Duration of In Vitro Maturation of Recipient Oocytes Affects Blastocyst Development of Cloned Porcine Embryos

2005 ◽  
Vol 7 (1) ◽  
pp. 35-44 ◽  
Author(s):  
Michael Hölker ◽  
Björn Petersen ◽  
Petra Hassel ◽  
Wilfried A. Kues ◽  
Erika Lemme ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Blastocyst Development

53 EFFECT OF THE TIME INTERVAL BETWEEN OVARY COLLECTION AND OOCYTE IN VITRO MATURATION ON EQUINE CLONED EMBRYO DEVELOPMENT

2010 ◽  
Vol 22 (1) ◽  
pp. 184
Author(s):  
A. Gambini ◽  
J. Jarazo ◽  
R. Olivera ◽  
D. Salamone
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Limiting Factor ◽  
Time Interval ◽  
Activation Process ◽  
Blastocyst Development ◽  
Sodium Pyruvate ◽  
Chi Square ◽  
Group I

The availability of viable equine oocytes is a limiting factor on in vitro embryo production; therefore, it is necessary to assess some of the variables that affect oocyte viability. The aim of our study was to evaluate one of those variables: the effect of time between the collection of the ovary and oocyte in vitro maturation. Ovaries of slaughtered mares were collected during the breeding season (Argentine, Southern hemisphere). They were separated in bags every half hour and treated separately after arriving at the laboratory. COCs were recovered by a combination of scraping and washing of all visible follicles with a syringe filled with DMEM supplemented with 1 mM sodium pyruvate and 15 IU mL-1 heparin. COCs were matured for 24 to 26 h in 3 groups, according to time interval: 4 to 7 (group I), 7 to 10 (II), and 10 to 12 (III) hours. The medium for maturation was TCM-199 supplemented with 10% fetal bovine serum (FBS), 1 μL mL-1 insulin-transferrin-selenium, 1 mM sodium pyruvate, 100 mM cysteamine, and 0.1 mg mL-1 of FSH at 39°C in a humidified atmosphere of 5% CO2 in air. The cumulus was removed by a trypsin treatment and vortexing in hyaluronidase (1 mg mL-1). Cloning and fusion procedures were performed following the zona-free technique described by Lagutina et al. (2007 Theriogenology 67, 90-98). Two experiments were carried out by using different activation protocols. In experiment 1, the activation process was 22 mM ionomycin in H-TALP for 4 min followed by 3h culture in 1.9 mM 6-DMAP in SOF, whereas in experiment 2, we used 8.7 mM ionomycin in H-TALP for 4 min followed by 4 h culture in 1 mM 6-DMAP and 10 mg mL-1 cycloheximide in SOF. Embryos were cultured in wells of well (WOW) system. Half of the medium was renewed on Day 3 with fresh SOF and on Day 5 with DMEM/F12 with 10% FBS. Cleavage was assessed 48 h after activation; the rate of blastocyst formation was recorded at Days 8 and 9. Results were compared using chi-square test (P < 0.05). In experiment 1, maturation rates were significantly different between group I (n = 135, 54.1%) and III (n = 94, 40.4%), group II did not differ from them (n = 138, 53%). Cleavage rates differed statistically between II (n = 44, 75%) and III (n = 27, 40.7%), but not with group I (n = 53, 98%). No significant differences were found in blastocyst development; however, we observed a certain tendency towards an increase in the blastocyst rate as the time interval was lower (I: 3/53, 5.7%; II: 1/44, 2.3%; III: 0/27, 0%). In experiment 2, there were no significant differences between group I and II in rates of maturation (n = 56, 59% v. n = 111, 44.5%), cleavage (n = 22, 91% v. n = 34, 82%) or blastocyst rates (1/22, 4.5% v. 7/34, 20.6%). We conclude that cloned equine embryo development, using the two activation protocols tested, is not affected when the time interval between ovary collection and oocyte IVM is within 4 to 10 h.

