29 EFFECT OF EMBRYO CULTURE LENGTH ON PRODUCTION OF CLONED TRANSGENIC GOATS

2013 ◽  
Vol 25 (1) ◽  
pp. 162 ◽  
Author(s):  
J. Hall ◽  
Q. Meng ◽  
B. R. Sessions ◽  
Z. Fan ◽  
X. Wang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Embryo Culture ◽  
Polar Body ◽  
In Vitro Production ◽  
Cell Stage ◽  
First Polar Body ◽  
Fetal Fibroblasts ◽  
Transgenic Goats ◽  
Adult Fibroblasts

The yield of blastocysts and hatched blastocysts using in vitro production (IVP) in goats are still low. The development of caprine embryos is frequently arrested at the 8- to 16-cell stage, indicating suboptimal culture conditions (Jimenez-Macedo et al. 2005 Theriogenology 64, 1249–1262). Our goal was to produce transgenic goats by somatic cell nuclear transfer (SCNT) and further determine whether the length of embryo culture has an effect on development to term. We compared the efficiency of transferring single-cell embryos 12 h post-activation to transferring 4- to 8-cell embryos cultured for 60 h post-activation. Nine transgenic goats from 2 cell lines were produced through SCNT. Somatic donor cells were obtained from 2 sources: adult fibroblasts and fetal fibroblasts. Adult fibroblasts were obtained from a transgenic doe skin biopsy. Fetal fibroblasts were isolated from a 25-day-old fetus and then electroporated with a pcDNA3.1DV5-MHC-TGF-β1cys33ser vector, followed by G418 selection, screening, and subsequent use for SCNT. Oocytes with >4 layers of cumulus cells were collected by slicing abattoir ovaries and matured in vitro for 21 to 23 h. After being denuded, oocytes presenting a first polar body were enucleated and received a donor cell from 1 of the 2 cell lines. Fused embryos were then activated for 5 min in 5 µM ionomycin, followed by 4 h in 2 mM DMAP with 5 µg of cycloheximide mL–1. Activated embryos were cultured in G1 medium with 5 mg of BSA mL–1 for either 12 or 60 h post-activation, followed by surgical transfer into the oviducts of recipients synchronized to show estrus within 12 h of SCNT. Overall, 376 embryos were transferred into 23 recipients. Pregnancy was examined by ultrasonography on Day 30 post-transfer. No pregnancy losses were observed after Day 30 of gestation. All kids were born live (42% of recipients receiving embryos cultured for 12 h gave birth, compared with only 9% when cultured for 60 h). The data (Table 1) suggest that a longer culture time in vitro significantly reduces viability of cloned embryos. Table 1.Twelve-hour versus 60-h embryo culture This work was supported by Utah Agricultural Experiment Station project no. 1100.

39 CLONAL OFFSPRING DERIVED FROM SEPARATED BLASTOMERES IN CYNOMOLGUS MONKEYS (MACACA FASCICULARIS)

2008 ◽  
Vol 20 (1) ◽  
pp. 100
Author(s):  
C. Iwatani ◽  
J. Okahara-Narita ◽  
J. Yamasaki ◽  
H. Tsuchiya ◽  
R. Torii
Keyword(s):  
Cell Lines ◽  
Zona Pellucida ◽  
Cynomolgus Monkey ◽  
Behavioral Science ◽  
Polar Body ◽  
Calf Serum ◽  
Es Cell ◽  
Cell Stage ◽  
Splitting Technique

