Roser Morató

Biotechnology, Cryobiology, Cell Biology



  • Reproduction Fertility and Development 10/2015; · 2.40 Impact Factor
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    ABSTRACT: In the present study we examined whether exposure to high concentrations of NaCl or sucrose before vitrification improves the cryotolerance of in vitro-matured bovine oocytes. In Experiment 1, oocytes were exposed to different concentrations of NaCl (375-1517 mOsm) or sucrose (375-812 mOsm) for 1h. On the basis of the results of this experiment, in Experiment 2 oocytes were exposed to 0.25% NaCl (375 mOsmol) or 2.77% sucrose (375 mOsmol) solution, vitrified and warmed. Microtubule and chromosome configurations were examined by immunocytochemistry. In Experiment 3, in vitro embryo development was assessed after vitrification of oocytes with or without 2.77% sucrose (375 mOsmol) pretreatment. There was a similar percentage of oocytes showing normal spindle configurations in the sucrose-pretreated and control groups. Higher rates of abnormal spindles were found in groups treated with NaCl or sucrose solutions with >375 mOsmol. After vitrification and warming, a significantly higher percentage of oocytes with normal chromosome configurations was recorded for oocytes exposed to 375 mOsmol sucrose solution before vitrification compared with the control vitrified oocytes. However, these percentages were significantly lower than those recorded in untreated controls. Cleavage and blastocyst rates were higher in non-vitrified than vitrified oocytes. In conclusion, pretreatment with 375 mOsmol NaCl or sucrose solution had no adverse effects on the spindle status of vitrified-warmed cow oocytes. However, sucrose pretreatment offered no benefits for embryo development.
    Reproduction Fertility and Development 04/2015; DOI:10.1071/RD14516 · 2.40 Impact Factor

  • Reproduction in Domestic Animals 01/2015; 50:126-127. · 1.52 Impact Factor
  • Roser Morató · Míriam Castillo-Martín · Marc Yeste · Sergi Bonet ·
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    ABSTRACT: The aim of our study was to assess whether the cryotolerance of in vitro-produced embryos could be influenced by the length of in vitro culture and size of blastocoel cavity before vitrification, using the pig as a model. For this purpose we analysed the cryoresistance and apoptosis rate of blastocysts at different stages of development as derived on Day 5 and 6 of in vitro culture. Blastocysts were subsequently vitrified, warmed and cultured for 24h. Re-expansion rates were recorded at 3 and 24h and total cell number and apoptotic cells were determined at 24h. Day-6 blastocysts showed the highest rates of survival after warming, which indicates higher quality compared with Day-5 blastocysts. Higher re-expansion rates were observed for expanded blastocysts and those in the process of hatching when compared with early blastocysts. Total cell number and apoptotic cells were affected by blastocyst stage, vitrification-warming procedures and length of in vitro culture, as expanding and hatching-hatched blastocysts from Day 6 presented higher percentages of apoptotic cells than fresh blastocysts and blastocysts vitrified at Day 5. Our findings suggest that the cryotop vitrification method is useful for the cryopreservation of porcine blastocysts presenting a high degree of expansion, particularly when vitrification is performed after 6 days of in vitro culture. Furthermore, these results show that faster embryo development underlies higher blastocyst cryotolerance and provide evidence that blastocoel cavity expansion before vitrification is a reliable index of in vitro-produced embryo quality and developmental potential.
