116 EFFECTS OF l-GLUTAMINE ON CRYOPRESERVATION OF IMMATURE BOVINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 167
Author(s):  
C. Yamada ◽  
M. D. Goissis ◽  
H. V. A. Caetano ◽  
A. R. S. Coutinho ◽  
M. E. O. A. Assumpção ◽  
...  
Keyword(s):  
Amino Acids ◽  
In Vitro Maturation ◽  
Complex Structure ◽  
Cumulus Cell ◽  
Epifluorescence Microscopy ◽  
Control Group ◽  
Maturation Medium ◽  
Cell Layers ◽  
Bovine Oocytes

The cryopreservation of bovine oocytes remains a challenge despite significant reported progress. Immature bovine oocytes have a complex structure and the conventional cryoprotectants (penetrating cryoprotectants, sugars, and macromolecules) appear to be not sufficient to preserve them efficiently during freezing. Studies on semen and fibroblast cryopreservation indicate that amino acids, particularly l-glutamine, protect enzymes during freezing and increase the post-thaw viability. Therefore, the amino acids may optimize oocyte cryopreservation when associated with conventional cryoprotectants. This work evaluated the effect of l-glutamine on cryopreservation of immature bovine oocytes after in vitro maturation. Oocytes with homogeneous cytoplasm and several cumulus cell layers from slaughterhouse ovaries were distributed randomly in three groups: non-vitrified control, vitrified control, and vitrified with l-glutamine. Oocytes from vitrified groups were exposed for 10 min to PBS + 10% FCS + 10% ethylene glycol (EG) + 0.25 m trehalose (T), and for 30 s to PBS + 10% FCS + 25% EG + 25% dimethylsulfoxide + 0.5 m T at room temperature, adding 80 mm l-glutamine for the third group. Oocytes were loaded into OPS and plunged in liquid nitrogen. For thawing, OPS were immersed in PBS + 10% FCS + 10% EG + 1 m T for three min. Oocytes werethen placed in PBS + 10% FCS + 0.5 m T and in PBS + 10% FCS, remaining three min in each solution. For in vitro maturation, oocytes were washed three times on holding medium (TCM-HEPES + FCS + pyruvate + gentamycin), washed three times in maturation medium (TCM-bicarbonate + FCS + pyruvate + gentamycin + hCG + FSH + estradiol), and cultured in microdrops (90 μL) of maturation medium covered with mineral oil at 38.5°C under 5% CO2 in air and high humidity for 24 h. Oocytes were denuded, fixed in paraformaldehyde and triton, stained with Hoechst 33342, and evaluated under epifluorescence microscopy. Oocytes at metaphase II were considered matured. The group vitrified with l-glutamine had a significantly higher maturation rate than the group vitrified without l-glutamine; however, both had significantly lower maturation rates than the non-vitrified control group. In conclusion, l-glutamine improved the viability of vitrified oocytes. Table 1. Oocyte maturation rates of non-vitrified control, vitrified control, and vitrified with glutamine groups This work was supported by FAPESP 03/08543-1.

scholarly journals LÍQUIDO FOLICULAR EQÜINO NA MATURAÇÃO IN VITRO DE OÓCITOS BOVINOS

2004 ◽  
Vol 9 (1) ◽  
Author(s):  
M.G.L. PINTO ◽  
M.I.B. RUBIN ◽  
C.A.M. SILVA ◽  
T.F. HILGERT ◽  
M.F. SÁ FILHO ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Mineral Oil ◽  
Control Group ◽  
Sodium Pyruvate ◽  
Vitro Fertilization ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Bovine Oocytes

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.

