37 THE EFFECTS OF A CLASS III HISTONE DEACETYLASE INHIBITOR (SIRTINOL) ON EARLY DEVELOPMENT OF PORCINE CLONED EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 166
Author(s):  
S.-S. Kwak ◽  
Y. Jeon ◽  
J. D. Yoon ◽  
S.-A. Cheong ◽  
E. Lee ◽  
...  
Keyword(s):  
Treatment Group ◽  
Embryonic Development ◽  
Trichostatin A ◽  
Cell Number ◽  
Total Cell ◽  
Class Iii ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Total Cell Number ◽  
Significant Difference

Previous studies have demonstrated that treatment of cloned embryos with trichostatin A (TSA) or scriptaid, inhibitors of class I and II histone deacetylases (HDAC), significantly enhanced their developmental competence. In the present study, we investigated the effects of sirtinol, an inhibitor of class III HDAC, on the embryonic development of porcine cloned embryos. Data were analyzed with SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) using Duncan’s multiple range test and all experiments were replicated at least 5 times. In experiment 1, 648 parthenotes were divided into 4 groups (0-, 6-, 12-, and 24-h sirtinol treatment after activation) to investigate optimal treatment time using 100 µM sirtinol. The cleavage rate of the 24-h treatment group (81.3%) was significantly (P < 0.05) decreased compared with the 12-h treatment group (88.4%) but there was no difference compared with the control (86.9%) and 6-h treatment groups (86.9%). The parthenotes treated with sirtinol for 12 h after activation had a significantly higher blastocyst formation rate and total cell number in blastocysts (50.5% and 66.9, respectively) than the control (39.4% and 54.1, respectively). In experiment 2, 806 cloned embryos were divided into 5 groups (0, 50, 100, 150, and 200 µM sirtinol treatment for 12 h after activation) to investigate optimal concentration. There was no significant difference in cleavage rate. The rate of blastocyst formation and total cell number in blastocysts were significantly (P < 0.05) improved by treatment with 150 µM sirtinol for 12 h after activation (28.8% and 51.0, respectively) compared with the control (17.5% and 37.1, respectively). The total cell number in blastocysts was also significantly increased in 50 and 200 µM groups (47.9 and 48.4, respectively) compared with the control (37.1). In experiment 3, we examined the effects of 150 µM sirtinol treatment for 12 h after activation with or without 5 nM TSA on in vitro embryonic development after somatic cell nuclear transfer. The rate of blastocyst formation was significantly improved in sirtinol-treated and TSA-treated groups (30.9 and 31.3%, respectively) but not in the sirtinol with TSA group (27.6%) compared with the control (21.7%). The total cell number in blastocysts was significantly increased by treatment with sirtinol and TSA together (73.9) compared with the control (49.0) but there was no difference in only sirtinol- (59.8) or TSA- (59.2) treated groups. There was no significant difference in cleavage rate among groups. Our results suggest that sirtinol improves the embryonic development of porcine cloned embryos and sirtinol with TSA synergistically increases the blastocyst quality. This work was supported by a grant from the Next-Generation BioGreen 21 program (no. PJ008121012011), Rural Development Administration, Republic of Korea.

250 HUMAN EXHALED AIR CAN EFFECTIVELY SUPPORT IN VITRO MATURATION OF PORCINE OOCYTES AND SUBSEQUENT IN VITRO DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 223
Author(s):  
Z. B. Cao ◽  
L. C. Sui ◽  
S. F. Ji ◽  
J. W. Chen ◽  
T. Gui ◽  
...  
Keyword(s):  
Formation Rate ◽  
Cell Number ◽  
Total Cell ◽  
High Oxygen ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Nuclear Maturation ◽  
Air Atmosphere ◽  
Significant Difference

