Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum‐free or serum‐containing media

Abstract

In this study, the quantitative fluctuation of cytoplasmic lipid droplets (LD) and cryotolerance were investigated in bovine embryos derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes developed in different culture systems using serum-free or serum-containing media. The serum-free cultures were grown using IVMD101 medium in conjunction with bovine cumulus/granulosa cell (BCGC) cocultures or IVD101 medium without BCGC cocultures, and the serum-containing cultures were grown in the presence of BCGC cocultures using HPM199 medium supplemented with 5% calf serum (HPM199 + CS). Large numbers of sudanophilic LD were present in the cytoplasm of bovine embryos from 2-cell to hatched blastocyst stages, and the number and size differed between the embryos cultured in serum-free and serum-supplemented media. In the embryos cultured in HPM199 + CS, large (2-6 microm in diameter) sudanophilic LD increased significantly from the morula to the blastocyst stages. Throughout the embryonic development, the embryos developed in serum-free cultures with and without BCGC cocultures had numerous sudanophilic LD, but most of these droplets were small (<2 microm in diameter) and large LD were less numerous than those embryos cultured in HPM199 + CS. Giant LD (>6 microm in diameter) were frequently observed in morulae and blastocysts (including early blastocysts) developed in HPM199 + CS. Electron microscopic observations demonstrated that large LD were abundant in the cytoplasm of trophoblast and embryonic (inner cell mass) cells of blastocysts cultured in HPM199 + CS. These large LD were identified as osmophilic LD, an indication that these lipid inclusions contained a significant proportion of unsaturated lipids. Many elongated mitochondria were found in embryos developed in IVMD101 and IVD101 at the morula and early blastocyst stages, whereas many of the mitochondria in the morulae developed in HPM199 + CS were of an immature form such as spherical or ovoid shape. The survival and hatching rates of embryos (morulae, early blastocysts, and blastocysts) produced in serum-free media (both IVMD101 and IVD101) after post-thaw culture were superior to those of embryos produced in serum-containing medium. These results showed that bovine embryos cultured in serum-containing medium abnormally accumulated cytoplasmic lipids into their cytoplasm and the excess accumulation of cytoplasmic LD in embryos may affect the cryotolerance of embryos.

Serum free embryo culture medium improves in vitro survival of bovine blastocysts to vitrification

“…Culture in the absence of serum results in less lipid accumulation in embryos [26]. A further finding is that embryos with less lipids, produced without serum in the medium, also cryopreserve more successfully that lipidrich embryos produced in the presence of serum [4,8,26].…”

“…Other works [8,26,43] showed that bovine embryos cultured without serum had more cryo-resistance, particularly when embryos were vitrified. Mucci et al [43] observed a lack of benefit from the serum on embryo development, probably as a consequence of differences in experimental conditions compared with other authors, such as the absence of co-culture during the whole development period, and the type and concentration of serum used.…”

“…A further finding is that embryos with less lipids, produced without serum in the medium, also cryopreserve more successfully that lipidrich embryos produced in the presence of serum [4,8,26]. The relationship between the lipid droplets (LD) accumulated in the embryo and its impaired ability to survive cryopreservation has been clearly demonstrated [7,26,44]. Abe et al [26] showed that bovine embryos cultured in serum containing media, abnormally accumulated lipids into their cytoplasm, although in vitro development was unaffected.…”

“…A higher development rate to the blastocyst stage is obtained from media supported with serum, but the serum effectiveness for the vitro embryo production might change considerably from one batch to another [23]. Furthermore, embryos produced with serum show alterations in their ultrastructure, impaired compaction, abnormal blastulation, aberrant mRNA expression profiles and large calf syndrome with greater incidences of stillbirths and deaths after birth [26][27][28]. Moreover, bovine-derived sera or proteins have recently been avoided especially in human in vitro production systems because of the appearance of bovine spongiform encephalopathy and a viral or prion contamination risk.…”

Does serum cause lipid-droplet accumulation in bovine embryos produced in vitro, during developmental days 1 to 4?

“…Crosier et al [15,16], Abe et al [3] and Abe and Hoshi [1] based on observations in morulae and blastocysts produced in vitro proposed that the presence of serum would have adverse effects on mitochondria structure, promoting lipid accumulation, thus reducing the quality and viability of the embryo. Frequently this hypothesis is used to explain survival`s decrease of embryos after cryopreservation treatments though it has not yet been proven.…”