Influence of oocyte selection, activation with a zinc chelator and inhibition of histone deacetylases on cloned porcine embryo and chemically activated oocytes development

Zygote ◽  
2020 ◽  
Vol 28 (4) ◽  
pp. 286-290
Author(s):  
Felipe L. Ongaratto ◽  
Paula Rodriguez-Villamil ◽  
Marcelo Bertolini ◽  
Daniel F. Carlson
Keyword(s):  
Histone Deacetylases ◽  
Sucrose Solution ◽  
Chemical Activation ◽  
Cell Number ◽  
Deacetylase Inhibitor ◽  
Blastocyst Quality ◽  
Zinc Chelator ◽  
Genomic Reprogramming ◽  
Oocyte Selection

SummaryThe aim of this study was to evaluate the effects of alternative protocols to improve oocyte selection, embryo activation and genomic reprogramming on in vitro development of porcine embryos cloned by somatic cell nuclear transfer (SCNT). In Experiment 1, in vitro-matured oocytes were selected by exposure to a hyperosmotic sucrose solution prior to micromanipulation. In Experiment 2, an alternative chemical activation protocol using a zinc chelator as an adjuvant (ionomycin + N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) + N-6-dimethylaminopurine (6-DMAP)) was compared with a standard protocol (ionomycin + 6-DMAP) for the activation of porcine oocytes or SCNT embryos. In Experiment 3, presumptive cloned zygotes were incubated after chemical activation in a histone deacetylase inhibitor (Scriptaid) for 15 h, with the evaluation of embryo yield and total cell number in day 7 blastocysts. In Experiment 1, cleavage rates tended to be higher in sucrose-treated oocytes than controls (123/199, 61.8% vs. 119/222, 53.6%, respectively); however, blastocyst rates were similar between groups. In Experiment 2, cleavage rates were higher in zygotes treated with TPEN than controls but no difference in blastocyst rates between groups occurred. For Experiment 3, the exposure to Scriptaid did not improve embryo development after cloning. Nevertheless, the total number of cells was higher in cloned zygotes treated with Scriptaid than SCNT controls. In conclusion, oocyte selection by sucrose as well as treatments with zinc chelator and an inhibitor of histone deacetylases did not significantly improve blastocyst yield in cloned and parthenotes. However, the histone deacetylases inhibitor produced a significant improvement in the blastocyst quality.

95 DEVELOPMENT OF PORCINE TWO-CELL EMBRYOS WITH OR WITHOUT ZONA PELLUCIDA AND SINGLE BLASTOMERES

2009 ◽  
Vol 21 (1) ◽  
pp. 148
Author(s):  
D. N. Q. Thanh ◽  
K. Matsukawa ◽  
M. Kaneda ◽  
S. Akagi ◽  
Y. Kanai ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Monozygotic Twins ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
First Polar Body ◽  
Blastocyst Quality ◽  
Single Blastomeres

