98 VITRIFICATION OF ZYGOTES SUPPORTS FULL-TERM DEVELOPMENT IN RABBITS

2008 ◽  
Vol 20 (1) ◽  
pp. 129
Author(s):  
J. Xu ◽  
J. Zhang ◽  
Y. Chen ◽  
M. Cater ◽  
X. Yang ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Genetic Material ◽  
Full Term ◽  
Human Reproduction ◽  
Blastocyst Development ◽  
Cold Surface ◽  
Cell Stage ◽  
Disease Study ◽  
The University

Rabbit can serve as an excellent model for human reproduction and relevant disease study. The objective of the study was to develop an effective procedure to preserve the rabbit genome by vitrifying zygotes. Sexually matured New Zealand White rabbits maintained under a 16 h light:8 h dark cycle were superovulated with a regime of two 0.3-mg, two 0.4-mg, and two 0.7-mg injections of FSH with an interval of 12 h between, followed by an injection of 200 IU hCG and mating. Presumptive zygotes were flushed from the oviducts and collected by midventral lapartomy 18 h after hCG injection. One-cell-stage embryos were assigned to pre-equilibration at 38.5�C in HEPES-buffered TCM199 supplemented with 20% FBS + 7.5% ethylene glycol (EG) (v/v) + 7.5% dimethylsulphoxide (DMSO) (v/v) (dehydration medium) for 3 min, and subsequently to 1.0 ml of vitrification medium (TCM199 supplemented with 20% FBS + 20% EG and 20% DMSO), as described by Vajta et al. (1998 Mol. Reprod. Dev. 51, 53–58), at room temperature for 3 min. Five to six embryos per group were then vitrified in a micro-droplet (approximately 1–2 µL) by directly dropping them into a thin layer of liquid nitrogen on the solid surface that generated a super cold surface for vitrification. Vitrified embryos were sequentially warmed, rehydrated in 20% FBS M199 with different concentrations of sucrose, and washed in 20% FBS M199 for 5 min. Warmed embryos were assigned either for further in vitro culture or for embryo transfer to test corresponding developmental potentials. Dutch rabbits were used as recipients in the procedure of asynchronous ovulation induction (22-h delay to zygote donors) by an intramuscular injection of 15 µg of GnRH per doe. Three or four warmed zygotes were surgically transferred into the recipients on the same day of thawing. Pregnancy was monitored by palpation and/or ultrasound on Days 14–16 post-embryo transfer (ET); C-sections were performed on Day 31 to retrieve full-term developed newborns. Preliminary results showed that a 96% (n = 35) post-warming survival rate was achieved with vitrified rabbit zygotes; subsequent cleavage and blastocyst development were 41.6 and 33.3%, respectively. Transfer of a total of 23 warmed embryos into 6 recipients resulted in 5 pregnancies (83.3%, 5/6). Five live kits (21.7%, 5/23) were delivered. Our study suggests that vitrification of rabbit zygotes is a feasible approach for preserving rabbit genetic material. The establishment of suitable conditions for the vitrification of rabbit zygotes could be useful as a model for vitrification of human 1-celled embryos. The authors thank the staff of the animal facility at the University of Connecticut for animal care and maintenance. This work was supported by NIH/NCRR-SBIR grant: 1R43RR020261-01.

Initial results with in vitro fertilization and embryo transfer at the University Central Hospital of Tampere, Finland

1986 ◽  
Vol 3 (4) ◽  
pp. 270-270
Author(s):  
Reijo Punnonen ◽  
Pentti K. Heinonen ◽  
Erkki Kujansuu ◽  
Kirsti Selander ◽  
Ralph Ashorn ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Transfer ◽  
Central Hospital ◽  
Vitro Fertilization ◽  
The University ◽  
Initial Results

The effects of brain-derived neurotrophic factor and metformin on in vitro developmental competence of bovine oocytes

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 187-193 ◽  
Author(s):  
So Gun Hong ◽  
Goo Jang ◽  
Hyun Ju Oh ◽  
Ok Jae Koo ◽  
Jung Eun Park ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Embryo Development ◽  
Neurotrophic Factor ◽  
Brain Derived Neurotrophic Factor ◽  
Early Embryo ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Significant Difference

