scholarly journals 316 SUPPLEMENTAL CYSTEINE PRESENCE DURING THE DECONDENSATION OF SPERM CHROMATIN IMPROVES FERTILIZATION AND BLASTOCYST FORMATION AFTER INTRACYTOPLASMIC SPERM INJECTION IN PIGS

2005 ◽  
Vol 17 (2) ◽  
pp. 308
Author(s):  
M. Katayama ◽  
T. Cantley ◽  
A. Rieke ◽  
B. Day
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Culture Media ◽  
Morphological Changes ◽  
Formation Rate ◽  
Sperm Chromatin ◽  
Blastocyst Formation ◽  
Fertilization Rates ◽  
Duration Time

The effect of a cysteine supplement in culture media for oocytes matured in vitro after intracytoplasmic sperm injection (ICSI) on fertilization and embryo development were examined. In the first experiment, sperm injected oocytes were cultured in NCSU23 (control) or NCSU23 supplemented with 0.57–3.71 mM cysteine (0.57–3.71 Cys) for 12 h after ICSI, and then fixed to observe pronuclear formation. In the second experiment, to examine the appropriate duration time of cysteine supplement to support fertilization, sperm-injected oocytes were transferred into NCSU23 following culture in NCSU23 supplemented with 1.71 mM cysteine for 1, 2, 3, 4, 5, 6, or 9 h after ICSI, and then fixed at 12 h. At the same time, morphological changes of sperm heads in oocytes cultured in NCSU23 (1.71 Cys) were observed. In the third experiment, to examine the developmental ability of ICSI embryos fertilized in NCSU23 (1.71 Cys), sperm injected oocytes were cultured under the following conditions for a total of 168 h; NCSU23 (control), NCSU23 (1.71 Cys) for 3 h followed by transfer into NCSU23 (1.71 Cys-3 h), NCSU23 (1.71 Cys) for 12 h followed by transfer in NCSU23 (1.71 Cys-12 h), or NCSU23 (1.71 Cys) (1.71 Cys). Data were pooled from at least five replicates. Values in each replicate were analyzed using one-way ANOVA. Significance of differences was assessed by Student's t-test. Culture with several concentrations of cysteine for 12 h showed that 1.71–3.71 Cys significantly (P < 0.05) increased fertilization rates above controls or 0.57 Cys (56–60%, 35%, or 48%, respectively). Culture for several duration times with 1.71 Cys showed that fertilization rates increased as the duration time increased to 3 h which was significantly (P < 0.05) higher than controls (68% and 34%, respectively), and culture times of greater than 3 h did not increase fertilization rates (58–68%). At 3 h, 59% of oocytes cultured in NCSU23 (1.71 Cys) had decondensed sperm heads and 16% of those had enlarged sperm heads. At 6 h, 50% of oocytes cultured in NCSU23 (1.71 Cys) had male pronuclei. Blastocyst formation rate in 1.71 Cys-3 h was 29% which was higher than for controls (20%). On the other hand, 1.71 Cys-12 h cultures showed low blastocyst formation rates, and continuous culture in NCSU23 (1.71 Cys) for 168 h (1.71 Cys) significantly (P < 0.05) decreased blastocyst rates (16% and 7%, respectively). We found that the supplement of 1.71 mM cysteine to NCSU23 for culture of oocytes after ICSI improved fertilization rates. However, the presence of 1.71 mM cysteine for 12 h or longer after ICSI had adverse effects on embryo development. Since 1.71 mM cysteine supplement for 3 h after ICSI improved blastocyst formation with the same fertilization rates as when supplemented for 12 h, the presence of cysteine only during the decondensation of sperm chromatin was found to be associated with the improvement of fertilization and also the promotion of blastocyst formation.

