Effect of different cryo-devices on in vitro maturation and development of vitrified-warmed immature buffalo oocytes.

The aim of the study was to identify a cryo-device that would be best suited for the vitrification of buffalo immature cumulus-oocyte complexes (COCs) as judged by viability and meiotic competence of the vitrified-warmed oocytes and their development ability following in vitro fertilization (IVF). The expression of oocyte secreting factors and their receptors (GDF9, BMP15, BMPR2, TGFBR1) and apoptosis related genes (BCL2, BAX, P53, C-MYC) were compared in vitrified-warmed oocytes after in vitro maturation. COCs from the ovaries of slaughtered buffaloes were vitrified in a combination of dimethyl sulfoxide, ethylene glycol, and sucrose using either a conventional straw (CS), open pulled straw (OPS), cryoloop (CL), hemistraw (HS) or cryotop (CT). The fresh COCs were exposed to vitrification and warming solutions as in other vitrification methods without plunging in to liquid nitrogen (EC). The viability of vitrified-warmed COCs, 2 h post warming in HS and CT was similar to fresh and EC groups but significantly higher than CS and OPS methods. The proportions of oocytes with first polar body after 24 h in vitro maturation were significantly higher in HS and CT methods than in CS, OPS and CL methods. The development ability of these vitrified-warmed oocytes to blastocyst stage following IVF in all vitrified groups was significantly lower than control and EC groups. Among the vitrified groups, the blastocyst rate in HS, CT and CL groups was significantly higher than in OPS and CS groups. It was also observed that the expression levels of GDF9, BMP15, BMPR2, TGFBR1, BCL2, BAX, P53 and C-MYC genes in vitrified-warmed COCs in CT, HS and CL groups were similar to control. The results indicated that HS, CT and CL are more suitable cryo-devices for vitrification of buffalo immature oocytes.

Mahesh YU ，Gibence HRW ，Shivaji S ，Rao BS ... -  《-》

Effect of vitrification on meiotic maturation and expression of genes in immature goat cumulus oocyte complexes.

The aim of the study was to evaluate meiotic maturation, and expression of genes coding for oocyte secreted factors (GDF9, BMP15, TGFBR1, and BPR2) and apoptosis (BCL2, BAX and P53) after vitrification of immature goat cumulus oocyte complexes (COCs) and in vitro maturation. COCs were vitrified in a solution containing ethylene glycol, dimethyl sulfoxide and sucrose using either a conventional straw (CS), open pulled straw (OPS), cryoloop (CL), hemistraw (HS) or cryotop (CT). Freshly collected COCs (Control), COCs exposed to vitrification and dilution solutions without cryopreservation (EC) and vitrified-warmed COCs were matured in vitro for 27h. The viability of vitrified-warmed COCs 2 h post warming and in vitro maturation was similar for CL, HS and CT. The proportion of oocytes that extruded a 1st polar body and reached TI/MII was significantly higher with CT and HS followed by CL, OPS and CS. Gene expression of GDF9, BMP15, BMPR2, BAX and P53 were comparable to control levels for OPS, CL, HS and CT. The gene expression pattern in CS vitrified COCs was by contrast changed in that GDF9, BMP15, TGFBR1 and BAX were up regulated and BMPR2, BCL2 and P53 down regulated. In conclusion immature goat COCs vitrified using CT and HS showed that viability, maturation rates and expression of genes coding for oocyte secreted factors and apoptosis were similar to non-vitrified controls.

Rao BS ，Mahesh YU ，Charan KV ，Suman K ，Sekhar N ，Shivaji S ... -  《-》

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 ... -  《-》

Meiotic maturation and developmental capability of ovine oocytes at germinal vesicle stage following vitrification using different cryodevices.