SummaryBrain-derived neurotrophic factor (BDNF) signalling via tyrosine kinase B receptors may play an important role in ovarian development and function. It has been reported that metformin elevates the activity of Tyrosine kinase receptors and may amplify BDNF signalling. The objective of this study was to investigate the effect of BDNF during in vitro maturation (IVM) and/or in vitro culture (IVC) (Experiment 1), and to evaluate the collaborative effect of BDNF and metformin treatment on the developmental competence of bovine in vitro fertilized (IVF) embryos (Experiment 2). In Experiment 1, BDNF, which was added to our previously established IVM systems, significantly increased the proportions of MII oocytes at both 10 ng/ml (86.7%) and 100 ng/ml (85.4%) compared with the control (64.0%). However, there was no statistically significant difference in blastocyst development between the control or BDNF-supplemented groups. In Experiment 2, in order to investigate the effect of BDNF (10 ng/ml) and/or metformin (10−5 M) per se, TCM-199 without serum and hormones was used as the control IVM medium. The BDNF (48.3%) and BDNF plus metformin (56.5%) significantly enhanced the proportions of MII oocytes compared with the control (34.4%). Although, BDNF or metformin alone had no effect in embryo development, BDNF plus metformin significantly improved early embryo development to the 8–16-cell stage compared with the control (16.5 vs. 5.5%). In conclusion, the combination of BDNF and metformin may have a collaborative effect during the IVM period. These results could further contribute to the establishment of a more efficient bovine in vitro embryo production system.

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.

Micromolar concentration of pentoxifylline improves development in vitro of hamster 8-cell embryos: conrmation of biological viability by embryo transfer

1997 ◽  
Vol 9 (7) ◽  
pp. 697 ◽  
Author(s):  
Rupasri Ain ◽  
P. B. Seshagiri
Keyword(s):  
Embryo Transfer ◽  
Embryo Development ◽  
Human Sperm ◽  
Blastocyst Development ◽  
Preimplantation Embryo Development ◽  
Embryo Culture Medium ◽  
Cell Embryo ◽  
Continuous Presence ◽  
Development In Vitro

The inßuence of the sperm motility stimulant pentoxifylline (PF) on preimplantation embryo development in hamsters was evaluated. Eight-cell embryos were cultured in hamster embryo culture medium (HECM)-2, with or without PF (0· 0233·6 mM). There was 90%, 37% and 29% inhibition of blastocyst development by 3·6 (used for human sperm), 0·9 and 0 ·45 mM PF, respectively. However, 23 µM PF (exposed to hamster oocytes during IVF) signicantly (P < 0·05) improved blastocyst development (63· 6% v. 51· 8%); morulae development was, however, not curtailed by 0·45 mM or 0·9 mM PF (51·8%±6·0 or 50·5%±11·3, respectively). Post-implantation viability of PF-treated embryos was assessed by embryo transfer; 43% of 80 PF-treated embryos implanted compared with 40% of 79 control embryos. Of the 9 recipients, 6 females delivered pups (19, i.e. 16% of transferred embryos or 53% of implanted embryos). These data show that in hamsters, continuous presence of PF at 0·45-3·6 mM is detrimental to 8-cell embryo development whereas 23 µM PF improves the development of embryos to viable blastocysts which produce live offspring.

27 CLONING OF ADULT PIGS USING SCRIPTAID TREATMENT AND PREOVULATORY EMBRYO TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 120
Author(s):  
M. Albornoz ◽  
C. Colato ◽  
N. El-Beyrouthi ◽  
F. Mellano ◽  
A. Mellano ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Cell Lines ◽  
Fibroblast Cell ◽  
Cloning Efficiency ◽  
Cell Stage ◽  
Expected Time ◽  
Reconstructed Embryos ◽  
Adult Cell ◽  
Skin Biopsies