177 Increasing in vitro embryonic development through improved oocyte maturation in cattle oocytes

2019 ◽  
Vol 31 (1) ◽  
pp. 213
Author(s):  
J. Keim ◽  
Y. Liu ◽  
I. Polejaeva
Keyword(s):  
Growth Factor ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cumulus Cell ◽  
Control Group ◽  
Control Medium ◽  
Maturation Medium ◽  
First Polar Body ◽  
Blastocyst Rate

In vitro maturation (IVM) is an important process in the in vitro production of embryos. It has been recently shown that 3 cytokines: fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1) have increased the efficiency of IVM, blastocyst production, and in vivo development in pig (Yuan et al. 2017 Proc. Natl. Acad. Sci. USA 114, E5796-E5804). In vitro maturation in medium supplemented with cytokines doubled the blastocyst rate and quadrupled the litter size when transferred. It was observed that the addition of cytokines to IVM medium had an effect on the regulation of pMAPK1/3, cumulus cell expansion, and transzonal projections in cumulus-oocyte complexes (COC). This study was designed to assess the effect of these 3 cytokines on IVM in bovine oocytes and their consecutive development to blastocyst. Intracellular glutathione level (GSH), frequently used as an indicator of metaphase II (MII) oocyte quality, was also evaluated. The COC were retrieved from abattoir-derived ovaries and matured for 21h in either our standard maturation medium [TCM-199 (Gibco/Life Technologies, Grand Island, NY, USA), containing 10% fetal bovine serum, 0.5µg mL−1 FSH, 5µg mL−1 LH, and 100U mL−1 penicillin/streptomycin] or maturation medium supplemented with 20ng mL−1 human LIF, 20ng mL−1 human IGF1, and 40ng mL−1 human FGF2. After IVM, COC were placed in fertilization medium and incubated with frozen-thawed sperm for 20h. Cumulus cells were removed from fertilized COC and cultured in SOF culture medium at 38.5°C in 5% CO2/humidified air. Cleavage and blastocyst rates were assessed at 48h and Day 8 post-IVF, respectively. To assess GSH level, MII oocytes were incubated in 20 µM CellTracker Blue CMF2HC (Thermo Fisher Scientific, Waltham, MA, USA) and observed under blue fluorescent light. All statistical analysis was performed using one-way ANOVA and data are presented as mean±s.e.m. The MII rate, assessed by the presence of the first polar body, was significantly higher in the maturation medium supplemented with cytokines compared with the control medium (167/202; 82.4±2.02% v. 136/198; 68.8±1.1%; P<0.05, 4 replicates). For IVF, no statistical difference was found in the cleavage rate between oocytes matured in the medium supplemented with cytokines compared with control medium (351/473; 74.3±4.86% v. 358/573; 63.9±4.03%; P>0.05, 5 replicates), respectively. However, a significant increase in blastocyst rate was observed in the cytokine-containing medium (64/351; 17.7±2.06%) compared with the control group (42/358; 11.0±1.96%; P<0.05, 5 replicates). Furthermore, our preliminary data indicate an increase in GSH in MII oocytes matured in the cytokine-containing medium. In conclusion, the addition of FGF2, LIF, and IGF1 to maturation media improves bovine IVM efficiency and quality of the MII oocytes, leading to a greater blastocyst development rate. Supported by RFBR (18-29-07089) and UAES (1343).

PSX-B-11 Comparing effects of FGF2 and IGF1 on the development competence of parthenogenetic activated bovine oocytes maturing in vitro

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 327-328
Author(s):  
Galina Singina
Keyword(s):  
Growth Factor ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Maturation Medium ◽  
Bovine Oocytes

Abstract The oocyte quality acquired during in vitro maturation (IVM) are the main limitative factors affecting the embryo production. The aim of the present research was to study effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1) during IVM of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-oocyte complexes (COC; n = 1176) were cultured for 22h in either standard maturation medium (TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 10 μg/ml FSH and 10 μg/ml LH; Control) or maturation medium supplemented with different concentrations (5–160 ng/ml) of FGF2 and IGF1. After IVM, matured oocytes activated by sequential treatment with ionomycin followed by DMAP and cyclohexamide and then cultured up to the blastocyst stage. The obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using DAPI and TUNEL staining. The data from 4 replicates (77–91 oocytes per treatment) were analyzed by ANOVA. Cleavage rates of activated oocytes did not differ between groups and ranged from 63.7 to 68.1%. The addition of 10, 20 and 40 ng/ml of FGF2 to the IVM medium led to an increase in the yield of blastocysts [from 19.6±1.8% (Control) to 35.2±3.4, 29.8±1.9 and 31.1±2.1%, respectively (P<0.05)] and in the total cell number in embryos that developed to the blastocyst stage (P<0.05). Meanwhile, the blastocyst yield and the total cell number in blastocysts in the IGF1-treated groups were similar to that in the control group. No effects of both growth factors on the proportion of apoptotic nuclei in blastocysts (5.3–7.1%) were observed. Thus, FGF2 (but not IGF1) are able to maintain competence for parthenogenetic development of bovine COC during their maturation invitro. Supported by RFBR (18-29-07089) and the Ministry of Science and Higher Education of Russia.

