Impact of semen cryopreservation season on in vitro embryo production of prepubertal goat oocytes.

Goat production is affected by reproductive seasonality. In vitro embryo production (IVEP) could overcome this effect. This study aimed to evaluate the impact of the season of semen collection/freezing on IVEP of prepubertal goat oocytes and on sperm quality and functionality concerning capacitation. Semen from six fertile bucks was collected, pooled and cryopreserved in spring and autumn and used for IVEP of oocytes recovered during the breeding season. Oocytes were IVM in TCM-199 with hormones, EGF and cysteamine; fertilized and cultured in BO-IVF and BO-IVC media (IVF Bioscience, UK). Semen samples were assessed at 0 and 3 h after culture in capacitating (BO-IVF, CAP) and non-capacitating conditions for sperm plasma membrane and acrosome integrity, mitochondrial membrane potential (MMP), intracellular calcium and plasma membrane lipid disorder. Blastocyst production was higher with spring sperm compared to autumn (12.0% vs. 2.1%, respectively; p < .05). After CAP, acrosome reaction and intracellular calcium were higher (p < .05) in spring than autumn sperm. No differences were found in other sperm parameters. In conclusion, seasonal variations in the IVEP of prepubertal goats could be linked to differences in sperm ability to undergo in vitro capacitation.

Oocyte Competence of Prepubertal Sheep and Goat Oocytes: An Assessment of Large-Scale Chromatin Configuration and Epidermal Growth Factor Receptor Expression in Oocytes and Cumulus Cells.

The oocyte competence of prepubertal females is lower compared to that of adults, mainly because they originate from small follicles. In adult females, the germinal vesicle (GV) and epidermal growth factor receptor (EGFR) have been associated with oocyte competence. This study aimed to analyze GV chromatin configuration and EGFR expression in prepubertal goat and sheep oocytes obtained from small (<3 mm) and large (≥3 mm) follicles and compare them with those from adults. GV chromatin was classified from diffuse to condensed as GV1, GVn, and GVc for goats and NSN, SN, and SNE for sheep. EGFR was quantified in cumulus cells (CCs) by Western blotting and in oocytes by immunofluorescence. Oocytes from prepubertal large follicles and adults exhibited highly condensed chromatin in goats (71% and 69% in GVc, respectively) and sheep (59% and 75% in SNE, respectively). In both species, EGFR expression in CCs and oocytes was higher in prepubertal large follicles than in small ones. In adult females, EGFR expression in oocytes was higher than in prepubertal large follicles. In conclusion, GV configuration and EGFR expression in CCs and oocytes were higher in the large than small follicles of prepubertal females.

Fatty Acids and Metabolomic Composition of Follicular Fluid Collected from Environments Associated with Good and Poor Oocyte Competence in Goats.

In goats, embryo oocyte competence is affected by follicle size regardless the age of the females. In previous studies we have found differences in blastocyst development between oocytes coming of small (<3 mm) and large follicles (>3 mm) in prepubertal (1−2 months-old) goats. Oocyte competence and Follicular Fluid (FF) composition changes throughout follicle growth. The aim of this study was to analyze 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,ß-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 adults; and (d) the percentage of Linoleic Acid, total MUFAs, PUFAs, n-6 PUFAs and n-6 PUFAs: n-3 PUFAs ratio were higher in prepubertal goats. Not significant differences were found in follicle size of prepubertal goats, despite the differences in oocyte competence for in vitro embryo production.

Effect of vitrification of in vitro matured prepubertal goat oocytes on embryo development after parthenogenic activation and intracytoplasmic sperm injection.

