Effects of two combinations of cryoprotectants on the in vitro developmental capacity of vitrified immature porcine oocytes.

This study evaluated two cryoprotectant (CPA) combinations, ethylene glycol (EG) + DMSO and EG + propylene glycol (PG), used for the vitrification of germinal vesicle (GV) porcine oocytes. In experiment 1, the equilibration of GV with the two CPA combinations increased (P < 0.05) the percentage of oocytes that degenerated after IVM (18.1 ± 2.3% and 19.4 ± 2.6% for EG + DMSO and EG + PG groups, respectively) compared with control oocytes (7.6 ± 1.3%). However, CPAs did not affect the fertilization or developmental parameters of the embryos. In experiment 2, the percentages of live vitrified-warmed GV oocytes at 2 hours after warming (EG + DMSO: 67.0 ± 2.3% and EG + PG: 57.6 ± 2.3%) were lower than those of fresh control GV oocytes (97.3 ± 0.7%). The percentage of degenerated oocytes after IVM was higher (P < 0.001) in vitrified-warmed oocytes (EG + DMSO: 59.8 ± 2.3% and EG + PG: 56.2 ± 2.6%) than in the control (1.6 ± 1.3). Fertilization efficiency was higher (P < 0.05) in the EG + PG (39.6 ± 2.4%) and control (42.0 ± 2.2%) groups than in the EG + DMSO (26.3 ± 7.7%) group. The cleavage and blastocyst formation rates of the EG + DMSO (25.9 ± 3.5% and 6.6 ± 2.5%, respectively) and EG + PG (20.2 ± 5.4% and 4.7 ± 1.6%, respectively) vitrification groups were lower (P < 0.001) than those observed in the control oocytes (53.4 ± 2.7% and 31.9 ± 1.7%, respectively). In conclusion, in the absence of vitrification, the toxic effects of both CPA combinations on the GV oocytes were minimal. Vitrification resulted in important losses in viability at each step of the in vitro embryo production procedure. However, the surviving oocytes were able to mature and be fertilized, although the fertilization efficiency in the EG + DMSO group was lower. Blastocysts formation was similar for both CPA combinations.

Nohalez A ，Martinez CA ，Gil MA ，Almiñana C ，Roca J ，Martinez EA ，Cuello C ... -  《-》

Effect of different vitrification solutions and cryodevices on viability and subsequent development of buffalo oocytes vitrified at the germinal vesicle (GV) stage.

The cryopreservation of immature oocytes would generate a readily available, non-seasonal source of female gametes for research and reproduction. In domestic animals, the most promising results on oocyte cryopreservation have been reported in cattle, few studies have been conducted on buffalo. The aim of the present study was to compare the use of different vitrification solutions and various cryodevices on viability and developmental competence of buffalo oocytes vitrified at the germinal vesicle (GV) stage. Cumulus oocyte-complexes (COCs) obtained at slaughterhouse from mature buffalo ovaries were randomly divided into three main groups and vitrified by using either straw or open pulled-straw (OPS) or solid surface vitrification (SSV) in a solution composed of either 20% ethylene glycol (EG) + 20% glycerol (GLY); VS1 or 20% EG + 20% dimethylsulfoxide (DMSO); VS2, respectively. Following vitrification and warming, viable COCs were matured in vitro for 22 h. Some COCs were denuded and stained with 1.0% aceto-orcein to evaluate nuclear maturation, whereas the others were fertilized and cultured in vitro for 7 days to determine the developmental competence. Although the recovery rate (64.9%) was the lowest in the oocytes vitrified by SSV using 20% EG + 20% DMSO as compared to the other groups, the best survival rate of the COCs was achieved in the same treatment (96.7%), which was significantly higher (P < 0.05) than those vitrified using traditional straws (71.8% in VS1 and 73.6% in VS2) or those vitrified using OPS and VS1 (73.9%). Furthermore, in the nuclear maturation test, the highest maturation rate (75.5%) was achieved in SSV vitrified COCs using 20% EG + 20% DMSO (VS2), which was similar to the controls (77.1%). Post IVF and embryo culture, the highest cleavage and blastocyst development rates were obtained in COCs vitrified in 20% EG + 20% DMSO using SSV (47.1% and 24.0%, respectively), which showed no difference from the controls (61.2% and 46.9%, respectively). Our results clearly show that the combination of SSV and 20% EG + 20% DMSO could be used effectively to vitrify GV stage buffalo COCs.

El-Shalofy AS ，Moawad AR ，Darwish GM ，Ismail ST ，Badawy ABA ，Badr MR ... -  《-》

Vitrification of porcine immature oocytes: Association of equilibration manners with warming procedures, and permeating cryoprotectants effects under two temperatures.

