scholarly journals Birth of pups after transfer of mouse embryos derived from vitrified preantral follicles

Reproduction ◽  
2002 ◽  
pp. 593-600 ◽  
Author(s):  
EC dela Pena ◽  
Y Takahashi ◽  
S Katagiri ◽  
EC Atabay ◽  
M Nagano
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Subsequent Development ◽  
Developmental Competence ◽  
Preantral Follicles ◽  
Cell Complexes ◽  
Vitrification Solution ◽  
First Time

Preantral follicles mechanically isolated from the ovaries of 12-day-old mice were exposed to 2 mol ethylene glycol l(-1) for 2 or 5 min and then to a vitrification solution containing 6 mol ethylene glycol l(-1) and 0.3 mol raffinose l(-1) for 0.5, 1.0 or 2.0 min before vitrification. The vitrified and fresh preantral follicles were treated with collagenase, and the oocyte-granulosa cell complexes (OGCs) obtained were cultured in vitro for 10 days in membrane inserts. Preantral follicles exposed to 2 mol ethylene glycol l(-1) for 5 min and then to the vitrification solution for 0.5 or 1.0 min showed the highest survival rates after warming. The follicular loss after warming was approximately 20%. After in vitro culture, the proportion of viable OGCs from the vitrified follicles was 10% lower than that of the fresh preantral follicles. There were no differences in the rates of maturation, fertilization and subsequent development to blastocysts between the oocytes derived from vitrified follicles and those derived from fresh preantral follicles; however, the developmental competence of the oocytes derived from both vitrified and fresh preantral follicles grown in vitro was lower than that of oocytes grown in vivo. One of the five recipient mice that received 20 blastocysts derived from vitrified preantral follicles gave birth to six live pups. The results of the present study demonstrate for the first time that mouse preantral follicles can be vitrified and that some of the embryos derived from vitrified preantral follicles can develop to live pups.

scholarly journals Transient meiotic arrest maintained by DON (6-diazo-5-oxo-l-norleucine) enhances nuclear/cytoplasmic maturation of porcine oocytes

Reproduction ◽  
2019 ◽  
Vol 158 (6) ◽  
pp. 543-554
Author(s):  
Hae-Jun Yang ◽  
Sanghoon Lee ◽  
Bo-Woong Sim ◽  
Pil-Soo Jeong ◽  
Seon-A Choi ◽  
...  
Keyword(s):  
Formation Rate ◽  
Developmental Competence ◽  
Cortical Granules ◽  
Meiotic Arrest ◽  
Cellular Survival ◽  
Cytoplasmic Maturation ◽  
First Time ◽  
A Current

The developmental competence of in vitro-matured oocytes is still lower than that of the in vivo-matured oocytes due to precocious meiotic resumption and inappropriate cytoplasmic maturation. Although numerous efforts have been attempted to accomplish better in vitro maturation (IVM) condition, only limited progress has been achieved. Thus, a current study was conducted to examine the effects of 6-diazo-5-oxo-l-norleucine (DON, an inhibitor of hyaluronan synthesis) during the first half period of IVM on nuclear/cytoplasmic maturation of porcine oocytes and subsequent embryonic development. Based on the observation of the nucleus pattern, metaphase II (MII) oocyte production rate in 1 µM DON group was significantly higher than other groups at 44 h of IVM. The 1 µM of DON was suggested to be optimal for porcine IVM and was therefore used for further investigation. Meiotic arrest effect of DON was maximal at 6 h of IVM, which was supported by the maintenance of significantly higher intra-oocyte cAMP level. In addition, increased pERK1/2 levels and clear rearrangement of cortical granules in membrane of MII oocytes matured with DON provided the evidence for balanced meiosis progression between nuclear and cytoplasmic maturation. Subsequently, DON significantly improved blastocyst formation rate, total cell numbers, and cellular survival in blastocysts after parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. Altogether, our results showed for the first time that 1 µM DON can be used to increase the yield of developmentally competent MII oocytes by synchronizing nuclear/cytoplasmic maturation, and it subsequently improves embryo developmental competence.

