Effects of supplemental progesterone on the development, metabolism and blastocyst cell number of bovine embryos produced in vitro

2011 ◽  
Vol 23 (2) ◽  
pp. 311 ◽  
Author(s):  
Jamie E. Larson ◽  
Rebecca L. Krisher ◽  
G. Cliff Lamb
Keyword(s):  
Present Experiment ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryos ◽  
Morula Stage ◽  
Metabolic Data ◽  
Embryo Metabolism ◽  
Number Of Cells

The objectives of the present experiment were to determine whether supplementation with progesterone (LO, 1 ng mL–1 or HI, 100 ng mL–1) during either the first (Culture-1, Day 1 to 3) or second (Culture-2, Day 4 to 7) phase of culture of in vitro-produced embryos alters embryo development, embryo metabolism or blastocyst cell number. The percentage of oocytes that cleaved, the percentage of cleaved embryos that developed to the morula stage or greater, the blastocyst stage or greater or the hatched blastocyst stage were similar among treatments. Quantities of glucose metabolised per blastocyst per hour were similar, but when metabolic data was normalised for numbers of cells in each blastocyst, the LO treatment during Culture-2 metabolised more glucose (P = 0.03) compared with all other treatments. Embryos receiving LO progesterone tended to have greater (P = 0.085) metabolism of glucose compared with embryos receiving HI progesterone. Quantities of pyruvate oxidised per blastocyst per hour, and per cell, were similar among treatments. The number of cells per blastocyst in the control group was increased (P = 0.039) compared with cells in progesterone-treated groups. In conclusion, supplementation with progesterone during the culture of in vitro-produced embryos does not appear to improve embryo characteristics.

75 EFFECT OF PHENAZINE ETHOSULFATE ON PORCINE BLASTOCYST DEVELOPMENT, APOPTOSIS, AND CRYOTOLERANCE AFTER OPEN PULLED STRAW VITRIFICATION

2008 ◽  
Vol 20 (1) ◽  
pp. 118
Author(s):  
B. Gajda ◽  
Z. Smorag ◽  
M. Bryla
Keyword(s):  
Cell Number ◽  
Developmental Competence ◽  
Control Group ◽  
Tunel Staining ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Morula Stage ◽  
Phenazine Ethosulfate ◽  
And Control

It is possible to improve the success of cryopreservation of in vitro-produced bovine embryos by modifying the embryos with the metabolic regulator phenazine ethosulfate (PES) (Seidel 2006 Theriogenology 65, 228–235). The PES treatment increased glucose matabolism, tended to increase the pentose phosphate pathway flux of glucose, and clearly reduced accumulation of lipids in cultured bovine embryos (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 597–607). It is known that porcine embryos have a considerably high content of lipids, and the success rates of their cryopreservation appear to be highly correlated with cytoplasmic lipid content. In our preliminary study, we observed that supplementation of NCSU-23 medium with PES has a positive effect on efficiency of pig blastocysts of good quality (Gajda et al.. 2007 Acta Biochim. Pol. 54(Suppl 1), 52 abst). In the present study, the effects of PES on pig blastocyst development, apoptosis, and survival after vitrification were investigated. In Exp. 1, porcine zygotes obtained from superovulated gilts were cultured in NCSU-23 medium supplemented with 0 (control), 0.025, 0.05, or 0.075 µm PES. The culture was performed at 39�C, with 5% CO2 in air, for 96–120 h. Embryo quality criteria were developmental competence (cleavage, morula stage, and blastocyst stage), cell number per blastocyst, and the degree of apoptosis as assessed by TUNEL staining. In Exp. 2, expanded blastocysts cultured with 0.025 µm PES were vitrified in a ethylene glycol and dimethyl sulfoxide mixture using open pulled straw (OPS) technology (Vajta et al. 1997 Acta Vet. Scand. 38, 349–352). After thawing, the blastocysts were cultured in vitro for re-expansion or transferred to synchronized recipients. Data were analyzed by chi-square test. There was a difference between the 0.025 µm PES-treated and the control group in percentage of cleaved embryos (99.0 and 91.4%, respectively; P < 0.05), between all experimental groups and control in percentage of morula stage (90.7, 87.8, 83.8, and 80.0%, respectively), and between 0.025 and 0.05 µm PES-treated and control in percentage of blastocyst rates (70.0, 75.5, and 65.7%, respectively). The number of cells and percentage of TUNEL-positive nuclei per blastocyst were lower in the PES-treated than in the control group. The survival rate of blastocysts after vitrification and thawing was enhanced in the presence of PES compared to that in the PES-free group (45.2 and 38.9%, respectively; P < 0.05). After transfer of 56 expanded blastocysts cultured with PES and vitrified into 3 recipients, two gilts were confirmed pregnant at 35 days of gestation. In conclusion, a higher blastocyst percentage with a low incidence of apoptosis was obtained in the presence of PES compared to control. These blastocysts also had an increased ability to survive cryopreservation.

