127 IN VITRO DEVELOPMENT OF IVF CRYOPRESERVED BOVINE EMBRYOS SUPPORTED BY TCM-199 OR SOFaa CULTURE MEDIUM

2007 ◽  
Vol 19 (1) ◽  
pp. 181
Author(s):  
A. C. Nicacio ◽  
W. B. Feitosa ◽  
M. Rovegno ◽  
R. Simões ◽  
J. S. de A. Gonçalves ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Embryo Development ◽  
Culture Medium ◽  
Cell Monolayer ◽  
Slow Freezing ◽  
Bovine Embryo ◽  
Quick Freezing ◽  
Slow Group ◽  
Hatching Rates

In vitro bovine embryo production is commercially applied around the world. However, the cryopreservation of these embryos is not yet possible, which raises difficulties for the expansion of this biotechnology. The aim of this work was to evaluate the influence of the culture media on embryo development after cryopreservation. Cumulus–oocyte complexes obtained from slaughterhouse bovine ovaries were in vitro-matured, fertilized, and cultured. A total of 600 expanded blastocysts (between 7 and 9 days of culture) were cryopreserved by slow freezing, quick freezing, or vitrification methods. For slow freezing (slow group), the embryos were exposed to 10% ethylene glycol (EG) for 10 min and cryopreserved at 1.2�C per minute. For quick freezing (quick group), the embryos were exposed to 10% EG for 10 min and to 20% EG + 20% glycerol (Gly) for 30 s. For vitrification (vitrification group), the embryos were exposed to 10% EG for 10 min and to 25% EG + 25% Gly for 30 s. The straws (quick and vitrification groups) were placed in nitrogen vapor (0.8 cm over the liquid nitrogen) for 2 min and then immersed in liquid nitrogen. The embryos were thawed in air for 10 s and in a water bath at 25�C for 20 s. For warming, embryos were washed in PBS + 0.2% BSA + 0.3 M sucrose for 3 min and in PBS + 0.2% BSA for 3 min. To evaluate development after thawing, the embryos were cultured on a granulosa cell monolayer with TCM-199 or SOFaa for 4 days. The embryos of the slow group showed re-expansion rates of 55.55% (55/99) and 29.00% (29/100), respectively, for TCM-199 and SOFaa. The quick group showed re-expansion rates of 4.85% (5/103) and 7.22% (7/97), and the vitrification group 10.89% (11/101) and 14.00% (14/100), respectively, for TCM-199 and SOFaa. The slow group showed hatching rates of 47.47% (47/99) and 11.00% (11/100), respectively; the quick group did not show hatching rates in either medium. The vitrification group showed hatching rates of 7.92% (8/101) and 6.00% (6/100), respectively, for TCM-199 and SOFaa. The results were analyzed by chi-square test, and all values were significant at P < 0.05. The slow group showed difference in re-expansion and hatching rates when the different media were compared. The quick and vitrification groups did not show differences in re-expansion and hatching rates when the different media were compared. The slow group showed higher re-expansion and hatching rates than the quick and vitrification groups. In conclusion, the culture medium influences embryo development after slow freezing, and the TCM-199 is more appropriate than SOFaa. This work was supported by FAPESP 04/05335-1

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<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

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>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

144 USE OF A NOVEL BOVINE EMBRYO CULTURE MEDIUM TO IMPROVE BLASTOCYST DEVELOPMENT AND SURVIVAL FOLLOWING VITRIFICATION

2010 ◽  
Vol 22 (1) ◽  
pp. 231
Author(s):  
J. Block ◽  
L. Bonilla ◽  
P. J. Hansen
Keyword(s):  
Amino Acids ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Fresh Embryos ◽  
Hatching Rates