233. Oxygen concentration during in vitro maturation of murine oocytes affects blastocyst cell lineage

2005 ◽  
Vol 17 (9) ◽  
pp. 91
Author(s):  
K. M. Banwell ◽  
M. Lane ◽  
D. L. Russell ◽  
K. L. Kind ◽  
J. G. Thompson
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Cell Lineage ◽  
Developmental Competence ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
Treatment Groups ◽  
Significant Difference ◽  
O2 Concentration

Follicular antral oxygen tension is thought to influence subsequent oocyte developmental competence. Despite this, in vitro maturation (IVM) is routinely performed in either 5 or 20% O2 and while low O2 has been shown to be beneficial to embryo development in many species, the effect of altering O2 concentration during IVM has not been adequately investigated. Here we investigated the effects of a range of O2 concentrations during IVM on meiotic maturation and subsequent embryo development after IVF. Ovaries from eCG-stimulated CBA F1 female mice (21 days) were collected and intact cumulus oocyte complexes (COCs) cultured for 17–18 h under 2, 5, 10 or 20% O2 (6% CO2 and balance of N2). Matured COCs were denuded of cumulus cells, fixed and stained (1% aceto-orcein) for visualisation of maturation status. No significant difference in maturation rates between treatment groups was observed. Following IVF (performed under 5% O2, 6% CO2 and balance of N2), no difference in fertilisation rates between treatment groups was observed in a randomly selected cohort 7 h post-fertilisation. There was also no significant difference in cleavage rates after 24 h or ability to reach blastocyst stage after 96 h, with a tendency (P = 0.079) for more blastocysts in 2% O2. However there was a significant increase in the number of trophectoderm cells present in the resulting blastocysts (P < 0.05) in the 2% O2 group (35 ± 2.1) compared to 20% O2 (25 ± 2.8). Our data suggests that O2 concentration during IVM does not influence nuclear maturation or subsequent fertilisation, cleavage and blastocyst development rates. However, maturation in 2% O2 significantly alters subsequent cell lineage within blastocysts to favour trophectoderm development. Such skewed trophectoderm cell number may influence embryo viability. Funded by NHMRC and NIH.

Relationship between follicle size and oocyte developmental competence in prepubertal and adult pigs

2007 ◽  
Vol 19 (7) ◽  
pp. 797 ◽  
Author(s):  
Melanie A. Bagg ◽  
Mark B. Nottle ◽  
David T. Armstrong ◽  
Christopher G. Grupen
Keyword(s):  
In Vitro Maturation ◽  
Fold Increase ◽  
Cell Number ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Parthenogenetic Activation ◽  
Follicle Size ◽  
Low Efficiency ◽  
The Relationship

The present study compared the distribution and steroid composition of 3-, 4- and 5–8-mm follicles on the surface of prepubertal and adult ovaries, and determined the relationship between follicle size and developmental competence of oocytes following parthenogenetic activation. The effect of 1 mm dibutyryl cAMP (dbcAMP) for the first 22 h of in vitro maturation (IVM) on the embryo development of prepubertal oocytes from the three follicle size cohorts was also determined. Compared with adult, prepubertal ovaries contained a higher proportion of 3-mm follicles (46 v. 72%, respectively), but a lower proportion of 4-mm (33 v. 22%, respectively) and 5–8-mm follicles (21 v. 6%, respectively). Adult follicular fluid (FF) contained 11-fold higher levels of progesterone (P4) than prepubertal FF, with similar levels observed between all adult follicle sizes. In prepubertal FF, the P4 concentration increased with follicle size from 3 to 4 to 5–8 mm. Rates of blastocyst development following parthenogenetic activation of adult oocytes from all three follicles sizes were similar (approximately 55%), whereas rates from prepubertal oocytes increased with increasing follicle size from 3 (17%) to 4 (36%) to 5–8 mm (55%). Treatment with dbcAMP for the first 22 h of IVM led to a 1.5-fold increase in the rate of blastocyst development for prepubertal oocytes from 3-mm follicles, but had no effect on prepubertal oocytes from the 4 and 5–8 mm classes. Mean blastocyst cell number increased with follicle size in prepubertal ovaries and was similar for all follicle sizes in adult ovaries. The present study demonstrates that the low efficiency of in vitro embryo production observed using prepubertal compared with adult pig oocytes is due to a greater proportion of 3-mm follicles on prepubertal ovaries, which contain oocytes of inferior developmental competence.

62 COMPARISON OF SUGARS, COMBINATIONS OF PERMEABLE CRYOPROTECTANTS, AND EQUILIBRATION REGIMENS FOR THE SOLID SURFACE VITRIFICATION OF IMMATURE PORCINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 124 ◽  
Author(s):  
T. Somfai ◽  
N. T. Men ◽  
H. Kaneko ◽  
J. Noguchi ◽  
S. Haraguchi ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Solid Surface ◽  
Embryo Culture ◽  
In Vitro Maturation ◽  
Blastocyst Development ◽  
Oocyte Vitrification ◽  
Morphological Evaluation ◽  
Development Rates ◽  
Significant Difference

