DNA methylation status of bovine blastocysts obtained from peripubertal oocyte donors.

In the dairy industry, the high selection pressure combined with the increased efficiency of assisted reproduction technologies (ART) are leading toward the use of younger females for reproduction purposes, with the aim to reduce the interval between generations. This situation could impair embryo quality, decreasing the success rate of the ART procedures and the values of resulting offspring. Young Holstein heifers (n = 10) were subjected to ovarian stimulation and oocyte collection at 8, 11, and 14 months of age. All the oocytes were fertilized in vitro with semen from one adult bull, generating three pools of embryos per animal. Each animal was its own control for the evaluation of the effects of age. The EmbryoGENE platform was used to compare the DNA methylation status of blastocysts obtained from oocytes collected at 8 versus 14 and 11 versus 14 months of age. Age-related contrast analysis identified 5,787 and 3,658 differentially methylated regions (DMRs) in blastocysts from heifers at 8 versus 14 and 11 versus 14 months of age, respectively. For both contrasts, the DMRs were distributed nonrandomly in the different DNA regions. The DNA from embryos from 8-month-old donors was more hypermethylated, while the DNA from embryos from 11-month-old donors displayed an intermediate phenotype. According to Ingenuity Pathway Analysis, the upstream regulator genes cellular tumor antigen p53, transforming growth factor ß1, tumor necrosis factor, and hepatocyte nuclear factor 4α are particularly associated with methylation sensitive targets, which were more hypermethylated in embryos from younger donors.

Transcriptomic evaluation of bovine blastocysts obtained from peri-pubertal oocyte donors.

Assisted reproduction technologies (ART) and high selection pressure in the dairy industry are leading towards the use of younger females for reproduction, thereby reducing the interval between generations. This situation may have a negative impact on embryo quality, thus reducing the success rate of the procedures. This study aimed to document the effects of oocyte donor age on embryo quality, at the transcriptomic level, in order to characterize the effects of using young females for reproduction purpose. Young Holstein heifers (n = 10) were used at three different ages for ovarian stimulation protocols and oocyte collections (at 8, 11 and 14 months). All of the oocytes were fertilized in vitro with the semen of one adult bull, generating three lots of embryos per animal. Each animal was its own control for the evaluation of the effects of age. The EmbryoGENE platform was used for the assessment of gene expression patterns at the blastocyst stage. Embryos from animals at 8 vs 14 months and at 11 vs 14 months were used for microarray hybridization. Validation was done by performing RT-qPCR on seven candidate genes. Age-related contrast analysis (8 vs 14 mo and 11 vs 14 mo) identified 242 differentially expressed genes (DEGs) for the first contrast, and 296 for the second. The analysis of the molecular and biological functions of the DEGs suggests a metabolic cause to explain the differences that are observed between embryos from immature and adult subjects. The mTOR and PPAR signaling pathways, as well as the NRF2-mediated oxidative stress response pathways were among the gene expression pathways affected by donor age. In conclusion, the main differences between embryos produced at peri-pubertal ages are related to metabolic conditions resulting in a higher impact of in vitro conditions on blastocyts from younger heifers.