    Reproduction Fertility and Development 12/2014; DOI:10.1071/RD14203 · 2.40 Impact Factor
  • N Arcarons · R Morató · J F W Spícigo · M A M M Ferraz · T Mogas ·
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    ABSTRACT: It has been previously described that a simple treatment with medium containing elevated NaCl or sucrose concentrations increases the cryotolerance and developmental competence of in vitro-matured porcine oocytes after vitrification and parthenogenetic activation (Lin et al. 2009 Reprod. Fertil. Dev. 21, 338-344). This work was designed to study whether the exposure to increased concentrations of NaCl or sucrose before vitrification improves cryotolerance of in vitro-matured bovine oocytes. In Experiment 1, in vitro-matured oocytes were exposed to different NaCl and sucrose concentrations (from 375 to 808 mOsm) for 1h. In Experiment 2, and according to the results obtained in the first experiment, oocytes were exposed to 375 mOsm NaCl or sucrose solution, vitrified, and warmed. Nontreated oocytes were used as controls. In both experiments, oocytes were fixed after treatment and microtubule, and chromosome distribution was analysed by immunocitochemistry. All statistical analyses were conducted with the IBM SPSS 19 for Windows (IBM corp., Chicago, IL). ANOVA was performed to analyse differences in meiotic spindle. Statistical significance was set at P<0.05. After exposure to 375 mOsm of NaCl or sucrose, similar percentages of oocytes showing normal chromosome distribution were obtained compared to the control group (83.4, 71.8, and 85.0%, respectively). Groups treated with higher concentrations (443 to 808 mOsm) triggered significantly lower proportions of normal spindles. After vitrification/warming, no significant differences were observed between nonvitrified oocytes (71.3%) and those treated with NaCl before vitrification/warming procedure (41.9%) when normal chromosome organisation was analysed. Significantly higher percentages of normal chromosome configuration were observed when oocytes were exposed to sucrose before vitrification (34.2%) compared with control-vitrified oocytes (23.3%). However, pretreatment with NaCl or sucrose before vitrification did not trigger significant differences in terms of percentages of normal microtubule configuration (41.9 and 32.9%, respectively) compared with control-vitrified oocytes (40.2 and 24.4%, respectively), although both treatments differed significantly from control (79.1 and 81.7%, respectively). In conclusion, this study showed that a 375-mOsm NaCl or sucrose pretreatment of bovine oocytes before vitrification did not have a deleterious effect on the organisation of the meiotic spindle of vitrified/warmed bovine oocytes. Further experiments are required to investigate whether in vitro-matured oocytes subjected to this osmotic treatment could improve their development competence after being vitrified/warmed.
    Reproduction Fertility and Development 12/2014; 27(1):116. DOI:10.1071/RDv27n1Ab46 · 2.40 Impact Factor
  • J F W Spricigo · N Arcarons · T Mogas · M A N Dode · R Morato ·
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    ABSTRACT: After cryopreservation, oocytes may suffer morphological and functional damage, due to the high cytoplasm lipid content and to the reactive oxygen species formation. The aim of this work was to evaluate the spindle configuration and the DNA fragmentation of vitrified/warmed oocytes after in vitro maturation (IVM) in a media supplemented with l-carnitine and/or resveratrol, a lipolytic and antioxidant agent, respectively. The IVM viable COC with at least 3 cumulus cell layers and homogenous cytoplasm were randomly distributed into 4 groups: (1) control: conventional IVM media with TCM-199, epidermal growth factor, and 10% FCS; (2) L-CAR: control media supplemented with 0.6mgmL(-1) of l-carnitine; (3) RES: control media supplemented with 1μMmL(-1) of resveratrol; and 4) L+R: control media supplemented with 0.6mgmL(-1) of l-carnitine and 1μMmL(-1) of resveratrol. After 22h of IVM, half of the COC from each group were vitrified and warmed, using the cryotop methodology. After warming, the oocytes were allowed to recover in their respective media for 2 additional hours. After 24h of IVM, oocytes from all treatments were completely denuded and fixed and stained using specific fluorescent probes. The microtubule/chromosome configuration and the DNA fragmentation were analysed by immunocytochemistry under a fluorescent microscope (A.40FL, Carl Zeiss, Oberkochen, Germany). All statistical analyses were conducted with IBM SPSS 19 (IBM; Chicago, IL, USA). ANOVA was performed to analyse differences in meiotic spindle configuration, and the Chi-squared test was used for DNA fragmentation. The significance level was 5%. Although vitrification may cause severe oocyte damage, IVM with l-carnitine alone or in association with resveratrol was able to reduce the percentage of abnormal spindle configurations (Table 1), whereas the addition of resveratrol alone or its association with l-carnitine reduced DNA fragmentation of IVM oocytes after a vitrification/warming process. These results indicate the IVM supplementation with RES and/or L-CAR could modify oocyte composition, increasing its cryotolerance. However further studies are required to confirm the beneficial effect of these molecular interactions.
