EVALUATION OF DEVELOPMENT AFTER CRYOPRESERVATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 136
Author(s):  
A. C. Nicacio ◽  
R. Simões ◽  
M. A. Peres ◽  
J. S. A. Gonçalves ◽  
F. F. Paula-Lopes ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Granulosa Cells ◽  
Embryo Cryopreservation ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Quick Freezing ◽  
Group 10 ◽  
Hatching Rates

The inefficiency of embryo cryopreservation protocols limits the broad use of in vitro production (IVP) of bovine embryos. The aim of this work was to identify the damage caused by cryopreservation and embryo culture of IVP bovine embryos after thawing by in vitro development before and after cryopreservation. Cumulus–oocyte complexes were in vitro-matured, fertilized, and cocultured on granulosa cells in SOF with amino acids (SOFaa) supplemented with FCS. Expanded blastocysts (n = 600) harvested on Days 7 to 9 were submitted to controlled freezing [controlled group: 10% ethylene glycol (EG) for 10 min and 1.2°C min–1 cryopreservation], quick-freezing [quick group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s], or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. The embryos of the control group were not exposed to cryoprotectant or cryopreservation method, and the hatching rate was evaluated on Day 12 post-insemination. The straws (quick and vitrification groups) were first placed on nitrogen vapor (0.8 cm over the liquid nitrogen) for 2 min and then immersed in liquid nitrogen. Embryos were thawed in air for 10 s followed by a 25°C water bath for 20 s. Embryos were rehydrated in PBS + 0.2% BSA + 0.3 m sucrose and PBS + 0.2% BSA for 3 min each. To evaluate development of frozen–thawed embryos, they were cocultured on granulosa cells in TCM-199 or SOFaa both supplemented with FCS for 4 days. Hatching rate of the control group was 46.1%. Data were analyzed by PROC MIXED model of SAS System for Windows®. For TCM-199, the controlled group hatching rate was 44.65 ± 5.94%, quick group did not hatch, and vitrification group showed hatching rates of 9.4 ± 6.8%. For SOFaa, the controlled group hatching rate was 11.6 ± 3.4%, embryos submitted to the quick group did not hatch, and the vitrification group showed hatching rates of 8.7 ± 4.5%. Values were significant at P < 0.05. The controlled group showed a difference compared with the other groups of cryopreservation in both media (TCM-199 and SOFaa). However, TCM 199 showed high rates of re-expansion and hatching. In conclusion, the culture medium influences embryo development after cryopreservation, and TCM-199 is more appropriate than SOFaa. Financial support by FAPESP (04/05335-1).

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

Zygote ◽  
2011 ◽  
Vol 20 (2) ◽  
pp. 117-122 ◽  
Author(s):  
Alessandra Corallo Nicacio ◽  
Renata Simões ◽  
Fabiola Freitas de Paula-Lopes ◽  
Flavia Regina Oliveira de Barros ◽  
Maria Angelica Peres ◽  
...  
Keyword(s):  
Culture Conditions ◽  
Slow Freezing ◽  
Control Group ◽  
Hatching Rate ◽  
Rapid Freezing ◽  
Bovine Embryos ◽  
Group 10 ◽  
Controlled Freezing ◽  
Hatching Rates

SummaryThe aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7–9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen–thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows®. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

scholarly journals 113COMPARISON OF TWO ETHYLENE GLYCOL EQUILIBRATION TREATMENTS FOR THE QUICK FREEZING OF IN VITRO-PRODUCED BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 178
Author(s):  
A.C. Nicácio ◽  
R. Simões ◽  
C. Yamada ◽  
H.V.A. Caetano ◽  
M.R.B. Mello ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Liquid Nitrogen ◽  
Granulosa Cell ◽  
Cell Monolayer ◽  
Slow Freezing ◽  
Bovine Embryos ◽  
Treatment Groups ◽  
Quick Freezing ◽  
Hatching Rates

