Fecundación in vitro : millorant la crioconservació

En el camp de la fecundació in vitro es poden utilitzar oòcits frescos o crioconservats (congelats). Els avantatges de la crioconservació són evidents, però té un desavantatge important : l'eficàcia de la fecundació és molt menor. Aquest treball ha explorat un mètode per estabilitzar els oòcits mitjançant Taxol en crioconservar-los, incrementant el percentatge d'èxit de la fecundació.En el campo de la fecundación in vitro se pueden utilizar ovocitos frescos o crioconservados (congelados). Las ventajas de la crioconservación son evidentes, pero tiene una importante desventaja: la eficacia de la fecundación es mucho menor. Este trabajo ha explorado un método para estabilizar los ovocitos mediante Taxol al crioconservarlos, incrementando el porcentaje de éxito de la fecundación.In the field of in vitro fertilisation, the oocytes used can be either fresh or cryopreserved. The advantages of cryopreservation are evident, but there is also a strong disadvantage: fertilisation efficiency is significantly lower. This work explores a method to stabilise the oocytes by using Taxol when cryopreserving them, which increases the success rate of fertilisation

Effect of cryoprotectant concentration on bovine oocyte permeability and comparison of two membrane permeability modelling approaches

Generalitat de Catalunya 2019 FI_B2 00055The plasma membrane permeability to water and cryoprotectant (CPA) significantly impacts vitrification efficiency of bovine oocytes. Our study was designed to determine the concentration-dependent permeability characteristics for immature (GV) and mature (MII) bovine oocytes in the presence of ethylene glycol (EG) and dimethyl sulphoxide (MeSO), and to compare two different modeling approaches: the two parameter (2P) model and a nondilute transport model. Membrane permeability parameters were determined by consecutively exposing oocytes to increasing concentrations of MeSO or EG. Higher water permeability was observed for MII oocytes than GV oocytes in the presence of both MeSO and EG, and in all cases the water permeability was observed to decrease as CPA concentration increased. At high CPA concentrations, the CPA permeability was similar for MeSO and EG, for both MII and GV oocytes, but at low concentrations the EG permeability of GV oocytes was substantially higher. Predictions of cell volume changes during CPA addition and removal indicate that accounting for the concentration dependence of permeability only has a modest effect, but there were substantial differences between the 2P model and the nondilute model during CPA removal, which may have implications for design of improved methods for bovine oocyte vitrification

In vitro maturation in the presence of Leukemia Inhibitory Factor modulates gene and miRNA expression in bovine oocytes and embryos

Altres ajuts: OECD Co-operative Research Programme: Biological Resource Management for Sustainable Agricultural Systems ; Natural Sciences and Engineering Research Council of CanadaMembers of the interleukin-6 (IL-6) family of cytokines are important for reproductive function that are mediated through changes in gene and miRNA expression. Herein, we characterized the expression of miR-21, miR-155, miR-34c and miR-146a in bovine oocytes and cumulus cells during in vitro maturation (IVM) with leukemia inhibitory factor (LIF), IL-6 and IL-11 or unsupplemented controls. LIF-exposed COCs showed higher expression of miR-21 and miR-155 in oocytes, whereas miR-146a expression was increased in oocytes matured with IL-6 and IL-11. In cumulus cells, miR-155 expression was elevated by all treatments while only LIF increased miR-21 expression. Based on these results, we next examined how LIF exposure during IVM affected oocyte competence, through IVF and the expression of specific genes in GV- and MII-oocytes, in 2- and 8-cell embryos, and in Day 8-blastocysts. LIF supplementation did not affect cleavage rate, blastocyst yield or several other developmental parameters, but did increase hatching rate. LIF suppressed DPPA3, ZAR1 and NPM2 expression in 2 cell- and/or 8-cell embryos. LIF increased the expression of KAT2A and HSPA1A in MII-oocytes, and that of HDAC1, KAT2A and HSP90AA1 and the BAX:BCL2L1 ratio in 2-cell embryos. In contrast, HDAC1, KAT2A and HSP90AA1 expression and BAX:BCL2L1 ratio was lower in 8-cell embryos derived from LIF oocytes. IVM with LIF also increased the expression of DNMT3A, HSPA1A and HSP90AA1 in blastocysts. In conclusion, supplementation with LIF during IVM was consistently associated with changes in the relative abundance of transcripts in mature bovine oocytes and in specific embryo developmental stages

Cryoprotectant role of exopolysaccharide ID1 in the vitrification/in-straw warming of in vitro-produced bovine embryos