75 ADDITION OF HYALURONAN TO A VITRIFICATION SOLUTION FOR IMMATURE BOVINE OOCYTES SELECTED BY BRILLIANT CRESYL BLUE

2013 ◽  
Vol 25 (1) ◽  
pp. 185
Author(s):  
P. Rodriguez Villamil ◽  
F. Ongaratto ◽  
M. Fernandez Taranco ◽  
G. A. Bó
Keyword(s):  
Ethylene Glycol ◽  
Solid Phase ◽  
Animal Health ◽  
Survival Rates ◽  
Control Group ◽  
Cresyl Blue ◽  
Development Rates ◽  
Vitrification Solution ◽  
Bovine Oocytes

An experiment was designed to evaluate the effect of brilliant cresyl blue (BCB) selection of immature oocytes and the addition of sodium hyaluronate (HA) to the vitrification solution on survival rates of bovine oocytes vitrified using solid-phase vitrification. Bovine cumulus–oocyte complexes (COC; n = 716) obtained from slaughterhouse ovaries were used in 6 replicates. Cumulus–oocyte complexes were washed in tissue culture medium 199 (TCM-199) and randomly allocated to 2 groups to be exposed to BCB stain (Sigma Chemical Company, St. Louis, MO, USA) for 90 min as described by Alm et al. (2005 Theriogenology 63, 2194–2205) or (control) maintained in Vigro holding medium (Bioniche Animal Health, Belleville, Canada) for 90 min (n = 220). Cumulus–oocyte complexes in the BCB group were selected based on their response to BCB as BCB+ (colored, n = 248) or BCB– (colorless, n = 248), whereas those in the control group were selected morphologically as described by Rodríguez-González et al. (2002 Theriogenology 57, 1397–1409). Oocytes from both BCB groups and 100 oocytes in the control group were vitrified by solid-phase vitrification as previously described by Rodriguez et al. (2012 Reprod. Fertil. Dev. 24, 132). The remaining 120 oocytes in the control group were not vitrified and were matured, fertilized, and cultured in vitro (in SOFaa in a controlled atmosphere) for 7 days. Vitrified oocytes were exposed to 10% ethylene glycol for 10 min, and 20% ethylene glycol + 0.2-M trehalose for 30 s, and then were subdivided to be exposed to 30% ethylene glycol + 0.5-M trehalose with or without 0.1 mg mL–1 HA (MAP 5, Bioniche Animal Health). Vitrified oocytes were stored in liquid nitrogen for at least one week and then placed directly into a 0.5-M sucrose solution (in TCM 199) at 37°C for 5 min, 0.25 M of sucrose for another 5 min, and finally TCM-199 and matured, fertilized, and cultured. Development rates (i.e. proportion of blastocysts) were examined on Day 7 after fertilization. Proportional data were first transformed by square root and then analyzed by ANOVA to detect the effect of replicate, type of oocyte (BCB+, BCB–, controls), and vitrified with or without HA or not vitrified as main effects, using the software Infostat (UNC, Argentina, 2010). There was a significant effect of oocyte type on blastocyst rate (P < 0.01) following vitrification (BCB+, 6.4 ± 0.4%. v. BCB–, 1.6 ± 0.6%). Control oocytes (not exposed to BCB) resulted in 3.0 ± 2.0% blastocysts following vitrification, which was lower to that obtained with the BCB+ oocytes. Vitrification also influenced development rates (3.0 ± 2.0 v. 32.0 ± 1.3%) for blastocysts produced from vitrified v. nonvitrified oocytes, respectively (P < 0.01). Furthermore, the use of HA in the vitrification solutions did not have a significant effect on development rates (4.7 ± 0.9 v. 3.3 ± 0.9%, for blastocysts obtained from vitrified oocytes with or without HA, respectively). In conclusion, the selection of oocytes by BCB increased the in vitro development rates of vitrified immature oocytes, whereas the use of HA in the vitrification solution did not improve the survival rates of vitrified oocytes.

