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.

62 EXPOSURE TO ETHYLENE GLYCOL AND DIMETHYL SULFOXIDE CAUSES ACTIVATION AND SPINDLE ANOMALIES IN BUFFALO (BUBALUS BUBALIS) OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 131 ◽  
Author(s):  
M. De Blasi ◽  
E. Mariotti ◽  
M. Rubessa ◽  
S. Di Francesco ◽  
G. Campanile ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Embryo Development ◽  
Sucrose Solution ◽  
Bubalus Bubalis ◽  
Meiotic Spindle ◽  
Blastocyst Development ◽  
Chi Square ◽  
Chi Square Test ◽  
Simple Exposure

Despite the increasing interest, buffalo oocyte cryopreservation is still inefficient, especially in terms of blastocyst development after IVF. The aim of this work was to evaluate chromatin and spindle organization of buffalo in vitro-matured oocytes after vitrification/warming by cryotop and after their simple exposure to cryoprotectants (CP). An overall amount of 251 COC was selected and matured in vitro. In the vitrification group, COC were first exposed to 10% ethylene glycol (EG) + 10% DMSO for 3 min, and then to 20% EG + 20% of DMSO and 0.5 m sucrose, loaded on cryotops, and plunged into liquid nitrogen within 25 s. Oocytes were warmed into a 1.25 m sucrose solution for 1 min and then to decreasing concentrations of sucrose (0.625 m, 0.42 m, and 0.31 m) for 30s each. In order to test CP toxicity, COC were simply exposed to the vitrification and warming solutions. Two hours after warming, oocytes were fixed and immunostained for microtubules using a method previously described (Messinger SM and Albertini DF 1991 J. Cell Sci. 100, 289–298), stained for nuclei with Hoechst, and examined by fluorescence microscopy. Fresh in vitro-matured oocytes were fixed and stained as controls. Data were analyzed by chi-square test; results are shown in Table 1. The percentages of MII oocytes in the control and vitrification groups were greater than in the toxicity group, in which a greater percentage of telophase II stage oocytes were found compared with both the control and vitrification groups, indicating occurrence of activation. Of the MII oocytes, both exposure to CP and vitrification procedures gave greater percentages of oocytes with abnormal spindle and abnormal chromatin configuration compared with the control. An unexpected datum was the evidence of a significant percentage of spontaneously activated oocytes in the toxicity group. We speculate that the lack of activation in the vitrification group may be related to the slowing down of metabolic activity subsequent to thermal shock, and hence, that activation after vitrification may occur later than 2 h post-warming. In conclusion, the simple exposure to CP causes activation of the COC and damage to the cytoskeleton similar to that induced by the whole vitrification protocol. The damages to the meiotic spindle and DNA fragmentation may lead to aneuploidy incompatible with subsequent embryo development and account for the poor embryo development currently recorded in buffalo. Table 1.Chromatin and spindle organization in oocytes vitrified and exposed to cryoprotectants

14 EQUINE CLONING AND EMBRYO AGGREGATION: EFFECT OF BOVINE, PORCINE, FELINE AND EQUINE OOPLAST

2012 ◽  
Vol 24 (1) ◽  
pp. 118
Author(s):  
A. Gambini ◽  
J. Jarazo ◽  
A. De Stefano ◽  
F. Karlanian ◽  
D. Salamone
Keyword(s):  
Embryo Development ◽  
Metaphase Plate ◽  
Donor Cell ◽  
Development Stage ◽  
Blastocyst Stage ◽  
Chi Square ◽  
Cell Stage ◽  
Aggregation Effect ◽  
Group I

