Cryopreservation of pufferfish sperm in small and large volumes: the effect on kinetic parameters, and fertilization and hatching rates
Juan F. Asturiano, Manabu
Yoshida, Luz Pérez, Victor Gallego
Abstract
Cryopreservation of fish gametes
has many potential applications for ecological, scientific and aquaculture
purposes, and cryobank management has been established for many freshwater and marine
fish. Nevertheless, there are no studies for the long-term conservation of
pufferfish (Takifugu alboplumbeus) sperm,
thus the objectives of this work were (i) to develop a new cryopreservation
protocol for pufferfish sperm using different sperm:extender ratios and vials;
and (ii) to assess the fertilization capacity of cryopreserved sperm. Results
showed that cryopreserved sperm samples showed excellent postthawing motility
results when they were frozen in straws, reaching values over 60% with both 1:20
and 1:50 sperm:extender dilution ratios. Samples frozen in cryotubes showed
slightly lower motility results (around 50%) than those obtained in the straws.
Due to the excellent results obtained in the cryopreservation trials, in vitro
fertilization trials were undertaken using different sperm:egg ratios with both
fresh and cryopreserved sperm. High fertilization (FR) and hatching (HR) rates
(over 90%) were reached using high 1:105
and 1:106 ratios. However, when the spermatozoa
amount was limited in the fertilization micro-environment (using lower
egg:sperm ratios of 1:103 and 1:104), the cryopreserved
sperm generated lower values of FR and HR than the fresh sperm. These results
suggest that for achieving high FR and HR, it is essential to use 10 times more
cryopreserved sperm than fresh sperm. We then recommend using egg:sperm ratios
of 1:104 and 105 (for fresh and cryopreserved sperm,
respectively) for "Takifugu"
species in order to optimize the amount of gametes collected during aquaculture
procedures. This study has laid the basis for the establishment of
cryopreservation protocols in pufferfish, that will be helpful for further reproduction
in captivity programs and genetic cryobanking.