The objective of the present study was to examine the feasibility of culturing porcine oocytes and embryos in vitro using the human exhaled lung air atmosphere. In Experiment 1, the effects of lung air atmosphere on nuclear maturation of prepubertal gilt oocytes and subsequent development in vitro of parthenogenetic-activated and somatic-cell-cloned embryos were explored. Abattoir-derived prepubertal gilt cumulus–oocyte complexes (COC) were matured in TCM-199 supplemented with 10 IU mL–1 of eCG, 10 IU mL–1 of hCG, 10 ng mL–1 of epidermal growth factor, and 10% porcine follicular fluid (pFF) for 40 to 44 h at 38.5°C, 100% humidity, and 5% CO2+20% O2 (high oxygen tension) or human exhaled air encapsulated in plastic, airtight bags (lung air) or 5% CO2+7% O2 (low oxygen tension) in the incubator. Nuclear maturation was evaluated by the presence of the 1st polar body. For parthenogenetic activation, denuded oocytes with the 1st polar body were selected and stimulated with a single 1.6-kV/cm, 100-μs direct current pulse followed by culture in porcine zygote medium-3. For NT, denuded metaphase II oocytes were enucleated, and then the donor cell was directly injected into the perivitelline space. After NT, reconstructed couplets were fused and activated electrically followed by treatment in 7.5 μg mL–1 of cytochalasin B and 10 μg mL–1 of cycloheximide for 4 to 6 h before culture in porcine zygote medium-3. We found no significant difference among groups in terms of nuclear maturation rate (66.5% v. 60.2%, 63.2%), cleavage rate (94.8% v. 94.2%, 85.2%), blastocyst formation rate (39.5% v. 40.3%, 32.5%), and total cell number (37 v. 38, 32). Moreover, as for porcine cloned embryo, no significant difference between the lung-air and high-oxygen (20% O2) groups was observed in the cleavage rate (88.3% v. 80.3%), blastocyst formation rate (7.3% v. 10.7%), and total cell number (34 v. 36). The above results indicated that porcine oocytes can be matured in vitro safely and efficiently using the human exhaled lung air atmosphere. In Experiment 2, in vitro developmental competence of porcine zona-free parthenogenetically activated embryos cultured in a lung air, low oxygen (5% O2), or high oxygen (20% O2) tension gas environment was studied. We found no obvious difference among the 3 groups regarding the rates of cleavage (83.0%, 83.6%, 82.8%), but blastocyst formation rate (26.8% v. 48.6%, 48.2%) and total cell number (23 v. 34, 29) in lung air were lower than those in the rest of the groups (P < 0.05). The results show that lung air could be an alternative for preparing a gas environment for in vitro culture of porcine zona-free parthenotes, although not an ideal alternative. Taken together, porcine oocytes and embryos can be cultured in vitro safely and efficiently using the human exhaled lung air atmosphere. Z. B. Cao and L. C. Sui contributed equally to this work. X. R. Zhang and Y. H. Zhang are the corresponding authors. This work was supported by NSFC (30700574), 863 (2008AA101003).

158 ASTAXANTHIN EFFECTS ON MATURATION, FERTILIZATION, AND DEVELOPMENT OF PORCINE OOCYTES CULTURED UNDER HEAT STRESS

2014 ◽  
Vol 26 (1) ◽  
pp. 193 ◽  
Author(s):  
L. T. K. Do ◽  
V. V. Luu ◽  
Y. Sato ◽  
M. Taniguchi ◽  
T. Otoi
Keyword(s):  
Heat Stress ◽  
Dna Fragmentation ◽  
Cell Number ◽  
Total Cell ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Control Group ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Maturation Medium ◽  
Significant Difference

