title: An efficient method for blocking the 1st mitotic cleavage of fish zygote using combined thermal treatment, exemplified by mud loach (Misgurnus mizolepis).
abstract: Blocking the first mitotic cleavage of the zygote is a key tool for chromosome-set manipulations in fish. We developed an improved method for inducing tetraploidy by blocking the mitosis with a combination of heat shock at 40.5 degrees C for 1, 2, or 3 min followed by cold shock at 1.5 degrees C for 30, 45, or 60 min. When applied during the first cleavage metaphase of mud loach (Misgurnus mizolepis) zygotes, the optimal combination was heat for 2 min followed by cold for 45 min. At 1 month, the frequency of 4N survivors and the yield from total eggs fertilized was 55.7 and 14.4%, respectively, compared to heat shock alone with 20.0% efficiency and 3.6% yield. The effectiveness of the procedure was confirmed by diploid mitotic gynogenesis using transgenic markers. The overall yield of homozygous diploids, 34.0%, was better than that for single heat shock, 17.3%. The tetraploids and homozygous diploids had higher early mortality than normal diploid controls. However, at 1 month, the viability of the tetraploids was the same as normal diploids. For gynogenetic diploids, the survival was similar to normal diploids after 3 months. The high efficiency of this new protocol extends the opportunity to study polyploidy in basic and applied research.
authors.affiliation: Department of Aquaculture, Pukyong National University, Busan, South Korea. yoonknam@pknu.ac.kr
authors.forename: Yoon Kwon
authors.initials: YK
authors.lastname: Nam
authors.forename: Geyong Cheol
authors.initials: GC
authors.lastname: Choi
authors.forename: Dong Soo
authors.initials: DS
authors.lastname: Kim
mesh_terms: D000818:Animals; D003531:Cypriniformes / Q000196:embryology* / Q000235:genetics; D004171:Diploidy; D006358:Hot Temperature; D008938:Mitosis; D011123:Polyploidy; D015053:Zygote / Q000166:cytology*