Evaluation of the numerical chromosomal abnormalities on in vitro early bovine embryos: effect of the cell co-culture with granulose cells

SEBASTIÁN DEMYDA PEYRÁS; MANUEL HIDALGO; JESUS DORADO; MIGUEL MORENO MILLÁN

Reno

Congreso; 40th Conference of the International Embryo Society; 2014

International Embryo Transfer Society

Chromosomal numerical abnormalities (CNA) were described as a major cause of developmental failures in IVP embryos. It has been described that CAN are influenced by the post-fertilization culture environment of the embryo. Furthermore, it was demonstrated that the use of different culture media affect the CAN rates. The addition of granulosa cells during early embryo development is a well-known procedure employed to simplify the culture of bovine in vitro produced and cloned embryos. This technique allows to avoid the use of culture environments saturated with N2 (tri-gas chambers). The aim of this study was to determine the effect of the addition of granulosa cells in the chromosomal abnormalities of in vitro produced cattle embryos. COCs were matured in TCM199 medium, supplemented with glutamine, sodium pyruvate, FSH, LH, estradiol and gentamicin during 20 h at 38.5ºC in a 5% CO2 humid atmosphere. Subsequently, matured oocytes were fertilized in IVF-TALP medium using 1x106 spz/mL, selected through a Percoll? gradient centrifugation. After fertilization, zygotes were divided in two groups and cultured in TCM-199 medium by 48h, with (TCM-GC) or without (TCM) the addition of 1x106 granulosa cells. These cells were obtained by centrifuging and washing the folicular fluid remaining from searching dishes and adjusted to the working concentration. After culture, a total of 106 early embryos (72 hpi) were cytogenetically evaluated following our standard laboratory techniques. Embryos showing normal development were individually fixed onto a slide, disaggregated into blastomeres with acetic acid and stained with giemsa solution. Chromosomal numerical abnormalities were evaluated by direct observation at x1250 magnification in a bright field microscope. Percentage of normal diploid embryos (D) and abnormal haploid (H), poliploidd (P) or aneuploid (A) embryos were determined. Results were statistically compared between treatments using a Z test for proportions. Results were : D = 81,4%, H = 7,2%, P = 7,2% and A = 3,6% in TCM and D = 84,3%, H = 3,9% , P = 9,8% and A = 1,9% in TCM-GC. No significant differences (p>0.05) were found between culture media in the chromosomal abnormality rates. According to our results, the use of somatic cells in coculture during embryo development has not influenced the appearance of abnormal complements in the produced embryos. This would allow the use of GC as a potential complement to simplify the techniques employed in the culture of bovine embryos until day 3.