91 A NOVEL TECHNIQUE FOR EVALUATION OF THE LIPID CONTENT IN PIG EMBRYOS

Abstract

A high level of lipids, mainly triglycerides and fatty acids, present in embryo cells in the form of lipid droplets is the major factor associated with low cryopreservation of porcine embryos. Previous results demonstrated that the low tolerance of pig embryos to cryopreservation can be increased through reduction of lipid droplet contents. Therefore, in order to improve cryopreservation techniques of porcine embryos, it is fundamental to establish proper culture conditions which ultimately will enable a decrease in lipid content. Unfortunately, there are no precise and efficient methods to evaluate the lipid contents of single pig embryos. Previously used stereological analysis combined with physical serial sectioning (Romek et al. 2007 Reprod. Domest. Anim. in press) is time-consuming, and measurement of triglyceride levels based on enzymatic hydrolysis eliminates other types of lipids from the analysis. Taking the above problems into account, we have developed a new method for evaluation of total lipid content in pig embryos. It is based on visualization of lipid droplets using the specific fluorescent dye Nile red and applying confocal scanning microscopy followed by the Cavalieri method. This method enables measurement of several stereological parameters, especially the volume density of lipid droplets per unit volume of cytoplasm Vv(fat,c), which quantifies most precisely the amount of intracellular lipid. The experiment was carried out on 2- to 4-cell and 8- to 16-cell pig embryos, morulae, blastocysts, and late blastocysts cultured in vitro. Embryos were developed from in vivo-produced zygotes to appropriate stages of development in North Carolina State University (NCSU) 23 medium. For each stage, ten of the embryos were examined. Embryos were denuded and fixed with 2% glutaraldehyde and 2% formaldehyde, stained with 100 nm Nile red (Molecular Probes, Leiden, The Netherlands), and analyzed by means of a confocal microscope LSM 510 Meta (Carl Zeiss MicroImaging GmbH, Göttingen, Germany). Serial optical sections of each individual embryo were measured by the point counting method, and then the Cavalieri method was used to estimate Vv(fat,). Vv(fat,c) values calculated for embryos at different stages of development were compared by one-way analysis of variance and Tukey's intervals. For cultured pig embryos, volume density of lipid droplets Vv(fat,c) significantly decreased during cleavage from 0.55 µm³ µm^–3 at the 2- to 4-cell-embryo stage to 0.46 µm³ µm^–3 at the blastocyst stage. The differences between lipid droplet volumes calculated for morulae, blastocysts, and late blastocysts were statistically significant. In conclusion, our new method is more precise, efficient, and quick in comparison to previously used ones. Moreover, we confirmed that the content of total lipids in cultured pig embryo is reduced during its development.

This research was funded by the State Committee for Scientific Research (Project No. 2 P06D 003 26) and Net of Reproduction Biotechnology.