Cysteamine supplementation during in vitro maturation and embryo culture: A useful tool for increasing the efficiency of bovine in vitro embryo production

2002 ◽  
Vol 62 (2) ◽  
pp. 203-209 ◽  
Author(s):  
D.G. de Matos ◽  
C. Herrera ◽  
R. Cortvrindt ◽  
J. Smitz ◽  
A. Van Soom ◽  
...  
Keyword(s):  
Embryo Culture ◽  
In Vitro Maturation ◽  
Embryo Production ◽  
In Vitro Embryo Production

186 SUPPLEMENTATION WITH LOW DOSES OF DIMETHYL SULFOXIDE DURING IN VITRO MATURATION RESULTS IN IMPROVED IN VITRO EMBRYO PRODUCTION IN CATTLE

2017 ◽  
Vol 29 (1) ◽  
pp. 201
Author(s):  
A. E. Ynsaurralde ◽  
M. Suvá ◽  
R. Bevacqua ◽  
S. Munilla ◽  
C. Luchetti ◽  
...  
Keyword(s):  
Dimethyl Sulfoxide ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Predictive Equation ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
First Polar Body ◽  
In Vitro Embryo Production

Oocyte in vitro maturation (IVM) is crucial for subsequent in vitro embryo production. It involves acquisition of competence for fertilization and embryo development. Therefore, its optimization could have a direct impact on in vitro embryo development. Dimethyl sulfoxide (DMSO) is commonly used as solvent or vehicle, but also increases the membrane permeability and behaves as a scavenger of cytotoxic free radicals. The aim of this study was to evaluate the effect of DMSO supplementation during bovine oocyte maturation on subsequent in vitro embryo development and to determine the optimal usage dose with no toxic effect. To this aim, cumulus-oocyte complexes were collected from slaughterhouse ovaries and IVM in TCM 199 containing 10% fetal bovine serum, 10 µg mL−1 of FSH, 0.3 mM sodium pyruvate, 100 mM cysteamine, and 2% antibiotic-antimycotic. The oocytes were incubated for 24 h at 6.5% CO2 in humidified air at 38.5°C. For Experiment 1, IVM medium was supplemented with DMSO at concentrations of 0, 0.1, 0.5, 1, or 10% (vol/vol) DMSO (n = 241, 195, 42, 192, 172 oocytes) and IVM rate was determined by presence of the first polar body. For Experiment 2, 0, 0.1, 0.25, 0.5, 0.75, 1, or 10% (vol/vol) DMSO (n = 446, 322, 65, 194, 77, 250, 39 oocytes) was supplemented to IVM medium and cleavage and blastocyst rates were determined to establish the optimal usage dose. In vitro fertilization was performed according to Brackett and Oliphant (1975), with 16 × 106 spermatozoa/mL for 5 h. Afterwards, presumptive zygotes were cultured in SOF for 7 days at 38.5°C and 5% O2. Cleavage and blastocyst rates were determined on Days 2 and 7, respectively. Results were statistically analysed using Fisher’s exact test by GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA). Also, the percentage of blastocyst was adjusted to DMSO concentration using the R software quadratic regression model. The optimum usage dose was determined by calculating the maximum of the estimated predictive equation. In vitro maturation in 10% DMSO resulted in significantly lower first polar body extrusion rates (0% = 74%a, 0.1% = 73%a, 0.5% = 83%a, 1% = 66%a, and 10% = 8%b; different letters indicate statistical differences) and lower cleavage rates (0% = 75%a, 0.1% = 77%a, 0.25% = 80%a, 0.5% = 79%a, 0.75% = 78%a, 1% = 77%a, and 10% = 3%b) than the other treatments. Furthermore, blastocyst production was higher for the 0.25 and 0.5% (vol/vol) supplemented DMSO groups (0% = 26%b, 0.1% = 37%ab, 0.25% = 40%a, 0.5% = 41%a, 0.75% = 34%ab, 1% = 23%b, and 10% = 0%c). The predictive equation results indicate that the maximum percentage of blastocysts is obtained with a concentration of 0.458% (vol/vol) of DMSO. In conclusion, DMSO supplementation during IVM of bovine oocytes had a positive effect on in vitro development. Further studies will be carried out to elucidate its mechanism of action.

