151 LIPID COMPOSITION OF FAT DROPLETS OF IN VIVO- AND IN VITRO PRODUCED PORCINE EMBRYOS

Abstract

Early-stage porcine embryos to peri-hatching blastocysts contain high levels of intracellular lipids in the form of fat droplets and are highly sensitive to cryopreservation. Recently, our sterological studies demonstrated that in embryos produced in vivo and cultured in vitro, the volume of lipid droplets significantly decreased from zygote to blastocyst. To date, however, there have been no reports concerning the type of lipids in pig embryos produced in vivo and in vitro. Thus, the objective of the present study was to investigate the lipid composition of fat droplets in pig embryos produced in vivo and in vitro. The experiment was carried out on pig zygotes produced in vivo and 2–4 and 8–16-cell embryos, morulae, blastocysts, and late blastocysts produced in vivo and in vitro. Embryos produced in vivo were obtained from superovulated gilts after flushing the oviduct or uterus. Embryos cultured in vitro were developed from zygotes produced in vivo. Embryos were cultured in vitro to appropiate stages of development in chemically defined medium, North Carolina State University (NCSU)-23. For analysis of the type of lipid in the fat droplets, embryos were fixed in 2.5% glutaraldehyde with the addition of 3 mM calcium chloride. The material was then embedded in Technovit 8100, cut into semi-thin sections, and analyzed by histochemical methods. Four techniques were used to detect different types of lipids: Churukian method with Oil red O, Cain method with Nile blue sulfate, Sudan black B, and osmium tetroxide methods. Fat droplets of embryos produced both in vivo and in vitro contained unsaturated hydrophobic lipids, free fatty acids, phospholipids, unsaturated esters, and triglycerides. Moreover, in the morula the total amount of lipids (especially the amount of free fatty acids and phospholipids) evidently decrease. The amount of the other unsaturated lipids decreased as early as the 2- to 4-cell stage. In conclusion, the content of different types of lipids in pig embryos is reduced during their development from zygote to blastocyst, and there are no differences in lipid composition of fat droplets between in vivo- and in vitro-produced porcine embryos. This research was funded by the State Committee for Scientific Research (Project No. 2 P06D 003 26).