141 BIOPSY TECHNIQUE IN BOVINE EMBRYOS PRODUCED IN VITRO AT EARLY STAGES OF DEVELOPMENT: EVALUATION OF QUALITY AND DEVELOPMENT CAPACITY

2008 ◽  
Vol 20 (1) ◽  
pp. 151
Author(s):  
J. Polisseni ◽  
M. O. Guerra ◽  
R. V. Serapião ◽  
M. M. Pereira ◽  
I. M. Folhadella ◽  
...  
Keyword(s):  
Preimplantation Genetic Diagnosis ◽  
Embryo Quality ◽  
Genetic Diagnosis ◽  
Blastocyst Stage ◽  
Chromosome Abnormalities ◽  
Control Group ◽  
Bovine Embryos ◽  
Blastocyst Rate ◽  
Number Of Cells

One of the causes of embryo mortality is chromosome abnormalities that occur during gametogenesis, fertilization, and embryo early development. Thus, a combination of morphological standards and techniques of molecular analyses could identify abnormal embryos. Preimplantation genetic diagnosis (PGD) is an emergent technology for use with farm animal embryos. With this procedure, blastomeres are removed by the biopsy of embryos at the 8- to 16-cell stage to provide cells for analyses of chromosome abnormalities prior to transfer. The aim of this study was to evaluate the effect of biopsy in bovine 8- to 16-cell embryos fertilized in vitro on embryo quality and subsequent development in vitro. A group of 706 oocytes were obtained from slaughterhouse ovaries, matured, and fertilized in vitro at 38.8�C with 95% humidified air and 5% CO2. The zygotes were semi-denuded and cultured in CR2aa medium under the same conditions as for in vitro fertilization. The rate of cleavage was 78.20%. Three days after fertilization, part of the 8- to 16-cell (298/706) embryos were distributed randomly across two groups: control (n = 103) and biopsy (n = 92) of blastomeres, and then returned to in vitro embryo culture to evaluate development until the blastocyst stage and the capacity to hatch. The amount of cells removed was one-fourth of the embryo. The blastocyst rate was evaluated on Day 8 after fertilization and the hatching rate on Day 10. Embryo morphology and quality were evaluated as previously described in the International Embryo Transfer Society manual (1998). To evaluate overall quality, embryos were stained on the 10th day of culture and the blastomeres were counted with the imaging software AxioVision 3.1 (Carl Zeiss, Feldbach, Switzerland). The blastocyst rate was analyzed by treatment groups with the chi-square test and the number of cells/embryo was analyzed by ANOVA with SAS (SAS Institute, Inc., Cary, NC, USA). The percentage of 8- to 16-cell embryos that developed to the blastocyst stage was similar (P > 0.05) between the control (66.0%, 68/103) and the biopsied (53.3%, 49/92) groups. Furthermore, no difference was noted in the hatching rates between the control group and the biopsied group (42.6%, 29/42 v. 44.9%, 22/49, respectively). Overall, no impact was detected on embryo quality from embryo biopsy with no difference in mean (�SE) blastocyst cell number between the control group (blastocysts: 67.1 � 3.1; expanded blastocysts: 100.7 � 6.9; hatched blastocysts: 189.9 � 16.1) and the biopsied group (blastocysts: 61.1 � 5.5; expanded blastocysts: 121.87 � 10.6; hatched blastocysts: 187.3 � 18.5). In conclusion, the biopsy used on 8- to 16-cell bovine IVF-derived bovine embryos does not affect the subsequent embryo development and number of cells/embryo or blastocyst, showing that it can be used to provide genetic material for preimplantation genetic diagnosis without affecting embryo quality. This work was supported financially by FAPEMIG.

