81 Effects of Day 5 or 6 HEPES-Buffered Transportation Culture Medium on Developmental Competence of Bovine In Vitro-Produced Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 179
Author(s):  
W. Choi ◽  
C. M. Owen ◽  
M. Barcelo-Fimbres ◽  
J. L. Altermatt ◽  
L. F. Campos-Chillon
Keyword(s):  
Lipid Content ◽  
Embryo Culture ◽  
Culture Medium ◽  
Nile Red ◽  
Developmental Competence ◽  
Fluorescent Intensity ◽  
Embryo Culture Medium ◽  
Thermo Fisher Scientific ◽  
And Control

Most in vitro-produced (IVP) bovine embryos are transferred fresh. Use of a HEPES/bicarbonate embryo culture medium for transportation would offer flexibility for embryo shipment and transfer. We hypothesized that embryos cultured for 36 (Day 6 embryos) or 60 h (Day 5 embryos) in a novel SCF1T medium (SOF for Conventional Freezing 1 supplemented with HEPES) would maintain developmental competence compared with bicarbonate-buffered medium SCF1 (control). In 5 replicates, IVP embryos were produced by aspirating cumulus–oocyte complexes (COC) from 2-to 8-mm follicles of abattoir ovaries. The COC (n = 1036) were matured for 23 h, fertilized with semen from 1 of 3 bulls, and cultured in SCF1 at 38.5°C, 5% CO2, 5% O2, and 90% N2 (Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130). Randomly, on Day 5 and 6 after fertilization, a subset of presumptive embryos were moved into 500-µL polystyrene vials containing 100 µL of SCF1T medium, covered with 300 µL of sterile mineral oil and cultured in a portable incubator (MicroQ iQ2, Scottsdale, AZ, USA) at 38.5°C for 60 and 36 h, respectively. On Day 7.5 post-fertilization, blastocyst rates were evaluated and embryos (n = 8) from each group were stained with 1 µg mL−1 Nile Red for lipid quantification, and 300 nM Mitotracker Red CMX-Rosamine (Thermo Fisher Scientific, Waltham, MA, USA) for mitochondrial polarity. Images were obtained with confocal microscopy and fluorescent intensity (AFU) was measured by Image J software (National Institutes of Health, Bethesda, MD, USA). Data were analysed by one-way ANOVA and means separated by Tukey’s HSD. Results (Table 1) indicate similar blastocyst rates and lipid content between embryos cultured for 36 to 60 h in SCF1T and control media (P > 0.05). However, mitochondrial polarity was lower in the Day 5 group (P < 0.05) compared with Day 6 and control groups. Results suggest that culturing embryos in SCF1T medium for 36 h maintains developmental competence compared with bicarbonate-buffered media and offers an alternative for shipment and transfer of IVP embryos. Studies involving evaluation of pregnancy rates of the present study are ongoing. Table 1.Effects of Day 5 or 6 SCF1T embryo culture medium on development, lipid content, and mitochondrial polarity

93 PRODUCTION OF LIVE PIGLETS BY CRYOPRESERVATION OF IN VITRO-PRODUCED PORCINE ZYGOTES

2008 ◽  
Vol 20 (1) ◽  
pp. 127
Author(s):  
T. Somfai ◽  
N. Kashiwazaki ◽  
M. Ozawa ◽  
J. Noguchi ◽  
H. Kaneko ◽  
...  
Keyword(s):  
Culture Medium ◽  
Aluminum Foil ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
High Rate ◽  
Cell Numbers ◽  
Embryo Culture Medium ◽  
Vitrification Solution ◽  
And Control