There are no reports of cloning by embryo splitting in the cynomolgus monkey, but production of genetically identical monkeys would have tremendous implications for biomedical research, especially for immunological studies, production of disease models, and behavioral science. Cloning would also reduce the number of animals required for the above research by increasing experimental reproducibility. In this study, we tried to produce cynomolgus monkey offspring by embryo splitting and embryo transfer. Controlled ovarian stimulation and oocyte recovery have been previously described by Torii et al. (2000 Primates 41, 39–47). Cumulus-free mature oocytes were fertilized by intracytoplasmic sperm injection. Single spermatozoa were individually immobilized by scoring their tails and picking them up with the injection pipette. The denuded oocyte was held by the holding pipette with the polar body in the 12 o'clock position. The injection pipette was then inserted at the 3 o'clock position and was introduced into the cytoplasm, breaking the ooplasmic membrane by pulling gently. One spermatozoon was injected into the cytoplasm. The injected oocytes were cultured at 38�C in 5% CO2, 5% O2 and 90% N2 in CMRL-1066 medium (Invitrogen, Grand Island, NY, USA) containing 20% calf serum (CS, Invitrogen) for 2–3 days. Splitting was performed using 4- to 7-cell-stage embryos. The zona pellucida was disrupted with acidic Tyrode's solution, and individual blastomeres were separated from the zona-free embryos by 0.25% trypsin-EDTA with added CaCl2 (<1 min). After transferring the zona-free embryos into TALP-HEPES medium, blastomeres were dissociated by pipetting with a 40–50 µm micropipette 4–5 times. These blastomeres were then transferred into empty zonae that had been produced from immature oocytes by the aspiration of ooplasm with a micromanipulator. Sixteen embryos underwent blastomere separation and a total of 33 split embryos were produced. After being cultured for 2–3 h in CMRL-1066 medium containing 20% CS, 30 of these split embryos, comprising 1–4 blastomeres each, were transferred into the oviducts of 23 fertile surrogate mothers at 0 to 5 days after ovulation. Pregnancy was confirmed in two animals (8.7%; 2/23) by ultrasound approximately 30 days after transfer. The pregnancies were uneventful and two normal healthy babies were born without any assistance 159 days after transfer. The low pregnancy rate could be due to the presence of fewer cells in the smaller split embryos, the ruptured zona pellucida, or the in vitro micromanipulation of embryos during blastomere separation and reconstruction. Here we report the first production of viable cloned offspring produced by blastomere separation in the cynomolgus monkey. Since we have previously succeeded in establishing ES cell lines from isolated blastomeres, in the future we will be able to produce genetically identical monkeys from a single 4- to 8-cell-stage embryo using those ES cell lines and the embryo splitting technique.

30 GENERATION OF CLONED TRANSGENIC GOATS WITH CARDIAC SPECIFIC OVEREXPRESSION OF TRANSFORMING GROWTH FACTOR β1

2013 ◽  
Vol 25 (1) ◽  
pp. 162 ◽  
Author(s):  
Q. Meng ◽  
J. Hall ◽  
H. Rutigliano ◽  
X. Zhou ◽  
B. R. Sessions ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Critical Role ◽  
Transforming Growth Factor Β1 ◽  
Transgenic Goat ◽  
First Polar Body ◽  
Fetal Fibroblasts ◽  
Transgenic Goats ◽  
Tgf Β1

Transforming growth factor β1 (TGF-β1) has a potent profibrotic function and is central to signaling cascades involved in interstitial fibrosis, which plays a critical role in the pathobiology of cardiomyopathy and contributes to diastolic and systolic dysfunction. In addition, fibrotic remodeling is responsible for generation of re-entry circuits that promote arrhythmias (Bujak and Frangogiannis 2007 Cardiovasc. Res. 74, 184–195). Due to the small size of the heart, functional electrophysiology of transgenic mice is problematic. Large transgenic animal models have the potential to offer insights into conduction heterogeneity associated with fibrosis and the role of fibrosis in cardiovascular diseases. The goal of this study was to generate transgenic goats overexpressing an active form of TGFβ-1 under control of the cardiac-specific α-myosin heavy chain promoter (α-MHC). A pcDNA3.1DV5-MHC-TGF-β1cys33ser vector was constructed by subcloning the MHC-TGF-β1 fragment from the plasmid pUC-BM20-MHC-TGF-β1 (Nakajima et al. 2000 Circ. Res. 86, 571–579) into the pcDNA3.1D V5 vector. The Neon transfection system was used to electroporate primary goat fetal fibroblasts. After G418 selection and PCR screening, transgenic cells were used for SCNT. Oocytes were collected by slicing ovaries from an abattoir and matured in vitro in an incubator with 5% CO2 in air. Cumulus cells were removed at 21 to 23 h post-maturation. Oocytes were enucleated by aspirating the first polar body and nearby cytoplasm by micromanipulation in Hepes-buffered SOF medium with 10 µg of cytochalasin B mL–1. Transgenic somatic cells were individually inserted into the perivitelline space and fused with enucleated oocytes using double electrical pulses of 1.8 kV cm–1 (40 µs each). Reconstructed embryos were activated by ionomycin (5 min) and DMAP and cycloheximide (CHX) treatments. Cloned embryos were cultured in G1 medium for 12 to 60 h in vitro and then transferred into synchronized recipient females. Pregnancy was examined by ultrasonography on day 30 post-transfer. A total of 246 cloned embryos were transferred into 14 recipients that resulted in production of 7 kids. The pregnancy rate was higher in the group cultured for 12 h compared with those cultured 36 to 60 h [44.4% (n = 9) v. 20% (n = 5)]. The kidding rates per embryo transferred of these 2 groups were 3.8% (n = 156) and 1.1% (n = 90), respectively. The PCR results confirmed that all the clones were transgenic. Phenotype characterization [e.g. gene expression, electrocardiogram (ECG), and magnetic resonance imaging (MRI)] is underway. We demonstrated successful production of transgenic goat via SCNT. To our knowledge, this is the first transgenic goat model produced for cardiovascular research. This work was supported by the Utah Science Technology and Research Initiative, Utah Multidisciplinary Arrhythmia Consortium.