This work studies the effect of vitrification of in vitro matured (IVM) prepubertal goat oocytes on: 1) oocyte damage assessed by reactive oxygen species (ROS) level and apoptosis and 2) embryo development after Intracytoplasmic sperm injection (ICSI) and Parthenogenic Activation (PA). Oocytes were IVM in supplemented TCM-199 for 22-24 h. Control group oocytes matured during 24 h were directly used for the analysis after IVM. Vitrified/warmed IVM-oocytes were vitrified after 22 h of IVM in 15% ethylene glycol (EG), 15% dimethyl sulfoxide (Me2SO) and 0.5 M sucrose and after subjected to warming procedure. Oocyte ROS level was measured by staining denuded IVM-oocytes with 10 µM 2'7' dichlorodihydrofluorescein diacetate. Apoptosis was analyzed by Annexin V (AV) Apoptosis Detection kit and Propidium iodide (PI) signal and oocytes were classified as: Live (AV- PI-), early apoptotic (AV+ PI-), dead non-apoptotic (AV- PI+) and necrotic (AV+ PI+). Developmental competence of vitrified/warmed oocytes was assessed by PA (5 min in 5 µM Ionomycin plus 4 h in 2 mM 6-Dimethylaminopurine), and by ICSI fertilization. Presumptive zygotes were in vitro cultured for 8 days in commercial media BO-IVC. Vitrified/warmed oocytes showed higher ROS levels (P < 0.0001), lower live oocytes (44 vs. 66%; P: 0.0025) and higher dead non-apoptotic oocytes (33 vs. 13% P: 0.023) compared to control. No differences were found on normal zygote formation (2 PN) (32 vs. 25%) or blastocyst development (0 vs. 4%) after ICSI fertilization. However, after PA, significant differences were found in cleavage rate (59 vs.78%; P < 0.0343) and blastocyst formation (1 vs. 25%; P < 0.0001). In conclusion, vitrification reduced oocyte competence by increasing dead oocytes and ROS levels.

Resveratrol supplementation during in vitro maturation improves embryo development of prepubertal goat oocytes selected by brilliant cresyl blue staining.

This study aimed to investigate the effect of resveratrol supplementation in maturation medium on the developmental ability and bioenergetic\oxidative status of prepubertal goat oocytes selected by brilliant cresyl blue (BCB). Oocytes collected from slaughterhouse-derived ovaries were selected by 13 µM BCB staining and classified as grown BCB+ and growing BCB- oocytes. All oocytes were matured in vitro in our conventional maturation medium and supplemented with 1 µM (BCB+R and BCB-R) and without (Control groups: BCB+C and BCB-C) resveratrol. After 24 h, IVM-oocytes were fertilized with fresh semen and presumptive zygotes were in vitro cultured for 8 days. Oocytes were assessed for blastocyst development and quality, mitochondrial activity and distribution, and levels of GSH, ROS, and ATP. BCB+R (28.3%) oocytes matured with resveratrol presented significantly higher blastocyst development than BCB+C (13.0%) and BCB- groups (BCB-R: 8.3% and BCB-C: 4.7%). Resveratrol improved blastocyst development of BCB-R oocytes at the same rate as BCB+C oocytes. No differences were observed in blastocyst quality among groups. GSH levels were significantly higher in resveratrol groups (BCB+R: 36554.6; BCB-R: 34946.7 pixels/oocyte) than in control groups (BCB+C: 27624.0; BCB-C: 27655.4 pixels/oocyte). No differences were found in mitochondrial activity, ROS level, and ATP content among the groups. Resveratrol-treated oocytes had a higher proportion of clustered active mitochondria in both BCB groups (BCB+R: 73.07%; BCB-R: 79.16%) than control groups (BCB+C: 19.35%; BCB-C: 40%). In conclusion, resveratrol increased blastocyst production from oocytes of prepubertal goats, particularly in better quality oocytes (BCB+).

Activin-A receptor expression patterns in prepubertal goat oocytes and derived embryos.

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 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 immunolabelled in all the cells of cleaved embryos. In blastocysts, while 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.

Effects of melatonin on oocyte developmental competence and the role of melatonin receptor 1 in juvenile goats.

Melatonin enhances in vitro embryo development in several species by improving the oocyte developmental competence during in vitro maturation (IVM). Melatonin has a wide range of actions, from scavenging reactive oxygen species (ROS) to regulating gene expression, and it can also act by way of melatonin receptors. The aim of this study was to determine the mechanism of action of melatonin during the IVM of juvenile goat oocytes and the role of the membrane receptors. Melatonin receptor 1 was immunolocalized in cumulus cells and oocytes before and after 24 hr of IVM. The effect of melatonin on oocyte developmental competence was tested in three experimental IVM groups: (a) control, (b) 10-7  M melatonin, and (c) 10-7  M melatonin +10-7  M luzindole (an inhibitor of both melatonin receptors). After IVM oocytes were assessed for ROS levels, mitochondrial activity, adenosine 5'-triphosphate (ATP) concentration and relative gene expression (ACTB, SLC1A1, SOD1, GPx1, BAX, DNMT1, GCLC and GDF9). IVM-oocytes were in vitro fertilized and cultured under conventional conditions. Blastocyst rate and quality (differential cell count) were assessed at 8 days post-fertilization. Melatonin decreased ROS levels, increased mitochondrial activity and ATP content and increased blastocyst quality compared to control group (55.8 vs. 30.4 inner cell mass ICM, p < 0.05). There was no effect on the relative gene expression due to treatment with melatonin. In conclusion, we have showed that melatonin improves oocyte developmental competence in juvenile goats by reducing ROS levels and improving mitochondrial activity.