228 IN VITRO EMBRYO PRODUCTION AFTER EXPOSURE OF BOVINE OOCYTES TO DIFFERENT TRANSPORTATION MEDIA AND PERIODS: PRELIMINARY RESULTS

2013 ◽  
Vol 25 (1) ◽  
pp. 262
Author(s):  
T. C. Rossi ◽  
J. Rossi ◽  
T. M. Miyauchi ◽  
C. A. C. Fernandes ◽  
L. S. A. Camargo ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Penicillin G ◽  
Controlled Atmosphere ◽  
Time Data ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Fetal Bovine ◽  
In Vitro Embryo Production ◽  
Main Effects

This study was designed to evaluate the effect of different oocyte transportation media and time. Immature oocytes were recovered from slaughterhouse bovine ovaries. Oocytes (n = 492) of quality grades I to III were randomly allotted to one of the following transportation media based on TCM-199, either buffered with HEPES (control) or buffered with NaHCO3 and added with FSH/LH (maturing). Both media were supplemented with pyruvate, penicillin G (10 000 IU), streptomycin (0.05 mg mL–1), and 10% fetal bovine serum (FBS). In control medium, the oocytes were kept for 1 or 8 h at 37°C, and thereafter were transferred to maturing medium until the maturation period (24 h) was completed, under controlled atmosphere (5% CO2) and temperature, in an incubator. The maturing oocytes were distributed in two types of equipment developed for oocyte and embryo transportation, with and without 5% CO2, and kept for 1, 8, or 24 h before maturation or fecundation (IVF) procedures. The oocytes kept for 24 h in the transportation device were placed directly for IVF. All procedures used for in vitro maturation, IVF, and in vitro culture were the same as those adopted for commercial in vitro embryo production at Biotran LTDA (Alfenas, Minas Gerais, Brazil). The cleavage rate was evaluated on Day 3 post-insemination, and the blastocyst production was evaluated on Day 7. The statistical model included the main effects of treatment (control and maturing with or without 5% CO2), time, replicate, and the interaction of media × time. Data (3 replicates) were analysed by ANOVA and differences were identified by Tukey’s test. The time before in vitro maturation at the incubator negatively (P < 0.007) affected cleavage rates (76.4 ± 16.9 v. 58.8 ± 13.2 and 52.2 ± 18.5%, respectively, for 1, 8, and 24 h). However, treatment had no effects (P = 0.3) on cleavage (66.9 ± 15.0, 57.8 ± 19.7, and 67.7 ± 19.8% for the control and maturation with and without 5% CO2, respectively). Similarly, blastocyst production rates differed (P < 0.04) between 1 h (33.4 ± 14.7) and 24 h (19.3 ± 17.0%), whereas blastocyst production at 8 h did not show significant effects (19.3 ± 17.0). Although it was not significant (P > 0.05), only 14.1% of the oocytes kept in medium 2 with a controlled atmosphere for 24 h became embryos. This difference was probably related to the trend in treatment effect (P = 0.06) and the lower rate of embryo production (22.2 ± 13.6%) in this treatment compared with the control (33.8 ± 18.1%) and maturation without 5% CO2 (29.8 ± 17.3%). These results showed that 24 h of transportation is detrimental for the oocyte development potential and that the buffered medium used in this study with 5% CO2 did not efficiently maintain embryo production. Supported by CNPq, CAPES, FAPEMIG, and Biotran LTDA.

144 EFFECTS OF CO-CULTURE WITH FIBROBLASTS AND OVIDUCT CELLS ON IN VITRO PRODUCTION OF PORCINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 219 ◽  
Author(s):  
X. N. Bui ◽  
T. H. Nguyen ◽  
V. L. Nguyen ◽  
K. Kikuchi ◽  
T. Nagai ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
In Vitro Maturation ◽  
Control Group ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Group 3 ◽  
In Vitro Embryo Production ◽  
Group 1

