Timing of the First Cleavage Division of the Mouse and the Duration of its Component Stages: A Study of Living and Fixed Eggs

1973 ◽  
Vol 12 (3) ◽  
pp. 799-808
Author(s):  
M. H. KAUFMAN
Keyword(s):  
Close Correlation ◽  
Time Lapse ◽  
Culture Conditions ◽  
Effect Of Temperature ◽  
Time Intervals ◽  
Cleavage Division ◽  
Cell Stage ◽  
Mitotic Time ◽  
Mouse Eggs

Fertilized mouse eggs were examined between 27 and 34 h after the superovulating injection of human chorionic gonadotrophin (HCG). Out of 1334 eggs examined, 432 were already at the 2-cell stage; the remaining 902 at the 1-cell stage were examined in detail. All chromosome preparations of the first cleavage mitosis were classified into groups corresponding with the stages of prometaphase (early and late), metaphase (early or ‘prechromatid’, ‘chromatid’ and ‘late chromatid’) and anaphase. An indirect estimate was made of the duration of the first cleavage mitosis and of its component stages from the incidence of stages observed at different time intervals after the HCG injection. Fertilized eggs were also observed at 37°C by time-lapse cine-photography and the interval between the disappearance of the pronuclei and the beginning of telophase of the first cleavage division was determined. The progress of eggs fertilized in vitro was also observed under normal culture conditions. A close correlation was observed between the indirect method of assessing the mitotic time and the direct values obtained from the studies on time-lapse and in vitro culture. The effect of temperature on the mitotic time was also examined by the time-lapse method.

Timing of the First Cleavage Division of Haploid Mouse Eggs, and the Duration of its Component Stages

1973 ◽  
Vol 13 (2) ◽  
pp. 553-566 ◽  
Author(s):  
M. H. KAUFMAN
Keyword(s):  
Polar Body ◽  
Time Lapse ◽  
Follicle Cells ◽  
Cleavage Division ◽  
Cell Stage ◽  
Cell Embryo ◽  
Second Polar Body ◽  
Polar Body Extrusion ◽  
Mouse Eggs

Mouse eggs were activated by treatment with hyaluronidase which removed the follicle cells, followed by culture in vitro, and examined at the first cleavage mitosis. Second polar body extrusion usually occurred and haploid parthenogenesis was initiated. Air-dried chromosome preparations were made between 11 and 15.5 h after activation. Out of the 308 eggs examined 74 had already progressed to the 2-cell stage; the remaining 234 at the 1-cell stage were examined in detail. All chromosome preparations of the first cleavage mitosis were classified into groups corresponding with the stages of prometaphase, metaphase (early or ‘pre-chromatid’, ‘chromatid’ and ‘late chromatid’) and anaphase. An indirect estimate was made of the duration of the first cleavage mitosis and of its component stages from the incidence of stages observed at different time intervals after activation. Similar eggs were also observed at 37 °C by time-lapse cine-photography and the interval between the disappearance of the pronucleus to the beginning of telophase of the first cleavage division was determined. The results of timing studies on the haploid eggs were compared with results obtained from similar observations on the first cleavage division of fertilized eggs which would of course normally be diploid. Artificially activated eggs with 2 pronuclei, resulting from second polar body suppression, were also examined, and serial chromosome preparations during mitosis showed that the 2 pronuclear chromosome groups unite on the first cleavage spindle and divide to give a hetero-zygous diploid 2-cell embryo.

Expression of the HSP 70.1 gene, a landmark of early zygotic activity in the mouse embryo, is restricted to the first burst of transcription

Development ◽  
1995 ◽  
Vol 121 (1) ◽  
pp. 113-122 ◽  
Author(s):  
E. Christians ◽  
E. Campion ◽  
E.M. Thompson ◽  
J.P. Renard
Keyword(s):  
Dna Replication ◽  
Mouse Embryo ◽  
Culture Conditions ◽  
Hsp 70 ◽  
Cell Stage ◽  
Embryonic Genome ◽  
Genome Activation ◽  
Alpha Amanitin ◽  
Zygotic Genome

