In vitro comparisons of two cryopreservation techniques for equine embryos: Slow-cooling and open pulled straw (OPS) vitrification (original) (raw)
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Reproduction, Fertility and Development
The success of in vitro maturation (IVM) has a significant impact on the oocytes ability to develop to blastocyst stage. The quality of the produced blastocysts can be evaluated using staining techniques. The aims of this study were (a) to compare the effect of different IVM media on embryo production rates, and (b) to utilize differential (DF) and TUNEL staining to evaluate the quality and cryosurvival of the produced blastocysts. Abattoir-derived oocytes were randomly divided into the IVM groups: (1) M199 IVM (n = 2305): TCM-199 with glutamax-I (GIBCO, Paisley, UK), 0.25 mM Na-pyruvate, 100 IU mL−1 penicillin, and 100 μg mL−1 streptomycin; (2) FBS IVM (n = 2484): M199 IVM medium with hormones (10 μg mL−1 LH, 2 μg mL−1 FSH, and 100 μg mL−1 β-estradiol), and 10% FBS (GIBCO, New Zealand); and (3) FAFBSA IVM (n = 2411): as group (2), but FBS was replaced with 4 mg mL−1 fatty acid free albumin. Fertilized oocytes were denuded and cultured in modified SOFaaci + 4 mg mL−1 FAFBSA in 5% O2...
Theriogenology, 1999
Embryo quality of in vitro-produced bovine blastocysts was assessed at several steps of a vitrification procedure in which glycerol and ethylene glycol l~~ere used as cryoprotectants (3-step equilibration with cryoprotectants followed by vitri&ation, dilution of tile &yoprotectants in 0.85 M rralactose then in embryo transfer freezing medium IETFI. and finally co-culture for periods): To visualize cell membrane alterations,-double staining-was perforGed using a cell permeant fluorochrome (bisbenzimide-BIS) and a nonpermeant one (propidium iodide-PI). In Experiment 1, the effect of the vitrification procedure on the hatching rate and total cell number was assessed 72 h after treatment. Hatching rate and the number of stained nuclei were decreased in comoarison with untreated embrvos when blastocvsts were exuosed to the whole orocedure with 0; without vitrification (respectively 42 and 53% vs 76% fbr hatching and 128+ 17 and 141 f 17 vs 226 + 13 for stained nuclei). In Experiment 2, the effect of ctyoprotectants and their dilution was evaluated on membrane permeability and total cell numbers at various steps of the vitrification procedure. Blastocysts exposed only to cryoprotectant solutic.la and stained immediatelv after dilution in eaiactose showed no modification. After dilution in ETF. the total number of-stained nuclei dicecreased, and the number of blastomeres showing membrane permeabilization (PI-stained) increased (respectively, 74 f 5 vs 110 + 5 and 32 i 2% vs 0.1 + 1.8%). In Experiment 3, we demonstrated that the total number of stained nuclei after ethanol fixation (membrane permeabilization) was higher when embryos treated up to dilution in ETF were stained with PI than when the same embryos were stained with BIS. This suggests that, for unknown reasons, some nuclei of the treated embryos were not stained with BIS. Membrane permeabilization and inability of BIS to stain some-nuclei were the most obvious alterations probably induced by osmotic shock at dilution. This hypothesis is supported by the fact that the introduction of a further dilution step in 0.42M galactose (Experiment 4) before dilution in ETF decreased the orouortion of cells Dermeant to PI and increased the hatching rate after 72 h of co-culture. Inco&lusion, double &ining with BIS and PI allowed for dis&imination between different types of cellular injuries after the various steps of our vitrification protocol. It represents a useful tool for adjusting equilibration and dilution conditions during a cryopreservation procedure. CD ,399 by Elsevier sdence Inc.
