Effects of cryopreservation of pronuclear‐stage rabbit zygotes on the morphological survival, blastocyst formation, and full‐term development after DNA microinjection

Abstract

The objectives of this study were to examine the freezing sensitivity of pronuclear-stage rabbit zygotes and to produce transgenic rabbits using the cryopreserved zygotes. Zygotes were cryopreserved either by one of two vitrification protocols or by one of the two conventional freezing protocols. The morphological survival rates of zygotes subjected to two-step freezing in 1.5 M ethylene glycol and 0.1 M sucrose (74%) or to vitrification in 7.2 M ethylene glycol and 1.0 M sucrose (81%) were higher than those subjected to freezing in 1.5 M DMSO (46%) or to vitrification in a mixture of 2.0 M DMSO, 1.0 M acetamide, and 3.0 M propylene glycol (41%). But the in vitro development into blastocysts of zygotes cryopreserved by vitrification (17%) or to a lesser extent by freezing (52%) was impaired, when compared to that of fresh control zygotes (89%). Next, a fusion gene composed from bovine aS1-casein promoter and a human GH structural gene (2.8 kb) was microinjected into the pronucleus of rabbit zygotes frozen-thawed in ethylene glycol and sucrose. Then, the presence of exogenous DNA in the genome of newborn offspring was determined by PCR. The post-injection survival of frozen zygotes (97%) was the same as that of fresh control zygotes (96%). However, of 18 offspring derived from 414 frozen-thawed and DNA-injected zygotes, no transgenic rabbits were produced. Of 52 offspring derived from 403 DNA-injected fresh zygotes, 3 transgenic rabbits were found. Here we report the first rabbit offspring resulting from zygotes cryopreserved at the pronuclear-stage, although the cryopreservation procedure employed must be improved if zygotes are to be used for systematic production of transgenic rabbits.

Live Piglets Derived from In Vitro-Produced Zygotes Vitrified at the Pronuclear Stage1

“…Cryopreservation of pronuclear-stage zygotes is routinely performed in humans [31]. Successful vitrification of zygotes has already been reported in a number of species, including the mouse [32][33][34], the rat [35,36], and the rabbit [37][38][39]. Cryopreservation of zygotes may have importance in the production of transgenic animals, because cryopreserved pronuclear-stage zygotes have been successfully used for DNA microinjection [34,36,[40][41][42].…”

Cryopreservation of female germ cells and ovarian tissues for fertility preservation

“…As a result, the low CPA concentration reduced the toxicity of the CPAs drastically and enabled easier manipulation of the sample. Another approach is the cryotop method, which uses a small vitrification volume and direct contact with liquid nitrogen [39]. It is widely used in Japan for embryo freezing.…”

“…It is widely used in Japan for embryo freezing. A variety of these techniques can be found in the literature [36][37][38][39][40][41][42] and have been applied with different degrees of success to several species of mammals, including humans.…”

Establishment of a SPF Colony on Human Apo(a) Transgenic Rabbits by Frozen-Thawed Embryo Transfer

“…As well as avoiding carrying living animals into another facility, we also need to ensure we prepare appropriate numbers of embryos and recipients when using frozen-thawed embryos for transfer. Until now, a large amount of research has been done on rabbit embryo cryopreservation [1,10,12,[17][18][19][20]. Fertilized 1-or 2-cell stage eggs were found to be difficult to freeze [10], whereas morula stage embryos were relatively easy to cryopreserve.…”

“…Until now, a large amount of research has been done on rabbit embryo cryopreservation [1,10,12,[17][18][19][20]. Fertilized 1-or 2-cell stage eggs were found to be difficult to freeze [10], whereas morula stage embryos were relatively easy to cryopreserve. Single or several mixed cryoprotectants such as DMSO, EG, glycerol and propanediol have been used in different embryo freezing procedures [12,17,20].…”