There is growing interest in the use of swine in biomedical research. Cloning from cultured somatic cells (SCNT) has been the preferred method to generate genetically modified swine models. In a recent report, swine cloning efficiency was increased by treatment of reconstructed embryos with the inhibitor of deacetylase enzymes Scriptaid (Zhao et al. 2010 Cel. Reprog. 12, 75). Also, the timing of SCNT-embryo transfer with respect to the recipient’s expected time of ovulation was shown to affect cloning efficiency, whereas preovulatory embryo transfer resulted in a higher rate of cloned piglets born compared to postovulatory embryo transfer (Petersen et al. 2008 Cloning Stem Cells 10, 355). Therefore, our objective was to combine Scriptaid treatment and preovulatory embryo transfer in the same protocol for swine cloning. Cumulus–oocyte complexes aspirated from 3- to 6-mm diameter follicles were matured in vitro under standard conditions (Martinez Diaz et al. 2010 Cel. Reprog. 12, 85) and used as host oocytes for SCNT. Fibroblast cell lines were established from skin biopsies collected from 2 adult boars and cultured in DMEM supplemented with 10% FBS and 1% antibiotics. Oocytes were micromanipulated in Tyrode’s lactate-pyruvate-HEPES medium supplemented with 7.5 μg mL–1 cytochalasin B (CB) and electrically fused using a single DC pulse of 1.6 kV cm–1 for 70 μs. Activation was performed using ionomycin (15 μM/5 min) followed by exposure to CB (7.5 μg mL–1) and cyclohexemide (10 μg mL–1) for 5 h in porcine zygote medium (PZM-3; Yoshioka et al. 2002 Biol. Reprod. 66, 112). Reconstructed embryos were exposed to 500 nM Scriptaid for 10 to 12 h starting after ionomycin treatment. Oocytes were then washed and cultured in PZM-3 medium until transfer. Peripubertal recipient gilts were synchronized by oral administration of altrenogest (Regu-Mate®; 20 mg day–1) for 12 days, followed by 1.000 IU eCG injected on the last day of altrenogest treatment and 500 IU hCG 72 h later. 1-cell stage embryos were transferred into the oviduct after ∼20 h from hCG injection or 22 h before the expected ovulation time. Pregnancy was confirmed and monitored by ultrasonography and parturition was induced by injecting PGF2α at Day 115 of pregnancy. A total of 840 reconstructed embryos were transferred into 10 gilts [average 84 (range 60–110) embryos/gilt]. 4 gilts (40%) were detected to be pregnant 4 weeks after transfer, and 2 (20%) delivered 1 (1100 g) and 2 (950 and 850 g) healthy cloned piglets. The number of embryos transferred to these 2 gilts was 85 and 70. These results confirm that Scriptaid treatment and preovulatory embryo transfer can be applied in the same cloning protocol to produce cloned piglets from adult cell lines. To our knowledge, these are the first cloned pigs produced in Latin America.

82 EARLY PORCINE EMBRYO ENERGY PREFERENCE AND SUBSEQUENT DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 170
Author(s):  
L. D. Spate ◽  
B. K. Redel ◽  
R. S. Prather
Keyword(s):  
Energy Source ◽  
Subsequent Development ◽  
Cell Number ◽  
Total Cell ◽  
Control Medium ◽  
Blastocyst Development ◽  
Total Cell Number ◽  
Cell Stage ◽  
Porcine Embryo

Early porcine embryo metabolism in vitro is not completely understood. It has been suggested that before embryo genome activation (4-cell stage), the preferred energy source of the embryo is pyruvate. In our porcine zygote culture medium (MU1), the energy sources are 0.2 mM pyruvate and 2.0 mM calcium lactate. Three experiments were performed with in vitro-matured and IVF embryos to examine the effect on blastocyst development after withholding pyruvate and/or lactate during the first 48 h of culture. In Experiment 1, embryos were cultured without lactate for 48 and then cultured to Day 6 in control medium containing lactate. Control embryos were cultured in medium with lactate starting after fertilization to Day 6. All data were analysed by using SAS 9.3 with a GENMOD procedure used for the blastocyst data and a GLM procedure used for the cell number data. On Day 6, the percentage of embryos that formed blastocysts was 30.2% for control and 26.5% for embryos cultured for 48 h without lactate (n = 490, 4 replications). The difference was not significant P > 0.05. In Experiment 2, embryos were cultured without pyruvate for 48 and then cultured to Day 6 in control medium containing pyruvate. Control embryos were cultured in medium with pyruvate starting after fertilization to Day 6. On Day 6, the percentage of embryos that formed blastocysts was 31.1% for control and 30.5% for embryos cultured for 48 h without pyruvate (n = 385, 3 replications). In Experiment 3, embryos were cultured in control medium for the first 48 h and then cultured to Day 6 in medium without pyruvate, thus forcing the embryos to use lactate instead of pyruvate. On Day 6, the percentage of embryos that formed blastocysts in the pyruvate free medium increased from 28.6%a ± 1.0 to 33.9%b ± 1.0; P ≤ 0.05 (n = 490, 4 replications) compared with the control and total cell number increased from 30.7a ± 1.5 to 41.3b ± 1.8 cells, respectively; P ≤ 0.05 (n = 65, 4 replications). The results from Experiments 2 and 3 were unanticipated as it was believed that the embryo would be more dependent on pyruvate for energy up to the blastocyst stage. We believed in Experiment 2 that from zygote to 4 cells the embryos were not as capable of using lactate and that removing the pyruvate would hinder further development. In Experiment 3, forcing the embryo to use lactate from Day 2 to Day 6 significantly improved blastocyst development and total cell number, suggesting that the embryo is not dependent on a specific energy source or that there are adequate pyruvate stores in the oocyte to 4-cell stage, to promote development to blastocyst. Funding was provided by Food for the 21st Century, the University of Missouri, and the National Institutes of Health (OD011140).

