42 Vitrification of invitro-produced feline embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 146
Author(s):  
D. Fuller ◽  
J. Herrick ◽  
J. Graham ◽  
J. Barfield
Keyword(s):  
Dimethyl Sulfoxide ◽  
Liquid Nitrogen ◽  
Room Temperature ◽  
Culture Medium ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Epididymal Sperm ◽  
Quality Grade ◽  
Developmental Rates ◽  
Species Specific

Preservation of feline embryos is useful in propagating endangered species, preserving valuable genetics, and supporting biomedical research. Although a wide variety of cryoprotectants (CP) and protocols are successfully used for vitrification of invitro-produced (IVP) embryos, there are often species-specific differences in viability of embryos post-warming. The purpose of this study was to evaluate the viability of IVP feline embryos after vitrification using two common CPs, propanediol (PrOH) or ethylene glycol (EG). Embryos were produced with oocytes and frozen-thawed epididymal sperm collected from local spay-neuter clinics using a published IVP protocol developed for producing domestic feline embryos. Day 7 early blastocysts (stage 5), blastocysts (stage 6), and expanded blastocysts (stage 7) were evaluated for quality (grade 1 or 2) and randomly assigned to one of three treatments: vitrification with PrOH (n=32), vitrification with EG (n=31), or control (n=47), which was allowed to continue in culture until Day 8. The vitrification protocol was as follows. The base medium for all vitrification media was a HEPES-buffered feline optimized culture medium (FOCMH). Embryos were placed in 0.5mL of equilibration medium (7.5% dimethyl sulfoxide, 7.5% PrOH or EG, 0.5M sucrose, 10% Ficoll, and 20% fetal calf serum (FCS)) for 5min at room temperature. Individual embryos were then moved to 20-μL drops of vitrification medium (15% dimethyl sulfoxide, 15% PrOH or EG, 0.5M sucrose, 10% Ficoll, and 20% FCS) at room temperature for 30s before being loaded onto Cryolock devices and plunged into liquid nitrogen. Warming was done using a 3-step process for all vitrified embryos. First, embryos were moved from liquid nitrogen directly to 0.5mL of 1M sucrose, 10% Ficoll, and 20% FCS at 37°C for 1min. Next, embryos were moved to 0.5mL of 0.5M sucrose, 10% Ficoll, and 20% FCS at 20°C for 3min. Finally, embryos were transferred to 0.5mL of FOCMH for 5min at 37°C. All warmed embryos were cultured in medium, optimized for feline embryos, with 5% FCS and evaluated for re-expansion of the blastocoele and progression in development at 24 and 48h. Results are from five replicates. Embryos vitrified in EG exhibited higher percentages of viable embryos 24h after warming (84%) than embryos vitrified in PrOH (59%; P<0.05). The continued embryonic growth of viable embryos after culture for 48h showed equivalent developmental rates, at 87, 96, and 100% for control, EG-treated, and PrOH-treated embryos, respectively (P>0.05). Results indicate EG is a more successful CP treatment for vitrification of feline embryos when evaluating viability 24h post-warming. We report a higher viability of embryos post-thaw than previous studies using the same CPs (Pope et al. 2012 Reprod. Domest. Anim. 47, 125). This may be due to the shorter exposure time to the CPs we used during the vitrification process. We conclude that EG and PrOH are effective CPs for Day 7 feline IVP embryos using this protocol. Further research is needed to increase treatment numbers and evaluate pregnancy rates from embryos transferred post-warming.

98 EIGHT-CELL STAGE DELINEATES CRYOTOLERANCE OF VITRIFIED RABBIT EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 208
Author(s):  
T.-A. Lin ◽  
C.-H. Chen ◽  
L.-Y. Sung ◽  
J.-C. Ju ◽  
E. Chen ◽  
...  
Keyword(s):  
Culture Medium ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Developmental Competence ◽  
Cell Stage ◽  
General Linear Model Analysis ◽  
Developmental Rates ◽  
Humidified Air ◽  
Rabbit Embryos ◽  
Hatching Rates

