150 The influence of follicular fluid extracellular vesicles on in vitro maturation of oocytes in the domestic cat

O desenvolvimento embrionário de oócitos bovinos maturados in vitro (MIV) foi avaliado em meio suplementado com líquido folicular eqüino (Lfe). Foram distribuídos 1045 oócitos em 11 repetições formando três grupos tratamentos (T1, T2, T3) e um controle (C). O meio de maturação utilizado foi o TCM-199 acrescido de piruvato de sódio, hormônio folículo estimulante recombinante (rFSHh) e hormônio luteinizante equino (LHe). Suplementou-se esse meio com 10% de soro de égua em estro para o grupo controle e para T1, T2 e T3, o meio foi suplementado com 5, 10, e 20% de LFe, respectivamente. Os oócitos foram maturados in vitro (MIV) por 24h. A fecundação in vitro (FIV) foi realizada em meio Talp-Fert. A MIV e a FIV foram realizadas em estufa a 39ºC com 5% de CO2 em ar e umidade saturada. Os zigotos foram cultivados em meio SOFaaci, sob óleo mineral no interior de bolsas plásticas gaseificadas. As taxas de clivagem e de blastocistos foram observadas diariamente (D), e em D7, foram superiores (P0,05) às do grupo controle. Em D9, a taxa de blastocistos do T2 foi superior (P0,05). O LFe, na concentração de 10% pode ser utilizado, em substituição ao soro de égua em estro para suplementar o meio de MIV de oócitos bovinos. Equine follicular fluid on in vitro maturation of bovine oocytes Abstract Embryo development of bovine oocytes was evaluated using maturation medium supplemented with equine follicular fluid (eFF). One thousand and forty five (1045) oocytes were distributed in 11 replications forming three treatment groups (T1, T2 e T3) and one Control (C). TCM-199 added with sodium pyruvate, rFSHh and LHe was used as maturation medium. This medium was supplemented with 10% estrous mare serum for Control group, and 5, 10, and 20% eFF, respectively, for T1, T2 e T3 groups. In vitro maturation (IVM) of all groups was performed during 24h. In vitro fertilization (IVF) was performed in TALP-FERT medium. IVM and IVF were carried out in an incubator at 39ºC with 5% CO2 in air and saturated humidity. Zygotes were cultured in SOFaaci medium, under mineral oil in gasified bags. Cleavage and blastocyst rates were daily observed (D), and at D7, were higher (P0.05) for those from control group. At D9, blastocyst rate of T2 was higher (P0.05). The eFF, at a 10% concentration, can replace the use of estrous mare serum to supplement the IVM medium of bovine oocytes.

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Effects of follicular fluid supplementation of in-vitro maturation medium on the fertilization and development of equine oocytes after in- vitro fertilization or intracytoplasmic sperm injection

Human Reproduction ◽

10.1093/humrep/12.12.2766 ◽

1997 ◽

Vol 12 (12) ◽

pp. 2766-2772 ◽

Cited By ~ 65

Author(s):

M. E. Dell'Aquila ◽

Y. S. Cho ◽

P. Minoia ◽

V. Traina ◽

G. M. Lacalandra ◽

...

Keyword(s):

In Vitro Fertilization ◽

Follicular Fluid ◽

Intracytoplasmic Sperm Injection ◽

In Vitro Maturation ◽

Vitro Fertilization ◽

Maturation Medium ◽

Fluid Supplementation

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243 EFFECT OF OOCYTE RECOVERY TECHNIQUES ON IN VITRO MATURATION OF SWINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab243 ◽

2009 ◽

Vol 21 (1) ◽

pp. 219

Author(s):

F. R. O. de Barros ◽

M. G. Marques ◽

M. D. Goissis ◽

M. A. Peres ◽

M. P. Milazzotto ◽

...