211 IN VITRO PRODUCTION OF SWAMP BUFFALO EMBRYOS BY INTRACYTOPLASMIC SPERM INJECTION: EFFECT OF CHEMICAL ACTIVATION TREATMENTS

2009 ◽  
Vol 21 (1) ◽  
pp. 203
Author(s):  
Y. Y. Liang ◽  
D. N. Ye ◽  
C. Laowtammathron ◽  
T. Phermthai ◽  
R. Parnpai
Keyword(s):  
Polar Body ◽  
Chemical Activation ◽  
Cumulus Cells ◽  
Success Rates ◽  
In Vitro Production ◽  
Cell Stage ◽  
Sham Injection ◽  
Swamp Buffalo ◽  
Second Polar Body

Intracytoplasmic spern injection (ICSI) in the buffalo has not yet been well examined. Several factors involved affect the success rates of this technique, particularly the postinjection activation procedure. The objective of this study was to evaluate the effects of chemical activation treatments on in vitro development of oocytes after ICSI. A single spermatozoa was injected into the cytoplasm of an in vitro-matured oocyte using a micromanipulator under an inverted microscope. The ICSI oocytes were assigned to the following chemical activation treatments: (1) exposed to 5 μm ionomycin (Io) in Emcare medium for 5 min and placed in Emcare medium for 3 h, or (2) exposed to 7% ethanol (EtOH) in Emcare medium for 5 min and placed in Emcare medium for 3 h. The treated oocytes that extruded a second polar body were then selected and cultured either in (A) 1.9 mm 6-dimethylaminopurine (6-DMAP) in mSOF medium for 3 h, or (B) 10 μg mL–1 of cychloheximide (CHX) for 5 h. The treated oocytes were further cultured in mSOF medium supplemented with 3 mg mL–1 of fatty acid-free BSA at 38.5°C under a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 for 2 d. Thereafter, 8-cell-stage embryos were selected and co-cultured with buffalo cumulus cells in mSOF medium at 38.5°C under a humidified atmosphere of 5% CO2 in air for another 5 d. The medium was changed daily and the development of embryos was recorded at the same time the medium was changed. The sham-injected oocytes were treated and cultured along with ICSI oocytes. With 8 replications for each activation treatment, 336 oocytes were used for ICSI. With 6 replications for each activation treatment, 211 oocytes were used for sham injection. The cleavage of ICSI oocytes treated with Io + 6-DMAP, EtOH + 6-DMAP, and EtOH + CHX was 76.2, 69.4, and 78.3%, respectively, which was significant higher (P < 0.01) than ICSI oocytes treated with Io + CHX (52.4%) and also significant higher (P < 0.01) than sham-injected oocytes in all treatments. The highest blastocyst rate was observed in ICSI oocytes treated with Io + 6-DMAP (28.6%), which was not significantly different from ICSI oocytes treated with EtOH + CHX (24.4%). The blastocyst rates of ICSI oocytes treated with Io + 6-DMAP and EtOH + CHX were significantly higher than ICSI oocytes treated with Io + CHX (5.9%) and EtOH + 6-DMAP (16.5%) and also were significantly higher than sham-injected oocytes in all treatments. In conclusion, our study demonstrated that activated ICSI of swamp buffalo oocytes with Io + 6-DMAP or EtOH + CHX gave the highest cleavage and blastocyst rates. This work was supported by the Thailand Research Fund and Suranaree University of Technology.

