Production of good-quality porcine blastocysts by in vitro fertilization of follicular oocytes vitrified at the germinal vesicle stage

We investigated survival, meiotic competence, cytoplasmic maturation, in vitro fertilization, and development of immature porcine (Sus scrofa) oocytes cryopreserved by a modified solid surface vitrification protocol. Cumulus-oocyte complexes (COCs) collected from follicles 3 to 6 mm in diameter in abattoir-derived ovaries of prepubertal gilts were either vitrified (Vitrified group), subjected to cryoprotectant treatment (CPA group), or used without any treatment (Control group). Oocyte viability was assayed by staining with fluorescein diacetate. Live oocytes were matured in vitro and their meiotic progression investigated by nuclear staining. In a series of experiments, the glutathione (GSH) content of in vitro-matured oocytes and viability of cumulus cells were assayed simultaneously. The in vitro-matured oocytes were also fertilized and cultured in vitro to assess their ability to be fertilized and to develop to the blastocyst stage, respectively. The proportion of viable oocytes in the Vitrified group was significantly lower than that in the CPA and Control groups (27.7%, 90.4%, and 100%, respectively). Among the three groups, there were no differences in meiotic competence, cumulus viability, and GSH levels at the end of in vitro maturation. Fertilization parameters (i.e., rates of male pronucleus formation, monospermy, and second polar body extrusion) were also similar among groups. However, comparison of the developmental abilities of oocytes in the Vitrified, CPA, and Control groups revealed that the Vitrified group had a significantly reduced ability to undergo first cleavage (34.4%, 63.3%, and 69.0%) and to develop to the blastocyst stage (5.1%, 25.5%, and 34.6%). The mean total cell numbers in blastocysts after 6 d of culture were not significantly different among the Vitrified, CPA, and Control groups (40.3, 42.8, and 43.4). In conclusion, despite low survival rates and impaired development in the Vitrified group, meiotic competence, cytoplasmic maturation, and subsequent fertilization characteristics of surviving germinal vesicle oocytes were unaffected by vitrification, and high-quality blastocysts were produced from vitrified immature oocytes.     

Comparison of cytoskeletal integrity,fertilization and developmental competence of oocytes vitrified before or after in vitro maturation in a porcine model

Aim of the study was to investigate the effect of vitrification on viability, cytoskeletal integrity and in vitro developmental competence after in vitro fertilization (IVF) of oocytes vitrified before or after in vitro maturation (IVM) using a pig model. Oocytes from abattoir-derived porcine ovaries were vitrified at either the germinal vesicle (GV) or metaphase II (MII) stage by modified solid surface vitrification (SSV). Oocyte viability was evaluated by stereomicroscopic observation whereas their nuclear stage and morphology of microtubules and F-actin were observed by confocal microscopy after immunostaining. Fertilization was assessed by orcein staining. The survival rate after vitrification was higher for MII-stage than for GV-stage oocytes. However, the ability of surviving oocytes to reach the MII stage after vitrification at the GV stage (GV-vitrified oocytes) was similar to that of control oocytes. Furthermore, after IVM, GV-vitrified oocytes had better spindle and F-actin integrity than oocytes vitrified at the MII stage (MII-vitrified oocytes). In accordance with this result, GV-vitrified oocytes had better ability to extrude the second polar body and support male pronucleus formation after in vitro fertilization (IVF), in comparison to MII-vitrified oocytes. Fertilization rates did not differ among groups. Finally, the ability of GV-vitrified oocytes to develop into embryos was superior to that of MII-vitrified oocytes. However, both vitrified groups showed reduced blastocyst development compared with the control group. In conclusion vitrification of porcine oocytes at the GV stage is advantageous in conferring better cytoskeletal organization and competence to develop to the blastocyst stage in comparison with vitrification at the MII stage.     