Effects of Different Types of Incubators on Embryo Development and Clinical Outcomes

2020 ◽  
Author(s):  
Ji Liu ◽  
Yan-Hua Zhou ◽  
Xiao-Xiao Wang ◽  
Ling-Xi Tong ◽  
Yan-Hong Li ◽  
...  
Keyword(s):  
Clinical Outcomes ◽  
Embryo Development ◽  
Culture Media ◽  
Controlled Trial ◽  
Formation Rate ◽  
Fertilization Rate ◽  
Vitro Fertilization ◽  
Water Jacket ◽  
Different Types

Abstract Background: Different types of incubators have been designed for gamete and embryo culture in the past few years. The main differences of these incubators are humidity, temperature and gas control system, which play important roles in regulating the steady state of culture media. The objective of this study was to compare the effects of different types of incubators (air jacket incubators and water jacket incubators) on embryo development and clinical outcomes in human in vitro fertilization (IVF).Methods: First, the physical performances of different incubators were tested by mimicking routine IVF procedures. After that, in a randomized controlled trial, 1013 cumulus oocyte complexes from 43 patients were equally divided into two groups, fertilized and cultured in two types of incubators to analyze the effects of different types of incubators on embryo development and clinical outcomes. Results: We found that temperature recovery time in the air jacket incubator was significantly shorter than that in water jacket incubator. Although the O2 recovering time was also significantly shorter in the air jacket incubator as compared with the water jacket incubator, no significant differences were observed in the CO2 recovering time between two groups, which was also verified by pH recovering time of culture media. Besides, the temperature of culture medium in the dish covered with oil recovered more quickly in the air jacket incubators than that in water jacket incubators. However, there were no significant differences observed in the fertilization rate, Day 3 high-quality embryo formation rate, blastocyst formation rate, good blastocyst rate and clinical outcomes between two groups.Conclusions: These results indicate that the microenvironment, especially the temperature, in air jacket incubator recover faster than that in conventional water jacket incubator, however, there were no significant differences in embryo development and clinical outcomes between two types of incubators.

22 EFFECT OF OOCYTE AGE ON THE DEVELOPMENT OF MOUSE PARTHENOGENETIC EMBRYOS AND EFFECT OF OXYGEN CONCENTRATION ON EMBRYO DEVELOPMENT IN VITRO

2006 ◽  
Vol 18 (2) ◽  
pp. 119
Author(s):  
H. Bagis ◽  
S. Arat ◽  
H. Odaman ◽  
A. Tas
Keyword(s):  
Embryo Development ◽  
Formation Rate ◽  
Cell Number ◽  
Blastocyst Formation ◽  
Group 4 ◽  
Mouse Embryo Development ◽  
O2 Concentration ◽  
Group 3 ◽  
Development In Vitro

The objective of this study was to investigate the effects of two parameters on mouse embryo development in vitro. These parameters were the effect of oocyte age on activation and the effect of O2 concentration in culture. In the first experiment, oocytes were recovered from superovutated mice at 15 h (group 1) or 20 h (group 2) after human chorionic gonadotropin (HCG) injection. All oocytes were activated for 6 h with 10 mM Sr2+ in Ca2+ free medium in the presence of 5 �g/mL of cytochalasin B. After activation, embryos were cultured in KSOM.aa medium for 4.5-5.5 days. Zygotes from naturally bred mice were used as control. Differences in blastocyst formation rate and blastocyst cell number among treatments were analyzed by one-way ANOVA after arcsin square transformation. In the first experiment, blastocyst formation rate in the first group was higher than in the second group (62.6% vs. 47.1%; P < 0.05). In addition, blastocyst cell number was also higher in the first group than in the second one (69.4 � 3.2 vs. 52.4 � 2.2; P < 0.05). However, both values were higher in control group (80%, 76.2 � 1.2; P < 0.05) than in the experimental groups. These results showed that young oocytes were activated more effectively than aged oocytes. In the second experiment, mouse zygotes were cultured in a humidified atmosphere of 5% CO2 in air (group 3) or 5% CO2, 5% O2, and 90% N2 (group 4). Blastocyst formation rate and blastocyst cell number of zygotes cultured in low O2 concentration (group 4) for 4.5 days were higher than for group 3 (76.3% vs. 56.4 and 69.0 � 3.4 vs. 52.8 � 2.3; P < 0.05). There was a significant difference in blastocyt formation rate of embryos for 5.5 days between the two groups (25.8% for group 4 vs. 14.4% for group 3; P < 0.05). This suggests that the embryos developed more slowly in high O2 concentration. These results showed that low O2 concentration provided a more suitable environment for mouse embryo development in vitro. The same experiment was repeated with parthenogenetic embryos recently in our laboratory. This study was supported by a grant from TUBITAK, Turkey (VHAG-1022).