    Reproduction Fertility and Development 12/2014; 27(1):115-6. DOI:10.1071/RDv27n1Ab45 · 2.40 Impact Factor
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    ABSTRACT: Aquaporins (AQPs) are integral membrane water channels that allow transport of water and small solutes across cell membranes. Although water permeability is known to play a critical role in mammalian cells, including spermatozoa, little is known about their localisation in boar spermatozoa. Two aquaporins, AQP7 and AQP11, in boar spermatozoa were identified by western blotting and localised through immunocytochemistry analyses. Western blot results showed that boar spermatozoa expressed AQP7 (25kDa) and AQP11 (50kDa). Immunocytochemistry analyses demonstrated that AQP7 was localised in the connecting piece of boar spermatozoa, while AQP11 was found in the head and mid-piece and diffuse labelling was also seen along the tail. Despite differences in AQP7 and AQP11 content between boar ejaculates, these differences were not found to be correlated with sperm quality in the case of AQP7. Conversely, AQP11 content showed a significant correlation (P<0.05) with sperm membrane integrity and fluidity and sperm motility. In conclusion, boar spermatozoa express AQP7 and AQP11, and the amounts of AQP11 but not those of AQP7 are correlated with sperm motility and membrane integrity.
    Reproduction Fertility and Development 10/2014; DOI:10.1071/RD14237 · 2.40 Impact Factor
  • N. Arcarons · R. Morato · M. A. Ferraz · T. Mogas ·

    12th International Congress of the; 10/2014
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    ABSTRACT: The low number of oocytes collected from unstimulated donors by ovum pick-up means that embryos produced from each individual female have to be cultured individually or in very small groups. However, it has been demonstrated that single-embryo culture is less efficient than embryo culture in groups. To overcome this limitation, we developed a direct embryo-tagging system, which allows the collective culture of embryos from different origins whilst preserving their pedigree. Presumptive bovine zygotes were tagged with eight wheat-germ agglutinin biofunctionalised polysilicon barcodes attached to the outer surface of the zona pellucida (ZP). Four different barcodes were used to encode groups of 20-25 embryos, which were then cultured in the same drop. Cleavage, Day-7 and Day-8 blastocysts and barcode retention rates were assessed. In addition, Day-7 blastocysts were vitrified and warmed. Barcode attachment to the ZP of bovine embryos affected neither in vitro embryo development nor post-warming survival of the tagged embryos. All the embryos maintained barcodes attached until Day 8 of culture (3.63±0.37 barcodes per embryo) and could be identified. In conclusion, identification of embryos by barcodes attached to the ZP is feasible and will allow the culture of embryos from different donors in the same drop.
