Papers by Dolors Izquierdo
Reproduction, Fertility and Development, 2016
Resveratrol (3,4′,5-trihydroxystilbene) is a phytoalexin identified in various plant species, par... more Resveratrol (3,4′,5-trihydroxystilbene) is a phytoalexin identified in various plant species, particularly in grapevine peel. It is a strong antioxidant, induces mitochondrial biogenesis and enhances Sirtuin 1 (SIRT1) activity by inhibiting phosphodiesterase. SIRT1 belongs to the family of NAD+-dependent histone deacetylates and has been shown to regulate several key cellular processes, including transcriptional silencing, aging, chromatin remodeling, and genomic stability, via deacetylation of p53, FoxO transcription factors, and nuclear factor kappa B (NF-κB). The aim of this study was to determine whether supplementation of the maturation and fertilisation medium with resveratrol influences bovine oocyte maturation and subsequent embryonic development and whether these effects are mediated via SIRT1 pathway. Three different resveratrol concentrations were used during in vitro maturation (IVM) and IVF. Cumulus-oocyte complexes (n = 2878) were collected from slaughterhouse ovaries ...
Theriogenology, Oct 1, 2005
The objective of this study was to compare the embryo development of prepubertal goat oocytes aft... more The objective of this study was to compare the embryo development of prepubertal goat oocytes after ICSI and IVF procedures. Three experiments were carried out to achieve this objective. (1) An analysis of the efficiency of ICSI with or without chemical stimulation (5 microM ionomycin for 5 min and 2 mM 6-DMAP for 4 h). In this experiment, Sham and parthenogenetic oocyte groups were used as controls. (2) According to the results from experiment 1, we investigated the nuclear stage of zygotes obtained with ICSI and IVF, and their further embryo development. (3) We compared two embryo culture media (G1.3/G2.3 and TCM199 with granulosa cells) on the embryo development of zygotes obtained from ICSI and IVF procedures. Experiment 1 demonstrated that prepubertal goat oocytes needed additional chemical stimulation, after conventional ICSI, to form zygotes with male and female pronuclei (2PN). Experiment 2 showed that significantly higher percentages of -zygotes were found in ICSI-oocytes than IVF-oocytes (40.0 and 25.1%, respectively; P < 0.005). The percentage of embryos obtained and developed beyond the 8-cell stage was significantly higher for ICSI than for IVF and parthenogenetic embryos (22.8, 10.3 and 3.8%, respectively; P < 0.05). Experiment 3 showed that G1.3/G2.3 medium improved the embryo development of ICSI- and IVF-oocytes compared to co-culture with granulosa cells in TCM medium. The highest percentage of embryo development beyond 8-16 cells was found in ICSI-oocytes cultured in G1.3/G2.3 medium. However, a reduced number of morulae were found in this study.
Small Ruminant Research, 2013
Abstract The aim of this study was to test the effect of insulin–transferrin–selenium (ITS), l -a... more Abstract The aim of this study was to test the effect of insulin–transferrin–selenium (ITS), l -ascorbic acid (AA) and different hormone concentrations at IVM on blastocyst development, MPF activity and ATP content in lamb oocytes. In this study we used three maturation media: conventional IVM medium (CM), growth medium (GM: CM + ITS + AA and low level of hormones) and modified CM (MM: CM + ITS + AA and conventional hormone concentration). Cumulus–oocyte complexes (COCs) were classified into two categories according to results from BCB staining: fully grown oocytes or BCB+ and growing oocytes or BCB−. A group of control oocytes (not BCB stained), BCB+ and BCB− were matured (IVM) for 24 h in CM (Treatments A, B and C, respectively). Also, BCB− oocytes were matured in four treatment groups: Treatment D: 12 h in GM plus 12 h in CM; Treatment E: 24 h in GM; Treatment F: 12 h in GM plus 12 h in MM; Treatment G: 24 h in MM. After IVM, oocytes were fertilized and cultured for 8 days and blastocyst development was assessed. Before and after IVM, a sample of each oocyte group was taken to evaluate MPF and ATP content. The BCB+ oocytes produced the highest blastocyst (13.3%) yield. BCB− oocytes did not show any statistically significant differences in blastocysts between treatments, D (5.9%), E (7.2%), F (2.9%) and G (3.9%) compared to Treatment C (4%). Results of MPF activity and ATP content assessed before and after IVM showed an increase in all oocytes (P
