scholarly journals Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes

Reproduction ◽  
2006 ◽  
Vol 131 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Mark G Larman ◽  
Courtney B Sheehan ◽  
David K Gardner
Keyword(s):  
Ethylene Glycol ◽  
Zona Pellucida ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Initial Increase ◽  
Cell Stage ◽  
Free Treatment ◽  
Zona Hardening ◽  
Free Media ◽  
Oocyte Freezing

Despite the success of embryo cyropreservation, routine oocyte freezing has proved elusive with only around 200 children born since the first reported birth in 1986. The reason for the poor efficiency is unclear, but evidence of zona pellucida hardening following oocyte freezing indicates that current protocols affect oocyte physiology. Here we report that two cryoprotectants commonly used in vitrification procedures, dimethyl sulfoxide (DMSO) and ethylene glycol, cause a large transient increase in intracellular calcium concentration in mouse metaphase II (MII) oocytes comparable to the initial increase triggered at fertilization. Removal of extracellular calcium from the medium failed to affect the response exacted by DMSO challenge, but significantly reduced the ethylene glycol-induced calcium increase. These results suggest that the source of the DMSO-induced calcium increase is solely from the internal calcium pool, as opposed to ethylene glycol that causes an influx of calcium across the plasma membrane from the external medium. By carrying out vitrification in calcium-free media, it was found that zona hardening is significantly reduced and subsequent fertilization and development to the two-cell stage significantly increased. Furthermore, such calcium-free treatment appears not to affect the embryo adversely, as shown by development rates to the blastocyst stage and cell number/allocation. Since zona hardening is one of the early activation events normally triggered by the sperm-induced calcium increases observed at fertilization, it is possible that other processes are negatively affected by the calcium rise caused by cryoprotectants used during oocyte freezing, which might explain the current poor efficiency of this technique.

scholarly journals 25 IN VITRO DEVELOPMENT OF AGGREGATED NUCLEAR TRANSFERRED EMBRYOS DERIVED FROM BOVINE CUMULUS CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 162
Author(s):  
S. Akagi ◽  
B. Tsuneishi ◽  
S. Watanabe ◽  
S. Takahashi
Keyword(s):  
Zona Pellucida ◽  
Cumulus Cells ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
In Vitro Development ◽  
Functional Peptide ◽  
Vitro Development

It has been reported that aggregation of two nuclear transfer (NT) mouse embryos shows an improvement in full-term development (Boiani M et al. 2003 EMBO J. 22, 5304–5312). In this study, we examined the effect of aggregation on in vitro development of bovine NT embryos. As donor cells for NT, cumulus cells of passage 3–5 were used following culture in serum-starved medium for 5–7 days. NT was performed as previously described (Akagi S et al. 2003 Mol. Reprod. Dev. 66, 264–272). NT embryos were cultured in a serum-free medium (IVD-101, Research Institute of Functional Peptide Co., Ltd., Shimojo, Yamagat, Japan). Eight-cell-stage embryos on Day 2 or 16- to 32-cell-stage embryos on day 4 were used for embryo aggregation after removal of the zona pellucida. A small depression was made in a 25-μL drop of TCM-199 with 50 μg/mL phytohemagglutinin (TCM199/PHA) or IVD-101 using a darning needle. Two or three NT embryos were placed into the depression in the drop of TCM199/PHA for 20 min. NT aggregates were then moved into the depression in the drop of IVD-101 and cultured until Day 7. In vitro development of NT aggregates was summarized in Table 1. There were no differences in the cell number and ICM ratio of blastocysts between non-aggregated zona-intact and zona-free embryos. All aggregates of three NT embryos developed to the blastocyst stage and the cell number of these blastocysts was significantly higher than that of non-aggregated NT blastocysts. These results indicate that removal of the zona pellucida does not affect the cell number and ICM ratio of blastocysts and that aggregates of three NT embryos can develop to blastocysts with high cell numbers which are equivalent to in vivo-derived embryos (166 ± 11, Knijn HM et al. 2003 Biol. Reprod. 69, 1371–1378). Table 1. Development, cell number, and ICM ratio of NT aggregates

