scholarly journals 104EFFECT OF HATCHING STATUS ON VITRIFICATION OF CLONED BOVINE BLASTOCYSTS

2004 ◽  
Vol 16 (2) ◽  
pp. 174
Author(s):  
C. Laowtammathron ◽  
T. Terao ◽  
C. Lorthongpanich ◽  
S. Muenthaisong ◽  
T. Vetchayan ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Sucrose Solution ◽  
Survival Rates ◽  
Nuclear Transplantation ◽  
Fibroblast Cells ◽  
Anova Test ◽  
University Of Technology ◽  
Vitrification Solution ◽  
Cloned Bovine

Bovine blastocysts produced by nuclear transplantation have mechanical slits in their zonae pellucidae, and therefore initiate hatching earlier than the non-manipulated embryos. The present study was undertaken to examine whether the hatching stage of cloned blastocysts is among the factors influencing their survival after vitrification and warming. Cloned bovine blastocysts were produced by using adult ear fibroblast cells as reported previously (Parnpai et al., 2002, Theriogenology 57, 443), except that fused couplets were co-cultured with bovine oviductal epithelial cells in mSOFaa medium supplemented with 0.1% linoleic acid-albumin (LAA)+0.2% BSA (Hochi et al., 1999, Theriogenology 52, 497–504). Hatching blastocysts harvested on Day 7 were classified into one of three groups according to the ratio of extruding embryonic diameter from zona (D2) to embryonic diameter inside the zona (D1); category-A: D2/D1=0.01–0.70; category-B: D2/D1=0.71–1.00; category-C: D2/D1=1.01–1.70. The blastocysts were first exposed to 10% DMSO+10% ethylene glycol in TCM199+20% FCS for 2min, and then equilibrated in 20% DMSO+20% ethylene glycol+0.5M sucrose with or without 10% Ficoll in TCM199+20% FCS for 30s. One to three blastocysts were placed on a Cryotop sheet (Kitazato Supply Co., Tokyo, Japan) and vitrified in liquid nitrogen. The samples were warmed in 0.5M sucrose solution for 2min and transferred into TCM199+20% FCS in five steps (5min per step). The post-warm survival of the blastocysts was assessed by in vitro culture for 24h. When Ficoll-free vitrification solution was used, post-warm survival rate of the category-A blastocysts (77%, 23/30) was not significantly different (ANOVA test) from those of category-B and category-C blastocysts (74%, 20/27; and 80%, 24/30; respectively). Inclusion of 10% Ficoll in the vitrification solution did not improve (ANOVA test) the post-warm survival rates of cloned blastocysts (category-A: 65%, 22/34; category-B: 54%, 15/28; category-C: 59%, 19/32). Groups of fresh nonsurgical embryos, vitrified with or without Ficoll, yielded 66.7% (4/6), 66.7% (2/3) and 40.0% (2/5), respectively, of recipients pregnant at 48 days of gestation. In conclusion, cloned bovine blastocysts, regardless of their hatching stages, were relatively resistant to cryopreservation by vitrification. (Supported by Thailand Research Fund and R&D Fund of Suranaree University of Technology.)

75 ADDITION OF HYALURONAN TO A VITRIFICATION SOLUTION FOR IMMATURE BOVINE OOCYTES SELECTED BY BRILLIANT CRESYL BLUE

2013 ◽  
Vol 25 (1) ◽  
pp. 185
Author(s):  
P. Rodriguez Villamil ◽  
F. Ongaratto ◽  
M. Fernandez Taranco ◽  
G. A. Bó
Keyword(s):  
Ethylene Glycol ◽  
Solid Phase ◽  
Animal Health ◽  
Survival Rates ◽  
Control Group ◽  
Cresyl Blue ◽  
Development Rates ◽  
Vitrification Solution ◽  
Bovine Oocytes