The objective of the present study was to determine whether culture of bovine embryos in a proprietary serum-free culture medium, Block-Bonilla-Hansen-7 (BBH-7), could improve development to the blastocyst stage and enhance survival following vitrification. For Exp. 1, embryos were produced in vitro and cultured in BBH-7 or modified synthetic oviductal fluid (mSOF; as in zygote 10:341 except with 10 μL mL-1 of nonessential amino acids, 20 μL mL-1 of essential amino acids, and 1 mg mL-1 of polyvinyl alcohol instead of albumin) in 5% (v/v) oxygen. Grade 1 expanded blastocysts were harvested at Day 7 post-insemination and vitrified using the open-pulled straw method (Vagta et al. 1998 Mol. Reprod. Dev. 51, 53-58). Vitrified embryos were thawed and cultured in vitro in either mSOF or BBH-7 supplemented with 10% fetal bovine serum and 50 μM dithiolthreitol. Re-expansion and hatching rates were recorded at 24, 48, and 72 h post-thaw. There was no effect of culture medium on cleavage rate. The proportion of oocytes that developed to the blastocyst and advanced blastocyst stages (expanded, hatching, and hatched) at Day 7 was higher (P < 0.001) for embryos cultured in BBH-7 than for embryos cultured in mSOF (41.9 ± 2.0 v. 14.7 ± 2.0% and 31.1 ± 1.3 v. 6.4 ± 1.3%, respectively). There was no effect of culture medium on re-expansion rates at 24, 48, and 72 h post-thaw or on hatching rates at 48 or 72 h. However, the proportion of embryos that were hatching or had hatched by 24 h post-thaw was higher (P < 0.001) for BBH-7 than for mSOF (59.0 ± 0.5 v. 26.7 ± 0.5%). For Exp. 2, late lactation and/or repeat breeder, lactating Holstein cows were synchronized for timed embryo transfer using the OvSynch-56 protocol. Embryos were produced in vitro and cultured in BBH-7 in 5% (v/v) oxygen. Vitrified embryos were produced as for Exp. 1. Fresh embryos were grade 1 expanded blastocysts harvested at Day 7 after insemination. A single embryo was transferred at Day 7 after putative ovulation to all cows with a corpus luteum confirmed by ultrasonography. Pregnancy was diagnosed at Day 28-30 of gestation by ultrasonography. There was no difference in the proportion of recipients that became pregnant after receiving either a fresh (7/18 = 39%) or vitrified (10/27 = 37%) embryo cultured in BBH-7. The results of the present study indicate that BBH-7 can be used to increase the proportion of oocytes that develop to the blastocyst stage. Moreover, the results demonstrate that vitrified embryos produced after culture in BBH-7 can achieve pregnancy rates similar to those obtained using fresh embryos. Support: USDA 2006-55203-17390 and Southeast Milk Checkoff Program.

Effects of amino acids on development in vitro of cleavage-stage bovine embryos into blastocysts

1996 ◽  
Vol 8 (5) ◽  
pp. 835 ◽  
Author(s):  
T Pinyopummintr ◽  
BD Bavister
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Embryo Development ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Development In Vitro ◽  
Defined Culture

Effects of amino acids on early bovine embryo development in vitro were examined using a chemically-defined, protein-free culture medium. Bovine embryos produced in vitro were cultured from 18 h to 72 h post insemination in a simple medium containing lactate as the only energy source except for the amino acid treatments. Subsequently, embryos were transferred to TCM-199 supplemented with serum for blastocyst development to substantiate their developmental competence. Treatments were: (1) non-essential amino acids from TCM-199 (NEA); (2) essential amino acids from TCM-199 (EA); (3) NEA+EA; (4) Eagle's minimum essential medium amino acids (MEM AA); (5) 11 amino acids present in HECM-6 (11 AA); and (6) 0.2 mM glutamine (GLN). A higher proportion of embryos (percentage of inseminated ova) cleaved to the > or = 8-cell stage by 72 h post insemination in NEA (56.7%), EA (41.2%), 11 AA (40.3%) and GLN (51.1%) than in either NEA+EA (30.0%) or MEM AA (33.1%). However, after transfer to complex medium, embryos that had developed in EA, as well as those in MEM AA or NEA+EA, produced significantly fewer blastocysts (37.1%, 34.4% and 25.6% respectively) than those in NEA (56.7%), GLN (48.9%) or 11 AA (37.7%). The ability of blastocysts to hatch from their zonae pellucidae was also affected by amino acid treatment during cleavage stages. The present study indicated that the addition of NEA or GLN or 11 AA to a chemically-defined culture medium during the cleavage phase of bovine embryo development increases their subsequent ability to reach the blastocyst stage. These data have implications for understanding the nutritional needs of bovine embryos produced in vitro and for optimizing the composition of culture media to support their development.