Heat stress can engender various disorders in reproductive functions such as impairment of oocyte maturation, fertilization, and embryonic development. Astaxanthin, an extremely common carotenoid, is a typical fat-soluble antioxidant that scavenges ROS and blocks lipid peroxidation. Moreover, astaxanthin has been shown to improve the development of embryos exposed to heat stress by a reduction in stress-inducible genes. This study was conducted to investigate the effects of astaxanthin supplementation on the meiotic competence, fertilization, and development of porcine oocytes exposed to high temperature (41°C) during maturation culture. Cumulus–oocyte complexes (COC) collected from ovaries were transferred into maturation medium supplemented with astaxanthin (0, 0.25, 0.5, or 1.0 ppm) and were then cultured for 46 h at 41°C or 38.5°C. After maturation culture, the COC were subjected to IVF and embryo culture to evaluate the fertility and development of oocytes. The total cell number and DNA fragmentation in the blastocysts were assessed using terminal deoxynucleotidyl transferase dUTP nick end labelling and Hoechst 33342 staining. The total numbers of oocytes matured at 41°C and 38.5°C in each treatment group were 432 to 470 and 426 to 444, respectively. Data were analysed using ANOVA, followed by Fisher's protected least significant difference test. Exposure to elevated temperatures during maturation culture significantly reduced the proportions of oocytes that reached metaphase II. When the COC were cultured in the maturation medium supplemented with 0.5 and 1.0 ppm of astaxanthin under heat stress conditions (41°C), the supplementation of astaxanthin significantly improved the proportions of maturation, fertilization, and blastocyst formation compared with the control group (0 ppm) (50–52%, 45–55%, and 11–12% v. 17, 25, and 6%, respectively). The supplementation of the maturation medium with 0.25 ppm of astaxanthin improved only blastocyst formation (9.6%). Similarly, the supplementation of astaxanthin at 1.0 ppm improved the proportions of maturation, fertilization, and blastocyst formation of oocytes matured at 38.5°C s compared with the control group (67, 57, and 18% v. 48, 33, and 12%, respectively). However, no beneficial effect of astaxanthin supplementation was found in the total cell number or DNA fragmentation in the blastocysts, irrespective of culture temperature. Our findings show that the supplementation of astaxanthin to maturation medium improves maturation, fertilization, and embryo development of porcine oocytes exposed to heat stress during maturation culture.

166 Paracrine Regulation of Epidermal Growth Factor-Like Factors in Oviduct Cells and its Effect on Oocyte Maturation and Subsequent Embryo Development

2018 ◽  
Vol 30 (1) ◽  
pp. 222
Author(s):  
S. H. Lee ◽  
E. M. N. Setyawan ◽  
B. C. Lee
Keyword(s):  
Growth Factor ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Cell Number ◽  
Total Cell ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Significant Difference ◽  
Epidermal Growth ◽  
Oviduct Cell

Progesterone (P4) and progesterone receptor signalling appears essential for maintenance of a proper cumulus cell expansion during the oocyte maturation by regulating the epidermal growth factor-like factors (EGF-F) related pathway during the ovulatory process. It is known that expression of EGF-F including amphiregulin (AREG), epiregulin (EREG), and betacellulin (BTC) is critical for cumulus–oocyte complex (COC) expansion and resumption of meiosis. Therefore, we hypothesised that oviduct cells might be involved in nonexclusive mechanisms of actions of P4 that in turn modulate oocyte meiosis resumption by regulating the levels of EGF-F. First, we added different concentrations of P4 (0, 0.5, 1, and 2 μg mL−1) to oviduct cell culture medium and assessed the effect of P4 on expression of AREG, EREG, and BTC in oviduct cells by immunocytochemical analysis. Then, the oviduct cells were used for co-culturing under the proper concentration of P4 with porcine oocytes. The COC were randomly cultured in 3 groups: (1) culturing without oviduct cells, (2) co-culturing with oviduct cells, and (3) co-culturing with oviduct cells treated with P4. After IVM, extrusion of the 1st polar body was observed under the microscope. To evaluate embryo development competence, the matured oocytes were activated with electrical stimulus and parthenotes were cultured in porcine zygote medium-5 for 7 days at 39°C, 5% CO2 and O2 in a humidified atmosphere. The cleavage and blastocyst formation rates were observed under the microscope to evaluate developmental competence. To count the total cell number of blastocysts, they were stained with 5 μg mL−1 of Hoechst 33342 for 10 min. The data were analysed by one-way ANOVA using GraphPad Prism 5.0 (GraphPad Inc., San Diego, CA, USA). Values are means ± standard error of mean (P < 0.05). Significantly higher levels of EGF-F were observed in oviduct cells treated with 1 μg mL−1 progesterone. The oocyte maturation rate of co-culture group treated with P4 (80.7 ± 1.6%) was significantly higher than that of the control (69.7 ± 2.1%). There was a significant difference between co-culture treated with P4 and the control in cleavage rate (67.2 ± 2.4% and 82.0 ± 1.6%). However, no significant difference was observed between the co-culture groups. The co-culture treated with P4 group showed significantly higher rate of blastocyst formation (37.7 ± 0.8%) and total cell number of blastocyst (72.8 ± 1.0) than control and co-culture groups. In conclusion, co-culturing with oviduct cell treated with P4 improved oocyte maturation and subsequent embryo development. Thus, we suggested that oviduct cells induce the expression of EGF-F under the treatment of P4, which has a beneficial effect on porcine oocyte development. This research was supported by NRF-20142A1021187, Korea IPET (#316002-05-2-SB010), RDA (#PJ010928032017) and Research Institute for Veterinary Science, the BK21 plus program.

