82 IN VITRO SURVIVAL OF PORCINE BLASTOCYSTS VITRIFIED USING THE CRYOLOGIC VITRIFICATION METHOD

2006 ◽  
Vol 18 (2) ◽  
pp. 149 ◽  
Author(s):  
L. Beebe ◽  
S. McIlfatrick ◽  
R. Ashman ◽  
M. Nottle
Keyword(s):  
Ethylene Glycol ◽  
Liquid Nitrogen ◽  
Direct Contact ◽  
Survival Rates ◽  
Genetic Material ◽  
Basic Medium ◽  
Embryo Cryopreservation ◽  
Fetal Bovine ◽  
And Storage

Porcine embryo cryopreservation is an important technology for the storage and transport of valuable genetic material. With many of the current vitrification and storage systems, such as the open pulled straws and microdrops, there is direct contact between the medium containing the embryos and the liquid nitrogen. This represents a possible contamination risk. One system with which there is no direct contact between the embryos and liquid nitrogen during the vitrification process is the Cryologic Vitrification System (CVM; Lindemans et al. Reprod. Fertil. Dev. 16, 174) which uses solid surface vitrification. Microdrops of vitrification medium containing the embryos are placed in contact with a metal block that has been precooled by partial submersion in liquid nitrogen, resulting in very rapid cooling rates. Blastocysts were collected surgically on day 5 of pregnancy from mature sows, and the embryos were randomly divided into two groups; each group was then vitrified and warmed with either of two previously published protocols except that the CVM replaced the open pulled straws plunged into liquid nitrogen in both protocols. The first method (OPS/CVM) was based on the open pulled straw method (Cuello et al. Theriogenology 61, 843-850), and used DMSO and ethylene glycol as cryoprotectants and TCM-199 as the basic medium. The second method (EG/CVM) used HEPES-buffered NCSU23 as the basic medium; the blastocysts were centrifuged prior to vitrification in ethylene glycol and polyvinylpyrrolidone (PVP) and the zona pellucida was removed immediately after warming (Cameron et al. Theriogenology 61, 1533-1543). Embryos were then cultured in NCSU23 +10% fetal bovine serum for 48 h at 38.5�C in an humidified atmosphere of 5% CO2, 5% O2, and 90% N2. Embryos that had reformed the blastocoel and continued to expand were considered to have survived. These were stained with Hoechst 33342 and the nuclei counted using fluorescence microscopy. There was no difference between the OPS/CVM or EG/CVM methods in either the survival rates (27/29; 93%, and 24/27; 89%, respectively) or the number of cells (mean � SEM; 109 � 6 and 112 � 6, respectively). The survival rates are comparable to previously published rates using these two methods and open pulled straws. These data suggest that the CVM can successfully replace the open pulled straws in these two protocols. However, transfer of vitrified and warmed embryos into recipients would be needed to confirm the viability of the surviving 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.

scholarly journals 123VITRIFICATION OF IN VITRO-PRODUCED BOVINE BLASTOCYSTS BY ADDITION OF CRYOPROTECTANT IN ONE STEP

2004 ◽  
Vol 16 (2) ◽  
pp. 184
Author(s):  
D.J. Walker ◽  
L.F. Campos-Chillon ◽  
G.E. Seidel
Keyword(s):  
Ethylene Glycol ◽  
Embryo Quality ◽  
Survival Rates ◽  
Defined Medium ◽  
Embryo Cryopreservation ◽  
Equilibration Time ◽  
One Step ◽  
Hatching Rates