Treatment with roscovitine and butyrolactone I prior to in vitro maturation alters blastocyst production

Zygote ◽  
2019 ◽  
Vol 28 (1) ◽  
pp. 24-31 ◽  
Author(s):  
Rosiara Rosária Dias Maziero ◽  
Carlos Renato de Freitas Guaitolini ◽  
Daniela Martins Paschoal ◽  
André Maciel Crespilho ◽  
Bianca Andriolo Monteiro ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Study Groups ◽  
Embryo Cryopreservation ◽  
Control Group ◽  
Bovine Embryos ◽  
Nuclear Maturation ◽  
Oocyte Meiosis ◽  
Maturation Index ◽  
Bovine Oocytes

SummaryThis study evaluated the effects of oocyte meiosis inhibitors roscovitine (ROS) and butyrolactone I (BL-I) on in vitro production of bovine embryos. Bovine oocytes were maintained in pre in vitro maturation (pre-IVM) with 25 µM ROS or 100 µM BL-I for 24 h to delay meiosis and for 24 h in in vitro maturation (IVM). Following this treatment, the nuclear maturation index was evaluated. All embryos degenerated following this procedure. In the second set of experiments, oocytes were maintained for 6 or 12 h in pre-IVM with the following three treatments: ROS (25 µM or 12.5 µM), BL-I (100 µM or 50 µM) or a combination of both drugs (6.25 µM ROS and 12.5 µM BL-I). Oocytes were cultivated for 18 or 12 h in IVM. When a meiosis-inducing agent was used during pre-IVM for 24 h, more degenerated oocytes were observed at the end of the IVM period. This effect decreased when the meiotic blocking period was reduced to 6 or 12 h. No significant differences were observed in the blastocyst production rate of oocytes in pre-IVM for 6 h with ROS, BL-I, or ROS + BL-I compared with that of the control group (P > 0.05). However, inhibition of oocytes for 12 h resulted in decreased embryo production compared with that in the controls (P < 0.05). There was no difference in the post-vitrification embryo re-expansion rate between the study groups, showing that the meiotic inhibition for 6 or 12 h did not alter the embryo cryopreservation process.

332 GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF) ENHANCES CUMULUS CELLS EXPANSION OF IN VITRO-MATURED BOVINE CUMULUS - OOCYTE COMPLEXES IN A CHEMICALLY DEFINED MEDIUM

2010 ◽  
Vol 22 (1) ◽  
pp. 322
Author(s):  
D. D. Bücher ◽  
M. A. Castro ◽  
M. E. Silva ◽  
M. A. Berland ◽  
I. I. Concha ◽  
...  
Keyword(s):  
Cell Viability ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Cell Number ◽  
Control Group ◽  
Cumulus Expansion ◽  
Gm Csf ◽  
Nuclear Stage