In the mouse, single blastomeres of the 2-cell embryos can develop into adult mice and occasionally both separated blastomeres can give rise to twin animals (reviewed by Tarkowski AK et al. 2001 Int. J. Dev. Biol. 45, 591–596). As a preliminary study for production of monozygotic twins from porcine 2-cell embryos, we investigated the effects of removal of zona pellucida and blastomere isolation at the 2-cell stage on subsequent development of parthenogenetic embryos. Oocytes with the first polar body were parthenogenetically activated after 44 h of in vitro maturation. Stimulated oocytes were then incubated in IVC-PyrLac (IVC medium with pyruvate and lactose) according to the method reported by Kikuchi K et al. (2002 Biol. Reprod. 66, 1033–1041). After 24 to 30 h of parthenogenetic activation, equally cleaved 2-cell embryos were selected and used for the experiments. Some 2-cell embryos were then treated with pronase to remove the zona pellucida and cultured individually as zona-free 2-cell embryos having 2 blastomeres in pair (ZF group), and single blastomeres were split from ZF group and cultured separately (SB group) in V-shaped microwells. In addition, intact 2-cell embryos were cultured individually without pronase treatment as a control group. After 24 h of in vitro culture, IVC-PyrLac was replaced by IVC-Glu (IVC with glucose). The blastocyst rates on Day 6 (Day 0 was defined as the day of electrical stimulation) in control, ZF, and SB groups did not differ (47.6, 50.0, and 42.1%, respectively). Nevertheless, blastocysts derived from the ZF (28.6 ± 3.0) and SB groups (25.9 ± 1.3) had a significantly lower total cell number than that of the control group (41.7 ± 3.2; P < 0.01 by ANOVA). Although the total cell number of blastocysts originating from single blastomeres was significantly lower than that in the intact embryos, the blastocyst formation rates were not different between them. This indicated the possibility of production of monozygotic twins from porcine 2-cell embryos divided into 2 single blastomeres. However, further research is needed to improve blastocyst quality descended from single blastomeres. In conclusion, the removal of the zona pellucida had a negative influence on blastocyst quality but did not affect the development of porcine embryos to the blastocyst stage.

50 EFFECT OF AGGREGATION OR FUSION ON DEVELOPMENT AND CELL NUMBER OF BOVINE EMBRYOS PRODUCED BY HANDMADE CLONING AND PARTHENOGENESIS

2008 ◽  
Vol 20 (1) ◽  
pp. 105
Author(s):  
E. S. Ribeiro ◽  
R. P. C. Gerger ◽  
L. U. Ohlweiler ◽  
I. Ortigari Jr ◽  
F. Forell ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Chemical Activation ◽  
Primary Cultures ◽  
Cell Number ◽  
Total Cell ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Vitro Development ◽  
Handmade Cloning

Cloning by somatic cell nuclear transfer has been associated with developmental abnormalities, with the level of heteroplasmy imposed by cell fusion being one of many potential determining factors. As the cytoplast exerts a key role in nuclear reprogramming, embryo aggregation is an alternative to minimize such negative effects during cloning. The aim of this study was to determine the effect of fusion of hemi-cytoplasts or aggregation of hemi-embryos on in vitro development and cell number of clone and parthenote embryos. Bovine cumulus–oocyte complexes (COCs) from slaughterhouse ovaries, after 17 h of IVM, were used for the production of parthenotes by chemical activation, and clone embryos by handmade cloning (HMC) (Vajta et al. 2003 Biol. Reprod. 68, 571–578). Following cumulus and zona removal, oocytes were manually bisected, followed by segregation of nucleated and enucleated hemi-cytoplasts by fluorescence using Hoechst stain. One or two enucleated hemi-cytoplasts were paired with an adult skin somatic cell from primary cultures (>90% confluence) and fused using a 25V AC pre-pulse, followed by a single 1.2 kV cm–1 DC pulse for 10 μs. Reconstructed clone structures and groups of zona-intact oocytes and nucleated hemi-cytoplasts were chemically activated in ionomycin and 6-DMAP. Clone and parthenote structures were in vitro-cultured in the WOW system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264) for 7 days, as follows: (G1) clone embryos reconstructed by aggregation of two hemi-embryos per WOW; or (G2) one embryo (two hemi-cytoplasts + cell) perWOW; and parthenote embryos composed of (G3) zona-intact oocytes cultured in wells; or aggregation of one (G4), two (G5), three (G6), or four (G7) nucleated hemi-cytoplasts per WOW. Fusion, cleavage (Day 2), and blastocyst (Day 7) rates, evaluated on a per WOW basis, were compared by the chi-square test (8 replications). Total cell number estimated by fluorescence (Hoechst stain) in blastocysts was analyzed by the Student t-test. Fusion rates of one hemi-cytoplast + cell (G1; 275/592, 46.5%) were lower than for two hemi-cytoplasts + cell (G2; 264/337, 78.3%). Cleavage rates were lower in G1 and G4 and higher in G6 and G7 than G2 and G3. A significant linear increase in blastocyst rates was observed in G5, G6, and G7. Total cell numbers were lower in parthenotes than in clones, except in G6 and G7. The lower fusion and cleavage rates after the aggregation of two clone hemi-embryos (G1) caused nearly a 50% reduction in the overall cloning efficiency. In addition, the aggregation of parthenogenetic hemi-embryos increased cleavage and blastocyst rates and cell number. However, aggregation of hemi structures did not improve blastocyst yield or cell number on a hemi-cytoplast basis. Table 1. In vitro development of parthenote or clone bovine embryos This work was supported by funding from CAPES/Brazil.