Vitrification combined with in-straw dilution may replace conventional cryopreservation of bovine embryos, but this requires further study for practicality. Our objectives were to compare three ethylene glycol concentrations (6, 7, and 8M) and two equilibration times (2.5 and 3.5min) for one-step addition of cryoprotectant. In vitro-matured oocytes from slaughterhouse ovaries, fertilized using sperm of 3 bulls, were cultured in chemically defined medium (CDM-1/CDM-2) plus FAF-BSA to produce 420 blastocysts. Day 7.5 embryos were placed into HCDM-2 (HEPES-buffered medium) and then transferred to a 6μL drop of vitrification solution (V) (6, 7, or 8M ethylene glycol, 0.5M galactose, and 18% w/v Ficoll 70 in HCDM-2). Immediately thereafter, 1cm column of DHCDM (0.5M galactose in HCDM-2) was drawn into a 0.25mL straw, followed by a 0.5cm column of air and another 7cm of DHCDM. Another 0.5cm column of air was aspirated before the 6μL of V (0.5cm) containing the embryos were aspirated; then 0.5cm of air followed. Finally, DHCDM was drawn until the first column came into contact with the cotton plug. Straws were then heat-sealed and plunged into liquid nitrogen slightly above the embryos after 2.5 or 3.5min equilibration. The rest of the straw was then submerged slowly. Straws were thawed in air for 10s and then in 37°C water for 20s. Straws were held at room temperature (24°C) for 4min before being expelled into HCDM-2. They were then placed into CDM-2+5% FCS for culture. Quality score (1=excellent, 2=fair, 3=poor), survival (S) as determined by expansion of blastocysts, and hatching (H) were assessed at 24 and 48h post-thaw. Data from 6 replicates (2/bull) were analyzed by ANOVA after arc sin transformation of percentage data. S and H responses were calculated as a percentage of non-frozen controls in the same replicate. Control survival and hatching rates were: 24S: 90%, 24H: 50%, 48S: 90%, 48H: 72%. Quality scores at both 24 and 48h were higher (P&lt;0.05) for 8M than 6M ethylene glycol (2.68 and 3.24 for 24h; 2.55 and 3.17 for 48h); values for 7M ethylene glycol were intermediate. Equilibration time had no effect on embryo quality (P&gt;0.1). Neither ethylene glycol concentration nor exposure time affected survival or hatching at 24 or 48h (P&gt;0.1). Survival rates (as a % of control) at 48h were: 8M: 57%, 7M: 55%, 6M: 36% and hatching: 8M: 39%, 7M: 30%, and 6M: 21%; 2.5min tended to be better than 3.5min for survival at 24h, hatching at 24h, survival at 48h, but not hatching at 48h (56% and 43%, 30% and 26%, 55% and 44%, 28% and 32% respectively). Higher concentrations of ethylene glycol proved beneficial in terms of embryo quality, with the same trend for survival and hatching rates. One-step addition of cryoprotectant for vitrification shows potential for simplifying embryo cryopreservation. However, further research is needed to produce more acceptable survival rates and to study vitrification of in vivo-produced embryos.

106 PREGNANCY RATE AFTER EMBRYO TRANSFER OF IN VIVO-PRODUCED OVINE EMBRYOS CRYOPRESERVED BY SLOW FREEZING OR VITRIFICATION

2010 ◽  
Vol 22 (1) ◽  
pp. 212
Author(s):  
N. Mucci ◽  
F. Hozbor ◽  
G. G. Kaiser ◽  
E. Sanchez ◽  
R. H. Alberio
Keyword(s):  
Ethylene Glycol ◽  
Pregnancy Rate ◽  
Liquid Nitrogen ◽  
Embryo Transfer ◽  
Slow Freezing ◽  
Embryo Cryopreservation ◽  
Chi Square ◽  
Chi Square Test

