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A novel technique of chimeric somatic cell cloning was applied to produce a transgenic rabbit (NT20). Karyoplasts of transgenic adult skin fibroblasts with Tg(Wap-GH1) gene construct as a marker were microsurgically transferred into one, previously enucleated, blastomere of 2-cell non-transgenic embryos, while the second one remained intact. The reconstructed embryos either were cultured in vitro up to the blastocyst stage (Experiment I) or were transferred into recipient-females immediately after the cloning procedure (Experiment II). In Experiment I, 25/102 (24.5%) embryos formed blastocysts from whole embryos and 46/102 (44.12%) embryos developed to the blastocyst stage from single non-operated blastomeres, while the reconstructed blastomeres were damaged and degenerated. Thirteen (12.7%) embryos did not exceed 3- to 4-cell stages and 18 (17.7%) embryos were inhibited at the initial 2-cell stage. Out of 14 blastocysts which were subjected to molecular analysis, the transgene was detected in the cells of 4 blastocysts. In Experiment II, 163/217 (75.0%) embryos were transferred into 9 pseudopregnant recipient-rabbits (an average of 18 embryos per recipient). Four recipient-females (44.4%) became pregnant and delivered a total of 24 (14.7%) pups. Molecular analysis confirmed that two pups (1.2%), one live and one stillborn, showed a positive transgene signal. Live transgenic rabbit NT20 appeared healthy and anatomically as well as physiologically normal. The results of our experiments showed that transgenic adult skin fibroblast cell nuclei, which have been introduced into the cytoplasmic microenvironment of single enucleated blastomeres from 2-cell stage rabbit embryos, are able to direct the development of chimeric embryos not only to the blastocyst stage but also up to term.

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Abstract. Bull effect on results of in vitro embryo production has been well documented. The aim of the present study was to find the relationship between quality of bull sperm chromatin and its effect on in vitro embryo production. Bovine in vitro matured oocytes were fertilized in vitro using capacitated spermatozoa (freshly ejaculated or frozen-thawed) of 12 bulls. Semen was simultaneously processed according to the sperm chromatin structure assay (SCSA) method and was analysed by flow cytometry. At least 3 replications of IVP with the same semen sample were done. The percentage of spermatozoa with abnormal chromatin ranged from 0.4% to 23.8%. All bulls used for the experiment were divided into three groups showing minimal (0.82% ± 6.82%), low (1.70% ± 15.82%) and high (18.16% ± 53.59%) percentages of spermatozoa with abnormal chromatin structure. Both cleavage rates and embryo development to the blastocyst stage were correlated significantly with sperm chromatin abnormalities and resulted in 23.1, 17.7 and 12.2% of blastocysts respectively for sperm with minimal, low and high percentages of chromatin abnormalities. The SCSA method may be used as a practical indicator of suitability of bull ejaculate for IVP purposes.