149 EFFECT OF RESVERATROL ANALOGUE ON DEVELOPMENT OF IN VITRO-FERTILIZED BOVINE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 183 ◽  
Author(s):  
T. A. Patrocínio ◽  
C. A. C. Fernandes ◽  
L. S. Amorim ◽  
J. R. Ribeiro ◽  
G. C. Macedo ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Culture Medium ◽  
Synthetic Analogue ◽  
Bovine Embryo ◽  
High Humidity ◽  
Cleavage Rate ◽  
Antioxidant Agents ◽  
Blastocyst Rate

Oxidative stress is one of the main effects of in vitro culture. Generation of reactive oxygen species (ROS) by embryos can be enhanced by the sub-optimal in vitro culture conditions and are associated with a delay in embryonic development. However, supplementation of culture medium with antioxidant agents can minimize the effects of ROS (Guérin et al. 2001 Hum. Reprod. Update 7, 175–189). Resveratrol is an example of a potent antioxidant, and modifications in its structure can improve its biological activity. This study evaluated the effect of AR33 (formula with patent pending), an analogue of resveratrol with high antioxidant activity, on embryo development. Bovine cumulus-oocyte complexes recovered from ovaries collected at the slaughterhouse were in vitro matured for 24 h and oocytes were in vitro fertilized for 20 h, both at 38.8°C under 5% CO2 in air and high humidity. Partially denuded presumptive zygotes were randomly distributed in 4 treatments (with 6 replicates): 0 µM (control, n = 347), 0.1 µM (n = 337), 0.5 µM (n = 277), and 2.5 µM (n = 343) of AR33. The base medium was SOFaa supplemented with 2.5% FCS and incubation conditions were 38.8°C under 5% CO2 in air and high humidity. Half of culture medium was renewed (feeding) at Day 3 and 5 post-fertilization. Cleavage was evaluated at Day 3 and blastocyst rates at Day 7 and 8 post-fertilization. Data were analysed by logistic regression considering the significance level of P < 0.05. Values are shown as mean ± SEM. Cleavage rate was higher (P < 0.05) for 2.5 µM (69.0 ± 4.4%) than for 0, 0.1, and 0.5 µM AR33 (62.1 ± 2.0%, 60.7 ± 5.9%, and 56.7 ± 5.8%, respectively). At Day 7, the blastocyst rate was similar (P > 0.05) among 0.1, 0.5, and 2.5 µM (18.1 ± 5.4%, 17.5 ± 2.9%, and 19.4 ± 3.3%, respectively) and all of them were higher (P < 0.05) than 0 µM AR33 (12.4 ± 2.5%). At Day 8, there was again no difference (P > 0.05) among 0.1, 0.5, and 2.5 µM AR33 (21.0 ± 5.0%, 18.4 ± 2.1%, and 24.6 ± 3.3%, respectively) but only 0.1 and 2.5 µM showed higher (P < 0.05) blastocyst rate than 0 µM AR33 (15.2 ± 2.5%). In conclusion, the synthetic analogue of resveratrol tested in this study can improve bovine embryo development in culture medium supplemented with 2.5% FCS under 5% CO2 in air. A concentration of 2.5 µM AR33 can be a choice for further studies. This study was supported by Fapemig, CAPES, and CNPq.

scholarly journals Culture system and long-term storage of culture media in the in vitro production of bovine embryos

2011 ◽  
Vol 59 (1) ◽  
pp. 129-139 ◽  
Author(s):  
Santiago Varga ◽  
Carmen Diez ◽  
Lina Fernández ◽  
Jenny Álvarez ◽  
Adelino Katchicualula ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture System ◽  
Culture Media ◽  
Calf Serum ◽  
Significant Loss ◽  
Bovine Embryo ◽  
In Vitro Production ◽  
Fresh Culture Medium ◽  
Hatching Rates

The optimum culture system for in vitro matured and fertilised oocytes still remains to be clarified. Culture media (CM) for mammalian embryos are routinely prepared fresh for use and preserved under refrigeration during one or two weeks. The purposes of this work were (1) to compare the efficiency of a synthetic oviduct fluid (SOF) with two different bovine serum albumin (BSA) concentrations (3 and 8 g/L) for the in vitro production of bovine blastocysts, (2) to test the effect of timing on adding fetal calf serum (FCS) to the SOF, and (3) to evaluate the effects on bovine embryo development of freezing and lyophilisation as procedures for preserving the SOF. Supplementation of SOF with 3 g/L BSA increased Day-7 blastocyst expansion rates (18.3 ± 1.6 vs. 14.4 ± 0.7; P < 0.05), although no differences in hatching rates were found. Addition of FCS to SOFaa (SOF with amino acids) medium supplemented with sodium citrate (SOFaaci) at 48 and at 72 h post-insemination (PI) allowed obtaining higher Day-6 embryo development rates than when FCS was added at 18 or 96 h PI (Day-6 morulae + blastocyst rate: 30.0 ± 1.1, 40.8 ± 1.1, 43.9 ± 2.3 and 39.3 ± 0.5 for FCS addition at 18, 48, 72 and 96 h, respectively). Hatching rates were significantly improved when serum was added at 72 h PI. Finally, both refrigeration and lyophilisation appeared as useful cryopreservation procedures for SOFaaci, although a significant loss of its ability to support embryo development, compared to the control fresh culture medium, was observed.