122 GLYCOSAMINOGLYCANS IMPROVE THE NUMBER OF BLASTOMERES IN ZONA-FREE RAT AND MOUSE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 161
Author(s):  
M. Okuyama ◽  
H. Funahashi
Keyword(s):  
Hyaluronic Acid ◽  
Early Development ◽  
Zona Pellucida ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Rat Embryos ◽  
Heparin Sulfate

In general, early mammalian embryos are enclosed by zona pellucida until the blastocyst stage. The possible role of zona pellucida could be to maintain the three-dimensional structure of cleaving embryos, as well as preventing blastomeres from attack by immune cells and miscellaneous germs. However, beneficial roles of the zona on early development of blastomeres are still unknown. The present study was undertaken to examine if the absence of zona pellucida of rat and mouse embryos at the 8-cell stage affected the early development to the blastocyst stage. Furthermore, we examined whether supplementation of culture medium with glycosaminoglycans, such as hyaluronic acid and heparan sulfate, improved the early development or not. In the first experiment, embryos at the 8-cell stage were collected from mated Wistar rats or ICR mice and then, directly or after removing zona pellucida by using acidity Tyrode’s solution, cultured in modified R1ECM or kSOM medium for 24 h in an atmosphere of 5% CO2 in air. Following culture, the incidence of blastocyst formation and the cell number of blastocysts were examined. In the second experiment, intact or zona-free 8-cell embryos were cultured in various concentration of hyaluronic acid (0, 65, 125, 250 μg mL–1) or heparin sulfate (0 or 15 μg mL–1) for 24 h. After culture, blastocyst formation and cell number of blastocyst were similarly examined. Statistical analyses was performed by one-way ANOVA with Bonferroni/Dunn’s post hoc test (significance, P < 0.05). All percentage data were subjected to arc-sine transformation before statistical analysis. Percentage of blastocyst formation and the mean cell number of the blastocyst were less when zona-free 8-cell rat and mouse embryos were cultured (72.1 ± 2.9% and 22.8 ± 0.7), as compared with control intact embryos (94.3 ± 3.5% and 30.6 ± 1.1). Supplementation with hyaluronic acid (250 μg mL–1) improved the blastocyst formation rate of rat embryos (86.4 ± 5.0%) and the cell number of blastocysts (28.8 ± 0.5) to the same level of zona-intact embryos. In mouse embryos, the same concentration of hyaluronic acid improved only the cell number of blastocysts (from 21.7 ± 0.7 to 28.9 ± 0.7) to the same level of zona-intact embryos (30.9 ± 0.7). When heparin sulfate was supplemented, the incidence of blastocyst formation of rat embryos did not improve, but the cell number of the blastocyst (31.1 ± 0.7) did improve to the similar level with zona-intact embryos (30.6 ± 1.1). These results indicate that the zona pellucida has a beneficial effect on the early development of rat and mouse embryos and suggest that glycosaminoglycans, such as hyaluronic acid and heparan sulfate, contribute to the beneficial effect.

282 IMPROVED QUALITY OF PORCINE BLASTOCYSTS BY AGGREGATION OF EMBRYOS AT THE 4 - 8-CELL STAGE

2006 ◽  
Vol 18 (2) ◽  
pp. 248
Author(s):  
S.-G. Lee ◽  
C.-H. Park ◽  
D.-H. Choi ◽  
H.-Y. Son ◽  
C.-K. Lee
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Transgenic Animals ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Vitro Fertilization

Use of blastocysts produced in vitro would be an efficient way to generate embryonic stem (ES) cells for the production of transgenic animals and the study of developmental gene regulation. In pigs, the morphology and cell number of in vitro-produced blastocysts are inferior to these parameters in their in vivo counterparts. Therefore, establishment of ES cells from blastocysts produced in vitro might be hindered by poor embryo quality. The objective of this study was to increase the cell number of blastocysts derived by aggregating 4–8-cell stage porcine embryos produced in vitro. Cumulus–oocyte complexes were collected from prepubertal gilt ovaries, and matured in vitro. Embryos at the 4–8-cell stage were produced by culturing embryos for two days after in vitro fertilization (IVF). After removal of the zona pellucida with acid Tyrode’s solution, one (1X), two (2X), and three (3X) 4–8-cell stage embryos were aggregated by co-culturing them in aggregation plates followed by culturing to the blastocyst stage. After 7 days, the developmental ability and the number of cells in aggregated embryos were determined by staining with Hoechst 33342 and propidium iodide. The percentage of blastocysts was higher in both 2X and 3X aggregated embryos compared to that of 1X and that of intact controls (Table 1). The cell number of blastocysts also increased in aggregated embryos compared to that of non-aggregated (1X) embryos and controls. This result suggests that aggregation might improve the quality of in vitro-fertilized porcine blastocysts by increasing cell numbers, thus becoming a useful resource for isolation and establishment of porcine ES cells. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the pluripotent cell population by staining for Oct-4 and to apply improved aggregation methods in nuclear-transferred (NT) porcine embryos. Table 1. Development, cell number, and ICM ratio of aggregated porcine embryos