Acord transformatiu CRUE-CSICThe cold-adapted bacterium Pseudomonas sp. ID1 produces the extracellular exopolysaccharide ID1 (EPS ID1) with cryoprotective activity. This study was designed to optimize the vitrification/in-straw warming protocol of in vitro-produced (IVP) blastocysts by adding EPS ID1 to the vitrification media. Day 7-expanded blastocysts were vitrified/warmed using the VitTrans device after the addition of 0 or 100 μg/mL EPS ID1 to the vitrification media. Blastocysts vitrified by the Cryotop method and fresh non-vitrified blastocysts served as controls. Outcomes were assessed in the warmed embryos in terms of survival rates and mRNA relative abundances of BAX, BCL2, GPX1, and CDX2 genes. No differences in survival rates were observed at 3 h post-warming between vitrification treatments. At 24 h post-warming, the addition of EPS prior to vitrification with the VitTrans device produced similar survival rates to Cryotop-vitrified embryos and similar hatching rates to fresh non-vitrified or Cryotop-vitrified embryos. No differences emerged in BCL2 gene expression. Lower BAX (p <.05) and higher GPX1 (p <.05) and CDX2 (p <.1) gene expression were observed in expanded and/or hatched blastocysts derived from VitTrans-EPS-vitrified embryos when compared to those from the non-supplemented group. In conclusion, addition of EPS not only promoted blastocyst survival and hatching after VitTrans vitrification/warming but also modified the expression of genes associated with better embryo quality

Exopolysaccharide ID1 Improves Post-Warming Outcomes after Vitrification of In Vitro-Produced Bovine Embryos

This study aimed to assess the cryoprotectant role of exopolysaccharide (EPS) ID1, produced by Antarctic Pseudomonas sp., in the vitrification of in vitro-produced (IVP) bovine embryos. IVP day 7 (D7) and day 8 (D8) expanded blastocysts derived from cow or calf oocytes were vitrified without supplementation (EPS0) or supplemented with 10 µg/mL (EPS10) or 100 µg/mL (EPS100) EPS ID1. The effect of EPS ID1 was assessed in post-warming re-expansion and hatching rates, differential cell count, apoptosis rate, and gene expression. EPS100 re-expansion rates were significantly higher than those observed for the EPS0 and EPS10 treatments, regardless of culture length or oocyte source. EPS100 hatching rate was similar to the one of the fresh blastocysts except for those D7 blastocysts derived from calf oocytes. No differences were observed among EPS ID1 treatments when the inner cell mass, trophectoderm, and total cell number were assessed. Although apoptosis rates were higher (p ≤ 0.05) in vitrified groups compared to fresh embryos, EPS100 blastocysts had a lower number (p ≤ 0.05) of apoptotic nuclei than the EPS0 or EPS10 groups. No differences in the expression of BCL2, AQP3, CX43, and SOD1 genes between treatments were observed. Vitrification without EPS ID1 supplementation produced blastocysts with significantly higher BAX gene expression, whereas treatment with 100 µg/mL EPS ID1 returned BAX levels to those observed in non-vitrified blastocysts. Our results suggest that 100 µg/mL EPS ID1 added to the vitrification media is beneficial for embryo cryopreservation because it results in higher re-expansion and hatching ability and it positively modulates apoptosis

A Shorter Equilibration Period Improves Post-Warming Outcomes after Vitrification and in Straw Dilution of In Vitro-Produced Bovine Embryos

This study was designed to the optimize vitrification and in-straw warming protocol of in vitro-produced bovine embryos by comparing two different equilibration periods, short equilibrium (SE: 3 min) and long equilibrium (LE: 12 min). Outcomes recorded in vitrified day seven (D7) and day eight (D8) expanded blastocysts were survival and hatching rates, cell counts, apoptosis rate, and gene expression. While survival rates at 3 and 24 h post-warming were reduced (p < 0.05) after vitrification, the hatching rates of D7 embryos vitrified after SE were similar to the rates recorded in fresh non-vitrified blastocysts. The hatching rates of vitrified D8 blastocysts were lower (p < 0.05) than of fresh controls regardless of treatment. Total cell count, and inner cell mass and trophectoderm cell counts were similar in hatched D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values regardless of treatment. The apoptosis rate was significantly higher in both treatment groups compared to fresh controls, although rates were lower for SE than LE. No differences emerged in BAX, AQP3, CX43, and IFNτ gene expression between the treatments, whereas a significantly greater abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE. A shorter equilibration vitrification protocol was found to improve post-warming outcomes and time efficiency after in-straw warming/dilution

