scholarly journals Effect of glycine and alanine supplementation on development of cattle embryos cultured in CRlaa medium with or without cumulus cells

1996 ◽  
Vol 5 (5) ◽  
pp. 503-508 ◽  
Author(s):  
Kristiina Bredbacka ◽  
Peter Bredbacka
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Bovine Embryo ◽  
Control Medium ◽  
Cell Numbers ◽  
Combined Supplementation ◽  
The Mean ◽  
Development In Vitro

The effect of alanine (1 mM) and glycine (10 mM) supplementation on bovine embryo development in vitro was investigated. Presumptive bovine zygotes, produced by in vitro maturation and insemination of oocytes, were cultured for 144 h in CRlaa medium in the absence (Experiments 1 and 2) or presence of cumulus cells (Experiment 3). In Experiment I, the proportion of morulae and blastocysts of cleaved embryos in glycine-supplemented medium was not different from that of the control medium (34% in both media); however, the cell numbers of morulae and blastocysts were significantly higher in the glycine-enriched medium (69.5 vs. 53.3, P = 0.016). In Experiment 2, addition of alanine did not improve the formation of morulae and blastocysts (13% vs. 21% in control medium), and the mean cell numbers in morulae and blastocysts were lower than those in the control group (34.3 vs. 68.7, P = 0.007). In the presence of cumulus cells, the combined supplementation of glycine and alanine increased the proportion of morulae and blastocysts over that in the control medium (31% vs. 14%, P = 0.003).

300 BLASTOCYST PRODUCTION BY IN VITRO MATURATION AND DEVELOPMENT OF PORCINE OOCYTES IN DEFINED MEDIA FOLLOWING INTRACYTOPLASMIC SPERM INJECTION

2007 ◽  
Vol 19 (1) ◽  
pp. 265
Author(s):  
Y. Kobayashi ◽  
M. Fukui
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
In Vitro Maturation ◽  
Subsequent Development ◽  
Basic Medium ◽  
Control Medium ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Defined Media ◽  
The Mean

Most culture media for in vitro production (IVP) of porcine embryos contain serum or bovine serum albumin (BSA); especially in the case of in vitro maturation (IVM) porcine follicular fluid is added. The present study was carried out to establish porcine-defined IVP. In Experiment 1, we investigated the efficacy of additional 0.6 mM cystine, 100 �M cysteamine (Cys), or cystine + Cys to a defined TCM-199 maturation medium with regard to the developmental competence of in vitro-matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The control medium was a modified TCM-199 containing 0.05% (w/v) polyvinyl alcohol (PVA). In Experiment 2, the effect of epidermal growth factor (EGF) addition to a modified porcine zygote medium (mPZM) for in vitro culture (IVC) medium was investigated on embryonic development following ICSI. As positive and negative controls, 0.3% BSA (mPZM-3) or 0.3% PVA (mPZM-4), respectively, was added to the basic medium. Five or 10 ng mL-1 of EGF was supplemented in the negative control medium (mPZM-4). All percentage data on cleavage and blastocyst development were subjected to arcsine transformation before statistical analysis using the STATVIEW program (Abacus Concepts, Berkeley, CA). The mean cell numbers per blastocyst (assessed by Giemsa staining method) were analyzed by one-way ANOVA, and the differences among the groups were also analyzed using Tukey-Kramer multiple comparison tests. In Experiment 1, there were no significant differences in the rates of cleavage (31.4 to 64.3%) and blastocyst formation (6.5 to 22.9%) among the treatment and control groups. The mean cell numbers per blastocyst ranged from 30 to 48 among the groups, without significant differences. In Experiment 2, the rates of cleavage (43.0 to 58.0%) and blastocyst formation (14.0 to 23.6%), and the mean cell numbers per blastocyst (30 to 37) were not significantly different among the groups. However, the addition of 5 ng mL-1 of EGF to the mPZM-4 resulted in a significantly (P d 0.05) higher blastocyst rate (48.6%) to cleaved embryos than the other two defined groups (mPZM-4 and mPZM-4 with 10 ng mL-1 EGF: 23.4% and 23.1%, respectively), but not significantly different with mPZN-3 (40.1%). The present results indicate that TCM-199 with added cysteamine (100 �M) can be used as a defined IVM medium for porcine oocytes, and further addition of cystine into the IVM medium is not necessary. The addition of 5 ng mL-1 of EGF to a defined IVC medium enhanced subsequent development after ICSI. These results show that porcine blastocysts can be produced by defined media throughout the steps of IVP (IVM, ICSI, and IVC). This study was partly supported by a grant-in-aid for Scientific Research (17580242) from the Japan Society for the Promotion of Science.