Developmental pattern of hexaploid mouse embryos produced by blastomere fusion of diploid and tetraploid embryos at the 2-cell stage

Zygote ◽  
2009 ◽  
Vol 17 (2) ◽  
pp. 125-130 ◽  
Author(s):  
Lei Lei ◽  
Na Guan ◽  
Yan-Ning Xu ◽  
Qing-Hua Zhang ◽  
Jing-Ling Shen ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Karyotype Analysis ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Lower Number ◽  
Mouse Embryos ◽  
Developmental Pattern ◽  
Cell Stage ◽  
Positive Expression

SummaryPolyploid mouse embryos are important models for understanding the mechanisms of cleavage and preimplantation development in mammals. In this study, hexaploid (6n) mouse embryos were produced by the electrofusion of blastomeres from diploid (2n) and tetraploid (4n) embryos at the 2-cell stage. Furthermore, the developmental pattern of hexaploid embryos was evaluated by blastocyst rate, cell number, karyotype analysis, cytoskeleton staining and Oct-4 immunofluorescence. The results showed that 72.7% of the hexaploid embryos were able to develop to the blastocyst stage, which is a lower number than that found with normal diploid embryos (98.0%, p < 0.05). The cell number in hexaploid blastocyst was 12.3 ± 2.0, which was less than that found in diploid or tetraploid blastocysts (41.2 ± 7.2; 18.4 ± 3.5). Karyotype analysis confirmed that the number of chromosomes in hexaploid embryos was 120. β-Tubulin and Oct-4 immunofluorescence indicated that the hexaploid blastocysts were nearly lacking inner cell mass (ICM), but some blastomeres did show Oct-4-positive expression.

scholarly journals Efficiency of Dimethyl Sulphoxide and Ethylene Glycol on Subsequent Development of Vitrified Awassi Sheep Embryos

2020 ◽  
Vol 25 (2) ◽  
pp. 60
Author(s):  
Omar R Mardenli ◽  
MS Mohammad ◽  
HA Hassooni
Keyword(s):  
Ethylene Glycol ◽  
Large Range ◽  
Survival Rates ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Dimethyl Sulphoxide ◽  
Three Solutions ◽  
Cell Stage ◽  
Awassi Sheep

The use of cryoprotectants in vitrification would reduce the critical damages to the embryos, thus increase the survival rates. This research was conducted in the laboratory of reproductive biotechnology at the faculty of Agriculture of Aleppo University.  The study aimed to evaluate the viability and survivability of early Syrian Awassi embryos under the influence of dimethyl sulphoxide (DMSO) and ethylene glycol (EG) following vitrification. Embryos were vitrified in three solutions of cryoprotectants (A: DMSO (3 ml), B: EG (3 ml), and C which was composed of a combination of DMSO (1.5 ml) and EG (1.5 ml)). After thawing, embryos that had been vitrified in C solution achieved the highest rates of cleavage (P&lt; 0.01) comparing with A and B solutions for 2-16 cell stage (50.00% Vs 30.77% and 36.36%), morula (9.00% Vs 44.44% and 40.00%) and blastocyst stage embryos (92.86% Vs 58.33% and 50.00%) respectively. Down to the hatching blastocyst stage, 2-16 cell stage vitrified embryos in C solution achieved an encouraging rate comparing with A and B solutions (39.20% Vs23.08% and 22.73% respectively). The rates of arrested embryos decreased significantly (P&lt; 0.05) after thawing across the three solutions especially the morula and blastocyst stage (0.00 and 3.70% respectively) (C solution). No significant differences were observed in the three types of embryos across all stages and solutions despite the large range among these rates. Given the apparent benefit of the participatory effect of cytoprotectants, it is advised to use a mixture of DMSO and EG (1:1) in vitrification of ovine embryos.