Three dimensional in vitro culture of preantral follicles following slow-freezing and vitrification of mouse ovarian tissue

To evaluate the effects slow-freezing and vitrification on three dimensional in vitro culture of preantral follicles, ovaries of 12–14 days old female NMRI mice were isolated and randomly assigned to fresh control, slow-freezing and vitrification groups. Slow-freezing was performed using programmable freezer. Vitrification was carried out in a medium consisting of ethylene glycol (EG) and dimethyl sulphoxide (Me₂SO) by needle immersion method. middle sized preantral follicles were mechanically isolated and cultured for 12 days in 0.7% sodium alginate gel. The follicles development and quantitative expression of oocyte specific genes (Bmp15, Gdf9, Fgf8) and the growth related genes (Igf1, Kit, Kit-l) were assessed after 1, 8 and 12 days of culture. Both cryopreserved groups showed reduction of follicular survival rates compared to the control group on days 8 and 12 of culture (P < 0.05). Antrum formation rates reduced in slow-freezing after 12 days of culture (P < 0.05). Evaluation of gene expression showed reduction of Bmp15, Gdf9, Fgf8, Kit and Kit-l during 12 days of culture (P < 0.05). Kit and Kit-l expression in slow-freezing group significantly reduced on day 8 of culture (p < 0.05). Igf1 expression was lower in slow-freezing group on 1st day of culture than vitrification and control groups (P < 0.05). Finally, intergroup comparison showed same expression pattern of genes after 12 days of culture. Thus, cryopreservation of mouse ovaries by both methods can preserve most developmental parameters and expression of maturation genes. However, vitrification is a better method for cryopreservation of mouse ovaries due to greater antrum formation and expression of growth related markers.     

Developmental competence of domestic cat oocytes from ovaries stored at various durations at 4 °C temperature

Temporal storage of ovaries can provide opportunity to rescue oocytes from ovaries of endangered felids. The objective of the study was to examine the effect of different storage periods (2, 24 and 48 h) of ovaries at 4 °C for maturation of cat oocytes in vitro. Ovaries were collected from 25 domestic cats at various stages of the estrous cycle by routine ovariohysterectomy following anesthesia at different local veterinary clinics, and maintained in physiological saline at 4 °C for 2, 24 or 48 h until oocytes recovery. Selected COCs were maturated at 38 °C for 48 h in four-well petri dishes, which included 500 μL modified synthetic oviduct fluid (mSOF) medium under mineral oil in a humidified 5% CO₂, 5% O₂, and 90% N₂ atmosphere incubator. After the in vitro maturation period, there were no differences between the rate of oocytes matured at MII stages in 2 and 24 h storage groups (50.7% and 48.2% respectively, p > 0.05). However, the same result for the 48 h group was significantly lower than the 2 and 24 h groups (28.0%, p < 0.001).Our results suggest that while 2 or 24 h storage of ovaries at 4 °C does not affect the meiotic competence of oocytes in vitro, 48 h storage of ovaries decrease the results dramatically.     

Factors affecting the survival,fertilization, and embryonic development of mouse oocytes after vitrification using glass capillaries

