a salinity of 30 – 32 ppt. Eggs were counted using appropriate dilutions in a Sedgwick–Rafter chamber. After counting, the eggs — arrested at prophase of meiosis
1 — were held in seawater at 258C and checked microscopically until germinal vesicle
Ž .
breakdown GVBD was observed, a sign that meiosis was ready to resume. At this stage, we assumed that the eggs were blocked in metaphase of meiosis 1. Eggs remained
in seawater at least 45 min, but no longer than 60 min during counting. A pool of sperm obtained by stripping three different diploid males was suspended in
1 mm FSW, sieved through a 25 mum screen, and used to fertilize the eggs. 2.3. Fertilization and treatment
Just before fertilization, eggs were checked for sperm contamination, i.e., for evidence of early development. Given the generally low fecundity observed among
triploid females, all the females presenting more than 1 million eggs were used, and all
the eggs fertilized. For each spawning, a diploid female was used as control, and the egg density adjusted to that of the triploid egg density. Depending on the experiment, the
egg density ranged from 1 to 5 million eggs by liter. Fertilization occurred at 258C in 1 mm FSW, and aliquots of the sperm suspension were added to obtain about 10 to 20
spermatozoa per egg. CB treatment consisted of adding 0.5 mg CB dissolved in 1 ml
Ž .
dimethyl sulfoxide DMSO per liter of seawater. At the end of the treatment, eggs were drained on a 25-mm Nytex screen, rinsed with 1 mm FSW, then soaked in 0.05
Ž .
DMSO vrv dissolved in 1-mm FSW for 20 min. After this rinse, eggs were recounted and poured into culture tanks.
In the first experiment, we attempted to produce tetraploid larvae based on techniques Ž
. described by Guo and Allen 1994b , without looking at the time of meiotic events. For
Ž .
these trials, pair matings a single male and female were used. After hydration, eggs Ž
. from triploids were treated with CB 0.5 mgrl from 5 to 20 min PF. Four trials were
attempted, using four different females. In the second experiment, we varied the duration of CB treatment to try to increase
the yields of tetraploids. These experiments were accomplished on five triploid females Ž
. replicate spawns , with eggs split into two batches for either a ‘‘short’’ or ‘‘long’’ CB
Ž .
treatment. Based on some work on timing of meiosis first polar body extrusion in Ž
. triploid eggs Eudeline et al., 2000 , we decided to use two treatments: short — from 15
to 35 min PF, corresponding approximately to half of the period required for the Ž
. expulsion of 50 first polar bodies PB 1 in triploid eggs; and long — from 7 to 43
min PF, a duration estimated to include about three quarters of that period. In our last experiment, we adapted the treatment to the timing of meiosis of
Ž .
individual females n s 8 . That is, for the eggs of each female, CB treatment was
Ž started when first signs of polar body extrusion were observed or at 12 min, if none
. were seen before and proceeded until 50 PB 1 were evident or until 37 min PF,
whichever came first. In order to observe 50 PB 1 extrusion, it was necessary to take a subsample of fertilized eggs prior to the addition of CB and hold them at the same water
temperature as the experimental group, observing meiotic events over time by repeated sampling. One trial of tetraploid induction was also attempted by mixing eggs from
seven different females whose fecundity ranged from 1.3 to 9.2 million eggs, the total number of treated eggs being 24.7 million. Treatments for this batch were based on the
biological criteria described above.
2.4. LarÕal culture Ž
. All batches of larvae were cultured in 200-l tanks American Plastics at a density of
Ž .
less than 10rml according to standard practices e.g., Breese and Malouf, 1975 . Rearing temperature ranged from 238C to 258C and salinity from 30 to 32 ppt. Survival
Ž .
of developing embryos to D-stage day 2 , day 8, and as spat was recorded in all groups when possible. The ploidy composition of the larvae was determined by flow cytometry
Ž .
Allen and Bushek, 1992 at day 2 and for the surviving spat after setting. Beginning approximately on day 15, and continuing until day 20 to 25, competent eyed larvae were
collected on a 240-mm Nytex screen. Competent eyed larvae were put in downwellers Ž
. 180 mm Nytex screen with microcultch for settlement until they reached a shell length
of approximately 700 – 800 mm. At this time, they were transferred to upwelling system and the densities adjusted according to need as spat grew.
2.5. Flow cytometry 2.5.1. LarÕae
Approximately 10,000 48-h-old larvae were sampled from each culture for flow Ž
. cytometric FCM analysis to determine the percentage of tetraploids produced. In
preparation for flow cytometry, larvae were concentrated into a 1 ml suspension, then pelleted by centrifugation at 1500 = g for 10 s in a microcentrifuge. Supernatant
Ž seawater was withdrawn and 0.5 ml of DAPIrdetergentrDMSO solution 146 mM
NaCl, 10 mM Tris, adjust to pH 7.4, 2 mM CaCl , 22 mM MgCl , 0.05g BSA, 0.1
2 2
. Triton X, 10 mgrml DAPI and 10 DMSO was added to the tube. Larvae were
resuspended by vortex. Larval suspensions were frozen at y808C, and, after at least 1 h, larvae were thawed and disaggregated by repeated aspiration with a 1-ml syringe fitted
with a 26G needle. Cell suspensions were passed through a 25-mm screen immediately before the assay. FCM analyses were accomplished on a Partec CA-II flow cytometer.
Analysis of larval samples yielded data from a population of larvae, about 10,000 prepared by disaggregation. The proportion of cells in each ploidy class was calculated
relative to the proportion of observations in all ploidy classes, after curve fitting with
Ž . Ž
. Modfit Verity Software House, Topsham, ME, USA
Allen and Bushek, 1992 . 2.5.2. Spat
When the spat reached about 1000 – 2000 mm, about 30 individual spat from each experiment were examined by flow cytometry to assess ploidy. Spat were placed into
separate tubes and crushed gently. About 0.5 ml of DAPI solution was added to each tube, and the contents vortexed and screened on a 25-mm Nytex screen before FCM
analysis.
3. Results