scholarly journals 152EFFECT OF REPLACEMENT OF PYRUVATE/LACTATE IN CULTURE MEDIUM WITH GLUCOSE ON PREIMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 198
Author(s):  
N.W.K. Karja ◽  
S. Medvedev ◽  
D. Fuchimoto ◽  
A. Onishi ◽  
M. Iwamoto ◽  
...  
Keyword(s):  
Energy Source ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Culture Period ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Energy Substrates ◽  
Anova Test

Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) reported that replacement of pyruvate and lactate with glucose, as energy substrates, at 48h of culture in IVC medium enhanced the quality of IVP porcine blastocysts. However, the exact time during early cleavage stages when the utilization of glucose as an energy source is optimal has not yet been determined. The purpose of this study was to examine the effects of glucose supplementation at different times of culture on the developmental competence of IVP porcine embryos. Porcine cumulus-oocytes complexes were matured in modified NCSU-37 solution and fertilized in vitro according to Kikuchi et al. All cultures were performed at 38.5°C, 5% O2, 5% CO2, and 90% N2. In experiment 1, after being fertilized (Day 0), putative zygotes (1158 in 6 trials) were cultured in NCSU-37 supplemented with 0.4% BSA, 0.17mM sodium pyruvate, and 2.73mM sodium lactate (IVC-pyr/lac). Embryos (30–50 in each group) were then transferred into NCSU-37 supplemented with 0.4% BSA and 5.55mM D-glucose (IVC-glu) at 24, 48, 72, 96, or 118h of culture. As control groups, putative zygotes (391) were cultured in IVC-pyr/lac or IVC-glu for the whole culture period. In experiment 2, after being fertilized, putative zygotes (543 in 4 trials, 30–50 in each group) were cultured in IVC-pyr/lac, and then were transferred into IVC-glu at 48h, 53h, 58h, or 63h of culture, because glycolytic activity of in vitro-derived porcine embryos was reported to increase around the 8-cell stage, and some embryos develop to that stage before 72h of culture in experiment 1. All embryos were cultured for 6 days, and then development to the blastocyst stage and number of cells per blastocyst were assessed. When IVF embryos were cultured in IVC pyr/lac for 24h or 48h and subsequently in IVC-glu until day 6 in experiment 1, the rates of blastocyst formation were significantly higher (P&lt;0.05, ANOVA test) than those of embryos cultured in IVC-pyr/lac for the whole culture period (24.4% and 23.0% v. 14.5%, respectively). However, when IVC pyr/lac was replaced with IVC-glu, there were no significant differences between the energy source replacement groups and the glucose-only group in terms of the proportions of cleavage, development to the blastocyst stage and mean cell number per blastocyst (P&gt;0.05, ANOVA test) (15.2%–24.4%, and 16.8%, respectively). Replacement of pyruvate and lactate with glucose at 58h of culture in experiment 2 significantly enhanced the rate of blastocyst formation (P&lt;0.05, ANOVA test) but not the mean cell number compared with zygotes in which the replacement was done at 48, 53, and 63h of culture (31.3% v. 20.6%, 20.8%, and 21.1%, respectively) (P&lt;0.05, ANOVA test). In conclusion, replacement of pyruvate and lactate with glucose as energy substrates was optimal at 58h of culture for the in vitro development of pig embryos to the blastocyst stage.

81 HIGH THROUGHPUT VITRIFICATION OF IN VIVO FERTILIZED AND IN VITRO CULTURED PORCINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 146
Author(s):  
C. N. Murphy ◽  
L. D. Spate ◽  
B. K. Bauer ◽  
R. S. Prather
Keyword(s):  
Ethylene Glycol ◽  
Uv Light ◽  
Cell Number ◽  
Total Cell ◽  
Swine Industry ◽  
Total Cell Number ◽  
Cell Stage ◽  
High Osmolarity