335 EFFECT OF VITAMIN E ON THE DEVELOPMENT OF BOVINE OOCYTES AFTER CHEMICAL ACTIVATION

2007 ◽  
Vol 19 (1) ◽  
pp. 283
Author(s):  
J. I. Park ◽  
Y. Jang ◽  
E. S. Lee
Keyword(s):  
Vitamin E ◽  
Apoptotic Cell ◽  
Chemical Activation ◽  
Calf Serum ◽  
Cell Number ◽  
Total Cell ◽  
Chi Square ◽  
Cell Numbers ◽  
Humidified Air

Oxidative stress is known to induce apoptotic cell death by reactive oxygen species (ROS) generated from in vitro culture systems. This study was conducted to evaluate the effect of Vitamin E (VitE), as antioxidant, on development of bovine embryos activated in vitro. Bovine ovaries were collected from slaughtered cows at a local abattoir. Oocytes were aspirated from follicles 3-8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, 100 mg/mL-1 l-cysteine, 20 mg/mL-1 sodium pyruvate, gonadotropins (250 IU each of eCG and hCG/mL), 10 mg/mL-1 epidermal growth factor, and 100 �M VitE. Oocytes were cultured at 38.9�C in 5% CO2 in humidified air. After 22 hours of culture, oocytes with polar bodies were selected and subjected to activation treatments. Oocytes were exposed to calcium ionomycin (5 �M for 5 min), followed by incubation with 6-DMAP (2 mM) for 3.5 hours in medium supplemented with or without VitE (100 �M). After activation, oocytes were cultured in mSOF medium containing 0.8% BSA at 38.9�C in 5% CO2, 5% O2 in humidified air for 7–8 days. Cell numbers were counted by the number of nuclei of blastocysts stained with Hoechst 33342, and apoptosis was detected by TUNEL assay using a MK500 kit (Takara Bio, Inc., Otsu, Shiga, Japan). Total cell and apoptotic cell number were determined under a fluorescence microscope. Data were analyzed using Student&apos;s t-test and chi-square test. The cleavage and blastocyst rates were significantly higher (P &lt; 0.05) after activation with VitE (78.1&percnt; and 16.3&percnt;, n &equals; 80) than without VitE (66.7&percnt; and 11.0&percnt;, n &equals; 60). Total cell numbers were also significantly higher (P &lt; 0.05) in blastocysts after activation with VitE (143.0 &plusmn; 34.02, n &equals; 21) than in those without VitE (127.63 &plusmn; 40.25, n &equals; 20). However, the percentage of TUNEL-positive (apoptotic) cells was similar between blastocysts activated with VitE (5.38 &plusmn; 2.22) and those without VitE (6.76 &plusmn; 1.98). The results of the present study demonstrate that vitamin E added to activation medium promoted further development of activated embryos, although its role in the alleviation of apoptosis remains unclear.