27 EFFECT OF A SPECIFIC INHIBITOR OF DOT1L ON PREIMPLANTATION DEVELOPMENT OF PORCINE SOMATIC CLONED EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 106
Author(s):  
J. Tao ◽  
Y. Zhang ◽  
D. Song ◽  
Y. Li ◽  
Y. Zhang
Keyword(s):  
Treatment Group ◽  
Specific Inhibitor ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Cleavage Rate ◽  
Total Cell Number ◽  
In Vitro Development ◽  
Vitro Development

EPZ004777 (EPZ), a specific inhibitor of DOT1L (a methyltransferase of H3K79), can significantly improve the generation and quality of mouse induced pluripotent stem cells [Onder et al. 2012 Nature 483(7391), 598–602), suggesting that H3K79 dimethylation (H3K79me2) is involved in controlling cell pluripotency. To date, however, it is unclear whether H3K79me2 regulates development competency of animal cloned embryos. Thus, we aimed to examine the dynamic changes of H3K79me2 in pre-implantation cloned embryos of pigs, and to explore effect of EPZ treatment of embryos on in vitro development fate in order to lay the foundation for revealing the role of H3K79me2 and mechanisms in controlling cell pluripotency. Porcine cloned embryos were treated immediately when fusion and activation stimuli were conducted, in vitro with porcine zygote medium (PZM)-3, including 0.5, 5, or 50 nM EPZ (S7353, Selleck Chemicals, Houston, TX, USA) and 1‰ DMSO (vol/vol, control group) for 24 h, respectively. Then, they were transferred into fresh PZM-3 without EPZ. We found that there was no significant difference in cleavage rate among groups, whereas the blastocyst rate of 0.5 nM EPZ group was higher than that of control group [28.97 ± 2.65% (28/96) v. 17.13 ± 2.69% (17/96)]. No obvious difference was observed for the total cell number of blastocyst among groups. We further treated the SCNT embryos with 0.5 nM EPZ for 0 (control group), 12, 24, and 36 h, respectively. No significant differences were found for cleavage rate among groups, whereas the blastocyst rates of the 12- and 24-h groups were significantly higher than that of control and 36-h groups [28.56 ± 3.51% (27/97), 28.34 ± 3.00% (25/88) v. 16.32 ± 1.93% (16/97), 17.93 ± 0.64% (18/100)]. Except for the remarkable decrease in the 36-h treatment group, no obvious difference was observed for the total cell number of blastocyst among the other 3 groups. All the above experiments were repeated at least 3 times. These results suggested that treatment of porcine SCNT embryos with 0.5 nM EPZ for 12 to 24 h could improve their development during the early stage. Then, we tested whether the EPZ favoured the in vitro development of porcine SCNT embryos by regulating H3K79me2 reprogramming. Porcine SCNT embryos were treated with 0.5 nM EPZ from the onset of electric activation and fusion stimuli was performed, and then the H3K79me2 signal (by immune-fluorescent staining) and expression of DOT1L (by RT-qPCR) at different development stages was analysed. We found that the H3K79me2 signal in control group (without EPZ treatment) decreased slowly from the time of electric stimulation to 4 hpa, and it disappeared in 8 hpa stage. In the EPZ treatment group, H3K79me2 signal started decreasing from 2 hpa, and disappeared in 8 hpa stage. The mRNA level of DOT1L in EPZ treatment group was lower than that in control group, although the difference was not significant. Taken together, treatment with EPZ at the appropriate concentration and for an appropriate time can improve the early in vitro development of pig SCNT embryos, probably by inhibiting expression of DOT1L and facilitating reprogramming of H3K79me2.Research was supported by NSFC No. 31272442.