The aim of this study was to compare two ethylene glycol (EG) equilibration procedures for the quick freezing of in vitro-produced bovine embryos. Cumulus-oocyte complexes (COCs) were collected from slaughterhouse ovaries. COCs were matured in TCM199 containing 10% of bovine fetal serum, LH, FSH and E2, and fertilized. Presumptive zygotes were co-cultured in TCM199 with a granulosa cell monolayer, at 39°C in humidified atmosphere of 5% CO2 in air. Grade 1, expanded blastocysts (n=761) were selected 7 and 9 days after insemination and randomly distributed to one of eight treatment groups. In Equilibration Procedure 1, embryos were exposed to 10% EG for 5 min, and then to 17%, 22% or 28% EG for 60s (respectively referred to as EG 17, EG 22 and EG 28). In Equilibration Procedure 2, embryos were exposed to the same EG solutions as in Equilibration Procedure 1, but the period of exposure was 10min to 10% EG and 30 s to EG 17, EG 22 and EG 28. In Equilibration Procedure 3 (slow-freezing controls), embryos were exposed to 10% EG for either 5 or 10min and then cryopreserved by slow-freezing method at 1.2°C/min. In all treatment groups, EG solutions were prepared in PBS+0.2% BSA, and embryos were exposed to EG solutions at 22°C. Embryos were loaded into 0.25mL straws and heat-sealed. Straws were cooled in liquid nitrogen vapor for 2min, and then plunged into and stored in liquid nitrogen. Straws were thawed in room temperature air for 10s, and then in 25°C water for 20s. Thawed embryos were diluted by transferring them into 0.5ml of PBS+0.2% BSA+0.3M sucrose for 3min, and then 0.5mL of PBS+0.2% BSA for 3min. Embryos were co-cultured on granulosa cell monolayer in TCM199 and evaluated after 24h for blastocyst re-expansion (EXP), and again at 48, 72 and 96h for hatching (HAT). A total of 724 in vitro-produced bovine blastocysts were used as controls to determine hatching rates. The results are presented in the table. Embryos exposed to 10% EG for 10min (Equilibration Procedure 1) yielded significantly higher rates of blastocyst re-expansion and hatching when compared to embryos exposed for 5min (Equilibration Procedure 2, P&lt;0.05). These results suggest that quick freezing of in vitro-derived bovine embryos may be an alternative to vitrification; however, additional studies are needed to optimize cryopreservation protocols and increase post-thaw survival. This project was supported by FAPESP (01/11266-4) Table 1 Effect of equilibration procedure on in vitro re-expansion and hatching rates of embryos cryopreserved by slow and quick freezing methods

106 EFFECT OF GLYCEROL AND ETHYLENE GLYCOL ON THE DEVELOPMENT OF IN VITRO BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 161
Author(s):  
A. C. Nicacio ◽  
R. Simões ◽  
M. A. Peres ◽  
J. S. A. Gonçalves ◽  
M. E. O. D'Ávila Assumpção ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Cell Monolayer ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Chi Square ◽  
Chi Square Test ◽  
Hatching Rates

The aim of this study was to evaluate the viability of in vitro-produced bovine embryos after exposure to different cryoprotectant solutions and cryopreservation. Bovine ovaries were collected at slaughterhouse and oocytes were matured, fertilized, and cultured in vitro. The embryos were co-cultured on a granulosa cell monolayer in SOF + 5% FCS and nonessential amino acids. In Experiment 1, expanded blastocysts were exposed to 10% ethylene glycol (EG) solution for 10 min (Group EG) or to 10% EG solution for 10 min and to 20% EG + 20% glycerol (Gly) solution for 30 s (Group EG/Gly). Cryoprotectants were diluted with PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min, and the hatching rate was evaluated after culture. In Experiment 2, after exposure, EG Group was cryopreserved by slow freezing procedure (1.2�C/min) and EG/Gly Group was vitrified on nitrogen vapor for 2 min. After thawing, cryoprotectants were diluted using PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min; hatching rate was evaluated after culture. As a control group for both experiments, non exposed embryos were cultured and evaluated for hatching rate. In Experiment 1, the hatching rates were 59.72% (43/72) for control, 62.38% (63/101) for EG, and 69.00% (69/100) for EG/Gly groups. In Experiment 2, hatching rates were 59.72% (43/72) for control, 15.22% (7/46) for EG, and 0.00% (0/46) for EG/Gly groups. Results were analyzed by chi-square test. In Experiment 1, no differences were observed among groups (P > 0.05) and in Experiment 2, differences were observed among control, EG, and EG/Gly groups (P < 0.05). In conclusion, the cryoprotectants were not deleterious to the development of in vitro bovine embryos until hatching, but the cryopreservation procedures decreased embryo viability. This work was supported by FAPESP 04/05335-1.