A number of groups have successfully vitrified rabbit embryos at morula or blastocyst (BL) stage. For earlier stages (1- to 8-celled), however, there are very limited studies and the results are generally unsatisfactory. In this study, we examined the survival and developmental competence of rabbit embryos vitrified at different preimplantational stages. Sexually matured female New Zealand White (NZW) rabbits were superovulated with our standard protocols, followed by mating with NZW males. At 18h post-hCG treatment, viable fertilized embryos were collected and cultured in 2.5% FBS B2 medium (Laboratories CCD, Paris, France) in 5% CO2 with humidified air at 38.5°C. A total of 691 rabbit embryos at pronuclear, 2-celled, 4-celled, 8-celled, and morula/early blastocyst (BL; Day 3) stages were vitrified by the open pulled straw (OPS) method in HEPES buffered TCM-199 medium containing 20% fetal calf serum, ethylene glycol, and dimethyl sulfoxide. After stored in liquid nitrogen for at least 1 month, embryos were sequentially warmed, rehydrated, and washed before culture in B2 culture medium. Survival and developmental rates were analyzed by general linear model analysis (SPSS 11.0, SPSS Inc., Chicago, IL, USA). Embryos vitrified at 8-celled stage or beyond showed greater survival, and expanded BL and hatching rates than those at pronuclear, 2-celled and 4-celled embryos (Table 1). In particular, the expanded and hatched BL rates were significantly higher in the 8-celled group than those in 4-celled group, suggesting that the 8-celled is the threshold stage to tolerate the vitrification procedure in rabbit embryos. In addition, the rates of expanded and hatched blastocysts in morula/BL group were still significantly higher than those in the 8-celled group. Our results may provide the proper timing of cryopreserving fertilized, transgenic, and/or cloned rabbit embryos at early stages for biomedical research. Table 1.Survival and developmental competence of rabbit embryos vitrified at different stages after thawing This study was supported by NIH1R43 RR023774-01A1 and 5R44HL091605-03.

scholarly journals Stimulierung von Kulturen embryonaler Rattenzellen durch eine Proteinfraktion aus fötalem Kälberserum / Stimulation of Embryonic Rat Cells in Culture by a Protein Fraction Isolated from Fetal Calf Serum

1971 ◽  
Vol 26 (10) ◽  
pp. 1045-1048 ◽  
Author(s):  
Dieter F. Hülser ◽  
Werner Frank
Keyword(s):  
Cell Cycle ◽  
Culture Medium ◽  
Fetal Calf Serum ◽  
Surface Membrane ◽  
Calf Serum ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Membrane Potential Difference ◽  
Stimulation Of

Normal embryonic rat cells incubated in serum-free medium accumulate in G1-phase of the cell cycle. On addition of a growth-stimulating protein isolated from fetal calf serum they are triggered to proceed through the cycle, and they resume DNA-synthesis 15 to 20 hours later. In this paper it is demonstrated that the surface membrane potential difference (PD) decreases immediately after changing serum-free medium against culture medium containing either calf serum or the isolated serum protein; the original PD is restored 2 to 3 hours later. Serumprotein without growthstimulating activity does not affect the PD.A permanent rat cell line which grows independently of serum also has been tested. The PD of these cells is not significantly influenced by calf serum.

Forskolin effect on the cryosurvival of in vitro-produced bovine embryos in the presence or absence of fetal calf serum

Zygote ◽  
2012 ◽  
Vol 22 (2) ◽  
pp. 146-157 ◽  
Author(s):  
Daniela Martins Paschoal ◽  
Mateus José Sudano ◽  
Midyan Daroz Guastali ◽  
Rosiára Rosária Dias Maziero ◽  
Letícia Ferrari Crocomo ◽  
...  
Keyword(s):  
Culture Medium ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Similar Rate ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Vitrification Solution ◽  
Oviductal Fluid