An experiment was designed to evaluate the effect of brilliant cresyl blue (BCB) selection of immature oocytes and the addition of sodium hyaluronate (HA) to the vitrification solution on survival rates of bovine oocytes vitrified using solid-phase vitrification. Bovine cumulus–oocyte complexes (COC; n = 716) obtained from slaughterhouse ovaries were used in 6 replicates. Cumulus–oocyte complexes were washed in tissue culture medium 199 (TCM-199) and randomly allocated to 2 groups to be exposed to BCB stain (Sigma Chemical Company, St. Louis, MO, USA) for 90 min as described by Alm et al. (2005 Theriogenology 63, 2194–2205) or (control) maintained in Vigro holding medium (Bioniche Animal Health, Belleville, Canada) for 90 min (n = 220). Cumulus–oocyte complexes in the BCB group were selected based on their response to BCB as BCB+ (colored, n = 248) or BCB– (colorless, n = 248), whereas those in the control group were selected morphologically as described by Rodríguez-González et al. (2002 Theriogenology 57, 1397–1409). Oocytes from both BCB groups and 100 oocytes in the control group were vitrified by solid-phase vitrification as previously described by Rodriguez et al. (2012 Reprod. Fertil. Dev. 24, 132). The remaining 120 oocytes in the control group were not vitrified and were matured, fertilized, and cultured in vitro (in SOFaa in a controlled atmosphere) for 7 days. Vitrified oocytes were exposed to 10% ethylene glycol for 10 min, and 20% ethylene glycol + 0.2-M trehalose for 30 s, and then were subdivided to be exposed to 30% ethylene glycol + 0.5-M trehalose with or without 0.1 mg mL–1 HA (MAP 5, Bioniche Animal Health). Vitrified oocytes were stored in liquid nitrogen for at least one week and then placed directly into a 0.5-M sucrose solution (in TCM 199) at 37°C for 5 min, 0.25 M of sucrose for another 5 min, and finally TCM-199 and matured, fertilized, and cultured. Development rates (i.e. proportion of blastocysts) were examined on Day 7 after fertilization. Proportional data were first transformed by square root and then analyzed by ANOVA to detect the effect of replicate, type of oocyte (BCB+, BCB–, controls), and vitrified with or without HA or not vitrified as main effects, using the software Infostat (UNC, Argentina, 2010). There was a significant effect of oocyte type on blastocyst rate (P < 0.01) following vitrification (BCB+, 6.4 ± 0.4%. v. BCB–, 1.6 ± 0.6%). Control oocytes (not exposed to BCB) resulted in 3.0 ± 2.0% blastocysts following vitrification, which was lower to that obtained with the BCB+ oocytes. Vitrification also influenced development rates (3.0 ± 2.0 v. 32.0 ± 1.3%) for blastocysts produced from vitrified v. nonvitrified oocytes, respectively (P < 0.01). Furthermore, the use of HA in the vitrification solutions did not have a significant effect on development rates (4.7 ± 0.9 v. 3.3 ± 0.9%, for blastocysts obtained from vitrified oocytes with or without HA, respectively). In conclusion, the selection of oocytes by BCB increased the in vitro development rates of vitrified immature oocytes, whereas the use of HA in the vitrification solution did not improve the survival rates of vitrified oocytes.

scholarly journals 116DEVELOPMENT INTO BLASTOCYSTS OF SWAMP BUFFALO OOCYTES AFTER VITRIFICATION AND NUCLEAR TRANSFER

2004 ◽  
Vol 16 (2) ◽  
pp. 180 ◽  
Author(s):  
R. Parnpai ◽  
C. Laowtammathron ◽  
T. Terao ◽  
C. Lorthongpanich ◽  
S. Muenthaisong ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Somatic Cell ◽  
Sucrose Solution ◽  
Nuclear Transplantation ◽  
Developmental Potential ◽  
Domestic Species ◽  
Reconstructed Embryos ◽  
University Of Technology ◽  
Before And After ◽  
Swamp Buffalo

