In silico-designed vitrification protocols: an approach to improve survival of in vitro produced-bovine embryos.

In brief: In silico predictions validated in this study demonstrate the potential for designing shorter equilibration protocols that improve post-warming re-expansion and hatching rates of D7 and D8 in vitro-produced bovine embryos. Our results benefit the livestock industry by providing a refined and reproducible approach to cryopreserving bovine embryos, which, in addition, could be useful for other mammalian species. Abstract: The cryopreservation of in vitro-produced (IVP) embryos is vital in the cattle industry for genetic selection and crossbreeding programs. Despite its importance, there is no standardized protocol yielding pregnancy rates comparable to fresh embryos. Current approaches often neglect the osmotic tolerance responses to cryoprotectants based on temperature and time. Hereby, we propose improved vitrification methods using shorter dehydration-based protocols. Blastocysts cultured for 7 (D7) or 8 days (D8) were exposed to standard equilibration solution (ES) at 25ºC and 38.5ºC. Optimized exposure times for each temperature and their impact on post-warming re-expansion, hatching rates, cell counts, and apoptosis rate were determined. In silico predictions aligned with in vitro observations, showing original volume recovery within 8 min 30 s at 25ºC or 3 min 40 s at 38.5ºC (D7 blastocysts) and 4 min 25 s at 25ºC and 3 min 15 s at 38.5ºC (D8 blastocysts) after exposure to ES. Vitrification at 38.5ºC resulted in D7 blastocysts re-expansion and hatching rates (93.1% and 38.1%, respectively) comparable to fresh embryos (100.0% and 32.4%, respectively), outperforming the 25ºC protocol (86.2% and 24.4%, respectively; P < 0.05). No differences were observed between D7 and D8 blastocysts using the 38.5ºC protocol. Total cell number was maintained for D7 and D8 blastocysts vitrified at 38.5ºC but decreased at 25ºC (P < 0.05). Apoptosis rates increased post-warming (P < 0.05), except for D8 blastocysts vitrified at 38.5ºC, resembling fresh controls. In conclusion, based on biophysical permeability data, new ES incubation times of 3 min 40 s for D7 blastocysts and 3 min 15 s for D8 blastocysts at 38.5ºC were validated for optimizing vitrification/warming methods for bovine IVP blastocysts.

Blastocoel fluid aspiration improves vitrification outcomes and produces similar sexing results of in vitro-produced cattle embryos compared to microblade biopsy.

The potential applications of in vitro-produced (IVP) cattle embryos are significantly enhanced when combined with genotype selection and cryopreservation techniques. While trophectoderm (TE) biopsies are frequently used for genotyping, cell-free DNA (cfDNA) found in blastocoele fluid (BF) arises as a less-invasive method. Moreover, the blastocoel collapse produced by BF aspiration could be beneficial for embryo cryotolerance. This study was conducted to test the BF as a source of cell free-DNA (cfDNA) and to compare the BF to the TE biopsy in terms of sexing efficiency/accuracy, embryo survival and gene expression after vitrification/warming. IVP day 7 expanded blastocysts were artificially collapsed by aspiration of BF (VIT-Collapsed) or biopsied (VIT-Biopsied). After sample collection, embryos were vitrified/warmed by the Cryotop method and individually cultured in vitro. Intact fresh non-vitrified and vitrified/warmed blastocysts served as Fresh Control and VIT-Control, respectively. After sex identification of BF or TE biopsies and the corresponding surviving embryos, amplification efficiency and sexing accuracy were assessed. There were no differences between the BF and TE biopsy samples in terms of sexing accuracy or efficiency. Although all vitrified groups showed lower post-warming re-expansion rates (p < 0.05), the blastocyst re-expansion rates in the VIT-Collapsed group were comparable to those in the Fresh Control group whereas biopsied blastocysts showed the lowest (p < 0.05) re-expansion rates. VIT-Collapsed blastocysts had hatching rates that were comparable to those of Fresh Control blastocysts but significantly higher than those of the other vitrification treatments. Proapoptotic gene BAX was overexpressed in VIT-Biopsied embryos, whereas BCL2 transcripts were more abundant in the VIT-Collapsed group. On the other hand, VIT-Biopsied embryos showed altered ATP1B1- and AQP3-mRNA levels. The analysis of the cfDNA present in the BF is an efficient, minimally invasive approach to sex IVP cattle embryos. Besides, the artificial collapse of blastocoel prior to vitrification resulted in higher re-expansion and hatching ability than when embryos were vitrified after being biopsied.

