Phosphatidylcholine and sphingomyelin profiles vary in Bos taurus indicus and Bos taurus taurus in vitro- and in vivo-produced blastocysts.

Lipid droplets, subspecies (Bos taurus indicus vs. Bos taurus taurus), and in vitro culture are known to influence cryopreservation of bovine embryos. Limited information is available regarding differences in membrane lipids in embryo, such as phosphatidylcholines (PC) and sphingomyelins (SM). The objective of the present study was to compare the profiles of several PC and SM species and relate this information to cytoplasmic lipid levels present in Nellore (B. taurus indicus) and Simmental (B. taurus taurus) blastocysts produced in vitro (IVP) or in vivo (ET). Simmental and IVP embryos had more cytoplasmic lipid content than Nellore and ET embryos (n = 30). Blastocysts were submitted to matrix-assisted laser desorption/ionization mass spectrometry. Differences in the PC profile were addressed by principal component analysis. The lipid species with PC (32:1) and PC (34:1) had higher ion abundances in Nellore embryos, whereas PC (34:2) was higher in Simmental embryos. IVP embryos had less abundant ions of PC (32:1), PC (34:2), and PC (36:5) compared to ET embryos. Moreover, ion abundance of PC (32:0) was higher in both Nellore and Simmental IVP embryos compared to ET embryos. Therefore, mass spectrometry profiles of PC and SM species significantly differ with regard to unsaturation level and carbon chain composition in bovine blastocysts due to subspecies and in vitro culture conditions. Because PC abundances of Nellore and Simmental embryos were distinct (34:1 vs. 34:2), as were those of IVP and ET embryos (32:0 vs. 36:5), they are potential markers of postcryopreservation embryonic survival.

Sudano MJ ，Santos VG ，Tata A ，Ferreira CR ，Paschoal DM ，Machado R ，Buratini J ，Eberlin MN ，Landim-Alvarenga FD ... -  《-》

Cryotolerance and global gene-expression patterns of Bos taurus indicus and Bos taurus taurus in vitro- and in vivo-produced blastocysts.

In a 2×2 factorial experimental design, embryo development, cryotolerance and global gene expression of Nellore (Bos taurus indicus) and Simmental (Bos taurus taurus) blastocysts produced in vitro (IVP) and in vivo (multiple ovulation derived embryo, MODE) were assessed. Blastocyst production was higher in Nellore than in Simmental (47.7±2.0% vs 27.0±2.0%) cows. The total numbers of ova or embryos recovered (5.5±0.9 vs 3.7±0.8) and transferable embryos (3.8±1.0 vs 2.3±0.8) per cow were not different between breeds. Simmental and MODE (34.6% and 38.5%, n=75 and 70) blastocysts had higher survival rates after cryopreservation compared with Nellore and IVP (20.2% and 18.1%, n=89 and 94) embryos, respectively. Differences between transcriptomes were addressed by principal-component analysis, which indicated that gene expression was affected by subspecies (158 genes), origin (532 genes) and interaction between both subspecies and origin (53 genes). Several functional processes and pathways relevant to lipid metabolism and embryo viability involving differentially expressed genes were identified. The lipid metabolism-related genes were upregulated in Simmental (AUH and ELOVL6) and IVP (ACSL3 and ACSL6) blastocysts. The expression profiles of genes related to mitochondrial metabolism (ATP5B), oxidative stress (GPX4), apoptosis (DAD1, DAP, PRDX2), heat shock (HSPA5), pregnancy (IFNT2, PAG2) and cell differentiation (KRT18) varied between experimental groups.

Sudano MJ ，Caixeta ES ，Paschoal DM ，Martins A Jr ，Machado R ，Buratini J ，Landim-Alvarenga FD ... -  《-》

Membrane lipid profile of in vitro-produced embryos is affected by vitrification but not by long-term dietary supplementation of polyunsaturated fatty acids for oocyte donor beef heifers.

Dietary rumen-protected polyunsaturated fatty acids (PUFAs) rich in linoleic acid (LA) may affect embryo yield, and LA can modulate the molecular mechanisms of lipid uptake in bovine blastocysts produced in vitro. In embryos, membrane lipids, such as phosphatidylcholines (PCs) and sphingomyelins (SMs), affect cryopreservation success. The aim of the present study was to evaluate embryonic developmental rates after the IVF of oocytes retrieved from Nellore heifers fed for approximately 90 days with rumen-protected PUFAs rich in LA. In addition, we evaluated embryo cryotolerance and the membrane structure lipid composition using matrix-assisted laser desorption ionisation mass spectrometry of fresh and vitrified embryos. Embryo development to the blastocyst stage (mean 43.2%) and embryo survival after vitrification and warming (mean 79.3%) were unaffected by diet. The relative abundance of one lipid species (PC ether (PCe; 38:2, which means that this lipid has 38 carbon atoms and 2 double bonds in the fatty acyl residues) was increased after PUFAs supplementation. However, 10 ions were affected by cryopreservation; ions consistent with PC 32:0, PC 34:1, SM 24:1, PC 40:6 or PC 42:9, PC plasmalogen (PCp) 44:10 or PC 42:7, triacylglycerol (TAG) 54:9 and a not assigned ion (m/z 833.2) were lower in blastocysts that survived to the cryopreservation process compared with fresh blastocysts, whereas the abundance of the ions PC 36:3 or PC 34:0, PCe 38:2 or PC 36:6 and PC 36:5 or PCe 38:1 were increased after cryopreservation. Thus, the results demonstrate that the mass spectrometry profiles of PC, SM and TAG species differ significantly in bovine blastocysts upon cryopreservation. Because the lipid ion abundances of fresh and vitrified-warmed embryos were distinct, they can be used as potential markers of post-cryopreservation embryonic survival.