In order to assess effects of vitrification on ovine oocytes at the germinal vesicle (GV) stage, the conventional plastic straw (CS), the open-pulled straw (OPS), and Cryoloop were used to vitrify ovine oocytes. Oocytes were randomly divided into five groups: (1) Control; (2) Oocytes exposed to vitrification and dilution solutions without any cryopreservation (toxicity); (3) Oocytes vitrified using CS (CS); (4) Oocytes vitrified using OPS (OPS), and (5) Oocytes vitrified using Cryoloop (Cryoloop). The viability, cumulus cell expansion, nuclear maturation after in vitro maturation (IVM), and developmental capability of vitrified oocytes following parthenogenetic activation (PA) or in vitro fertilization (IVF) were assessed. The pretreatment in the vitrification and dilution solutions without any freezing or thawing did not adversely influence oocytes. The viability of vitrified oocytes were significantly declined compared to unfrozen oocytes (P < 0.05). The viability of oocytes vitrified using open-pulled straws or Cryoloop was significantly higher than that in the CS group (P < 0.05). After IVM, the percentage of oocytes reaching to the metaphase II (MII) stage was significantly higher with Cryoloop and OPS following by CS. However, the in vitro maturing percentage of vitrified oocytes was significantly less than that of unfrozen oocytes (P < 0.05). After PA, the developmental capability of vitrified oocytes was significantly decreased compared to unfrozen oocytes. The cleavage rate of oocytes vitrified using conventional plastic straws was significantly less than those of the other freezing groups (P < 0.05). The cleaving capability of oocytes vitrified using Cryoloop was significantly increased compared to the OPS group. However, there was no significant difference existing amongst the freezing groups as concerning the blastocyst rate. Following IVF, the developmental capability of vitrified oocytes was severely damaged compared to that of unfrozen oocytes. The cleavage rate of oocytes vitrified with Cryoloop was similar to that of oocytes vitrified with open-pulled straws. However, the cleavage rate of vitrified oocytes in the CS group was significantly less than that in the OPS or Cryoloop group (P < 0.05). None of oocytes vitrified using conventional plastic straw developed to the blastocyst stage following IVF. There was no significant difference existing between OPS and Cryoloop with respect to the blastocyst rate. After staining with cFDA and PI, cumulus cells surrounding oocytes were partly damaged by vitrification and thawing while the membrane of vitrified oocyte still remained intact. In conclusion, vitrification can seriously damage ovine immature oocytes and cumulus cells surrounding oocytes, which may subsequently affect their developmental capability. Finally, this study further proves that increasing the freezing and thawing velocity benefits survival of vitrified immature oocytes.

Quan GB ，Wu GQ ，Wang YJ ，Ma Y ，Lv CR ，Hong QH ... -  《-》

Effect of Disaccharide Inclusion in Vitrification and Warming Solutions on Developmental Competence of Vitrified/Warmed Germinal Vesicle Stage Buffalo Oocytes.

Cryopreservation of immature oocyte is a potential strategy for preserving the female germline, providing a non-seasonal, easily accessible source for reproduction and science. Exposure of oocytes to high concentrations of cryoprotectants during vitrification is toxic and can negatively impact the fertilization ability and development of vitrified/warmed oocytes. 1) to evaluate the effects of exposure of buffalo germinal vesicle (GV) oocytes to different vitrification solutions (VS), either supplemented with or without sucrose, on cumulus expansion and nuclear maturation following IVM; and 2) to compare the effects of sucrose and trehalose in the warming solution on developmental competence of buffalo oocytes vitrified at the GV-stage. Cumulus oocyte complexes (COCs) obtained at slaughter from mature buffalo ovaries were randomly assigned into five groups: control - directly subjected to IVM); VS1 group - exposed to 20% ethylene glycol (EG) + 20% glycerol (GLY) + 0.5 M sucrose; VS2 group - exposed to 20% EG + 20% GLY; VS3 group - subjected to 20% EG+20% dimethyl sulfoxide (DMSO) + 0.5 M sucrose; and VS4 group - subjected to 20% EG+20% DMSO. Following cryoprotectant dilution, viable oocytes were matured in vitro for 22 h; cumulus expansion and nuclear maturation were then evaluated (Experiment 1). COCs were vitrified by solid surface vitrification (SSV) in a solution composed of 20% EG + 20% DMSO (VS4). Following vitrification, COCs were warmed in a solution composed of either sucrose or trehalose in decreasing concentrations (1 M, 0.5 M and 0.25 M). Morphologically viable oocytes were matured, fertilized and cultured in vitro. Cleavage and blastocyst rates were evaluated at 30 h and day 7 post-insemination (p.i.), respectively (Experiment 2). Exposure of GV-buffalo oocytes to different cryoprotectant combinations did not significantly affect cumulus expansion following IVM. However, nuclear maturation rate (oocytes at MII) was significantly higher (P<0.05) in the groups exposed to sucrose-free vitrification solutions (VS2 and VS4) and not significantly different from the control. Compared with the control group, the cleavage and blastocyst rates were significantly (P<0.05) lower in oocytes vitrified and then warmed in a solution containing trehalose; whilst this was not the case when sucrose was present in the solution. Our results suggest that exposure of buffalo GV-oocytes to sucrose-free vitrification solutions improved nuclear maturation after IVM. Moreover, warming of vitrified buffalo oocytes in sucrose-based solution improved preimplantation development following IVM and IVF compared to trehalose based media.

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