The low number of horse slaughterhouses is one of the reasons for the limited availability of horse oocytes for research in cloning. The aim of our study was to assess the capability of equine, bovine, porcine, or feline ooplast to produce cloned embryos when equine cells are used as donor nuclei and to evaluate if embryo aggregation improves their development. Oocytes from mentioned species were collected from ovaries derived from slaughterhouses, except for cat ovaries that were obtained from ovariectomized queens. Oocytes were matured in TCM199 supplemented following standard protocols for each species. After maturation, cumulus and zona pellucida were removed. Enucleation was performed by aspiration of the metaphase plate under ultraviolet light. Donor cell and ooplast were attached by phytohemagglutinin treatment and then electrofused. Activation protocols were ionomycin for 4 min, except for porcine, which were electrically activated, followed by culture in 1.9 mM 6-DMAP for bovine, feline and porcine, except for equine: 1 mM 6-DMAP with 5 mg mL–1 of cycloheximide. Reconstructed embryos (RE) were cultured in SOF in the well of well system in 2 different groups: only one RE per well (1X) and three RE per well (3X, aggregated embryos, AE). Blastocysts derived from homospecific clones were transferred to synchronized mares. Cleavage and maximum development stage achieved of all experimental groups were assessed. In vitro development was compared using the chi-square test. In group 1X, a total of 64, 49, 38 and 145 RE were performed for porcine, bovine, feline and equine, respectively and in group 3X, 88, 48, 48 and 195 RE. Cleavage of cloned embryos ranged from 67 to 87%. Aggregated of homospecific equine clones showed the highest blastocyst rates (1X: 5.5%, 3X: 34%) and after embryo transfer (4 recipients for each group), an ongoing pregnancy (day 300, at the time of submission) was only achieved with aggregated embryo confirming the positive effect of embryo aggregation in these clones. The stages with higher developmental arrest of heterospecific nonaggregated embryos were 2 to 4 cells for porcine ooplast (23/64, 36%) and 4 to 8 cells for bovine and feline ooplast (37/49, 75% and 18/38, 47%, respectively). Blastocyst stage was only reached using feline ooplast (group I: 2/38, 5.26% and group II: 2/16, 12.5%). Heterospecific aggregated clones were able to achieve 16-cell stage, showing statistic differences compared with group 1X. As we reported previously, embryo aggregation shows benefits for homospecific equine clones, although more studies are needed to clarify if aggregation of heterospecific clones has the same effect. All heterospecific ooplasm was able to support embryo development. The stage of major developmental arrests was similar to embryonic genomic activation stage. Our results suggest that cat oocyte seems to be the best receptor to support equine cloned embryo development.

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.

332 EFFECT OF DIFFERENT PIG OOCYTE ACTIVATION PROTOCOLS ON EMBRYO DEVELOPMENT IN SOF AND NCSU-23

2007 ◽  
Vol 19 (1) ◽  
pp. 281 ◽  
Author(s):  
I. Lagutina ◽  
G. Lazzari ◽  
C. Galli
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Cell Number ◽  
Additional Treatment ◽  
Oocyte Activation ◽  
Blastocyst Development ◽  
Chi Square ◽  
Parthenogenetic Embryo ◽  
Pig Oocyte