Keyword(s):

Follicular Fluid ◽

Recovery Rate ◽

In Vitro Maturation ◽

Metabolic Stress ◽

Water Bath ◽

Epifluorescence Microscopy ◽

Nuclear Maturation ◽

Oocyte Recovery ◽

Centrifuge Tube

The aim of this study was to compare 2 different techniques to obtain swine oocytes from abattoir ovaries. Ovaries were washed in saline at 35°C and submitted to slashing or aspiration, simultaneously. For the slashing group, ovaries were held with a hemostat inside a beaker containing 35 mL of HEPES-buffered Tyrode’s media (HbT) and follicles (2–6 mm) were incised with a scalpel. For every 5 slashed ovaries, HbT-containing follicular fluid was transferred to 50-mL centrifuge tubes. For the aspiration group, follicles (2–6 mm) were aspirated using an 18-gauge needle and a 5-mL syringe. The follicular fluid of each ovary was transferred to a 50-mL centrifuge tube. Tubes from both techniques were placed in a water bath at 35°C for 15 min to allow settling of the cumulus–oocyte complexes (COC). The supernatant was removed and the sediment was resuspended in HbT and placed in water bath at 35°C for an additional 15 min. The sediment was resuspended in 15 mL of HbT and COC were recovered under stereomicroscopy. Oocytes were in vitro matured for 44 h in TCM-199 added with 10% porcine follicular fluid (PFF) and hormones (LH and FSH) at 38.5°C, 5% CO2 and high humidity. The oocyte recovery rate of each technique was determined by the ratio between the number of COC and ovaries used. To verify nuclear maturation by epifluorescence microscopy (Zeiss), oocytes were fixed, permeabilized, and incubated in 10 μg mL–1 of RNAse for 30 min and in 10 μg mL–1 of propidium iodide for 10 min. Heat shock protein 70 (HSP70) content was assessed as described in Kawarsky and King (2001 Zygote 9(3), 39–50) to verify the metabolic stress. Data were analyzed by ANOVA and Tukey’s test using the software Statistica for Windows. A level of 5% was considered significant in all assessments. The oocyte recovery rate (COC/ovary) was higher for the slashing group (2.665 ± 0.38) compared with the aspiration group (1.762 ± 0.15). The percentage of oocytes that reached the germinative vesicle (GV) stage (h 0 of maturation) did not differ between groups (100 ± 0 and 86.66 ± 13.36, slashing and aspiration group, respectively). The same was observed for the percentage of oocytes that reached the metaphase II stage (MII, after 44 of maturation; 79.99 ± 9.74 and 96.00 ± 4.00, slashing and aspiration group, respectively). Moreover, no difference at pixel quantification of HSP70 was observed between groups (256.50 ± 42.42 and 238.61 ± 71.18, slashing and aspiration group, respectively). In conclusion, the slashing procedure provided a better oocyte recovery rate compared with the aspiration of ovaries. This technique does not affect nuclear maturation, because no differences were observed regarding the percentage of oocytes that reached the GV and MII stages. In addition, it does not affect HSP70 content, suggesting that the slashing of ovaries does not increase the basal stress of oocytes in an in vitro-maturation system.

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208 EFFECT OF GDF8 AND SB-431542 ON PORCINE OOCYTE DURING IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab208 ◽

2016 ◽

Vol 28 (2) ◽

pp. 235

Author(s):

J. D. Yoon ◽

E. Lee ◽

S.-H. Hyun

Keyword(s):

Treatment Group ◽

Embryonic Development ◽

Follicular Fluid ◽

In Vitro Maturation ◽

Transforming Growth Factor ◽

Formation Rate ◽

Transforming Growth Factor Β ◽

Nuclear Maturation ◽

Porcine Oocyte

Growth differentiation factor-8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. SB-431542 (SB) is a specific inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors such as ALK4, ALK5, and ALK7. The purpose of this study is investigation of the effects of GDF8 and SB on porcine oocytes during in vitro maturation and subsequent embryonic development. We first performed ELISA to detect GDF8 concentrations in follicular fluid for each size of follicle; sizes were as follows: small (<3 mm), medium (>3 mm and <6 mm), and large (>6 mm) follicle. After detection of the GDF8 concentration in follicular fluid, we investigated the effect of GDF8 and SB treatment during in vitro maturation (IVM) on nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels, and embryonic development after IVF and parthenogenetic activation (PA). Data were analysed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science, IBM, New York, NY, USA) mean ± SEM. The ELISA result showed different concentrations of GDF8 for each grade of follicular fluid: small, 0.479 ng mL–1; medium, 0.668 ng mL–1; and large, 1.318 ng mL–1. During the IVM process, 1.318 ng mL–1 of GDF8 and 5 ng mL–1 of SB were added to the maturation medium as control, SB, SB+GDF8, and GDF8 treatment groups. After 44 h of IVM, GDF8 group (90.4%) showed a significantly higher nuclear maturation rate than control and SB+GDF8 groups (85.4 and 81.7%). The SB group (78.9%) showed significantly reduced nuclear maturation rate compared with control (P < 0.05). The GDF8 treatment group showed a significant decreased intracellular ROS and increased GSH levels compared with other groups (P < 0.05). The SB+GBF8 treatment group showed a significantly better cytoplasmic maturation than the SB treatment group. In the PA embryonic development analysis, the GDF8 treatment group showed a significantly higher blastocyst formation rate compared with other groups (47.9, 37.2, 46.4, and 58.7% respectively; P < 0.05). In the IVF embryonic development analysis, the GDF8 treatment groups showed significantly higher blastocyst formation rate compared with the SB group (28.2 and 42.2%, respectively; P < 0.05). In conclusion, treatment with GDF8 during porcine oocyte IVM improved the embryonic developmental competence via increased cytoplasmic maturation and led to better oocyte maturation from the ALK receptor inhibition by SB.