scholarly journals 311EFFECT OF OOCYTE MATURATION MEDIA ON THE SPEED OF MEIOTIC PROGRESSION AND BLASTOCYST DEVELOPMENT OF BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 275
Author(s):  
D. Fischer ◽  
J. Bordignon ◽  
C. Robert ◽  
D. Betts
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Immunofluorescence Microscopy ◽  
Chromosomal Abnormalities ◽  
Culture Media ◽  
Blastocyst Formation ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
Meiotic Progression

Environment is crucial for in vitro development of gametes and embryos. The recent progression of culture media towards defined conditions brought to surface the impact of different medium supplements on oocyte and embryo development. In this work we evaluate the effect of various oocyte culture media on bovine oocyte maturation and subsequent embryo development. Bovine cumulus-oocyte complexes were recovered from slaughterhouse ovaries and matured in vitro in either TCM-199 (Gibco) or SOF (Synthetic Oviduct Fluid) media supplemented with BSA (fatty acid-free) or serum (fetal bovine serum). Oocytes from each treatment group were denuded and fixed at 18, 20, 22, 24, 26 and 28h post-maturation (p.m.). Oocyte meiotic progression was monitored in each of the groups (n=28–40 oocytes/group) by immunofluorescence microscopy of chromatin. Oocytes matured in SOF showed a slower rate of meiotic progression when compared to the other groups, with the highest percentage of oocytes reaching the MII stage by 28h p.m. (60.71% SOF-BSA, 71.43% SOF-Serum). The fastest developmental rate was observed in oocytes matured in TCM-serum (77.15% at 24h p.m.) followed by oocytes matured in TCM-BSA (74.29% at 26h p.m.). In order to evaluate the effect of nuclear maturation on chromosome segregation, chromosomal organization of MII oocytes was evaluated by immunofluorescence microscopy within each media group (n=26–31 oocytes/group) at 18, 22 and 26h p.m.. No chromosomal abnormalities were found at 18h p.m.. Both media supplemented with BSA induced lower frequencies of chromosomal abnormalities (0 to 3.23%) and (3.57 to 7.69%) for SOF and TCM, respectively, when compared to their serum-supplemented counterparts (7.14 to 11.54%) and (10 to 10.71%) for SOF and TCM, respectively at 22 and 26h p.m.. Remarkably, the maturation medium and its supplements influenced the speed of blastocyst development. For this experiment, oocytes were matured in TCM-BSA, TCM-Serum, SOF-BSA or SOF-serum, fertilized in vitro in a TALP-base media supplemented with BSA and cultured in SOF-BSA. Blastocyst development was assessed at 7, 8 and 9 days of culture. Cleavage rates were similar between the groups (84–90%), whereas development rates to blastocyst stage varied among treatment groups. Maturation in SOF-BSA induced a delay in blastocyst formation that reached its highest percentage only on day 9 of culture (30.8%); moreover, blastocyst development was carried over until Day 12. When oocytes were matured in the presence of serum, the number of blastocysts did not increase after Day 8 of culture (26.6%, TCM-serum). These results provide evidence of a severe impact of oocyte culture media on the nuclear maturation of oocytes and their subsequent embryonic development after IVF. Moreover, the difference in the rate of oocyte maturation and blastocyst formation emphasizes the necessity for reviewing and adapting current protocols to new systems such as SOF-BSA. [Research funded by NSERC and OMAF of Canada.]