203 EQUINE EMBRYO IN VITRO DEVELOPMENT AFTER INTRACYTOPLASMIC SPERM INJECTION FOLLOWED BY CHEMICAL ACTIVATION

2012 ◽  
Vol 24 (1) ◽  
pp. 214
Author(s):  
J. Jarazo ◽  
A. Gambini ◽  
A. De Stefano ◽  
L. Muredas ◽  
J. G. Oriol ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Chemical Activation ◽  
Chi Square ◽  
Group 4 ◽  
Blastocyst Quality ◽  
The Difference ◽  
Chi Square Analysis ◽  
Vitro Development

Intracytoplasmic sperm injection (ICSI) is an alternative method for producing in vitro-fertilized embryos in horses. Some authors have suggested that using the piezo drill to inject the spermatozoon is required to obtain acceptable blastocyst rates after ICSI. In order to avoid the use of this equipment, the aim of our study was to evaluate 4 different chemical activation protocols and their effect on embryo development. Cumulus–oocyte complexes were recovered from ovaries of slaughtered mares. The maturation medium was DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 1 μL mL–1 of insulin-transferrin-selenium, 1 mM sodium pyruvate, 100 mM cysteamine and 0.1 mg mL–1 of FSH at 39°C in a humidified atmosphere of 6.5% CO2 in air for 24 h. The ICSI was carried out in 20-μL droplets of TALP-HEPES with a 9-μm pipette, using frozen-thawed spermatozoa from 1 stallion. Spermatozoa were held separate in 100-μL droplets of Modified Whittens. Motile spermatozoa were aspirated and transferred to a 5-μL drop of 7% (v/v) polyvinylpyrrolidone, where 1 sperm was immobilized by swiping the injection pipette across its tail; then, the sperm was injected into the oocyte. All injected oocytes were subjected to 8.7 μM ionomycin for 4 min, followed by 1 of 3 further activation treatments: (1) 4-h culture in 1 mM 6-DMAP and 10 mg mL–1 of cycloheximide, starting 3 h after ionomycin; (2) 5-h culture in 10 mg mL–1 of cycloheximide, starting 10 min after ionomycin; (3) An extra incubation with 5 mM ionomycin for 4 min, starting 3 h after ionomycin. Some injected oocytes were left without a further activation protocol (group 4). After activation, injected oocytes were cultured in 100-μL droplets of DMEM/F12 with 5% of FBS at 39°C in a humidified atmosphere of 5% O2, 5% CO2 and 90% N2. Cleavage (48 h after activation) and blastocyst formation (7–8 days) of all experimental groups were assessed. Culture medium was renewed on Day 3 with fresh DMEM/F12 with 5% of FBS. At Day 9, the zona pellucida of some blastocysts was removed and the blastocysts were maintained in culture until Day 15. Blastocyst growth was determined every 24 h. Statistical differences (using chi-square analysis) were observed in cleavage with treatments 1 and 3 when compared to the other groups (1: 30/52, 58%; 2: 8/40, 20%; 3: 9/25, 36%; and 4: 10/38, 26%). There was no difference on blastocyst rates based on injected oocytes (1: 5/52, 9.6%; 2: 2/40, 5%; 3: 1/25, 4%; and 4: 2/38, 5.3%). On Day 7, blastocyst quality did not differ among treatments and on Day 15, blastocysts from groups 3 and 4 reached 1130 μm and 4300 μm, respectively. Despite the difference observed in cleavage, this work suggests that equine blastocysts could be obtained with all of the activation protocols, without the use of the piezo drill. Further studies are required to assess the effect of chemical activation on in vivo development of produced blastocysts to confirm that they are not parthenogenetic. We are grateful to Mr. Willem Melchior, La Vanguardia Polo Club for some financial support and encouragement to undertake this project.

Effect of nucleocytoplasmic ratio on the in vitro porcine embryo development after in vitro fertilization or parthenogenetic activation

Zygote ◽  
2021 ◽  
pp. 1-7
Author(s):  
Nguyen Thi Men ◽  
Thanh Quang Dang-Nguyen ◽  
Tamas Somfai ◽  
Hiep Thi Nguyen ◽  
Junko Noguchi ◽  
...  
Keyword(s):  
Formation Rate ◽  
Cell Number ◽  
Control Group ◽  
Total Cell Number ◽  
Vitro Fertilization ◽  
Blastocyst Quality ◽  
Porcine Embryo ◽  
Oocyte Cytoplasm