Several factors affect nuclear transfer success. These include efficient parthenogenetic activation and embryo culture medium that should efficiently support pre-implantation development of good quality blastocysts. We investigated pig oocyte activation and embryo development in SOFaa in response to ionomycin (Io = 5 µM Io for 4 min; Io° = 15 µM Io for 20 min) and electric impulse (EL; one 30-µs pulse of DC 1.5 kV cm−1 in the presence of 50 µM Ca) in combination with 2 mM 6-DMAP or 10 µg mL−1 cycloheximide (CHX) +5 µg mL−1 cytochalasin B (CB) for 4 h. In addition, we studied the effect of elevated (1 mM) (Cheong et al. 2002 Mol. Reprod. Dev. 61, 488) in comparison with 50 µM Ca during EL activation on embryo development in SOFaa and NCSUaa-23. Porcine oocytes were recovered from slaughtered donors and matured in vitro for 44 h in DMEM-F12 supplemented with 10% FCS, 0.05 IU LH and FSH (Menogon®, Ferring, Milan, Italy), 0.3 mM cystine, 0.5 mM cysteamine, 50 ng mL−1 long-EGF, 100 ng mL−1 long-IGF1, 5 ng mL−1 bFGF (Sigma-Aldrich, Milan, Italy) in 5% CO2 at 38.5°C. The rates of cleavage, blastocyst formation (BL) and BL cell number on Day 7 (BL-D7) were recorded. All experiments were done with 3 replicates. The data were compared by chi-square test. There was no difference in the ability of Io (all groups) and EL + CB activated oocytes to cleave, whereas the additional treatment of EL-activated oocytes with DMAP and CHX + CB significantly increased cleavage. Io activation resulted in poor blastocyst development in comparison with all EL-activated groups (see Table 1). When calcium levels were elevated during EL activation, significantly more embryos developed in SOFaa (35.6%, n = 191 vs. 26%, n = 192; P &lt; 0.05), but no differences were observed with culture in NCSUaa-23 (about 56%). The BL rate was significantly higher in NCSUaa-23 vs. SOFaa (55.9%, n = 68 vs. 34.8%, n = 69, respectively); however, the BL total cell number was significantly higher in SOFaa (58 ± 18, n = 40 vs. 86 ± 35, n = 56, respectively; P &lt; 0.05). In conclusion, we have found that SOFaa and NCSUaa-23 differ in ability to support pig parthenogenetic embryo development. EL activation combined with elevated Ca significantly increased the embryo developmental capacity in SOFaa but not in NCSUaa-23. NCSUaa-23 was more efficient for embryo culture, whereas SOF produced BLs of higher quality. Table 1.Effect of activation protocol on the development of pig parthenogenetic embryos in SOFaa This work was supported by grants ISS-CS11 and Fondazione Cariplo.

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

scholarly journals In Vitro Maturation with Leukemia Inhibitory Factor Prior to the Vitrification of Bovine Oocytes Improves Their Embryo Developmental Potential and Gene Expression in Oocytes and Embryos

2020 ◽  
Vol 21 (19) ◽  
pp. 7067
Author(s):  
Meritxell Vendrell-Flotats ◽  
Tania García-Martínez ◽  
Iris Martínez-Rodero ◽  
Manel Lopez-Bejar ◽  
Jonathan LaMarre ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
Embryo Development ◽  
Leukemia Inhibitory Factor ◽  
In Vitro Maturation ◽  
Expression Patterns ◽  
Inhibitory Factor ◽  
Blastocyst Development ◽  
Developmental Potential

Oocyte cryopreservation has a significant impact on subsequent embryonic development. Herein, we investigated whether supplementing in vitro maturation medium with Leukemia Inhibitory Factor (LIF) prior to vitrification affects embryo development and gene expression at different embryo developmental stages. A panel of genes including maternal effect, epigenetics, apoptosis and heat stress was relatively quantified. The results show reduced cleavage rates after vitrification, regardless of the LIF treatment. Although not statistically different from control-vitrified oocytes, oocyte apoptosis and the blastocyst yield of LIF-vitrified oocytes were similar to their non-vitrified counterparts. Vitrification increased oocyte ZAR1, NPM2 and DPPA3 gene expression while its expression decreased in LIF-vitrified oocytes to similar or close levels to those of non-vitrified oocytes. With a few gene-specific exceptions, vitrification significantly increased the expression of DNMT3A, HDAC1, KAT2A, BAX and BCL2L1 in oocytes and most stages of embryo development, while comparable expression patterns for these genes were observed between LIF-vitrified and non-vitrified groups. Vitrification increased HSPA1A expression in oocytes and HSP90AA1 in 2-cell embryos. Our data suggest that vitrification triggers stage-specific changes in gene expression throughout embryonic development. However, the inclusion of LIF in the IVM medium prior to vitrification stimulates blastocyst development and several other developmental parameters and induces oocytes and embryos to demonstrate gene expression patterns similar to those derived from non-vitrified oocytes.

