Production of a cloned calf from a fetal fibroblast cell line

Mello, Marco Roberto Bourg de; Caetano, H. V. M.; Marques, Mariana Groke; Padilha, Mônica Soares Botelho; Garcia, José Fernando; Milazzotto, Marcella Pécora; Assumpção, Mayra Elena Ortiz D’Ávila; Lima, A. R. R.; Nicacio, Alessandra Corallo; Mendes, C. M.; Oliveira, V.P.; Visintin, José Antônio

doi:10.1590/s0100-879x2003001100006

Cited by 8 publications

(5 citation statements)

References 12 publications

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“…CCB and CCD have been widely used to prevent extrusion of pseudo PBs of G0/G1 somatic donors during the SCNT procedure [3]. With a similar purpose, exposure to these chemicals at different times, such as 1, 5 or 6 hr, have also been attempted in bovine [1,11,20,24,38]. In the present study, groups 1-4 showed extrusion rates ranging from 53-67%, whereas group 5 exhibited a significantly low rate of 22%.…”

Section: Discussionmentioning

confidence: 69%

Parthenogenetic Development and Ploidy following Various Chemical Activation Regiments of Bovine Oocytes

Ock

Rho

2008

The Journal of Veterinary Medical Science

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ABSTRACT. Bovine oocytes treated using various combinations of 5 µM ionomycin (Ion), 10 µg/ml cycloheximide (CHX) and 5 µg/ml cytochalsin B (CCB) were evaluated to determine developmental rates and ploidy status. The groups were comprised of metaphase II oocytes exposed to Ion for 5 min (Group 1); Ion treatment followed by CHX for 5 hr (Group 2); Ion treatment followed by CHX/CCB for 1 hr and CHX for 4 hr (Group 3); Ion treatment followed by CHX/CCB for 3 hr and CHX for 2 hr (Group 4); and Ion treatment followed by CHX/CCB for 5 hr (Group 5). Group 5 exhibited significantly (P<0.05) lower rates of second polar body (PB) extrusion and higher rates of cleavage and blastocyst development. At 8 hr after Ion treatment, the eggs in groups 2, 3 and 5 were divided into 2 subgroups based on the presence or absence of the second PB and were assessed for cleavage rate and ploidy at the two-cell stage. The cleavage rates did not differ among the activation treatments or between the presence and absence of the second PB in all groups. The diploid rate was significantly (P<0.05) higher in group 5 than in groups 2 and 3. However, the diploid rate in blastocyst-stage parthenotes did not differ among groups 2, 3 and 5. Consequently, oocyte activation by CHX/CCB for 5 hr after Ion treatment could enhance diploid parthenogenetic development in bovine. KEY WORDS: bovine parthenote, chemical activation, ploidy, polar body.J. Vet. Med. Sci. 70(11): 1165-1172, 2008 In bovine, oocyte activation is a major factor for successful production of reconstructed embryos by somatic cell nuclear transfer (SCNT) [34], parthenogenesis and intracytoplasmic sperm injection [23]. During normal fertilization in mammalian species, sperm penetration into the oocyte triggers intracellular Ca 2+ oscillation followed by inactivation of the maturation promoting factor (MPF) composed of the catalytic subunit of cdc2p34 protein kinase and the regulatory subunit cyclin B1/B2 for about 2 hr [37] [29,30], that prevent MPF re-accumulation by reducing cdc2 kinase activity [2] have also been applied. DMAP treatment of metaphase II oocytes induces diploid activation by preventing chromosomal separation and extrusion of the second polar body (PB) [19]. However, CHX does not prevent chromosomal segregation and second PB extrusion, resulting in haploid development. When cytochalasin D (CCD) was added in the medium, chromosomal segregation manifested, but the cytokinesis that induces second PB extrusion was prohibited in CHX-treated oocytes, leading to diploid development with two pronuclei in activated oocytes [4,19]. The combined treatment of ionomycin with DMAP for oocyte activation could induce an abnormal pattern of karyokinesis during the first cell cycle [8], and thus, CHX has widely been used as an alternative approach [19]. Moreover, in cloning, oocyte activation using CHX produces healthy live offspring in cattle [12,17], but the live offspring rate is still low [5].Campbell et al. [3] proposed the hypothesis that when donor cells at G1 stage are reconstructed...

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Section: Discussionmentioning

confidence: 69%

Parthenogenetic Development and Ploidy following Various Chemical Activation Regiments of Bovine Oocytes

Ock

Rho

2008

The Journal of Veterinary Medical Science

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“…Samples were collected at a local slaughterhouse in Sao Paulo. Finally, bovine clones were produced by somatic cell NT according to the methods of Mello et al (2003), Yamazaki et al (2005) and Miglino et al (2007). For analyses, only specimens without clear signs of resorption or regression were used.…”

Section: Sample Collectionmentioning

confidence: 99%

Development of bovine embryos derived from reproductive techniques

Alberto

Meirelles

Perecin

et al. 2013

Reprod. Fertil. Dev.

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Assisted reproduction techniques have improved agricultural breeding in the bovine. However, important development steps may differ from the situation in vivo and there is a high mortality rate during the first trimester of gestation. To better understand these events, we investigated the development of embryos and fetal membranes following fixed-time AI (FTAI), IVF and nuclear transfer (NT). The onset of yolk-sac development was not normal in cloned embryos. Later steps differed from conditions in vivo in all three groups; the yolk-sac was yellowish and juxtaposed with the amniotic membrane. Vascularisation of the chorioallantoic membrane was relatively late and low in NT gestations, but normal in the others. The overall development of the embryos was normal, as indicated by morphology and regression analysis of growth rate. However, NT conceptuses were significantly smaller, with the livers in some embryos occupying the abdominal cavity and others exhibiting heart abnormalities. In conclusion, the yolk-sac and the cardiovascular system seem to be vulnerable to morphogenetic alterations. Future studies will focus on gene expression and early vascularisation processes to investigate whether these changes may be responsible for the high incidence of intrauterine mortality, especially in clones.

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“…On day 7 after fusion, embryo quality was evaluated and grades 1 and 2 blastocysts were transferred to recipients. For further details of the protocols, see Mello et al [22] and Yamazaki et al [23].…”

Section: Enucleation Nucleus Transfer and Culturementioning

confidence: 99%