Theriogenology, 2004
The objectives of this study were to identify an improved in vitro cell-free embryo culture system and to compare post-warming development of in vitro produced (IVP) bovine embryos following vitrification versus slow freezing. In Experiment 1, non-selected presumptive zygotes were randomly allocated to four medium treatments without co-culture: (1) SOF þ 5% FCS for 9 days; (2) KSOM þ 0:1% BSA for 4 days and then KSOM þ 1% BSA to Day 9; (3) SOF þ 5% FCS for 4 days and then KSOM þ 1% BSA to Day 9; and (4) KSOM þ 0:1% BSA for 4 days and then SOF þ 5% FCS to Day 9. Treatment 4 (sequential KSOM-SOF culture system) improved (P > 0:05) morulae (47%), early blastocysts (26%), Day-7 blastocysts (36%), cell numbers, as well as total hatching rate (79%) compared to KSOM alone (Treatment 2). Embryos cultured in KSOM þ BSA alone developed slowly and most of them hatched late on Day 9, compared to other treatments. In Experiment 2, the sequential KSOM-SOF culture system was used and Day-7 blastocysts were subjected to following cryopreservation comparison: (1) vitrification (VS3a, 6.5 M glycerol); or (2) slow freezing (1.36 M glycerol). Warmed embryos were cultured in SOF with 7.5% FCS. Higher embryo development and hatching rates (P < 0:05) were obtained by vitrification at 6 h (71%), 24 h (64%), and 48 h (60%) post-warming compared to slow freezing (48, 40, and 31%, respectively). Following transfer of vitrified embryos to synchronized recipients, a 30% pregnancy rate was obtained. In conclusion, replacing KSOM with SOF after 4 days of culture produced better quality Theriogenology 62 (
Theriogenology, 2001
Cryopreservation of equine embryos with conventional slow-cooling procedures has proven challenging. An alternative approach is vitrification, which can minimize chilling injuries by increasing the rates of cooling and wanning. The open pulled straw (OPS) and cryoloop have been used for very rapid cooling and warming rates. The objective of this experiment was to compare efficacy of vitrification of embryos in OPS and the cryoloop to conventional slow cool procedures using 0.25 mL straws.
Effect of culture system on survival rate of vitrified bovine embryos produced in vitro
Cryobiology, 2009
This study was designed to evaluate the effect of in vitro culture system on bovine blastocyst yield and quality after vitrification. In Experiment 1, IVM/IVF zygotes were allocated to three culture conditions: (I) Oviductal cells-SOF (OCM-SOF); (II) Oviductal cells-TCM (OCM-TCM); and (III) SOF for 8 days. There was no significant difference between blastocyst rates among groups. In Experiment 2, the IVP-blastocysts in three above culture conditions were vitrified within groups segregated according to age (Day 7 and 8) and blastocoelic cavity size (early and expanded blastocysts). A trend of higher survival rate was obtained in vitrified/warmed early blastocysts compared with expanded ones, so that the difference in OCM-TCM group was significant (P < 0.001). Higher survival and hatching rates (P < 0.001) were obtained in OCM-SOF and OCM-TCM groups (co-culture) compared with SOF group and the age of blastocyst had no effect on post-thaw survival and hatching rates. In Experiment 3, after staining of blastocysts, in fresh blastocysts the highest number of trophectoderm cells was observed in OCM-TCM group and the number of inner cell mass (ICM) was higher in co-culture groups than SOF group (P < 0.001). In vitrified/warmed blastocysts the number of ICM and trophectoderm cells in co-culture groups was higher than SOF group (P < 0.001) except for the ICM of expanded blastocysts. In conclusion, in our culture conditions, the blastocyst yield is not influenced by culture system, while the cryotolerance of IVP-blastocysts is positively influenced by the presence of somatic cells. Moreover, the expanded blastocysts are more susceptible to cryoinjury than early blastocysts.
The aim of this study was to compare in vitro survival rates of in vivo and in vitro-produced bovine embryos by slow freezing or solid surface vitrification. In vivo-produced blastocysts (n = 210) and in vitroproduced blastocysts (n = 445) were randomly allocated in two cryopreservation groups. Group 1-embryos were exposed to 1.5 M ethylene glycol (EG) for 5 min, loaded in 0.5 ml straws, frozen at-6.5ºC and seeded. After 10 min of equilibration, straws were cooled at-0.6ºC/min until-35ºC, and then plunged into liquid nitrogen (-196ºC). Group 2-embryos were exposed to a 15% EG + 0.25 M trehalose solution for 1 min and then a 30% EG + 1 M trehalose solution for 30 sec to be vitrified using the Cryologic Vitrification Method (CVM ®). After at least one week of storage, embryos in the slow freezing group were thawed in a water bath at 30°C for 12 sec and then placed in holding medium for 5 min and transferred into SOF culture media. Vitrified embryos were placed directly into a 0.25 M sucrose solution for 5 min then cultured in SOF medium. Re-expansion and hatching rates were evaluated at 24 and 72 h, respectively. In vivo-produced embryos had higher (P < 0.01) re-expansion (179/210, 81% vs. 244/445, 54%) and hatching rates (159/210, 72% vs. 177/445, 39%) than in vitro-produced embryos, regardless of the cryopreservation method. However, re-expansion and hatching rates were higher (P < 0.01) for in vitro-produced vitrified embryos (155/223, 69% and 132/223, 59%) than in vitro-produced embryos cryopreserved by slow freezing (89/222, 40% and 45/222, 20%). Although similar re-expansion rates were obtained with in vivoproduced embryos cryopreserved by the two systems, hatching rates tended to be lower (P = 0.09) with in vivo-produced embryos that were vitrified as compared to slow freezing. In conclusion, solid surface vitrification improved the cryosurvival rates of in vitroproduced embryos compared to the conventional slow freezing procedure.