scholarly journals 122COMPARISON OF TWO CRYOPROTECTANT DILUTION TREATMENTS FOR QUICK FROZEN IN VITRO-PRODUCED BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 183
Author(s):  
J.A. Visintin ◽  
A.C. Nicácio ◽  
C. Yamada ◽  
H.V.C. Amaral ◽  
R. Simões ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Granulosa Cell ◽  
Sucrose Solution ◽  
Cell Monolayer ◽  
Bovine Embryos ◽  
Treatment Groups ◽  
Nitrogen Vapor ◽  
Quick Freezing ◽  
Hatching Rates

The aim of this study was to compare the viability of in vitro-produced bovine embryos following quick freezing in ethylene glycol (EG) and subsequent dilution of EG by either a two- or a three-step procedure. Cumulus-oocyte complexes (COCs) were collected from slaughterhouse ovaries. COCs were matured in TCM199 containing 10% bovine fetal serum, LH, FSH and E2, and fertilized. Presumptive zygotes were co-cultured in TCM199 with a granulosa cell monolayer, at 39°C in humidified atmosphere of 5% CO2 in air. Grade 1, expanded blastocysts (n=544) were selected 7 and 9 days after insemination and randomly distributed to one of three EG equilibration treatment groups. Embryos were exposed to 10% EG for 10min, and then to 17%, 22% or 28% EG for 30s (respectively referred to as EG 17, EG 22 and EG 28). In all treatment groups, EG solutions were prepared in PBS+0.2% BSA, and embryos were exposed to EG solutions at 22°C. Embryos were loaded into 0.25-mL straws which were then heat-sealed. Straws were cooled in liquid nitrogen vapor for 2min, and then plunged and stored in liquid nitrogen. Straws were thawed in room temperature air for 10s, and then in 25°C water for 20s. The thawed embryos of the EG 17, EG 22 and EG 28 groups were randomly assigned to one of two EG dilution procedures. Two-step dilution consisted of transfer of embryos into PBS+0.2% BSA+0.3M sucrose solution for 3min, and then PBS+0.2% BSA for 3min. Three-step dilution consisted of transfer of embryos into PBS+10% EG+0.2% BSA+0.3M sucrose for 3min, PBS+0.2% BSA+0.3M sucrose for 3min, and then PBS+0.2% BSA for 3min. Embryos were co-cultured on a granulosa cell monolayer in TCM199 and evaluated after 24h for blastocyst re-expansion (EXP), and again at 48, 72 and 96h for hatching (HAT). A total of 724 in vitro-produced bovine blastocysts were used as controls to determine hatching rates. The results are presented in the Table. No significant differences were found between the two- and three-step dilution procedures (P&gt;0.05) for in vitro-produced bovine embryos cryopreserved by quick freezing. This project was supported by FAPESP (01/11266-4). Table 1 In vitro re-expansion and hatching rates (%) of rapidly frozen embryos after two- or three-step dilution

Treatment with roscovitine and butyrolactone I prior to in vitro maturation alters blastocyst production

Zygote ◽  
2019 ◽  
Vol 28 (1) ◽  
pp. 24-31 ◽  
Author(s):  
Rosiara Rosária Dias Maziero ◽  
Carlos Renato de Freitas Guaitolini ◽  
Daniela Martins Paschoal ◽  
André Maciel Crespilho ◽  
Bianca Andriolo Monteiro ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Study Groups ◽  
Embryo Cryopreservation ◽  
Control Group ◽  
Bovine Embryos ◽  
Nuclear Maturation ◽  
Oocyte Meiosis ◽  
Maturation Index ◽  
Bovine Oocytes