313 PRE-IN VITRO MATURATION WITH CYCLIC AMP AND CYCLIC GMP MODULATORS IMPROVES DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 245 ◽  
Author(s):  
N. W. Santiquet ◽  
A. F. Greene ◽  
W. B. Schoolcraft ◽  
R. L. Krisher
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Subsequent Development ◽  
Developmental Competence ◽  
Nuclear Maturation ◽  
Oocyte Developmental Competence ◽  
Oocyte Collection ◽  
Short Period

In vitro maturation (IVM) of cumulus-oocyte complexes (COC) results in oocytes with reduced quality and is still not as efficient as in vivo maturation in most species. One hypothesis that could explain the low developmental competence of oocytes following IVM is that the oocytes resume meiosis too quickly after being retrieved from the follicles. Studies in mice and bovine have shown that a short period of prematuration in the presence of cAMP modulators, before IVM, enhances oocyte developmental competence. Moreover, other studies have recently demonstrated that cGMP is also a crucial molecule involved in meiotic resumption. Here, our objective was to examine the effect of a cGMP modulator in combination with a cAMP modulator during a short period of prematuration on mouse oocyte nuclear maturation and subsequent embryo development following IVF. The COC were collected (6 replicates) from 2-month-old outbred CF1 mice 48 h after PMSG (5 IU) injection in the presence (pre-IVM) or absence (control) of cGMP and cAMP modulators. Pre-IVM COC (n = 184) were then placed in prematuration medium that also contained these cGMP and cAMP modulators. After 2 h, pre-IVM COC were washed and transferred to our in-house prepared, completely defined IVM medium (Paczkowski et al. 2014 Reprod.) for the remaining 16 h of culture; 10 oocytes per 50 µL drop under oil, at 37°C in 7.5% CO2 and 6.5% O2 due to the increased altitude at our location. Control COC (n = 161) were matured in the same IVM medium under identical conditions for 18 h, without prematuration. After IVM, oocytes were fixed for assessment of nuclear maturation, or fertilized and cultured in vitro and subsequent development (96 and 112 h) was recorded (Paczkowski et al. 2014 Reprod.). Results were analysed by ANOVA. A short 2-h prematuration period in the presence of cGMP and cAMP modulators had no impact on oocyte nuclear maturation to metaphase II after IVM or on embryo cleavage after IVF. However, pre-IVM treatment improved the developmental competence of the oocyte, as demonstrated by increased embryo development. More (P < 0.02) blastocysts (96 h of culture) and hatched blastocysts (112 h of culture) developed in the pre-IVM treatment compared to control (31.0 ± 3.4 v. 19.9 ± 3.2%; 31.5 ± 3.4 v. 19.9 ± 3.2%, respectively). In conclusion, a combination of cGMP and cAMP modulators during oocyte collection and a subsequent short pre-IVM improves oocyte developmental competence and could therefore be a potential tool to improve embryo yield following IVM.

Co-culture of buffalo (Bubalus bubalis) preantral follicles with antral follicles: a comparative study of developmental competence of oocytes derived from in vivo developed and in vitro cultured antral follicles

Zygote ◽  
2012 ◽  
Vol 21 (3) ◽  
pp. 286-294 ◽  
Author(s):  
G. Taru Sharma ◽  
Pawan K. Dubey ◽  
Amar Nath ◽  
G. Saikumar
Keyword(s):  
Growth Factor ◽  
Bubalus Bubalis ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Term Survival ◽  
Preantral Follicles ◽  
Antral Follicles ◽  
Epidermal Growth