Cell co-culture during in vitro maturation or embryo culture has been reported as a method to improve the efficiency of maturation or embryo development (Kidson et al. 2003 Theriogenology 59, 1889; Romar et al. 2005 Anim. Reprod. Sci. 85, 287). Here, we present the impact of different methods of co-culture with mouse embryonic fibroblasts or oviduct epithelial cells on in vitro embryo production in pigs. Cumulus–oocyte complexes (COC) were collected from follicles with diameter larger than 3 mm and used for in vitro embryo production based on the method of Kikuchi et al. 2002 (Biol. Reprod. 66, 1033) with minor modifications. There were 8 groups; group 1: maturation and embryo culture without cell co-culture (control group); group 2: maturation in the presence of fibroblasts; group 3: embryo culture in the presence of fibroblasts; group 4: both maturation and embryo culture in the presence of fibroblasts; group 5: maturation in the presence of oviduct cells; group 6: embryo culture in the presence of oviduct cells; group 7: both maturation and embryo culture in the presence of oviduct cells; group 8: both maturation and embryo culture in the presence of both fibroblast and oviduct cells. In vitro maturation (IVM) was carried out at 39oC under 5% CO2 in air for 44 h using NCSU-37 as basic medium. Matured oocytes were inseminated using epididymal frozen semen in IVF medium modified Pig-FM supplemented with 2 mM caffeine and 5 mg mL–1 bovine serum albumin (Kikuchi et al. 2002). The percentage of cleaved embryos and percentage of cleaved embryos which developed to the compact morula and early blastocyst stage were recorded. Results were analysed by one-way ANOVA followed by Dunnett’s test. To investigate the effects of co-culture with mouse embryonic fibroblasts and oviduct epithelial cells on oocyte maturation, some COCs cultured in groups 1, 2, 5, and 8 were fixed to assess their nuclear maturation to the metaphase II stage. The rate of matured oocytes in the groups 2, 5, and 8 was 76.85 ± 3.39% (n = 102), 79.11 ± 3.75% (n = 64), and 81.84 ± 3.93% (n = 66), respectively; these rates were increased significantly compared to the group 1 (55.87 ± 1.88%, n = 94; P < 0.05). The effect of co-culture on the fertilization and embryo development is shown in Table 1. Our results indicate that co-culture increases the rates of embryonic cleavage in all groups by comparison with the control group. However, a significant increase in the rate of morula-blastocyst was only observed when embryos were co-cultured with fibroblasts or when both maturation and culture were performed in co-culture with either fibroblasts or oviduct cells (groups 3, 4, 7, and 8). The most important increase in morula-blastocyst rate was recorded for the group of embryos co-cultured with fibroblasts (group 3). In conclusion, the co-culture with fibroblast or oviduct cells during maturation can improve oocyte maturation and cleavage rate, while co-culturing the embryos with fibroblasts seems sufficient to improve both the cleavage and the morula-blastocyst rates. Table 1.Effect of co-culture on the fertilization and embryo development in pig Supported by a grant from the NAFOSTED VN.

183 IN VITRO EMBRYO PRODUCTION: A TOOL TO PRESERVE THE THREATENED WOOD BISON (BISON BISON ATHABASCAE)

2016 ◽  
Vol 28 (2) ◽  
pp. 222
Author(s):  
M. P. Cervantes ◽  
J. M. Palomino ◽  
M. Anzar ◽  
R. J. Mapletoft ◽  
G. Mastromonaco ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Animal Health ◽  
Developmental Competence ◽  
Bison Bison ◽  
High Humidity ◽  
Embryo Production ◽  
Wood Bison ◽  
In Vitro Embryo Production

In vitro embryo production is being developed as a tool to restore genetic diversity and eliminate endemic disease in wood bison. In a recent study in wood bison, we found that more oocytes reached maturity after 30 h v. 24 h of in vivo maturation following hCG treatment (Cervantes et al. 2014 Reprod. Fertil. Dev. 26, 199). An additional 4 h of in vitro maturation after an in vivo maturation period of 30 h also had a positive effect on developmental competence. The present study was designed to test the hypothesis that extending the in vivo maturation time (i.e. extending the interval between hCG treatment and cumulus-oocyte complex (COC) collection) from 30 to 34 h will improve in vitro embryo production in wood bison. Follicular wave development was synchronised among female wood bison (n = 28, 6 to 10 years old) by transvaginal follicular ablation. The study was done in 4 replicates (n = 7 bison per replicate). Bison were given FSH 1 day (300 mg) and 3 days (100 mg) after ablation for ovarian superstimulation, and hCG (2500 IU) 5 days after ablation to induce COC maturation in vivo. Bison were divided randomly into 2 groups (n = 14/group) in which COC were collected transvaginally at either 30 h or 34 h after hCG treatment. Expanded COC from the 30 h group were fertilised after 4 h of in vitro maturation, while expanded COC from the 34 h group were fertilised immediately. Oocytes and sperm were co-incubated (Day 0 = day of fertilization) for 18 h at 38.5°C in 5% CO2 in air and high humidity. Presumptive zygotes were cultured in 4-well dishes containing 500 μL well–1 of CR1aa medium at 38.5°C, 5% CO2, 5% O2, 90% N2 and high humidity, and assessed on Days 3, 7, and 8 (Day 0 = day of fertilization). Data were compared between groups by Chi-squared analysis. No effect of replicate was found. Compared to the 30 h group, the 34 h group had a greater cleavage rate [55/74 (74%) v. 49/86 (57%); (P < 0.05)], and a greater blastocyst rate on Day 7 [25/74 (34%) v. 9/86 (10%); (P < 0.05)] and Day 8 [(40/74 (54.1%) v. 32/86 (37.2%); (P < 0.05)]. We concluded that an extended period of in vivo maturation is beneficial for embryo production after in vitro fertilization in wood bison. We thank Vetoquinol Canada for providing FSH (Folltropin-V) and hyaluronan (MAP-5) and thank Merck Animal Health for hCG (Chorulon).

