49 EFFECT OF REPEATED CELL FREEZINGS ON PREGNANCY RATE OF BOVINE NUCLEAR TRANSFER DERIVED EMBRYOS

Cell line cryopreservation is nowadays one of the most useful tools in somatic cell nuclear transfer. Although this technique guarantees the genetic storage for an unlimited period of time, many studies have shown that it produces different kinds of cellular damage such as DNA fragmentation (Men et al. 2003 Mol. Reprod. Dev. 64, 245–250) and ultrastructural cell anomalies (Taddei et al. 2001 Cryobiology 42, 244–555; Nardid et al. 1997 Cryobiology 34, 107–113). The aim of the present study was to evaluate how repeated cell freezing/thawing processes could affect the pregnancy rate of bovine nuclear transfer-derived embryos. Two adult fibroblast cell lines from different animals were separated into two groups according to the number of freezing/thawing processes they went through (1 vs. 3). For both groups, the first freezing process was performed with cells from passage 1. Cells from passages 3 and 4 were used for the second and third freezings, respectively. The time interval between thawing and next freezing was 20 days. Cells were harvested at 80% confluence using trypsin, and cryopreservation was performed in D-MEM with 35% FCS and 10% DMSO. Enucleation and nuclear transfer (NT) were performed as described by Cibelli et al. (1998 Science 280, 1256–1258) with modifications. For both groups, cells from the same number of passages were used for the NT assays (between passages 2 and 7). Cytoplasts were activated using 5 µM ionomycin for 4 min and the couplets were subsequently fused. The fused units were cultured in 10 µg/mL cycloheximide and 5 µg/mL cytochalasin B for 6 h. Embryo culture was performed at 38.5°C in a 5% O2, 5% CO2, 90% N2 atmosphere, in 50-µL drops of KSOM. On Day 3 of culture, the KSOM was supplemented with 2% FCS and 0.2 mM glucose. After 6–7 days, the embryos were non surgically transferred to synchronized recipients. Pregnancy at 30 and 60 days was recorded by ultrasonography using an Aloka 500® scanner (Aloka Co., Tokyo, Japan). Data were analyzed by ANOVA (InfoStat, Austin, TX, USA) (Table 1). The results show an association between the number of cell freezing/thawing processes and a higher pregnancy loss at 60 days. This could be related to the cellular damages caused by multiple cryopreservation procedures, which could lead to chromosomal abnormalities in the donor cells and thus in the nuclear transfer (NT) embryos and pregnancies derived from them. Further studies should be done in order to evaluate the chromosomal status of the cell lines used in this work. Table 1. Numbers of cell freezings/thawings and their effect on pregnancy rate of bovine NT-derived embryos

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