Cryotop and solid surface vitrification are frequently used methods for the cryopreservation of porcine oocytes. These methods differ not only in the vitrification carrier but also in the cryoprotectant (CPA) treatment including the type of sugar, permeable CPA (pCPA) combinations, and the equilibration regimen. This study compared the distinct points of CPA treatment of these 2 methods to determine the optimum CPA treatment for the solid surface vitrification of immature porcine oocytes. We vitrified and warmed follicular cumulus-oocyte complexes by our method (Somfai et al. 2014 PLoS One 9, e97731). In each experiment, the vitrification solution consisted of 50 mg mL–1 polyvinyl pyrrolidone, 0.3 M of the actual sugar, and 35% [v/v] in total of the actual pCPA combination (depending on the experiment). After warming, the cumulus-oocyte complexes were subjected to in vitro maturation, IVF, and embryo culture (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). Oocyte survival was assessed after IVF by morphological evaluation, and live oocytes were subjected to in vitro embryo culture. Cleavage and blastocyst rates were calculated from cultured oocytes on Day 2 (Day 0 = IVF) and Day 6, respectively. Each experiment was replicated at least 3 times. Results were analysed by ANOVA. In Experiment 1, we compared trehalose (n = 416) and sucrose (n = 440) as supplementations during vitrification and warming (0.3 M and 0.4 M of each, respectively). There was no significant difference between oocytes vitrified with trehalose or sucrose in terms of survival, cleavage, and blastocyst development (83.2% v. 80.3%, 39.7% v. 42.4%, and 3.6% v. 5.9%, respectively). Thus, vitrification and warming media were supplemented with sucrose thereafter. In Experiment 2, we compared 1 : 1 combinations of ethylene glycol with propylene glycol (EG+PG group, n = 452) and ethylene glycol with dimethyl sulfoxide (EG+DMSO group, n = 465) used as pCPA for equilibration (4% [v/v] pCPA in total for 15 min) and vitrification (35% [v/v] pCPA in total for 30 s). Oocyte survival rate was higher (P < 0.05) in the EG+PG group compared with the EG+DMSO group (73.8% v. 51.1%, respectively); however, cleavage and blastocyst development rates of surviving oocytes were not significantly different between the 2 groups (30.5% v. 44.5% and 4.1% v. 6.3%, respectively). In Experiment 3, we compared an equilibration treatment in 4% [v/v] of EG+PG for 13 to 15 min (regimen A, n = 368) with an equilibration in 15% [v/v] of EG+PG for 5 to 7 min (regimen B, n = 363) for oocyte vitrification. Survival, cleavage, and blastocyst development rates were higher (P < 0.01) for oocytes vitrified using regimen A compared with those vitrified using regimen B (82.5% v. 22.7%, 24.0% v. 7.7%, and 3.2% v. 0%, respectively). In conclusion, trehalose and sucrose are equally effective during vitrification and warming, the combination of EG+PG as pCPA is superior to EG+DMSO, and equilibration in 4% pCPA for 13 to 15 min is superior to that in 15% pCPA for 5 to 7 min for the vitrification of immature porcine oocytes.This work was partly supported by JSPS KAKENHI Grant Number 26870839.

315 SELECTION OF BOVINE OOCYTES BY BRILLIANT CRESYL BLUE BEFORE IN VITRO MATURATION IMPROVES BLASTOCYST DEVELOPMENT

2007 ◽  
Vol 19 (1) ◽  
pp. 273 ◽  
Author(s):  
A. Sugulle ◽  
S. Katakawa ◽  
S. Yamamoto ◽  
S. Oomori ◽  
I. Itou ◽  
...  
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Control Group ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
Bovine Oocytes ◽  
Selection Of