Short-term treatment with 6-DMAP and demecolcine improves developmental competence of electrically or Thi/DTT-activated porcine parthenogenetic embryos

Zygote ◽  
2010 ◽  
Vol 19 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Sol Ji Park ◽  
Ok Jae Koo ◽  
Dae Kee Kwon ◽  
Ma Ninia Limas Gomez ◽  
Jung Taek Kang ◽  
...  
Keyword(s):  
Formation Rate ◽  
Cell Number ◽  
Term Treatment ◽  
Total Cell ◽  
Blastocyst Formation ◽  
Short Term ◽  
Cleavage Rate ◽  
Short Term Treatment ◽  
Total Cell Number ◽  
Long Term Treatment

SummaryTreatment with 6-dimethylaminopurine (6-DMAP) or demecolcine (DE) for several (at least 2) hours after artificial activation is known to improve in vitro development of porcine embryos. However, several reports have also shown that treatments with these chemicals induce apoptosis. The aim of this study was to find out whether short-term treatment with 6-DMAP and DE combined with electrical or thimerosal/dithiothreitol (Thi/DTT) activation had a beneficial effect on development of parthenogenetically activated porcine oocytes. We additionally treated embryos with 6-DMAP (2 mM) and/or DE (0.4 μg/ml) for a short time (40 min) after an electrical pulse (EP) or Thi/DTT. As a result, short-term treatment with 6-DMAP and DE successfully induced development of electrically or Thi/DTT-activated porcine parthenogenetic embryos with no significant difference in cleavage rate, blastocyst formation rate and total cell number compared with long-term treatment. To find optimal activation protocol, cleavage rate, blastocyst formation rate and total cell number were compared between EP and Thi/DTT treatments. Thi/DTT + 6-DMAP + DE showed significantly higher blastocyst formation rate (36.1 ± 3.5%) and total cell number (46.9 ± 1.0) than other groups (EP + 6-DMAP + DE, EP + Thi/DTT + 6-DMAP + DE: 23.3 ± 3.0%, 42.2 ± 1.1 and 17.2 ± 2.7%, 36.7 ± 1.5, respectively). In conclusion, this study demonstrates that short-term treatment with 6-DMAP and DE is as effective as the standard long-term treatment and Thi/DTT + 6-DMAP + DE exerts a synergistic effect.

scholarly journals 260 REPLACEMENT OF PVA WITH FETAL BOVINE SERUM IMPROVES FORMATION AND HATCHING OF PORCINE BLASTOCYSTS PRODUCED IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 280 ◽  
Author(s):  
K. Yoshioka ◽  
C. Suzuki ◽  
H. Rodriguez-Martinez
Keyword(s):  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Cell Number ◽  
Total Cell ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Significant Difference ◽  
Fetal Bovine ◽  
Hatching Rates

Porcine embryos, derived from in vitro maturation and fertilization, were used to investigate the effects of timing of serum inclusion and PVA replacement in the medium for in vitro culture (IVC) on rates of blastocyst formation and hatching. In Experiment 1, presumptive zygotes at 20 h post-insemination (hpi) or cleaved embryos obtained by culture in porcine zygote medium (PZM-5) containing 3 mg mL−1 polyvinyl alcohol (PVA) at 48 or 96 hpi were further cultured in either PZM-5 containing PVA or PZM-5 where PVA was replaced by 1%, 5%, or 10% fetal bovine serum (FBS) until Day 6 (Day 0 = the day of in vitro insemination). Supplementation with 1% to 10% FBS at 20 and 48 hpi reduced (P < 0.05; by ANOVA and Fisher's PLSD test) blastocyst rates on Days 5 (0% to 1%) and 6 (3% to 6%) compared with PVA supplementation (4% and 22%, respectively). However, addition of 10% FBS at 96 hpi increased (P < 0.05) blastocyst rates (30%) on Day 5 compared with PVA (11%) and 1% FBS (15%); there was no significant difference among treatments in rates of blastocyst formation on Day 6 (24% to 40%). The total number of blastomeres in Day 6 blastocysts did not differ among treatments at any timing of serum supplementation (26.5 to 48.3 cells). In Experiment 2, presumptive zygotes were cultured from 20 to 96 hpi in PVA medium, and the cleaved embryos were later transferred into PZM-5 containing PVA, or 1%, 5%, or 10% FBS for another 4 days. Hatching rates of embryos on Days 7 and 8 were significantly higher (P < 0.05) in PZM-5 where PVA was replaced with 10% FBS (15% and 20%, respectively) than those in PZM-5 containing PVA (1% and 5%, respectively). Moreover, the total cell number in hatching/hatched blastocysts on Day 8 were significantly greater (P < 0.05) in medium containing 10% FBS (135.1 cells) than that in PVA medium (77.0 cells). In Experiment 3, at 130 hpi, blastocysts derived from IVC with PZM-5 containing PVA were transferred into PZM-5 containing PVA, 3 mg mL−1 bovine serum albumin (BSA) or 10% FBS for another 2 days. Hatching rates of blastocysts on Days 6, 7 and 8 were significantly higher (P < 0.05) in PZM-5 where PVA was replaced with 10% FBS (12%, 56%, and 64%, respectively) than those in PZM-5 containing PVA (0%, 12%, and 20%, respectively) and BSA (0%, 12%, and 20%, respectively). Moreover, the total cell number in hatching/hatched blastocysts on Day 8 were significantly greater (P < 0.05) in medium containing 10% FBS (138.7 cells) than that in PVA (71.7 cells) and BSA medium (70.7 cells). The results indicate that the timing of serum inclusion in the culture medium markedly affects porcine embryo development in vitro and that replacement of PVA with FBS in PZM-5 at 96 hpi or later improves the subsequent development of embryos to the hatching/hatched blastocyst stage. This work was supported by MAFF, Japan, and STINT and FORMAS, Sweden.