Cryobiology, 2014
The aim of this work was to evaluate the protective effect of catalase (CAT) on frozen/thawed ibe... more The aim of this work was to evaluate the protective effect of catalase (CAT) on frozen/thawed ibex epididymal sperm recovered post mortem, and to detect any harmful effect this might have on sperm fertilisation capacity. Epididymal spermatozoa were diluted using a Tris-citric acid-glucose medium (TCG) composed of 3.8% Tris (w/v), 2.2% citric acid (w/v), 0.6% glucose (w/v), 5% glycerol (v/v), and 6% egg yolk (v/v). Sperm masses from the right epididymis were diluted with TCG medium, while those from the left were diluted with TCG medium supplemented with 200IU/mL CAT. Heterologous in vitro fertilisation (IVF) was used to assess the fertilisation capacity of this sperm. The addition of CAT to the extender did not improve frozen/thawed sperm variables. Moreover, a reduced fertilisation capacity was detected: sperm diluted with TCG provided 25.5% 2PN zygotes, while just 13.2% was recorded for that diluted with TCG-CAT (P<0.01). The percentage of cleaved embryos at 48hpi was higher (P<0.01) with the TCG sperm than with the TCG-CAT sperm (16.7% vs. 7.6%). The use of 200IU/mL CAT as an additive cannot, therefore, be recommended for the preservation of ibex epididymal sperm. Other antioxidants should, however, be tested in both this and related wild mountain ungulates.
Reproduction in Domestic Animals, 2012
The aim of this study was to test the effect of insulin-transferrin-selenium (ITS) and L-ascorbic... more The aim of this study was to test the effect of insulin-transferrin-selenium (ITS) and L-ascorbic acid (AA) supplementation and the hormonal level during in vitro maturation (IVM) of small oocytes from pre-pubertal goat on the blastocyst yield and quality. Concretely, we used four maturation media: conventional IVM medium (CM), growth medium (GM: CM+ITS+AA and low level of hormones), modified CM (mCM: CM with low level of hormones) and modified GM (mGM: CM+ITS+AA and normal level of hormones). Cumulus-oocyte complexes (COCs) were classified into two categories according to oocyte diameter: <125 μm and ≥ 125 μm. Large oocytes were matured 24 h in CM (Treatment A). Small oocytes were matured randomly in six experimental groups: Treatment B: 24 h in CM; Treatment C: 12 h in GM and 12 h in CM; Treatment D: 24 h in mGM; Treatment E: 12 h in mGM and 12 h in CM; Treatment F: 12 h in mCM and 12 h in CM; and Treatment G: 12 h in GM and 12 h in mGM. After IVM, oocytes were fertilized and cultured for 8 days. The blastocyst quality was assessed by the survival following vitrification/warming and the mean cell number. When different maturation media were combined, the blastocyst rate did not improve. The large oocytes produced the highest blastocysts yield. However, the culture of small oocytes in GM (53.3%) enhanced the post-warming survival of blastocysts compared to large oocytes matured in CM (35.7%). In conclusion, IVM of pre-pubertal goat small oocytes in GM would be useful to improve the quality of in vitro-produced blastocysts.
International Journal of Molecular Sciences
In goats, embryo oocyte competence is affected by follicle size, regardless of the age of the fem... more In goats, embryo oocyte competence is affected by follicle size, regardless of the age of the females. In previous studies, we found differences in blastocyst development between oocytes coming of small (< 3 mm) and large follicles (> 3 mm) in prepubertal (1–2 month-old) goats. Oocyte competence and follicular fluid (FF) composition changes throughout follicle growth. The aim of this study was to analyze the fatty acids (FAs) composition and metabolomic profiles of FF recovered from small and large follicles of prepubertal goats and follicles of adult goats. FAs were analyzed by chromatography and metabolites by 1H-nuclear magnetic resonance (1H-NMR) spectrometry. The results showed important differences between adult and prepubertal follicles: a) the presence of α,β-glucose in adult and no detection in prepubertal; b) lactate, -N-(CH3)3 groups and inositol were higher in prepubertal; c) the percentage of linolenic acid, total saturated fatty acids and n-3 PUFAs were higher in...