Linoleic (LA) and linolenic (ALA) acid concentrations in follicular fluid of prepubertal goats and their effect on oocyte in vitro maturation and embryo development.

In this study we assessed the concentration of linoleic acid (LA) and linolenic acid (ALA) in follicular fluid of prepubertal goats according to follicle size (<3mm or ≥3mm) by gas chromatography and tested the addition of different LA and ALA (LA:ALA) concentration ratios (50:50, 100:50 and 200:50µM) to the IVM medium on embryo development, mitochondrial activity, ATP concentration and relative gene expression (RPL19, ribosomal protein L19; SLC2A1, facilitated glucose transporter 1; ATF4, activating transcription factor 4; GPX1, glutathione peroxidase 1; HSPA5, heat-shock protein family A 70 kDa; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DNMT1, DNA methyltransferase 1; GCLC, glutamate-cysteine ligase catalytic subunit; SOD1, superoxide dismutase 1). Oocytes were in vitro matured, fertilised or parthenogenetically activated and zygotes were cultured following conventional protocols. LA concentration ranged from 247 to 319µM and ALA concentration from 8.39 to 41.19µM without any effect of follicle size. Blastocyst production from the different groups was: control FCS (22.33%) and BSA (19.63%), treatments 50:50 (22.58%), 100:50 (21.01%) and 200:50 (9.60%). Oocytes from the 200:50 group presented higher polyspermy and mitochondrial activity compared with controls and the rest of the treatment groups. No differences were observed in ATP concentration or relative expression of the genes measured between treatment groups. In conclusion, the low number of blastocysts obtained in the 200:50 group was caused by a high number of polyspermic zygotes, which could suggest that high LA concentration impairs oocyte membranes.

Beneficial effects of melatonin on in vitro embryo production from juvenile goat oocytes.

Melatonin is a universal antioxidant that improves in vitro embryo production in several species. The aims of this study were to determine the melatonin concentration in the ovarian follicular fluid (FF) of juvenile goats and the effect of melatonin during in vitro maturation (IVM) on embryo development. The FF melatonin concentration was 0.57--1.07×10-9 M, increasing with follicular diameter. Oocytes were matured, fertilised and cultured under conventional conditions. Blastocyst development, embryo quality and levels of reactive oxygen species (ROS) and reduced glutathione were assessed. In Experiment 1 different melatonin concentrations (10-3, 10-7, 10-9, 10-11 M) were added to the IVM medium, which contained cysteamine as antioxidant, and no differences were observed. In Experiment 2, melatonin (10-7 M) was tested in the presence or absence of cysteamine (experimental groups: melatonin, cysteamine, melatonin+cysteamine, non-antioxidant). The melatonin group presented a higher blastocyst rate than the non-antioxidant group (28.9 vs 11.7%; P<0.01) and a higher total cell number than the cysteamine group (225.1 vs 129.0; P<0.05). Oocytes from the melatonin and cysteamine groups had lower ROS levels than those from the non-antioxidant group. This study shows that melatonin is an interesting tool for improving oocyte competence in juvenile goats as it increases embryo production and quality.

miRNAs Regulation and Its Role as Biomarkers in Endometriosis.

MicroRNAs (miRNAs) are small non-coding RNAs (18-22 nt) that function as modulators of gene expression. Since their discovery in 1993 in C. elegans, our knowledge about their biogenesis, function, and mechanism of action has increased enormously, especially in recent years, with the development of deep-sequencing technologies. New biogenesis pathways and sources of miRNAs are changing our concept about these molecules. The study of the miRNA contribution to pathological states is a field of great interest in research. Different groups have reported the implication of miRNAs in pathologies such as cancer, diabetes, cardiovascular, and gynecological diseases. It is also well-known that miRNAs are present in biofluids (plasma, serum, urine, semen, and menstrual blood) and have been proposed as ideal candidates as disease biomarkers. The goal of this review is to highlight the current knowledge in the field of miRNAs with a special emphasis to their role in endometriosis and the newest investigations addressing the use of miRNAs as biomarkers for this gynecological disease.