Effect of vitrification technique and assisted hatching on rabbit embryo developmental rate

Zygote ◽  
2009 ◽  
Vol 17 (1) ◽  
pp. 57-61 ◽  
Author(s):  
M. Popelková ◽  
Z. Turanová ◽  
L. Koprdová ◽  
A. Ostró ◽  
S. Toporcerová ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Assisted Hatching ◽  
Hatching Rate ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Significant Difference ◽  
Rabbit Embryos

SummaryThe aim of the study was to determine the efficiency of two vitrification techniques followed by two assisted hatching (AH) techniques based on post-thaw developmental capacity of precompacted rabbit embryos and their ability to leave the zona pellucida (hatching) during in vitro culture. The total cell number and embryo diameter as additional markers of embryo quality after warming were evaluated. In vivo fertilized, in vitro cultured 8–12-cell rabbit embryos obtained from superovulated rabbit does were cryopreserved by two-step vitrification method using ethylene glycol (EG) as cryoprotectant or by one-step vitrification method with EG and Ficoll (EG+Ficoll). Thawed embryos were subjected to enzymatic or mechanical AH. Vitrified EG group showed significantly lower (P < 0.05) blastocyst rate (22.5%) and hatching rate (15%) than those vitrified with EG + Ficoll (63 and 63% resp.) and that of control (97 and 97% respectively). Significantly lower values of total cell number (P < 0.05) as well as embryo diameter (P < 0.01) in EG group compared with EG + Ficoll and control group were recorded. No significant difference was found in developmental potential of warmed embryos treated by either mechanical or enzymatic AH. The present study demonstrates that the EG + Ficoll vitrification protocol provides superior embryo survival rates over the EG vitrification protocol for 8–12-cell stage precompacted rabbit embryos. No positive effect of either mechanical or enzymatic AH on the post-thaw viability and quality of rabbit embryos in vitro was observed.

171 Effect of progesterone and prolactin on the developmental competence of bovine oocytes during the terminal phase of in vitro maturation

2019 ◽  
Vol 31 (1) ◽  
pp. 210
Author(s):  
G. Singina ◽  
E. Shedova ◽  
T. Taradajnic ◽  
V. Konnova ◽  
E. Tsyndrina
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Terminal Phase ◽  
Cleavage Rate ◽  
Total Cell Number ◽  
One Step ◽  
Group 2 ◽  
The One