Cryopreservation of mammalian oocytes is an important way to provide a steady source of materials for research and practice of parthenogenetic activation, in vitro fertilization, and nuclear transfer. However, oocytes cryopreservation has not been common used, as there still are some problems waiting to be solved on the repeatability, safety, and validity. Then, it is necessary to investigate the damage occurred from vitrification and find a way to avoid or repair it. In this study, mouse mature oocytes were firstly pretreated in different equilibrium media, such as 5% ethylene glycol (EG) + 5% dimethyl sulfoxide (DMSO), 10% EG + 10% DMSO, and 15% EG + 15% DMSO in TCM199 supplemented with 20% fetal calf serum (FCS), for 1, 3, and 5 min, respectively, and then oocytes were transferred into vitrification solution (20% EG, 20% DMSO, 0.3 M sucrose, and 20% FCS in TCM199, M2, Dulbecco’s phosphate buffered saline, and 0.9% saline medium, respectively) and immediately loaded into glass capillaries to be plunged into liquid nitrogen. After storage from 1 h to 1 wk, they were diluted in stepwise sucrose solutions. The surviving oocytes were stained for cortical granule, meiotic spindles, and chromosomes. Oocytes without treatments were used as controls. The results showed that oocytes pretreated in 5% EG +5% DMSO group for 3–5 min or in 10% EG + 10% DMSO group for 1–3 min were better than other treatments. Oocytes vitrified in TCM199 as basic medium showed higher survival and better subsequent embryonic development than other groups. When the concentration of FCS in vitrification solution reduced below 15%, the rates of survival, fertilization, and developing to blastocyst declined dramatically. The inner diameter (0.6 mm) of glass capillaries and amount of vitrification solution (1–3 μl) achieved more rapid cooling and warming and so reduce the injury to oocytes. Cropreservation led to the exocytosis of cortical granule of oocytes (about 10%) and serious disturbance of microtubules and chromosomes. With 2 h incubation, the microtubules could repolymerize and the rate of fertilization in vitro was much higher than those of 1 and 3 h incubation groups. In conclusion, the protection of basic medium and FCS to oocytes during cryopreservation and sufficient cooling and warming rates using glass capillaries have profound effects on oocytes survival and subsequent embryonic development competence. The appropriate time for fertilization in vitro may be related to the recovery of spindles after incubation and avoiding ageing in the whole process.     

Effects of vitrification for germinal vesicle and metaphase II oocytes on subsequent centromere cohesion and chromosome aneuploidy in mice

The present study examined the effect of vitrification on oocyte aneuploidy and centromere cohesion. Firstly, germinal vesicle (GV) and in vitro matured oocytes (metaphase II, MII) were vitrified by open-pulled straw method. Secondly, thawed GV oocytes were matured in vitro to detect the aneuploidy rate and the sister inter-kinetochore (iKT) distance (in situ spreading and immunofluorescent staining). The results revealed that the sister iKT distance and the aneuploidy rate in eggs matured from vitrified-thawed GV oocytes were higher than that from in vivo matured, in vitro matured, and in vitro matured frozen oocytes (0.47 ± 0.03 vs. 0.33 ± 0.01 vs. 0.33 ± 0.02 vs. 0.34 ± 0.01 μm; P < 0.01 and 22.9% vs. 6.5% vs. 5.8% vs. 11.8%; P < 0.05, respectively). Furthermore, the percentage of sister chromosome pairs whose sister iKT distances were higher than 0.9 μm in eggs matured from vitrified-thawed GV oocytes (8.7%) was higher than that from in vivo matured (1.6%), in vitro matured (1.6%), and in vitro matured frozen oocytes (2.3%) (P < 0.05). The sister iKT distance was associated with centromere cohesion. To investigate whether vitrification of GV oocytes deteriorated centromere cohesion by affecting cohesin complex formation, thawed and fresh GV oocytes were used to detect the cohesin subunits (SMC1β, STAG3, SMC3, and REC8) mRNA expression (quantitative real-time polymerase chain reaction). The relative expression of three cohesin subunits (SMC1β, STAG3, and SMC3) was significantly decreased in GV oocytes after vitrification. In conclusion, vitrification of GV oocytes may result in the subsequent deterioration of centromere cohesion and an increase in the aneuploidy rate. MII oocytes may be the ideal candidate to avoid aneuploidy for fertility cryopreservation.     

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.     

Viability of zebrafish (Danio rerio) ovarian follicles after vitrification in a metal container