Summary This study was conducted to examine whether the nuclear to cytoplasmic (N/C) ratio had any influence on the timing of embryo compaction and blastocoel formation, as well as formation rate and quality of blastocyst. First, we produced embryos with increased N/C ratio by removal of approximately one-third of the cytoplasm and with decreased N/C ratio by doubling the oocyte cytoplasm with an enucleated oocyte. The initiation of compaction and cavitation in reduced cytoplasm group was significantly earlier (P < 0.05) compared with the control and doubled cytoplasm groups. The rate of blastocysts in the reduced cytoplasm and doubled cytoplasm groups was significantly lower (P < 0.05) compared with the control group. Blastocyst quality in terms of total cell number in the reduced cytoplasm group was significantly lower (P < 0.05) compared with the doubled cytoplasm group, but not different from the control group. Next, we produced embryos with various N/C ratios by oocyte fusion combined with cytochalasin D treatment. The onset of compaction and cavitation in the 2N/2C group (decreased N/C ratio) was significantly delayed (P < 0.05) or had the tendency to be delayed (P = 0.064), respectively, compared with the control group (2N/1C). A significantly higher rate of blastocyst was observed in the 4N/2C group compared with the 1N/1C group (P < 0.05) but not different from the remaining groups. These results demonstrated that an increase in N/C ratio caused an earlier occurrence of morula compaction and blastocyst formation in both in vitro fertilization (IVF) and parthenogenetically activated pig embryos.

362 DIFFERENT IN VITRO DEVELOPMENT AFTER AGGREGATION OF BOVINE AND FELINE PARTHENOGENETIC EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 269
Author(s):  
A. De Stefano ◽  
A. Gambini ◽  
D. Salamone
Keyword(s):  
Cumulus Cells ◽  
Cell Number ◽  
Total Cell ◽  
In Vitro Development ◽  
Cell Numbers ◽  
Blastocyst Quality ◽  
Vitro Development ◽  
Parthenogenetic Embryos

Embryo aggregation has been shown to improve embryo development in several species. However, the effects seem to be different among species. Thus, the aim of this study was to compare the effect of embryo aggregation over in vitro development and blastocyst quality of bovine and feline parthenogenetic (PA) embryos. To this aim, bovine cumulus-oocyte complexes (COC) were collected from slaughterhouse ovaries, whereas cat ovaries were obtained from ovariectomized animals. The COC were in vitro matured in TCM199 supplemented following standard protocols for each species. After 24 h, cumulus cells and zona pellucidae were removed. Matured oocytes were selected and activated by 5 µM ionomycin treatment for 4 min followed by incubation in 1.9 mM 6-DMAP. Bovine and feline PA embryos were cultured in SOF medium in the well of well system in two different groups: only one PA embryo per microwell (1X); and three PA embryos per microwell (3X, aggregated embryos). Cleavage and blastocyst rates from all groups were assessed at Days 2 and 7, respectively. Size of blastocysts was measured at Day 7 using a millimetre eyepiece, and total cell number was determined by Hoechst 33342 staining. Blastocyst rates and embryo size were analysed by Fisher's test (P < 0.05) and total cell numbers by Kruskal–Wallis test with Dunn's correction (P < 0.05). Statistical differences were found in PA blastocyst rates between experimental groups (1X: 15/104, 24.6% v. 3X: 27/37, 62.2% for feline; and 1X: 21/113, 19.4% v. 3X: 20/32, 62.5% for bovine), but no differences were found between species. In addition, there was no statistical difference in the number of blastocysts obtained per oocyte used in any of the experimental groups. Bovine aggregated PA blastocysts were significantly larger than non-aggregated embryos (>200 microns, 1X: 2/20, 10% v. 3X: 9/19, 47.4%), but no differences were found in cell number. On the other hand, cat aggregated PA blastocysts had significantly higher cell numbers (1X: 122.4 ± 79.66 cells v. 3X: 259.8 ± 137.1 cells), but no differences were found in blastocyst size. This observation can contribute in the understanding of embryo physiology, suggesting that benefits of embryo aggregation in parthenogenic embryos vary among these species.