248 IN VITRO EMBRYO PRODUCTION USING IN VIVO-MATURED OOCYTES COLLECTED TRANSVAGINALLY FROM WOOD BISON (BISON BISON ATHABASCAE)

2015 ◽  
Vol 27 (1) ◽  
pp. 213
Author(s):  
M. P. Cervantes ◽  
J. M. Palomino ◽  
M. Anzar ◽  
R. J. Mapletoft ◽  
G. Mastromonaco ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Animal Health ◽  
Calf Serum ◽  
Developmental Competence ◽  
High Humidity ◽  
Embryo Production ◽  
Wood Bison ◽  
In Vitro Embryo Production

Reproductive technologies are being developed to help conserve the genetic diversity of wood bison, a threatened species. To date, the efficiency of in vitro embryo production in bison is very low and appears to be related to inadequate in vitro conditions for oocyte maturation. Recently, we have attempted to circumvent the problem by inducing oocyte maturation in vivo and found that more than one-third of superstimulated oocytes collected 30 h after administration of hCG were at metaphase II (Cervantes et al. 2013 Reprod. Fertil. Dev. 25, 283; Cervantes et al. 2014 Reprod. Fertil. Dev. 26, 199). We hypothesise that additional maturation time in vitro, after in vivo maturation, will allow the remaining oocytes to reach the MII stage, and thus improve in vitro embryo production in wood bison. The objective of this study was to determine the effect of an additional 4 h of in vitro maturation on the developmental competence of oocytes collected 30 h after hCG treatment. Wood bison cows (n = 24) were superstimulated by the administration of 300 mg of FSH (Folltropin-V) diluted in 0.05% hyaluronan on the day of follicular wave emergence and 100 mg of FSH in hyaluronan 2 days later. Bison were administered 2500 IU of hCG (Chorulon) IM 2 days after the last dose of FSH. Transvaginal ultrasound-guided follicle aspiration was performed 30 h after hCG treatment to collect cumulus-oocyte complexes (COC). Expanded COC (with no evidence of degeneration) were selected and assigned randomly to 2 groups (n = 38 COC/group) in which IVF was done immediately, or after 4 h of in vitro maturation in TCM 199 with 5% calf serum, 5 μg mL–1 pLH, 0.5 μg mL–1 pFSH, and 0.05 μg mL–1 gentamicin, at 38.5°C, 5% CO2 and high humidity. In vitro fertilization (Day 0) was done with frozen-thawed wood bison semen (dose 5 × 106 sperm mL–1) in Brackett-Oliphant medium at 38.5°C, 5% CO2, and high humidity. Presumptive zygotes were cultured in CR1aa plus 5% calf serum, at 38.5°C and in 5% CO2, 5% O2, and 90% N2 and high humidity. Cleavage was recorded on Day 3, and blastocyst formation was recorded on Days 7 and 8. Cleavage and blastocyst rates (calculated from the total number of oocytes submitted to IVF) were compared between groups by chi-square analysis. No difference was detected between groups (immediate fertilization v. after an additional 4 h in vitro) in cleavage rate on Day 3 (55.3 v. 60.5%, respectively, P = 0.82), or blastocyst rate on Day 7 (13.2 v. 23.7%, respectively, P = 0.37). However, the blastocyst rate on Day 8 was higher in the COC group exposed to an additional 4 h of in vitro maturation (18.4 v. 44.7%, respectively, P = 0.03). Results support the hypothesis that an additional short period of in vitro maturation improves the developmental competence of oocytes collected after 30 h of in vivo maturation.We thank Bioniche Animal Health for providing FSH (Folltropin-V) and hyaluronan (MAP-5), and Merck Animal Health for hCG (Chorulon).

Towards the use of microfluidics for individual embryo culture

2010 ◽  
Vol 22 (1) ◽  
pp. 32 ◽  
Author(s):  
R. L. Krisher ◽  
M. B. Wheeler
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Physical Culture ◽  
Embryo Production ◽  
Mammalian Embryo ◽  
Future Directions ◽  
Initial Work ◽  
In Vitro Embryo Production

Mammalian embryo development is still relatively inefficient in vitro. Much research has been conducted on the chemical environment, or culture medium, surrounding the embryo, but little attention has been given to the actual physical culture environment, which has changed very little over the years. The application of microfluidics to embryo production in vitro is a tantalising approach that may alleviate some of the limits that traditional microdrop culture places on embryo development and research into gamete and embryo physiology. These devices may lead to enhanced in vitro embryo development and quality by more closely mimicking the in vivo environment. Initial work in this area is promising and gives us proof-of-principle that these unique microfluidic systems may indeed be applicable to in vitro culture of gametes and embryos. The present paper reviews the advantages of microfluidics for in vitro embryo production: how the platforms are manufactured, the current uses of microfluidics in assisted reproduction, static v. dynamic culture environments, individual gamete and embryo culture and the future directions of microfluidic application to in vitro embryo production and manipulation. Finally, preliminary data from our laboratory using a new microfluidic well insert for porcine, bovine and murine embryo culture is discussed.

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