SummaryThe objective of this study was to assess the viability and cryotolerance of zebu embryos produced in vitro with or without the addition of fetal calf serum (FCS) and forskolin (F). Embryos produced in vivo were used as a control. Presumptive zygotes were cultured in modified synthetic oviductal fluid supplemented with amino acids (SOFaa), bovine serum albumin (BSA) and with (2.5%) or without (0%) FCS. On day 6 of growth, the embryos from each group were divided into treatments with or without 10 μM F to induce embryonic lipolysis, comprising a total of four experimental groups: 2.5% FCS, 0% FCS, 2.5% + F and 0% + F. For vitrification, embryos were exposed to vitrification solution 1 (5 M EG (ethylene glycol)) for 3 min and then transferred to vitrification solution 2 (7 M EG, 0.5 M galactose solution and 18% (w/v) Ficoll 70) before being introduced to liquid nitrogen. The presence of FCS in the culture medium resulted in the production of embryos with a similar rate of damaged cells compared with in vivo-produced embryos. After vitrification, the 2.5% FCS group had a significantly higher rate of damaged cells when compared with the other groups (P < 0.05). The results of this experiment indicated that the omission of FCS and the addition of forskolin do not have deleterious effect on embryo production rates. In addition, embryos produced in the presence of FCS had greater sensitivity to cryopreservation, but this effect was reversed when forskolin was added to the medium, which improved embryo survival without affecting embryo development and quality after vitrification.

scholarly journals Assessment of LDL-Receptor Function in Human Lymphocytes with Lipoprotein Depleted Fetal Calf Serum and Pravastatin in the Culture Medium

1992 ◽  
Vol 20 (1) ◽  
pp. 53-56
Author(s):  
Seiji IWATA ◽  
Nagahiko SAKUMA ◽  
Hiroyuki HIRATA ◽  
Takayoshi ICHIKAWA ◽  
Yoshinori NOGUCHI ◽  
...  
Keyword(s):  
Culture Medium ◽  
Fetal Calf Serum ◽  
Human Lymphocytes ◽  
Calf Serum ◽  
Ldl Receptor ◽  
Receptor Function

44 THE EFFECT OF SUCROSE CONCENTRATION FOR SINGLE-STEP DILUTION ON THE VIABILITY OF CRYOTOP-VITRIFIED IN VITRO-PRODUCED BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 115
Author(s):  
S. Kondo ◽  
K. Imai ◽  
O. Dochi
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Liquid Nitrogen ◽  
Sucrose Concentration ◽  
Calf Serum ◽  
Single Step ◽  
Bovine Embryos ◽  
Vitrification Solution ◽  
Phosphate Buffered Saline

The aim of this study was to test sucrose concentrations for single-step dilution on the viability of vitrified in vitro-produced bovine embryos. Blastocysts (n = 173, 7 to 8 days after fertilization) were vitrified using the Cryotop (Kitazato, Tokyo, Japan) method placement by incubating the blastocysts in Dulbecco's phosphate buffered saline supplemented with 20% calf serum, 7.5% ethylene glycol, and 7.5% dimethyl sulfoxide for 3 min and then transferring into vitrification solution (Dulbecco's phosphate buffered saline supplemented with 20% calf serum, 16.5% ethylene glycol, 16.5% dimethyl sulfoxide, and 0.5 M sucrose). Each embryo was placed on a Cryotop with minimum volume of vitrification solution, and then the Cryotop was plunged into liquid nitrogen. Total time from placement in vitrification solution to plunging into liquid nitrogen was 1 min. The blastocysts were warmed by incubation in the single-step dilution medium for 5 min [0 M sucrose (n = 42), 0.25 M sucrose (n = 44), 0.5 M sucrose (n = 43), and 1.0 M sucrose (n = 44)] at 38.0°C. After dilution, the embryos were washed in TCM-199 supplemented with 20% calf serum and 0.1 mM β-mercaptoethanol and were cultured for 72 h in the same medium at 38.5°C in an atmosphere of 5% CO2. The rates of re-expanded blastocysts and hatched blastocysts were determined at 24 and 72 h after warming, respectively. Data were analysed using the chi-squared test. The percent of re-expanded blastocysts at 24 h after warming in dilution medium supplemented with any level of sucrose was significantly higher (P < 0.05) than in blastocysts warmed without sucrose (Table 1). The hatched blastocyst rate of embryos at 72 h after warming in dilution medium with 0.5 M sucrose was significant higher than that with no sucrose. There were no differences in hatched blastocyst rates between the sucrose concentrations supplemented to the dilution medium. These results suggest that embryos vitrified by the Cryotop method can be diluted in single-step dilution using 0.25, 0.5, or 1.0 M sucrose supplemented to the medium. Table 1.The effect of sucrose concentration for single-step dilution on the viability of Cryotop vitrified in vitro-produced bovine embryos

