87 BACK TO THE DRAWING BOARD: IN VITRO CULTURE OF BOVINE EMBRYOS FOR FREEZING

2012 ◽  
Vol 24 (1) ◽  
pp. 156
Author(s):  
C. Vigneault ◽  
S. L. Underwood ◽  
V. Fournier ◽  
D. Bousquet ◽  
J. Belanger ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture System ◽  
Culture Media ◽  
Pregnancy Rates ◽  
Female Reproductive Tract ◽  
Slow Freezing ◽  
Direct Transfer ◽  
Bovine Embryos ◽  
Very High

Despite hundreds of scientific papers published, no system has resulted in in vitro embryos comparable to those produced in vivo. We hypothesised that assembling the most pertinent elements of IVF studies into one system would result in a highly efficient in vitro culture system. Here we report the in vitro production of bovine embryos using a culture system with strict environmental conditions that produces very-good-quality embryos at high rates. This system consists of a sequential culture system with media composition based on recent reports that characterise the bovine female reproductive tract (Hugentobler et al. 2007 Mol. Reprod. Dev. 74, 445–454; Hugentobler et al. 2007 Theriogenology 68, 538–548; Hugentobler et al. 2008 Mol. Reprod. Dev. 75, 496–503). This system uses a 3-step culture media to prevent toxicity resulting from ammonium accumulation and nutrient depletion and also to adjust the component concentrations to support embryo needs at different developmental stages. Fatty acid-free BSA is used as the protein source and the culture is in droplets under high-quality paraffin oil at 38.5°C under 6.8% CO2, 5% O2 and 88.2% N2. Numerous other aspects were investigated to limit embryo stresses (Lane et al. 2008 Reprod. Fertil. Dev. 20, 23–32) during manipulations, including the use of mini-incubators and very-high-purity gas combined with stringent laboratory practices. In the first year using this new embryo production system, 2839 oocytes were fertilized, resulting in a transferable blastocyst rate of 51%. Of the 1448 embryos produced, 779 were transferred fresh at our facility with pregnancy rates of 55 and 49% at 28 and 60 days, respectively. Pregnancy rates were directly related to the quality of the embryos transferred as 61% of grade 1 embryos transferred induced a pregnancy at Day 28, compared with 41% of grade 2 embryos. Pregnancy induction is not the only indication of good embryo quality. As is well-documented, in vitro-produced bovine embryos do not tolerate slow freezing, so vitrification was applied to surmount this intolerance. However, this is difficult to apply to industry because direct transfer of vitrified embryos is challenging. We hypothesised that the improvement of embryo culture would result in embryos that could tolerate slow freezing. Grade 1 blastocysts (n = 229) were frozen in 1.6M ethylene glycol and 0.1 M sucrose using standard slow freezing procedures. A very high proportion (91%) of frozen–thawed in vitro-produced embryos re-expanded after 24 h of culture with a good quality inner cell mass. Subsequently, 45 grade 1 blastocysts were frozen and transferred, giving pregnancy rates of 58% at Day 60. In conclusion, combining good-quality culture media and conditions resulted in the production of in vitro embryos that were very efficient at inducing pregnancies and tolerating slow freezing, which makes it now possible to consider direct transfer of frozen in vitro-produced bovine embryos.

31 Evaluation of in vitro-produced bovine embryos with conventional and SexedULTRA-4M X and Y chromosome-bearing semen: survival after slow freezing for direct transfer

2022 ◽  
Vol 34 (2) ◽  
pp. 250
Author(s):  
H. Álvarez-Gallardo ◽  
M. Kjelland ◽  
M. Pérez-Martínez ◽  
A. Velázquez-Roque ◽  
F. Villaseñor-González ◽  
...  
Keyword(s):  
Y Chromosome ◽  
Slow Freezing ◽  
Direct Transfer ◽  
Bovine Embryos