Fecundación in vitro : millorant la crioconservació

En el camp de la fecundació in vitro es poden utilitzar oòcits frescos o crioconservats (congelats). Els avantatges de la crioconservació són evidents, però té un desavantatge important : l'eficàcia de la fecundació és molt menor. Aquest treball ha explorat un mètode per estabilitzar els oòcits mitjançant Taxol en crioconservar-los, incrementant el percentatge d'èxit de la fecundació.En el campo de la fecundación in vitro se pueden utilizar ovocitos frescos o crioconservados (congelados). Las ventajas de la crioconservación son evidentes, pero tiene una importante desventaja: la eficacia de la fecundación es mucho menor. Este trabajo ha explorado un método para estabilizar los ovocitos mediante Taxol al crioconservarlos, incrementando el porcentaje de éxito de la fecundación.In the field of in vitro fertilisation, the oocytes used can be either fresh or cryopreserved. The advantages of cryopreservation are evident, but there is also a strong disadvantage: fertilisation efficiency is significantly lower. This work explores a method to stabilise the oocytes by using Taxol when cryopreserving them, which increases the success rate of fertilisation

Testis-Specific Protein Y-Encoded (TSPY) Is Required for Male Early Embryo Development in Bos taurus

TSPY is a highly conserved multi-copy gene with copy number variation (CNV) among species, populations, individuals and within families. TSPY has been shown to be involved in male development and fertility. However, information on TSPY in embryonic preimplantation stages is lacking. This study aims to determine whether TSPY CNV plays a role in male early development. Using sex-sorted semen from three different bulls, male embryo groups referred to as 1Y, 2Y and 3Y, were produced by in vitro fertilization (IVF). Developmental competency was assessed by cleavage and blastocyst rates. Embryos at different developmental stages were analyzed for TSPY CN, mRNA and protein levels. Furthermore, TSPY RNA knockdown was performed and embryos were assessed as per above. Development competency was only significantly different at the blastocyst stage, with 3Y being the highest. TSPY CNV and transcripts were detected in the range of 20-75 CN for 1Y, 20-65 CN for 2Y and 20-150 CN for 3Y, with corresponding averages of 30.2 ± 2.5, 33.0 ± 2.4 and 82.3 ± 3.6 copies, respectively. TSPY transcripts exhibited an inverse logarithmic pattern, with 3Y showing significantly higher TSPY. TSPY proteins, detected only in blastocysts, were not significantly different among groups. TSPY knockdown resulted in a significant TSPY depletion (p < 0.05), with no development observed after the eight-cell stage in male embryos, suggesting that TSPY is required for male embryo development

Embryo development of prepubertal goat oocytes fertilised by intracytoplasmic sperm injection (ICSI) according to oocyte diameter

The aim of this study was to evaluate embryo development of prepubertal goat oocytes fertilised by ICSI according to their diameter. Three experiments were carried out to achieve this objective. In all experiments, oocytes were matured in TCM199 supplemented with hormones, cysteamine and serum for 27 h at 38.58 C. In Experiment 1, we studied the nuclear stage of goat zygotes produced by conventional ICSI and IVF using 20 nM ionomycin plus 10 m M heparin as sperm treatment. A group of Shaminjected oocytes was used as control. Results showed differences in the percentage of 2 PN (zygotes with male and female pronuclei) between ICSI, IVF and Sham (40.9, 26.6 and 3.0%, respectively; P 135 mm), fertilised by ICSI and cultured for 192 h. Results showed a positive correlation between oocyte diameter and embryo development (morulae + blastocysts: Group A: 0%; Group B: 6.2%; Group C: 46.4% and Group D: 33.3%). In conclusion, sperm treatment with ionomycin plus heparin using the conventional ICSI protocol improved fertilisation rates in comparison to IVF. Oocytes smaller than 125 mm were unable to develop up to blastocyst stage

Embryo development of prepubertal goat oocytes fertilised by intracytoplasmic sperm injection (ICSI) according to oocyte diameter

The aim of this study was to evaluate embryo development of prepubertal goat oocytes fertilised by ICSI according to their diameter. Three experiments were carried out to achieve this objective. In all experiments, oocytes were matured in TCM199 supplemented with hormones, cysteamine and serum for 27 h at 38.58 C. In Experiment 1, we studied the nuclear stage of goat zygotes produced by conventional ICSI and IVF using 20 nM ionomycin plus 10 m M heparin as sperm treatment. A group of Shaminjected oocytes was used as control. Results showed differences in the percentage of 2 PN (zygotes with male and female pronuclei) between ICSI, IVF and Sham (40.9, 26.6 and 3.0%, respectively; P 135 mm), fertilised by ICSI and cultured for 192 h. Results showed a positive correlation between oocyte diameter and embryo development (morulae + blastocysts: Group A: 0%; Group B: 6.2%; Group C: 46.4% and Group D: 33.3%). In conclusion, sperm treatment with ionomycin plus heparin using the conventional ICSI protocol improved fertilisation rates in comparison to IVF. Oocytes smaller than 125 mm were unable to develop up to blastocyst stage