Aggregation of Leopardus geoffroyi hybrid embryos with domestic cat tetraploid blastomeres

Reproduction ◽  
2021 ◽  
Vol 161 (5) ◽  
pp. 539-548
Author(s):  
Matteo Duque Rodriguez ◽  
Andrés Gambini ◽  
Laura D Ratner ◽  
Adrian J Sestelo ◽  
Olinda Briski ◽  
...  
Keyword(s):  
Endangered Species ◽  
Cell Number ◽  
Domestic Cat ◽  
Control Group ◽  
Embryonic Cells ◽  
Total Cell Number ◽  
Blastocyst Quality ◽  
Developmental Rates ◽  
Cat Model

Heterospecific embryo transfer of an endangered species has been carried out using recipients from related domestic females. Aggregation of an embryo from an endangered species with a tetraploid embryo from the species to be transferred could improve the development of pregnancy to term. The main objective of the present study was to analyze embryo aggregation in domestic cat model using hybrid embryos. For this purpose, we compared in vitro development of synchronic (Sync) or asynchronic (Async) and asynchronic with a tetraploid (Async4n) aggregation of domestic cat IVF embryos. Furthermore, aggregated blastocyst quality was analyzed by evaluation of the total cell number, cell allocation by mitotrackers staining of embryonic cells, expression of Oct4, Nanog, Sox2, Cdx2 genes, number of OCT4+ nuclei, and presence of DNA fragmentation. Additionally, the developmental rates of Async4n aggregation of domestic cat with Leopardus geoffroyi hybrid (hLg) embryos were evaluated. Async aggregation increased blastocyst cell number and the number of OCT4+ nuclei as compared to non-aggregated diploid (2n) and tetraploid (4n) embryos. Moreover, blastocysts produced by Async4n aggregation showed reduced rates of fragmented DNA. No differences were found in the expression of the pluripotent genes, with exception of the Cdx2 expression, which was higher in 4n and aggregated embryos as compared to the control group. Interestingly, hybrids embryos derived by Async4n aggregation with domestic cat embryos had similar rates of blastocysts development as the control. Altogether, the findings support the use of two-cell-fused embryos to generate tetraploid blastomeres and demonstrate that Async4n aggregation generates good quality embryos.

24 EFFECT OF TREATMENT WITH TRICHOSTATIN A ON IN VITRO DEVELOPMENT OF BOVINE NUCLEAR-TRANSFERRED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 130 ◽  
Author(s):  
S. Akagi ◽  
K. Fukunari ◽  
K. Matsukawa ◽  
S. Watanabe ◽  
S. Takahashi
Keyword(s):  
Trichostatin A ◽  
Chemical Activation ◽  
Calcium Ionophore ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Fibroblast Cells ◽  
In Vitro Development ◽  
Vitro Development