187 INCREASED NUMBER OF EXPANDED BOVINE BLASTOCYSTS IN SOF AND CR1 RELATIVE TO KSOM

2007 ◽  
Vol 19 (1) ◽  
pp. 210
Author(s):  
D. M. Kohl ◽  
R. L. Monson ◽  
L. E. Enwall ◽  
J. J. Rutledge
Keyword(s):  
Gene Expression ◽  
Culture Media ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Culture Conditions ◽  
Pregnancy Rates ◽  
Embryo Morphology ◽  
Bovine Embryos ◽  
Chi Square Analysis

Assessment of morphological stage grade is a subjective procedure. Stage grade is of vital importance to, among other things, recipient synchrony for the purpose of establishing successful pregnancies. Asynchronous embryo transfer has led to decreases in pregnancy rates (Farin et al. 1995 Biol. Reprod. 52, 676–682) and has been implicated in contributing to large offspring syndrome (Young et al. 1996 Theriogenology 45, 231). Differences in embryo kinetics based on culture conditions have been well documented (Mello et al. 2005 Reprod. Fert. Dev. 17, 221 abst). Whether such differences are the result of species, breed, metabolic stress, sire effects, or separation from an in vivo environment has yet to be determined. The correlation between oxygen respiration rates and embryo morphology as well as embryo diameter in bovine embryos produced in vitro has shown promise in the development of a more objective predictor of embryo quality and perhaps pregnancy initiation (Lopes et al. 2005 Reprod. Fert. Dev. 17, 151 abst). As well, recent examination of gene expression patterns of in vitro-derived bovine embryos seems to indicate that longer periods of in vitro culture are associated with lower rates of embryo survival (Lonergan et al. 2006 Theriogenology 65, 137–152). We hypothesize that differences do exist in the number, rate, and morphological appearance of blastocysts and that these parameters are in large part based on culture conditions in vitro. The objective of this experiment was to determine the timing and distribution of blastocyst formation of in vitro-produced bovine embryos cultured in SOF8, CR18AA, and KSOM8, under a standard incubation environment. Bovine ovaries from a local abattoir were aspirated and matured for 18-22. Oocytes were fertilized with frozen-thawed Percoll-separated semen from a Holstein bull. Presumptive zygotes were vortexed to remove cumulus cells and placed into 3 different culture media in a highly humidified atmosphere containing 20% oxygen, 5% carbon dioxide, and compressed air at 38.5�C. Embryos were evaluated specifically at 168 h post-insemination (Day 7) and assigned a morphological stage grade (IETS) to determine fixed time point differences. A total of 6 complete replicates were performed. Only embryos exhibiting the presence of a blastocoel at this time were documented (early blast, mid-blast, expanded blast). At 168 h post-insemination, there were no significant differences in the total number of embryos reaching early or mid-blast stage in any of the media. However, chi-square analysis revealed an increase in the number of expanded blastocysts in SOF (n = 813) and CR1 (n = 838) treatments compared to KSOM (n = 824; P < 0.0001). Expanded blastocysts in SOF were also greater in number than in CR1 (P < 0.05). Embryo selection based on development to the expanded blastocyst stage on Day 7 may prove useful in increasing pregnancy rates, and may validate qualitative correlations based on oxygen consumption and gene expression profiles for embryos produced in vitro.

Cryopreservation and direct transfer of in vitro produced bovine embryos: A comparison between vitrification and slow-freezing

1998 ◽  
Vol 49 (1) ◽  
pp. 170 ◽  
Author(s):  
M.W. Lane ◽  
T.J. Ahern ◽  
I.M. Lewis ◽  
D.K. Gardner ◽  
T.T. Peura
Keyword(s):  
Slow Freezing ◽  
Direct Transfer ◽  
Bovine Embryos

A continuous flow, perifusion culture system for 8- to 16-cell bovine embryos derived from in vitro culture