Oocyte cryopreservation in the domestic species is still at the experimental stage, but recent studies indicated that vitrification characterized by ultra-rapid cooling rate is promising for cryopreservation of bovine oocytes. In the present study, denuded buffalo oocytes were vitrified by minimum volume cooling procedure (Kuwayama and Kato, 2000, J Assist Reprod Genet 17, 477) after IVM or after IVM and enucleation, and developmental potential into blastocysts of the post-warm oocytes after somatic cell nuclear transplantation was examined. Cumulus-oocyte complexes were matured, denuded, and enucleated as described previously (Parnpai et al., 1999, Buffalo J 3, 371–384). The presumptive metaphase-II (M-II) oocytes before and after enucleation were first equilibrated in 7.5% DMSO+7.5% ethylene glycol+20% FCS in TCM199 for 10 min, and then exposed to 15% DMSO+15% ethylene glycol+0.5M sucrose+20% FCS in TCM199 for 1min. Five oocytes were placed on a Cryotop sheet (Kitazato Supply Co., Tokyo, Japan) and vitrified in liquid nitrogen. The samples were warmed in 0.5M sucrose solution for 5min, directly transferred into TCM199+20% FCS, and kept at room temperature for 1h before being used for a cloning experiment. The post-warm oocytes were fused with ear skin fibroblasts by two DC pulses (26V, 17μs) and activated with 7% ethanol for 5min and then 10μg/mL cycloheximide and 1.25μg/mL cytochalasin-D for 5h. The reconstructed embryos were cultured in mSOFaa+0.2% BSA+0.1% linoleic acid albumin for 2 days, and then co-cultured with bovine oviductal epithelial cells for an additional 5 days. Post-warm morphological survival of M-II oocytes (80%, 187/235) was similar to that of enucleated oocytes (75%, 158/212). Vitrified M-II oocytes were successfully enucleated (96%, 136/142) as were fresh control oocytes (88%, 143/162). Fusion rates of M-II oocytes vitrified before and after enucleation (81%, 94/116 and 78%, 106/136, respectively) were also similar to those of fresh oocytes (81%, 100/123). Percentages of reconstructed embryos developing into hatching blastocysts on Day 7 were 5% (5/91), 6% (6/103), and 8% (8/99) in the groups of oocytes vitrified before and after enucleation, and of fresh control oocytes, respectively (ANOVA tests were not significant different). These results indicate that swamp buffalo oocytes cryopreserved by ultra-rapid vitrification procedure can be used successfully for subsequent somatic cell nuclear transplantation. (Supported by Thailand Research Fund and R&amp;D Fund of Suranaree University of Technology)

scholarly journals Birth of pups after transfer of mouse embryos derived from vitrified preantral follicles

Reproduction ◽  
2002 ◽  
pp. 593-600 ◽  
Author(s):  
EC dela Pena ◽  
Y Takahashi ◽  
S Katagiri ◽  
EC Atabay ◽  
M Nagano
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Subsequent Development ◽  
Developmental Competence ◽  
Preantral Follicles ◽  
Cell Complexes ◽  
Vitrification Solution ◽  
First Time

Preantral follicles mechanically isolated from the ovaries of 12-day-old mice were exposed to 2 mol ethylene glycol l(-1) for 2 or 5 min and then to a vitrification solution containing 6 mol ethylene glycol l(-1) and 0.3 mol raffinose l(-1) for 0.5, 1.0 or 2.0 min before vitrification. The vitrified and fresh preantral follicles were treated with collagenase, and the oocyte-granulosa cell complexes (OGCs) obtained were cultured in vitro for 10 days in membrane inserts. Preantral follicles exposed to 2 mol ethylene glycol l(-1) for 5 min and then to the vitrification solution for 0.5 or 1.0 min showed the highest survival rates after warming. The follicular loss after warming was approximately 20%. After in vitro culture, the proportion of viable OGCs from the vitrified follicles was 10% lower than that of the fresh preantral follicles. There were no differences in the rates of maturation, fertilization and subsequent development to blastocysts between the oocytes derived from vitrified follicles and those derived from fresh preantral follicles; however, the developmental competence of the oocytes derived from both vitrified and fresh preantral follicles grown in vitro was lower than that of oocytes grown in vivo. One of the five recipient mice that received 20 blastocysts derived from vitrified preantral follicles gave birth to six live pups. The results of the present study demonstrate for the first time that mouse preantral follicles can be vitrified and that some of the embryos derived from vitrified preantral follicles can develop to live pups.

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

62 EXPOSURE TO ETHYLENE GLYCOL AND DIMETHYL SULFOXIDE CAUSES ACTIVATION AND SPINDLE ANOMALIES IN BUFFALO (BUBALUS BUBALIS) OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 131 ◽  
Author(s):  
M. De Blasi ◽  
E. Mariotti ◽  
M. Rubessa ◽  
S. Di Francesco ◽  
G. Campanile ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Embryo Development ◽  
Sucrose Solution ◽  
Bubalus Bubalis ◽  
Meiotic Spindle ◽  
Blastocyst Development ◽  
Chi Square ◽  
Chi Square Test ◽  
Simple Exposure