Activation of the mouse embryonic genome at the 2-cell stage is characterized by the synthesis of several alpha-amanitin-sensitive polypeptides, some of which belong to the multigenic hsp 70 family. In the present work we show that a member of this family, the HSP 70.1 gene, is highly transcribed at the onset of zygotic genome activation. Transcription of this gene began as early as the 1-cell stage. Expression of the gene continued through the early 2-cell stage but was repressed before the completion of the second round of DNA replication. During this period we observed that the level of transcription was modulated by in vitro culture conditions. The coincidence of repression of HSP70.1 transcription with the second round of DNA replication was not found for other transcription-dependent polypeptides synthesized at the 2-cell stage.

In vitro development of haploid mouse embryos produced by bisection of one-cell fertilized eggs

Development ◽  
1977 ◽  
Vol 38 (1) ◽  
pp. 187-202
Author(s):  
Andrzej K. Tarkowski
Keyword(s):  
X Chromosome ◽  
Culture Conditions ◽  
Extrinsic Factors ◽  
Cell Stage ◽  
Glass Needle ◽  
Androgenetic Embryos ◽  
Haploid Embryos ◽  
Vitro Development ◽  
Further Development

F1(CBA × C57BL'10) mouse eggs originating from spontaneous or induced ovulation and fertilized by CBA-T6T6 or PO spermatozoa were bisected with a glass needle into halves each containing a pronucleus. This technique offers a unique opportunity of producing both androgenetic and gynogenetic haploid embryos from one egg. Out of 600 operated eggs, in 406 (67·7%) both halves survived. During 96 h of culture in vitro the fragments were inspected once daily and finally examined in air-dried preparations. Eighty-seven per cent of halves underwent first cleavage but their further development was to a large extent affected by extrinsic factors connected with experimental procedure (mainly by suboptimal and variable culture conditions) and by the origin of eggs (those from spontaneous ovulation being superior). For this reason developmental capabilities of egg halves were assessed in a selected group of pairs in which at least one partner reached the stage of four or more blastomeres. The observed ratio between pairs with both or only one sister embryo developing successfully suggests that androgenetic embryos carrying Y rather than X chromosome can cleave twice but do not survive beyond 4-cell stage. None of the metaphase plates from older embryos contained a Y chromosome. These observations imply that the X chromosome is genetically active during early cleavage and that a full haploid set is required for preimplantation development to be completed. Formation of blastocysts varied from batch to batch, with an average of 12·8% and maximal incidence of 29·5% . In 34 pairs both fragments developed beyond the 4-cell stage but in only one case did both form blastocysts. Haploid blastocysts were composed of 27 cells on average which was about a half of the number of cells in control diploid zona-free whole eggs. Ten out of 51 embryos with metaphase plates proved to be haploid/diploid mosaics.

Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection

2019 ◽  
Vol 31 (12) ◽  
pp. 1862 ◽  
Author(s):  
N. A. Martino ◽  
G. Marzano ◽  
A. Mastrorocco ◽  
G. M. Lacalandra ◽  
L. Vincenti ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Room Temperature ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Group 13 ◽  
Time Lapse Imaging

Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22–27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.

173 HINDERING OF CLEAVAGE TIMING IN BOVINE PARTHENOTES DURING THE HOT SEASON

2007 ◽  
Vol 19 (1) ◽  
pp. 203 ◽  
Author(s):  
A. Aroyo ◽  
S. Yavin ◽  
Z. Roth ◽  
A. Arav
Keyword(s):  
Thermal Stress ◽  
Embryonic Development ◽  
Elevated Temperatures ◽  
Time Lapse ◽  
Cold Season ◽  
Seasonal Effects ◽  
Cell Stage ◽  
Developmental Potential ◽  
Hot Season