1996 ◽  
Vol 46 (8) ◽  
pp. 1441-1450 ◽  
Author(s):  
J.M. Lim ◽  
B.C. Reggio ◽  
R.A. Godke ◽  
W. Hansel
Keyword(s):  
In Vitro Culture ◽  
Continuous Flow ◽  
Culture System ◽  
Bovine Embryos

scholarly journals A novel embryo culture media supplement that improves pregnancy rates in mice

Reproduction ◽  
2017 ◽  
Vol 153 (3) ◽  
pp. 327-340 ◽  
Author(s):  
A R Highet ◽  
T Bianco-Miotto ◽  
K G Pringle ◽  
A Peura ◽  
S Bent ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Embryo Culture ◽  
Mouse Strain ◽  
Culture Media ◽  
Blastocyst Stage ◽  
Pregnancy Rates ◽  
Female Reproductive Tract ◽  
Reproductive Capacity ◽  
Blastocyst Development

The preimplantation embryoinvivois exposed to numerous growth factors in the female reproductive tract, which are not recapitulated in embryo culture mediain vitro. The IGF2 and plasminogen activator systems facilitate blastocyst development. We hypothesized that the addition of IGF2 in combination with urokinase plasminogen activator (uPA) and plasminogen could improve rates of blastocyst hatching and implantation in mice. B6BcF1 and CBAB6F2 mouse embryos were divided into one of four supplemented culture media treatment groups: (1) control (media only); (2) 12.5 nM IGF2; (3) 10 µg/mL uPA and 5 µg/mL plasminogen; or (4) a combination of IGF2, uPA and plasminogen treatments. Embryo development to blastocyst stage and hatching were assessed before transfer to pseudopregnant recipient females and implantation, pregnancy rates and postnatal growth were assessed. After 90.5 h of culture, IGF2 + U + P treatment increased the percentage of B6BcF1 embryos that were hatching/hatched and percentage developing to blastocyst stage compared with controls (P < 0.02). Following B6BcF1 embryo transfer, IGF2 + U + P treatment increased implantation sites at day 8 of pregnancy compared with controls (P < 0.05). Replication in the CBAB6F2 mouse strain showed significant improvements in pregnancy rates at days 8 and 18 but not in blastocyst development. No adverse effects were seen on gestational age, litter size or birthweight, or the reproductive capacity of offspring of IGF2 + U + P treated embryos. For embryos susceptible to detrimental effects ofin vitroculture, IGF2, uPA and plasminogen supplementation of culture media can improve pregnancy success, but the effect of treatment is dependent on the mouse strain.

scholarly journals Effects of Delipidation with Forskolin During In Vitro Culture of Bovine Embryos and Recipient Synchronization on Pregnancy Rates.

2010 ◽  
Vol 83 (Suppl_1) ◽  
pp. 677-677 ◽  
Author(s):  
Moises Barceló-Fimbres ◽  
Alfredo Anchondo-Garay ◽  
Esther López-Franco ◽  
Sara García-Quiñonez ◽  
Javier Antillón-Ruiz ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Pregnancy Rates ◽  
Bovine Embryos

168 CORRELATION OF DEVELOPMENT KINETICS AND SEX OF IN VITRO-PRODUCED BOVINE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 196
Author(s):  
A. R. Buzzo ◽  
A. R. Pupulim ◽  
J. Mazucheli ◽  
F. V. Meirelles ◽  
I. P. Emanuelli
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Proteinase K ◽  
Bovine Embryos ◽  
Male And Female ◽  
Stage Of Development ◽  
Males And Females ◽  
Kinetics Of