Animal reproduction, 2016
Previous reports in our laboratory (Catala 2014. Rep. fert. & dev.; 27(1):216-7) showed significa... more Previous reports in our laboratory (Catala 2014. Rep. fert. & dev.; 27(1):216-7) showed significant differences in blastocyst production of prepubertal goat oocytes after IVF in autumn (7%) and winter (33%). In the present study we have compared the effect of autumn and winter on cleavage rate and blastocyst production of lamb oocytes after IVF, ICSI and parthenogenetic activation (PA). For this purpose, prepubertal sheep (4 month) ovaries were collected in autumn (22 september to 21 december) and winter (22 december to 21 march) from a local slaughterhouse and transported to the laboratory in sterile dulbecco’s (PBS) held at 34-37oC. COC’s were obtained by slicing the ovarian surface in Hepes-TCM199 medium. Afterwards, 30 COC’s were selected and cultured in 100 µL drop of maturation medium (TCM 199 supplemented with hormones and 10% FBS (fetal bovine serum)) for 24 h at 38.5oC in 100% humidified atmosphere and 5% CO2.Matured oocytes were subjected to the different IVEP techniques. ...
Animal reproduction, 2016
The aim of this study was compare the hormonal status (Estradiol and Progesterone) from follicles... more The aim of this study was compare the hormonal status (Estradiol and Progesterone) from follicles of different sizes (diameter) from prepuberal and adult ewes. Eleven ovaries from adults (n = 7) and prepuberal ewes (n = 4; from two to three months old), were obtained from abattoir and transported to the laboratory in PBS at 34 to 37o C. These follicles were dissected and grouped as: 1) smaller (S: from 2.0 to 2.9 mm) and; 2) large (L: ≥ 3mm) for prepuberal (P) and adults (A) ewes/group. The dissected follicles were carefully cut with microscissor and follicular fluid was recovered into vial of 200 mL and centrifugated at 10000 x g for 15 minutes. The supernatant was stored and analysed by radioimmunoassay to detect Estradiol (E2) and Progesterone (P4) concentrations. Steroids were analysed by radioimmunoassay using ImmuChem Double Antibody P4 and E2 125I RIA kit (MP Biomedicals™, Santa Ana, California, USA). The intra-assay and inter-assay coefficient of variation and the limit of s...
PLOS ONE, 2019
In vitro embryo production success in juvenile animals is compromised due to their intrinsic lowe... more In vitro embryo production success in juvenile animals is compromised due to their intrinsic lower oocyte quality. Conventional in vitro maturation (IVM) impairs oocyte competence by inducing spontaneous meiotic resumption. A series of experiments were performed to determine if maintaining meiotic arrest during a pre-maturation culture phase (pre-IVM) prior to conventional IVM improves oocyte competence of juvenile-goat (2 months old) cumulusoocyte complexes (COCs). In experiment 1, COCs were cultured with C-type natriuretic peptide (CNP; 0, 50, 100, 200 nM) for 6 and 8 h. Nuclear stage was assessed, revealing no differences in the incidence of germinal vesicle (GV) breakdown. In experiment 2, the same CNP concentrations were assessed plus 10 nM estradiol, the known upstream agonist activating expression of NPR2, the exclusive receptor of CNP. CNP (200 nM) plus estradiol increased the rate of oocytes at GV stage at 6 h compared to control group (74.7% vs 28.3%; P<0.05) with predominantly condensed chromatin configuration. In experiment 3, relative mRNA quantification revealed NPR2 expression was down-regulated after pre-IVM (6 h). In experiment 4, analysis of transzonal projections indicated that pre-IVM maintained cumulus-oocyte communication after oocyte recovery. For experiments 5 and 6, biphasic IVM (6 h pre-IVM with CNP and estradiol, plus 24 h IVM) and control IVM (24 h) were compared. Biphasic IVM increased intra-oocyte glutathione and decreased ROS, up-regulated DNA-methyltransferase 1 and pentraxin 3 expression and led to an increase in rate of blastocyst development compared to control group (30.2% vs 17.2%; P<0.05). In conclusion, a biphasic IVM, including a pre-IVM with CNP, maintains oocyte meiotic arrest for 6 h and enhances the embryo developmental competence of oocytes from juvenile goats.