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine that stimulates proliferation, differentiation and function in different cells types. We have previously demonstrated (Bücher DD et al. 2008 Reprod. Dom. Anim. 43 (Suppl. 3), 146 abst.) that both subunits of GM-CSF receptor are expressed in granulosa cells from antral follicles in bovine ovaries. Also, we determined that the cytokine enhances glucose uptake through facilitative hexose transporters in granulosa cells in primary culture. The goals of the present study were to characterize the expression of GM-CSF receptor in cumulus cells and oocytes from bovine antral follicles and to determine its effects on in vitro-matured bovine COCs in a chemically defined medium. To determine the presence of a and |5 subunits of GM-CSF receptor, COCs were aspirated from follicles <8 mm in diameter, fixed, and submitted to immunocytochemistry. To study the effect of GM-CSF on in vitro maturation of oocytes, COCs (n =481) were cultured using serum-free medium (SOF) containing 0, 1, 10, and 100 ng mL-1 of human recombinant GM-CSF (R&D Systems, Inc., Minneapolis, MN, USA) for 22 h at 39°C, 5% CO2 in humidified air. Nuclear stage, cumulus expansion, cumulus cell number, and viability were analyzed after in vitro maturation. Cumulus expansion was assessed using the cumulus expansion index (CEI) (Fagbohun C and Down S 1990 Biol. Reprod. 42, 413-423). Nuclear stage was evaluated using aceto-orcein stain. To determine cumulus cell viability and number, COCs (n = 10-12 per group) were transferred into an Eppendorf tube and cumulus cells were removed by vortexing for 3 min, stained with trypan blue and counted with a hemocytometer. The study was conducted in 6 replicates. Data from cumulus expansion and cell number were analyzed by Kruskal-Wallis analysis. Data for nuclear stage and cell viability were analyzed by chi-square analysis and one way ANOVA, respectively. Both receptor subunits were present in cumulus cells and oocytes from COCs. COCs cultured in 10 and 100 ng mL-1 GM-CSF had CEI scores (0.8 and 1.22, respectively) greater (P < 0.01) than controls (0.2), but the proportion of COCs displaying second metaphase did not differ (P = 0.5) among treatment groups. GM-CSF at a concentration of 100 ng mL-1 increased (P < 0.01) cumulus cell viability by more than 20% compared to the control group. Similarly, GM-CSF at concentrations of 10 and 100 ng mL-1 increased (P < 0.05) cumulus cell number by more than 20% and 45%, respectively, from the control group. The use of a specific inhibitor of PI3 kinase (Ly294002; 10 and 100 μM) blocked the stimulatory effect of GM-CSF on cumulus expansion, cell viability, and cell number. In conclusion, the results of the study suggest a plausible modulator role of GM-CSF in the metabolism and function of cumulus cells and oocytes during in vitro maturation. Funding from Faculty of Veterinary Sciences, Universidad Austral de Chile, MECESUP AUS-0005, AUS-0601, and DID D-2006-24 and from Universidad Católica de Temuco, research grant 2007 DGI-CDA-04.

165 FETAL CALF SERUM INFLUENCES CYCLIC GMP PATHWAY, WHICH IN TURN AFFECTS THE LIPID CONTENT IN IN VITRO-MATURED BOVINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 196
Author(s):  
K. R. L. Schwarz ◽  
R. C. Botigelli ◽  
F. C. Castro ◽  
M. R. Chiaratti ◽  
C. L. V. Leal
Keyword(s):  
Lipid Content ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Control Group ◽  
Maturation Medium ◽  
Cgmp Pathway ◽  
Bovine Oocytes ◽  
Synthesis And Degradation

The sensitivity of IVP embryos to cryopreservation is often associated with lipid accumulation in the cytoplasm induced by the presence of fetal calf serum (FCS) during culture. Intracellular levels of cyclic (c)AMP and cGMP are involved in the regulation of lipolysis in adipocytes; high levels stimulate lipolysis whereas low levels lead to lipogenesis. Both nucleotides are present in bovine oocytes, together with the enzymes for their synthesis and degradation. The aim of this study was to analysis the effect of FCS on the cGMP pathway and the influence of cGMP on cytoplasmic lipids in bovine oocytes. In experiments 1 and 2, cumulus–oocyte complexes (COC) were cultured for 24 h in maturation medium with different proportions of FCS (2 and 10%) and a control group was matured with 0.4% BSA. After this period, transcripts for cGMP pathway were assessed by real-time PCR (GUCY1B3 and PDE5, cGMP synthesis and degradation enzymes, respectively; experiment 1) in oocytes and cumulus cells, and cGMP levels were measured in COC using commercial enzyme immunoassay kits (EIA; experiment 2). In experiments 3 and 4, COC were matured for 24 h with 0.4% BSA and different concentrations of the phosphodiesterase (PDE)5 inhibitor (0, 10–7, and 10–5 M sildenafil) to inhibit cGMP degradation and a control group was matured with 0.4% BSA. The nucleotide levels were measured in COC (experiment 3) and the oocytes were stained with Nile Red (1 μg mL–1) for evaluation of lipid content (experiment 4). Statistical analyses were performed by ANOVA followed by Tukey post hoc test using SAS software (SAS Institute Inc., Cary, NC, USA). Data for gene expression from 5 replicates and for cGMP measurements and lipid content from 3 replicates were log10-transformed into before analyses. The level of significance was 5%. The presence of FCS reduced GUCY1B3 expression in both cells and increased PDE5A in cumulus cells (P < 0.05). In experiment 2, the groups treated with 2 (0.64 fmol/COC) and 10% FCS (1.04 fmol/COC) showed decreased cGMP levels compared with control (9.46 fmol/COC; P < 0.05). In experiment 3, inhibition of PDE5A increased cGMP levels in the treated groups (36 and 56 fmol/COC for 10–7 and 10–5 M sildenafil, respectively) compared with control (9.5 fmol/COC; P < 0.05). Therefore, sildenafil showed inverse effects compared with FCS (experiment 2). In experiment 4, oocytes treated with 10–7 and 10–5 M sildenafil showed a reduced lipid content compared with controls (11.6 ± 9.4 v. 13.9 μm2 fluorescence intensity, respectively; P < 0.05). The results suggest that FCS in maturation medium affects the cGMP pathway, interfering with the transcription of genes that control its levels, which in turn results in nucleotide reduction. Inhibition of PDE5 increases cGMP levels and reduces the lipid content of oocytes, indicating that changes in this pathway caused by FCS may affect lipid metabolism of oocytes. More studies are underway to better understand this mechanism. The authors acknowledge FAPESP 2012/00170-0 for financial support.