To date, considerable progress has been achieved in in vitro production (IVP) technologies in cattle; however, developmental potentials of oocytes matured in vitro remain low compared with in vivo-matured oocytes. Thus, a better understanding of different aspects of oocyte maturation may allow us to increase the embryo development rate. Our study was aimed to assess the effects of progesterone (P4) and prolactin (PRL) on the bovine oocyte developmental competence. Bovine cumulus-enclosed oocytes (CEO) were matured using either one-step or two-step maturation conditions. For the one-step protocol, CEO were cultured for 24h in TCM-199 supplemented with 10% fetal calf serum (FCS), 10μg mL−1 porcine FSH, and 10μg mL−1 ovine LH (standard medium). For the two-step procedure, CEO were first cultured for 16h in the standard medium (n=1263) and then transferred to 1 of 3 experimental media and cultured for additional 8h in either absence or presence of either P4 (50 ng mL−1) or bovine PRL (50ng mL−1). The 3 media tested in the two-step maturation were (1) TCM-199 containing 10% FCS (group 1), (2) TCM-199 containing 3mg mL−1 BSA (group 2), or (3) Fert-TALP medium supplemented with 6mg mL−1 BSA (group 3). Fert-TALP was selected because it can potentially be used throughout maturation and fertilization. Following in vitro maturation, all oocytes underwent an IVF/in vitro culture procedure as described previously (Singina et al. 2014 Reprod. Fertil. Devel. 26, 154). The embryo development was evaluated at Days 2 and 7 for cleavage and blastocyst rates. In addition, obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using 4′,6-diamidino-2-phenylindole (DAPI) and TUNEL staining. The data from 4 to 5 replicates (113-159 oocytes per treatment) were analysed by ANOVA. For oocytes matured for 24h in the one-step culture, the cleavage rate, blastocyst rate, total cell number, and apoptotic nuclei per blastocyst were 66.1±1.1, 23.7±2.0, 71.4±9.1, and 4.8±1.2%, respectively. For the two-step culture, the cleavage rate did not differ from that of the one-step culture system, ranging from 64.8 to 76.5%. Also, no effects of the two-step systems were observed on total cell number (63.0-78.8) or the proportion of apoptotic nuclei (3.3-5.3%) at the blastocyst stage. The culture of CEO in group 1 (without the supplements) had a reduced blastocyst rate (17.4±0.4%; P&lt;0.05) compared with the standard one-step maturation group, and the addition of P4 (but not PRL) improved the blastocyst yield (P&lt;0.05). Furthermore, when P4 (but not PRL) was added to group 2 and group 3 media, blastocyst rates increased significantly (32.9±3.1 and 32.8±2.7%, respectively) compared with those of the one-step group (P&lt;0.05), but did not differ from those of untreated groups 2 and 3 (26.2±2.7 and 30.0±3.0%, respectively). Our data indicate that P4 supplementation during the terminal phase of two-step IVM can enhance the developmental competence of bovine oocytes and that the nature of this effect depends on the composition of IVM medium, whereas no effect of PRL supplementation was observed. The study was supported by RFBR (No. 17-29-08035).

65 EFFECT OF TRICHOSTATIN A AND 5-AZA-2'-DEOXYCYTIDINE TREATMENT OF DONOR CELLS, FUSED EMBRYOS, OR BOTH ON THE DEVELOPMENTAL COMPETENCE, QUALITY, AND EPIGENETIC STATUS OF CLONED BUFFALO (BUBALUS BUBALIS) EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 162
Author(s):  
M. Saini ◽  
N. L. Selokar ◽  
H. Agrawal ◽  
S. K. Singla ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Species Conservation ◽  
Bubalus Bubalis ◽  
Cell Number ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Epigenetic Modifiers ◽  
Donor Cells ◽  
Significant Difference ◽  
Transgenic Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology in buffalo for multiplication of elite animals, species conservation, and production of transgenic embryos for therapeutic applications. However, the cloning efficiency obtained in this species is very low, which might be due to improper reprogramming of donor cells after SCNT. Treatment of donor cells or fused embryos or both with epigenetic modifiers might be a suitable approach to improve the ability of donor cells to be reprogrammed. The present study was aimed at examining the effects of treatment of donor cells (24 h before SCNT) or fused embryos (10 h post-electrofusion) or both with 50 nM TSA + 7.5 nM 5-aza-dC on the developmental competence, quality, and epigenetic status of buffalo embryos produced by hand-made cloning (HMC) as described earlier (Saini et al. 2014 Reprod. Fertil. Dev. doi: 10.1071/RD14176). The percentage data were analysed using SYSTAT 12.0 (SPSS Inc., Chicago, IL, USA) after arcsine transformation. Differences between means were analysed by one-way ANOVA followed by Fisher’s least significant difference test. The blastocyst rate was significantly higher (P < 0.05) and the apoptotic index was significantly lower (P < 0.05) in embryos produced from donor cells or fused embryos or both treated with TSA + 5-aza-dC than that of controls (Table 1). However, the cleavage rate and the total cell number were not significantly different among all the groups. The global level of H3K18ac, examined by immunofluorescence staining, was higher (P < 0.05) and that of H3K27me3 was lower (P < 0.01) in blastocysts produced from donor cells or fused embryos or both treated with TSA + 5-aza-dC than that of controls. These results show that treatment of donor cells, fused embryos, or both with TSA + 5-aza-dC improves the developmental competence and quality, and alters the epigenetic status of buffalo embryos produced by HMC. However, the effects of treatment with these epigenetic modifiers on the pregnancy rate require further studies. Table 1.Effect of treatment of donor cells, fused embryos, or both with 50 nM TSA + 7.5 nM 5-aza-dC on the developmental competence and level of apoptosis in cloned embryos