Blastocyst production by in vitro maturation and development of porcine oocytes in defined media following intracytoplasmic sperm injection

Zygote ◽  
2007 ◽  
Vol 15 (2) ◽  
pp. 93-102 ◽  
Author(s):  
M. Kobayashi ◽  
S. Asakuma ◽  
Y. Fukui
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Subsequent Development ◽  
Control Group ◽  
Control Medium ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Defined Media ◽  
Significant Difference ◽  
The Mean

SummaryThe present study was carried out to establish porcine defined IVP. In Experiments 1 and 2, we investigated the efficacy of additional 0.6 mM cystine and/or 100 µM cysteamine (Cys) to a defined TCM199 maturation medium with regard to the intracellular glutathione (GSH) concentration and the developmental competence of in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The control medium was a modified TCM199 containing 0.05% (w/v) polyvinyl alcohol (PVA). Cys and/or cystine were added to the control medium. The control group and immature oocytes (presumptive germinal vesicle oocytes; GV) were prepared for GSH assay. In Experiment 3, the efficacy of epidermal growth factor (EGF) addition to a modified porcine zygote medium (mPZM) for in vitro culture (IVC) medium was investigated on embryonic development and the mean cell number of blastocysts following ICSI. As a positive or negative control, 0.3% BSA (mPZM-3) or 0.3% PVA (mPZM-4), respectively, was added to the base medium. The defined IVC medium was supplemented with 5 or 10 ng/ml EGF. In Experiment 1, no significant difference was found in the rates of cleavage (31.4–64.3%) and blastocyst formation (6.5–22.9%) among the treatment and control groups. The mean cell numbers per blastocyst ranged from 30 to 48 among the groups without significant differences. However, in Experiment 2, the intracellular GSH concentrations in the oocytes cultured in the medium supplemented with 100 µM Cys (9.6 pmol/oocyte) or Cys + cystine (9.9 pmol/oocyte) were significantly (p < 0.05) higher than the control (2.5 pmol/oocyte) and 0.6 mM cystine (6.5 pmol/oocyte) groups, but not different from the GV group (9.0 pmol/oocyte). The GSH concentration in the cystine group was also significantly (p < 0.05) higher than that in the control group, but not different from the GV group. In Experiment 3, the rates of cleavage and blastocyst formation and the mean cell numbers of blastocysts were not significantly different among the groups. However, the addition of 5 ng/ml EGF into the mPZM-4 resulted in a significantly (p < 0.05) higher blastocyst rate per cleaved embryo than the other two defined groups (mPZM-4 + 5 ng/ml: 48.6%, mPZM-4 and mPZM-4 +10 ng/ml: 23.4% and 23.1%, respectively).The present results indicate that the addition of Cys to a defined medium for in vitro maturation (IVM) of porcine oocytes increases intracellular GSH concentration. Further addition of cystine into the IVM medium containing 100 µM Cys is not necessary and TCM199 plus Cys (100 µM) could be used as a defined IVM medium for porcine oocytes. The addition of 5 ng/ml EGF to a defined IVC medium has enhanced subsequent development after ICSI. This study shows that porcine blastocysts can be produced by defined media throughout the steps of IVP (IVM, ICSI and IVC).

scholarly journals 46 Effect of the addition of α-tocopherol to in vitro maturation media of bovine oocytes

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 2-3
Author(s):  
Theisy P Acosta Pérez
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Chi Square ◽  
Cumulus Expansion ◽  
Minimum Concentration ◽  
Maturation Rate ◽  
Chi Square Test ◽  
System Data

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P &gt;0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

249 FOLLICLE-STIMULATING HORMONE AND DIBUTYRYL cAMP TREATMENTS DURING IN VITRO MATURATION IMPROVE THE DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 204
Author(s):  
R. Oishi ◽  
Y. Isaji ◽  
H. Imai ◽  
M. Yamada
Keyword(s):  
In Vitro Maturation ◽  
Basal Medium ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Medium ◽  
Dibutyryl Camp ◽  
Mouse Oocytes ◽  
Junctional Communication