Effect of trichostatin A on fertilization and embryo development during extended culture of mouse oocyte

Zygote ◽  
2011 ◽  
Vol 20 (1) ◽  
pp. 27-32 ◽  
Author(s):  
Byung Chul Jee ◽  
Jun Woo Jo ◽  
Jung Ryeol Lee ◽  
Chang Suk Suh ◽  
Seok Hyun Kim ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Embryo Development ◽  
Trichostatin A ◽  
Formation Rate ◽  
Control Group ◽  
Blastocyst Formation ◽  
Developmental Potential ◽  
Extended Culture ◽  
Mouse Oocytes

SummaryWe performed this study to investigate the effect of histone deacetylase inhibition during extended culture of in vitro matured mouse oocytes. In vitro matured mouse (BDF1) oocytes were cultured in vitro for 6, 12, and 24 h, respectively, and then inseminated. During in vitro culture for 6 and 12 h, two doses of trichostatin A (TSA), a histone deacetylase inhibitor, were added (100 nM and 500 nM) to the culture medium and the oocytes were then inseminated. During the 24-h in vitro culture, two doses of TSA were added (100 nM and 500 nM) to the medium and the oocytes were activated with 10 mM SrCl2. After the 6-h culture, the fertilization rate was similar to that of the control group, but the blastocyst formation rate was significantly decreased. After the 12-h culture, both the fertilization and blastocyst formation rates were significantly decreased. After the 24-h culture, total fertilization failure occurred. In the oocytes cultured for 6 and 12 h, the fertilization and blastocyst formation rates did not differ between the TSA-supplemented and control groups. Although extended culture of the mouse oocytes significantly affected their fertilization and embryo development, TSA supplementation did not overcome their decreased developmental potential.

Enhancement of Developmental Competence of Immature Oocytes Supplemented with Growth Factors in Culture Media 

2021 ◽  
Author(s):  
Sonia B. Umdor ◽  
M. Karunakaran ◽  
D.K. Mandal ◽  
A. Santra ◽  
Subrata K. Das
Keyword(s):  
Growth Factors ◽  
Culture Media ◽  
Formation Rate ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Mammalian Development ◽  
Cleavage Rate ◽  
Early Mammalian Development ◽  
Excellent Source

Background: In vitro embryo production is a valuable tool for understanding early mammalian development, therapeutic applications, excellent source for research in the field of developmental biology and production of valuable animals. The purpose of this study is to improve the production of in vitro cattle embryos using fibroblast and platelet derived growth factor as media supplement. Methods: Ovaries were collected from local abattoir in 0.9% saline (30-35°C) supplemented with antibiotics. Cumulus oocyte complexes were aspirated, washed 5-6 times and placed in maturation media supplemented with growth factors and cultured in 5% CO2 incubator at 38.5°C with maximum humidity. After 24 h oocytes were co-incubated with in vitro capacitated sperms for fertilization for 15-18 h and then presumptive zygotes were cultured for embryo development. Cleavage was observed after 40-42 h and embryos were co-cultured with oviductal cells for 7-9 days. Result: The highest cleavage and blastocyst formation rates were 55.93 ± 4.75, 57.06 ± 4.78, 51.24 ± 4.12 and 3.26 ±1.53, 2.42 ± 1.02, 2.70 ± 1.17 in FGF (1ng ml-1), PDGF (10 ng ml-1) and in combination of FGF and PDGF (1ng ml-1 each) respectively. It can be concluded that PDGF (10 ng ml-1) enhanced cleavage rate and FGF (1ng ml-1) enhanced blastocyst formation rate.

scholarly journals Adjustment of redox potential in the culture media equivalent to the redox potential in follicular fluid improves in vitro embryo development and blastocyst formation

2018 ◽  
Vol 110 (4) ◽  
pp. e169
Author(s):  
I. Maldonado Rosas ◽  
A. Agarwal ◽  
L.G. Villar ◽  
J.F. Solorzano Vazquez ◽  
F.M. Perez Bernal ◽  
...  
Keyword(s):  
Redox Potential ◽  
Embryo Development ◽  
Follicular Fluid ◽  
Culture Media ◽  
Blastocyst Formation

125 INTRACYTOPLASMIC SPERM INJECTION (ICSI)-BASED MOUSE EMBRYO ASSAY: CHOICE OF EMBRYO CULTURE SYSTEM OUTWEIGHS THE EFFECT OF FERTILIZATION PROCEDURE ON EMBRYO DEVELOPMENT