Heat stress is a major contributing factor to low fertility among dairy cattle, as reflected by the dramatic reduction in conception rate during the hot months. The effects of thermal stress on oocyte competence and embryonic development have been well documented. However, timing of embryonic cleavage, which may be considered a parameter for the identification of good-quality embryos, and its association with elevated temperatures have not been studied. Two experiments were performed to examine and characterize seasonal effects (i.e. thermal stress) on cleavage timing of bovine parthenogenetic embryos. Oocytes were aspirated from ovaries collected at the local abattoir in 2 seasons: cold (Dec–Apr) and hot (May–Nov). Matured oocytes were chemically activated (ionomycin followed by 6-DMAP) and cultured in vitro; cleavage timing to the 2- and 4-cell stages was observed and documented. The one-way ANOVA procedure was used for statistical analysis. In the first experiment (n = 5416 oocytes), cleavage was documented at specific time points during development post-activation. The peak in embryonic development to the 2-cell stage was earlier (22 to 27 vs. 27 to 40 h after activation) and the cleavage rate higher (39 vs. 21%; P < 0.0001) during the cold season relative to the hot season, respectively. Similarly, the peak in 4-cell-stage development was also observed earlier (46–52 vs. 52–70 h after activation) and corresponded with a higher proportion of developing embryos (33 vs. 21%; P < 0.0001) during the cold season as compared to the hot season, respectively. These results indicate that embryonic development is delayed and a lower proportion of embryos cleaved during the hot season. To better understand the delay in cleavage timing, a second experiment (n = 308 oocytes) was performed through two consecutive hot seasons. A time-lapse system (EmbryoGuard; IMT, Ltd., Ness-Ziona, Israel) was employed to collect accurate data on the first cleavage division, known to be indicative of embryo quality. The time-lapse system was pre-programmed to take photos at 1-h intervals such that culture dishes did not need to be removed from the incubator. Similar to the pattern noted for the hot season in the first experiment, a wide distribution of cleavage timing (18-40 h after activation) was observed. Further analysis revealed that embryos cleaved in 2 distinct waves: cleavage timing of the first wave (18 to 25 h after activation) was characterized by a time frame similar to that in the cold season, suggesting good-quality embryos; however, the second wave, from 27 to 40 h after activation, presented a delay in cleavage timing, suggesting that these late-cleaving embryos are of inferior quality. Taken together, the results of the 2 experiments lead to the assumption that oocytes harvested from lactating cows during the hot season are of reduced developmental potential, which may be explained, in part, by the pattern of 2 cleavage waves. Furthermore, cleavage timing appears to be a good indicator of embryo potential and may increase the chances of selecting better in vitro-derived embryos during the hot season for embryo transfer.

242 PROMISING TWO-STEP EVALUATION SYSTEM FOR SELECTING HIGH DEVELOPMENTAL COMPETENCE IN VITRO FERTILIZED EMBRYOS IN CATTLE USING WELL-OF-THE-WELL DISH

2015 ◽  
Vol 27 (1) ◽  
pp. 210
Author(s):  
M. Taniai ◽  
M. Takayama ◽  
O. Dochi ◽  
K. Imai
Keyword(s):  
Evaluation System ◽  
Evaluation Method ◽  
Calf Serum ◽  
Time Lapse ◽  
Developmental Competence ◽  
Bovine Embryo ◽  
Chi Square ◽  
Cell Stage ◽  
Chi Square Test

