Crucial surviving aspects for vitrified <i>in vitro</i>-produced bovine embryos

Sudano, Mateus José; Paschoal, D. M.; Rascado, Tatiana da Silva; Crocomo, Letícia Ferrari; Magalhães, Luis Carlos Oña; Martins, A.; Machado, R.; Landim-Alvarenga, F. C.

doi:10.1017/s0967199412000196

Cited by 18 publications

(13 citation statements)

References 41 publications

(91 reference statements)

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“…Improvements to cryosurvival after vitrification were observed with the use of inhibitors of fatty acid synthesis [ 19 , 21 , 63 ], lipolytic agents [ 64 , 65 ], antioxidants [ 66 ] or molecules capable of modulating the molecular mechanisms of lipid uptake [ 67 , 68 ]. A possible explanation for our absence of positive results on cryopreservation in relation to embryos produced with lower lipid content, may be related with other factors that influence their quality and consequently, affecting embryo sensitivity to vitrification [ 16 ].…”

Section: Discussionmentioning

confidence: 99%

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The role of cGMP as a mediator of lipolysis in bovine oocytes and its effects on embryo development and cryopreservation

et al. 2018

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This study aimed to determine the influence of cyclic guanosine 3’5’-monophosphate (cGMP) and cGMP-dependent kinase (PKG) during in vitro maturation (IVM) on lipolysis-related parameters in bovine cumulus-oocyte complexes (COCs), and on embryo development and cryosurvival. COCs were matured with cGMP/PKG modulators and assessed for metaphase II rates (MII), cGMP levels, lipid content in oocytes (OO), transcript abundance for genes involved in lipolysis (ATGL) and lipid droplets (PLIN2) in cumulus cells (CC) and OO, and presence of phosphorylated (active) hormone sensitive lipase (HSLser563) in OO. Embryo development, lipid contents and survival to vitrification were also assessed. Phosphodiesterase 5 inhibition (PDE5; cGMP-hydrolyzing enzyme) with 10-5M sildenafil (SDF) during 24 h IVM increased cGMP in COCs (56.9 vs 9.5 fMol/COC in untreated controls, p<0.05) and did not affect on maturation rate (84.3±6.4% MII). Fetal calf serum (FCS) in IVM medium decreased cGMP in COCs compared to bovine serum albumin (BSA) + SDF (19.6 vs 66.5 fMol/COC, respectively, p<0.05). FCS increased lipid content in OO (40.1 FI, p<0.05) compared to BSA (34.6 FI), while SDF decreased (29.8 and 29.6 FI, with BSA or FCS, respectively p<0.05). PKG inhibitor (KT5823) reversed this effect (38.9 FI, p<0.05). ATGL and PLIN2 transcripts were detected in CC and OO, but were affected by cGMP and PKG only in CC. HSLser563 was detected in OO matured with or without modulators. Reduced lipid content in embryos were observed only when SDF was added during IVM and IVC (27.6 FI) compared to its use in either or none of the culture periods (34.2 FI, p<0.05). Survival to vitrification was unaffected by SDF. In conclusion, cGMP and PKG are involved in lipolysis in OO and possibly in CC and embryos; serum negatively affects this pathway, contributing to lipid accumulation, and cGMP modulation may reduce lipid contents in oocytes and embryos, but without improving embryo cryotolerance.

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Section: Discussionmentioning

confidence: 99%

“…As reported in adipocytes, the elevation of cAMP levels by forskolin, stimulates lipolysis and reduces the amount of lipids in porcine oocytes and embryos [ 14 , 15 ] and bovine oocytes [ 16 ]. In these studies, reduced lipids led to an improvement in cryotolerance.…”

Section: Introductionmentioning

confidence: 98%

The role of cGMP as a mediator of lipolysis in bovine oocytes and its effects on embryo development and cryopreservation

et al. 2018

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“…This impairment occurs due to the physical and chemical damages induced during the cryopreservation process (Overstrom, 1996;Baguisi et al, 2000). Sudano et al (2012a) reported the effects of this damage by comparing the apoptosis rate caused by the stress of cryopreservation between fresh and vitrified blastocysts. In this study, there was a 2.4-fold increase (P < 0.0001) in the apoptosis rate of vitrified (49.4 ± 1.9) in relation to fresh embryos (20.8 ± 1.1).…”

Section: Cryopreservation Of Bovine Embryosmentioning

confidence: 99%

“…Studies have shown that the fetal calf serum (FCS) concentration affects the number of cytoplasmic lipid droplets of embryos (Leroy et al, 2005;Sudano et al, 2012a). Moreover, in vitro embryos cultured in a serum-free medium had decreased lipids and higher cryotolerance (Pereira and Marques, 2008).…”

Section: Strategies To Increase the Cryotolerance Of In Vitro Embryosmentioning

confidence: 99%

Cryopreservation of in vitro-produced embryos: challenges for commercial implementation