188 IMPROVEMENT OF IN VITRO CANINE OOCYTE MATURATION BY OVIDUCTAL SECRETOME

2017 ◽  
Vol 29 (1) ◽  
pp. 202 ◽  
Author(s):  
A. Lange-Consiglio ◽  
C. Perrini ◽  
P. Esposti ◽  
F. Cremonesi
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Follicular Development ◽  
Cumulus Cells ◽  
Chi Square ◽  
Cell Layers ◽  
Hoechst Staining

The in vitro maturation of canine oocyte is problematic because it is difficult to reproduce the oviducal microenvironment where the in vivo maturation occurs. Because cells are able to communicate with each other by paracrine action, oviducal cells could be in vitro cultivated to obtain the conditioned medium (CM) consisting of soluble factors and microvesicles (MV), which represent a carrier for nonsoluble molecules including microRNA. The aim of the present work was to investigate the effect of the addition of CM or MV, secreted by oviducal cells, to the canine in vitro maturation medium. To generate CM, cells from oviducts of 3 animals in late oestrus were cultured for 5 days at 38.5°C in a humidified atmosphere of 5% CO2. Supernatants were collected, pooled, centrifuged at 2500 × g, and stored at −80°C. Microvesicles were obtained by ultracentrifugation of CM at 100,000 × g for 1 h at 4°C and measured for concentration and size by a Nanosight instrument. Ovaries were obtained from 50 healthy domestic bitches (1–4 years old) of different breeds that underwent ovariectomy regardless of the oestrous cycle. Cumulus-oocyte complexes were released by slicing the ovarian cortex with a scalpel blade, and only Grade 1 cumulus-oocyte complexes (darkly granulated cytoplasm and surrounded by 3 or more compact cumulus cell layers) 110 to 120 µm in diameter were selected for culture. Maturation was performed at 38.5°C in a humidified atmosphere of 5% CO2 and 5% of O2 in bi-phasic systems: 24 h in SOF with 5.0 μg mL−1 of LH followed by 48 h in SOF supplemented with 10% of oestrous bitch serum and 10% CM or 50, 75, 100, or 150 × 106 MV mL−1 labelled with PKH-26. Control was the same medium without CM or MV. Oocytes were observed under a fluorescent microscope to detect metaphase II (MII), by Hoechst staining, and the incorporation of MV. Statistical analysis was performed by chi-square test. Results show that canine oviducal cells secreted MV of 234 ± 23 nm in size, underling that these MV fall within the shedding vesicles category. The incorporation of labelled MV occurred at first in cumulus cells, at 48 h of maturation, and then, at 72 h, in oocyte cytoplasm. These MV had a positive effect on maturation rate (MII) at the concentration of 75 and 100 × 106 MV mL−1 compared with CM and control (20.34 and 21.82 v. 9.09 and 3.95%, respectively). The concentration of 150 × 106 MV mL−1 provided only 9.26% of MII. To understand the role of MV, we assessed the expression of 3 microRNA (miRNA-30b, miR-375, and miR-503) that are involved in some key pathways (WNT, MAPK, ERbB, and TGFβ) regulating follicular development and meiotic resumption. The lower rate of MII with the higher concentration of MV is possibly due to the high level of miR-375, which recent literature shows to suppress the TGFβ pathway, leading to impaired oocyte maturation. In conclusion, the oviducal MV, or specific microRNA, are involved in cellular trafficking during oocyte maturation, and their possible use in vitro could facilitate the exploitation of canine reproductive biotechnologies.