MicroRNA expression profile in endometriosis: its relation to angiogenesis and fibrinolytic factors.

STUDY QUESTION: Could an aberrant microRNA (miRNA) expression profile be responsible for the changes in the angiogenic and fibrinolytic states observed in endometriotic lesions? SUMMARY ANSWER: This study revealed characteristic miRNA expression profiles associated with endometriosis in endometrial tissue and endometriotic lesions from the same patient and their correlation with the most important angiogenic and fibrinolytic factors. WHAT IS ALREADY KNOWN?: An important role for dysregulated miRNA expression in the pathogenesis of endometriosis is well documented. However, to the best of our knowledge, there are no reports of the relationship between angiogenic and fibrinolytic factors and miRNAs when endometrial tissue and different types of endometriotic lesions from the same patient are compared. STUDY DESIGN, SIZE, DURATION: Case-control study that involved 51 women with endometriosis and 32 women without the disease (controls). PARTICIPANTS/MATERIALS, SETTING, METHODS: The miRNA expression profiles were determined using the GeneChip miRNA 2.0 Affymetrix array platform, and the results were analysed using Partek Genomic Suite software. To validate the obtained results, 12 miRNAs differentially expressed were quantified by using miRCURY LNA™ Universal RT microRNA PCR. Levels of vascular endothelial growth factor (VEGF-A), thrombospondin-1 (TSP-1), urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) proteins were quantified by ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: Patient endometrial tissue showed significantly lower levels of miR-202-3p, miR-424-5p, miR-449b-3p and miR-556-3p, and higher levels of VEGF-A and uPA than healthy (control) endometrium. However, tissue affected by ovarian endometrioma showed significantly lower expression of miR-449b-3p than endometrium from both controls and patients, and higher levels of PAI-1 and the angiogenic inhibitor TSP-1. A significant inverse correlation between miR-424-5p and VEGF-A protein levels was observed in patient endometrium, and an inverse correlation between miR-449b-3p and TSP-1 protein levels was observed in ovarian endometrioma. Peritoneal implants had significantly higher levels of VEGF-A than ovarian endometrioma samples. LIMITATIONS, REASONS FOR CAUTION: Functional studies are needed to confirm the specific targets of the miRNAs differently expressed. WIDER IMPLICATIONS OF THE FINDINGS: Differences in miRNA levels could modulate the expression of VEGF-A and TSP-1, which may play an important role in the pathogenesis of endometriosis. The higher angiogenic and proteolytic activities observed in eutopic endometrium from patients might facilitate the implantation of endometrial cells at ectopic sites. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by research grants from ISCIII-FEDER (PI11/0091, Red RIC RD12/0042/0029), Consellería de Educación-Generalitat Valenciana (PROMETEO/2011/027), Beca de Investigación Fundación Dexeus para la Salud de la Mujer (2011/0469), and by Fundación Investigación Hospital La Fe (2011/211). A.B-B. has a Contrato Posdoctoral de Perfeccionamiento Sara Borrell-ISCIII (CD13/00005). J.M-A. has a predoctoral grant PFIS-ISCIII (FI12/00012). The authors have no conflicts of interest to declare.

MicroRNA signatures in hereditary breast cancer.