Although slow freezing is the method of choice to cryopreserve in vivo-produced ovine embryos, vitrification has became an alternative procedure mostly developed for in vitro-produced bovine embryos. The aim of this work was to compare pregnancy rates after cryopreservation of in vivo-produced ovine embryos with slow freezing or open pulled straw (OPS) vitrification method. Ewes were synchronized using intravaginal sponges containing 60 mg of medroxyprogesterone acetate for 14 d. Superovulation was performed using a total dose of 176 IU of ovine FSH (Ovagen), in 6 decreasing doses (i.m.) from Day 12 to 14 of treatment (Day 0 = sponge placing). Ewes were hand mated with 2 rams of proven fertility. Embryos were recovered 6 days after estrous detection by surgical procedure, evaluated under stereomicroscope, and randomly assigned to the cryopreservation treatments. Slow freezing was performed in D-PBS supplemented with 1.78 M ethylene glycol, 0.1 M sucrose, 4 mg mL-1 of BSA, and 20% serum. Embryos were loaded into 0.25-mL plastic straws and placed into a -7°C methanol bath chamber. After seeding embryos were cooled to -35°C at a rate of 0.5°C/min and then stored in liquid nitrogen. Thawing was performed by placing the straws in a 30°C water bath for 30 sec. Vitrification was performed by using the OPS method (Vajta et al. 1998) with minor modifications. Embryos were incubated in D-PBS supplemented with 1.78 M ethylene glycol, 1.3 M DMSO for 3 min and then transferred for 25 s in vitrification solution of D-PBS with 3.56 M ethylene glycol, 2.6 M DMSO, and 0.5 M sucrose, loaded in a 1 mL drop in the OPS, and immediately submerged into and stored in liquid nitrogen. Warming was performed in D-PBS plus 0.25 M sucrose for 5 min and then into D-PBS plus 0.15 M sucrose for another 5 min. Before embryo transfer, the presence of corpus luteum (CL) was detected by laparoscopic examination. One embryo per recipient was surgically transferred in the apical extreme of the uterine horn ipsilateral to the CL. Pregnancies were determined by ultrasonography 41 days after embryo transfer. Data were analyzed using the chi-square test. We found 47.8% pregnancy rate using slow freezing (11/23) and 43.5% pregnancy rate using OPS vitrification (10/23). Statistical differences were not detected (P = 0.09). We conclude that vitrification by OPS system, with minor modifications, is a suitable procedure for in vivo-produced ovine embryo cryopreservation.

83 VITRIFICATION OF IMMATURE AND IN VITRO-MATURED HORSE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 149 ◽  
Author(s):  
L. Bogliolo ◽  
F. Ariu ◽  
I. Rosati ◽  
M. T. Zedda ◽  
S. Pau ◽  
...  
Keyword(s):  
Survival Rate ◽  
Ethylene Glycol ◽  
Survival Rates ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Hoechst 33342 ◽  
Nuclear Maturation ◽  
And Control ◽  
Cryopreservation Protocol

Few attempts have been carried out to cryopreserve equine oocytes, and an effective cryopreservation protocol is not defined yet. Studies were conducted to compare the viability of immature and in vitro-matured horse oocytes vitrified by the minimal volume cooling (MVC) cryotop vitrification method (Kuwayama et al. 2005 Reprod. BioMed. Online 11, 300–308). Oocytes were recovered from slaughterhouse ovaries and divided, on the basis of the morphology of cumulus cells, into cumulus-expanded (CE) and cumulus-compacted (CC) oocytes. Groups of CC and CE oocytes were vitrified immediately after recovery [germinal vesicle (GV) stage] or matured in vitro (IVM) and cryopreserved at the MII stage as follows: oocytes were incubated 30 min in TCM-199 + 20% FCS + 10% ethylene glycol (EG) + 10% DMSO, followed by 20 min in TCM-199 + 20% FCS + 20% EG + 20% DMSO + 0.25 M sucrose, loaded in cryotops (2 µL), and plunged into liquid nitrogen. Warming was performed at 38.5°C by washing the oocytes in TCM-199 + 20% FCS with decreasing sucrose concentrations (1.25 M, 0.62 M, 0.31 M). After warming oocytes cryopreserved at the GV stage were matured in vitro for 24 h (CE) or 36 h (CC) in TCM-199 + 10% FCS + FSH, LH each at (0.1 UI/mL) + cysteamine, fixed, and stained with glycerol-Hoechst 33342 to assess nuclear maturation. Oocytes vitrified at the MII stage were in vitro cultured for 2 h to evaluate their morphological survival on the basis of the presence of an intact zona pellucida and membrane. Nonvitrified oocytes undergoing the same maturation protocol were used as controls. Results (Table 1) indicated that the survival rate of oocytes vitrified at the GV stage, after IVM, was similar between CE and CC oocytes (43.6% vs 42.6%). Significantly (P < 0.01) higher numbers of vitrified CE MII oocytes (52.9%) survived, compared to CC (34.8%), after 2-h culture. The percentages of viable MII oocytes from CE and CC GV vitrified oocytes were 43.6% and 40.9% respectively and were comparable to those from vitrified MII oocytes (CE, 52.9%; CC, 34.8%) and control oocytes (CE, 56.4%; CC, 53.3%). In conclusion, the results of this study showed that vitrification by the MCV Cryotop method of horse oocytes at either the GV or the MII stage allows a similar number of viable mature oocytes to be recovered. Table 1. Maturation and survival rates of immature and mature equine oocytes vitrified by the MCV Cryotop method