Developmental capacity of mechanically bisected mouse morulae and blastocysts

1990 ◽  
Vol 2 (6) ◽  
pp. 683 ◽  
Author(s):  
ZJ Wang ◽  
A Trounson ◽  
M Dziadek
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Implantation Rate ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Morula Stage ◽  
Developmental Capacity ◽  
Number Of Cells

Mouse embryos were mechanically bisected at the morula, early blastocyst or expanded blastocyst stages of development and cultured in vitro to the expanded blastocyst stage. Their capacity for postimplantation development was assessed after transfer to pseudopregnant foster mice. Embryos bisected at blastocyst stages had a higher survival rate in vitro than those bisected at the morula stage. Half-embryos had approximately half the number of cells at the blastocyst stage as control embryos, but the proportion of cells in the inner cell mass (ICM) was unaltered. The implantation rate of blastocysts derived from bisected embryos was only slightly lower than that of control embryos, but bisected embryos had a significantly reduced capacity to form fetuses. Histological analyses showed that failure to form a fetus is due to the absence of egg cylinder development, which correlates with the reduced number of cells in the ICM of bisected embryos. Postimplantation viability of half-embryos was significantly higher when blastocysts were transferred to Day-3 rather than Day-4 pseudopregnant recipients, presumably because of an increase in cell number in vivo prior to implantation.

scholarly journals 136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Direct Role ◽  
Treatment Groups ◽  
And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

PSX-B-11 Comparing effects of FGF2 and IGF1 on the development competence of parthenogenetic activated bovine oocytes maturing in vitro

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 327-328
Author(s):  
Galina Singina
Keyword(s):  
Growth Factor ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Maturation Medium ◽  
Bovine Oocytes

Abstract The oocyte quality acquired during in vitro maturation (IVM) are the main limitative factors affecting the embryo production. The aim of the present research was to study effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1) during IVM of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-oocyte complexes (COC; n = 1176) were cultured for 22h in either standard maturation medium (TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 10 μg/ml FSH and 10 μg/ml LH; Control) or maturation medium supplemented with different concentrations (5–160 ng/ml) of FGF2 and IGF1. After IVM, matured oocytes activated by sequential treatment with ionomycin followed by DMAP and cyclohexamide and then cultured up to the blastocyst stage. The obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using DAPI and TUNEL staining. The data from 4 replicates (77–91 oocytes per treatment) were analyzed by ANOVA. Cleavage rates of activated oocytes did not differ between groups and ranged from 63.7 to 68.1%. The addition of 10, 20 and 40 ng/ml of FGF2 to the IVM medium led to an increase in the yield of blastocysts [from 19.6±1.8% (Control) to 35.2±3.4, 29.8±1.9 and 31.1±2.1%, respectively (P&lt;0.05)] and in the total cell number in embryos that developed to the blastocyst stage (P&lt;0.05). Meanwhile, the blastocyst yield and the total cell number in blastocysts in the IGF1-treated groups were similar to that in the control group. No effects of both growth factors on the proportion of apoptotic nuclei in blastocysts (5.3–7.1%) were observed. Thus, FGF2 (but not IGF1) are able to maintain competence for parthenogenetic development of bovine COC during their maturation invitro. Supported by RFBR (18-29-07089) and the Ministry of Science and Higher Education of Russia.