    Reproduction Fertility and Development 06/2014; 26(5):645-52. DOI:10.1071/RD13066 · 2.40 Impact Factor
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    ABSTRACT: One of the major obstacles for the vitrification of mature porcine oocytes with ethylene glycol is their low permeability to this cryoprotectant, which results in osmotic stress-induced cell damage and low survival. Pig blastocysts, on the other hand, show enhanced water and cryoprotectant permeability, which has been related to the transcriptional activation of aquaporin-3 (AQP3) channels at this stage of development. In this study, we asked if expression of cRNAs encoding two aquaglyceroporins, human AQP3 (hAQP3) or the zebrafish Aqp3b-T85A mutant, in porcine oocytes can increase their permeability. Microinjection of germinal-vesicle-stage oocytes with enhanced green fluorescent protein (EGFP) or AQP3 cRNAs resulted in the expression of the corresponding proteins in ∼26% of the metaphase-II stage oocytes at 40-44 h of in vitro culture; co-injection of EGFP cRNA appeared to be a suitable marker for oocyte selection since all EGFP-positive oocytes also expressed the corresponding aquaporin. Using this method, we found that mature oocytes co-expressing EGFP and hAQP3 or EGFP and Aqp3b-T85A showed approximately a twofold increase of the hydraulic conductivity (Lp ) with respect non-injected or EGFP alone-injected oocytes in a 0.43 M sucrose or 1.3 M ethylene glycol solution, whereas the ethylene-glycol permeability (PEG ) of EGFP + hAQP3 and EGFP + Aqp3b-T85A oocytes was 6.7- and 12-fold higher, respectively, than control oocytes. These data demonstrate that the artificial expression of aquaglyceroporins in porcine metaphase-II oocytes improves their permeability, and that the zebrafish Aqp3b-T85A mutant is more efficient than the human channel at increasing the oocyte permeability to ethylene glycol. Mol. Reprod. Dev. © 2014 Wiley Periodicals, Inc.
    Molecular Reproduction and Development 05/2014; 81(5). DOI:10.1002/mrd.22310 · 2.53 Impact Factor
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    ABSTRACT: The aim of the present study was to determine the effect of l-ascorbic acid on embryo quality and gene expression of porcine blastocysts after supplementations of in vitro culture medium and/or vitrification–warming media. Embryo quality, in terms of total cell number (TCN), DNA fragmentation and peroxide levels, together with the relative transcript abundance of BCL-2 associated X protein (BAX), BCL2-like 1 (BCL2L1), POU class 5 homeobox 1 (POU5F1) and heat shock protein 70 (HSPA1A), was analysed. In Experiment 1, gene expression and embryo quality of fresh blastocysts were evaluated after culture with or without l-ascorbic acid; no significant differences were observed between the groups. In Experiment 2, blastocysts cultured with or without l-ascorbic acid were vitrified using two different vitrification solutions, supplemented or not with l-ascorbic acid. Supplementation of culture and vitrification media significantly enhanced survival rates and reduced peroxide levels. No significant differences i
    Reproduction Fertility and Development 04/2014; DOI:10.1071/RD14078 · 2.40 Impact Factor
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    ABSTRACT: The aims of the present study were to: (1) evaluate the effect of vitrification and warming on quality parameters and expression levels of pluripotency, apoptotic and stress genes in in vitro-produced (IVP) porcine blastocysts; and (ii) determine the correlation between these parameters. To this end, total cell number, DNA fragmentation, peroxide levels and the relative transcript abundance of BCL-2 associated X protein (BAX), BCL2-like 1 (BCL2L1), heat shock protein 70 (HSPA1A), POU class 5 homeobox 1 (POU5F1), superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2) were analysed in fresh and vitrified IVP blastocysts. The results suggest that vitrification procedures have no effect on total cell number and gene expression of BAX, BCL2L1, SOD1 and SOD2 or the BAX : BCL2L1 ratio. Nevertheless, a significant increase in DNA fragmentation (2.9 ± 0.4% vs 11.9 ± 2.0%) and peroxide levels (80.4 ± 2.6 vs 97.2 ± 3.1) were seen in vitrified compared with Day 7 fresh blastocysts. In addition,
    Reproduction Fertility and Development 04/2014; DOI:10.1071/RD13405 · 2.40 Impact Factor
  • Roser Morató · Teresa Mogas ·
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    ABSTRACT: Two experiments were designed to test the use of a new device designed to vitrify and in-straw warm in vitro produced (IVP) embryos, which can potentially be used for their direct transfer to recipient females in field conditions. In experiment 1, IVP embryos from both prepubertal and adult animals were vitrified on cryotops and warmed in steps (1 M, 0.5 M and 0 M sucrose; protocol W3) or directly in 0.5 M (protocol W1/0.5) or 0 M sucrose (protocol W1/0). Similar survival rates were recorded 24h after warming for calf embryos irrespective of the warming procedure (W3: 79.2%, W1/0.5: 62.5%, W1/0: 66.7%). For cow embryos, survival rates at 24h post-warming were significantly higher when embryos were warmed using the W3 (85.7%) or W1/0.5 (89.1%) protocols compared to the W1/0 protocol (70.5%). In experiment 2, IVP embryos were vitrified on the new designed device followed by their in-straw cryoprotectant (0.5 M sucrose) dilution/warming and different warming temperatures (45°C, 50°C, 60°C and 70°C) were tested. When warming solution passed through the new vitrification/warming device at 45°C, 61.5% of blastocysts were fully re-expanded or hatched at 24h post-warming, being not significantly different to the control (65%). Other warming temperatures triggered significantly lower survival rates at 24h post-warming. No significant differences were detected in total cell numbers and blastocyst apoptosis indices in response to vitrification followed by warming at 45°C respect to the control. Our findings indicate that the new device allows vitrification and in-straw warming of IVP bovine embryos, being a useful option for their direct transfer in field conditions.
