It is shown that the complexation of Eu(fod)3 with the MTPA ester of trans‐4‐tert‐butylcyclohexanol is very similar to that of ketones with Eu(fod)3. It is found to be more likely that the MTPA moiety can exist as two rotamers, suggesting that earlier models for correlating LIS and absolute configuration are inadequate.
The production of excessive levels of reactive oxygen species can be a major problem during in vitro embryo culture. Although studies have shown that supplementation with exogenous antioxidants can improve embryo quality, the results are controversial among researchers. In this study, we examined the effects of different concentrations of β-mercaptoethanol (β-ME) added to the culture media, on cleavage rates, the quality and developmental competence of in vitro-produced bovine embryos. The embryos were produced in vitro as described previously (Van Hoeck et al., 2013). Briefly, in total, 753 grade I cumulus–oocyte complexes (COC) from 2- to 6-mm-diameter follicles were matured in groups of 50 in 500 μL of TCM with 20 ng mL–1 EGF for 24 h, fertilized in groups of 100 in 500 μL of fertilization medium for 20 h (5% CO2, 38.5°C). Presumptive zygotes were denuded and randomly assigned to 4 treatments with different concentrations of β-ME: 0 μM (control), 50 μM, 100 μM, and 150 μM. They were cultured in groups of ±25 in 50 μL of SOF supplemented with ITS (10 μg mL–1 insulin; 5.5 μg mL–1 transferrin; 6.7 ng mL–1 selenium) and 2% BSA and covered with mineral oil (5% O2, 5% CO2, 38.5°C). At 48 h post-insemination (p.i.), cleavage rate was evaluated and expressed as the number of cleaved embryos on total number of oocytes. At Day 7 p.i., blastocyst rate was determined (number of blastocysts on total number of oocytes), blastocysts were fixed in 4% paraformaldehyde, and total cell number was determined by DAPI staining. Data were analysed by ANOVA and post hoc test. Comparable cleavage rates were obtained in treated groups: control (80.8%), 50 μM (77.7%), 100 μM (77.9%), and 150 μM (73.6%; P > 0.05). Also, no significant effect of treatment could be found on blastocyst rates: control (36%), 50 μM (36.5%), 100 μM (38.4%), and 150 μM (30.4%). The total cell number per blastocyst increased significantly (P < 0.05) using 100 μM of β-ME compared with the controls (158.0 ± 24.3 v. 123.2 ± 9.72, respectively). These results suggest that the inclusion of 100 μM β-ME during in vitro embryo culture could be used for production of high quality bovine blastocysts.
The enzymatic "in vitro" reduction of 2-chlorocyclohexanone in a coupledsubstrate coenzyme recycling system (ketoneethanol -NAD' -HLAD) delivers optically pure (1S,2S)-2-chlorocyclohexanol. The optical purity is shown by "F-NMR of the (R)-MTPA esters. The absolute configuration is mainly determined by ORD of the (2S)-2-chlorocyclohexanone.
In humans, diets rich in carbohydrates and saturated fatty acids are common and the subsequent altered metabolism has been linked to reduced fertility. Also, modern dairy cows are fed milk-stimulating diets rich in starch and fatty acids. The pre-implantation embryo is vulnerable to nutritionally induced changes in its micro-environment. We have shown that a dietary induced hyperlipidemia has detrimental consequences on development, quality and gene expression patterns of the pre-implantation embryo (Leroy et al. 2010 Hum. Reprod. 25, 768–778). Hyperlipidemia was induced by feeding starch- and saturated-fat-rich diets and the collected serum was added during bovine embryo culture. In the present study, we hypothesized that changing the saturated into a polyunsaturated fat source could alleviate these negative effects. We furthermore hypothesized that the sequence in which the different fat sources are given can affect embryo development and quality. Therefore, in the first setup: bovine zygotes (n = 1104; 4 replicates) were cultured in SOF medium supplemented with 10% serum collected from 3 synchronized heifers after 3 successive dietary treatments each fed during 4 weeks: control serum after a hay-based maintenance diet, saturated serum (SAT1) after a carbohydrate rich diet supplemented with saturated fatty acids (twice maintenance, C16 : 0, palmitic acid, 4.5% total fat), or unsaturated serum (UNSAT1) after a carbohydrate-rich diet supplemented with unsaturated fatty acids (twice maintenance, C18 : 3, linolenic acid, 4.45% total fat). In the second setup (n = 1483; 5 replicates): bovine zygotes were cultured in SOF medium supplemented with 10% serum from 3 synchronized heifers successively fed the 3 same dietary treatments in different order: control, unsaturated (UNSAT2), or saturated serum (SAT2). Day 7 blastocyst developmental competence (binary logistic regression), total cell number, and apoptotic cell ratio (ACR) (mixed model ANOVA) were evaluated. Supplementation of SAT1 serum in culture significantly reduced blastocysts from cleaved zygotes (36.7% v. 44.7%) and significantly increased ACR (0.1% ± 0.05 v. 0.06% ± 0.04) compared to controls (P < 0.05). Zygotes cultured in UNSAT1 displayed a significant higher cell number than control and SAT1 blastocysts (126.4 ± 25.7 v. 120.4 ± 24.3 and 108.3 ± 15.5, respectively; P ≤ 0.05) and lower ACR compared to SAT1 blastocysts (0.06% ± 0.03 v. 0.1% ± 0.05; P < 0.05). However, UNSAT1 zygotes showed a tendency for reduced development into blastocysts compared to control zygotes (P = 0.05). By contrast, UNSAT2 serum significantly improved blastocysts development from cleaved zygotes (40.0% v. 26.7%) and led to a lower ACR (0.06% ± 0.04 v. 0.1% ± 0.05) compared to SAT2 embryos (P < 0.05). In conclusion, our study confirmed the negative effects diets rich in starch and saturated fat on pre-implantation embryo development and quality. Changing the fat source to polyunsaturated eliminated these negative effects. Furthermore, we showed that the order in which different fat types are fed affects the zygote’s ability to sustain further development.
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