168 EFFECT OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS DURING OOCYTE IN VITRO MATURATION ON BOVINE EMBRYOS (Gyr×HOLSTEIN) QUALITY

2016 ◽  
Vol 28 (2) ◽  
pp. 214
Author(s):  
G. R. Leal ◽  
C. A. S. Monteiro ◽  
H. F. R. A. Saraiva ◽  
A. J. R. Camargo ◽  
P. M. S. Rosa ◽  
...  
Keyword(s):  
Lipid Content ◽  
In Vitro Maturation ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Bovine Embryos ◽  
Tropical Conditions ◽  
Significant Difference ◽  
Number Of Cells

In vitro embryo production (IVP) is an important tool for cattle breeding. Brazilian dairy systems are based on Gyr × Holstein crossbreds, which integrates adaptability to tropical conditions and milk production. Quality determines the oocyte proportion that will develop to blastocyst stage, and although the lipid content is important in oocyte development, a high concentration in embryos is associated with cryotolerance reduction, making this a relevant issue for IVP systems. The in vitro maturation system (IVM) simulated physiological oocyte maturation (SPOM) mimics the physiological maturation events by using cyclic adenosine monophosphate (cAMP) modulators, which promote the increase of oocyte competence. Among the modulators, Forskolin has lipolytic properties. The aim of this study was to evaluate the effect of the SPOM system (Albuz 2010 Hum. Reprod. 25, 12) on bovine embryos (Gyr × Holstein) regarding their total number of cells (TNC) and lipid content. Oocytes were obtained by ovum pick-up from Gyr cows in 5 replications. After selection, they were randomly divided into 2 groups: SPOM (S) and control (C). The IVM lasted 24 h for group C (TCM 199 medium without FBS) in culture oven at 38.5°C, 5% CO2 in atmospheric air and high humidity. In the SPOM system, oocytes were in pre-IVM [TCM 199 medium + 100 µM Forskolin + 500 µM 3-isobutyl-1-methylxanthine (IBMX)] for 2 h and followed for extended IVM (TCM 199 medium + 20 µM cilostamide) for 28 h under the same conditions as control group. After IVM, oocytes were fertilised with semen from a single Holstein bull that was prepared by Percoll gradient method in Fert-TALP medium (Bioklone® Animal Reproduction, São Paulo, Brazil) for 22 h and transfered to culture droplets, where they remained for 7 days (n = 10–13 per group). The lipid content analysis was performed by staining with Oil red and the stained area fraction of each embryo was measured using software ImageJ (NIH, Bethesda, MD, USA). The TNC was measured after being stained with Hoechst 33342 and results were analysed by Student's t-test in Instat GraphPad program, with a 5% significance level. There was no significant difference (P > 0.05) between embryos from both groups on TNC (group S: 88.9 ± 28.0A; group C: 101.6 ± 29.1a) and lipid content (group S: 0.93 ± 12:18A; group C: ±0.15 to 0.96) analysis. Some studies have shown there is a beneficial effect on embryo quality when using this system; however, our results demonstrated that there was no effect on total number of cells using our conditions. Some authors have also demonstrated a reduction in embryo lipid content using Forskolin during in vitro culture. Our results suggest that the time of Forskolin exposure was not enough to ensure lipolytic action on the structures produced from oocytes (Gyr) treated in pre-IVM. It was concluded that the SPOM system had no effect on TNC and lipid content of Gyr/Holstein embryos. Financial support from FAPERJ and CAPES is acknowledged.

99 In Vitro Development of Alpaca Embryos Obtained by Bisection

2018 ◽  
Vol 30 (1) ◽  
pp. 189
Author(s):  
L. Landeo ◽  
R. S. Molina ◽  
M. E. Zuñiga ◽  
T. R. Gastelu ◽  
C. Sotacuro ◽  
...  
Keyword(s):  
Amino Acids ◽  
Streptomyces Griseus ◽  
Petri Dish ◽  
Cumulus Cells ◽  
Essential Amino Acids ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Morula Stage