119 LEPTIN ENHANCED THE DEVELOPMENT OF BUFFALO (BUBALUS BUBALIS) EMBRYO CULTURED IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 159 ◽  
Author(s):  
Y. Q. Lu ◽  
D. N. Ye ◽  
M. Zhang ◽  
S. S. Lu ◽  
K. H. Lu
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Bubalus Bubalis ◽  
Development Rate ◽  
Blastocyst Development ◽  
Mediterranean Countries ◽  
Technical Factors ◽  
Development In Vitro

Buffalo is an important livestock resource in many Asian and Mediterranean countries. In vitro embryo production (IVEP) and transfer of the embryos to produce calves with high genetic merit would be of great interest in buffalo species. The efficiency of the IVEP in buffalo is low compared to that in bovine. It may be due to the reproductive physiology of buffalo or the technical factors in IVEP procedures. Recent research revealed that supplementation of leptin in the in vitro culture (IVC) medium could significantly increase embryo development (2005 Mol. Cell Endocrinol. 229, 141–147; 2006 Reproduction 132, 247–256). In this study, the effect of leptin on buffalo embryo development in vitro was assessed by supplementation of the leptin into the IVC medium. Methods: Buffalo oocytes were aspirated form 2 to 6 mm follicles from slaughterhouse ovaries and washed in TCM199 and once more with in vitro maturation (IVM) medium (TCM199, 5% ECS, 15 μg mL–1 FSH). Oocytes with compact cumulus cells were matured in IVM medium at 38.5°C, 5% CO2 for 22–22 h. The frozen–thawed buffalo sperm underwent a centrifugation in Percoll gradient to remove the dead sperm. Ten to 15 matured oocytes were added to a drop of 40 μL modified Tyrode’s medium supplemented with 0.6% BSA, 2.0 mm caffeine and 20 μg mL–1 Heparin. Concentration of sperm added into the fertilization medium was 1 to 2 million per mL. Eight to 10 h after insemination, the presumptive zygotes were transferred to IVC medium (TCM199, 10% newborn cow serum) supplemented with 0 ng mL–1 (control), 10 ng mL–1, 100 ng mL–1 or 500 ng mL–1 of leptin. Cleavage and blastocyst development rate was recorded on Day 2 and Day 6 to 8 after insemination. The experiment was repeated 10 times, and a total of 831 oocytes were used with the IVF procedures. The results revealed that the cleavage rates in the group of 0 ng mL–1, 10 ng mL–1, 100 ng mL–1 and 500 ng mL–1 of leptin were 50.1 ± 3.5%, 55.0 ± 1.3%, 50.0 ± 1.8% and 52.9 ± 2.2%, respectively. No statistical difference was observed regarding cleavage rates between treatments (P > 0.05). The percentage of oocytes developing to blastocysts in the group of 10 ng mL–1 and 100 ng mL–1 leptin were 26.1 ± 1.5% and 23.5 ± 1.2%, respectively, significantly higher than that of 17.5 ± 2.1% in the control (P < 0.05). The blastocyst development rate in the group of 500 ng mL–1 leptin was 20.9 ± 1.4%, less than that of 10 ng mL–1 (P < 0.05). In conclusion, the results of this study indicated that supplementation of leptin in the IVC medium could enhance the blastocyst development in buffalo species and the optimal concentration of leptin in the present procedures was 10 ng mL–1. This work was jointly supported by National Science and Technology Supporting Program (No. 2006BAD04A18), Guangxi Science Foundation (0832012) and Guangxi University Key Research Program (No. 2005ZD05).

