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

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

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.

65 Comparison of SexedULTRA-4M™ X and Y chromosome-bearing semen versus conventional semen for the in vitro production of bovine embryos

2022 ◽  
Vol 34 (2) ◽  
pp. 268
Author(s):  
A. Velázquez-Roque ◽  
H. Álvarez-Gallardo ◽  
M. Kjelland ◽  
M. Pérez-Martínez ◽  
F. Villaseñor-González ◽  
...  
Keyword(s):  
Y Chromosome ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Vitro Production

Offspring resulting from direct transfer of cryopreserved bovine embryos produced in vitro in chemically defined media

2002 ◽  
Vol 69 (3-4) ◽  
pp. 151-158 ◽  
Author(s):  
H.J. Hernandez-Fonseca ◽  
S. Sirisathien ◽  
P. Bosch ◽  
H.S. Cho ◽  
J.D. Lott ◽  
...  
Keyword(s):  
Direct Transfer ◽  
Bovine Embryos ◽  
Chemically Defined Media ◽  
Defined Media

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 Cryopreservation of in vitro-produced bovine embryos by vitrification: In pursuit of a simplified, standardized procedure that improves pregnancy rates to promote cattle industry use

2020 ◽  
Vol 36 (3) ◽  
pp. 251-270
Author(s):  
Van Do ◽  
Andrew Taylor-Robinson
Keyword(s):  
Slow Rate ◽  
Survival Rates ◽  
Slow Freezing ◽  
Embryo Cryopreservation ◽  
Human Embryos ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cattle Industry

The goal of cryopreservation is to retain the original stage of gametes and embryos after they have endured cooling and warming. Slow freezing is a standard method for in vivo-derived bovine embryo cryopreservation, threefifths of such embryos being frozen by this method globally. However, it is evident that slow freezing is not efficient for cryopreserving in vitro-produced bovine embryos. Hence, only one-third of in vitro-produced bovine embryos are cryopreserved. Vitrification is a preferred method for storage of human embryos; consequently, it has been explored as a novel means to store in vitro-produced bovine embryos, for which it shows considerable promise as an alternative to slow freezing. This is due to several reasons: vitrification is often less time-consuming than slow freezing; it does not need expensive slow rate freezing machines; and it has been proven to have comparatively higher survival rates. Yet, in the cattle industry vitrification continues to present shortcomings, such as possible toxicity of vitrification solutions and failure to standardize methods, which pose a challenge for its application to in vitro-produced bovine embryos. Therefore, determining the most suitable procedure is crucial to make vitrification more practical in commercial settings.

scholarly journals 97 ASSESSMENT OF VIABILITY OF IN VITRO PRODUCED BOVINE EMBRYOS FOLLOWING VITRIFICATION BY CVM OR SLOW FREEZING WITH ETHYLENE GLYCOL AND TRIPLE TRANSFER

2005 ◽  
Vol 17 (2) ◽  
pp. 199 ◽  
Author(s):  
B. Peachey ◽  
K. Hartwich ◽  
K. Cockrem ◽  
A. Marsh ◽  
A. Pugh ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Slow Freezing ◽  
High Survival ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Frozen Embryos ◽  
Morula Stage ◽  
Fresh Embryos

Vitrification has become the method of choice for the preservation of in vitro derived embryos of a number of species, and several methods of vitrification have been developed. One such method, the cryoLogic vitrification method (CVM) yields high survival rates of warmed embryos (Lindemans W et al. 2004 Reprod. Fertil. Dev. 16, 174 abst). In this study, the post-warm viability of bovine IVP embryos following either vitrification using CVM or slow freezing using ethylene glycol (EG) was compared. In addition, the survival of embryos following triple transfer to synchronized recipients was measured and the embryo (“e”) and recipient (“r”) contributions to embryo survival was determined using the “er” model for embryo survival (McMillan WH et al. 1998 Theriogenology 50, 1053–1070). Bovine IVP methods were those of van Wagtendonk et al. 2004 Reprod. Fertil. Dev. 16, 214 (abst). On day 7 of culture (Day 0 = IVF), Grade 1 and 2 embryos that had reached at least the late morula stage were selected for vitrification (20% DMSO, 20% ethylene glycol) or freezing in 1.5 M ethylene glycol + 0.1 M sucrose (0.5°C/min to −35°C). Following storage in LN2 for at least 24 h the embryos were thawed, the cryoprotectant removed, and the embryos cultured for 72 h in mSOF medium under 5% CO2, 7% O2, 88% N2. The number of hatching embryos was recorded at 24-h intervals. In addition, blastocyst and expanded blastocyst embryos were thawed and immediately transferred nonsurgically to recipients (three embryos of the same grade to each recipient) on Day 7 of a synchronized cycle (Day 0 = heat). The recipients were ultrasound-scanned for the presence of, and number of, fetuses on Days 35 and 62, respectively. The invitro assessment of 148 CVM and 230 EG frozen embryos indicated that more vitrified than EG embryos hatched by 72 h (73% vs. 62%; CVM vs. EG, χ2 = 4.5, P < 0.05). Overall, more Grade 1 embryos hatched than Grade 2 (74% vs. 60%, χ2 = 7.2, P < 0.01). CVM embryos (105) were triple-transferred to 35 recipients, and EG embryos (30) were triple-transferred to 10 recipients. Recipient pregnancy rates at Day 62 were 80% and 70%, respectively. Overall embryo survival was 38.5% (41% for CVM and 30% for EG). The overall calculated “e” and “r” values were 0.39 and 1.0 (“e”: 0.42 and 1.0, and “r”: 0.31 and 1.0, respectively, CVM and EG groups). Survival rates of CVM embryos to Day 62 (41%) were slightly lower than that previously obtained for fresh embryos produced using an identical IVP procedure (44% – van Wagtendonk AM 2004).