The aim of this study was to evaluate the association of equilibration manners with warming procedures, and the different permeating cryoprotectants (pCPAs) effects under two temperatures, in terms of survival, maturation and subsequent parthenogenetic development of porcine immature oocytes after Cryotop vitrification. In Experiment 1, oocytes were equilibrated by exposure to 5% (v/v) ethylene glycol (EG) for 10 min (EM1) or stepwise to 7.5% (v/v) and 15% (v/v) EG for 2.5 min respectively (EM2). Warming procedures were performed in 1.0 M sucrose for 1 min, then in 0.5 and 0.25 M sucrose for 2.5 min respectively (WP1), or in 0.5, 0.25 and 0.125 M sucrose each step for 2 min (WP2), or in 0.25, 0.125 and 0.063 M sucrose each step for 2 min (WP3). After 2 h of warming, the survival rate of oocytes treated by EM1 and WP1 was significantly higher (P < 0.05) than that of the other groups. Moreover, a similar proportion of survival and nuclear maturation in all vitrified groups was obtained after completion of the IVM. No significant difference in blastocyst development was observed among vitrified groups except the group treated by EM2 and WP3. In Experiment 2, oocytes were vitrified by using EG alone, EG combined with dimethyl sulphoxide (EG + DMSO) or propylene glycol (EG + PROH) as pCPAs under 25 °C and 39 °C. The percentages of cryosurvival and nuclear maturation were similar in all vitrified groups. Under 25 °C, the embryo development and total cell numbers of blastocysts were not significantly different among EG, EG + DMSO and EG + PROH groups. However, the application of EG + PROH at 39 °C resulted in significantly decreased both cleavage and blastocyst formation rates. In conclusion, our data showed that equilibration manner and warming procedure affect the cryosurvival of porcine immature oocytes, and the combination of pCPAs cannot give a better cryopreservation outcome whether 25 °C or 39 °C. Notably, the Cryotop vitrification accompanied by our modified strategy for porcine immature oocytes could achieve high survival and respectable blastocyst production.

Wu G ，Jia B ，Quan G ，Xiang D ，Zhang B ，Shao Q ，Hong Q ... -  《-》

Optimization of cryoprotectant treatment for the vitrification of immature cumulus-enclosed porcine oocytes: comparison of sugars, combinations of permeating cryoprotectants and equilibration regimens.

Somfai T ，Men NT ，Noguchi J ，Kaneko H ，Kashiwazaki N ，Kikuchi K ... -  《-》

Effect of different combinations of cryoprotectants on in vitro maturation of immature buffalo (Bubalus bubalis) oocytes vitrified by straw and open-pulled straw methods.

This study was designed to evaluate effects of different combinations of cryoprotectants on the ability of vitrified immature buffalo oocytes to undergo in vitro maturation. Straw and open-pulled straw (OPS) methods for vitrification of oocytes at the germinal vesicle stage also were compared. The immature oocytes were harvested from ovaries of slaughtered animals and were divided into three groups: (i) untreated (control); (ii) exposed to cryoprotectant agents (CPAs); or (iii) cryopreserved by straw and OPS vitrification methods. The vitrification solution (VS) consisted of 6 m ethylene glycol (EG) as the standard, control vitrification treatment, and this was compared with 3 m EG + 3 m dimethyl sulfoxide (DMSO), 3 m EG + 3 m glycerol, and 3 m DMSO + 3 m glycerol. Cryoprotectants were added in two steps, with the first step concentration half that of the second (and final) step concentration. After warming, oocyte samples were matured by standard methods and then fixed and stained for nuclear evaluation. Rates of MII oocytes exposed to CPAs without vitrification were lower (54.3 +/- 1.9% in EG, 47.5 +/- 3.4% in EG + DMSO, 36.8 +/- 1.2% in EG + glycerol and 29.9 +/- 1.0% in DMSO + glycerol; p < 0.05) than for the control group (79.8 +/- 1.3%). For all treatments in each vitrification experiment, results were nearly identical for straws and OPS, so all results presented are the average of these two containers. The percentages of oocytes reaching telophase-I or metaphase-II stages were lower in oocytes cryopreserved using all treatments when compared with control. However, among the vitrified oocytes, the highest maturation rate was seen in oocytes vitrified in EG + DMSO (41.5 +/- 0.6%). Oocytes cryopreserved in all groups with glycerol had an overall low maturation rate 19.0 +/- 0.6% for EG + glycerol and 17.0 +/- 1.1% for DMSO + glycerol. We conclude that the function of oocytes was severely affected by both vitrification and exposure to cryoprotectants without vitrification; the best combination of cryoprotectants was EG + DMSO for vitrification of immature buffalo oocytes using either straw or OPS methods.

Mahmoud KG ，Scholkamy TH ，Ahmed YF ，Seidel GE Jr ，Nawito MF ... -  《-》