SummaryThis study evaluated the effects of oocyte meiosis inhibitors roscovitine (ROS) and butyrolactone I (BL-I) on in vitro production of bovine embryos. Bovine oocytes were maintained in pre in vitro maturation (pre-IVM) with 25 µM ROS or 100 µM BL-I for 24 h to delay meiosis and for 24 h in in vitro maturation (IVM). Following this treatment, the nuclear maturation index was evaluated. All embryos degenerated following this procedure. In the second set of experiments, oocytes were maintained for 6 or 12 h in pre-IVM with the following three treatments: ROS (25 µM or 12.5 µM), BL-I (100 µM or 50 µM) or a combination of both drugs (6.25 µM ROS and 12.5 µM BL-I). Oocytes were cultivated for 18 or 12 h in IVM. When a meiosis-inducing agent was used during pre-IVM for 24 h, more degenerated oocytes were observed at the end of the IVM period. This effect decreased when the meiotic blocking period was reduced to 6 or 12 h. No significant differences were observed in the blastocyst production rate of oocytes in pre-IVM for 6 h with ROS, BL-I, or ROS + BL-I compared with that of the control group (P > 0.05). However, inhibition of oocytes for 12 h resulted in decreased embryo production compared with that in the controls (P < 0.05). There was no difference in the post-vitrification embryo re-expansion rate between the study groups, showing that the meiotic inhibition for 6 or 12 h did not alter the embryo cryopreservation process.

239 USE OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS IN IN VITRO PRODUCTION OF BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 209
Author(s):  
T. Fanti ◽  
N. M. Ortega ◽  
R. Garaguso ◽  
M. J. Franco ◽  
C. Herrera ◽  
...  
Keyword(s):  
Phosphodiesterase Inhibitor ◽  
Basal Medium ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Oocyte Competence

In vitro embryo production systems (IVP) try to emulate and enhance molecular events that occur in in vivo reproductive systems in order to increase, not only the number of embryos generated, but also their quality. Despite advances, IVP processes are still inefficient compared with in vivo systems. Several studies have attributed this deficiency to a lack of oocyte competence due to spontaneous premature resumption of meiotic maturation in the oocyte following the removal from its follicular environment. Therefore, our objective was to increase oocyte competence avoiding premature resumption of meiosis by using cyclic adenosine monophosphate modulators. Cumulus-oocyte complexes (COC) were obtained from ovaries of slaughterhouses, washed, and randomly allocated in 2 culture systems. Oocytes in the control group (IVM) were cultured for a period of 24 h in basal medium TCM-199 with EGF (1 µg mL–1) supplemented with rhFSH (25 mIU mL–1). Oocytes in the biphasic in vitro maturation (b-IVM) group were cultured for 2 h in a basal medium supplemented with a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 500 µM), and an activator of adenylate cyclase (forskolin, 100 µM). Subsequently, COC were washed and cultured in basal medium supplemented with cilostamide (20 µM) and rhFSH (25 mIU mL–1) for 24 h. Maturation rates were analysed and IVF was performed with a dose of 1 × 106 sperm cells mL–1 in IVF-SOF medium. The presumptive zygotes were cultured in continuous-single-culture medium (Irvine) supplemented with 8 mg mL–1 of BSA until they reached the blastocyst stage. No significant differences in maturation, cleavage, and cryotolerance were observed between b-IVM and IVM groups (P > 0.05; Table 1). This study showed that b-IVM produced a significant increase in IVP compared with the control (IVM) at Days 7 and 8 (P < 0.01). Blastocyst hatching rate was significant (P < 0.05) for both treatment and day of analysis. The b-IVM group yielded an increase of 10 and 7.5% at Days 7 and 8, respectively, of IVP. The biphasic maturation showed an improvement in quality regarding the control group, in the timing analysis of production, and hatching percentages, and these results show that the use of cyclic adenosine monophosphate modulators in the oocyte maturation process enhances oocyte competence, which is reflected in increased productivity and embryo quality. We propose this treatment as an alternative to the standard protocols currently used in IVP of bovine embryos. Table 1.Effect of treatment on maturation, cleavage, and cryotolerance

47 A New Device and Method for Successful Vitrification of In Vitro-Produced Ovine Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 163
Author(s):  
S. Ledda ◽  
J. M. Kelly ◽  
S. K. Walker ◽  
Y. Natan ◽  
A. Arav
Keyword(s):  
Survival Rate ◽  
Embryo Transfer ◽  
Blastocyst Stage ◽  
Embryo Cryopreservation ◽  
Control Group ◽  
Hatching Rate ◽  
High Survival ◽  
Embryo Survival ◽  
New Device

