An efficient method for blocking the 1st mitotic cleavage of fish zygote using combined thermal treatment, exemplified by mud loach (Misgurnus mizolepis).
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.
Nam
Yoon Kwon
YK
Department of Aquaculture, Pukyong National University, Busan, South Korea. yoonknam@pknu.ac.kr
Choi
Geyong Cheol
GC
Kim
Dong Soo
DS
D000818:Animals; D003531:Cypriniformes / Q000196:embryology* / Q000235:genetics; D004171:Diploidy; D006358:Hot Temperature; D008938:Mitosis; D011123:Polyploidy; D015053:Zygote / Q000166:cytology*