This study aims to identify signatures of miR associated with hereditary, BRCA1 or BRCA2 mutation positive breast cancer (BC), and non-hereditary BC, either sporadic (SBC) or non-informative (BRCAX). Moreover, we search for signatures associated with tumor stage, immunohistochemistry and tumor molecular profile. Twenty formalin fixed paraffin embedded (FFPE) BCs, BRCA1, BRCA2, BRCAX and SBC, five per group were studied. Affymetrix platform miRNA v.3.0 was used to perform miR expression analysis. ER, PR, HER2 and Ki67 protein expression was analyzed by immunohistochemistry. BRCA1, BRCA2 and RASSF1 methylation analysis, AURKA copy number variations, and BRCA1 and BRCA2 deletions, were studied by MLPA. We validated eight of the miR selected by the arrays in 77 BCs by qRT-PCR. The miR profiles associated with tumor features were studied applying the Sparse Partial Least Squares Discriminant Analysis. MiR discrimination capability to distinguish hereditary and non-hereditary BC was analyzed by the discriminant function. With 15 out of 1,733 hsa-miRs, it was possible to differentiate the four groups. BRCA1, BRCA2 and SBC were associated with clusters of hyper-expressed miRs, and BRCAX with hypo-expressed miRs. Hsa-miR-4417 and hsa-miR-423-3p expressions (included among the eight validated miRs) differentiated 70.1 % of hereditary and non-hereditary BCs. We found miR profiles associated with tumor features like node involvement, histological grade, ER, PR and HER2 expression. Regarding molecular parameters, we only found a weak association of miRs in BC harboring losses in AURKA. We conclude that array miR expression profiles can differentiate the four study groups using FFPE BC. However, miRs expression estimated by qRT-PCR differentiates only hereditary and non-inherited BCs. The miR expression array is a simple and rapid approach that could be useful to facilitate the identification of those SBC carrying genetic or epigenetic changes in BRCA genes responsible of BRCA-like phenotype. These patients could benefit from the treatment with PARP inhibitors.

Brilliant Cresyl Blue stain selects largest oocytes with highest mitochondrial activity, maturation-promoting factor activity and embryo developmental competence in prepubertal sheep.

The aim of this study was to test the Brilliant Cresyl Blue (BCB) stain to select prepubertal sheep oocytes for in vitro blastocyst production. Oocyte diameter, mitochondrial activity, maturation-promoting factor (MPF) activity and mRNA relative expression (RE) of genes related to metabolism (ATPase Na(+)/K(+) transporting α 1 (ATP1A1) and cytochrome c oxidase subunit 1 (COX1)) and constitutive function of the cell (cytoplasmic polyadenylation-element-binding protein (CPEB) and S100A10) were assessed. Immature oocytes were exposed to different BCB concentrations (13, 26, 39 and 52  µM) and classified according to their cytoplasm colouration as grown BCB+ (blue cytoplasm) and growing BCB- (colourless cytoplasm). Staining oocytes with 13  µM BCB during 60  min allows selection of (BCB+) the largest (123.66  µm) and most competent oocytes to develop to the blastocyst stage (21%) with a higher number of cells (69.71 ± 6.19 s.e.m.) compared with non-stained BCB- oocytes (106.82  µm, 9% and 45.91 ± 3.35 s.e.m. respectively). Mitochondrial activity, assessed by MitoTracker Orange CMTMRos probe, was significantly higher in BCB+ than in BCB- oocytes after in vitro maturation (3369 and 1565  AU respectively). MPF activity was assessed by CDC2 kinase activity assay showing significantly higher activity at metaphase II stage in BCB+ than in BCB- oocytes (1.479 ± 0.09 and 1.184 ± 0.05 optical density respectively). The genes analysed in this work, ATP1A1, COX1, CPEB and S100A 10, did not show significant effect in mRNA RE between BCB selected oocytes. In conclusion, BCB stains larger and more competent oocytes to develop to the blastocyst stage with more active mitochondria and MPF activity and higher blastocyst cell number.

Vitrification of in vitro produced goat blastocysts: effects of oocyte donor age and development stage.

This study examines the effectiveness of the cryotop vitrification method for the cryopreservation of goat blastocysts. To determine the effects of embryo development stage and donor age on in vitro survival rates, good-quality blastocysts from adult and prepubertal goats were sorted into non-expanded, expanded, hatching and completely hatched. In vitro produced (IVP) blastocysts were derived from prepubertal goat oocytes by slicing of ovaries from slaughtered animals while adult goat oocytes were collected by the laparoscopic ovum pick up (LOPU) method. Blastocysts were vitrified/warmed using the cryotop technique. Survival rates were determined in terms of blastocoele re-expansion at 3 and 20 h post-warming. For prepubertal goats, survival rates at 3h post-warming were significantly higher when expanded blastocysts (78.3%) were vitrified/warmed compared to hatched blastocysts (57.4%), whereas non-expanded (62.5%) or hatching blastocysts (71.4%) showed similar rates. For adult goats, survival rates were significantly higher after warming in expanded (36.4%), hatching (75%) or hatched (50%) blastocysts when compared to non-expanded (0%) blastocysts. When survival rates were assessed at 20 h post-warming, no differences were observed when we compared non-expanded (45.8%), expanded (56.5%), hatching (64.3%) and hatched (50.5%) blastocysts from prepubertal goats; and for blastocysts from adult goats, survival rates were only significantly lower for the non-expanded stage (0%) compared to the other stages. For adult versus prepubertal blastocysts at the same developmental stage, our data indicate significantly higher survival rates at 3 h post-warming for non-expanded and expanded blastocysts from prepubertal goats over their counterparts from adult goats. At 20 h post warming, survival rates were only higher for non-expanded blastocysts from prepubertal goats versus adult goats. Collectively, our data reveal that blastocysts produced in vitro from prepubertal goats return similar survival rates regardless of their development stage, whereas blastocysts derived from adult goats are best for vitrification at the expanded, hatching or hatched stage.