28 Effect of deuterium oxide on bovine oocyte cryotolerance

2019 ◽  
Vol 31 (1) ◽  
pp. 140
Author(s):  
F. Salerno ◽  
M. Rubessa ◽  
B. Gasparrini ◽  
M. Wheeler
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Bovine Serum ◽  
In Vitro Maturation ◽  
Deuterium Oxide ◽  
Control Group ◽  
Crystal Formation ◽  
Cleavage Rate ◽  
Maturation Medium

It is known that cryopreservation triggers spindle disassembly, increased aneuploidy risk, decreased post-thaw survival, fertilization, and embryo development. We hypothesised that a treatment with D2O before vitrification would slow down oocyte metabolism and reduce ice crystal formation by replacing water inside the cells. The aim of the study was to evaluate the effect of a 4-h treatment with different D2O concentrations (0, 3, 15, and 30%) on cryotolerance of bovine in vitro-matured oocytes. Abattoir-derived bovine oocytes were matured in vitro for 20h in TCM-199 medium with 15% of bovine serum (BS), 0.5µg mL−1 of FSH, 5µg mL−1 of LH, 0.8mM l-glutamine, and 50µg mL−1 of gentamicin at 39°C with 5% of CO2 and randomly divided into 5 experimental groups. A group of non-vitrified oocytes was used as the fresh oocyte control group, whereas the remaining oocytes were incubated for 4h in in vitro maturation medium with 0% (vitrified control; n=205), 3% (n=205), 15% (n=205), and 30% D2O (n=205) before vitrification. The experiment was repeated 4 times. Oocytes were denuded in HEPES-buffered TCM-199 (H199)+5% BS and vitrified using a cryotop freezing straw. The oocytes were incubated in 200μL of H199+20% BS with 7.5% ethylene glycol and 7.5% dimethyl sulfoxide for 3min. After that, oocytes were collected in 50μL of H199+20% fetal bovine serum with 15% ethylene glycol+15% dimethyl sulfoxide and 0.5M sucrose for 20s and plunged into LN2. One month later, oocytes were warmed in thawing media with decreasing concentrations of sucrose (1.35M to 0.31M) and then placed into in vitro maturation medium for 2h before IVF. Matured oocytes were IVF and cultured according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). Cleavage and blastocyst rates were evaluated after 7 days of culture. Data were analysed using the GLM procedure of SPSS (SPSS Inc., Chicago, IL, USA). The least statistical difference post-hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. As expected, both cleavage [60.5±4.6 (fresh control); 36.9±2.6 (0% D2O); 46.3±3.7 (3% D2O); 31.6±2.4 (15% D2O); and 24.4±2.6 (30% D2O)] and blastocyst rates [25.7±0.8 (fresh control); 9.0±0.8 (0% D2O); 9.0±0.7 (3% D2O); 3.6±0.2 (15% D2O); and 4.3±0.8 (30% D2O)] decreased in all vitrified groups compared with the fresh control group. Within vitrified oocytes, cleavage rate increased (P&lt;0.05) with 3% D2O treatment compared with the other groups. However, pretreatment with higher (15-30%) D2O concentrations decreased (P&lt;0.05) blastocyst rates of vitrified-warmed oocytes. In conclusion, a pretreatment with low concentrations (3%) of D2O improved the cleavage rate of bovine vitrified-warmed oocytes, suggesting a potential beneficial effect, whereas deleterious effects were observed using the higher concentrations. Therefore, further studies are required to assess a potential use of D2O to improve oocyte cryotolerance, likely testing different incubation times.