Bovine IVF embryos are evaluated morphologically using light microscopy just before transfer. However, this evaluation method is subjective, and an objective method with more certainty is needed. Sugimura et al. (PLoS ONE 2012 7, e36627) reported a promising system for selecting healthy IVF bovine embryo by using time-lapse cinematography and 5 prognostic factors. This study was to investigate the efficacy of a 2-step evaluation system of IVF embryos using microscopy for selecting high developmental competence IVF embryos. Cumulus-oocyte complexes (COC) were collected by ovarian follicular aspiration (2 to 5 mm diameter) obtained from a local abattoir. The COC (n = 488) were matured in TCM-199 medium supplemented with 5% calf serum (CS) and 0.02 IU mL–1 of FSH at 38.5°C for 20 h in an atmosphere of 5% CO2 (20 COC 100 µL–1 droplets). After 10 h of gametes co-culture (5.0 × 106 sperm cells mL–1), the presumptive zygotes were cultured in 125 µL of CR1 aa medium supplemented with 5% CS in well of-the-well culture dishes (AS ONE, Japan; 25 zygotes well–1) at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 for 9 days. Two-step evaluations of embryos were done at 27 and 55 h post-IVF (hpi). In the first step of evaluation, cleavage patterns at 27 hpi were categorized as mono-cell, 2-cell with even blastomeres and without fragments (normal cleavage), 2-cell with uneven blastomeres, and ≥3 blastomeres. During the second step of evaluation, embryos were classified by their number of blastomeres (2 to 5 cells, 6 to 8 cells, and >8 cells) and the absence or presence of multiple fragments (<20 or >20%) at 55 hpi. The data were analysed by chi-square test. The blastocyst rate (BL%) of embryos cleaved before 27 hpi (56.6%, n = 106) was higher (P < 0.01) than those of embryos cleaved after 27 hpi (37.0%, n = 235). A greater percentage (P < 0.05) of 2-cell embryos with normal cleavage (68.0%, n = 50) developed to blastocysts than from with =3 blastomeres at 27 hpi (40.6%, n = 32). Superior BL% (P < 0.01) was obtained from embryos categorized as 6- to 8-cell stage (58.6%, n = 140) and >8 cell stage (70.6%, n = 25) compared with those embryos at the 2- to 5-cell stage at 55 hpi (26.1%, n = 176). Embryos with no fragments (58.0%, n = 467) had higher BL% (P < 0.01) compared with those with <20% fragments (30.7%, n = 127) and having with >20% fragments (17.5%, n = 25) at 55 hpi. The highest of BL% was observed in embryos showing a normal cleavage to 2-cells with at 27 hpi and having >6 cells with no fragments at 55 hpi (95.2%, n = 21, P < 0.01). These results demonstrate that the 2-step evaluation system at 27 and 55 hpi using microscopy is an effective method for selecting IVF embryos with high developmental competence.

Study of Morphokinetics in Day 3 Embryo with Implantation Potential and Effect of Sperm Cryopreservation on Embryogenesis

2017 ◽  
Vol 8 (2) ◽  
pp. 61-67
Author(s):  
Harsha K Bhadarka ◽  
Nayana H Patel ◽  
Kruti B Patel ◽  
Nilofar R Sodagar ◽  
Yuvraj D Jadeja ◽  
...  
Keyword(s):  
Time Lapse ◽  
Mean Value ◽  
Primary Objective ◽  
Sperm Cryopreservation ◽  
Cell Stage ◽  
Frozen Semen ◽  
Significant Difference ◽  
Cryopreserved Sperm ◽  
Day 3 Embryo

ABSTRACT Aim In recent past, many studies had come up with the combination of time-lapse (TL) imaging of embryo morphokinetics as a noninvasive means for improving embryo selection and in vitro fertilization (IVF) success. The primary objective of the study was to find out if there is significant variation in morphokinetics of embryos with different implantation potential and also to study the effect of sperm freezing on time points of embryogenesis events in embryos with implantation potential. Materials and methods Kinetic data and cycle outcomes were analyzed retrospectively in 142 patients who had undergone IVF/intracytoplasmic sperm injection (ICSI) cycles using semen with normal parameters and embryo transfer (ET) on day 3. For the surety of specificity of morphokinetics, only cases with single ET cycles were included in the study. Timing of specific events, from the point of ICSI, was determined using TL imaging. Kinetic markers like time to syngamy (t-pnf), t2, time to two cells (c), 3c (t3), 4c (t4), 5c (t5), 8c (t8), tMor, CC2, CC3, t5–t2, t5–t4, s1, s2, and s3 were calculated. The cleavage synchronicity from the 2–8 cell stage (CS2–8), from 4 to 8 cell stage (CS4–8), and from 2 to 4 cell stage (CS2–4) were calculated as defined elsewhere. Deoxyribonucleic acid replication time ratio (DR) was also included in the comparison. Analysis of variance test was used for comparison of the mean timing of cell division and cell cycle intervals. Results Morphokinetics t-pnf, t2, t8, CC2, S2, S3, CS2–8, CS4–8, and CS2–4 differed significantly between embryos with and without implantation potential, when embryos were developed using fresh semen, while t3, t4, t5, CC2, S2, t5–t2, CS2–4, and DR differed significantly between the embryos with and without implantation potential when frozen semen was used. No significant difference was found in mean value of any of the above-stated parameters when comparison was done between implanted embryos fertilized by either fresh or cryopreserved sperm. Conclusion Many morphokinetics parameters of embryogene­sis vary significantly between embryos with different ability to implant; therefore, the criteria developed in our IVF lab can be useful for selection of suitable embryo even at day 3 of development with more chances of implantation. Clinical significance Study indicates necessity of development of individualized selection model based on morphokinetics for every IVF lab and also confirms freezing as an important tool for fertility preservation of males as it does not affect events of embryogenesis. How to cite this article Bhadarka HK, Patel NH, Patel KB, Sodagar NR, Jadeja YD, Patel NH, Patel MN, Patel AV, Patel DH, Patel JS. Study of Morphokinetics in Day 3 Embryo with Implantation Potential and Effect of Sperm Cryopreservation on Embryogenesis. Int J Infertil Fetal Med 2017;8(2):61-67.