One barrier to successfully making embryo transfer viable in the swine industry is an inability to consistently cryopreserve oocytes and embryos. This process is made difficult by the high lipid content of porcine oocytes and embryos. The objective of this study was to test the in vivo fertilized embryo’s sensitivity to vitrification. Gilts were inseminated on the first day of standing oestrus (Day 0) and then again 12 h later. On Day 2 the oviducts and tip of the uterine horns were flushed with PVA-treated TL-HEPES and 2-cell stage embryos were collected and placed into PVA-treated TL-HEPES and centrifuged at 17 000 × g. The treatment groups were 1) 300 mOsmo centrifuged for 6 min, 2) 500 mOsmo centrifuged for 6 min, 3) 500 mOsmo centrifuged for 12 min, and 4) 500 mOsmo centrifuged for 18 min. After centrifugation the embryos were transferred to Porcine Zygote Medium 3 (PZM3) and cultured to Day 6 or 7 at which point blastocysts were vitrified using 10% DMSO, 10% ethylene glycol in M199 supplemented with 20% FBS (holding medium) for 2 min. Embryos were transferred to holding media with 20% DMSO and 20% ethylene glycol and drawn into an open pulled straw via capillary reaction; it was then submerged into LN2. Embryos were thawed using a step down concentration of 0.33 mM and then 0.2 mM sucrose in holding media each for 6–7 min and then were moved to holding medium alone for 6 to 7 min. The embryos were washed in PZM3, then transferred to 500 μL of PZM3 and cultured for 18 h. Re-expanded embryos were observed, and the nuclei of all embryos were stained with Biz-benzimide and visualised with UV light to determine total cell number. After the embryos were centrifuged and cultured, there was no difference in development to blastocyst (SAS Institute, Cary, NC, USA; Proc GLM) with a mean percentage blastocyst of 85.1% and an N of 54, 51, 53, and 51, respectively, for each treatment. After thawing, percentage of embryos re-expanded was 23.5a, 26.4a,b, 43.2a,b, and 45.6b, respectively. Data was analysed using a PROC GLM in SAS (P < 0.05), with 37, 43, 30, and 36 embryos in each group, respectively. No difference in total cell number across treatments was detected after analysis using PROC GLM in SAS (P < 0.05) with a mean cell number of 29.0. These data suggest that in vivo matured and fertilized blastocysts can survive high osmolarity treatment, centrifugation, and vitrification. The data also show that a high osmolarity treatment centrifuged for 18 min leads to a greater number of re-expanded embryos post-thaw, which may be attributed to better separation of the lipid. Funded by the NIH NCRR R21RR025879 and Food for the 21st Century.

299. Sensitivity of embryos to an environmental stressor, ammonium, is dependent on stage of temporal exposure

2005 ◽  
Vol 17 (9) ◽  
pp. 127
Author(s):  
D. L. Zander ◽  
J. G. Thompson ◽  
M. Lane
Keyword(s):  
Gene Expression ◽  
Glucose Metabolism ◽  
Glucose Uptake ◽  
Early Stage ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Stages Of Development ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Stage Embryo

Extended embryo culture in vitro may cause increased cellular perturbations resulting in poorer developmental outcomes. Exposure of embryos to ammonium throughout the entire pre-implantation period decreased cell number and ICM development, increased apoptosis and perturbs glucose metabolism. The aim of this study was to examine the relative susceptibility of the pre- and post-compaction stage embryo to these perturbations resulting from temporal exposure to ammonium. Mouse embryos (n = 350 per treatment) were collected from F1 female mice. Embryos were exposed to either control medium or medium with 300 μM ammonium for the entire culture period. Temporal treatments involved culture with or without ammonium, from the zygote to 2-cell stage, 2-cell to 8-cell stage, or the 8-cell to the blastocyst stage. At the blastocyst stage, ICM development, apoptosis, gene expression and glucose metabolism were assessed. Differences between treatments were determined using generalised linear modelling and LSD post-hoc tests. Exposure to ammonium at any stage did not affect blastocyst development. Exposure to ammonium pre-compaction significantly decreased both blastocyst and ICM cell number while these were unaffected when exposure occurred post-compaction. Levels of apoptosis were significantly increased when exposure to ammonium was continual to the blastocyst stage (6.5% compared to control 2.4%, P < 0.05) or from the zygote to the 2-cell stage (5.8%, P < 0.05). However, apoptosis was not altered during post-compaction exposure (2.8%). Glucose uptake was decreased by culture with ammonium at all stages of development (P < 0.001). Gene expression of GLUT1 in the blastocyst was not altered by ammonium while GLUT3 expression was significantly reduced by exposure at all stages of development (P < 0.01). The data presented suggests that the pre-compaction stage embryo is most susceptible to ammonium stress and the effects of this early stage exposure appear irreversible. Intriguingly, glucose uptake and GLUT3 expression at the blastocyst stage appear to be markers of ammonium exposure.