104 DEVELOPMENTAL POTENTIAL OF CLONED EMBRYOS FROM ADULT AND FETAL OVINE SOMATIC CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 169
Author(s):  
H. M. Zhou ◽  
Y. Chen
Keyword(s):  
Skin Fibroblast ◽  
Polar Body ◽  
Subsequent Development ◽  
Fibroblast Cells ◽  
Fetal Skin ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
First Polar Body ◽  
Fetal Fibroblasts

This study reconstructed embryos using adult and fetal skin fibroblast cells as donor karyoplasts and ovine enucleated oocytes as recipient cytoplasts for comparing the developmental potential of the reconstructed embryos. Ovine ovaries were collected at a local slaughterhouse and the cumulus–oocyte complexes (COCs) were extracted from antral follicles 2 to 5 mm in diameter. A group of 20 to 30 COCs were matured in a 50-�L microdrop of maturation medium that was composed of TCM-199 supplemented with 20% FBS, 10 �g mL-1 FSH, 20 �g mL-1 LH, and 1.5 �g mL-1 17β-estradiol under mineral oil in a 35-mm petri dish in humidified atmosphere of 5% CO2 in air at 38.5�C for 18–22 h. Then oocytes with extruded first polar body (MII) were selected and enucleated for use as recipient cytoplasm. Adult and fetal ovine skin tissues were cut into small pieces (1 mm3), transferred to a 25-mL culture flask containing 2 mL DMEM-F12 medium supplemented with 10% FBS, and then incubated by using explant tissue culture in humidified atmosphere of 5% CO2 in air at 37�C for 5 to 7 days. The medium and unattached epithelial cells were discarded. The attached fibroblast cells were digested by 0.25% trypsin in D-Hanks solution at 37�C for 5 min and dispersed by pipetting. The cell suspensions were transferred to a centrifuge tube and centrifuged at 100g for 10 min. Subsequently, the recovered cells were subcultured for 4–6 passages and then frozen in DMEM-F12 medium containing 10% dimethyl sulfoxide (DMSO) and 20% FBS in liquid nitrogen. The fibroblast cells were serum-starved in DMEM-F12 supplemented with 0.5% FBS for 3 to 5 days and transferred into a micromanipulation drop consisting of H-M199 supplemented with 10% FBS and 5 �g mL-1 cytochalasin B for use. The adult and fetal skin fibroblast cells were injected into the recipient cytoplasm. The fusion of fibroblast cells into the recipient cytoplasm was induced by electrofusion (1500 V cm-1 for 40 �s two times with an interval of 0.125 s). The fused oocytes were activated by 5 mM mL-1 ionomycin with 2 mM mL-1 6-dimethylaminopurine (6-DMAP). A group of 6–10 of the activated reconstructed embryos were co-cultured with ovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% FBS in a 20-�L microdrop for 168 h. The results indicated that 76.0% (596/784) and 75.5% (249/330) of the nuclear transfer couplets were successfully fused from adult fibroblasts and fetal fibroblasts, respectively; 76.2% (454/596) and 79.5% (198/249) of the fused oocytes cleaved within 48 h after activation for adult and fetal, respectively; 26.9% (122/454) and 28.3% (56/198) of the cleaved oocytes developed to morula or/and blastocyst embryo stages, respectively. This study demonstrated that the ovine somatic cell transferred embryos were initiated for cell cycle of mitosis and underwent subsequent development to morula/blastocyst embryo stage in vitro, and that there were no statistical differences (P &gt; 0.05) in developmental capacity between the cloned embryos from adult and fetal skin fibroblast cells.

