Does the use of natriuretic peptide type-C in in vitro culture of bovine embryos alter the in vitro and in vivo characteristics of its criotolerance?

Abstract

In the last decade, there were several technical advances on the in vitro production of bovine embryos. However, the cryopreservation of those embryos is not consistent with the general improvements of the technique. One of the main factors related to the low post-cryopreservation survival rate is their distinct lipid profile and content. The lipids are present as droplets in the cytoplasm and in the cell membrane of the embryos. Therefore, some attempts to add or remove molecules in the In Vitro Culture (IVC) medium have already been described. Their aims were to reduce or to alter the lipid content, which resulted in improvements of the embryos submitted to the cryopreservation. The use of the natriuretic peptide type-C (CNP) - a modulator of intracellular cAMP and cGMP concentrations - has already been described in in vitro maturation or pre-maturation. However, there is a scarce information about its use to change the lipid profile of the cells during the IVC of bovine embryos. The aim with this work is to elucidate the interaction of CNP with bovine embryos and, especially, its potential effect on the alteration of the cellular lipid profile and content of the embryo. Also, CNP effects will be evaluated on the in vitro and in vivo following the embryo cryopreservation. The in vitro produced embryos will be submitted to one of the CNP concentrations (100 nM and another to be established) on the day 5 of the IVC. To evaluate the concentration and lipid profile, the embryos will be submitted to Sudan Black B and MRM-profiling techniques. To characterize the competence and quality of the CNP derived embryos, the transcript abundance of some related genes to the lipid and energy metabolism, embryonic quality, and CNP system will be analyzed. The putative CNP receptor (NPR2) will be immunolocalized in the cell membrane of morulae and blastocysts (Exp. I). The CNP concentration that is best will be used in experiments II and III. To evaluate the cryotolerance (Exp. II), embryos treated with CNP will be submitted to the cryopreservation by two different techniques: slow curve method and vitrification and, after warming, the rates of re-expansion and hatching will be evaluated. In experiment III, in vitro produced embryos (derived from OPU collected oocytes) will be allocated in four groups (2x2 factorial that is fresh transfer or submitted to cryopreservation, and with or without CNP supplementation in the IVC). They will be evaluated after the embryo transfer in recipients for the potential of pregnancy establishment. It is expected a deeper understanding about the CNP supplementation effects on the profile and lipid content of bovine embryos and, consequently, their cryotolerance and pregnancy establishment. (AU)