scholarly journals Development of Preimplantation Mouse Embryos in vivo and in vitro

1982 ◽  
Vol 35 (2) ◽  
pp. 187 ◽  
Author(s):  
GM Harlow ◽  
P Quinn
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cell Stage ◽  
Preimplantation Mouse ◽  
Development In Vitro

The culture conditions for the development in vitro of (C57BL/6 X CBA) F2 hybrid two-cell embryos to the blastocyst stage have been optimized. Commercially available pre-sterile disposable plastic culture dishes supported more reliable development than re-usable washed glass tubes. The presence of an oil layer reduced the variability in development. An average of 85 % of blastocysts developed from hybrid two-cell embryos cultured in drops of Whitten's medium under oil in plastic culture dishes in an atmosphere of 5% O2 : 5% CO2 : 90% N2 ? The time taken for the total cell number to double in embryos developing in vivo was 10 h, and in cultured embryos 17 h. Embryos cultured in vitro from the two-cell stage to blastocyst stage were retarded by 18-24 h in comparison with those remaining in vivo. Day-4 blastocysts in vivo contained 25-70 cells (mean 50) with 7-28 (mean 16) of these in the inner cell mass. Cultured blastocysts contained 19-73 cells (mean 44) with 8-34 (mean 19) of these in the inner cell mass. In the uterine environment, inner-cell-mass blastomeres divided at a faster rate than trophectoderm blastomeres and it is suggested that a long cell cycle is associated with terminal differentiation. Although cultured blastocysts and inner cell masses contained the same number of cells as blastocysts and inner cell masses in vivo, the rate of cell division in cultured inner cell masses was markedly reduced.

scholarly journals Controlled spermatozoa–oocyte interaction improves embryo quality in sheep

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Debora Agata Anzalone ◽  
Luca Palazzese ◽  
Marta Czernik ◽  
Annalaura Sabatucci ◽  
Luca Valbonetti ◽  
...  
Keyword(s):  
Embryo Quality ◽  
Time Frame ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Free Oxygen Radicals ◽  
High Concentration ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Set Up

AbstractThe current protocols of in vitro fertilization and culture in sheep rely on paradigms established more than 25 years ago, where Metaphase II oocytes are co-incubated with capacitated spermatozoa overnight. While this approach maximizes the number of fertilized oocytes, on the other side it exposes them to high concentration of reactive oxygen species (ROS) generated by active and degenerating spermatozoa, and positively correlates with polyspermy. Here we set up to precisely define the time frame during which spermatozoa effectively penetrates and fertilizes the oocyte, in order to drastically reduce spermatozoa-oocyte interaction. To do that, in vitro matured sheep oocytes co-incubated with spermatozoa in IVF medium were sampled every 30 min (start of incubation time 0) to verify the presence of a fertilizing spermatozoon. Having defined the fertilization time frame (4 h, data from 105 oocytes), we next compared the standard IVF procedures overnight (about 16 h spermatozoa/oocyte exposure, group o/nIVF) with a short one (4 h, group shIVF). A lower polyspermic fertilization (> 2PN) was detected in shIVF (6.5%) compared to o/nIVF (17.8%), P < 0.05. The o/nIVF group resulted in a significantly lower 2-cell stage embryos, than shIVF [34.6% (81/234) vs 50.6% (122/241) respectively, P < 0.001]. Likewise, the development to blastocyst stage confirmed a better quality [29% (70/241) vs 23.5% (55/234), shIVF vs o/nIVF respectively] and an increased Total Cell Number (TCN) in shIVF embryos, compared with o/n ones. The data on ROS have confirmed that its generation is IVF time-dependent, with high levels in the o/nIVF group. Overall, the data suggest that a shorter oocyte-spermatozoa incubation results in an improved embryo production and a better embryo quality, very likely as a consequence of a shorter exposure to the free oxygen radicals and the ensuing oxidative stress imposed by overnight culture.

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