SummaryThe present study was undertaken to examine whether the presence of antral follicles (AFs) affects the survival, growth and steroidogenesis of preantral follicles (PFs) and compare the maturation and developmental competence of buffalo oocytes derived from in vivo developed and in vitro cultured AFs. Two experiments were carried out. In experiment I, PFs (200–250 μm) were isolated and cultured with or without AFs (3–5 mm) in TCM-199 medium that contained 10% fetal bovine serum (FBS), 1% insulin transferin selenium (ITS), 20 ng/ml epidermal growth factor (EGF), 0.5 μg/ml follicle-stimulating hormone (FSH) and 100 ng/ml insulin-like growth factor (IGF)-I. In experiment II, in vitro developmental competence was compared for the cumulus–oocyte complexes (COCs) recovered from in vivo developed and in vitro cultured AFs. Survival, growth, development of antrum, accumulation of estradiol and progesterone was (P < 0.05) higher when PFs were co-cultured with AFs. Developmental competence of both types of follicular oocytes did not differ significantly in terms of maturation and cleavage rate, but morula and blastocyst production rate were (P < 0.05) higher with in vivo developed AFs as compared with the in vitro cultured antral follicular oocytes. In conclusion, co-culture of PFs with AFs supports long-term survival and growth of buffalo PFs and this co-culture system plays a dual role for in vitro production of embryos as well as understanding the relationship between developing PFs and AFs.

40 THE EFFECT OF EXPOSURE TIME ON TOXICITY OF VITRIFICATION SOLUTION ON PORCINE CUMULUS–OOCYTE COMPLEXES BEFORE IN VITRO MATURATION

2017 ◽  
Vol 29 (1) ◽  
pp. 127 ◽  
Author(s):  
R. Appeltant ◽  
T. Somfai ◽  
E. C. S. Santos ◽  
K. Kikuchi
Keyword(s):  
Ethylene Glycol ◽  
Propylene Glycol ◽  
Exposure Time ◽  
In Vitro Maturation ◽  
Subsequent Development ◽  
Control Group ◽  
Dibutyryl Camp ◽  
Porcine Oocyte ◽  
Vitrification Solution

Although offspring have been produced from porcine cumulus-oocyte complexes (COC) vitrified at the immature stage (Somfai et al. 2014 PLoS One 9, e97731), embryo development rates have remained low. Numerous vitrification protocols are reported with a wide variation in the applied exposure time to the vitrification solution. Because cryoprotectants in the vitrification solution can be detrimental to the oocytes and their subsequent development, it is important to verify the effect of their exposure time to the COC. In this study, we compared the development of a control group with 3 toxicity control (TC) groups in which COC were exposed to the vitrification solution for 30 s, 1 min, or 1.5 min (TC1, TC2, and TC3, respectively) at 38.5°C. Before exposure, the COC were rinsed and equilibrated in 7 µg mL−1 cytochalasin B. The equilibration solution consisted of 2% (vol/vol) ethylene glycol + 2% (vol/vol) propylene glycol and the vitrification solution contained 17.5% (vol/vol) ethylene glycol + 17.5% (vol/vol) propylene glycol, 50 mg mL−1 polyvinylpyrrolidone and 0.3 M sucrose. The COC were not exposed to liquid nitrogen. After washing in a warming solution of 0.4 M sucrose at 42°C, COC were washed in a sucrose gradient from 0.2 to 0.0 M. Subsequently, the COC were subjected to in vitro maturation in porcine oocyte medium. During the first 20 h of in vitro maturation, the porcine oocyte medium was supplemented with 10 IU mL−1 eCG, 10 IU mL−1 hCG, 1 mM dibutyryl cAMP, and 10 ng mL−1 epidermal growth factor. Then, the medium was replaced with dibutyryl cAMP-free porcine oocyte medium for an additional 28 h. After in vitro maturation, oocytes were parthenogenetically activated (Day 0) and cultured for 7 days in porcine zygote medium. Survival, nuclear maturation, cleavage, and blastocysts rates (Days 6 and 7) were assessed. All parameters were statistically analysed by binary logistic regression. Only the survival rate of TC3 was significantly lower than that of the control group (89.2 v. 95.6%). Exposure to cryoprotectants significantly decreased maturation rates in TC1, TC2, and TC3 compared with the control (72.6%, 75.2%, 76.3% v. 86.1%). Cleavage rates were significantly lower in TC2 and TC3 than that in the control (82.8% and 81.7% v. 92.9%). Concerning blastocyst rates on Day 6 and Day 7 of in vitro culture, only TC1 could reach the same level as the control, expressed on the total number of activated oocytes (54.6% v. 67.7%, and 64.0% v. 72.9%, respectively) as well as expressed on the cleaved oocytes (61.4% v. 72.4% and 72.0% v. 78.0%, respectively). Consequently, despite the reduced maturation rate, TC1 provides the same quantity of blastocysts from matured oocytes as the control. In conclusion, exposure to the vitrification solution for longer than 30 s has toxic effects on COC and therefore is not recommended for vitrification. R. Appeltant is an International Research Fellow of the JSPS Japan (P15402).

