CRYOPRESERVATION OF MALE GAMETES OF THE PACIFIC WHITE SHRIMP, LITOPENAEUS VANNAMEI
B. Subramanian; C.L. Browdy
Abstract :
Development of a viable technique for cryogenic
storage of male gametes of cultivable crustaceans would facilitate reduction
in the number of adult males maintained in commercial hatcheries, greatly
reducing feed costs. Furthermore, the technique would provide for selective
breeding programs and inter-hatchery gamete transfer. In the present study,
we report our findings on the suitability of application of cryopreserved
male gametes for artificial insemination in in
vitro fertilization in Litopenaeus vannamei. We have used sperm mass,
as extracted from the spermatophores and sperm suspension for our
experiments. Spermatophores were collected by manual ejaculation and
suspended in the extender media (filtered sea water, Ca++ free artificial
sea water). Sperm mass, separated from the spermatophores was subjected to
cryoprotectant treatment (dimethyl sulfoxide, ethylene glycol, glycerol and
sucrose, alone or as mixtures) for 15 minutes and frozen either in 1.8mL
polypropylene cryovials or in ½ cc plastic straws in a programmable freezer
at rates ranging from –1 to –10°C/min. Additionally, the sperm mass was
gently homogenized in a tissue grinder, pelleted and re-suspended in the
extender media to form a sperm suspension. After morphological assessments,
the free spermatozoa (~106 sperm/ml cryoprotectant medium) were also frozen.
Freezing proceeded to –40°C, where from the samples were cooled rapidly
to –150°C and plunged into liquid nitrogen for overnight storage. Thawing
was accomplished by immersing the cryovials and straws in water bath
maintained at room temperature. Optimal protocols involved freezing at rates
if –1 to –3°C in 1.25M glycerol and 2M ethylene glycol. Addition of
sucrose (0.3M) marginally improved post-thaw survival. Sperm morphology,
biostain assays and activation studies using divalent cation ionophore
A23187 and egg water were used as indices for post-thaw survival. The
percentage of abnormal sperm increased to 49% in the frozen-thawed samples,
when compared to 31% in the unfrozen cryoprotectant-treated controls.
Frozen-thawed sperm pellets, recovered from –40°C, used for artificial
insemination yielded 28% hatching when compared to 69% in controls. We are
investigating the possibility of attaching the cryopreserved sperm pellets
on to the female sternum using natural binders like gum arabic and
cyanoacrylate glues.
(Waddell Mariculture Center, South Carolina
Department of Natural Resources, POB 809, Bluffton, SC 29910, USA, e-mail: subraman@musc.edu)