Theriogenology, 2003
This study evaluated the efficiency and toxicity of two cryopreservation methods, solid-surface vitrification (SSV) and cryoloop vitrification (CLV), on in vitro matured oocytes and in vivo derived early stage goat embryos. In the SSV method, oocytes were vitrified in a solution of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4% trehalose. Microdrops containing the oocytes were cryopreserved by dropping them on a cold metal surface that was partially immersed in liquid nitrogen. In the cryoloop method, oocytes were transferred onto a film of the CLV solution (20% DMSO, 20% EG, 10mg/ml Ficoll and 0.65 M sucrose) suspended in the cryoloop. The cryoloop was then plunged into the liquid nitrogen. In vivo derived embryos were vitrified using the same procedures. The SSV microdrops were warmed in a solution of 0.3M trehalose and those vitrified with CLV were warmed with incubation in 0.25 and 0.125 M sucrose. Oocytes and embryos vitrified by the SSV method had a significantly lower survival rate than the control (60 and 39% versus 100%, respectively; P&amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05), while the survival rate of CLV oocytes and embryos (89 and 88%, respectively) did not differ from controls. Cleavage and blastocyst rates of the surviving vitrified oocytes (parthenogenetically activated) and embryos (cultured for 9 days) were not significantly different (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;0.05) from the control nor did they differ between vitrification methods. Embryos vitrified with the CLV method gave rise to blastocysts (2/15). Our data demonstrated that the two vitrification methods employed resulted in acceptable levels of survival and cleavage of goat oocytes and embryos.
Vitrification of early-stage bovine and equine embryos
Theriogenology, 2009
The objectives of this study were to: (1) determine an optimal method and stage of development for vitrification of bovine zygotes or early embryos; and (2) use the optimal procedure for bovine embryos to establish equine pregnancies after vitrification and warming of early embryos. Initially, bovine embryos produced by in-vitro fertilization (IVF) were frozen and vitrified in 0.25 mL straws with minimal success. A subsequent experiment was done using two vitrification methods and super open pulled straws (OPS) with 1-or 8-cell bovine embryos. In Method 1 (EG-O), embryos were exposed to 1.5 M ethylene glycol (EG) for 5 min, 7 M ethylene glycol and 0.6 M galactose for 30 s, loaded in an OPS, and plunged into liquid nitrogen. In Method 2 (EG-DMSO), embryos were exposed to 1.1 M ethylene glycol and 1.1 M dimethyl sulfoxide (DMSO) for 3 min, 2.5 M ethylene glycol, 2.5 M DMSO and 0.5 M galactose for 30 s, and loaded and plunged as for EGO. Cryoprotectants were removed after warming in three steps. One-and eight-cell bovine embryos were cultured for 7 and 4.5 d, respectively, after warming, and control embryos were cultured without vitrification. Cleavage rates of 1-cell embryos were similar (P > 0.05) for vitrified and control embryos, although the blastocyst rates for EGO and control embryos were similar and higher (P < 0.05) than for EG-DMSO. The blastocyst rate of 8-cell embryos was higher (P < 0.05) for EGO than EG-DMSO. Therefore, EGO was used to cryopreserve equine embryos. Equine oocytes were obtained from preovulatory follicles. After ICSI, injected oocytes were cultured for 1-3 d. Two-to eight-cell embryos were vitrified, warmed and transferred into recipient's oviducts. The pregnancy rate on Day 20 was 62% (5/8) for equine embryos after vitrification and warming. In summary, a successful method was established for vitrification of early-stage bovine embryos, and this method was used to establish equine pregnancies after vitrification and warming of 2-to 8-cell embryos produced by ICSI.
A system to evaluate the quality of frozen embryos through short-term culture
Animal Reproduction Science, 2008
The aim of the present study was to evaluate a culture system as a non-invasive approach intended for assessing the viability of recently thawed embryos prior to transfer. Embryos (n = 51) were collected seven days after insemination out of 20 cows that had been treated to synchronize estrus and induce superovulation. Embryos were classified as good, fair, and poor and frozen. All embryos were cultured in McCoy © medium. Morphology was monitored for a period of 24 h to register the development stage every 30 min for the first 2 h, and every hour thereafter. A sample of four embryos of each classification was separated at 4 h, another four at 12 h, and the remaining seven at 24 h and the degree of apoptosis was determined for all the embryos using the TUNEL technique. Embryos of good and fair quality did not undergo major detrimental changes in development even after 7 h of incubation, whereas poor quality embryos experienced changes as early as 2 h after incubation. Good quality embryos invariably had fewer numbers of apoptotic cells than those of fair and poor quality suggesting that embryo culture can be a useful method to assess viability and to confirm the quality of thawed embryos previously stored in liquid nitrogen prior to transfer.