To advance the use of embryo vitrification technology in veterinary practice, we developed a system in which embryo vitrification, warming, and dilution can be performed within a straw. An in-straw embryo cryopreservation method reduces the need for equipment and technical skills and can facilitate direct embryo transfer to the uterus. This study proposes the use of a new device named “Sarah” that is designed to permit all in-straw embryo cryopreservation procedures. Ovine in vitro-produced (IVP) embryos were vitrified at either early blastocyst stage (EB, n = 65, 6 days post-IVF) or fully expanded blastocyst stage (FB, n = 168, 7 days post-IVF). The vitrification procedure using Sarah constituted a 0.25-mL straw with a capsule having 50-µm pores inserted at one end. Embryos at each stage (EB and FB) were divided into 2 subgroups and vitrified by 1 of 2 methods: (1) multi-step (MS) group-a straw containing 2 embryos was sequentially loaded vertically into 1.5-mL tubes containing 6 different vitrification solutions: 10, 20, 40, 60, 80, or 100% ES (with 100% ES being 7.5% DMSO +7.5% EG + 20% FCS in TCM-199; 90 s each step) followed by 30 s each in 75 and 100% VS (100% VS being 18% DMSO +18% EG + 0.5 M trehalose + BSA in TCM-199); and (2) two-step (TS) group-the straw (2 embryos/straw) was loaded with 100% of ES (5 min), followed by 100% VS solution for 30 s. For both methods, at the end of the preparation steps, the straws were plunged directly into liquid N2. Non-vitrified embryos were maintained in in vitro culture as a control group (n = 102). The warming procedure consisted of placing the straws directly into 5-mL tubes containing 100, 50, 25% WS (WS = 1 M sucrose in TCM-199+ 20% FCS) at 38.6°C (for first solution) and at room temperature for all the rest (5 min each), before being placed into the holding medium. Embryos were recovered from the straws, incubated at 38.6 C in 5% CO2 in air in TCM 199 + 5% FCS, and evaluated for blastocoel re-expansion, embryo survival, and hatching rate at 2, 14, 48 h post-warming. Blastocyst re-expansion (2 h) after warming increased as the developmental stage progressed and was not affected by the vitrification method. In fact, it was significantly (P < 0.05) higher for FB vitrified in the MS and TS methods (77.90% and 71.25%, respectively) compared with the EB method (62.5% and 48.50%, respectively). At 24 h, survival rate of vitrified FB was significantly higher (P < 0.05) in the MS system (95.35%) compared with those in TS (86.25%). Survival rates of FB embryos for both methods (MS and TS) were significantly higher (P < 0.001) than EB embryos vitrified in MS (56.25%) and TS (56.55) methods. After 48 h of culture, the hatching rate for FB vitrified in the MS system (87.21%) was comparable with TS (77.5%) and control (85.3%) groups but significantly higher (P < 0.001) than vitrified EB in MS (43.75%) and TS (36.36%). In conclusion, we showed that a high survival rate of IVP embryos can be achieved by this new in-straw vitrification and warming device (“Sarah”), with hatching rates in vitro comparable with that of control fresh embryos. This method has the potential for use in direct embryo transfer in field conditions.

133 EFFECTS OF ACTIVINA ON mRNA EXPRESSION IN IN VITRO FERTILIZED BOVINE EMBRYOS CULTURED FROM CHEMICALLY DEFINED TWO-STEP CULTURE MEDIUM

2008 ◽  
Vol 20 (1) ◽  
pp. 147
Author(s):  
J. E. Park ◽  
G. Jang ◽  
H. J. Oh ◽  
S. G. Hong ◽  
I. S. Yang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Early Stage ◽  
Activin A ◽  
Developmental Competence ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Defined Culture