Developmental kinetics of in vitro produced bovine embryos: the effect of sex, glucose and exposure to time-lapse environment

Zygote ◽  
2001 ◽  
Vol 9 (2) ◽  
pp. 105-113 ◽  
Author(s):  
J. Peippo ◽  
M. Kurkilahti ◽  
P. Bredbacka
Keyword(s):  
Sex Determination ◽  
Video Recording ◽  
Time Lapse ◽  
Recording System ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Potential ◽  
Kinetics Of ◽  
Time Lapse Video

In this study, a simple time-lapse video recording system was used to compare developmental kinetics of female and male bovine embryos produced in vitro. Following embryo sex determination, the timing of each cleavage up to the 4-cell stage was compared between the sexes from the videotapes after culture in the presence and absence of glucose. In the second experiment, the consequences of exposure to a time-lapse video recording (TL) environment were studied by culturing embryos further until day 7 in an incubator, followed by collection and sex determination of morulae and blastocysts. In the absence of glucose, female embryos cleaved earlier than male ones. In the presence of glucose, however, male embryos cleaved earlier than female ones. There was no difference in the number of morulae/blastocysts in the absence of glucose, but in the presence of glucose more male than female embryos reached the morula and blastocyst stage. Exposure to the TL environment itself also had a sex-related effect, being more detrimental to male than female embryos. The difference in the number of functional X chromosomes between the sexes during early preimplantation development could explain these findings. In females, an increased capacity for oxygen radical detoxification through the pentose phosphate pathway could result in a reduced cleavage rate. Furthermore, glucose may influence the expression of enzymes located on the X chromosome. According to these results, a simple time-lapse video recording system is suitable for investigating embryo developmental kinetics and perhaps for the selection of embryos with the greatest developmental potential.

scholarly journals Dynamics of cytoplasm and cleavage divisions correlates with preimplantation embryo development

Reproduction ◽  
2018 ◽  
Vol 155 (1) ◽  
pp. 1-14 ◽  
Author(s):  
Robert Milewski ◽  
Marcin Szpila ◽  
Anna Ajduk
Keyword(s):  
Time Lapse ◽  
Preimplantation Development ◽  
Embryonic Cells ◽  
High Quality ◽  
Cell Stage ◽  
Preimplantation Embryo Development ◽  
Non Invasive ◽  
Image Velocimetry ◽  
Time Lapse Imaging