Cryopreservation of ovarian tissue has been studied for female germline preservation of farm animals and endangered mammalian species. However, there are relatively few reports on cryopreservation of fish ovarian tissue and especially using vitrification approach. Previous studies of our group has shown that the use of a metal container for the cryopreservation of bovine ovarian fragments results in good primordial and primary follicle morphological integrity after vitrification. The aim of this study was to assess the viability and in vitro development of zebrafish follicles after vitrification of fragmented or whole ovaries using the same metal container. In Experiment 1, we tested the follicular viability of five developmental stages following vitrification in four vitrification solutions using fluorescein diacetate and propidium iodide fluorescent probes. These results showed that the highest viability rates were obtained with immature follicles (Stage I) and VS1 (1.5 M methanol + 4.5 M propylene glycol). In Experiment 2, we used VS1 to vitrify different types of ovarian tissue (fragments or whole ovaries) in two different carriers (plastic cryotube or metal container). In this experiment, Stage I follicle survival was assessed following vitrification by vital staining after 24 h in vitro culture. Follicular morphology was analyzed by light microscopy after vitrification. Data showed that the immature follicles morphology was well preserved after cryopreservation. Follicular survival rate was higher (P < 0.05) in vitrified fragments, when compared to whole ovaries. There were no significant differences in follicular survival and growth when the two vitrification devices were compared.     

Effects of trehalose co-incubation on in vitro matured prepubertal ovine oocyte vitrification

Our aim was to evaluate if loading prepubertal ovine oocyte with trehalose would impact on their further developmental potential in vitro and if it would improve their survival to vitrification procedures. COCs matured in vitro with (TRH) or without (CTR) 100mM trehalose were tested for developmental potential after in vitro fertilization and culture. Trehalose uptake was measured by the antrone spectrophotometric assay. No differences were recorded between the two experimental groups in fertilization rates (91.1 CTR vs 92.5% TRH), cleavage rates calculated on fertilized oocytes (96.1 CTR vs 95.4% TRH), first cleavage kinetic (56.1 CTR vs 51% TRH), and blastocyst rates (14.3 CTR vs 13.0% TRH). Anthrone assay revealed that in TRH group trehalose concentration/oocyte was 2.6microM. MII oocytes were then vitrified using cryoloops in TCM 199 containing 20% FCS, sucrose 0.5M, 16.5% Me(2)SO, 16.5% EG and plunged in LN(2). After warming, oocytes from TRH and CTR groups were tested for membrane integrity using the propidium iodide (PI)/Hoechst differential staining, and for developmental ability after in vitro fertilization. Trehalose in maturation medium affected membrane resistance (P<0.01) to vitrification/warming but not fertilization and cleavage rates. The differential staining showed a lower number of PI positive cells in TRH group compared to CTR one (14.3 vs 24.7%, respectively). Fertilization rates and cleavage rates did not differ between the two groups (55.3 and 41% for TRH and 47.7 and 41.7% for CTR, respectively). In conclusion trehalose in maturation medium stabilizes cell membranes during vitrification/warming of prepubertal ovine oocytes but does not affect fertilization and cleavage rates after warming.     

Cryopreservation of human failed maturation oocytes shows that vitrification gives superior outcomes to slow cooling

This study investigated whether failed maturation oocytes could be used to evaluate different cryopreservation procedures. A total of 289 failed maturation oocytes (GV and MI stages), obtained from 169 patients undergoing IVF treatment (mean age 33.84 ± 5.0) were divided into two different slow-cooling groups (1.5 mol/l 1,2-propanediol + 0.2 mol/l sucrose in either NaCl (group A) or choline chloride (ChCl) (group B) based cryopreservation solutions) and one vitrification group (15% ethylene glycol + 15% dimethyl sulphoxide). Survival rate, in vitro maturation (IVM) rate, fertilization and developmental rate of cryopreserved oocytes were assessed. Regardless of the stage at which cryopreservation was performed (GV + MI), the slow cooling with ChCl based medium always gave significantly lower survival rate than the slow cooling in NaCl based medium (p = 0.01) and vitrification (p < 0.001). An extended study also showed statistically reduced survival rate between slow-cooling NaCl based medium and vitrification (p < 0.05). Global results of in vitro maturation and fertilization showed worse results between both slow-cooling NaCl and ChCl based media versus vitrification. In conclusion, for oocytes that had failed to mature, vitrification gave better survival, maturation, fertilization and also cleavage rates than the slow-cooling protocols. Four cells embryos were obtained only from vitrified in vitro matured MI oocytes.     

Effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes

The effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes was investigated. Oocytes with compact cumulus cells were cultured for 0, 6, 12 and 24 h in TCM199 supplemented with 5% fetal bovine serum (FBS) in 3% CO2 in air. The oocytes were first exposed to 20% ethylene glycol solution and were subjected to vitrification in a solution containing 40% ethylene glycol, 18% Ficoll-70 and 0.3 M sucrose. After warming in 20 degrees C water, oocytes which had been vitrified at less than 24-h of IVM were again cultured to complete the 24-h of IVM period. Oocytes were then incubated with frozen-thawed spermatozoa in Brackett and Oliphant (BO) medium containing 60 micrograms/ml heparin and 0.25% BSA for 20 h. In vitro fertilization rates of oocytes vitrified-warmed at 0, 6, 12 and 24-h IVM were 75.2, 68.0, 82.0 and 72.4%, respectively, comparable to the rates for unvitrified control oocytes (80.6%). A higher incidence of polyspermic fertilization was observed in oocytes vitrified at 24-h IVM (44.9 vs 22.6% in the control group, P < 0.05). Vitrification of oocytes at 12-h IVM seemed to be better than that of other IVM groups, since the normal fertilization rate of all treated oocytes was the highest (36.0%) among the vitrification groups. Developmental competence of the oocytes following vitrification and in vitro fertilization (12-h IVM group) was examined by cell-free culture of presumptive zygotes up to 9 d in modified synthetic oviduct fluid (mSOF) in 5% CO2, 5% O2 and 90% N2. The cleavage rate of zygotes from vitrified oocytes 48 h after insemination was 29.8%, which was lower than that of the control group (57.0%, P < 0.05). Development to blastocysts from the vitrified oocytes (4.8%) was much lower than that of the control group (27.0%, P < 0.05). These results indicate that cryopreservation of bovine oocytes by vitrification may be affected by their maturation stage in vitro, and that developmental competence to blastocysts of cleaved oocytes following vitrification may be impaired compared with unvitrified control oocytes.     

Immature cat oocyte vitrification in open pulled straws (OPSs) using a cryoprotectant mixture

Cryopreservation of gametes is an important tool in assisted reproduction programs to optimise captive breeding programmes of selected felid species. In this study the vitrification was evaluated in order to cryopreserve the immature domestic cat oocytes by assessing the survival of cumulus-oocyte complexes (COC), and the development competence after IVM and IVF by fresh cat epididymal sperms. From a total of 892 COC obtained from queens after ovariectomy were divided into two groups: Experiment 1 for viability evaluation (150 vitrified and 100 control COC) and Experiment 2 for assessing the developmental competence (414 vitrified and 228 control COC). The viability was evaluated by double staining with carboxyfluorescein and Trypan blue, while the developmental competence was evaluated by in vitro maturation (IVM), in vitro fertilisation (IVF) by fresh epididymal spermatozoa and in vitro culture (IVC). The vitrification was performed in OPS into sucrose medium (1 M sucrose in HSOF + 6% BSA) containing dimethyl sulfoxide (DMSO) (16.5% final concentration) and ethylene glycol (EG) (16.5% final concentration) as cryoprotectants. Percentage of non-viable COC was significantly higher in Experimental 1 vs Control 1 (11% vs 54.5%; P < 0.01), while cleavage rate were significantly lower for vitrified oocytes (Experimental 2) than control 2 (18.6% vs 48.2%; P < 0.01). Blastocyst rate on day 8 was higher for control oocytes than vitrified counterparts (4.3% vs 20.6% P < 0.01). This vitrification protocol ensured a development to blastocyst stage and it is the first report of development of vitrified GV COC.     