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206 EFFECT OF ADDITION OF FOLLICULAR FLUID OR GROWTH DIFFERENTIATION FACTOR-9 ON IN VITRO MATURATION OF PORCINE OOCYTES DENUDED 20h AFTER THE START OF IN VITRO MATURATION

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab206 ◽

2016 ◽

Vol 28 (2) ◽

pp. 234

Author(s):

P. Ferré ◽

T. T. M. Bui ◽

M. T. Tran ◽

T. Wakai ◽

H. Funahashi

Keyword(s):

Follicular Fluid ◽

In Vitro Maturation ◽

Cumulus Cells ◽

Developmental Competence ◽

Dapi Staining ◽

Experimental Conditions ◽

Nuclear Maturation ◽

Differentiation Factor ◽

Growth Differentiation Factor 9

The interruption of communication between oocyte and cumulus cells (CC) can trigger meiotic resumption and exogenous additives, such as follicular fluid (FF) and growth differentiation factor-9 (GDF9), can improve oocyte quality and the developmental competence. This study was undertaken to examine if the absence and presence of FF from medium follicles (MF; 3–6 mm in diameter) or recombinant human GDF9 (Biovision, Milpitas, CA, USA) during the first or/and second half of in vitro maturation (IVM) had any effects on IVM of oocytes from small follicles (SF; 0.5–2 mm in diameter) or MF when the oocytes were denuded at 20 h after the start of IVM. Cumulus-oocyte complexes (COC) were aspirated from SF or MF of slaughtered prepubertal gilt ovaries. Groups of ~30 COC were cultured in a 300-μL drop of porcine oocyte medium containing 50 µM β-mercaptoethanol (mPOM) with or without 10% (v/v) FF and/or 100 ng mL–1 GDF9 at 39°C and 5% CO2 in air. During the first 20 h after the start of IVM, the medium was supplemented with 1 mM dibutyryl c-AMP, 10 IU mL–1 eCG and 10 IU mL–1 hCG. After the first period of IVM, the CC surrounding the oocytes were removed and the denuded oocytes continued culture for IVM with or without FF or/and GDF9 in the absence of dibutyryl c-AMP and gonadotropins in the same medium for another 24 h. At the end of IVM, meiotic progression of the oocytes was examined by DAPI staining. Statistical analyses from at least 4 replicates data were performed by a 2-way ANOVA and a Tukey’s multiple comparisons test. Removal of CC 20 h after the start of IVM significantly improved the incidence of mature oocytes derived from SF (59.2–64.1% v. 41.6–43.1% in controls, P < 0.05) but not from MF (73.1–78.5% v. 70.6–71.8% in controls), whereas regardless of supplementation with FF or GDF9, the maturation rates were always significantly higher in the denuded oocytes from MF (72.4–83.6%) than SF (57.8–66.2%; P < 0.05). Despite of the origin of COC (SF or MF), maturation rates of oocytes denuded 20 h after the start of IVM were not affected by supplementation with FF or GDF9 during the first and/or second half of IVM (P > 0.05). In summary, CC removal from COC 20 h after the start of IVM promotes nuclear maturation of oocytes from SF. Exogenous additives such as GDF9 and follicular fluid from MF do not seem to affect the promotion of nuclear maturation in our experimental conditions.

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