Effect of the time interval between fusion and activation on nuclear state and development in vitro and in vivo of bovine somatic cell nuclear transfer embryos

• Published in: Reproduction (Cambridge, England) Vol. 131; no. 1; pp. 45 - 51
• Main Authors: Aston, K I, Li, G P, Hicks, B A, Sessions, B R, Pate, B J, Hammon, D, Bunch, T D, White, K L
• Format: Journal Article
• Language: English
• Published: Colchester Society for Reproduction and Fertility 01-01-2006; Journals of Reproduction and Fertility, Ltd; Portland
• Subjects: Animals; Biological and medical sciences; blastocyst; Calcium - metabolism; Cattle; Cell Nucleus - metabolism; Cell Nucleus - ultrastructure; Cells, Cultured; chromatin; Chromatin - ultrastructure; Cloning, Organism - methods; cytoplasm; Cytoplasm - metabolism; Early stages. Segmentation. Gastrulation. Neurulation; Electric Stimulation; Embryo Transfer; Embryology: invertebrates and vertebrates. Teratology; Embryonic Development; Female; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Ionomycin - pharmacology; Ionophores - pharmacology; morula; Nuclear Transfer Techniques; oocytes; Oocytes - metabolism; Oocytes - ultrastructure; Parthenogenesis; Pregnancy; pregnancy rate; pronucleus; somatic cells; Time Factors; Embryonic development; Chromatin; Melting; Cell nucleus; Egg activation; Assisted procreation; Ox; In vitro; Vertebrata; Mammalia; Animal; Transfer; Artiodactyla; Ungulata
• ISSN: 1470-1626; 1741-7899
• DOI: 10.1530/rep.1.00714

This study indicated that prolonged exposure of donor cell nuclei to oocyte cytoplasm before activation results in abnormal chromatin morphology, and reduced development to compacted morula/blastocyst stage in vitro. However, after transfer of embryos to recipients, there was no difference in pregnancy rates throughout gestation. Chromatin morphology was evaluated for embryos held 2, 3, 4 and 5 h between fusion and activation. In embryos held 2 h, 15/17 (88.2%) embryos contained condensed chromosomes, while only 12/24 (50.0%) embryos held 3 h exhibited this characteristic. The proportion of embryos with elongated or fragmented chromosomes tended to increase with increased hold time. While 15/19 (78.9%) of embryos held 2 h developed a single pronucleus 6 h after activation, only 8/22 (36.4%) had one pronucleus after a 4-h hold. Embryos held 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 h cleaved at rates of 207/281 (73.7%), 142/166 (85.5%), 655/912 (71.8%), 212/368 (57.6%), 406/667 (60.9%), 362/644 (56.2%) and 120/228 (52.6%) respectively. Further development to compacted morula/blastocyst stage occurred at rates of 78/281 (27.8%), 42/166 (25.3%), 264/912 (28.9%), 79/368 (21.5%), 99/667 (14.8%), 94/644 (14.6%) and 27/228 (11.8%) respectively. Embryos held less than 2.5 h between fusion and activation established pregnancies in 18/66 (27.3%) of recipients, while embryos held over 2.5 h established pregnancies at a rate of 17/57 (29.8%). This study indicates that holding bovine nuclear transfer embryos less than 2.5 h between fusion and activation results in improved nuclear morphology and increased development to compacted morula/blastocyst stage, and results in pregnancy rates equivalent to embryos held over 2.5 h.