Cryopreservation of human failed maturation oocytes shows that vitrification gives superior outcomes to slow cooling

This study investigated whether failed maturation oocytes could be used to evaluate different cryopreservation procedures. A total of 289 failed maturation oocytes (GV and MI stages), obtained from 169 patients undergoing IVF treatment (mean age 33.84 ± 5.0) were divided into two different slow-cooling groups (1.5 mol/l 1,2-propanediol + 0.2 mol/l sucrose in either NaCl (group A) or choline chloride (ChCl) (group B) based cryopreservation solutions) and one vitrification group (15% ethylene glycol + 15% dimethyl sulphoxide). Survival rate, in vitro maturation (IVM) rate, fertilization and developmental rate of cryopreserved oocytes were assessed. Regardless of the stage at which cryopreservation was performed (GV + MI), the slow cooling with ChCl based medium always gave significantly lower survival rate than the slow cooling in NaCl based medium (p = 0.01) and vitrification (p < 0.001). An extended study also showed statistically reduced survival rate between slow-cooling NaCl based medium and vitrification (p < 0.05). Global results of in vitro maturation and fertilization showed worse results between both slow-cooling NaCl and ChCl based media versus vitrification. In conclusion, for oocytes that had failed to mature, vitrification gave better survival, maturation, fertilization and also cleavage rates than the slow-cooling protocols. Four cells embryos were obtained only from vitrified in vitro matured MI oocytes.     

Rescue of vitrified-warmed bovine mature oocytes by short-term recovery culture with resveratrol

Resveratrol, a well-known antioxidant, has been reported to protect mouse metaphase-II (M − II) stage oocytes from vitrification injuries when used as a treatment during a series of vitrification processes. The present study was conducted to investigate whether short-term treatment of post-warm bovine mature oocytes with resveratrol can increase blastocyst formation rate following in vitro fertilization and culture. Bovine denuded M − II oocytes were vitrified-warmed using Cryotop® or nylon mesh (pore size = 37 μm) as a cryodevice. The post-warm oocytes were treated for 2 h with 1 μM resveratrol in recovery culture medium. The resveratrol treatment had no harmful influence on morphological survival and cleavage rate of the oocytes vitrified-warmed with Cryotop® or nylon mesh. In the Cryotop® vitrification series, blastocyst formation rate of resveratrol-treated post-warm oocytes (39.0%) was not significantly different from that of non-treated post-warm oocytes (31.7%). However in the nylon mesh vitrification series, there was a significant increase in the blastocyst yield (42.4% vs. 31.3%, P < 0.05) when post-warm oocytes were treated with resveratrol. Blastocyst yield from fresh control oocytes was 49%. Levels of reactive oxygen species were comparable between post-warm and fresh control M − II oocytes, and decreased in oocytes after recovery culture with resveratrol. Mitochondrial activity of post-warm oocytes was restored to the pre-vitrification level during the recovery culture regardless of resveratrol supplementation. Thus, short-term recovery culture with resveratrol can rescue bovine M − II oocytes vitrified-warmed on a nylon mesh cryodevice.     

Developmental competence of mature yak vitrified–warmed oocytes is enhanced by IGF-I via modulation of CIRP during in vitro maturation

The objective of this study was to investigate whether developmental competence of mature vitrified–warmed yak (Bos grunniens) oocytes can be enhanced by supplemented insulin-like growth factor I (IGF-1) during in vitro maturation (IVM), and its relationship with the expression of cold-inducible RNA-binding protein (CIRP). In experiment 1, immature yak oocytes were divided into four groups, and IVM supplemented with 0, 50, 100 and 200 ng/mL IGF-1 was evaluated; the mRNA and protein expression levels of CIRP in mature oocytes in the four groups were evaluated using quantitative real-time PCR and western blotting analyses. In experiment 2, the mature yak oocytes in the four groups were cryopreserved using the Cryotop (CT) method, followed by chemical activation and in vitro culture for two days and eight days to determine cleavage, blastocyst rates, and total cell number in the blastocysts. Mature yak oocytes without vitrification served as a control group. The outcomes were as following: (1) the expression of CIRP in the matured oocytes was up-regulated in the IGF-1 groups and was highest expression was observed in the 100 ng/mL IGF-1 treatment group. (2) In the vitrified–warmed groups, the rates of cleavage and blastocyst were also highest in the 100 ng/mL IGF-1 treatment group (81.04 ± 1.06%% and 32.16 ± 1.01%), which were close to the rates observed in groups without vitrification (83.25 ± 0.85% and 32.54 ± 0.34%). The rates of cleavage and blastocyst in the other vitrified–warmed groups were 70.92 ± 1.32% and 27.33 ± 1.31% (0 ng/mL); 72.73 ± 0.74% and 29.41 ± 0.84% (50 ng/mL); 72.43 ± 0.61% and 27.61 ± 0.59% (200 ng/mL), respectively. There was no significant difference in the total cell number per blastocysts between the vitrified–warmed groups and group without vitrification. Thus, we conclude that the enhancement in developmental competence of mature yak vitrified–warmed oocytes after the addition of IGF-1 during IVM might result from the regulation of CIRP expression in mature yak oocytes prior to vitrification.     