288 PRELIMINARY RESULTS OF THE USE OF TUMOR CELLS OF A DOG TO CONDITION BOVINE EMBRYO IN VITRO CULTURE MEDIUM

2010 ◽  
Vol 22 (1) ◽  
pp. 301
Author(s):  
M. Moreno Millán ◽  
J. Ocaña Quero ◽  
L. J. De Luca ◽  
M. Hidalgo Prieto ◽  
J. Dorado Martín ◽  
...  
Keyword(s):  
Conditioned Medium ◽  
Embryo Development ◽  
Culture Medium ◽  
Calf Serum ◽  
Development Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Humid Atmosphere ◽  
Preliminary Results

The aim of this study was to analyze the response of bovine embryos fertilized in vitro to a tumor dog cell- conditioned TCM-199 culture medium used in the development stage. Two hundred ninety-five oocytes obtained from slaughterhouse ovaries were cultured in groups of 20 cells at 39°C in TCM-199 medium droplets with 20% estrous cow serum (ECS) for 24h in a humid atmosphere containing 5% CO2. After incubation, these oocytes were fertilized with a 1 × 106 sperm cell dose, previously capacitated by a 1-h incubation in SP-TALP medium under usual conditions, by incubation in small petri dishes with 1 mL of Fert-TALP medium for 24 h under the conditions described previously. Alleged fertilized oocytes were randomly separated into 2 groups. A control group (n = 144) was cultured in TCM-199 medium with 10% fetal calf serum (FCS) and the other (n = 151) was cultured in TCM-199 medium previously conditioned by incubation for 72 h with 1 × 106 cells of a complex adenocarcinoma grade 1 obtained from a 6-year-old boxer dog and sterilized by filtration with 0.2-(im membrane. After 72 h of culture, the zygotes were harvested and prepared for a quick microscope observation. Alleged zygotes were classified as “not fertilized,” “divided,” or “degenerated.” Statistical differences (P < 0.01) were found among both groups with a greater number of normal divided embryos (65.2%) in the conditioned medium group and a lower number (30.4%) in the control group. The number of normal divided embryos in the conditioned medium after 72 h of incubation was similar to that achieved by current methods. These preliminary results are consistent with results obtained by other researchers in which growth factors derived from established cell lines can be used to condition culture medium for in vitro bovine embryo development. Further studies will be necessary to clarify how these factors contribute to in vitro embryo development. Spanish Retinta Breed Asociation, Spain.

scholarly journals 248 EMBRYO DEVELOPMENT IN MICRODROPS AND MICROCHANNELS: COMPARISON OF NCSU23 SEQUENTIAL AND NON-SEQUENTIAL CULTURE MEDIUM

2005 ◽  
Vol 17 (2) ◽  
pp. 274
Author(s):  
A.S. Lima ◽  
C.E. Ferguson ◽  
M.B. Wheeler
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Culture Media ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
The Other ◽  
Cell Stage ◽  
Development Rates ◽  
Hatching Rates