Paradoxical effects of kisspeptin: it enhances oocyte in vitro maturation but has an adverse impact on hatched blastocysts during in vitro culture

2012 ◽  
Vol 24 (5) ◽  
pp. 656 ◽  
Author(s):  
Islam M. Saadeldin ◽  
Ok Jae Koo ◽  
Jung Taek Kang ◽  
Dae Kee Kwon ◽  
Sol Ji Park ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Formation Rate ◽  
Early Period ◽  
Threshold Effect ◽  
Developmental Competence ◽  
Control Group ◽  
Maturation Medium ◽  
Paradoxical Effects

Kisspeptin (Kp) is best known as a multifunctional peptide with roles in reproduction, the cardiovascular system and cancer. In the present study the expression of kisspeptin hierarchy elements (KISS1, GNRH1 and LHB) and their receptors (KISS1R, GNRHR and LHCGR, respectively) in porcine ovary and in cumulus–oocyte complexes (COCs) were investigated, as were its effects on the in vitro maturation (IVM) of oocytes and their subsequent ability to sustain preimplantation embryo competence after parthenogenetic electrical activation. Kp system elements were expressed and affected IVM of oocytes when maturation medium was supplemented with 10–6 M Kp. Oocyte maturation, maternal gene expression (MOS, GDF9 and BMP15), blastocyst formation rate, blastocyst hatching and blastocyst total cell count were all significantly increased when oocytes were matured in medium containing Kp compared with the control group (without Kp). A Kp antagonist (p234) at 4 × 10–6 M interfered with this hierarchy but did not influence the threshold effect of gonadotrophins on oocyte maturation. FSH was critical and permissive to Kp action on COCs by increasing the relative expression of KISS1R. In contrast, Kp significantly increased apoptosis, the expression of pro-apoptotic gene, BAK1, and suppressed trophoblast outgrowths from hatched blastocysts cultured on feeder cells. The present study provides the first functional evidence of the Kp hierarchy in porcine COCs and its role in enhancing oocyte maturation and subsequent developmental competence in an autocrine–paracrine manner. However, Kp supplementation may have a harmful impact on cultured hatched blastocysts reflecting systemic or local regulation during the critical early period of embryonic development.

scholarly journals Use of Melatonin in the In Vitro Production of Bovine Embryos

2020 ◽  
Vol 21 ◽  
Author(s):  
Alan da Silva LIRA ◽  
Ricardo de Macedo CHAVES ◽  
Felipe de Jesus MORAES JUNIOR ◽  
Sergio Henrique COSTA JUNIOR ◽  
Brenda Karine Lima do AMARAL ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Control Group ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Maturation Medium ◽  
Significant Difference ◽  
Maturation Rate ◽  
Vitro Production

ABSTRACT We aimed to assess the effects of melatonin in the in vitro production of bovine embryos. Our experiment was conducted at the Laboratório de Reprodução Animal of the Universidade Estadual do Maranhão. The cumulus-oocyte complexes (COCs) were distributed among treatments at concentrations of 0, 10-1, 10-3 and 10-5 µMol/L melatonin. Our experiment was further divided into two: the first was to assess the effect of different concentrations of melatonin (treatments) on the maturation rate of COCs, and the second was to assess the effects of melatonin treatments on the in vitro production of bovine embryos. The results from the first experiment demonstrated no significant difference between the in vitro maturation rate of the cultivated COCs in treatments with melatonin. In the second experiment, however, melatonin treatments yielded statistically higher cleavage, morula and blastocyst rates in the 10-5 µM group (52.9%, 52.9%, and 35.3%, respectively), and lower rates in the 10-1 µM group (19.5%, 19.5% and 7.8%, respectively), compared to the others. The control group (no melatonin) and the 10-3 µM group showed similar results. We concluded that supplementation of melatonin in the in vitro maturation medium resulted in no improvement in the oocyte maturation rate, but in the in vitro production of embryos at different concentrations, the 10-5 µM group displayed better results, but with no improvement in the variables (P < 0.05).

Sign in / Sign up

Export Citation Format

Share Document