Restoration of the loach, Misgurnus anguillicaudatus, from cryopreserved diploid sperm and induced androgenesis

Abstract

In the present study, we evaluated the feasibility of using cryopreserved diploid sperm as a repository genebank for the loach, Misgurnus anguillicaudatus, along with a rederivation strategy utilising induced-androgenesis. Firstly, we evaluated three types of media for egg inactivation: Hank's saline solution + 0.5% bovine serum albumin (BSA), Ringer's solution + 0.5% BSA, and masu salmon seminal plasma. Haploid and diploid sperm were taken from diploid and tetraploid loaches, respectively. Fresh and cryopreserved haploid or diploid sperm were then used to fertilize intact or UVirradiated eggs from wild diploid females. The irradiation media evaluated here successfully maintained the egg quality over 2 hours. Fertilization and hatching rates of eggs fertilized with cryopreserved diploid sperm were 11.68 ± 6.74% and 7.14 ± 6.29%respectively, compared to 63.51 ± 10.68% and 45.19 ± 16.2% for intact eggs fertilized by fresh haploid sperm. All-male inheritance was confirmed by determination of larval morphology, ploidy status and microsatellite genotypes of putative androgenetic progeny.

Improvement of gamete quality and its short-term storage: an approach for biotechnology in laboratory fish

“…In general, warm-water species presents low storage ability after ovulation (Rizzo et al, 2003;Nguenga et al, 2004). Better storage are found in some cold-water species including salmonids (Barrett, 1951;Withler and Morley, 1968;Poon and Johnson, 1970;Jensen and Alderdice, 1984;Babiak and Dabrowski, 2003;Niksirat et al, 2007), sturgeons (Billard et al, 2004;Sohrabnezhad et al, 2006), flatfishes (Suquet et al, 1999) and walleye (Dietrich et al, 2012) although it is dependent on species and storage conditions (Rothbard et al, 1996;Linhart et al, 2001;Yasui et al, 2010). Based on our results, we conclude that oocytes from yellowtail tetra cannot be stored successfully, although other procedures may be used in future works including solutions with additives that may improve storage ability as was observed in the other warm-water fish such as the tilapia (Harvey and Kelley, 1984).…”

“…For evaluation of sperm motility, 1 µl of diluted sperm was pipetted on a microscope slide previously coated with 0.1% bovine serum albumin and the sperm was activated by a 30-fold dilution with distilled water. Then, we evaluated the total motility, progressive motility and duration of progressive motility using the same criteria of our previous studies Yasui et al, 2008Yasui et al, , 2009Yasui et al, , 2010.…”

“…Studies regarding chromosome-set manipulation, embryology, transgenesis, nuclear transplantation and chimerism requires a precise fertilization timing for successful manipulation (Tanaka et al, 2009;Yasui et al, 2010Yasui et al, , 2011. The timing for gamete activation is necessary for those techniques because manipulation must take place within a few minutes after fertilization (Nam et al, 2004;Yasui et al, 2010). For induction of polyploid fish, temperature or pressure shocks must be applied before the first cell cleavage few minutes after gamete activation (Piferrer et al, 2009).…”

Androgenesis: where males hijack eggs to clone themselves

“…There has been some success with this technique with sperm harvested from cadavers (e.g. [101,102]), and one could imagine that in an emergency situation one might clone an endangered species in the eggs of a common farmed one [103].…”

Production of loach (Misgurnus anguillicaudatus) germ-line chimera using transplantation of primordial germ cells isolated from cryopreserved blastomeres1

“…Previously, we cryopreserved loach sperm (Yasui et al, 2008(Yasui et al, , 2009) and reconstituted 58 viable progeny by combining cryopreserved diploid sperm and artificially-induced androgenesis 59 (Yasui et al, 2010). However, the resultant androgenotes exhibited low survival rates and were 60 predominantly males due to unknown mechanisms (Fujimoto et al, 2010a).…”