The objective of this study was to evaluate the in vitro developmental competence of alpaca embryos bisected at different embryonic stages. Gametes were obtained from ovaries and testes collected from a local abattoir. Cumulus-oocyte complexes (COC) were recovered (n = 120) by aspiration of ovarian follicles using a 5-mL syringe with an 18-gauge needle. Then, COC with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured in TCM-199 supplemented with 10% FCS, FSH (0.02 IU [JM1] [P2] [P3]), and 0.01 mg mL−1 oestradiol 17β [JM4] for 26 h at 38.5°C and 5% CO2 in air. After in vitro maturation, COC were placed in a 30-mL Petri dish containing FERT-TALP solution for 30 min. Then, epididymal alpaca spermatozoa (3 × 106 mL−1) were added to the dish and co-incubated with the COC for 20 h at 38.5°C and 5% CO2 in air. Motile epididymal sperm were selected by swim-up method centrifuged for 15 min at 350 × g in 2 mL of SPERM-TALP supplemented with 6 mg mL−1 of fatty-acid-free BSA. Sperm pellet was extended and culture in 5% CO2 in air at 38.5°C for 45 min. Thirty-three viable embryos at different stages [2-cells (n = 6), 8-cells (n = 15), and morulae (n = 12)] were bisected into approximately equal halves using a micro-surgical blade. The embryos were previously treated with 2 mg mL−1 of protease from Streptomyces griseus (P 8811, Sigma, St. Louis, MO, USA) for 2 min to remove the zona pellucida. After bisection, the demi-embryos were cultivated in in vitro culture (IVC) medium containing 0.036 mg mL−1 sodium pyruvate, 0.146 mg mL−1 l-glutamine, 1% essential amino acids, 0.5% nonessential amino acids, and supplemented with 10% FCS using the well-of-the-well system. The demi-embryos were incubated for 7 days (changing the media every 48 h) in 5% CO2 in air at 38.5°C. Additional embryos (n = 60) were obtained using the same conditions described above and used as a control group (unmanipulated). We obtained 66 demi-embryos [2-cells (n = 12), 8-cells (n = 30), and morulae (n = 24)] after bisection that were considered for IVC. From 12 demi-embryos bisected at 2-cell and 30 bisected at 8-cell stages, 3 (25%) and 30 (100%) reached the morula stage respectively. However, they did not develop any further. Interestingly, 18 demi-embryos bisected in morula reached the blastocyst stage (80%). For unmanipulated embryos, we obtained 42% (25/60), 35% (21/60), 32% (19/60), and 28% (17/60) of cleavage, morulae, and blastocyst and hatched blastocyst rates, respectively. In conclusion, alpaca embryos bisected at earlier stages (less than 8-cell) are not suitable to produce blastocysts. The earliest stage to produce blastocyst from bisected alpaca embryos is the morula stage.

135 SHORT-TERM CRYOPRESERVATION OF VITRIFIED MOUSE MORULAE AT - 79°C

2007 ◽  
Vol 19 (1) ◽  
pp. 185
Author(s):  
Y. Takagi ◽  
M. Shimizu ◽  
M. Morimura ◽  
S. Yokomizo ◽  
K. Hara ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Blastocyst Stage ◽  
Control Group ◽  
Embryo Viability ◽  
Chi Square ◽  
Significant Difference ◽  
Morula Stage ◽  
Chi Square Test ◽  
Student’S T