32 EFFECTS OF FUSION/ACTIVATION METHODS ON DEVELOPMENT OF EMBRYOS PRODUCED BY NUCLEAR TRANSFER OF PORCINE FETAL FIBROBLASTS

2007 ◽  
Vol 19 (1) ◽  
pp. 134
Author(s):  
P. Q. Cong ◽  
E. S. Song ◽  
E. S. Kim ◽  
Z. H. Li ◽  
Y. J. Yi ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Metaphase Plate ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Pulse Number ◽  
Donor Cells ◽  
First Polar Body ◽  
Fetal Fibroblasts

Pigs have become increasingly important in the field of biomedical research, and interest has grown in the use of transgenic cloned pigs as potential xenograft donors. The present study were carried out to investigate the effects of intensity of DC pulse, number of DC pulses, and equilibration before fusion/activation on developmental ability of porcine embryos derived from nuclear transfer. Porcine cumulus-oocyte complexes (COCs) were cultured in modified TCM-199 (mTCM-199) medium for 44 h at 38.5�C, 5% CO2 in air. After in vitro maturation (IVM), metaphase II oocytes were selected for enucleation. Porcine fetal fibroblasts were obtained from a porcine fetus on Day 35 of gestation as donor cells. Oocytes were enucleated by removing, with a micropipette, the first polar body along with adjacent cytoplasm containing the metaphase plate; then a donor cell was injected in contact with the cytoplasm of each oocyte. In experiment 1, several different fusion/activation intensities (two DC pulses of 0.4, 0.8, 1.2, 1.6, and 2.0 kV cm-1 for 30 �s) were carried out to investigate the effect on the development of nuclear transfer embryos. In experiment 2, the reconstructed oocytes were fused and activated with 1, 2, or 3 DC pulses of 1.2 kV cm-1 for 30 �s. In experiment 3, reconstructed oocytes were equilibrated in mTCM-199 medium at 38.5�C, 5% CO2 for 0, 1, 2, 3, 4, 5, and 6 h. After equilibration, the reconstructed oocytes were fused and activated with one DC pulse of 1.2 kV cm-1 for 30 �s in fusion medium. The reconstructed embryos were transferred into PZM-3 medium containing 0.3% BSA for further culture. The rates of embryo cleavage and development of blastocyst stage were evaluated at 48 h and 6-7 days, respectively. The cell numbers of blastocysts were counted by using Hoechst 33342 epifluorescence staining. Data were analyzed by ANOVA and Duncan

scholarly journals 196 IN VITRO DEVELOPMENT OF RECONSTRUCTED WATER BUFFALO (BUBALUS BUBALIS) OOCYTES AFTER FETAL SKIN FIBROBLAST CELL NUCLEAR TRANSFER

2005 ◽  
Vol 17 (2) ◽  
pp. 248
Author(s):  
C.R. Meena ◽  
S.K. Das
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Polar Body ◽  
Fibroblast Cell ◽  
Skin Fibroblasts ◽  
Developmental Competence ◽  
Fetal Skin ◽  
Cell Stage ◽  
First Polar Body

