89 Phenazine Ethosulfate and Fetal Calf Serum Effect in the Ultrastructure and Development of in Vitro-Produced Bovine Embryos

Abstract

Over the past decades, there have been great advances in in vitro production (IVP) systems, with improved culture methods and new knowledge regarding embryo physiology, ultrastructure and morphology. Currently, the major obstacle associated with the extensive use of this technology is the great sensitivity of IVP embryos to cryopreservation. According to the literature, the reduced cryotolerance of IVP embryos is frequently associated with their high lipid content. Although is not clear until now how the lipid accumulation occurs, it may be influenced by the use of undefined culture media, supplemented with fetal calf serum (FCS); or as a result of embryo energy metabolism abnormalities that affect mitochondrial function, leading to the decrease in both the embryo quality and survival after cryopreservation. In this context, phenazine ethosulfate (PES), a reducer of NADPH electrons, which favours pentose–phosphate pathways and also inhibits the fatty acids synthesis, has been used to increase IVP embryo cryotolerance (Sudano et al. 2011 Theriogenology 75, 1211–1220). The aim of the present study was to evaluate the phenazine ethosulfate and FCS effect in the ultrastructure of IVP bovine embryos. A 2 × 2 factorial experiment design was used to test 2 FCS concentrations (0 or 10%) and the addition of PES (without or with PES) in the culture media. Slaughterhouse ovaries were used to obtain oocytes which were matured and fertilized in vitro (Day 0). Presumptive zygotes (n = 1440) were divided in 4 culture media: SOFaa without FCS; SOFaa without FCS + 0.3 μM PES (started on Day 4); SOFaa + 10% FCS; SOFaa + 10% FCS + 0.3 μM PES (started on Day 4). Embryo development was evaluated after 7 days under standard culture conditions (at 38.5°C in atmosphere of 5% O2, 5% CO2 and 90% N2). Transmission electron microscopy (TEM) was performed on Day-7 blastocysts from each group (n = 5) through standard protocol. For the statistical analysis, the arcsine transformation was applied to blastocyst percentage data and submitted to the ANOVA, followed by Tukeys' test through PROC GLM (SAS Institute Inc., Cary, NC, USA). In the absence of significant interactions, only main effect means are presented. The blastocyst production was not affected (P = 0.47) by the use of PES (42.7 ± 3.2 vs 39.3 ± 3.2, respectively for control and PES Day 4). The addition of 10% of FCS increased (P < 0.0001) the percentage of blastocysts (48.9 ± 3.2 vs 33.0 ± 3.2, respectively, for 10% and 0% of FCS). The ultrastructure analysis showed similar features in embryos from all studied groups. However, embryos cultured in the absence of FCS presented fewer and smaller lipid droplets. Moreover, embryos cultured without FCS presented more cellular debris in the perivitelinic space and in the blastocoele, indicating loss of blastomeres. The use of PES was able to reduce lipid droplets and increase the mitochondrial number in serum-produced embryos. Therefore, the PES decreased lipid content and increased mitochondrial number without affecting the development and ultrastructure of IVP bovine embryos. FAPESP 09/54513-3, 10/09922-0.

Lipid Droplet Analysis Using In Vitro Bovine Oocytes and Embryos

“…In the present work, oocytes supplemented with SR contained the lowest amount of lipid droplets, and similar results were obtained by Sudano et al. (). Sudano et al.…”

“…FCS is currently the most commonly used product in in vitro embryo production; however, it also causes alterations in embryos subjected to this procedure (Sudano et al. ), such as excess lactate production, the presence of dark and granulated cells in the ICM (Bavister et al. ); Krisher et al.…”

“…Currently, serum-derived synthetic products are frequently used in an attempt to establish a defined culture serum for both oocyte maturation and embryo (Moore et al 2007). FCS is currently the most commonly used product in in vitro embryo production; however, it also causes alterations in embryos subjected to this procedure (Sudano et al 2011), such as excess lactate production, the presence of dark and granulated cells in the ICM (Bavister et al 1992); Krisher et al 1999), an increase in apoptotic cells (Byrne et al 1999), minor protein synthesis (Kuran et al 2001), decrease in the ICM/trophoblastic cell ratio as well as the number of union complexes between embryo cells (Shamsuddin and Rodriguez-Martinez 1994) and an abnormal accumulation of cytoplasmic lipid droplets (Shamsuddin and Rodriguez-Martinez 1994); Thompson 1995); Abe et al 1999a,b). However, the underlying mechanism of why these lipids accumulate in the cytoplasm remains unknown; yet, it is believed that it may be a result of two processes: (i) the embryo is capable of absorbing lipids from the culture environment, particularly under conditions in which a rich lipid source (serum) exists, and (ii) it is postulated that this lipid accumulation is a result of the lack of mitochondrial capability to metabolize lipid complex via beta oxidation (De La Torre-Sanchez et al 2006).…”

“…Some characteristics such as the loss of blastomeres and lack of embryonic development are indicators that these in vitro ‐produced embryos are highly sensitive to the cryopreservation process, which is reflected in the low conception rates compared to those obtained from in vivo ‐produced embryos (Sudano et al. ). The cause of the lipid accumulation in the in vitro ‐produced embryos is still unknown; however, many studies have attributed this effect to the use of foetal calf serum (Abe et al.…”

“…This reduced cryotolerance is frequently associated with the accumulation of lipid droplets in the cytoplasm of the embryos (Abe et al 2002;Rizos et al 2002;De La Torre-Sanchez et al 2006;Mucci et al 2006;Barcelo-Fimbres and Seidel 2007). Some characteristics such as the loss of blastomeres and lack of embryonic development are indicators that these in vitro-produced embryos are highly sensitive to the cryopreservation process, which is reflected in the low conception rates compared to those obtained from in vivo-produced embryos (Sudano et al 2011). The cause of the lipid accumulation in the in vitro-produced embryos is still unknown; however, many studies have attributed this effect to the use of foetal calf serum (Abe et al 2002;Mucci et al 2006;Barcelo-Fimbres and Seidel 2007), a common protein supplement in embryonic developmental cultures (Abe et al 2002;Rizos et al 2002;De La Torre-Sanchez et al 2006;Mucci et al 2006;Seidel 2007, 2007).…”