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.

Impact of equilibration duration combined with temperature on the outcome of bovine oocyte vitrification.

The cryopreservation of mammalian oocytes and embryos has become an integral part of assisted reproduction in both humans and veterinary species. However, the methods used to cryopreserve bovine oocytes still have significant shortcomings. A wide variety of approaches has been used to try to improve and optimize methods of cryopreservation. However, these procedures employed are not always designed to specifically take account of the osmotic tolerance response of the cells according to the temperature and time of cryoprotectant (CPA) addition. When these properties are considered, optimal procedures for the addition of CPAs can be designed proactively. Based on in silico and in vitro osmotic observations, we propose shorter dehydration-based protocols at different temperatures (25°C vs. 38.5°C) towards defining an improved cryopreservation method. In vitro matured oocytes were exposed to equilibration solution (ES) at 25°C and 38.5°C and effects of optimized exposure times for each temperature were determined prior to vitrification/warming on oocyte spindle configuration, DNA fragmentation, and further embryo development. Upon exposure to standard ES (7.5% dimethyl sulfoxide + 7.5% ethylene glycol in TCM199 medium + 20% fetal bovine serum), original oocyte volume was recovered within 2 min 30 s at 38.5°C and 5 min 30 s at 25°C. In vitro matured oocytes were then exposed to the aforementioned cryoprotectants at both temperature/duration conditions and vitrified/warmed. While similar percentages of oocytes exhibiting a normally configured spindle and DNA fragmentation were observed in the fresh control group and oocytes vitrified at 38.5°C, significantly higher apoptosis rate and lower percentages of normal spindle configuration were observed in oocytes vitrified at 25°C when compared to control fresh oocytes. Similar cleavage rates and blastocyst yields were observed in the vitrified/38.5°C and fresh controls, while these rates were lower in vitrified/25°C. These results revealed that the limitation of the exposure time of the oocytes to the ES to the point of osmotic equilibrium volume recovery could be a more efficient approach to prepare them for vitrification. Therefore, exposure time to ES to 2 min 30 s at 38.5 °C appears to improve the quality of vitrified/warmed oocytes by protecting spindle integrity and reducing DNA fragmentation thus improving blastocyst rates and embryo quality.

The Role of Aquaporin 7 in the Movement of Water and Cryoprotectants in Bovine In Vitro Matured Oocytes.

Aquaglyceroporins are known as channel proteins, and are able to transport water and small neutral solutes. In this study, we evaluate the effect of exposure of in vitro matured bovine oocytes to hyperosmotic solutions containing ethylene glycol (EG), dimethyl sulfoxide (Me2SO) or sucrose on the expression levels of AQP3, AQP7 and AQP9. Moreover, we studied whether artificial protein expression of AQP7 in bovine oocytes increases their permeability to water and cryoprotectants. Exposure to hyperosmotic solutions stimulated AQP3 and AQP7 but not AQP9 expression. Oocytes exposed to hyperosmotic Me2SO solution exhibited upregulated AQP3 expression, while AQP7 expression was upregulated by EG hyperosmotic exposure. Microinjection of oocytes at the germinal vesicle stage with enhanced green fluorescent protein (EGFP) or EGFP+AQP7 cRNAs resulted in the expression of the corresponding proteins in ≈86% of the metaphase-II stage oocytes. AQP7 facilitated water diffusion when bovine MII oocytes were in presence of Me2SO solution but not EG or sucrose solution. However, the overexpression of this aquaporin did not increase membrane permeability to Me2SO or EG. In summary, cryoprotectant-induced increase of AQP3 and AQP7 expression could be one of the mechanisms underlying oocyte tolerance to hyperosmotic stress. Water diffusion appears to be improved when AQP7 overexpressed oocytes are exposed to Me2SO, shortening the time required for oocytes to achieve osmotic balance with cryoprotectant solutions.