The present study was undertaken to explore the feasibility of using buffalo fetal skin fibroblasts as donor nuclei and to determine the developmental competence of embryos following transfer of these nuclei to in vitro-matured enucleated buffalo oocytes. Skin cells were isolated from 1–2-month-old fetuses, obtained from an abattoir, by enzymatic digestion (0.5% w/v trypsin + 0.05% w/v collagenase in Dulbecco's PBS) for 15–20 min. The cells were washed four times with Dulbecco's PBS and then once with RPMI-1640 medium + 10% FBS by centrifugation at 600g. The cells were then cultured in the same medium in a CO2 incubator (5% CO2 in air) at 38.5°C for 2–3 days. COCs collected from slaughterhouse buffalo ovaries were subjected to IVM in the IVM medium (TCM-199 + 1 μg mL−1 E-β + 5 μg mL−1 FSH-P + 10 μg mL−1 LH + 10% FBS) for 22–24 h in a CO2 incubator (5% CO2 in air) at 38.5°C. Oocytes were denuded with 0.1% trypsin followed by repeated pipetting and then enucleated by aspirating the first polar body and 10–15% of nearby cytoplasm with a micromanipulator. Two different types of donor cells (growing cells and those arrested with cytochalasin-B) were used for reconstruction of oocytes. The reconstructs were electrofused and incubated in the activation medium (TCM-199 + 8 μg mL−1 cytochalasin-B + 10% FBS) for 4 h. These were then cultured in IVC medium (TCM-199 + 10% FBS) in a CO2 incubator (5% CO2 in air) at 38.5°C for 48 h. Next, the cleaved embryos were co-cultured with buffalo oviductal cells in embryo development medium. Out of 119 denuded matured oocytes which were enucleated and reconstructed with growing cells, 78 (65.5%) were electrofused, activated and cultured, out of which 4 (5.1%) reconstructs cleaved and developed to the 2-cell stage, 3 (3.8%) reached the 4-cell stage, and 1 (1.3%) reached the 8-cell stage. In the synchronized group, out of 100 denuded matured oocytes which were reconstructed with cytochalasin-B blocked cells, 40 (40%) were electrofused, activated, and cultured, out of which 4 (10%) developed to the 2-cell stage, 3 (7.5%) to the 4-cell stage, 2 (5.0%) to early morula stage, and 1 (2.5%) to blastocyst stage. These results suggest that buffalo fetal skin fibroblasts could be used as donor nuclei for the production of buffalo embryos after nuclear transfer to enucleated in vitro-matured buffalo oocytes.

scholarly journals 142 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS CULTURED IN KSOM, CR1AA, OR KSOM/CR1aa

2005 ◽  
Vol 17 (2) ◽  
pp. 221 ◽  
Author(s):  
M.R.B. Mello ◽  
C.E. Ferguson ◽  
A.S. Lima ◽  
M.B. Wheeler
Keyword(s):  
Embryo Culture ◽  
Cumulus Cells ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Cell Stage ◽  
In Vitro Development ◽  
Significant Difference ◽  
Developmental Rates ◽  
Vitro Development

In vitro embryo culture is an important step of in vitro production of bovine embryos. It has been shown that IVF-derived bovine embryos cultured in KSOM or CR1aa have high development rates. In our laboratory, we have observed that 8-cell embryos are morphologically superior when embryos are cultured in KSOM whereas blastocysts are morphologically superior when embryos are cultured in CR1aa. Based on these observations, we hypothesized that development of IVF-derived bovine embryos can be improved by sequential use of these media (KSOM and CR1aa). The aim of this experiment was to compare the in vitro development of bovine embryos cultured in KSOM, CR1aa or KSOM/CR1aa supplemented with BSA at Day 0 and BSA and FBS at Day 3. In order to accomplish the sequential culture, fertilized oocytes where cultured in KSOM to the 8-cell stage and then transferred to CR1aa for further development. Oocytes were purchased from Bomed (Madison, WI, USA), and after 22 hours of maturation were fertilized with frozen-thawed semen for 5 hours at 39°C in 5% CO2. After fertilization, the presumptive zygotes were denuded from cumulus cells by votexing and were randomly allotted to one of 3 treatments: (1) cultured only in KSOM (n = 110), (2) cultured only in CR1aa (n = 102), and (3) cultured in KSOM in the first 3 days and then in CR1aa from Day 3 to Day 9 (n = 110). The embryo culture was carried out in 50-μL droplets of medium that were placed in an airtight modular incubator filled with 5% CO2, 5% O2 and 90% N2. The embryos were evaluated on Days 6 to 9 post insemination. All embryo developmental rates were calculated from presumptive zygotes. The Day 6 morula rates were 52%, 40%, and 47% for KSOM, CR1aa, and KSOM/CR1aa, respectively. The Day 7 blastocyst rates for KSOM (40%), CR1aa (25%), and KSOM/CR1aa (30%) were not significantly different; however, Day 9 hatched blastocyst rates were significantly higher (P < 0.05) for KSOM (22%) compared to CR1aa (9%) but not different from KSOM/CR1aa (14%). Regarding embryo quality, Day 7 transferable embryos rates (Grade 1 and Grade 2) were 35%, 25%, and 30%, respectively for KSOM, CR1aa, and KSOM/CR1aa; however, no significant difference was observed. These results indicate that IVF-derived bovine embryos can develop in KSOM, CR1aa, or KSOM/CR1aa with no significant difference among morula, blastocyst and hatched blastocyst rates. However, the combination of KSOM and CR1aa during in vitro culture did not decrease the morula and blastocyst rates.