44 IN VITRO AND IN VIVO SURVIVABILITY ON VITRIFIED EMBRYOS OBTAINED BY BOVINE SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 131
Author(s):  
K. Kaneyama ◽  
S. Kobayashi ◽  
S. Matoba ◽  
Y. Hashiyada ◽  
K. Imai ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Survival Rates ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Nuclear Transplantation ◽  
Embryo Cryopreservation

Although many studies have been conducted on somatic cell nuclear transfer, there are only a few reports on cryopreservation of reconstructed embryos after nuclear transplantation. The objective of this study was to examine in vitro or in vivo development of vitrified blastocysts obtained by nuclear transfer. Nuclear transfer was carried out according to the procedure of Goto et al. (1999 Anim. Sci. J. 70, 243–245), and conducted using abattoir-derived oocytes and cumulus cells derived by ovum pickup from Holstein and Japanese Black cows. Embryos were vitrified as described by Saito et al. (1998 Cryobiol. Cryotech. 43, 34–39). The vitrification solution (GESX solution) was based on Dulbecco's PBS containing 20% glycerol (GL), 20% ethylene glycol (EG), 0.3 M sucrose (Suc), 0.3 M xylose (Xyl), and 3% polyethylene glycol (PEG). The blastocysts were equilibrated in three steps, with 10% GL, 0.1 M Suc, 0.1 M Xyl, and 1% PEG for 5 min (1); with 10% GL, 10% EG, 0.2 M Suc, 0.2 M Xyl, and 2% PEG for 5 min (2) and GESX solution (3). After transfer to GESX, equilibrated embryos were loaded to 0.25-mL straws and plunged into liquid nitrogen for 1 min. The vitrified blastocysts were warmed in water (20°C) and diluted in 0.5 M and 0.25 M sucrose for 5 min each. Equilibration and dilution procedures were conducted at room temperature (25–26°C). After dilution, the vitrified blastocysts were cultured in TCM-199 supplemented with 20% fetal calf serum and 0.1 mM β-mercaptoethanol at 38.5°C under gas phase of 5% CO2 in air. In Experiment 1, survival rates after vitrification were compared between the nuclear transfer and the IVF blastocysts. Survival rates of vitrified nuclear transfer blastocysts (n = 60, Day 8) at 24 and 48 h were 70.0% and 56.7%, respectively, and those of vitrified IVF blastocysts (n = 41) were 82.9% and 82.9%, respectively. There were no significant differences in survival rates at 24 and 48 h between the two groups. In Experiment 2, one (VIT-single) or two (VIT-double) vitrified and one (nonVIT-single) or two (nonVIT-double) nonvitrified reconstructed blastocysts per animal were transferred into Holstein dry cows. The result of Experiment 2 is shown in Table 1. This experiment demonstrated that the vitrification method in this study can be used for cloned embryo cryopreservation but the production rate should be improved. Table 1. Comparison of survival rates of vitrified or nonvitrified cloned embryos after transfer

71 VITRIFICATION OF BOVINE OOCYTES IN MEDIA WITH GLYCEROL + ETHYLENE GLYCOL BEFORE AND AFTER IN VITRO MATURATION

2008 ◽  
Vol 20 (1) ◽  
pp. 116
Author(s):  
L. G. Devito ◽  
C. B. Fernandes ◽  
H. N. Ferreira ◽  
F. C. Landim-Alvarenga
Keyword(s):  
Ethylene Glycol ◽  
Liquid Nitrogen ◽  
In Vitro Maturation ◽  
Calf Serum ◽  
Quiescent State ◽  
Developmental Potential ◽  
Immature Oocytes ◽  
Nitrogen Vapor ◽  
Bovine Oocytes