scholarly journals 119CRYOPRESERVATION OF BIOPSIED BOVINE EMBRYOS PRODUCED IN VITRO USING ARABINOGALACTAN AND 1.5M ETHYLENE GLYCOL

2004 ◽  
Vol 16 (2) ◽  
pp. 182
Author(s):  
B. Shangguan ◽  
N. Yang ◽  
R. Vanderwal ◽  
M.D. Darrow
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Field Trials ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Ag Addition ◽  
Freezing Medium ◽  
Frozen Embryos

Arabinogalactan (AG) in combination with 1.5M ethylene glycol (EG) has been used successfully in cryopreserving biopsied in vivo bovine embryos (Darrow, 2002 Theriogenology 57(1), 531). This study was undertaken to investigate the efficiency of AG addition in a freezing medium (FM) to cryopreserve biopsied bovine embryos produced in vitro (IVP). Blastocysts of grade 1 were collected at Days 7 and 8 post-insemination. After biopsy with a small blade, embryos were transferred to CR1aa medium and cultured for 2 hours (h) before being frozen. In experiment 1, a group of unbiopsied embryos were handled in a manner similar to that used for the biopsied embryos. Embryos were frozen using either 1.5M EG+0.1M sucrose (EG+) (AB Technology, Pullman, WA, USA) or a FM containing 1.5M EG and different concentrations of AG (AG1, 2 and 3, courtesy of AB Technology). Embryos remained in FM for 10 (exp.1), 5 (exp.2), 5 and 10 (exp.3) or 5, 10, and 20 (exp.4) minutes before being loaded into a freezer and cooled down to −35°C at 0.3°C/min. Frozen embryos were thawed (35°C, 20 seconds) and cultured in CR1aa at 38.5°C for 3 days. Embryo survival rates (S%) were recorded at 24, 48 and 72h post-thawing. Data were compared with t-test or ANOVA procedures using SigmaStat 3.0. Results from exp.1 (Table) indicate that biopsied and unbiopsied embryos survived well in EG+ or AG2. While the biopsy procedure did not affect the post-thaw S% of embryos in either FM, no significant differences were observed between embryos frozen with EG+ and AG2 (P=0.055). Reducing or increasing AG concentration in FM by 2-fold (AG1 and 3, respectively) did not significantly affect the post-thaw S% at 24h (EG+, 80.0%, n=133; AG1, 83.3%, n=135; AG2, 71.4%, n=137 and AG3, 75.0%, n=135; P=0.217, exp.2). However, shortened exposure from 10 to 5 minutes to AG2 resulted in an improvement in S% at 24h, from 35.7% (n=80) to 61.4% (n=82, P&lt;0.05; exp.3). When AG1 (=0.5×AG2) was used in the FM the S% at 24h after different exposure times was not significant (5 minutes, 77.8%, n=179; 10 and 20 minutes, 66.7%, n=179 and 183; P=0.472, exp.4). This study demonstrates that addition of AG to the FM effectively sustains the viability of biopsied IVP embryos during freezing and any potential harmful impact of AG on embryo survival can be minimized by reducing AG concentration or the time of embryo exposure to AG prior to freezing. Further studies are needed to determine optimal AG concentration. Currently, field trials are underway to evaluate the ability of AG medium to promote pregnancies from frozen, biopsied IVP embryos. Table 1 Post-thaw survival rates of biopsied IVP embryos frozen in ethylene glycol with sucrose (EG+) and a FM containing arabinogalactan (AG2). Data are means±SEM

