Nuestro último artículo, publicado en Reviews in Fish Biology and Fisheries

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.

doi: 10.1007/s11160-024-09912-9