Successful cryopreservation of in vitro-produced porcine zygotes is reported in the present study. Follicular oocytes were collected from prepubertal gilts. They were matured (IVM), fertilized (IVF), and cultured (IVC) in vitro (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). Ten or 23 h after IVF, the oocytes were centrifuged at 10 000g at 37�C for 20 min to permit visualization of pronuclei. Zygotes with two or three pronuclei were selected under stereomicroscope and used for solid surface vitrification (SSV). Briefly, after equilibration in 4% ethylene glycol (EG) for 15 min, zygotes were washed in vitrification solution (35% EG, 5% polyvinyl pyrrolidone, and 0.3 m trehalose), and then dropped with about 2 µL vitrification solution onto the dry surface of aluminum foil floating on the surface of liquid nitrogen (LN2). Microdroplets were transferred into cryotubes and stored in LN2. During warming, vitrified droplets were transferred in warming solution (0.4 m trehalose) at 37�C for 1 min, and then consecutively transferred for 1-min periods into 0.2 m, 0.1 m, or 0.05 m trehalose solutions. Survival of vitrified/warmed zygotes was determined by their morphology. To assess their developmental competence, vitrified (SSV), cryoprotectant-treated (CT), and untreated (control) zygotes were cultured in vitro for 6 days. There was no difference in developmental competence between control and CT zygotes in terms of cleavage rates (88.1% and 86.1%, respectively), blastocyst rates (23.2% and 20.8%, respectively), and blastocyst cell numbers (38.0 � 2.0 and 41.2 � 1.7, respectively). The rate of live zygotes after SSV and warming was similar to that of the control (93.4% and 100%, respectively). Cleavage rates (71.7% and 86.3%, respectively) and blastocyst rates (15.8% and 24.5%, respectively) of SSV were significantly reduced after vitrification compared to control (P < 0.05, ANOVA). Blastocyst cell numbers of SSV and control embryos were similar (41.2 � 3.4 and 41.6 � 3.3, respectively). There was no difference in developmental ability between zygotes cryopreserved at an early (10 h after IVF) or late (23 h after IVF) pronuclear stage. When embryo culture medium was supplemented with 1 µm of the antioxidant glutathione, development of cryopreserved zygotes to the blastocyst stage did not differ significantly from that of the control zygotes (18.6% and 22.1%, respectively). To test their ability to develop to term, 150 vitrified zygotes were transferred into a recipient, resulting in pregnancy and the production of five live piglets. These data demonstrate that a high rate of porcine zygotes could be successfully cryopreserved at the pronuclear stage, preserving their full developmental competence.

In vitro development of equine nuclear transfer embryos: effects of oocyte maturation media and amino acid composition during embryo culture

Zygote ◽  
2003 ◽  
Vol 11 (1) ◽  
pp. 77-86 ◽  
Author(s):  
Y.H. Choi ◽  
Y.G. Chung ◽  
S.C. Walker ◽  
Westhusin M.E. ◽  
K. Hinrichs
Keyword(s):  
Amino Acids ◽  
Embryo Culture ◽  
Nuclear Transfer ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Embryo Culture Medium ◽  
Igf I

This study was conducted to evaluate the effects of insulin-like growth factor I (IGF-I) and other media factors during oocyte maturation, and the presence of different compositions of amino acids in embryo culture medium, on the development of equine embryos. Oocytes recovered from slaughterhouse-derived ovaries were matured in vitro for 24 h and those with a polar body were subjected to intracytoplasmic sperm injection (ICSI) or nuclear transfer with adult fibroblasts (NT). For ICSI embryos, there were no significant differences in rates of morphological cleavage, cleavage with normal nuclei or average nucleus number at 96 h post-ICSI between the absence and presence of IGF-I in maturation medium, or between embryos cultured in G1.2 or a modified CZB medium (CZB-C). Embryos produced by interspecies NT (equine donor cells into bovine cytoplasts) also showed no difference in cleavage rate or average nucleus number whether cultured in G1.2 or in CZB-C. The rates of cleavage, cleavage with normal nuclei and average nucleus number of equine NT embryos were not significantly different among oocytes matured in M199 with FSH in the presence or absence of IGF-I, or in EMMI medium, which contains IGF-I, epidermal growth factor, steroid hormones, FSH and LH. There were no differences in development of equine NT embryos cultured in any of three amino acid treatments (with or without non-essential amino acids, or containing taurine, hypotaurine and cysteine only). The cleavage rate and average nucleus number of parthenogenetically activated oocytes (treated similarly to NT oocytes but not enucleated or subjected to donor cell injection) were significantly (p < 0.05) higher than those for NT embryos. These results indicate that the presence of IGF-I or of EMMI medium during in vitro maturation of equine oocytes does not have a beneficial effect on their developmental competence as assessed at 96 h. Presence or absence of non-essential amino acids in embryo culture medium does not affect development of NT embryos within the first 96 h of culture. Factors associated with enucleation or nuclear transfer decrease the developmental competence of equine NT embryos. CZB-C medium may be used for culture of equine embryos with results similar to those obtained with G1.2 medium, thus providing a base medium that may be modified for further study of culture requirements of equine embryos.