    Cryobiology 04/2014; 68(2). DOI:10.1016/j.cryobiol.2014.02.010 · 1.59 Impact Factor
  • Miriam Castillo-Martín · Sergi Bonet · Roser Morató · Marc Yeste ·
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    ABSTRACT: The present study sought to determine the effect of adding L-ascorbic acid (AC) to (1) in vitro culture medium and (2) vitrification and warming solutions on redox status and developmental ability and quality of IVP porcine embryos. In both experiments, embryo quality was analysed in terms of total cell number (TCN), DNA fragmentation, intracellular peroxide levels and expression of three oxidative stress-related genes: glutathione peroxidase 1 (GPX1), superoxide dismutase 1 (SOD1) and 2 (SOD2). In the first experiment, fresh blastocysts were found to upregulate SOD1 expression when cultured with medium supplemented 100 μM AC. No differences were found between culture groups in the other analysed parameters, In the second experiment, blastocysts cultured with or without AC were divided into two groups: vitrified and warmed with solutions containing 0 or 100 μM AC. Addition of AC during culture and vitrification-warming upregulated the expression of GPX1 and SOD1 genes, enhanced survival rates and decreased peroxide levels at 24 h post-warming. In addition, peroxide levels were negatively correlated with relative GPX1- and SOD1-transcript abundances, whereas GPX1 was positively correlated with embryo survival at 24h post-warming. No effects of AC-supplementation were seen for TCN, DNA fragmentation or relative SOD2-transcript abundance in vitrified blastocysts. In conclusion, the addition of AC to culture and vitrification-warming media increases gene expression of antioxidant enzymes SOD1 and GPX1. This appears to improve redox balance and is suggested to ultimately enhance embryo cryosurvival.
    Cryobiology 03/2014; 68(3). DOI:10.1016/j.cryobiol.2014.03.001 · 1.59 Impact Factor
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    ABSTRACT: The present study examined the relationship between the relative amount of high motile sperm and sperm-oocyte interactions obtained from Holstein bull ejaculates. Post-thaw sperm motility was analyzed using a computer-assisted sperm analyzer system and evaluated to determine the sperm motility subpopulations. Adhesion and penetration of zona pellucida (ZP) and pronucleus formation using post-thawed samples (15 ejaculates form 5 different bulls) with different percentages of sperm in the subpopulation with the fastest and most progressive subpopulation (subpopulation 4 [SP4]) were analyzed. The correlation between the proportion of sperm in SP4 and the number of spermatozoa bound to the zona pellucida (ZBA), the penetration rate, and the rate of pronucleus formation were calculated. A significant (P < 0.05) and positive correlation was found between the number of spermatozoa bound to the zona pellucida, the penetration rate, and the rate of pronucleus formation with the proportion of sperm in SP4 (r = 0.79, r = 0.66, and r = 0.63, respectively). Our results suggest that this specific high motile and progressive subpopulation is positively and significantly correlated with the ability of a thawed bull semen sample to interact properly with the oocyte and its extracellular vestments. These findings emphasize the relevance of analyzing semen subpopulation composition to predict bull sperm fertilizing ability and to select Holstein bulls for breeding purposes.