2013 ◽  
Vol 25 (1) ◽  
pp. 209
Author(s):  
C. Schwarzer ◽  
T. C. Esteves ◽  
S. Le Gac ◽  
V. Nordhoff ◽  
S. Schlatt ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Mouse Embryo ◽  
Embryo Culture ◽  
Intracytoplasmic Sperm Injection ◽  
Culture Medium ◽  
Culture Media ◽  
Blastocyst Formation ◽  
The Absolute ◽  
Mouse Embryo Assay

Human embryo culture media, intended for assisted reproductive technologies (ARTs), are released for clinical use if they pass the mouse embryo assay (MEA). This assay prescribes that at least 70% of in vivo fertilized mouse 1-cell embryos form blastocysts, in order to grant the culture medium approval. In the fertility clinic, however, human embryos undergo more manipulation than their MEA counterparts through, for example, fertilization by intracytoplasmic sperm injection (ICSI); further, only a minority of the embryos transferred to the uterus goes on to establish gestations. In this context, we asked if the results of the MEA only depend on the type of in vitro culture, or are also affected by the method of fertilization. Superovulated B6C3F1 mouse oocytes were fertilized by ICSI using C57Bl/6 sperm. Pronuclear-stage eggs were allocated to four developmental environments: two ART culture protocols (HTF/MultiBlast, Irvine Scientific; ISM1/ISM2, Origio), standard mouse culture medium (KSOM(aa), made in-house) and the oviduct of pseudopregnant CD1 mice. As control for the invasive manipulation, pronuclear-stage eggs were generated by mating (B6C3F1 × C57Bl/6) and cultured in KSOM(aa) medium. Embryos were recovered from culture or from the CD1 uterus and scored for blastocyst formation at 96 h of development (Table 1). For these blastocysts, we determined the number of total, inner cell mass (ICM), and trophectoderm (TE) cells (Table 1) by confocal immunofluorescence microscopy (Schwarzer et al. 2012 doi:10.1093/humrep/des223). Our results show that ART culture protocols applied to mouse ICSI embryos are not equivalent in supporting blastocyst formation. Based on blastocyst rates, the ranking observed here after ICSI, reflects the ranking reported by us for IVF embryos (Schwarzer et al. 2012); that is, KSOM(aa) > HTF/MultiBlast > oviduct > ISM1/2. This similarity suggests that the effect of in vitro culture on mouse development exceeds the effect of ICSI, provided gametes are of good quality. From the analysis of cell numbers, we note that while the ICM/TE ratios are not of easy interpretation, the absolute numbers of cells in the ICM draw a clear line between the environment of the oviduct and those of culture media. Irrespective of the ICM/TE ratio, only the oviduct environment secures 8 cells in the ICM (Table 1). Soriano and Jaenisch (1986 Cell 46, 19–29) reported that 8 cells of the ICM are set aside to give rise to the body of a mouse. In summary, the current MEA is a valuable assay to assess the quality of culture medium, however, its refinement is necessary to better model the adaptive properties of embryo culture when different methods of fertilization are applied. Until the MEA is extended into postimplantation development, as we advocate (Schwarzer et al. 2012), the absolute numbers of cells in the ICM may be a better gauge of embryo quality than the blastocyst rates. Table 1.Mouse embryo assay outcomes after ICSI

376 FAILURE OF SPERM DECONDENSATION AFTER INTRACYTOPLASMIC SPERM INJECTION IN SWAMP BUFFALO (BUBALUS BUBALIS)

2010 ◽  
Vol 22 (1) ◽  
pp. 344
Author(s):  
V. Chankitisakul ◽  
A. Tharasanit ◽  
K. Thaseephoo ◽  
M. Techakumphu
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Chemical Activation ◽  
Formation Rate ◽  
Calcium Ionophore ◽  
Developmental Competence ◽  
Ionophore A23187 ◽  
Blastocyst Formation ◽  
Swamp Buffalo ◽  
Pronuclear Formation

