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P1.28 Comparison of morphological quality of fetal fibroblasts fsed in nuclear transfer for swine embryo production for xenotransplantation

Ernesto Goulart, Brazil

Professor - Doctor 1
Departament of Genetics and Evolutionary Biology
University of Sao Paulo

Abstract

Comparison of morphological quality of fetal fibroblasts fsed in nuclear transfer for swine embryo production for xenotransplantation

Ligiane Leme1, Georgina Hasteinreiter1, Débora Rodrigues1, Tainah Moraes1, Priscila T. Silva1, Guilherme Meskauckas1, Thiago Mantovani1, Ludmilla V. Garcia3, Luiz G. C. Munhoz3, Michelle S. Araujo3, Vitor Leão3, Luciano Brito3, Mayana Zatz2,3, Silvano M. A. Raia3, Ernesto Goulart2,3.

1Embryology - XenoBR, Sao Paulo University, São Paulo, Brazil; 2Genetics, Sao Paulo University, São Paulo, Brazil; 3XenoBR, Sao Paulo University, São Paulo, Brazil

Centro de Pesquisa sobre o Genoma Humano e Células Tronco - USP. Faculdade de Medicina - USP.

The production of cloned pigs via somatic cell nuclear transfer (SCNT) is a promising approach to generate organs suitable for xenotransplantation. The quality of donor cells, particularly oocytes and fetal fibroblasts (FF), plays a crucial role in the success of cloning procedures. This study aims to compare morphological quality of FF used in SCNT and how it influences embryo development. FF were cultured until 80-95% confluence and evaluated based on morphological criteria: cell shape (rounded), size (<15 micrometers), and population homogeneity. Each parameter was scored on a scale from 0-1 (poor), 2-3 (medium), to 4-5 (good), and a mean score was adopted. Only medium- and good- scored cells were used for cloning. Additionally, cumulus cells (CC) were employed as a control group to assess the impact of cell type on cloning efficiency, and parthenogenetic (PTN) embryos were used as second control to compare oocyte quality. Oocytes were also evaluated using the same morphological scoring criteria. Post-reconstruction and chemical activation, embryos were cultured in vitro, and developmental competence was assessed by cleavage and blastocyst formation rates on day 7. Medium- and high-scores were compared, data were analyzed by chi-square, considering P<0.05 as significant. Notably, the PTN group exhibited difference in embryo development based on oocyte quality, with high-scored oocytes resulting in higher cleavage (111/116; 95.7%) and blastocyst rates (78/116; 67.2%), compared to medium-scored oocytes (20/25; 80.0% and 9/25; 36.0%), respectively (P<0.005). No difference was observed between high- and medium-quality FF: cleavage rates were 120/193 (62.2%) vs. 60/106 (56.6%), and blastocyst rates were 27/193 (14.0%) vs. 18/106 (17.0%). Similarly, no significant differences were found when comparing CC groups: cleavage rates of 65/106 (61.3%) vs. 26/46 (56.5%), and blastocyst rates of 18/106 (17.0%) vs. 6/46 (13.0%). Likewise, no significant differences were observed between FF and CC. These findings suggest that, FF morphology did not markedly impact developmental success beyond the influence of oocyte quality and consequently does not appear to influence swine SCNT success. Nevertheless, cells with optimal appearance, appropriate size, and homogeneous populations are preferred because they facilitate the micromanipulation procedures. Maintaining cells at 80-95% confluence is indicative of cell cycle arrest in the G1/G0 phase, which likely contributes to consistent results across experiments. These findings highlight that, within the parameters tested, selecting high-quality donor cells based on morphology may be less critical than ensuring proper cell cycle status. Still, choosing healthy, well-characterized donor cells remains important for optimizing cloning efficiency in xenotransplantation applications.

References:

[1] Xenotransplantation
[2] SCNT
[3] Swine cloning
[4] Embryo production

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