113 EVALUATION OF DIFFERENT CRYOPROTECTANT AND FORSKOLIN IN THE CULTURE MEDIUM FOR IMPROVING THE EFFICACY OF VITRIFICATION OF BOS INDICUS IN VITRO-DERIVED EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 215 ◽  
Author(s):  
B. V. Sanches ◽  
B. D. O. Filho ◽  
J. H. F. Pontes ◽  
A. C. Basso ◽  
M. L. G. Meirinhos ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Lipid Droplets ◽  
Culture Medium ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Calf Serum ◽  
Genetic Material ◽  
Embryo Cryopreservation ◽  
Hatching Rate

Embryo cryopreservation is an essential method for the biotechnology of reproduction. This is the safest option for interchange of genetic material for research and commercial purposes. For cattle, Brazil has become the leading country in the world for the number of in vitro-produced embryos, using mostly Bos indicus animals. However, considering the in vitro method of embryo production, field results have shown a lower resistance to cryopreservation for B. indicus when compared with Bos taurus embryos. A possible explanation for this is a great concentration of lipid droplets in the cytoplasm of cells fromB. indicus embryos. The objective of this study was to compare 2 cryoprotectants (Propanediol and DMSO) to vitrification and evaluate the effect of adding 10 μM forskolin to the SOF medium for embryo culture before cryopreservation. For all the experiments, ovaries from slaughtered Nelore Bos indicus donors were recovered and maintained at 30 to 35°C in NaCl solution until recovery of the COC. Embryos submmited to vitrification were expanded blastocysts at Day 7 of in vitro culture. In the first experiment embryos were first incubated in 10% ethylene glycol (EG) plus 10% DMSO dissolved in holding medium (TCM-HEPES with 20% calf serum) for 1 min and then transferred to droplet of 20% EG plus 20% DMSO in holding medium and 0.5 M sucrose for 20 s before immersing in liquid nitrogen (n = 107; group EG + DMSO). For the group EG + Propanediol (EG + PRO; n = 96), blastocysts were placed in 10% EG plus 10% PRO in holding medium for 1 min and then transferred to a droplet of 20% EG plus 20% PRO in holding medium and 0.5 M sucrose for 20 s before immersing in liquid nitrogen. Both treatments were performed using the Cryotop system. Results were compared with embryos (n = 118) not submitted to cryopreservation. The evaluation was done by the hatching rate of blastocysts at Day 9, being higher (86.4%) for embryos not cryopreserved, when comparing with 77.1% for group EG + PRO and 72.9% for group EG + DMSO (P < 0.05). In the second experiment, Day 5 embryos obtained in vitro from Nelore donors were cultured using SOF medium with 10 μM forskolin (n = 112) or not (control; n = 101), being all submitted to cryopreservation using Cryotop and the same vitrification method for group EG + DMSO. Results were compared with embryos cultured with SOF medium and not submitted to cryopreservation (n = 96). The evaluation was performed by considering hatching rate at Day 9, being higher (85.4%) for not cryopreserved, when compared with 63.3% for control and 70.5% for forskolin group (P < 0.05). Considering embryos submitted to cryopreservation, the hatching rate was higher (P < 0.05) for the forskolin group.

54 Effect of Different Cryopreservation Protocols for Sheep Embryonic Stem Cells

2018 ◽  
Vol 30 (1) ◽  
pp. 166
Author(s):  
N. Ibraimova ◽  
A. Seisenbayeva ◽  
Y. Toishibekov
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Fetal Calf Serum ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Embryonic Stem ◽  
Slow Freezing