During the preimplantation stage, embryo development occurs in a maternal environment within the oviducts and uterine horns. It has been speculated that both the embryo itself and the maternal reproductive tract provide paracrine factors that influence embryo development (Jones et al. 2006 Reproduction 132(5), 799–810). Activins are known for FSH releasers, and several previous studies have reported that activin subunits and activin receptors mRNA were expressed in oocytes, zygotes, and oviduct (Yoshioka et al. 1998 Reprod. Fertil. Dev. 10(3), 293–298; Gandolfi et al. 1995 Mol. Reprod. Dev. 40(3), 286–291). The purposes of the present study were Experiment 1) to evaluate the effects of activin A on developmental competence of bovine embryos derived from two-step defined culture medium (Lim et al. 2007 Theriogenology 67(2), 293–302) and Experiment 2) to analyze the effects of activin A on transcriptional level of the genes in IVF embryos. Cumulus–oocyte complexs were harvested from ovaries obtained from a local slaughter house, matured, and fertilized in vitro. In vitro fertilized zygotes cultured in media supplemented with activin A in the early stage at the concentrations of 0, 10, or 100 ng mL–1 or in the later stage medium at the concentrations of 0, 10, or 100 ng mL–1. Data were analyzed using the Statistical Analysis System (SAS) program. In Exp. 1, although the development competence of embryos that cultured with activin A in the early stage medium was not significantly different, development to blastocysts on day 8 in the later stage medium with 100 ng mL–1 activin A was significantly higher than the control group [22.4% (54/264) v. 34.7% (76/233); P < 0.05]. Hatching rate of blastocyst on day 8 was significantly higher in the presence of 100 ng mL–1 activin A in the later stage culture medium compared with the control group [9.3% (5/54) v. 22.4% (17/76); P < 0.05]. In Exp. 2, the relative expression of 3 genes (Na/KATPase, E-cad, Glut-1) related to blastocyst hatching and implantation was analyzed. The relative abundance (ratio to GAPDH mRNA) of gene transcripts in blastocysts was measured by conventional semi-quantitative reverse transcription-polymerase chain reaction. The expression level of the Na/K ATPase, E-cad, and Glut-1 gene were higher in the presence of activin A in the culture medium compared with the control group. In conclusion, this study suggests that activin A during the later stage of in vitro bovine embryo development can enhance the developmental competence of preimplantation embryos, increase the hatching rate, and affect expression level of genes related to hatching and implantation in defined culture medium. This study was financially supported by KOSEF (grant ? M10625030005-07N250300510) and the Korean MOE, through the BK21 program for Veterinary Science.

113 EVALUATION OF DIFFERENT CRYOPROTECTANT AND FORSKOLIN IN THE CULTURE MEDIUM FOR IMPROVING THE EFFICACY OF VITRIFICATION OF BOS INDICUS IN VITRO-DERIVED EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 215 ◽  
Author(s):  
B. V. Sanches ◽  
B. D. O. Filho ◽  
J. H. F. Pontes ◽  
A. C. Basso ◽  
M. L. G. Meirinhos ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Lipid Droplets ◽  
Culture Medium ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Calf Serum ◽  
Genetic Material ◽  
Embryo Cryopreservation ◽  
Hatching Rate

Embryo cryopreservation is an essential method for the biotechnology of reproduction. This is the safest option for interchange of genetic material for research and commercial purposes. For cattle, Brazil has become the leading country in the world for the number of in vitro-produced embryos, using mostly Bos indicus animals. However, considering the in vitro method of embryo production, field results have shown a lower resistance to cryopreservation for B. indicus when compared with Bos taurus embryos. A possible explanation for this is a great concentration of lipid droplets in the cytoplasm of cells fromB. indicus embryos. The objective of this study was to compare 2 cryoprotectants (Propanediol and DMSO) to vitrification and evaluate the effect of adding 10 μM forskolin to the SOF medium for embryo culture before cryopreservation. For all the experiments, ovaries from slaughtered Nelore Bos indicus donors were recovered and maintained at 30 to 35°C in NaCl solution until recovery of the COC. Embryos submmited to vitrification were expanded blastocysts at Day 7 of in vitro culture. In the first experiment embryos were first incubated in 10% ethylene glycol (EG) plus 10% DMSO dissolved in holding medium (TCM-HEPES with 20% calf serum) for 1 min and then transferred to droplet of 20% EG plus 20% DMSO in holding medium and 0.5 M sucrose for 20 s before immersing in liquid nitrogen (n = 107; group EG + DMSO). For the group EG + Propanediol (EG + PRO; n = 96), blastocysts were placed in 10% EG plus 10% PRO in holding medium for 1 min and then transferred to a droplet of 20% EG plus 20% PRO in holding medium and 0.5 M sucrose for 20 s before immersing in liquid nitrogen. Both treatments were performed using the Cryotop system. Results were compared with embryos (n = 118) not submitted to cryopreservation. The evaluation was done by the hatching rate of blastocysts at Day 9, being higher (86.4%) for embryos not cryopreserved, when comparing with 77.1% for group EG + PRO and 72.9% for group EG + DMSO (P < 0.05). In the second experiment, Day 5 embryos obtained in vitro from Nelore donors were cultured using SOF medium with 10 μM forskolin (n = 112) or not (control; n = 101), being all submitted to cryopreservation using Cryotop and the same vitrification method for group EG + DMSO. Results were compared with embryos cultured with SOF medium and not submitted to cryopreservation (n = 96). The evaluation was performed by considering hatching rate at Day 9, being higher (85.4%) for not cryopreserved, when compared with 63.3% for control and 70.5% for forskolin group (P < 0.05). Considering embryos submitted to cryopreservation, the hatching rate was higher (P < 0.05) for the forskolin group.