Expression of mRNA encoding leukaemia inhibitory factor (LIF) and its receptor (LIFRβ) in buffalo preimplantation embryos produced in vitro: markers of successful embryo implantation

Zygote ◽  
2012 ◽  
Vol 21 (2) ◽  
pp. 203-213 ◽  
Author(s):  
S. Eswari ◽  
G. Sai Kumar ◽  
G. Taru Sharma
Keyword(s):  
Culture Media ◽  
Total Cell ◽  
Inhibitory Factor ◽  
Preimplantation Embryos ◽  
Leukaemia Inhibitory Factor ◽  
Hatching Rate ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Significant Difference

SummaryThe objective of this study was to evaluate the effect of supplementation of recombinant leukaemia inhibitory factor (LIF) in culture media on blastocyst development, total cell number and blastocyst hatching rates and the reverse transcription-polymerase chain reaction analysis of preimplantation buffalo embryos to determine whether they contain the LIF-encoding mRNA and its beta receptor (LIFRβ) genes in different stages of preimplantation buffalo embryos. Cumulus–oocyte complexes retrieved from slaughterhouse buffalo ovaries were matured in vitro and fertilized using frozen buffalo semen. After 18 h of co-incubation with sperm, the presumptive zygotes were cultured in modified synthetic oviductal fluid without (control) or with rhLIF (100 ng/ml). There was no significant difference in the overall cleavage rate up to morula stage however the development of blastocysts, hatching rate and total cell numbers were significantly higher in the LIF-treated group than control. Transcripts for LIFRβ were detected from immature, in vitro-matured oocytes and in the embryos up to blastocyst stage, while transcripts for the LIF were detected from 8–16-cell stage up to blastocyst, which indicated that embryo-derived LIF can act in an autocrine manner on differentiation process and blastocyst formation. This study indicated that the addition of LIF to the embryo culture medium improved development of blastocysts, functional (hatching) and morphological (number of cells) quality of the blastocysts produced in vitro. The stage-specific expression pattern of LIF and LIFRβ mRNA transcripts in buffalo embryos indicated that LIF might play an important role in the preimplantation development and subsequent implantation of buffalo embryos.

Some effects of genotype and composition of the culture medium on the development of mouse zygotes in vitro

1993 ◽  
Vol 5 (4) ◽  
pp. 405 ◽  
Author(s):  
ZF Du ◽  
RG Wales
Keyword(s):  
Culture Medium ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Reciprocal Crosses ◽  
Maternal Genome ◽  
Zygote Development ◽  
Mouse Zygotes ◽  
Better Than

The effects of EDTA and the presence of glucose and glutamine in CZB medium on the development of mouse zygotes of different genotype were investigated. Although 30-80% of zygotes (depending on the cross) passed the 2-cell stage in EDTA-free medium, the addition of a low concentration of EDTA was necessary in these experiments to obtain blastocysts in culture. In reciprocal crosses between outbred (Qs), inbred (DBA/2) and hybrid (B10D2F1) stock, there was evidence of a strong influence of the maternal genome on zygote development, with those from B10D2F1 females performing best irrespective of sire. A paternal influence on development was also evident but the most successful sire varied with the genotype of female used and reciprocal crosses differed greatly in the ability of the resultant zygote to develop in culture. For zygotes recovered from Qs females, CZB medium containing glucose and glutamine supported development to the blastocyst stage better than did medium devoid of these substrates. Tests with embryos from B10D2F1 females indicated that the presence of glucose for the whole or for part of the incubation period stimulated blastocyst development. However, the addition of glutamine to the medium in these tests had no significant effect on the development of blastocysts.

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