Leão BCS ，Rocha-Frigoni NAS ，Nogueira É ，Cabral EC ，Ferreira CR ，Eberlin MN ，Accorsi MF ，Neves TV ，Mingoti GZ ... -  《-》

Cryosurvival and pregnancy rates after exposure of IVF-derived Bos indicus embryos to forskolin before vitrification.

In vitro-produced (IVP) bovine embryos are more sensitive to cryopreservation than their in vivo counterparts due to their higher lipid concentrations, whereas Bos indicus IVP embryos are even more sensitive than Bos taurus IVP embryos. To examine the effects of a lipolytic agent, before vitrification of Bos indicus IVP embryos, on embryo survival, viability, and pregnancy rates, two experiments were conducted. In experiment 1, Bos indicus (Nelore) embryos were produced from abattoir-derived ovaries and allocated into two groups. In the treatment group, 10 μM of forskolin was added to the in vitro culture medium on Day 5 and incubated for 48 hours. On Day 7 of culture, IVP-expanded blastocysts from both the control (n = 101) and treatment (n = 112) groups were vitrified with ethylene glycol and DMSO via the Cryotop procedure. Although there was no significant difference between the rates of blastocoel reexpansion and hatching of the embryos exposed to forskolin (87.5% and 70.5%, respectively) compared with the control embryos (79.2% and 63.3%, respectively), the numerically superior rates of the embryos exposed to forskolin led to another experiment. In experiment 2, blastocysts produced from the ovum pick up were exposed or not exposed to the lipolytic agent and vitrified as in experiment 1. Embryos treated with forskolin had higher pregnancy rates than the control group (48.8% vs. 18.5%). In view of these results, 1908 Bos indicus embryos were produced from ovum pick up, exposed to the lipolytic agent, and blastocysts were transferred to recipients, and the pregnancy rates of the embryos of various breeds were compared. The mean pregnancy rate obtained was 43.2%. All data were analyzed by chi-square or by binary logistic regression (P ≤ 0.05). In conclusion, treatment with forskolin before vitrification improved cryotolerance of Bos indicus IVP embryos, resulting in good post-transfer pregnancy rates.

Sanches BV ，Marinho LS ，Filho BD ，Pontes JH ，Basso AC ，Meirinhos ML ，Silva-Santos KC ，Ferreira CR ，Seneda MM ... -  《-》

Effects of heat stress on development, quality and survival of Bos indicus and Bos taurus embryos produced in vitro.

Heat stress is an important cause of poor development and low survival rates in bovine embryos. Experiments were conducted to test the hypothesis that Bos indicus embryos are more resistant to heat stress than are Bos taurus embryos. In experiment 1, Nelore and Jersey embryos from oocyte pick-up-derived oocytes were submitted to heat stress (96 hours post-insemination, 41 °C, 6 hours), developmental ratios were assessed at Day 7 (Day 0 = day of fertilization), and blastocysts were frozen for RNA extraction. Experiment 2 evaluated expression of COX2, CDX2, HSF1, and PLAC8 in previously frozen blastocysts. In experiment 3, Nellore and Angus embryos from oocyte pick-up-derived oocytes were submitted to heat stress (96 hours post-insemination, 41 °C, 12 hours) and transferred to recipients on Day 7. In experiment 4, embryos developed as in experiment 3 were fixed for Terminal deoxynucleotidyl transferase dUTP nick end labeling labeling and total cell counting. In experiment 1, heat stress decreased the percentage of Jersey oocytes that became blastocysts, but had no effect on Nellore embryos (34.6%, 25.0%, 39.5%, and 33.0% for Jersey control, Jersey heat-stressed, Nellore control, and Nellore heat-stressed oocytes, respectively; P < 0.05). In experiment 2, heat stress decreased (P < 0.05) expression of CDX2 and PLAC8, with higher expression of these genes in Nellore embryos than in Jersey embryos. Heat stress also decreased (P < 0.05) expression of COX2 in Jersey embryos, but had no effect on Nellore embryos. Expression of HSF1 was decreased (P < 0.05) by heat stress in both breeds, with a greater effect in Nellore embryos. In experiment 3, heat stress tended (P = 0.1) to decrease the percentage of pregnancies among cows (Day 30 to 35) that received Angus embryos. In experiment 4, heat stress increased (P < 0.05) the percentage of apoptotic blastomeres, but had no breed-specific effects. In addition, Nellore embryos had fewer (P < 0.05) Terminal deoxynucleotidyl transferase dUTP nick end labeling- positive blastomeres than did Angus embryos. We concluded that the detrimental effects of heat stress were dependent upon embryo breed and were more evident in Bos taurus embryos than in Bos indicus embryos.

Silva CF ，Sartorelli ES ，Castilho AC ，Satrapa RA ，Puelker RZ ，Razza EM ，Ticianelli JS ，Eduardo HP ，Loureiro B ，Barros CM ... -  《-》