Generation of Live Piglets from Cryopreserved Oocytes for the First Time Using a Defined System for In Vitro Embryo Production

We report the successful piglet production from cryopreserved oocytes for the first time by using a simple, high capacity vitrification protocol for preservation and a defined system for in vitro embryo production. Immature cumulus-oocyte complexes (COCs) from prepubertal gilts were vitrified in microdrops and stored in liquid nitrogen. After warming, COCs were subjected to in vitro maturation (IVM), fertilization (IVF), and subsequent culture (IVC). Adjusting warmplate temperature to 42°C during warming prevented temperature drops in a medium below 34.0°C and significantly increased the percentage of oocyte survival and thus blastocyst yields obtained from total vitrified oocytes compared with that of warming at 38°C (87.1% vs 66.9% and 4.4% vs 2.7%, respectively). Nuclear maturation and fertilization of oocytes were not affected by vitrification and warming temperature. Blastocyst development on day 7 (day 0 = IVF) of the surviving oocytes after warming at 38°C and 42°C was not different but lower (P<0.05) than those of non-vitrified control oocytes (4.6%, 5.2% and 17.9%, respectively). However, blastocyst cell numbers in the control and vitrified groups were similar irrespective of warming temperature. Omitting porcine follicular fluid (pFF) from IVM medium (POM) did not affect maturation, fertilization and embryo development of vitrified-warmed oocytes. Transfer of blastocysts obtained on day 5 from vitrified oocytes matured either with or without pFF into 4 recipients (2 for each group) resulted in 4 pregnancies and the delivery of a total of 18 piglets. In conclusion, optimization of warming temperature was a key factor for achieving high survival rates, and surviving oocytes could be utilized in vitro using defined media. Using these modifications, live piglets could be obtained from cryopreserved oocytes for the first time.     

The presence of synthetic polymers in the maturation medium affects the cryotolerance and developmental capacity after parthenogenic activation of vitrified goat oocytes