251 IS THERE A GAP BETWEEN NUCLEAR AND CYTOPLASMIC MATURATION IN IN VITRO-MATURED EQUINE OOCYTES?

2015 ◽  
Vol 27 (1) ◽  
pp. 215
Author(s):  
E. Claes ◽  
K. Smits ◽  
C. De Schauwer ◽  
B. Leemans ◽  
E. Wydooghe ◽  
...  
Keyword(s):  
Fluorescent Dyes ◽  
Polar Body ◽  
Cortical Granules ◽  
In Vitro Production ◽  
General Belief ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Cytoplasmic Maturation ◽  
Specific Stimulation

It is a general belief that as soon as the oocyte is recovered from the follicular environment, the nuclear maturation starts spontaneously in vitro, while specific stimulation for the cytoplasmic maturation is lacking (Gilchrist and Thompson 2007 Theriogenology 67, 6–15; Albuz et al. 2010 Hum. Reprod. 25, 2999–3011). As both nuclear and cytoplasmic maturation are required to prepare the oocyte for successful fertilization and embryonic development, a defective cytoplasmic maturation might be an important cause of low blastocyst rates in vitro (Albuz et al. 2010 Hum. Reprod. 25, 2999–3011). Nuclear and cytoplasmic maturation can be evaluated using fluorescent dyes. Assessment of nuclear maturation is typically based on the visualisation of chromatin, whereas cytoplasmic maturation is evaluated by the localization of cytoplasmic organelles [i.e. the cortical granules (CG)]. Equine oocytes were recovered from ovaries of slaughtered mares. After in vitro maturation (IVM; Smits et al. 2010 Vlaams Diergen. Tijds. 79, 134–138), oocytes were fixed and permeabilized. Subsequently, CG were labelled by incubation in 10 µg mL–1 FITC-labelled lens culinaris agglutinin during 15 min at RT. Chromatin was counterstained to verify the nuclear status with 20 µg mL–1 Hoechst 33342 during 15 min at RT. Stained oocytes with no or dispersed chromatin were classified as degenerated. Based on the absence or presence of the first polar body (PB), non-degenerated oocytes were either classified as nuclear immature (MI, no PB present) or nuclear mature (MII, PB present). The non-degenerated oocytes were further subdivided in 3 categories based on the migration of the CG: 1) cytoplasmic immature oocytes with (clusters of) CG randomly distributed throughout the ooplasm, 2) oocytes in transition stage with progressing CG migration to the oocyte cortex, and 3) cytoplasmic mature oocytes with a clearly visible CG monolayer just underneath the oolemma. The mean and standard deviation of nuclear and cytoplasmic parameters were calculated using Excel (Excel 2007, Microsoft Corp., Redmond, WA, USA). In 3 replicates, 86.6 ± 2.75% of all oocytes (131/151) demonstrated a corresponding nuclear and cytoplasmic maturation pattern (MI corresponding to CG1 and 2; MII corresponding to CG3). Only 12.0 ± 2.82% of the oocytes (16/133) revealed a cytoplasmic maturation pattern (CG 1 or 2) that lagged behind the nuclear maturation (MII). On the other hand, 22.2 ± 9.8% of the oocytes (4/18) were already cytoplasmic (CG3) but not yet nuclear matured (MI). Since most of the equine in vitro matured oocytes exhibited, surprisingly, a corresponding nuclear and cytoplasmic maturation pattern, it can be concluded that the gap between the nuclear and cytoplasmic maturation in vitro is less important than is generally believed. Consequently, the IVM step is not the main obstacle to increase the efficiency of the in vitro production process in horses.

