García-Ximéne Fernando, Escribá María José
Departamento de Ciencia Animal, Universidad Politécnica de Valencia, Spain.
Theriogenology. 2002 Mar 1;57(4):1319-25. doi: 10.1016/s0093-691x(01)00718-x.
The female parthenogenetic haploid embryos can be stored long-term by cryopreservation. Briefly, rabbit haploid parthenotes at the 32-cell stage were produced by electroactivation and in vitro culture. At this embryonic stage, parthenotes were individually cryopreserved by a slow-freezing procedure. After thawing, every embryo was disaggregated and blastomeres used as haploid maternal donors of nuclei. These nuclei were transferred to androgenetic haploid hemizygotes, obtained by female pronuclear removal offertilizedova. In the firstexperiment, 38 out of 87 reconstructedheteroparental diploid zygotes reachedthe hatched blastocyst stage invitro. In the second experiment, ourpurpose was toobtain live pups from each frozen-thawed parthenote. Viable offspring (at least one live pup at delivery) were obtained from five out of seven frozen-thawed haploid morula used as donors, with three live hemiclones being the highest number of pups produced from a single thawed parthenote. These results indicate that the rabbit female gametic endowment can be successfully stored by cryopreservation of parthenogenetic haploid embryos.
雌性孤雌生殖单倍体胚胎可通过冷冻保存进行长期储存。简要来说,通过电激活和体外培养产生处于32细胞期的兔单倍体孤雌胚。在此胚胎阶段,通过慢速冷冻程序对孤雌胚进行单独冷冻保存。解冻后,将每个胚胎打散,卵裂球用作单倍体细胞核的母源供体。这些细胞核被转移到通过去除受精卵雌原核获得的雄核发育单倍体半合子中。在第一个实验中,87个重构的异源二倍体合子中有38个在体外发育到孵化囊胚阶段。在第二个实验中,我们的目的是从每个冻融孤雌胚中获得活仔。从7个用作供体的冻融单倍体桑椹胚中的5个获得了存活后代(分娩时至少有1只活仔),单个解冻孤雌胚产生的活仔数量最多为3只半克隆仔。这些结果表明,通过冷冻保存孤雌生殖单倍体胚胎可以成功储存兔雌性配子资源。