Particular attention is required to improve cryopreservation of embryonic stem cells (ESC) and study their characteristics. Stem cells were obtained from the inner cell mass of Day 5-6 blastocysts. The ESC were then cultured on mTeSR™1 medium (Stemcell Technologies, Cambridge, MA, USA). We studied the survival of ESC after slow freezing and vitrification. Slow freezing was carried out using a Planer Kryo 360-3.3 freezer (Planer plc, Sunbury-on-Thames, United Kingdom), using various cryoprotectants: 1.5 M dimethyl sulfoxide (Me2SO), 1.5 M ethylene glycol (EG), or 1.5 M propylene glycol (PG). Six vitrification solutions (VS) were used to vitrify ESC: VS1 = 20% Me2SO + 20% EG + 0.5 M sucrose; VS2 = 20% Me2SO + 20% PROH + 0.5 M sucrose; VS3 = 20% EG + 20% PG + 0.5 M sucrose; VS4 = 20% Me2SO + 20% EG + 0.5 M sucrose + 10% FCS; VS5 = 20% Me2SO + 20% PROH + 0.5 M sucrose + 10% FCS; and VS6 = 20% EG + 20% PG + 0.5 M sucrose + 10% FCS. For the dehydration of cells and the addition of vitrification solutions, a 3-step equilibration was used. The proliferative properties of the cells were determined using an Apel PD-303S spectrophotometer (Apel Co. Ltd., Kawaguchi, Japan), using an MTT test (staining with methylthiazolyl-diphenyl tetrazolium). After slow freezing, the highest percentage of frozen–thawed cells proliferating was observed when using 1.5 M EG (P > 0.05). At the same time, the highest cell doubling after thawing was observed when using 1.5 M EG, and 1.5 M Me2SO. After vitrification, the highest percentage of proliferation was observed in the VS2 and VS4 groups (49.7 ± 3.2% and 53.2 ± 3.8%, respectively). It should be noted that the addition of fetal calf serum to the vitrification solution also increased the proliferation of ESC after vitrification and thawing.

137 PHENAZINE ETHOSULFATE AND FETAL CALF SERUM EFFECTS ON THE DEVELOPMENT AND APOPTOSIS OF IN VITRO PRODUCED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 173
Author(s):  
M. J. Sudano ◽  
D. M. Paschoal ◽  
T. S. Rascado ◽  
L. C. O. Magalhães ◽  
L. F. Crocomo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Fetal Calf Serum ◽  
Bos Indicus ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Blastocyst Quality ◽  
Phenazine Ethosulfate

Phenazine ethosulfate (PES) is a metabolic regulator that inhibits fatty acid synthesis and favours the pentose-phosphate pathway. Supplementation of fetal calf serum (FCS) during culture has been correlated with the reduction of quality of in vitro produced bovine embryos (IVPE). The aim of the present study was to evaluate embryo development and apoptosis in blastocysts after the supplementation of PES and FCS in culture medium of IVPE. Oocytes (N = 4320) were matured and fertilized in vitro (Day 0). The zygotes (Bos indicus) were cultured in SOFaa medium with 4 concentrations of FCS (0, 2.5, 5, and 10%) and with the use or not of 0.3 μM PES from Day 4 (after 96 h of embryo culture). Embryo development was evaluated after 7 days of culture. Apoptosis in blastocysts (N = 60–80) was accessed through TUNEL reaction. Embryos (Bos indicus) recovered from superstimulated cows were used as in vivo control (n = 15). Data were analysed by ANOVA followed by LSD using PROC GLIMMIX (SAS; SAS Institute Inc., Cary, NC, USA) means ± SEM. Increasing FCS concentration in the culture media did not change cleavage (86.7 ± 1.7, 82.3 ± 1.6, 86.3 ± 1.4, 87.0 ± 1.5, P > 0.05) and augmented blastocyst production (30.5 ± 2.5a, 41.8 ± 2.4b, 40.5 ± 2.6b, 47.2 ± 2.8b, P < 0.05), respectively, for 0, 2.5, 5, and 10%. Additionally, increasing FCS concentration increased apoptosis in blastocysts (13.8 ± 1.2b, 19.1 ± 1.8b, 20.7 ± 1.9bc, 28.4 ± 2.3c, P < 0.05, respectively, for 0, 2.5, 5, and 10%). The addition of PES from Day 4 in the culture medium did not affect (P > 0.05) cleavage (87.0 ± 1.3 and 84.4 ± 1.3), blastocyst production (42.0 ± 2.8 and 43.0 ± 2.0), and apoptosis in blastocysts (20.7 ± 2.0b and 18.9 ± 2.1b), respectively, for control and PES Day 4 groups. Independent of FCS withdrawal or PES addition to culture medium, the in vivo control group presented the lowest apoptosis rate (6.3 ± 1.1a). Therefore, increasing FCS concentration augmented embryo development and reduced blastocyst quality. However, the addition of 2.5% of FCS in the culture medium increased the embryo development without the reduction of blastocyst quality. Moreover, the PES supplementation from Day 4 did not affect embryo development and blastocyst quality. São Paulo Research Foundation – FAPESP.

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