Approaches to improve the culture medium for in vitro production (IVP) of bovine embryos have been continuous because of the high commercial demand and a portion of this attempts the production of female cattle (dairy cows and stud cattle). However, in some embryonic in vitro culture systems, the development kinetics is faster in male than in female embryos (Avery 1992 Mol. Reprod. Dev. 32, 265–70; Xu 1992 Mol. Reprod. Dev. 31, 249–50). The aim of this work was to relate the kinetics of blastocyst expansion with the production rates of male and female embryos. Cumulus–oocyte complexes (n = 917; classes I and II) of cows from a slaughterhouse were matured with TCM-199 bicarbonate and 10% FCS (38.5°C, 5% CO2) for 24 h and fertilized with frozen-thawed semen in TALP-IVF medium for 18 h. Presumptive zygotes were culture in SOF medium supplemented with 10% FSB (5% O2, 38.5°C). Seven days after IVF, embryos were divided in 2 groups according to their kinetic stage of development: nonexpanded blastocysts (n = 175), or hatched and expanded blastocysts (n = 146). Hence, embryos were individually frozen in LN and stored in cryotubes. After thawing, Proteinase K (16 mg mL–1) was added to each tube and the tubes were incubated for 60 min at 37°C. Proteinase was denatured at 98°C for 10 min and the contents of each tube were divided into 2 samples (A and B) and subjected to the PCR technique. Two pairs of primers for the specific sequence of the Y chromosome were used to amplify the sequence of 210 and 250 bp for the male bovine and 1 pair of primers was used for the autosomal bovine sequence with a 280-bp fragment. Female embryos with a 280-bp product were observed in sample A and none were observed in sample B. The presence of 2 amplicons (280 and 210 bp) in sample A and 1 amplicon of 250 bp in sample B indicated that the embryo was male. A chi-square test was used to evaluate homogeneity. An analysis of the percentage of males and females between the experimental groups was performed by logistic regression and significance was considered when P < 0.05. There was no difference in the proportions of males and females in the nonexpanded blastocyst group (49.71 and 50.29%; P > 0.05). In the hatched and expanded blastocyst group, the proportion of males (65.75%) was statistically different from the proportion of females (34.25%); that is, the chance of the embryo being male was twice as high (P < 0.0038). These results suggest that there is a difference in the kinetics of embryo development between male and female embryos and that blastocyst expansion can point that out. In vitro culture media with FCS support the development of expanded male blastocysts. Further research in culture medium modifications (FCS, the energy source, amino acids and others) are needed to respond to the trend in the production of sex-defined embryos.

138 TIME-LAPSE CINEMATOGRAPHY-COMPATIBLE INJECTION-MOLDED MICROWELL CULTURE SYSTEM FOR TRACKING THE DEVELOPMENT OF INDIVIDUAL BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 173
Author(s):  
S. Sugimura ◽  
T. Akai ◽  
T. Somfai ◽  
M. Hirayama ◽  
Y. Aikawa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Culture System ◽  
Cell Mass ◽  
Calf Serum ◽  
Time Lapse ◽  
Pregnancy Rates ◽  
Cleavage Pattern ◽  
Bovine Embryos ◽  
Physiological Level

We have developed a polystyrene-based well of-the-well system (WOW) using injection moulding to track individual embryos throughout culture using time-lapse cinematography (TLC). The WOW-cultured bovine embryos following in vitro fertilization (IVF) were compared with conventional droplet (control)-cultured embryos on in vitro and in vivo development. Twenty-five of zygotes were cultured in each culture system containing 125 μL of CR1aa medium supplemented with 5% calf serum for 168 h after IVF. No differences between control- and WOW-cultured embryos were observed during development to the blastocyst stage. Morphological quality, inner cell mass (ICM), and trophectoderm (TE) cell numbers and post-vitrification survival rates were not different between control- and WOW-derived blastocysts; however, incidence of apoptosis in the ICM and TE cells was reduced in WOW culture (P < 0.01). Oxygen consumption in WOW-derived blastocysts was closer to physiological level than that of control-derived blastocysts. Moreover, WOW culture improved embryo viability, as indicated by increased pregnancy rates at days 30 and 60 after embryo transfer (P < 0.05). The TLC monitoring was performed to evaluate the cleavage pattern and the duration of the first cell cycle of embryos from oocytes collected by ovum pick-up; correlations with success of pregnancy were determined. Logistic regression analysis indicated that the cleavage pattern correlated with success of pregnancy (P < 0.05), but cell cycle length did not. Higher pregnancy rates (66.7%) were observed for animals in which transferred blastocysts had undergone normal cleavage, identified by the presence of 2 blastomeres of the same size without fragmentation, than among those with abnormal cleavage (33.3%). These results suggest that our microwell culture system is a powerful tool for producing and selecting healthy embryos and for identifying viability biomarkers. This work was supported by the Research and Developmental Program for New Bio-Industry Initiatives.