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

The 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 (Me2SO), 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 Me2SO or EG. Higher water permeability was observed for MII oocytes than GV oocytes in the presence of both Me2SO 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 Me2SO 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.

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.

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

Members 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.

Glutathione Ethyl Ester Protects In Vitro-Maturing Bovine Oocytes against Oxidative Stress Induced by Subsequent Vitrification/Warming.

This study aimed to examine whether the addition of glutathione ethyl ester (GSH-OEt) to the in vitro maturation (IVM) medium would improve the resilience of bovine oocytes to withstand vitrification. The effects of GSH-OEt on spindle morphology, levels of reactive oxygen species (ROS), mitochondrial activity and distribution, and embryo developmental potential were assessed together with the expression of genes with a role in apoptosis (BAX, BCL2), oxidative-stress pathways (GPX1, SOD1), water channels (AQP3), implantation (IFN-τ) and gap junctions (CX43) in oocytes and their derived blastocysts. Vitrification gave rise to abnormal spindle microtubule configurations and elevated ROS levels. Supplementation of IVM medium with GSH-OEt before vitrification preserved mitochondrial distribution pattern and diminished both cytoplasmic and mitochondrial ROS contents and percentages of embryos developing beyond the 8-cell stage were similar to those recorded in fresh non-vitrified oocytes. Although not significantly different from control vitrified oocytes, vitrified oocytes after GSH-OEt treatment gave rise to similar day 8-blastocyst and hatching rates to fresh non-vitrified oocytes. No effects of GSH-OEt supplementation were noted on the targeted gene expression of oocytes and derived blastocysts, with the exception of GPX1, AQP3 and CX43 in derived blastocysts. The addition of GSH-OEt to the IVM medium before vitrification may be beneficial for embryo development presumably as the consequence of additional anti-oxidant protection during IVM.

In Vitro Maturation with Leukemia Inhibitory Factor Prior to the Vitrification of Bovine Oocytes Improves Their Embryo Developmental Potential and Gene Expression in Oocytes and Embryos.

Oocyte cryopreservation has a significant impact on subsequent embryonic development. Herein, we investigated whether supplementing in vitro maturation medium with Leukemia Inhibitory Factor (LIF) prior to vitrification affects embryo development and gene expression at different embryo developmental stages. A panel of genes including maternal effect, epigenetics, apoptosis and heat stress was relatively quantified. The results show reduced cleavage rates after vitrification, regardless of the LIF treatment. Although not statistically different from control-vitrified oocytes, oocyte apoptosis and the blastocyst yield of LIF-vitrified oocytes were similar to their non-vitrified counterparts. Vitrification increased oocyte ZAR1, NPM2 and DPPA3 gene expression while its expression decreased in LIF-vitrified oocytes to similar or close levels to those of non-vitrified oocytes. With a few gene-specific exceptions, vitrification significantly increased the expression of DNMT3A, HDAC1, KAT2A, BAX and BCL2L1 in oocytes and most stages of embryo development, while comparable expression patterns for these genes were observed between LIF-vitrified and non-vitrified groups. Vitrification increased HSPA1A expression in oocytes and HSP90AA1 in 2-cell embryos. Our data suggest that vitrification triggers stage-specific changes in gene expression throughout embryonic development. However, the inclusion of LIF in the IVM medium prior to vitrification stimulates blastocyst development and several other developmental parameters and induces oocytes and embryos to demonstrate gene expression patterns similar to those derived from non-vitrified oocytes.