Despite the increasing interest, buffalo oocyte cryopreservation is still inefficient, especially in terms of blastocyst development after IVF. The aim of this work was to evaluate chromatin and spindle organization of buffalo in vitro-matured oocytes after vitrification/warming by cryotop and after their simple exposure to cryoprotectants (CP). An overall amount of 251 COC was selected and matured in vitro. In the vitrification group, COC were first exposed to 10% ethylene glycol (EG) + 10% DMSO for 3 min, and then to 20% EG + 20% of DMSO and 0.5 m sucrose, loaded on cryotops, and plunged into liquid nitrogen within 25 s. Oocytes were warmed into a 1.25 m sucrose solution for 1 min and then to decreasing concentrations of sucrose (0.625 m, 0.42 m, and 0.31 m) for 30s each. In order to test CP toxicity, COC were simply exposed to the vitrification and warming solutions. Two hours after warming, oocytes were fixed and immunostained for microtubules using a method previously described (Messinger SM and Albertini DF 1991 J. Cell Sci. 100, 289–298), stained for nuclei with Hoechst, and examined by fluorescence microscopy. Fresh in vitro-matured oocytes were fixed and stained as controls. Data were analyzed by chi-square test; results are shown in Table 1. The percentages of MII oocytes in the control and vitrification groups were greater than in the toxicity group, in which a greater percentage of telophase II stage oocytes were found compared with both the control and vitrification groups, indicating occurrence of activation. Of the MII oocytes, both exposure to CP and vitrification procedures gave greater percentages of oocytes with abnormal spindle and abnormal chromatin configuration compared with the control. An unexpected datum was the evidence of a significant percentage of spontaneously activated oocytes in the toxicity group. We speculate that the lack of activation in the vitrification group may be related to the slowing down of metabolic activity subsequent to thermal shock, and hence, that activation after vitrification may occur later than 2 h post-warming. In conclusion, the simple exposure to CP causes activation of the COC and damage to the cytoskeleton similar to that induced by the whole vitrification protocol. The damages to the meiotic spindle and DNA fragmentation may lead to aneuploidy incompatible with subsequent embryo development and account for the poor embryo development currently recorded in buffalo. Table 1.Chromatin and spindle organization in oocytes vitrified and exposed to cryoprotectants

38 Vitrification of prepubertal lamb spermatogonia using a novel vitrification system

2019 ◽  
Vol 31 (1) ◽  
pp. 145 ◽  
Author(s):  
S. Ledda ◽  
S. Pinna ◽  
S. Nieddu ◽  
D. Natan ◽  
A. Arav ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
In Vitro Culture ◽  
Dimethyl Sulfoxide ◽  
Fertility Preservation ◽  
Testicular Tissue ◽  
Gonadal Tissue ◽  
New Device ◽  
Vitrification Solution ◽  
Tissue Cryopreservation

Vitrification is a method extensively used for preserving oocytes and embryos and is also gaining acceptance for preserving gonadal tissue. Cryopreservation of spermatogonial stem cells is an applicable method for young males seeking fertility preservation before starting a treatment or can be a tool for genetic preservation of rare or high-value animals. The aim of this work was to evaluate the cryopreservation of testicular tissue from young lambs by vitrification using a new device named E.Vit (FertileSafe, Ness Ziona, Israel) that permits all cryopreservation procedures to be performed in straw. The new device consists of a 0.3-mL straw (Cryo Bio System, IMV, L’Aigle, France) with a capsule containing 50-µm pores inserted at one end. Testicular tissue extracts were prepared from testes of slaughtered lambs (n=10, 40 days old), opened by sagittal sectioning with a microblade and collecting small pieces of testicular tissue (1mm3) from the middle part of the rete testis. Three pieces of gonadal tissue were inserted into each E.Vit device. Each straw was sequentially loaded vertically in two 1.5-mL microtubes, which contained the following solutions: first, the equilibrating solution (7.5% dimethyl sulfoxide+7.5% ethylene glycol+20% FCS in TCM-199) for 6min, followed by 90min in the vitrification solution (18% dimethyl sulfoxide+18% ethylene glycol+0.5M Trehalose+BSA in TCM-199). After exposure to the equilibrating solution and vitrification solution, the solutions were removed and the straws were directly loaded into LN2. The warming procedure consisted of placing the straws directly into 5-mL tubes containing 100, 50, and 25% warming solution (1M sucrose in TCM-199+20% FCS) at 38.6°C for 5min each before arrival into the holding medium. Samples were recovered from the straws incubated at 38.6°C in 5% CO2 in air in TCM 199+5% FCS and evaluated at 0 and 2h post-warming for viability using trypan blue staining. Expression of a panel of specific genes (SOD2, HSP90b, BAX, POUF5/OCT4, TERT, CIRBP, KIF11, AR, FSHR) was analysed by real-time PCR in cryopreserved tissue in vitro cultured for 2h post-warming (2hV), in fresh controls immediately after tissue dissection (0hF), and after 2h of in vitro culture (2hF). The majority of cells survived after vitrification, although viability immediately after warming (0hV: 56%±1.45) or after 2h of in vitro culture (IVC) (2hV: 54±7%) was significantly lower compared with non-cryopreserved fresh controls (0hF: 89%±1.45; ANOVA P&lt;0.05). Expression analysis showed specific patterns for the different genes. Notably, BAX transcript abundance was not affected by vitrification or IVC, indicating an acceptable level of stress for the cells. The genes HSP90b and CIRBP were down-regulated in 2hF but increased in 2hV, as expected. Expression of SOD1 and OCT4 was altered by vitrification but not by IVC. Conversely, expression of TERT, KIF11, and AR was affected by both IVC and cryopreservation (ANOVA P&lt;0.05). This novel protocol for testicular tissue cryopreservation of prepubertal animals may be a promising strategy for fertility preservation and can contribute as a new approach in the development of large-scale biodiversity programs.