171 LASER-ASSISTED ZONA PELLUCIDA HATCHING IN FROZEN - THAWED BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 244 ◽  
Author(s):  
M. K. Chiasson ◽  
J. A. Carter ◽  
K. R. Bondioli ◽  
R. A. Godke ◽  
G. T. Gentry
Keyword(s):  
Calf Serum ◽  
Pregnancy Rates ◽  
Water Bath ◽  
Assisted Hatching ◽  
Direct Transfer ◽  
Pulse Treatment ◽  
Bovine Embryos ◽  
Hatching Rates

Incomplete zona hatching or failure of the zona to rupture compromises post-transfer embryo viability and conceptus development. Assisted hatching prior to the transfer of frozen-thawed bovine embryos has been proposed as a means to increase recipient pregnancy rates. The objective of this study was to determine if laser-assisted hatching would improve in vivo derived frozen-thawed bovine embryo hatching rates. In Exp. 1, direct-transfer beef cattle embryos were air-thawed for 15 s, placed in a 30°C water bath for 15 s, then held in TALP-HEPES, evaluated for stage and grade (1 = good to 3 = poor) and randomly applied to treatments. Embryos (n = 156) received either 2 or 3 symmetrical rents 40% through the outer zona surface using the XYClone diode laser (Hamilton Thorne, Beverly, MA, USA) at 90% power with a 600 μs pulse (Treatment A) or remained zona intact (Treatment B). Embryos were then cultured in vitro in CR1aa supplemented with 10% calf serum at 39°C in 5% CO2 and 5% O2 for 4 d. Embryo hatching rates were 47% for Treatment A and 53% for Treatment B. In Exp. 2, in vivo produced, nonsurgically collected direct-transfer Hereford embryos (n = 64) were utilized. In Exp. 3, in vivo produced nonsurgically collected glycerol frozen Brangus embryos (n= 46) were utilized. Embryos utilized in Exp. 2 and 3 were air-thawed for 15 s, placed in a 30°C water bath for 15 s, and then held in 1 M sucrose for 7 min. Embryos were then held in phosphate-buffered saline with 10% calf serum (Exp. 2) or ViGRO Holding Plus (Bioniche, Pullman, WA, USA) (Exp. 3), evaluated for stage and grade before being randomly assigned to either Treatment A or B. Embryos received either 3 symmetrical rents 40% through the outer zona surface using the XYClone laser at 90% power with a 600-μs pulse (Treatment A) or remained zona intact (Treatment B). Embryos were transferred nonsurgically (1 embryo/female) by the same technician into synchronized mixed breed recipient beef cows on Day 7 of the estrous cycle. Pregnancy status was determined at 35 days and 60 days via ultrasonography. In Exp. 2, treatment did not affect 60 day pregnancy rates across embryo grades 1, 2, and 3. Also, treatment did not affect pregnancy rates at 35 or 60 days (41% and 28% for Treatment A and 44% and 41% for Treatment B, respectively). Likewise, there was no difference in calving rate for recipients confirmed pregnant at 60 days for Treatment A (89%) and Treatment B (77%). In Exp. 3, treatment did not affect 60 day pregnancy rates across embryo grades 1, 2, and 3. Pregnancy rates at 35 and 60 days were not affected by treatment (65% and 65% for Treatment A and 76% and 59% for Treatment B, respectively). Calving rates for those recipients in Exp. 3 were not available at the time of abstract preparation. Based on the data presented herein, it does not appear that laser-assisted hatching with the XYClone laser increases the number of in vivo derived frozen-thawed embryos that hatch following in vitro culture or increase pregnancy rates after transfer to recipient cattle.