161 LIPID CONTENT, RESISTANCE TO CRYOPRESERVATION, AND SEX RATIO OF IN VITRO BOVINE BLASTOCYSTS PRODUCED IN A SERUM-FREE SYSTEM

2006 ◽  
Vol 18 (2) ◽  
pp. 188
Author(s):  
F. George ◽  
C. Daniaux ◽  
G. Genicot ◽  
F. Focant ◽  
B. Verhaeghe ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Lipid Content ◽  
Culture Medium ◽  
Nile Red ◽  
Embryo Cryopreservation ◽  
Hatching Rate ◽  
Free System ◽  
Serum Free

In vitro-produced (IVP) bovine blastocysts are known to be more sensitive to cryopreservation than their in vivo counterparts. Removing serum from the culture medium decreases sanitary risk and could improve embryo resistance to cryopreservation by preventing the accumulation of intracellular lipids. Our objectives were to evaluate the lipid content, resistance to cryopreservation, and sex ratio of IVP embryos cultured in a serum-free system. Oocytes from slaughterhouse ovaries were matured in a serum-free enriched medium (Donnay et al. 2004 Reprod. Fertil. Dev. 16, 274) and cultured in 5% O2 in modified SOF supplemented with 5% FCS (FCS) or with insulin-transferrin-selenium (ITS) and 0.1 mg/mL polyvinylpyrrolidone (PVP) (ITS-PVP) or 4 mg/mL BSA (ITS-BSA) (Daniaux et al. 2005 Reprod. Fertil. Dev. 17, 217). Day 5 morulae were stained with the fluorescent dye Nile Red in order to evaluate their lipid content (Genicot et al. 2005 Theriogenology 63, 1181). Day 7 blastocysts (diameter ≥160 µm) were selected, classified according to their size, and frozen in HEPES-SOF containing 1.5 M ethylene glycol, 0.1 M sucrose, and 1.8 mg/mL wheat peptones (George et al. 2002 Reproduction 29, 51). The lipid content was significantly lower in morulae cultured in ITS-BSA compared with the two other media (320 ± 10 arbitrary fluorescence units vs. 383 ± 12 in FCS and 406 ± 10 in ITS-PVP; n = 271; ANOVA2: P < 0.01). After cryopreservation, a higher total hatching rate was found 24 h post-thawing in blastocysts cultured in ITS-BSA and for both serum-free conditions at 48 h (Table 1). In particular, embryos ≤180 µm cultured in FCS were less resistant to cryopreservation than embryos of the same size produced without serum. Expanded blastocysts cultured in ITS-BSA were sexed by PCR (Grisart et al. 1995 Theriogenology 43, 1097) and a higher proportion of male embryos was found (62.7%; n = 51). In conclusion, a complete serum-free system was set up from oocyte maturation to embryo cryopreservation that gave high quality embryos resistant to cryo-preservation. Embryos produced in ITS-BSA presented a lower lipid content, but a shift of the expanded blastocyst sex ratio toward males was observed. Table 1. Hatching rates post-thawing as a function of the blastocyst size and the culture medium

scholarly journals Genetic influence on the reduction in bovine embryo lipid content by L-carnitine

2016 ◽  
Vol 28 (8) ◽  
pp. 1172 ◽  
Author(s):  
Luis Baldoceda ◽  
Dominic Gagné ◽  
Christina Ramires Ferreira ◽  
Claude Robert
Keyword(s):  
Lipid Content ◽  
Culture Medium ◽  
Cell Damage ◽  
Mitochondrial Activity ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Intracellular Lipid ◽  
Embryo Culture Medium

The decreased rate of pregnancy obtained in cattle using frozen in vitro embryos compared with in vivo embryos has been associated with over-accumulation of intracellular lipid, which causes cell damage during cryopreservation. It is believed that the higher lipid content of blastomeres of bovine embryos produced in vitro results in darker-coloured cytoplasm, which could be a consequence of impaired mitochondrial function. In this study, l-carnitine was used as a treatment to reduce embryonic lipid content by increasing metabolism in cultured bovine embryos. We have observed previously that in vivo embryos of different dairy breeds collected from cows housed and fed under the same conditions differed in lipid content and metabolism. As such, breed effects between Holstein and Jersey were also examined in terms of general appearance, lipid composition, mitochondrial activity and gene expression. Adding l-carnitine to the embryo culture medium reduced the lipid content in both breeds due to increased mitochondrial activity. The response to l-carnitine was weaker in Jersey than in Holstein embryos. Our results thus show that genetics influence the response of bovine embryos to stimulation of mitochondrial metabolism.