The in vitro culture systems used to produce pig embryos generally result in few embryos developing to the blastocyst stage. The use of pyruvate (pyr) and lactate (lac) during the culture of zygotes to the 8-cell stage followed by glucose (glu) supplementation replacing pyr and lac appears to be beneficial for embryo development in the pig. The aim of this study was to compare the embryo development rates from pig oocytes fertilized with and without cumulus cells in 100-μL microdrops (MD) and cultured in 100-μL MD or microchannels (MC), using NCSU23 containing 8 mg/mL of BSA and supplemented with (1) glu or (2) pyr/lac or (3) pyr/lac for the first three days and then with just glu for the remainder of culture period (pyr/lac-glu). Sow oocytes were matured in TCM199 supplemented with gonadotropins for the first 22 h, and for an additional 22 h without hormones. After 44 h of maturation, oocytes were placed in MD of modified tris-buffered medium to be fertilized using 3 × 105 sperm/mL. Oocytes were divided into two groups for fertilization: with and without cumulus cells. Following 6 h of fertilization, all inseminated oocytes were washed, divided into groups of 15, allotted to the three culture media treatment groups as described above, and incubated in either MD or MC. With the exception of one treatment there were no significant differences in development rates among embryos cultured in MD or MC, hence data were pooled from these two culture devices. Only oocytes fertilized without cumulus cells and cultured in pyr/lac in MC appeared to have lower rates of blastocyst formation (11.67%) than those cultured in MD (26.67%) in the same culture medium. When the six treatments were compared, oocytes fertilized with cumulus cells and cultured in glu had significantly higher (P < 0.05) blastocyst rates and hatching rates compared with the other treatments, with the exception of those fertilized without cumulus cells and cultured in pyr/lac-glu. There were no significant differences among other treatments in Day 7 blastocyst or in Day 9 hatching rates. In conclusion, both culture devices can be used to reach similar blastocyst rates with different treatments. In this experiment, the removal of cumulus cells before fertilization appeared to enhance embryo development in vitro when sequential media are used. On the other hand, the presence of cumulus cells before fertilization seems to enhance embryo development when non-sequential glu medium is used. Table 1. Embryo development rates on Day 9 for three different culture treatments

48 EFFECTS OF LIPID METABOLIC REGULATORS DURING BOVINE EMBRYO CULTURE ON BLASTOCYST DEVELOPMENT AND CRYOSURVIVAL

2014 ◽  
Vol 26 (1) ◽  
pp. 138 ◽  
Author(s):  
A. Ruiz ◽  
P. J. Hansen ◽  
J. Block
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Blastocyst Stage ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Phenazine Ethosulfate ◽  
Metabolic Regulators ◽  
Hatching Rates

The overall objective was to determine the effects of addition of lipid metabolic regulators during embryo culture on blastocyst development and survival following cryopreservation. For Experiment 1, embryos produced in vitro were cultured in 5% (vol/vol) oxygen in SOF-bovine embryo 1 (SOF-BE1) medium supplemented with or without 100 μM trans-10,cis-12 conjugated linoleic acid (CLA) and 0.3 μM phenazine ethosulfate (PES). Treatment with CLA began at the initiation of culture, whereas treatment with PES began at Day 3 after insemination. At Day 7 after insemination, the proportion of oocytes that developed to the blastocyst and advanced blastocyst (expanded, hatching, or hatched) stages was recorded. Blastocysts and expanded blastocyst-stage embryos were harvested and slow frozen in 1.5 M ethylene glycol. Embryos were thawed and then cultured for 72 h in SOF-BE1 medium containing 10% (vol/vol) fetal bovine serum and 50 μM dithiothreitol. Re-expansion and hatching rates were recorded at 24, 48, and 72 h. Addition of CLA had no effect on embryo development, whereas PES reduced (P < 0.01) development to the blastocyst (26.0 ± 0.8 v. 22.1 ± 0.8%) and advanced blastocyst (19.2 ± 0.9 v. 14.4 ± 0.9%) stages. Blastocysts cultured in the presence of CLA had higher (P < 0.05) re-expansion rates at 24, 48, and 72 h (50.8 ± 3.7 v. 65.7 ± 3.7%, 57.2 ± 4.0 v. 72.0 ± 4.05%, and 57.2 ± 4.0 v. 72.0 ± 4.0%, respectively). Addition of CLA tended (P < 0.07) to increase the hatching rate at 24 h and did increase (P < 0.05) the hatching rate at 48 h (12.4 ± 1.3 v. 16.2 ± 1.3% and 39.0 ± 3.2 v. 50.0 ± 3.2%, respectively). Treatment with PES had no effect on re-expansion rates but reduced (P < 0.05) hatching rates at 24 and 48 h (18.2 ± 1.3 v. 10.3 ± 1.3 and 50.2 ± 3.2 v. 38.8 ± 3.2%, respectively). There was no interaction between CLA and PES affecting embryo development or cryosurvival. For Experiment 2, embryos were produced in vitro as in Experiment 1 and cultured in SOF-BE1 medium with or without 3.03 mM L-carnitine (LC) and 10 μM forskolin (FK). Treatment with LC began at the initiation of culture and treatment with FK began at Day 6. All other methods were as described for Experiment 1. Addition of LC did not affect development to the blastocyst stage but reduced (P < 0.05) development to the advanced blastocyst stage (21.0 ± 1.2 v. 17.1 ± 1.2%). Treatment with FK had no effect on embryo development to the blastocyst or advanced blastocyst stages. Blastocysts cultured in the presence of LC had increased (P < 0.05) re-expansion rates at 24, 48, and 72 h (60.2 ± 2.0 v. 78.0 ± 2.0%, 62.9 ± 1.2 v. 83.3 ± 1.2%, and 63.0 ± 2.4 v. 82.8 ± 2.4%, respectively) and hatching rates at 48 and 72 h (48.6 ± 4.3 v. 64.1 ± 4.3% and 59.6 ± 3.0 v. 78.5 ± 3.0%, respectively). There was no effect of FK on cryosurvival and no interaction between LC and FK affecting embryo development or cryosurvival. In conclusion, blastocyst yield was not improved by any of the lipid metabolic regulators tested. Cryosurvival was enhanced by addition of CLA and LC but FK reduced survival following freezing. There were no additive effects of either CLA and PES or LC and FK for blastocyst yield or cryosurvival.Support was provided by USDA AFRI Grant 2010-85122-20623.