The morphological identification of immature oocytes has commonly been used to select the bovine oocytes for IVF. However, &lt;30% of the recovered oocytes reach the blastocyst stage after fertilization, and this is probably due to the quality of the oocytes at the beginning of maturation. The brilliant cresyl blue (BCB) stain determines the activity of glucose-6-phosphate dehydrogenase, an enzyme synthesized in growing oocytes. The aim of this study was to evaluate the effect of the BCB stain on the selection of bovine oocytes and on the subsequent embryo development for in vitro production (IVP). Cumulus–oocyte complexes (COCs) were collected by the aspiration of 2- to 6-mm follicles. A total of 559 oocytes were divided into 2 groups: (1) a control group, immediately cultured, and (2) a BCB-incubated group. After 90 min of BCB staining (Pujol et al. 2004 Theriogenology 61, 735–744), the oocytes were divided into oocytes with blue cytoplasm (BCB+) and oocytes without blue cytoplasm (BCB−). The COCs were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 mg mL−1 FSH at 38.5°C under an atmosphere of 5% CO2 in air. The matured COCs were inseminated with 5 × 106 sperm mL−1. After 18 h of gamete co-culture, the presumed zygotes were cultured in CR1aa supplemented with 5% CS for 9 days at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2. Embryonic development was evaluated at 48 h after IVF (proportion of ≥5-cell stage, the total cleavage rates) and on Days 7 to 9 (blastocyst rate). The experiment was replicated 5 times, and the data were analyzed by a chi-square test and ANOVA. The results are presented in Table 1. The proportion of embryos with ≥5-cell stage was significantly higher (P &lt; 0.01) in the BCB+ group than in the BCB− group, but not in the control group. The total cleavage rate for the BCB+ embryos was significantly higher than that of either the BCB− or the control group (P &lt; 0.01). There were also significant differences (P &lt; 0.01) in the blastocyst development between the BCB+ and BCB− embryos and between the BCB− and the control embryos (P &lt; 0.05). This result showed that the selection of bovine oocytes by BCB staining before in vitro maturation may be useful for selecting oocytes that are developmentally competent up to Day 9 for IVP. Table 1.Effect of selection of oocytes by brilliant cresyl blue (BCB) staining on the subsequent embryo development of in vitro-matured/in vitro-fertilized bovine embryos

257 DEVELOPMENT OF HEAT-STRESSED OVA MATURED IN THE PRESENCE OF A PROSTAGLANDIN F2ALPHA RECEPTOR ANTAGONIST

2009 ◽  
Vol 21 (1) ◽  
pp. 226
Author(s):  
A. M. Ward ◽  
F. N. Schrick ◽  
R. R. Payton ◽  
E. Peixoto ◽  
J. L. Edwards
Keyword(s):  
Heat Stress ◽  
Receptor Antagonist ◽  
Fixed Effects ◽  
In Vitro Maturation ◽  
Elevated Temperatures ◽  
Block Design ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Blastocyst Development

Studies in the literature have shown that cumulus–oocyte complexes produce PGF2α, that ova and cumulus cells have PGF2α receptors, and that PGF2α addition to maturing ova, above what would normally be produced, decreases blastocyst development. Because previous studies have shown elevated systemic and tissue levels of PGF2α as a consequence of heat stress, it was hypothesized that detrimental effects of exposing maturing ova to elevated temperatures may be mediated in part through heat-induced increases in PGF2α. To test this hypothesis, cumulus–oocyte complexes were matured at 38.5 or 41.0°C in the presence of a PGF2α receptor antagonist (AL-8810). Preattachment embryo development of AL-8810-treated ova was compared with development of ova matured in media with or without diluent (DMSO added at the same concentration as AL-8810; diluent and developmental controls, respectively), resulting in 6 total treatment combinations. Data were analyzed as a randomized block design (blocking on oocyte collection date) with fixed effects of maturation temperature, AL-8810, and the respective interaction included in the statistical model. In experimental replicates in which the effects of heat stress decreased blastocyst development greater than 10% (n = 14), a significant maturation temperature × AL-8810 interaction was noted when evaluating blastocyst development (P = 0.05). Specifically, when ova were heat stressed during the first 12 h of in vitro maturation, blastocyst development was reduced in developmental and diluent controls (26.2 v. 18.8 and 24.4 v. 19.9, respectively; SEM = 1.6). In contrast, when ova were matured under the same conditions but in the presence of a PGF2α receptor antagonist, the effects of heat stress to reduce blastocyst development after in vitro fertilization were no longer observed (22.5 v. 22.5; SEM = 1.6). When using abattoir-derived ovaries, it is not uncommon to collect, on occasion, ova that are developmentally challenged (i.e. blastocyst development is less than the 20 to 50% expected). In this experiment, this occurred on 5 occasions. Data from these experimental replicates were analyzed and reported separately because previous efforts had shown that the responsiveness of ova to changes in culture environment differs depending on the level of developmental competence. Relevant to this study, addition of AL-8810 to developmentally challenged ova matured under thermoneutral conditions increased cleavage (60.4 v. 55.4%, respectively; P = 0.06) and blastocyst development (17.7 v. 13.7%, respectively; P = 0.07). In summary, data illustrate that developmentally challenged ova, heat-stressed or otherwise, are susceptible to detrimental effects of PGF2α. The ability to increase blastocyst development approaching or exceeding the values expected for competent ova suggests the usefulness of a PGF2α receptor antagonist during in vitro maturation to improve the efficiency of in vitro production procedures.

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