The high level of cyclic adenosine monophosphate (cAMP), which is provided to the oocytes from cumulus cells via gap junctional complexes in cumulus-enclosed oocytes (CEOs), is known to contribute to meiotic arrest at the germinal vesicle (GV) stage of CEOs. However, whether intraoocyte cAMP during the period of in vitro maturation (IVM) affects postfertilization developmental competence of mouse oocytes still remains unclear. The aim of this study was to examine the effects of FSH or dibutyryl cAMP (dbcAMP) treatment during IVM on in vitro development of mouse oocytes after in vitro fertilization (IVF). Whether a junctional association between cumulus cells and the oocyte would be essential for a cytoplasmic maturation-promoting effect was also examined. CEOs were isolated from and eCG-primed 3-week-old ICR mouse by rupturing preovulatory follicles with needles in M16 medium with 5% FCS and essential and nonessential amino acids (basal medium). IVM media used were basal medium without (control) or with 100 µm dbcAMP or 1 IU mL–1 FSH. Carbenoxolone (100 µm, CBX), an inhibitor of gap junction, was used to inhibit a junctional association between cumulus cells and the oocyte. Denuded oocytes (DOs) were prepared by repeatedly pipetting in basal medium with 0.2% hyaluronidase. CEOs and DOs were cultured in IVM media at 37�C under 5% CO2 in air for 16.5 h, and then transferred to TYH medium (a modified Krebs-Ringer bicarbonate medium) containing 0.4% BSA, followed by insemination with capacitated sperm. After 6 h of IVF, inseminated oocytes were cultured in KSOM medium with 0.3% BSA. Development to the 2-cell and blastocyst stages was estimated at 24 h and 120 h after IVF, respectively. All experiments were done in 3 replicates, and the statistical analysis was carried out by ANOVA and Fisher's protected least-squares difference (PLSD) test. When CEOs were matured in IVM media, the rates of postfertilization development to the 2-cell and blastocyst stages of oocytes matured in the control medium were very low(29% and 13%, respectively), whereas those of oocytes matured with FSH or dbcAMP significantly increased (FSH: 61% and 52%, dbcAMP: 63 and 57%, respectively, v. control; P < 0.05). Next, when CEOs were matured in basal medium with 1 IU mL–1 FSH and 100 µm CBX, the developmental rate to the 2-cell stage (56%) was similar to that in medium with FSH alone (61%) but the rate to the blastocyst stage (40%) was little lower compared with that in medium with FSH alone (52%), although not significantly different (P > 0.05). Furthermore, when DOs were matured in IVM media, the developmental rates to the blastocyst stage after IVF of the oocytes matured with FSH or dbcAMP significantly increased (FSH: 25%, dbcAMP: 15%; P < 0.05) compared with those in control medium (7%). Taken together, it is suggested that increasing the concentration of intraoocyte cAMP during the IVM period is important to improve the developmental competence after IVF of mouse oocytes, and that the competence is acquired in part in a cumulus-oocyte junctional communication-independent manner.

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.

321 OOCYTE DIAMETER INFLUENCES THE MEIOTIC RESUMPTION AND PROGRESSION INDUCED BY OKADAIC ACID IN DOG

2006 ◽  
Vol 18 (2) ◽  
pp. 268
Author(s):  
F. Ariu ◽  
L. Bogliolo ◽  
I. Rosati ◽  
M. T. Zedda ◽  
S. Pau ◽  
...  
Keyword(s):  
Okadaic Acid ◽  
In Vitro Maturation ◽  
Mammalian Species ◽  
Cumulus Cells ◽  
Meiotic Maturation ◽  
Control Group ◽  
Oocyte Diameter ◽  
Treatment Groups ◽  
Different Diameters

The acquisition of meiotic competence, in the bitch as in many other mammalian species, is related to the oocyte diameter. This study was designed to determine the effect of okadaic acid (OA), a potent inhibitor of seronine/threonine 1 and 2A phosphatases, on meiotic resumption and progression in canine oocytes with different diameters. In two experiments, healthy cumulus-oocytes complexes were collected from ovaries of bitches at various stages of the estrous cycle and divided, by diameters, into three treatment groups for in vitro maturation: <110 �m, 110-120 �m, and >120 �m. In Experiment 1, oocytes were pre-incubated for 1 h in TCM-199 + 20% estrous canine serum (SCE) + cysteamine + OA (0.5 �M). Then, oocytes were cultured for 48 h in the same medium without OA at 38.5�C, 5% CO2 in air. As a control group, oocytes were matured in vitro under the same conditions but without pre-incubation with OA. In Experiment 2, to determine if the effect of OA is mediated by cumulus cells, >120 �m oocytes were denuded from cumulus cells, incubated with or without OA, and cultured in vitro as previously described. At 48 h, all oocytes were stained and fixed with glycerol-Hoechst 33342 to assess the stage of meiotic maturation. In Experiment 1, OA induced a significantly higher incidence of meiotic resumption in oocytes <110 �m (16/108, 14.8%; P < 0.05) and 110-120 �m (70/130, 53.8%; P < 0.01) as compared to that of oocytes in the <110 �m and 110-120 �m control groups (2/58, 3.4%; 24/82, 29.3%). The percentage of oocytes in the 110-120 �m OA group that underwent in vitro maturation to metaphase II (MII) was significantly higher than in the 110-120 �m control group (18/130, 13.8% vs. 4/82, 4.9%, respectively; P < 0.05). In contrast, smaller oocytes (<110 �m) did not develop to MII with or whitout OA. Meiotic resumption rate of >120 �m OA group (64/78, 82.0%) was similar to the >120 �m control group (56/72, 77.8%), but a significantly higher proportion of the oocytes pre-incubated with OA progressed to MII than did the control oocytes (40/78, 51.3% vs. 12/72, 16.7%, respectively; P < 0.01). Low rates of meiotic resumption were observed in denuded >120-�m oocytes with (7/63, 11.1%) or without OA (7/55, 12.7%) and none of them progressed to MII. In conclusion, the results of the present study indicate that treatment of fully grown (>120 �m) oocytes with okadaic acid at the onset of in vitro maturation can result in a higher frequency of meiotic maturation than previously reported. Also, we determined that the beneficial effect of okadaic acid was mediated by cumulus cells.