133 EVALUATION OF OOCYTE DONOR SOURCE AND CULTURE MEDIUM ON DOMESTIC CAT EMBRYO DEVELOPMENT RATES

2011 ◽  
Vol 23 (1) ◽  
pp. 171
Author(s):  
A. J. Pearks Wilkerson ◽  
R. D. Landry ◽  
C. R. Long
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Culture Medium ◽  
Cytochalasin B ◽  
Culture Media ◽  
Domestic Cat ◽  
Bovine Embryo ◽  
Development Rates ◽  
L1 And L2

The use of assisted reproductive technology (ART), including in vitro maturation (IVM) and embryo culture, is well established in several species, including canine and feline culture systems. Embryo production conditions tend to be specific for each species and prepared in unique formulations by laboratory. However, the increasing numbers of commercially available media allows for new comparisons in companion animal systems. Therefore, a goal of this study was to compare the development rates of feline parthenotes cultured in a commercially available bovine embryo culture medium with those cultured in a published 3-step domestic cat-specific system. In addition, the source of ovaries utilised for oocyte retrieval was evaluated as a factor in development rates. Ovaries from 2 locations (L1 and L2) were collected on the same day, and harvested oocytes were held in meiotic arrest medium containing 25 μM roscovitine for 14 to 18 h. Oocytes were incubated in maturation medium for 24 h before cumulus cell removal with vigorous pipetting in 0.4% hyaluronidase, and a subset of each group was fixed and stained to determine meiotic maturation rates (n = 76 and 55 for L1 and L2, respectively). Following activation (day 0) by a single course of three 50-μs electric pulses at 1.2 kV cm–1 in 0.3 M mannitol, 0.1 mM CaCl2, and 0.1 mM MgSO4, parthenotes from each source were randomly divided to culture medium treatment of Bovine Evolve medium (Zenith Biotech, Guilford, CT, USA) with 4 mg mL–1 BSA (n = 209) or IVC-1 medium n = 269; (Pope et al. 2009 Theriogenology 71, 864–871), each containing 10 μg mL–1 cycloheximide and 7.5 μg mL–1 cytochalasin B. After a 4-h activation treatment, parthenotes were moved to culture media without cycloheximide and cytochalasin B for embryo development. All parthenotes in IVC-1 medium were moved to IVC-1a medium on day 2. On day 5, both sets of parthenotes were moved to culture media containing 10% heat-inactivated FBS instead of BSA. On day 7, all parthenotes were fixed and stained with Hoechst to determine cell number. No differences were seen in maturation rates between L1 and L2 (56.3 ± 9.5 v. 54.7 ± 9.5, respectively). However, cleavage rates tended to differ, and proportion of embryos greater than 64 cells was different (60.7 ± 5.8 v. 78.3 ± 5.8, P = 0.056 and 3.0 ± 3.1 v. 19.7 ± 3.1, P < 0.005; respectively). We hypothesised that the physical condition of the ovary donors may have affected development rates because cats from L1 tended to be feral animals, whereas cats from L2 were mostly privately owned. Bovine Evolve was similar to IVC-1 medium for cleavage, 32-cell, and 64-cell development rates (74.2 ± 6.7 v. 64.8 ± 6.7; 24.0 ± 7.5 v. 31.8 ± 7.5; 10.7 ± 4.8 v. 12.0 ± 4.8, respectively; P > 0.05). These results indicate that commercially available culture medium can support in vitro development, even if the commercial medium is developed for a different species, but that source of cat ovaries should be considered in feline ART.

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