Embryos of various species are successfully vitrified and cryopreserved in liquid nitrogen (&lt;−150°C). Like the preservation of frozen somatic cells cooled by dry ice (−79°C), the cryopreservation of embryos at −79°C is useful for a reduction in the shipping costs. The purpose of this study was to evaluate the effect of the cryopreservation period at −79°C on the in vitro embryo viability of vitrified mouse morulae after thawing. Morula-stage mouse embryos were collected from superovulated ICR donors 70 h after hCG injection. The embryos were exposed first to 5% DMSO + 5% ethylene glycol (EG) in Dulbecco's PBS + 20% FCS (mPBS) for 2 min, and then equilibrated for 20–30 s in a vitrification solution composed of 10% DMSO + 10% EG + 0.6 M sucrose in mPBS. The embryos were loaded onto cryoloops (Lane et al. 1999 Nat. Biotech. 17, 1234–1236) and plunged directly into liquid nitrogen. The cryoloops were placed in 1.2-mL cryotubes and stored in a −79°C freezer for 1–7 days. The embryos were warmed by passing through 4 dilution media and rinsed with mWM culture medium. They were then cultured at 37°C in 5% CO2 for 44 h. Non-cryopreserved embryos and embryos cryopreserved in liquid nitrogen served as controls. Data were analyzed by the chi-square test and the Student's t-test. Results are shown in Table 1. There was no significant difference (P &gt; 0.01) in the developmental abilities to the blastocyst stage of the vitrified embryos that were cryopreserved at −79°C for 1 day, 3 days, and 5 days, the embryos cryopreserved in liquid nitrogen, and the non-vitrified control. The blastocyst rate of embryos was significantly lower (P &lt; 0.01) for the Day 7 group than for the control group. The cell numbers of blastocysts were significantly lower (P &lt; 0.01) for the Day 1, Day 3, Day 5, and Day 7 groups than for the control group. This study suggests that vitrified mouse morulae can be successfully cryopreserved at −79°C for 5 days. Table 1. Effect of the cryopreservation period on the viability of vitrified mouse morulae preserved at −79°C

123 EFFECTS OF TAURO URSODEOXYCHOLIC ACID ON DEVELOPMENT OF BOVINE EMBRYOS FROM IN VITRO FERTILIZATION

2015 ◽  
Vol 27 (1) ◽  
pp. 153 ◽  
Author(s):  
F. Lu ◽  
Z. Li ◽  
Z. Ruan ◽  
X. Liu ◽  
S. Du ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Fetal Bovine Serum ◽  
Cell Number ◽  
Control Group ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Qrt Pcr ◽  
Apoptosis Index ◽  
Fetal Bovine

Endoplasmic reticulum stress (ERS) is a novel apoptotic pathway and plays an important role for embryonic development. Tauro ursodeoxycholic acid (TUDCA) is a specific chemical chaperone that can inhibit ERS. In this study, we investigated the effects of TUDCA on the development and mRNA expression of ERS-related genes in bovine embryos from IVF in order to improve the efficiency of embryo in vitro culture. Bovine oocytes collected from ovaries at slaughter were cultured in the maturation medium (TCM-199 + 26.2 mmol L–1 NaHCO3 + 5 mmol L–1 HEPES + 5% fetal bovine serum) for 24 h and fertilized in vitro with bovine sperm. After fertilization, the embryos were respectively placed into the medium (TCM-199 + 3% fetal bovine serum) containing different concentrations of TUDCA (0, 100, 250, 500, 1000 μmol L–1) and cultured in the 5% CO2 at 38.5°C. Blastocyst development was evaluated after 7 days of culture, and then the total cell number and apoptosis index of blastocysts were detected with TUNEL. In addition, X-box binding protein 1 (XBP-1) of embryos at 2-cell, 4-cell, morula, and blastocyst stages was detected with RT-PCR, and the change of the mRNA expression of ERS-related (Grp78, Ire1, Chop) and apoptosis-related (Bax, Bcl-2) genes in blastocyst collected at 7 days of culture were analysed by QRT-PCR. A total of 1336 oocytes were used in this study, and each experimental group comprised 6 replicates. The results revealed that the splicing of XBP-1 was present during the development of bovine embryos, and especially obvious at the 4-cell, morula, and blastocyst stages. When embryos were cultured in medium with different concentrations of TUDCA, compared with the control group (0 μmol L–1), more embryos developed to blastocyst stage with 500 μmol L–1 TUDCA (31.86 ± 7.32% v. 21.11 ± 8.05%; P < 0.05), but the cleavage rate was not significantly different among groups (P > 0.05). The result for TUNEL found that when adding 500 μmol L–1 TUDCA to culture, the bovine embryos significantly improved the total cell number of blastocysts (110. ± 15.21 v. 102.3 ± 8.62; P < 0.05), and the apoptosis index of blastocysts was markedly decreased (3.71 ± 0.91 v. 5.36 ± 1.92; P < 0.05) relative to the control group. Moreover, the result of QRT-PCR analysis showed that treating embryos with 500 μmol L–1 TUDCA significantly reduced the mRNA expression level of Ire1 and Chop genes (P < 0.05) and up-regulated the expression of anti-apoptotic Bcl-2 gene (P < 0.05), while down-regulated the expression of pro-apoptotic Bax gene (P < 0.05). Furthermore, XBP-1 splicing in blastocysts also abated after embryos were treated with 500 μmol L–1 TUDCA. In conclusion, ERS occurs in bovine embryos during in vitro culture, but treating embryos with 500 μmol L–1 TUDCA may reduce ERS to facilitate embryonic development. This work was funded by the China High Technology Development Program (2011AA100607), China Natural Science Foundation (31072033), and Guangxi Science Foundation (2011GXNSFA018084, 2012GXNSFFA060004).