It has been reported that 5 or 50 nM trichostatin A (TSA) treatment after somatic cell nuclear transfer (NT) improves the success rate of mouse cloning (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189). In this study, we examined the effect of TSA treatment on the in vitro development of bovine NT embryos. As donor cells for NT, bovine fibroblast cells of passages 3 to 5 were used following culture in serum-starved medium for 5 to 7 days. Oocytes were enucleated after in vitro maturation in TCM-199 supplemented with 10% fetal bovine serum. Enucleated MII oocytes were fused with fibroblast cells by a DC pulse of 25 V/150 µm for 10 µs in Zimmerman mammalian cell fusion medium. Fused oocytes were activated by 10 µM calcium ionophore for 5 min, followed by incubation with 2.5 µg mL−1 cytochalasin D, 10 µg mL−1 cycloheximide, and 5 or 50 nM TSA for 1 h, and then cycloheximide and 5 or 50 nM TSA for 4 h. After chemical activation, NT embryos were cultured in IVD-101 (Research Institute of Functional Peptide Co., Ltd., Yamagata, Japan) with 5 or 50 nM TSA for 10 h and subsequently cultured in IVD-101 without TSA. Control NT embryos were cultured in the same medium without TSA after fusion. After in vitro culture for 8 days, blastocyst formation and cell numbers of blastocysts were examined. The fusion rate of enucleated oocytes with fibroblast cells was 81% (199/247). In vitro development of NT embryos is summarized in Table 1. There were no differences in the cleavage rate and development rate to the blastocyst stage of NT embryos among control, and 5 and 50 nM TSA treatments. The cell number of 50 nM TSA-treated NT embryos at the blastocyst stage was higher than that of control NT embryos without TSA treatment. In conclusion, 50 nM TSA treatment for 15 h after activation did not affect the in vitro developmental competence, but increased total cell number in bovine NT embryos. These results suggest that TSA treatment may improve the quality of blastocysts in bovine NT. Table 1. Effects of TSA treatment on in vitro development of NT embryos derived from fibroblast cells

48 DEVELOPMENTAL COMPETENCE OF CLONED OR PARTHENOGENETICALLY ACTIVATED PORCINE EMBRYOS: EFFECT OF DIAMETER OF PREPUBERTAL GILT OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 130
Author(s):  
J. Li ◽  
J. Adamsen ◽  
R. Li ◽  
H. Pedersen ◽  
Y. Liu ◽  
...  
Keyword(s):  
Chemical Activation ◽  
Cell Number ◽  
Visual Observation ◽  
Total Cell ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Total Cell Number ◽  
Mean Diameter ◽  
Blastocyst Rate

One of the primary factors influencing the developmental ability of cloned embryos is the oocyte′s diameter (Hirao et al. 1994 J. Reprod. Fertil. 100, 333–339). However, the oocyte donor's age (i.e. its sexual maturity) is also important to consider, because a high proportion of immature oocytes can be expected (Ikeda and Takahashi 2003 Reprod. Fertil. Dev. 15, 215–221). The present study was to investigate the effect of diameter of oocytes collected from prepubertal gilts weighing 100 to 120 kg on the developmental ability of cloned and parthenogenetically activated (PA) embryos. Cumulus–oocyte complexes collected from ovaries of prepubertal gilts were in vitro matured for 42 to 44 h as described for sow oocytes (Li et al. 2008 Theriog 70, 800–808). After removal of the cumulus cells, the matured oocytes were sorted into 2 groups based on visual inspection: large (L) and small (S) oocytes, whereas non-sorted oocytes were used as control (C). In addition, 1 batch from each of the 3 groups of oocytes had their mean size measured. Subsequently, all 3 groups were used for handmade cloning (HMC; Li et al. 2009 Reprod. Domest. Anim. 44, 122–127) or parthenogenetic activation (PA; Kragh et al. 2005 Theriogenology 64, 1536–1545). Then a chemical activation with 5 μg mL–1 cytochalasin B and 10 μg mL–1 cycloheximide in PZM-3 medium was applied for 4 h on both HMC and PA embryos. Finally, the activated embryos were washed and cultured in PZM-3 medium using the WOW system. The embryo development was evaluated by cleavage rate (Day 2), blastocyst rate (Day 6), and total cell number in blastocysts. Data were analysed by ANOVA with single factor in Excel (Microsoft Excel 2007, Redmond, WA, USA). The results showed (Table 1) that by simple visual observation, oocytes could be easily sorted into the following groups: L group (mean diameter 110 μm, from 105 to 116 μm), S group (mean diameter 101 μm, from 93 to 106 μm) and C group (mean diameter 107 μm, from 93 to 116 μm). Cleavage rates and total cell number were similar in the 3 groups. However, the blastocyst rate in L group either for HMC or PA was higher than S group. The data confirm that prepubertal gilt oocytes are useful for cloning and PA, but developmental rates can be increased by selection of large oocytes by simple visual observation. Table 1.Data analysis results

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