PRELIMINARY OBSERVATIONS ON CRYOPRESERVATION OF
EMBRYOS AND LARVAE OF THE PACIFIC WHITE SHRIMP, LITOPENAEUS VANNAMEI
B. Subramanian; C.L. Browdy
Abstract:
The presentation details our observations on
cryopreservation of embryonic, naupliar and zoeal stages of Litopenaeus
vannamei. Embryos and larvae were obtained by spawning high health females
in the maturation facility at Waddell Mariculture Center, South Carolina,
USA. The animals were maintained on a shifted photoperiod to make allowances
for manipulation of experimental material during the daylight hours. The
methods employed for sourcing, spawning and harvesting are described. The
tolerance of embryos, nauplii and zoeae to chilling and cryoprotectant
treatment were evaluated prior to freezing. No embryonic stage tolerated
chilling to 4°C for a period beyond 60 min and the hatchery performance of
larvae obtained from cryoprotectant-treated/frozen embryos was dilatory.
However, nauplii (N5) and zoeae (Pz1) endured chilling and cryoprotectant
treatment and were suitable for subsequent experimentation. The results on
chill tolerance and cryoprotectant toxicity are presented. Based on the
toxicity and chill tolerance trials, N5 and Pz1 were selected as stages for
cryopreservation and experimented using a cocktail containing a quaternary
permeating mixture with ethylene glycol, propylene glycol and dimethyl
sulfoxide. Additionally, polyethylene glycol 8000, was used as a
non-permeating cryoadditive. Samples in ½ cc straws were conventionally
cooled/frozen using a programmable freezer at rates of –0.3, -1 and –5°C/min
to intermediate sub-zero temperatures of –10, -20, -30, -40 and –50°C
and given isothermal holds (t</= 30 min) before thawing. An additional
batch of samples from –50°C were cooled rapidly to –180°C, plunged
into liquid nitrogen and held for an hour before thawing. After thawing to
room temperature, the samples were cultured for 24 hours to assess survival.
Post-thaw survival was near total in the larvae that were frozen to –10°C
at the different rates tried, while a dramatic drop in survival (20%) was
noticed in the samples frozen –20°C at –5°C/min. Although nauplii and
zoeae recovered upon thawing when exposed to –30, -40 and –50°C, they
failed to metamorphose and subsequently died after 6 hr culture at room
temperature. Interestingly, 11% of the nauplii and 14% of the zoeae plunged
into liquid nitrogen demonstrated feeble twitching movements immediately
after thawing. We are furthering the studies to optimise the freezing
protocols in order to develop a banking facility for genetically improved
penaeid shrimp stock.
(Waddell Mariculture Center, South Carolina
Department of Natural Resources, POB 809, Bluffton, SC 29910, USA, e-mail: subraman@musc.edu)