26 EFFECT OF TREATMENT OF BOVINE DONOR CELLS WITH MOUSE EMBRYONIC STEM CELL EXTRACT ON THE DEVELOPMENT OF EMBRYOS AFTER NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 119
Author(s):  
S. Akagi ◽  
E. Mizutani ◽  
Y. Inaba ◽  
M. Kaneda ◽  
T. Somfai ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Control Group ◽  
In Vitro Development ◽  
Donor Cells ◽  
Effect Of Treatment ◽  
Vitro Development

The efficiency of somatic cell cloning is very low, probably because of incomplete reprogramming of the somatic cell nucleus. In recent studies, it is suggested that transient exposure of donor somatic cells to mouse embryonic stem cell (ESC) extract enhances pluripotency of the cells in vitro (Bru et al. 2008 Exp. Cell Res. 314, 1634–1642; Xu et al. 2009 Anat. Rec. 292, 1229–1234). In the present study, we examined the effect of treatment of donor cells with mouse ESC extract on the in vitro development of bovine NT embryos. First, in order to examine effect of treatment of donor cells with streptolysin O (SLO), which reversibly permeabilizes the plasma membrane, we compared the in vitro development of NT embryos using donor cells treated with 5 μg mL–1 SLO (SLO group) and untreated donor cells (control group). As donor cells for NT, bovine fibroblast cells of passages 3 to 5 were used. Fibroblasts were treated with 5 μg mL–1 SLO for 45 min, and then incubated for resealing in DMEM including 2 mM CaCl2 for 60 min. NT was performed as previously described (Akagi et al. 2003 Mol. Reprod. Dev. 66, 264–272). After in vitro culture for 8 days, blastocyst formation and cell number of blastocysts were examined. There were no significant differences between SLO and control groups in the fusion rate (80% and 72%, respectively), cleavage rate (60% and 65%, respectively), developmental rate to the blastocyst stage of NT embryos (31% and 28%, respectively), and blastocyst cell number (127 ± 6 and 112 ± 14, respectively). These results suggest that SLO treatment of donor cells has no negative effect on the in vitro development of NT embryos. Next, we examined the in vitro developmental ability of NT embryos using donor cells treated with mouse ESC extract (ES extract group). After SLO treatment for 45 min, permeabilized fibroblast cells were treated with mouse ESC extract for 45 min, and then incubated in DMEM including 2 mM CaCl2 for 60 min, and used for producing NT embryos. There were no differences between ES extract and control groups in the fusion rate (68% and 69%, respectively), cleavage rate (86.7% and 80.6%, respectively), and developmental rate to the blastocyst stage of NT embryos (39.8% and 43.5%, respectively). The cell number of NT embryos at the blastocyst stage in ES extract group (201 ± 30) was significantly (t-test; P < 0.05) higher than that in control group (140 ± 14). In conclusion, treatment of bovine donor cell with mouse ESC extract did not affect the in vitro developmental ability of NT embryos, but improved the quality of blastocysts.

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