73 Cytokine addition does not increase developmental competence of invitro-produced bovine embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 162
Author(s):  
C. M. Helland ◽  
M. Barcelo-Fimbres ◽  
L. F. Campos-Chillon
Keyword(s):  
Lipid Content ◽  
Culture Media ◽  
Nile Red ◽  
Cell Number ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Lipid Levels ◽  
Fluorescent Intensity ◽  
Mitotracker Red ◽  
Main Effects

Recently in porcine, the addition of 3 cytokines (FGF2, LIF, and IGF1) improved oocyte maturation, quadrupling the number of piglets born per oocyte collected (Yuan et al. 2017 Proc. Natl. Acad. Sci. USA 114, E5796-E5804). We hypothesised that in the bovine, addition of these cytokines to maturation (MCyt) and culture media (CCyt) would lower lipid content and increase mitochondrial activity, representing an improved developmental competence when compared with standard maturation (MCon) and culture (CCon) conditions. The experimental design was a 2 (MCon and MCyt)×2 (CCon and CCyt) factorial in 8 replicates, testing the interactions of each maturation medium with each culture medium. Invitro-produced embryos produced aspirating cumulus-oocyte complexes (COCs) from 2 to 8mm follicles of abattoir ovaries. The COCs (n=2156) were matured for 23h in MCon or MCyt media at 6% CO2 in air, fertilised with semen from one of two bulls, and cultured in CCon or CCyt media at 38.5°C, 6% CO2, 5% O2, and 89% N2. On Day 7.5 post-fertilisation, blastocyst rates were evaluated and embryos (n=4/replicate/group) were stained with 1µgmL−1 Nile Red for lipid quantification or 300 nM MitoTracker Red CMX-Rosamine for mitochondrial polarity. Images were obtained with confocal microscopy and fluorescent intensity (AFU) was measured by Image J software (National Institutes for Health) adjusted per cell number. Data were analysed by GLM using ANOVA and l.s.d. with SAS (SAS Institute Inc.). Results (Table 1) indicated similar cleavage and blastocyst rates between all groups (P&gt;0.05). The combination of MCon×CCon resulted in higher mitochondrial activity than any other combination (P&lt;0.05). The MCon×CCyt showed the highest lipid levels, whereas MCyt×CCyt showed the lowest lipid levels (P&lt;0.05). Results suggest that the combination of MCyt×CCyt media produces the lowest lipid levels, whereas the MCon×CCon media lead to the highest mitochondrial activity. The addition of cytokines to both maturation and culture media maintains competence and lowers lipid content; however, it also seems to lower mitochondrial activity. Cryopreservation studies and evaluation of pregnancy rates are ongoing. Table 1.Oocyte and embryo developmental competence matured and cultured in control and cytokine-added media Treatment Oocytes (n) Cleavage (%) Blastocysts per oocyte (%) Nile Red per cell (AFU) MitoTracker per cell (AFU) Maturation main effects MCon 1000 96.8±0.4 29.9±2.7 36.1±2.1a 385.1±65.8a MCyt 1156 96.0±0.4 26.2±2.7 30.0±2.1b 209.1±65.8b Culture main effects CCon 1036 96.2±0.4 28.0±2.7 33.1±2.1 392.0±65.8a CCyt 1120 97.7±0.4 28.1±2.7 33.0±2.1 202.2±65.8b Interactions MCon×CCon 461 96.6±0.8 27.1±3.7 33.6±3.0ab 559.0±91.1a MCon×CCyt 539 95.3±0.9 29.5±2.7 38.6±3.0a 211.2±91.1b MCyt×CCon 575 95.4±0.6 27.4±2.1 32.7±3.0bc 224.9±91.1b MCyt×CCyt 581 95.6±0.9 23.2±2.9 27.4±3.0c 193.1±91.1b a-cValues with different superscript in the same column differ (P&lt;0.05).

scholarly journals Serum free embryo culture medium improves in vitro survival of bovine blastocysts to vitrification

2008 ◽  
Vol 69 (8) ◽  
pp. 1013-1021 ◽  
Author(s):  
E. Gómez ◽  
A. Rodríguez ◽  
M. Muñoz ◽  
J.N. Caamaño ◽  
C.O. Hidalgo ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture Medium ◽  
Serum Free ◽  
Embryo Culture Medium ◽  
In Vitro Survival
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