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 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).

114 VITRIFICATION OF BOVINE EMBRYOS IN MEDIUM WITH POLYVINYL ALCOHOL REPLACING BSA

2006 ◽  
Vol 18 (2) ◽  
pp. 165
Author(s):  
D. J. Walker ◽  
G. E. Seidel Jr
Keyword(s):  
Ethylene Glycol ◽  
Polyvinyl Alcohol ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Sodium Hyaluronate ◽  
Blastocyst Stage ◽  
Embryo Survival ◽  
Better Than

Embryos vitrified in medium supplemented with 4.25 μg/mL sodium hyaluronate (SH) and 0.1% polyvinyl alcohol (PVA) survived vitrification better than embryos vitrified in medium supplemented with 0.25% FAF-BSA (Walker and Seidel 2005 Reprod. Fert. Dev. 17, 153). The purpose of the present study was to determine if the small amount of SH was beneficial to in vitro survival and to examine the effects of different concentrations of PVA in vitrification solutions. Day 7 blastocysts (n = 360) were produced in vitro with semen from three bulls, two replicates each. Cryoprotectant solutions were prepared in a 2 × 3 factorial combination with two SH concentrations (0 or 4.25 μg/mL) and three PVA concentrations (0.05, 0.1%, or 0.2%). For vitrification, embryos were placed into chemically defined HEPES-buffered medium (HCDM-2) at room temperature (22–24°C) and then transferred to V1 (5 m ethylene glycol in HCDM-2) for 3 min. Next, embryos were placed in a 6 μL drop of V2 (7 m ethylene glycol, 0.5 m galactose, and 18% w/v Ficoll 70 in HCDM-2) for 45 s. During these 45 s, dilution medium (0.5 m galactose in HCDM-2) was aspirated into 0.25-mL straws, followed by the 6 μL drop of V2 plus embryos and a final short column of dilution medium. When 45 s had elapsed, the heat-sealed end of straw was dipped into liquid nitrogen to cover the embryo, and then the remainder of the straw was immersed slowly. Straws were thawed in air for 10 s and then in 37°C water for 20 s. Next, straws were shaken like a clinical thermometer four times to mix columns, and held in 37°C water for 10 min before embryos were expelled, rinsed and cultured in CDM-2 + 5% FCS. At 48 h, embryo survival (as determined by expansion of blastocysts), embryo quality (1 = excellent, 2 = fair, 3 = poor), inner cell mass (ICM) quality (1 = large and compact, 2 = clearly visible, 3 = not discernable) and blastocyst stage (5 = early, 6 = full, 7 = expanded, 8 = hatching, 9 = hatched) were evaluated and replicate averages were analyzed by ANOVA. Neither bull nor SH concentration nor PVA concentration significantly affected any response (P > 0.10). Averaged over PVA concentrations, vitrification of embryos in 0 μg/mL or 4.25 μg/mL SH resulted in similar survival rates (67% vs. 62%, respectively). When averaged over SH concentrations, 0.2% PVA had a numerically higher survival rate of blastocysts as compared to 0.1% or 0.05% (71% vs. 63% and 60%, respectively). The main effects of 0 μg/mL SH and 0.2% PVA also resulted in numerically higher, but nonsignificant improvements in quality score, ICM score and blastocyst stage as compared to the other doses of SH and PVA. Vitrification of Day 7 in vitro-produced bovine blastocysts in medium containing 0.2% PVA in the absence of SH resulted in a subclass mean of 80% embryo survival. Results of this experiment show no benefit of 4.25 μg/mL SH and that 0.2% PVA may be slightly better than 0.05% or 0.1% in terms of embryo survival. Therefore, our results indicate that 0.2% PVA can be used alone as an effective alternative to animal products in this vitrification procedure for in vitro-derived bovine blastocysts.

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