scholarly journals Survival rates of mouse blastocyst vitrified in dimethylformamide based solutions associated with ethylene glicol or 1-2 propanediol

2011 ◽  
Vol 41 (11) ◽  
pp. 1985-1990 ◽  
Author(s):  
Paula Rodriguez Villamil ◽  
Felipe Ledur Ongaratto ◽  
Daniela Scherer da Silva ◽  
Berenice de Avila Rodrigues ◽  
Jose Luiz Rodrigues
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
The Other ◽  
Developmental Competence ◽  
Time Interval ◽  
Equilibrium Solutions ◽  
Mouse Blastocyst ◽  
In Vitro Development ◽  
Vitro Development

The aim of this study was to determine the effect of dimethylformamide (DF) associated with ethylene glycol (EG) or 1-2 propanediol (PROH) during vitrification, on the in vitro development of mouse blastocysts. Cryoprotectant toxicity was evaluated exposing embryos into three different equilibrium solutions (ES) composed by DF, EG or PROH mixtures (10% v/v of each) in mPBS + 0.5% PVA at different interval times (1, 3 and 10min). In a second experiment, embryos were exposed to the same ES (either 1 or 3min), following for the three respectively vitrification solutions (VS) (20% v/v of each) for 30s. After 72 hours of in vitro culture, embryo hatching and expansion rates were similar for the ES1 and ES2 equilibration solutions during the time interval of 1 or 3min. However embryos exposed for 10 min to the DF equilibration solutions, had lower survival rates than EG-PROH solution (P<0.01). Furthermore, survival rates for embryos exposed to DF-PROH (ES+VS) were lower than embryos exposed to the other solutions (P<0.01). Blastocyst vitrification was performed with the three ES+VS (for 1min and 30s, respectively), using glass micropipettes (GMP). Survival rates were lower for blastocysts vitrified with DF solutions (3%-3/108 and 17.1%-19/111) (P<0.01) than with PROH+EG vitrification solutions (69%-73/105). In conclusion, DF as a cryoprotectant into vitrification solutions have deleterious effects on the in vitro developmental competence of vitrified mouse blastocysts.

scholarly journals 104EFFECT OF HATCHING STATUS ON VITRIFICATION OF CLONED BOVINE BLASTOCYSTS

2004 ◽  
Vol 16 (2) ◽  
pp. 174
Author(s):  
C. Laowtammathron ◽  
T. Terao ◽  
C. Lorthongpanich ◽  
S. Muenthaisong ◽  
T. Vetchayan ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Sucrose Solution ◽  
Survival Rates ◽  
Nuclear Transplantation ◽  
Fibroblast Cells ◽  
Anova Test ◽  
University Of Technology ◽  
Vitrification Solution ◽  
Cloned Bovine

