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.

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.

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.

Effect of oocyte quality on blastocyst development after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in a sheep model.

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 (BCB+) 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 (BCB-) 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 BCB+ and BCB- 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 BCB- oocytes. CONCLUSION(S): Brilliant cresyl blue-stained oocytes show more adenosine triphosphate content than BCB- oocytes. Results from IVF were affected by the oocyte quality while ICSI did not produce differences in embryo development or blastomere numbers.