235 A COMPARISON OF PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES SYNCHRONISED DURING MATURATION BY CYCLOHEXIMIDE OR cAMP

2009 ◽  
Vol 21 (1) ◽  
pp. 215
Author(s):  
W. C. Chen ◽  
J. Zhu ◽  
P. Fisher ◽  
D. Amarnath ◽  
K. H. S. Campbell
Keyword(s):  
Time Window ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Chi Square ◽  
Parthenogenetic Development ◽  
High Level

In vitro maturation of porcine oocytes is characterized by a high level of asynchrony between oocytes. Previous studies reported that cycloheximide (CHX) and 3′, 5′-cyclic AMP (cAMP) synchronize porcine oocytes and improve development to blastocyst stage following IVF or have been used for somatic cell nuclear transfer (SCNT) (Ye et al. 2005 Biol. Reprod. 72(2), 399–406; Betthauser et al. 2000 Nat. Biotechnol. 18(10), 1055–1059). We previously reported that cAMP was more effective than CHX in synchronizing porcine oocyte maturation, producing MII oocytes in a shorter time window and providing a more homogenous population for future SCNT studies (Chen et al. 2008 SRF conference, 2008 abst, p34). Here we compared parthenogenetic development of porcine oocytes synchronized by these two treatments. Selected cumulus–oocyte complexes (COC) obtained from slaughtered gilts were randomly divided into three groups and cultured at 39°C, 5% CO2 in air in modified NCSU-23 medium (with 1 μm glutathione, 1 mm cysteine, 5 mg L–1 insulin, 10 ng mL–1 epidermal growth factor, 10% (v/v) porcine follicular fluid, 1% essential and 0.5% nonessential amino acids) ± hormones (10 IU mL–1 PMSG and 10 IU mL–1 hCG): (1) with hormones for the first 22 h and then without hormones until 44 h; (2) with hormones and 5 μg mL–1 CHX for 12 h, and then with hormones but no CHX until 44 h; (3) with hormones and 1 mm cAMP for 22 h, and then without hormones and cAMP until 44 h. Parthenogenetic development of cycloheximide and cAMP treated oocytes was compared by cleavage rate at 48 h postactivation (hpa) and blastocyst formation at 168 hpa. No significant differences were observed in the frequency of cleavage (96.7 ± 2.1% v. 81.4 ± 11.6% v. 84.5 ± 5.7%), development to blastocyst (28.3 ± 11.4% v. 27.1 ± 5.7% v. 32.8 ± 5.3%) between control, CHX or cAMP treated oocytes, respectively (chi-square test, P > 0.05). However, total cell number was significantly higher in the CHX group than cAMP group (42.7 ± 4.1 v. 31.8 ± 2.0, respectively; t-test, P < 0.05). The results demonstrate that synchronization of porcine oocytes by treatment with CHX or cAMP does not affect subsequent parthenogenetic development if judged by the blastocyst formation, although the meaning of the difference of total cell numbers between CHX and cAMP treatments is still unclear.

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