76 Tetrahydrofuran does not have Embryo Toxic Effects on In Vitro-Produced Bovine Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 176
Author(s):  
M. M. R. Chowdhury ◽  
I. Khan ◽  
A. Mesalam ◽  
K.-L. Lee ◽  
J.-Y. Hwang ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Bovine Serum ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Control Group ◽  
Bovine Embryos ◽  
Sodium Pyruvate ◽  
Fetal Bovine ◽  
The Impact

In vitro embryo developmental potentials are still suboptimal compared with in vivo potential due to the challenge of various unknown stressors that must be overcome by in vitro-cultured oocytes. To improve existing embryo developmental potentials, many chemicals have been treated in maturation media by dissolving in toxic substances such as dimethyl sulfoxide (DMSO) or other carrier molecule. The foremost effort of this study was to investigate the impact of the solvent tetrahydrofuran (THF) on the cytotoxicity of in vitro embryo production (IVP). The experiment was completed within 8 replicates. Statistical analyses were performed using SPSS version 22.0 (IBM/SPSS, Armonk, NY, USA), a one-way ANOVA followed by multiple pairwise comparisons (Tukey’s test), and Duncan’s multiple range post hoc test. The level of statistical significance was considered P < 0.05. Oocytes were cultured in vitro maturation media (IVM) followed by in vitro fertilization (IVF), in vitro culture media 1 (IVC1), and in vitro culture media 2 (IVC2). Composition of the media was as follows: IVM medium was TCM-199 supplemented with 10% (v/v) fetal bovine serum, 1 µg mL−1 oestradiol-17β, 10 µg mL−1 FSH, 0.6 mM cysteine, and 0.2 mM sodium pyruvate. The IVC1 medium consisted of CR1-aa supplemented with 44 µg mL−1 sodium pyruvate, 14.6 µg mL−1 glutamine, 10 IU mL−1 penicillin, 0.1 mg mL−1 streptomycin, 3 mg mL−1 BSA, and 310 µg mL−1 glutathione. The IVC2 medium was the same composition as IVC1 except that BSA was replaced with 10% (v/v) fetal bovine serum. The final concentration of the optimized (0.5 µM) THF in culture medium was 0.4%. When coculturing with 0.5 µM THF in the IVM stage, the cleavage rate (58.65 ± 1.90% v. 56.87 ± 1.68%) was not significantly different, but the blastocyst rate (35.21 ± 1.44% v. 28.34 ± 2.11%) was significantly higher compared with the control group. The TUNEL assay confirmed that apoptotic nuclei in THF group were significantly reduced compared with the control group (2.32 ± 0.14 v. 5.65 ± 0.12). The total cell number of trophectoderm (TE) in control and THF groups was 115.34 ± 0.98 and 132.13 ± 1.55, and that of the inner cell mass (ICM) was 29.67 ± 0.40 and 39.94 ± 0.44, respectively. However, the ICM:TE ratio in control and treated blastocysts was 1:3.34 and 1:3.9, which was not statistically significant. Immunocytochemistry analysis (using antibodies to IKBKB, NFkB, COX2, CASP9, and CASP3) demonstrated that THF supplementation significantly attenuated expression of these proteins. The quantitative recerse transcription PCR data established that relative mRNA expression level of the anti-apoptotic gene BCL2 was up-regulated, whereas that of COX2, iNOS, BAX, IKBKB, NFkB, CASP9, and CASP3 were significantly down-regulated in the THF treated group compared with the control. In conclusion, 0.5 µM THF supplement in the IVM media did not have injurious effects on in vitro-cultured bovine embryos. This work was supported by grant from the Next-Generation BiogGeen21 (No. PJ01107703), IPET (No. 315017-5 and 117029-3), Allergy free cat (Co.. Felix Pets) and BK21plus.

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