The cryopreservation process aims to keep the cellular metabolism in a quiescent state for an indeterminate length of time. In mammals, oocyte cryopreservation success is important for the establishment of genetic banks. The objective of the present experiment was to evaluate the effect of vitrification on oocyte meiotic ability and the integrity of the metaphase plate in immature and in vitro-matured bovine oocytes. Bovine cumulus–oocytes complexes (COCs) were harvested from slaughterhouse ovaries and randomly divided into 3 groups: (G1) non-vitrified oocytes subjected to in vitro maturation, (G2) immature oocytes vitrified and then subjected to in vitro maturation after warming, and (G3) in vitro-matured oocytes subjected to vitrification. For in vitro maturation, oocytes were incubated for 22 h in 5% CO2 in air in TCM-199 with fetal calf serum, estradiol, LH, FSH, pyruvate, and gentamicin. For vitrification, the oocytes were exposed to the cryoprotectors in three steps: solution 1 containing 1.4 m glycerol in PBS for five min, and then solution 2 containing 1.4 m glycerol and 3.6 m ethylene glycol in PBS for another five min. After exposure to the second solution, the oocytes were transferred to 30-µL drops of solution 3 containing 3.4 m glycerol and 4.6 m ethylene glycol, loaded (5 oocytes per straw) in less than 1 min into 0.25-mL straws between two columns of 0.5 m galactose in PBS separated by two air bubbles, and immediately set in liquid nitrogen vapor. After 1 min of equilibration in liquid nitrogen vapor, the straws were immersed in liquid nitrogen. Warming was performed by holding the straws for 10 s in air, followed by 10 more s in a water bath at 20–22�C. The straws were then shaken 5 to 8 times to mix the bubbles (movement similar to that for a thermometer) and left horizontally for 6 to 8 min at room temperature. The rates of metaphase II and degeneration were analyzed by ANOVA followed by the Student t-test. The oocytes were stained with 100 µg mL–1 Hoechst 33342 and examined in an inverted microscope equipped with fluorescent light (UV filters 535 and 617 mm). Three different routines were realized with a total of 90 oocytes per group. The metaphase II rates in G1 (48/90, 53.3%) and G3 (42/90, 46.6%) were statistically the same (P e 0.05), but were higher (P d 0.05) than in G2 (0/90, 0%). The degeneration rates were: G1 (18/90, 20%), G2 (77/90, 85.6%), and G3 (7/90, 7.8%). The vitrification procedure damaged mainly the immature oocytes, since in the G2 the degeneration rate was higher and the oocytes were not able to resume meiosis. Meanwhile, when oocytes were vitrified after in vitro maturation, the metaphase II rate was similar to the one observed in IVM oocytes not subjected to vitrification. This indicates that the vitrification procedure performed in this experiment did not damage the structure of the metaphase II plate. However, more studies are necessary to predict the developmental potential of these in vitro-matured oocytes.

51 Effect of Cryoprotectant Exposure Time on Development of Vitrified-Warmed Immature Equine Oocytes

2018 ◽  
Vol 30 (1) ◽  
pp. 164
Author(s):  
H. S. Canesin ◽  
J. G. Brom-de-Luna ◽  
Y.-H. Choi ◽  
A. M. Pereira ◽  
G. G. Macedo ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Propylene Glycol ◽  
Exposure Time ◽  
In Vitro Maturation ◽  
Initial Exposure ◽  
Exact Test ◽  
Fetal Bovine ◽  
Follicle Aspiration ◽  
Meiotic Competence