    Theriogenology 01/2014; 81(8). DOI:10.1016/j.theriogenology.2014.01.033 · 1.80 Impact Factor
  • M. Castillo-Martin · S. Bonet · R. Morato · T. Mogas · M. Yeste ·

    Reproduction in Domestic Animals 01/2014; 47:90-90. · 1.52 Impact Factor
  • M. Castillo-Martin · M. Yeste · R. Morato · J. Miro · S. Bonet ·

    Reproduction in Domestic Animals 01/2014; 49:60-60. · 1.52 Impact Factor
  • S Hammami · D Izquierdo · M G Catalá · M T Paramio · R Morató ·
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    ABSTRACT: The present study was designed to evaluate the effect of activin-A during the in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on nuclear maturation, blastocyst yield and blastocyst quality of prepubertal goat oocytes. In Experiment 1, three groups of oocytes were used during the IVM of prepubertal goat oocytes to determine the optimal concentration of recombinant human activin-A added to the maturation medium. Cumulus-oocyte complexes were matured in an IVM medium containing 0, 10 and 100 ng/ml (groups A0, A10 and A100), fertilized and in vitro cultured using standard procedures. In Experiment 2, the addition of 10 ng/ml activin-A at IVM (A10A0), IVC (A0A10) or IVM+IVC (A10A10) was studied and compared with the control group (A0A0). Results of the first experiment demonstrated that the addition of activin-A yielded similar percentages of maturation (⩽71.0%) and blastocyst formation rates (⩽24.9%) than the control group (A0). Experiment 2 showed that exposure of prepubertal goat oocytes to an IVC medium containing 10 ng/ml activin-A (A0A10) significantly increased the rates of development to the blastocyst stage, as compared with the control group (A0A0) (19.5±2.21% v. 13.1±2.37%, respectively; P<0.05). With regard to the blastocyst quality, total number of cells, inner cell mass (ICM) and trophectoderm of prepubertal goat embryos produced in the presence of activin-A did not differ significantly among experimental groups. In summary, these results indicate that supplementation of the IVC medium with activin-A enhances embryo development of prepubertal goat oocytes.
    animal 10/2013; 8(1):1-8. DOI:10.1017/S1751731113001936 · 1.84 Impact Factor
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    ABSTRACT: Our aim was to determine the effect of ejaculates with different motile sperm subpopulations structure on sperm-oocyte binding ability. Fifteen ejaculates from five separate bulls were collected and cryopreserved through a standard protocol. Post-thaw sperm motility was analyzed by using a CASA system and evaluated to identify sperm subpopulations as described by Mui~no et al. (Ani Repr Sci 109; 27–39, 2008). Adhesion to zona pellucida of post-thawed samples with low, medium and high percentages of sperm included in the subpopulation with the fastest and most progressive spermatozoa (T4) were tested through a zona binding assay (ZBA). For this purpose, in vitro maturated cow oocytes were denuded and transferred in groups of 10 to a 45 ll drop of fertilization medium. A 5 ll aliquot of sperm suspension (10 9 106 spz/ml) was added to each drop. After 19– 20 hpi, the oocytes were fixed and stained with DAPI. Statistical differences between means were analyzed by Tukey’s test and correlations by Spearman rank correlation. We found a significant difference (p < 0.05) in the number of spermatozoa bound to zona pellucida among the ejaculates for the same bull and between different bulls. A significant correlation (r = 0.79, p < 0.01) was found between the ZBA and the precise percentage of sperm pertaining to the T4 subpopulation. Our results suggest that the precise motile sperm subpopulations structure is closely related with the ability of a thawed bull semen sample in binding to the ZP.
    Reproduction in Domestic Animals 09/2013; 48(1):77. · 1.52 Impact Factor
  • Arcarons N · Morató R · Ferraz M · Mogas T ·

    AETE, Istambul, Turkey; 09/2013

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