Bovine blastocysts produced by nuclear transplantation have mechanical slits in their zonae pellucidae, and therefore initiate hatching earlier than the non-manipulated embryos. The present study was undertaken to examine whether the hatching stage of cloned blastocysts is among the factors influencing their survival after vitrification and warming. Cloned bovine blastocysts were produced by using adult ear fibroblast cells as reported previously (Parnpai et al., 2002, Theriogenology 57, 443), except that fused couplets were co-cultured with bovine oviductal epithelial cells in mSOFaa medium supplemented with 0.1% linoleic acid-albumin (LAA)+0.2% BSA (Hochi et al., 1999, Theriogenology 52, 497–504). Hatching blastocysts harvested on Day 7 were classified into one of three groups according to the ratio of extruding embryonic diameter from zona (D2) to embryonic diameter inside the zona (D1); category-A: D2/D1=0.01–0.70; category-B: D2/D1=0.71–1.00; category-C: D2/D1=1.01–1.70. The blastocysts were first exposed to 10% DMSO+10% ethylene glycol in TCM199+20% FCS for 2min, and then equilibrated in 20% DMSO+20% ethylene glycol+0.5M sucrose with or without 10% Ficoll in TCM199+20% FCS for 30s. One to three blastocysts were placed on a Cryotop sheet (Kitazato Supply Co., Tokyo, Japan) and vitrified in liquid nitrogen. The samples were warmed in 0.5M sucrose solution for 2min and transferred into TCM199+20% FCS in five steps (5min per step). The post-warm survival of the blastocysts was assessed by in vitro culture for 24h. When Ficoll-free vitrification solution was used, post-warm survival rate of the category-A blastocysts (77%, 23/30) was not significantly different (ANOVA test) from those of category-B and category-C blastocysts (74%, 20/27; and 80%, 24/30; respectively). Inclusion of 10% Ficoll in the vitrification solution did not improve (ANOVA test) the post-warm survival rates of cloned blastocysts (category-A: 65%, 22/34; category-B: 54%, 15/28; category-C: 59%, 19/32). Groups of fresh nonsurgical embryos, vitrified with or without Ficoll, yielded 66.7% (4/6), 66.7% (2/3) and 40.0% (2/5), respectively, of recipients pregnant at 48 days of gestation. In conclusion, cloned bovine blastocysts, regardless of their hatching stages, were relatively resistant to cryopreservation by vitrification. (Supported by Thailand Research Fund and R&amp;D Fund of Suranaree University of Technology.)

scholarly journals 123VITRIFICATION OF IN VITRO-PRODUCED BOVINE BLASTOCYSTS BY ADDITION OF CRYOPROTECTANT IN ONE STEP

2004 ◽  
Vol 16 (2) ◽  
pp. 184
Author(s):  
D.J. Walker ◽  
L.F. Campos-Chillon ◽  
G.E. Seidel
Keyword(s):  
Ethylene Glycol ◽  
Embryo Quality ◽  
Survival Rates ◽  
Defined Medium ◽  
Embryo Cryopreservation ◽  
Equilibration Time ◽  
One Step ◽  
Hatching Rates