Use of a novel polydimethylsiloxane well insert to successfully mature, culture and identify single porcine oocytes and embryos

2014 ◽  
Vol 26 (3) ◽  
pp. 375 ◽  
Author(s):  
Ye Yuan ◽  
Melissa Paczkowski ◽  
Matthew B. Wheeler ◽  
Rebecca L. Krisher
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
In Vitro Maturation ◽  
Transcript Abundance ◽  
Blastocyst Development ◽  
Developmental Potential

The objective of this study was to evaluate the efficacy of a novel polydimethylsiloxane (PDMS) well-insert system for oocyte in vitro maturation (IVM) and in vitro embryo culture (IVC) in pigs. The PDMS well inserts, consisting of multiple microwells with connecting microchannels, resulted in equivalent blastocyst development compared with standard microdrop culture for IVC. These PDMS well inserts were then evaluated for IVM or IVC in a rocking versus static environment. The rocking environment during both oocyte IVM and embryo culture had detrimental effects on oocyte and embryo development compared with a static environment. Importantly, blastocyst development of oocytes and embryos cultured in the PDMS well inserts in the static environment was equivalent to that of standard microdrops. Further analysis of transcript abundance in blastocysts produced from these different environments revealed that the PDMS well-insert system may produce more viable embryos. In conclusion, this PDMS well-insert system can successfully mature oocytes and culture embryos in an individually-identifiable manner without compromising, and perhaps enhancing, developmental potential.

scholarly journals 291 EFFECT OF DIFFERENT TRANSPORT TEMPERATURES (+4°C, +32°C) ON IN VITRO MATURATION OF OOCYTES COLLECTED FROM CATTLE AND SHEEP OVARIES

2005 ◽  
Vol 17 (2) ◽  
pp. 296
Author(s):  
O.B. Ozdas ◽  
M. Tas ◽  
U. Cirit ◽  
M. Evecen ◽  
K. Demir ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Developmental Stages ◽  
Chi Square ◽  
Medium Temperature ◽  
Factors Affecting ◽  
Group I ◽  
Significant Difference ◽  
Temperature Group ◽  
C 32

At present, blastocyst rates in embryos obtained from in vitro maturation of oocytes, and their fertilization and culture, is still not at the desired level. One of the most important problems encountered in in vitro culture studies is seen in the maturation period of oocytes until they reach the fertilizable level. Transport time of the ovaries and, in particular, temperature of the transport medium used are among the factors affecting complete maturation. The aim of this study was to determine the effects of different transport temperatures (4°C, 32°C) of sheep and cattle ovaries on the in vitro maturation of oocytes. Two experimental groups were formed in the study. Sheep and cattle ovaries were put into saline solution at 32°C. The ovaries were transported at the same temperature (Group I) or at 4°C following a 10-min incubation at room temperature (Group II), in 2–4 h to the laboratory (n = 6). For each group, oocytes were collected from ovaries using the dissection method and selected oocytes were matured in their own group in 700 μL TCM-199 (supplemented with pyruvate, LH, FCS) for 23 h at a gas atmosphere of 5% CO2, 5% O2, 90% N2 and at 38.8°C. At the end of maturation, oocytes were cleansed from their cumulus oophorus cells and fixed in acetic acid-ethyl alcohol (1:3) for 48 h. The developmental stages until MII of oocytes stained with aceto-orcein were then examined under the phase contrast microscope. The chi-square test was used for statistical analysis (Table 1). While oocytes obtained from sheep ovaries transported at +32°C reached the MII stage at a faster rate compared to those at +4°C (P < 0.001), no statistically significant difference was observed between the maturation to the MII stage of oocytes obtained from cattle ovaries transported at +4°C and +32°C. As a result of this study, while it was established that cattle ovaries could be transported at both +4°C and +32°C and that there was no difference in oocyte maturation, a medium temperature of +4°C was determined to be unsuitable for transporting sheep ovaries. Table 1. Stages of development in sheep and cattle oocytes after 23 h of culture This work was supported by Istanbul University.

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