101 EFFECT OF NITROCOOLER NEGATIVE PRESSURE AND RECOVERY INTERVAL ON CRYOTOLERANCE OF BOVINE IN VITRO-PRODUCED EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 210 ◽  
Author(s):  
J. C. Mezzalira ◽  
L. U. Ohlweiler ◽  
M. Urio ◽  
S. G. Neto ◽  
L. R. Marinho ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Negative Pressure ◽  
Short Exposure ◽  
Time Interval ◽  
Hatching Rate ◽  
Chi Square ◽  
Embryo Survival ◽  
Pressure Stress ◽  
Hatching Rates

Exposing bovine embryos and porcine oocytes to hydrostatic pressure has been shown to increase cryosurvival, possibly by a resulting expression of stress tolerance proteins. This study aimed to evaluate the effect of the negative pressure stress condition (a 5-min-long embryo exposure to a negative pressure) and the interval between vacuum exposure and vitrification (40 min or 2 h) on survival of bovine in vitro-produced (IVP) embryos. The negative pressure was achieved with the same apparatus used previously for the cryopreservation of embryos (Nitrocooler; Mezzalira et al. 2009 Reprod. Fert. Dev. (1) 134), in which a negative pressure (vacuum) is applied to liquid nitrogen to increase the cooling rate through the slush phenomenon, except that in this study, the vacuum was applied to the chamber without liquid nitrogen, at room temperature. Grades 1 and 2 bovine IVP expanded blastocysts were allocated to 1 of 5 experimental groups: embryos in vitro-cultured as fresh (control) or after vitrification (Vitri); and embryos subjected to the negative pressure for 5 min and then in vitro-cultured as fresh (NP-fresh) or after vitrification performed 40 min (NP-Vitri-40 min) or 2 h (NP-Vitri-2h) following the vacuum exposure. Embryos were vitrified in pulled glass micropipettes in a solution with 20% ethylene glycol + 20% dimethylsulfoxide + 20% fetal bovine serum and rewarmed in decreasing sucrose concentrations (Mezzalira et al. 1999 Acta Scientiae Veterinariae 27 262-262). In vitro culture was carried out in all treatments for 72 h for the assessment of re-expansion and hatching rates (Table 1), which were analyzed by the chi-square test, for P < 0.05. No differences in re-expansion rates were observed between groups. However, the vitrification of embryos after 2 h of exposure to a 5-min-long negative pressure (NP-Vitri-2h) improved embryo survival expressed by a higher hatching rate than for embryos vitrified without vacuum exposure (Vitri) or after 40 min following the 5-min-long exposure to vacuum. In addition, hatching rates in group NP-Vitri-2h were similar to those for fresh embryos (control and NP-fresh). Our results indicated that a short exposure of embryos to a negative pressure can improve cryotolerance following vitrification, which is dependent on the time interval between NP exposure and cryopreservation. Bovine IVP embryos should be allowed to recover for at least 2 h after NP exposure before the increase in cryotolerance is achieved. Table 1.Effect of negative pressure on re-expansion and hatching rates of fresh and vitrified

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