Vitrification combined with in-straw dilution may replace conventional cryopreservation of bovine embryos, but this requires further study for practicality. Our objectives were to compare three ethylene glycol concentrations (6, 7, and 8M) and two equilibration times (2.5 and 3.5min) for one-step addition of cryoprotectant. In vitro-matured oocytes from slaughterhouse ovaries, fertilized using sperm of 3 bulls, were cultured in chemically defined medium (CDM-1/CDM-2) plus FAF-BSA to produce 420 blastocysts. Day 7.5 embryos were placed into HCDM-2 (HEPES-buffered medium) and then transferred to a 6μL drop of vitrification solution (V) (6, 7, or 8M ethylene glycol, 0.5M galactose, and 18% w/v Ficoll 70 in HCDM-2). Immediately thereafter, 1cm column of DHCDM (0.5M galactose in HCDM-2) was drawn into a 0.25mL straw, followed by a 0.5cm column of air and another 7cm of DHCDM. Another 0.5cm column of air was aspirated before the 6μL of V (0.5cm) containing the embryos were aspirated; then 0.5cm of air followed. Finally, DHCDM was drawn until the first column came into contact with the cotton plug. Straws were then heat-sealed and plunged into liquid nitrogen slightly above the embryos after 2.5 or 3.5min equilibration. The rest of the straw was then submerged slowly. Straws were thawed in air for 10s and then in 37°C water for 20s. Straws were held at room temperature (24°C) for 4min before being expelled into HCDM-2. They were then placed into CDM-2+5% FCS for culture. Quality score (1=excellent, 2=fair, 3=poor), survival (S) as determined by expansion of blastocysts, and hatching (H) were assessed at 24 and 48h post-thaw. Data from 6 replicates (2/bull) were analyzed by ANOVA after arc sin transformation of percentage data. S and H responses were calculated as a percentage of non-frozen controls in the same replicate. Control survival and hatching rates were: 24S: 90%, 24H: 50%, 48S: 90%, 48H: 72%. Quality scores at both 24 and 48h were higher (P&lt;0.05) for 8M than 6M ethylene glycol (2.68 and 3.24 for 24h; 2.55 and 3.17 for 48h); values for 7M ethylene glycol were intermediate. Equilibration time had no effect on embryo quality (P&gt;0.1). Neither ethylene glycol concentration nor exposure time affected survival or hatching at 24 or 48h (P&gt;0.1). Survival rates (as a % of control) at 48h were: 8M: 57%, 7M: 55%, 6M: 36% and hatching: 8M: 39%, 7M: 30%, and 6M: 21%; 2.5min tended to be better than 3.5min for survival at 24h, hatching at 24h, survival at 48h, but not hatching at 48h (56% and 43%, 30% and 26%, 55% and 44%, 28% and 32% respectively). Higher concentrations of ethylene glycol proved beneficial in terms of embryo quality, with the same trend for survival and hatching rates. One-step addition of cryoprotectant for vitrification shows potential for simplifying embryo cryopreservation. However, further research is needed to produce more acceptable survival rates and to study vitrification of in vivo-produced embryos.

Promotion of glucose utilization by insulin enhances granulosa cell proliferation and developmental competence of porcine oocyte grown in vitro

Zygote ◽  
2016 ◽  
Vol 25 (1) ◽  
pp. 65-74 ◽  
Author(s):  
Nobuhiko Itami ◽  
Yasuhisa Munakata ◽  
Koumei Shirasuna ◽  
Takehito Kuwayama ◽  
Hisataka Iwata
Keyword(s):  
Granulosa Cell ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Oocyte Development ◽  
Energy Status ◽  
Cell Complexes ◽  
Porcine Oocyte ◽  
High Insulin

SummaryIn vitro culture of the oocyte granulosa cell complexes (OGCs) from early antral follicles (EAFs) shows granulosa cell (GC) proliferation, but to a lesser extent than that observed in vivo during follicle development. As the number of GCs closely relates to energy sufficiency of the oocytes, enhancement of GC proliferation influences oocyte development. GC proliferation depends on glycolysis and insulin-mediated AKT/mTOR signaling pathway; therefore, addition of culture medium containing insulin and glucose may potentially promote GC proliferation and hence improve oocyte development. In the present study, we assessed the effect of exogenous insulin and glucose concentration on GC proliferation and oocyte energy status as well as developmental abilities of porcine oocytes grown in vitro. In the presence of 5.5 mM of glucose (Low), a comparison of 10 versus 20 μg/ml insulin showed that high insulin enhanced GC proliferation but exhausted glucose from the medium, which resulted in low energy status including lipid and adenosine triphosphate of the oocyte. Whereas, in the presence of 20 μg/ml insulin, medium with 11 mM glucose (High) enhanced GC proliferation and oocyte energy status as well as developmental ability up to the blastocyst stage. Considering that there was no difference in OGCs development observed with medium (10 μg/ml insulin) containing 5.5 versus 11 mM glucose, we concluded that the combination of high insulin and glucose enhanced GC proliferation and energy status of oocytes as well as the developmental ability of the oocytes grown in vitro.

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