Effective methods for cryopreservation of equine oocytes have not yet been established. Vitrification involves use of high cryoprotectant (CPA) concentrations, which can be cytotoxic. Thus, it is critical to determine a CPA concentration and exposure time able to protect the cell during cooling but with a minimal toxicity. Using a rapid non-equilibrating system, we fixed the time in the first, lower CPA concentration solution (V1) at 40 s, based on the time to maximal shrinkage. We then evaluated different exposure times in the final vitrification solution (V2). Cumulus-oocyte complexes (COC) were collected from slaughterhouse-derived ovaries and held overnight in commercial embryo holding medium. Fetal bovine serum was used as the base medium (BM). In experiment 1, COC were held in BM, incubated in V1 (2% propylene glycol + 2% ethylene glycol) for 40 s followed by incubation in V2 (17.5% propylene glycol + 17.5% ethylene glycol + 0.3 M trehalose) for 0, 45, 75, or 110 s, and then loaded in groups of 6 to 10 oocytes on a 75-µm steel mesh and plunged into liquid nitrogen. Warming was performed in decreasing trehalose concentrations in BM: 0.4 M (60-70 s), 0.2 M (5 min), 0.1 M (5 min), 0.05 M (5 min), and 0 M. After warming, oocytes were cultured for in vitro maturation (IVM) and evaluated after staining with Hoechst 33258. Differences between treatments were analysed by Fisher’s exact test. The maturation (metaphase II, MII) rate of the Control (non-vitrified oocytes; 38.8%, 31/80) was similar to that of the 75-s treatment (34.8%, 16/46; P = 0.71), and higher (P < 0.05) than those of the 0, 45, and 110 s treatments (0.0%, 0/10; 11.4%, 4/35; and 3.6%, 1/28; respectively). In experiment 2, timings in V2 focusing around 75 s were evaluated. The COC were collected and vitrified as for experiment 1, except that time in V2 was 50, 60, 70, 80, 90, or 100 s. The vitrified COC were then shipped to the intracytoplasmic sperm injection (ICSI) laboratory. After warming and IVM, oocytes were subjected to ICSI and embryo culture. Control oocytes were recovered by transvaginal follicle aspiration. The MII rate of the Control (60%, 33/55) was similar (P > 0.05) to that of the 60- and 70-s treatments (38.9%, 7/18, and 35.3%, 6/17, respectively), and higher (P < 0.05) than those of the 50-, 80-, 90-, and 100-s treatments (5.6 to 31.6%). The cleavage rates were 94% (31/33) for the Control and 71 to 100% for vitrified oocytes (P > 0.05). No blastocyst was produced from vitrified oocytes compared with 8/33 (24.2%) for Control. This work demonstrates that a rapid, non-equilibrating vitrification technique using a 40-s initial exposure and 70- to 80-s final exposure to CPA is associated with maintenance of meiotic competence of immature equine oocytes; however, further work is required to optimize embryonic development with this method. Research supported by the Clinical Equine ICSI Program and the Link Equine Research Fund, Texas A&M University.

scholarly journals Effect of Drying Process, Encapsulation, and Storage on the Survival Rates and Gastrointestinal Resistance of L. salivarius spp. salivarius Included into a Fruit Matrix

2020 ◽  
Vol 8 (5) ◽  
pp. 654
Author(s):  
Ester Betoret ◽  
Noelia Betoret ◽  
Laura Calabuig-Jiménez ◽  
Cristina Barrera ◽  
Marco Dalla Rosa
Keyword(s):  
Survival Rate ◽  
Physicochemical Properties ◽  
Freeze Drying ◽  
Molecular Mechanisms ◽  
Survival Rates ◽  
In Vitro Digestion ◽  
Hot Air ◽  
Freeze Dried ◽  
And Storage

In a new probiotic food, besides adequate physicochemical properties, it is necessary to ensure a minimum probiotic content after processing, storage, and throughout gastrointestinal (GI) digestion. The aim of this work was to study the effect of hot air drying/freeze drying processes, encapsulation, and storage on the probiotic survival and in vitro digestion resistance of Lactobacillus salivarius spp. salivarius included into an apple matrix. The physicochemical properties of the food products developed were also evaluated. Although freeze drying processing provided samples with better texture and color, the probiotic content and its resistance to gastrointestinal digestion and storage were higher in hot air dried samples. Non-encapsulated microorganisms in hot air dried apples showed a 79.7% of survival rate versus 40% of the other samples after 28 days of storage. The resistance of encapsulated microorganisms to in vitro digestion was significantly higher (p ≤ 0.05) in hot air dried samples, showing survival rates of 50–89% at the last stage of digestion depending on storage time. In freeze dried samples, encapsulated microorganisms showed a survival rate of 16–47% at the end of digestion. The different characteristics of the food matrix after both processes had a significant effect on the probiotic survival after the GI digestion. Documented physiological and molecular mechanisms involved in the stress response of probiotic cells would explain these results.