Do embryonic polar bodies commit suicide?

Zygote ◽  
2012 ◽  
Vol 22 (1) ◽  
pp. 10-17 ◽  
Author(s):  
Dušan Fabian ◽  
Štefan Čikoš ◽  
Pavol Rehák ◽  
Juraj Koppel
Keyword(s):  
Polar Body ◽  
Point Of View ◽  
Embryonic Cells ◽  
Cell Stage ◽  
Polar Bodies ◽  
First Polar Body ◽  
External Conditions ◽  
Living Organisms ◽  
Second Polar Body

SummaryThe extrusion and elimination of unnecessary gametic/embryonic material is one of the key events that determines the success of further development in all living organisms. Oocytes produce the first polar body to fulfill the maturation process just before ovulation, and release the second polar body immediately after fertilization. The aim of this study was to compile a physiological overview of elimination of polar bodies during early preimplantation development in mice. Our results show that three-quarters of the first polar bodies were lost even at the zygotic stage; the 4-cell stage embryos contained only one (second) polar body, and the elimination of second polar bodies proceeded continuously during later development. Both first and second polar bodies showed several typical features of apoptosis: phosphatidylserine redistribution (observed for the first time in the first polar body), specific DNA degradation, condensed nuclear morphology, and inability to exclude cationic dye from the nucleus during the terminal stage of the apoptotic process. Caspase-3 activity was recorded only in the second polar body. From the morphological point of view, mouse polar bodies acted very similarly to damaged embryonic cells which have lost contact with their neighboring blastomeres. In conclusion, polar bodies possess all the molecular equipment necessary for triggering and executing an active suicide process. Furthermore, similarly as in dying embryonic cells, stressing external conditions (culture in vitro) might accelerate and increase the incidence of apoptotic elimination of the polar bodies in embryos.

scholarly journals In vitro production of bovine blastocyst with oocyte collected from abattoir ovary

2016 ◽  
Vol 45 (1) ◽  
pp. 31-35
Author(s):  
GK Deb ◽  
SR Dey ◽  
TN Nahar ◽  
MYA Khan ◽  
MM Rahman
Keyword(s):  
Polar Body ◽  
Cumulus Cell ◽  
Morphological Characteristics ◽  
Bovine Embryo ◽  
In Vitro Production ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Development Rates ◽  
First Polar Body

This study was designed to adopt in vitro embryo production (IVP) protocol using abattoir ovary. Ovaries were collected from local abattoir; cumulus-oocyte-complexes (COCs) were aspirated from 3 to 8 mm diameter follicles using a 10 ml disposable syringe attached with a 21G needle. The COCs were selected based on morphological characteristics and selected COCs were transferred into in vitro maturation (IVM) medium for 22 to 24 hrs. The matured COC were fertilized in vitro (IVF) using fresh semen capacitated through incubation with heparin sodium salt (20 ?g/ml). After IVF, the presumed denuded zygotes were cultured in in vitro culture medium-I (IVC-I) for 3 days. After 3 days, the ?8 cell embryos were transferred into IVC-II medium until day 8 of embryonic development (day 0: day of IVF). Cleavage and blastocyst development rates were evaluated at day 3 and day 7. The maturation rates of COC were examined through detection of first polar body and cumulus cell expansion. Results showed that 74.16 ± 5.49% of the total immature COCs were matured as detected by the presence of first polar body. The diameter of matured COC was 2.21 times higher than that of the immature COC. Moreover, about 64.30 ± 6.71% COC showed full expansion of their cumulus cell. The cleavage rate of presumed zygotes was 62.05 ± 7.07%. Among the cleaved embryos, 26.67 ± 11.78%, 10.84 ± 6.13%, 22.51 ± 9.57% and 39.98 ± 5.25% embryos were at 2-, 4-, 8- and 16 to 32-cell embryonic stages, respectively at day 3. Blastocyst development rates were 14.95 ± 4.39%. This study inferred that the BLRI adopted culture system supports development of bovine embryo in vitro.Bang. J. Anim. Sci. 2016. 45 (1): 31-35

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