Efficient one-step direct transfer to recipients of thawed bovine embryos cultured in vitro and frozen in chemically defined medium

2020 ◽  
Vol 146 ◽  
pp. 39-47 ◽  
Author(s):  
Enrique Gómez ◽  
Susana Carrocera ◽  
David Martín ◽  
Juan José Pérez-Jánez ◽  
Javier Prendes ◽  
...  
Keyword(s):  
Defined Medium ◽  
Direct Transfer ◽  
Chemically Defined Medium ◽  
Bovine Embryos ◽  
One Step

50 SURVIVAL OF SEXED IVF-DERIVED BOVINE EMBRYOS FROZEN AT DIFFERENT PREIMPLANTATION STAGES OF DEVELOPMENT

2017 ◽  
Vol 29 (1) ◽  
pp. 132
Author(s):  
L. Ferré ◽  
C. Fresno ◽  
M. Kjelland ◽  
P. Ross
Keyword(s):  
Cytochalasin B ◽  
Cell Monolayer ◽  
Cumulus Cell ◽  
Slow Freezing ◽  
Treatment Level ◽  
Bovine Embryos ◽  
Low Oxygen ◽  
Stages Of Development ◽  
Cell Layers

The ability to freeze in vitro-produced bovine embryos with a high post-thaw viability is still problematic and hampers logistics of on-farm embryo transfer. The objectives of this experiment were to compare different stages of development, freezing methods, and addition of cytoskeletal stabilisers (cytochalasin-B) before freezing. Ovaries were collected from an abattoir and oocytes aspirated from 2- to 6-mm follicles. Cumulus-oocyte complexes containing compact and complete cumulus cell layers were selected and matured in groups of 50 in 400 µL of M199 medium supplemented with ALA-glutamine (0.1 mM), Na pyruvate (0.2 mM), gentamicin (5 µg mL−1), EGF (50 ng mL−1), ovine FSH (50 ng mL−1), bLH (3 µg mL−1), cysteamine (0.1 mM), and 10% fetal bovine serum (FBS) for 22 to 24 h. Fertilization (Day 0) was done using female sex-sorted semen selected with a discontinuous density gradient and diluted to a final concentration of 1 × 106 sperm/mL. Synthetic oviductal fluid (SOF)-FERT medium was supplemented with fructose (90 µg mL−1), penicillamine (3 µg mL−1), hypotaurine (11 µg mL−1), and heparin (20 µg mL−1). After 18 h, presumptive zygotes were denuded and cultured in groups of 15 to 20 in 50-µL drops of SOF-BSA for 7 days. On Day 3.5 post-fertilization, 3% FBS was added. Low oxygen tension (5% O2) was used for culture. Morulae were selected at Day 5.5–6, blastocysts at Day 6–6.5, and expanded blastocysts at Day 6.5–7. Embryo harvesting for each stage was performed from a dedicated drop/dish and discarded in order to avoid further embryo stage collections. Grade 1 morulae, blastocysts, and expanded blastocysts were selected for freezing and placed randomly into 2 groups: slow-freezing and vitrification. Before freezing, half of the embryos from each stage were exposed to cytochalasin-B for 45 min. The slow freezing protocol consisted of 1.5 M ethylene glycol (EG) + 20% FBS + 0.4% BSA, and the cooling rate was 0.5°C/min. Slow-frozen embryo thawing was performed by exposing the 0.25-mL straws to air (23°C) for 10 s and then underwater at 35°C for 1 min. The vitrification (Cryo-Top) medium was 15% (vol/vol) EG + propylene glycol. Vitrified embryos were thawed in a solution of H199 + 20% FBS and 0.25 M sucrose at 39°C. Thawed embryos from both groups were cultured in SOF-BSA + 10% FBS under cumulus/granulosa cell monolayer co-culture. Embryo assessment involved post-thaw survival (0 h), re-expansion, and hatching of the zona pellucida (72 h). Three replicates were performed for each treatment level. Fisher’s l.s.d. test with Bonferroni correction was used to determine treatment differences (P < 0.05). The post-thaw survival, re-expansion, and hatching results showed that either expanded blastocysts (84.7 ± 3.2%, 74.1 ± 3.9%, and 60.9 ± 4.4%) or blastocysts (81.7 ± 3.5%, 69.6 ± 4.2%, and 55 ± 4.6%) were preferred (P < 0.05) embryo stages for cryopreservation compared with morulae (67.6 ± 4.4%, 52.5 ± 4.6%, and 33.2 ± 4.3%). Vitrification and cytochalasin-B pre-freezing exposure (61.3 ± 3.6% and 56.6 ± 3.8%) provided better (P < 0.05) hatching results compared with slow-freezing and without cytochalasin-B (37.8 ± 3.6% and 42.5 ± 3.7%).

In vitro survival of in vitro produced (IVP) bovine embryos frozen or vitrified by techniques suitable for direct transfer

1995 ◽  
Vol 43 (1) ◽  
pp. 197 ◽  
Author(s):  
A. Dinnyés ◽  
C. Carolan ◽  
P. Loneragan ◽  
L. Solti ◽  
A. Massip ◽  
...  
Keyword(s):  
Direct Transfer ◽  
Bovine Embryos ◽  
In Vitro Survival
Sign in / Sign up

Export Citation Format

Share Document