Survival and apoptosis rates after vitrification in cryotop devices of in vitro-produced calf and cow blastocysts at different developmental stages.

Two experiments were designed to determine the ability of in vitro-cultured blastocysts at different stages of development to survive the vitrification procedure using cryotop devices. Day 7 and Day 8 embryos were classified as non-expanded, expanded or hatching and/or hatched blastocysts. In the first experiment, we examined the survival rate of vitrified-warmed blastocysts after 3 h incubation in synthetic oviducal fluid (SOF) medium. In the second experiment, vitrified-warmed blastocysts were evaluated using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) technique to detect nuclei with damaged DNA. In both experiments, results for cow and calf blastocysts were compared. No differences in survival rates were observed after vitrification of Day 8 expanded (52.4%) and hatched (50%) cow blastocysts or Day 8 expanded (54.5%) and hatched (59.4%) calf blastocysts. When embryos were vitrified on Day 7, survival rates of 78.4% and 66.7% were observed after warming expanded and hatched cow blastocysts, respectively, compared with rates of 80% and 76.9%, respectively, for calf blastocysts. Lowest survival rates were recorded for non-expanded blastocysts (26%-54%) compared with the other developmental stages, particularly those vitrified at Day 8 (

Effect of crocetin added to IVM medium for prepubertal goat oocytes on blastocyst outcomes after IVF, intracytoplasmic sperm injection and parthenogenetic activation.

Crocetin is an active constituent of saffron recently used as antioxidant for embryo culture. The aim of this study was to test the effect of crocetin added in the in vitro maturation (IVM) of prepubertal goat oocytes on the embryo development after in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and parthenogenetic activation (PA). Cumulus-oocyte complexes (COCs) were released from slaughterhouse ovaries of prepubertal goats and in vitro matured in supplemented TCM 199 medium during 24 h without (control group) and with crocetin. In Experiment 1, we evaluated the effect of the IVM supplementation with 0 µM (control), 0.5 µM, 1 µM and 2 µM of crocetin on the blastocyst development after IVF. No significant differences were obtained on blastocyst formation among groups (12, 7, 10, 11%; respectively). Although the blastocyst total cell number was higher in 1 µM crocetin group (150.7 cells) compared to the control (105.5), 0.5 µM (116.2) and 2 µM (93.7) crocetin groups, no significant differences were detected. In experiment 2, we assessed the effect of 1 µM crocetin supplementation in the IVM medium on the oocyte GSH level, ROS level and mitochondrial activity. ROS was significantly higher in the control than in the crocetin group (P < 0.05), but no differences in GSH level and mitochondrial activity were observed. In experiment 3, we evaluated the effect of 1 µM crocetin on the blastocyst development of oocytes after ICSI and PA. No statistical differences were found on blastocyst rate or cell number. However, compared with control, crocetin groups led to higher cleavage (59 vs. 67%, respectively, P = 0.09) and blastocyst rates (19 vs. 12%, respectively; P = 0.12) after ICSI. Although crocetin reduced ROS levels in prepubertal goat oocytes, it did not have a significant effect on oocyte embryo developmental competence.

Small Ruminants: Prepubertal Oocyte Donors.

In vitro embryo production using oocytes of prepubertal females is named JIVET (juvenile in vitro embryo technologies). The aim of the JIVET is to increase genetic gain by shortening the generation interval in breeding programs. In this chapter we describe the methodology currently used in our laboratory for producing in vitro embryos from prepubertal sheep (90-120 days old) and goats (30-45 days old).Briefly, we obtain cumulus-oocyte complexes (COCs) from slaughtered female ovaries. These COCs are in vitro matured in TCM199 with 10% (v/v) fetal bovine serum (FBS) at 38.5 °C in a humidified air atmosphere with 5% CO2 for 24 h. The next day we collect ejaculates from males of proven fertility and select the most motile spermatozoa by density gradient. Following coincubation with mature COCs, presumptive zygotes and embryos are cultured in SOF for 8 days at 38.5 °C, 90% N2, 5% CO2, 5% O2, and 100% humidity to obtain blastocysts.