Comparison of two different methods for the vitrification of hatched blastocysts from the dromedary camel (Camelus dromedarius)

2005 ◽  
Vol 17 (5) ◽  
pp. 523 ◽  
Author(s):  
J. A. Skidmore ◽  
M. Billah ◽  
N. M. Loskutoff
Keyword(s):  
Polyethylene Glycol ◽  
Survival Rate ◽  
Ethylene Glycol ◽  
Liquid Nitrogen ◽  
Survival Rates ◽  
Camelus Dromedarius ◽  
Dromedary Camel ◽  
Vitrification Solution

The uteri of 32 donor camels were flushed non-surgically on Day 6, 7 or 8 after ovulation and a total of 184 embryos was recovered. Sixty Day 6 embryos and 61 Day 7 embryos were vitrified or frozen ultrarapidly using open pulled straws and a modified version of the Vajta protocol. These embryos were subjected to concentrations of either 10% and 20% or 20% and 40% ethanediol as the cryoprotectant before being loaded into open pulled straws (OPS) and plunged into liquid nitrogen. All embryos were subsequently thawed and rehydrated either directly into holding media or into holding media containing 0.2 m sucrose and were incubated for 5 or 10 min before being transferred to holding media before transfer to recipients. Although the survival rate of the embryos immediately after thawing was high (OPS 20%/40% ethanediol resulted in 97% and 100% survival for Day 6 and Day 7 embryos, respectively; OPS 10%/20% ethanediol resulted in 90% and 70% survival for Day 6 and Day 7 embryos, respectively), after 2 h in culture, survival rates had decreased to 46% and 53% for Day 6 and Day 7 embryos, respectively, using OPS 10%/20% and 53% and 63% for Day 6 and Day 7 embryos, respectively, using OPS 20%/40%; however, none of the embryos transferred resulted in a viable fetus. A further 63 embryos (Day 6: n = 31; Day 7: n = 16; Day 8: n = 16) were subsequently exposed to vitrification solution (20% glycerol + 20% ethylene glycol + 0.3 m sucrose + 0.375 m glucose + 3% polyethylene glycol) in three steps and after loading into 0.25 mL straws were plunged into liquid nitrogen. However, a much greater percentage of the Day 7 and Day 8 embryos (43.8% and 81.2% respectively) were fractured or torn after warming and none of the 12 intact embryos transferred resulted in a pregnancy. Better survival rates immediately after thawing and rehydration were obtained with the smaller Day 6 embryos (94%), which resulted in a total of eight fetuses from the 21 embryos transferred.

scholarly journals Fertility Preservation in Female Cancer Patients

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Chung-Hoon Kim ◽  
Gyun-Ho Jeon
Keyword(s):  
Cancer Patients ◽  
Fertility Preservation ◽  
Survival Rates ◽  
Young Patients ◽  
Gynecologic Surgery ◽  
Embryo Cryopreservation ◽  
Future Fertility ◽  
Female Cancer Patients ◽  
Gestational Surrogacy

With improved survival rates among cancer patients, fertility preservation is now being recognized as an issue of great importance. There are currently several methods of fertility preservation available in female cancer patients and the options and techniques via assisted reproduction and cryopreservation are increasing, but some are still experimental and continues to be evaluated. The established means of preserving fertility include embryo cryopreservation, gonadal shielding during radiation therapy, ovarian transposition, conservative gynecologic surgery such as radical trachelectomy, donor embryos/oocytes, gestational surrogacy, and adoption. The experimental methods include oocyte cryopreservation, ovarian cryopreservation and transplantation, in vitro maturation, and ovarian suppression. With advances in methods for the preservation of fertility, providing information about risk of infertility and possible options of fertility preservation to all young patients with cancer, and discussing future fertility with them should be also considered as one of the important parts of consultation at the time of cancer diagnosis.

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