Intracytoplasmic sperm injection (ICSI) has been intensively used to examine the early events of gamete activation, but few studies have been reported for swamp buffalo. The first objective (Exp. 1) was to compare the developmental competence of oocytes after ICSI using either live or dead frozen-thawed spermatozoa. Matured oocytes were fertilized by ICSI using live (n = 148) or dead (n = 151) spermatozoa, followed by chemical activation using calcium ionophore (A23187) and cyclohexamide (CHX) in SOF medium. In vitro fertilization (n = 149) served as thecontrol. Cleavage rate was recorded on Day 2 and blastocyst formation rate was evaluated on Day 7. The second objective (Exp. 2) was to examine the effects of ICSI and activation regime on the decondensation of buffalo spermatozoa. A total of 148 matured oocytes were subjected to ICSI. The sperm-injected oocytes (n = 87) were then activated using the activation protocol as described in Exp. 1. The ICSI oocytes without activation (n = 61) and sham-injected oocytes with activation (n = 35) were used as controls. Nuclear changes of presumptive zygotes were mor- phologically evaluated for pronuclear formation using 4′,6-diamidino-2-phenylindole procedure and epifluorescent microscopy at 18 h post-ICSI. Statistical differences were determined among the groups using chi-square test. In Exp. 1, the results showed that the percentages of cleavage and blastocyst formation rate were 79.7, 77.0, and 41.6% and 33.8, 30.5, and 14.8% in the live sperm, dead sperm, and IVF groups, respectively. Embryo development rates did not significantly differ between ICSI groups; however, these rates were significantly higher than in the IVF group (P < 0.05). In Exp. 2, the pronuclear formation rate was significantly higher in the ICSI with chemical (70.1%) and sham injection with chemical (60.6%) groups than in the ICSI without chemical group (3.2%; P < 0.01). However, most of the presumptive zygotes with pronuclear formation from the ICSI with chemical activation group showed only intact sperm heads instead of the full male pronuclear formation. Our study suggests that the chemical activation directly affected the female pronuclear formation and embryo development but that it was not associated with the male pronuclear formation. It is postulated that ICSI oocytes that developed to cleavage and blastocyst stages underwent parthenogenesis after chemical activation. This work was supported by TRF-MAG (MRG-WII515S056) and CHE-TRF Senior Research Fund (RTA5080010).

scholarly journals In vitro maturation of porcine oocytes with retinoids improves embryonic development

2008 ◽  
Vol 20 (4) ◽  
pp. 483 ◽  
Author(s):  
C. Almiñana ◽  
M. A. Gil ◽  
C. Cuello ◽  
I. Caballero ◽  
J. Roca ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Formation Rate ◽  
Control Treatment ◽  
In Vitro Fertilisation ◽  
Blastocyst Formation ◽  
Maturation Rate ◽  
And Control

In the present study, the effects of retinoid metabolite administration during in vitro maturation (IVM) on oocyte maturation, parameters of in vitro fertilisation (IVF) and embryo development were examined. Varying concentrations of 9-cis retinoic acid (RA; 0, 5, 50 and 500 nm; Experiment 1) and all-trans retinol (ROH; 0, 125, 1250 and 12 500 nm; Experiment 2) were included in the maturation medium. Cumulus–oocyte complexes were matured in vitro and inseminated with frozen–thawed spermatozoa. Presumptive zygotes were cultured for 16 h to assess IVF parameters or for 7 days to assess embryo development and quality. In Experiment 1, the oocyte maturation rate to metaphase II was significantly decreased (P < 0.001), with values below 5%, in the presence of the highest concentration of RA (500 nm). However, 5 and 50 nm RA had no effect compared with control. Treatment with 5 nm RA improved the blastocyst development rate (P < 0.001). In Experiment 2, the oocyte maturation rate did not differ between 125 and 1250 nm ROH treatment groups and control. However, treatment with 12 500 nm ROH was deleterious because no matured oocytes were observed following the treatment. The penetration rate was lower in the group treated with 1250 nm ROH compared with the 125 nm ROH-treated and control groups, but the blastocyst formation rate did not differ among the three groups. In conclusion, 5 nm RA in the IVM medium significantly increased the blastocyst formation rate, suggesting that RA may play an important role during IVM.

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