Biphasic in vitro maturation with C-type natriuretic peptide enhances the developmental competence of juvenile-goat oocytes.

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) cumulus-oocyte 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.

Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on spindle and chromosome configuration and embryo development.

Two experiments were designed to assess the effectiveness of cryopreserving bovine MII oocytes using cryotops as the carrier system for vitrification. In the first experiment, we examined the developmental competence of oocytes after: (i) vitrification in open-pulled straws (OPS method); or (ii) vitrification in <0.1mul medium droplet on the surface of a specially constructed fine polypropylene strip attached to a plastic handle (Cryotop method). In the second experiment, warmed oocytes that had been vitrified in OPS or cryotops were fixed to analyze spindle and chromosome configuration. In all experiments both cow and calf oocytes were used. Significantly different fertilization rates were observed between the vitrification groups: 31.5% and 20.2% for the cow and calf oocytes vitrified in OPS, respectively, versus 46.1% and 46.4% for the oocytes vitrified using cryotops. After in vitro fertilization, 3.8% of the calf oocytes and 5.3% of the cow oocytes developed to the blastocyst stage. All blastocysts from vitrified oocytes resulted from the Cryotop method. A significantly lower percentage of the OPS-vitrified calf oocytes showed a normal spindle configuration (37.8%) compared to control fresh oocytes (69.9%), while normal spindle and chromosome configurations were observed in a significantly higher proportion of the cryotop-vitrified calf oocytes (60.2%). For the cow oocytes, 60.6% in the OPS group and 60.3% in the Cryotop group exhibited a normal morphology after warming. These findings suggest the cryotop system is a more efficient carrier for vitrification than OPS for the cryopreservation of bovine oocytes.

Total RNA and protein content, Cyclin B1 expression and developmental competence of prepubertal goat oocytes.

The aim of this study was to examine the relationship between the developmental competence of oocytes and their total RNA and protein contents, and the level of Cyclin B1 transcription. Ovaries from prepubertal goats were collected from a slaughterhouse. Oocytes were recovered by slicing and those with two or more layers of cumulus cells and homogenous cytoplasm were matured in vitro (20-25 oocytes per drop) for 27 h. Both before and after IVM, samples of oocytes were denuded and categorised into four group treatments by diameter (<110 microm, 110-125 microm, 125-135 microm; >135 microm), separated into sub-groups of 10 oocytes per treatment-replicate and stored in liquid nitrogen until total RNA content analysis by spectophotometry, total protein content analysis by a colorimetric assay and Cyclin B1 transcription analysis by RT-PCR. For the study of developmental competence, the rest of the matured oocytes were fertilised in vitro in groups of 20-25 for 24 h. Presumptive zygotes were denuded, sorted into the four categories of diameter noted above, and placed into culture drops in groups of 18-25 for in vitro culture. Cleavage rate was evaluated at 48 hpi and embryo development at 8 d post-insemination. There were four replicates of each treatment for each assay or evaluation point of the experiment. There were no significant differences between the size categories of oocytes at collection in total RNA content, total protein content and Cyclin B1 mRNA. There were significant differences (P<0.05) in the expression of Cyclin B1 before IVM with oocytes in the >135 mm diameter category having the highest value for this variant. There were no significant differences in these characteristics between the categories of oocyte diameter after IVM except in respect of total RNA content, which was lower for the largest size of oocytes (>135 microm; mean+/-S.D.=12.3+/-1.84 ng/oocyte) than the other three size groups (19.2+/-1.38-22.1+/-4.44 ng/oocyte; P<0.05). Significant differences (P<0.05) in cleavage rate were observed between the different oocyte size categories (<110 microm, 3.0%; 110-125 microm, 32%; 125-135 microm, 50%; >135 microm, 73%). Only oocytes >125 microm diameter developed to the blastocyst stage (125-135 microm, 7%; >135 microm, 10%). This study showed that the RNA content and the Cyclin B1 RNA expression of prepubertal goat oocytes, and their development to embryos varied between the different size categories of the oocytes.