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&lt;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&gt;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&lt;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).

197 EFFECTS OF PRE-IN VITRO MATURATION WITH CAFFEINE ON BOVINE OOCYTE DEVELOPMENTAL CAPACITY

2016 ◽  
Vol 28 (2) ◽  
pp. 229
Author(s):  
S. M. B. Ulloa ◽  
J. Heinzmann ◽  
D. Herrmann ◽  
U. Baulain ◽  
K.-G. Hadeler ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Developmental Competence ◽  
Differential Staining ◽  
Control Group ◽  
Dependent Manner ◽  
Meiotic Arrest ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Camp Levels

High cyclic adenosine monophosphate (cAMP) concentrations are critical for maintaining oocyte meiotic arrest in vivo. For in vitro maturation (IVM), the oocyte is released mechanically from the follicle, which induces a significant drop in intra-oocyte cAMP levels, triggering non-physiological meiotic resumption. It has been proposed that modulation of cAMP before IVM can increase bovine blastocyst rates in vitro. Caffeine is a nonspecific competitive phosphodiesterases (PDE) inhibitor and can inhibit meiotic resumption of oocytes due to maintenance of cAMP levels. It has been reported that gamete treatment with caffeine can increase developmental potential. The current study evaluated the effects of pre-in vitro maturation culture with different concentrations of caffeine on meiotic progress, developmental rates and blastocyst cell numbers. Bovine ovaries were collected from a local abattoir. A total of 6648 cumulus-oocyte complexes were obtained by slicing. Caffeine was used in 5 different concentrations (1, 5, 10, 20, and 30 mM) during slicing, searching, and 2 h pre-IVM culture. A control group, with 2 h pre-IVM without caffeine (0 mM) and a standard control were also included. Oocytes were washed either after standard or pre-IVM treatments and cultured for 24 h in vitro without caffeine. After IVM, oocytes were fertilised in vitro for 19 h, and zygotes were cultured in vitro for 8 days until the blastocyst stage. Subsets of oocytes were fixed in 2% glutaraldehyde at 9, 20, and 24 h after IVM. Hoechst staining was performed to evaluate nuclear status of matured oocytes. Cleavage and blastocyst formation rates were evaluated at Days 3 and 8 after IVF. Expanded blastocysts from all treatments were submitted to differential staining. One-way ANOVA from R software was applied to evaluate differences in cleavage and blastocysts rates and blastocyst cell numbers. Fisher’s exact test complemented by Bonferroni correction was used to determine meiotic progress. Caffeine maintained oocytes in meiotic arrest after 9 h of IVM in a concentration-dependent manner (germinal vesicle: 79.0%, 92.2%, 66.7%, 55.1%, 56.9%, 43.9%, 30.2%, respectively, for 30, 20, 10, 5, 1, 0 mM and standard; P < 0.016). Cleavage rates were similar in all treatments; however 30 mM caffeine decreased blastocyst rates (Table 1; P < 0.05). The number of cells did not differ significantly among in vitro treatments (Table 1; P > 0.05). Developmental competence was not affected by 2 h pre-IVM culture. Caffeine supplementation before IVM delayed resumption of meiosis and affected embryo development. Table 1.In vitro developmental competence of oocytes treated with different caffeine concentrations before IVM

Effect of oviduct-conditioned medium and of cumulus cells on bovine embryo development in vitro

1992 ◽  
Vol 37 (1) ◽  
pp. 256 ◽  
Author(s):  
P. Mermillod ◽  
C. Boccart ◽  
C. Wils ◽  
A. Massip ◽  
F. Dessy
Keyword(s):  
Conditioned Medium ◽  
Embryo Development ◽  
Cumulus Cells ◽  
Bovine Embryo ◽  
Development In Vitro
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