Sanches¹,

Zangirólamo²,

Silva³

et al. 2017

Anim Reprod

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In the last several years, the high demand for embryo production has resulted in the need to study new methods to make the cryopreservation of bovine embryos produced in vitro more efficient. Despite the advantages offered by in vitro embryo production (IVEP), the major challenge to its greater dissemination is to improve embryonic survival after cryopreservation. Embryos that are produced in vitro are less resistant to cryopreservation compared to those produced in vivo, which is due to the higher accumulation of lipids in their cells, among other factors. In this context, changes in the culture conditions such as the addition of lipolytic chemical substances and the adjustment of fetal calf serum in the medium have been proposed to decrease the lipid amount within the embryos. Several years ago, vitrification allowed good results for in vitro produced (IVP) embryos because of its simplicity, speed and low cost. More recently, another technique applied to simplify the embryo post-thawing rehydration step in vivo, direct transfer (DT), is a strategy that has proven to be of interest in helping to overcome limitations to the cryopreservation of in vitro produced embryos. DT has been performed by commercial laboratories, ensuring good embryo viability after thawing. However, commercial and operational limitations prevent the large-scale use of these techniques. Thus, this review aims to discuss the use of strategies to improve the postcryopreservation survival capacity and the aspects to be overcome so that the cryopreservation of IVP embryos becomes a well-established and commercially applicable technique in addition to presenting new guidelines for embryo transfer (ET) programs from a better selection of recipients.

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“…In vitro production (IVP) of bovine embryos was performed as previously described [22,31], unless otherwise noted. Only oocytes with three or more compact layers of cumulus cells and homogeneous cytoplasm were used.…”

Section: Embryo Productionmentioning

confidence: 99%

160 Lipidome Signatures in Early Bovine Embryo Development

Sudano

Rascado

Tata

et al. 2016

Reprod. Fertil. Dev.

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Mammalian pre-implantation embryonic development is a complex, conserved, and well-orchestrated process involving dynamic molecular and structural changes. Understanding the membrane lipid profile fluctuation during this crucial period is fundamental to address cellular and molecular mechanisms governing embryogenesis. A full understanding of stage-specific lipid signatures in early bovine embryo development is, however, still lacking. The aim of the present work was to characterise stage-specific changes in lipid profiles during early bovine embryonic development. Immature oocytes were recovered from slaughterhouse-derived bovine ovaries and assigned among 5 in vitro developmental stages for lipid characterisation: immature oocytes, 2-cell embryos (32–40 h post-insemination), 8 to 16-cell embryos (72 h post-insemination), morulas (120 h post-insemination), and blastocysts (168 h post-insemination). Two different culture media were used for in vitro embryo production, SOFaaci medium supplemented with 2.5% of serum and serum-free SOF-BE1 medium. Cytoplasmic lipid droplets content and membrane phospholipids profiles for each development stage were assessed by lipid staining (Nile red; n = 5–9) and matrix-assisted laser desorption ionization as a mass spectrometry imaging (MALDI-MS; n = 5–9), respectively. For statistical analysis, univariate and multivariate models were used to compare lipid droplets content and membrane phospholipids profiles. Cytoplasmic lipid droplets content increased from minimum in the immature oocyte stage to maximum at the morula stage, followed by a sharp drop at the blastocyst stage (58.4 ± 10.5ac, 62.5 ± 9.4ac, 85.9 ± 8.2a, 148.3 ± 7.4b, 37.4 ± 9.9c of fluorescence intensity per embryo area, respectively, for immature oocyte, 2-cells, 8 to 16-cells, morulas, and blastocysts; abcP < 0.05). More cytoplasmic lipid droplets were detected in morulas and blastocyts cultured in SOFaaci than in SOF-BE1 (morulas, 162.6 ± 11.3 v. 137.1 ± 9.2 of fluorescence intensity per embryo area, respectively, P < 0.05; blastocysts, 49.9 ± 9.9 v. 20.7 ± 9.9 of fluorescence intensity per embryo area, respectively, P < 0.05). Characteristic dynamic changes of unsaturation level, acyl chain length and class composition (phosphatidylcholines, sphingomyelins, and phosphatidylethanolamines) of phospholipid profiles were observed during early embryo development. This study provides a comprehensive analysis, involving lipid staining and mass spectrometry evaluation, of stage-specific lipid signatures during bovine in vitro embryo development. These results may be useful for assessing the role of specific lipid species during important events of embryogenesis. Research was supported by CNPq, FAPESP, and FAPERGS.

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Crucial surviving aspects for vitrified in vitro-produced bovine embryos

Cited by 18 publications

References 41 publications

The role of cGMP as a mediator of lipolysis in bovine oocytes and its effects on embryo development and cryopreservation

The role of cGMP as a mediator of lipolysis in bovine oocytes and its effects on embryo development and cryopreservation

Cryopreservation of in vitro-produced embryos: challenges for commercial implementation

160 Lipidome Signatures in Early Bovine Embryo Development

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