In vitrofertilization has become increasingly popular as an infertility treatment. In order to improve efficiency of this procedure, there is a strong need for a refinement of existing embryo assessment methods and development of novel, robust and non-invasive selection protocols. Studies conducted on animal models can be extremely helpful here, as they allow for more extensive research on the potential biomarkers of embryo quality. In the present paper, we subjected mouse embryos to non-invasive time-lapse imaging and combined the Particle Image Velocimetry analysis of cytoplasmic dynamics in freshly fertilized oocytes with the morphokinetic analysis of recordings covering 5 days of preimplantation development. Our results indicate that parameters describing cytoplasmic dynamics and cleavage divisions independently correspond to mouse embryo’s capacity to form a high-quality blastocyst. We also showed for the first time that these parameters are associated with the percentage of abnormal embryonic cells with fragmented nuclei and with embryo’s ability to form primitive endoderm, one of the cell lineages differentiated during preimplantation development. Finally, we present a model that links selected cytoplasmic and morphokinetic parameters reflecting frequency of fertilization-induced Ca2+-oscillations and timing of 4-cell stage and compaction with viability of the embryo assessed as the total number of cells at the end of its preimplantation development. Our results indicate that a combined analysis of cytoplasmic dynamics and morphokinetics may facilitate the assessment of embryo’s ability to form high-quality blastocysts.

278 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION AND SUPERSTIMULATION ON THE NORMALITY OF BOVINE EMBRYOS PRODUCED IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 296 ◽  
Author(s):  
K. Imai ◽  
T. Somfai ◽  
M. Ohtake ◽  
Y. Inaba ◽  
Y. Aikawa ◽  
...  
Keyword(s):  
Cell Division ◽  
Development Program ◽  
Time Lapse ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
In Vitro Production ◽  
Cell Stage ◽  
Follicular Wave ◽  
Significant Difference

We previously reported that follicular wave synchronization by dominant follicle removal on Day 5 and the start of a superstimulatory treatment on Day 7 after ovum pick-up (OPU) was effective to increase oocyte quality (Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). The present study was designed to examine the effect of superstimulatory treatment-induced follicular wave synchronization on quality of embryos obtained by OPU and in vitro production. Japanese Black cows were reared under the same feeding and environmental conditions and 2 OPU sessions were conducted in each cow. The first OPU session was performed in 7 cows at arbitrary days of estrous cycle using a 7.5-MHz linear transducer with needle connected to an ultrasound scanner. Then, follicles larger than 8 mm in diameter were aspirated and CIDR was inserted on Day 5 (the day of first OPU session = Day 0). The cows then received 30 mg of FSH twice a day from Days 7 to 10 in decreasing doses (4, 4, 3, 3, 2, 2, 1, 1 mg per shot) by i.m. injections. Cloprostenol (PGF; 0.75 mg) was administered in the morning of Day 9. The second OPU session was performed 48 h after PGF administration (Day 11) and only follicles larger than 5 mm in diameter were aspirated. The CIDR was removed from the cows just before OPU. Grade 1 and 2 cumulus oocyte complexes were in vitro matured, fertilized (IVF), and cultured as described by Imai et al. (2006 J. Reprod. Dev. 52, Suppl. S19-29). Some zygotes were fixed and stained to check their sperm penetration. Embryo development was monitored by time-lapse cinematography for 168 h after IVF. Cleavage pattern of embryos was classified morphologically into normal and abnormal (including those with multiple fragments, protrusions, 3 to 4 blastomeres, and uneven cell division) groups at their first cleavage. Normal penetration rate of second OPU session was significantly (P < 0.05) higher than that of the first OPU session. There were no differences in the mean percentage of total blastocyst and grade 1 blastocyst rates between the first (45.2 and 46.9%, respectively) and second (47.5 and 41.8%, respectively) OPU sessions. However, the rates of blastocysts developing from embryos that were beyond the 4-cell stage at 48 h after IVF was significantly (P < 0.05) higher after the second OPU session (81.2%) than after the first OPU session (67.4%). Furthermore, a significant difference (P < 0.05) was found in the rates of normal cleavage at the first cell division in embryos that developed to the blastocyst stage between the first and second OPU sessions (53.3% and 73.9%, respectively). These results indicate that superstimulatory treatment-induced follicular wave synchronization improved the normality of fertilization and development of cattle oocytes obtained by OPU. This work was supported by the Research and Development Program for New Bio-industry Initiatives.

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