Reproduction in Domestic Animals, 2019
This study examines the presence of activin IIA and IIB receptors (ActR-IIA and ActR-IIB) by West... more This study examines the presence of activin IIA and IIB receptors (ActR-IIA and ActR-IIB) by Western blotting and immunocytochemistry in immature and IVM-oocytes, 2 to 8-cells embryos and blastocysts from prepubertal goats. Western blotting revealed that activin Accepted Article This article is protected by copyright. All rights reserved. receptors are synthesized during oocyte maturation and embryo development. In the immunocytochemistry experiments, no immunostaining for either receptor was detected in oocytes while both receptors were immunolabeled in all the cells of cleaved embryos. In blastocysts, whilst ActR-IIA expression appeared evenly distributed in the two cell lineages, inner cell mass and trophectoderm, the ActR-IIB immunosignal was restricted mainly to the inner cell mass. Our findings reveal the presence of activin type II receptors (ActR-IIA and ActR-IIB) in in vitro matured prepubertal goat oocytes and blastocyst-stage embryos. The expression of these receptors could be a key factor in understanding differences between competent and incompetent oocytes.
Fertility and Sterility, 2012
Objective: To compare blastocyst production, after IVF and ICSI, from sheep oocytes of various qu... more Objective: To compare blastocyst production, after IVF and ICSI, from sheep oocytes of various quality. Sham-injected oocytes and parthenogenetic activated oocyte groups were considered as control. Design: Prospective experimental study. Setting: University. Animal(s): Three-to 6-month-old sheep. Intervention(s): Oocyte quality was assessed with the use of brilliant cresyl blue (BCB) stain. Adenosine triphosphate content was measured. Intracytoplasmic sperm injection and IVF were performed and blastocyst development and cell numbers were analyzed. Main Outcome Measure(s): Adenosine triphosphate content, embryo development and blastomere numbers. Result(s): After IVF, BCB-stained (BCBþ) oocytes developed up to the blastocyst stage at higher percentages and with more cells per embryo (24.1% vs 4.0% and 69.7 vs 43.9, respectively) than unstained (BCBÀ) oocytes. Using intracytoplasmic sperm injection, no differences were found in blastocyst production (14.3% vs 11.8%) and number of cells per embryo (71.1 vs 54.3) between BCBþ and BCBÀ oocytes. Adenosine triphosphate content was higher before in vitro maturation than after in both types of oocytes. Brilliant cresyl blue-stained oocytes had more adenosine triphosphate content than BCBÀ oocytes. Conclusion(s): Brilliant cresyl blue-stained oocytes show more adenosine triphosphate content than BCBÀ oocytes. Results from IVF were affected by the oocyte quality while ICSI did not produce differences in embryo development or blastomere numbers. (Fertil Steril Ò 2012;97:1004-8. Ó2012 by American Society for Reproductive Medicine.
Domestic Animal Endocrinology, 2021
Reproductive Technologies in Animals, 2020
Abstract Reproductive technologies in goats are and will be the key to improve milk and meat prod... more Abstract Reproductive technologies in goats are and will be the key to improve milk and meat production. In this chapter we discuss on the principal methodologies and the results derived from the application of new reproductive technologies in goats such as in vitro embryo production, gamete and embryo cryopreservation, embryo sexing, and gamete culture. Finally, transgenesis and cloning methodologies will be discussed as a strategic approach in order to use goats as bioreactors for therapeutical recombinant proteins.
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Papers by Dolors Izquierdo