The purpose of this present study is to assess if addition of the synthetic polymers in maturation medium can influence cryotolerance and subsequently embryonic development of mammalian oocytes. We examined the roles of two polymers, including polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP), on in vitro maturation (IVM), embryonic developmental capacity, and cryotolerance of goat oocytes. The present study includes two parts. At first, goat cumulus−oocyte complexes (COCs) were matured in a medium supplemented with 10% fetal bovine serum (FBS), 3 mg/ml PVP, or 1 mg/ml PVA, respectively. Data of oocyte with first polar body, cleavage, and blastocyst following parthenogenetic activation (PA) were recorded. Secondly, after maturation in the above medium, oocytes were vitrified using the Cryotop technique and then the morphology, cleavage and blastocyst formation of vitrified oocytes have been checked. The results demonstrated that the adding of PVP or PVA in maturation medium can't affect IVM of goat oocytes in comparison with FBS, as concern cumulus cell expansion, first polar body formation, and embryonic development. Additionally, without plunging into liquid nitrogen, only exposure to the vitrification and warming solutions cannot also influence the quality of oocytes, in terms of morphology, cleavage, and blastocyst formation. However, after IVM with synthetic polymers and vitrification, the ratio of oocytes with standard morphology in PVP or PVA group was only 59.47% ± 3.56% or 54.86% ± 5.19%, respectively, and was significantly less than that in the FBS group (89.37% ± 4.52%, P < 0.05). Furthermore, the cleavage ratio of oocytes in PVP or PVA group was 37.41% ± 4.17% or 27.71% ± 3.91% and was considerably less than that in the FBS group (64.97% ± 4.69%, P < 0.05). In addition, the cleavage ratio in PVP group was statistically higher than that in PVA group (P < 0.05). In terms of blastocyst development, a significant difference was observed between the synthetic polymer group and the FBS group (24.96% ± 3.62%, P < 0.05). However, the blastocyst ratio in the PVA group (7.51% ± 1.68%) was statistically less than the PVP groups (13.20% ± 4.59%, P < 0.05) and the FBS group (P < 0.05). In conclusion, two potential serum replacements, either PVP or PVA, can support IVM and embryonic development of goat oocytes at the concentration used in this study. But IVM with synthetic polymers supplemented to maturation medium may reduce the cryotolerance of oocytes. Additionally, the supportive function of PVP on embryonic development of vitrified oocytes might be better than that of PVA.     

Animal oocyte and embryo cryopreservation

Cryopreservation of oocytes and embryos is a crucial step for the widespread and conservation of animal genetic resources. However, oocytes and early embryos are very sensitive to chilling and cryopreservation and although new advances have been achieved in the past few years the perfect protocol has not yet been established. All oocytes and embryos suffer considerable morphological and functional damage during cryopreservation but the extent of the injury as well as differences in survival and developmental rates may be highly variable depending on the species, developmental stage and origin (for example, in vitro produced or in vivo derived, micromanipulated or not). Currently, there are two methods for gamete and embryos cryopreservation: slow freezing and vitrification. We have experienced both techniques but vitrification has become a viable and promising alternative to traditional approaches especially when dealing with in vitro produced or micromanipulated embryos and oocytes. Recently new strategies based on emerging studies in the field of lipid research have been used to reduce intracellular lipid content in bovine in vitro produced embryos and therefore increase their tolerance to micromanipulation and cryopreservation. The addition of a conjugated isomer of linoleic acid, the trans-10, cis-12 octadecadienoic acid to embryo culture medium more than twice improved embryo post-thawing viability after micromanipulation and vitrification. Vitrification was also used for the cryopreservation of embryos belonging to the Portuguese Animal Germplasm Bank project presently running at our facilities. Presented at the International Consensus Meeting “New Horizons in Cell and Tissue Banking” on May 2007 at Vale de Santarém, Portugal.     

Developmental competence of frozen-thawed yak (Bos grunniens) oocytes followed by in vitro maturation and fertilization

In the present study, we examined the ability of immature germinal vesicle (GV) and subjected to in vitro matured (MII) yak oocytes to survive after cryopreservation as well as their subsequent development following in vitro maturation and fertilization. Both GV and MII oocytes were cryopreserved by using two different vitrification solutions (VS); VS-I contained 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in TCM-199 + 20% (v/v) fetal calf serum (FCS) whereas VS-II contained 40% EG + 18% Ficoll + 0.5 M sucrose in TCM-199 + 20% FCS. The percentage of oocytes found to be morphologically normal was greater (P < 0.01) in VS-I group than in VS-II group. Rates of cleavage (30.6–42.2%) and blastocyst formation (2.9–8.9%) did not differ among groups, but were lower than in unfrozen control (55.7% and 25.4%, P < 0.01). These results show that a combination of EG and DMSO or EG, Ficoll and sucrose can be used to cryopreserve yak oocytes in French straws.     

Developmental competence and gene expression of immature oocytes following liquid helium vitrification in bovine

The objective of this study was to develop an effective ultra-rapid vitrification method and evaluate its effect on maturation, developmental competence and development-related gene expression in bovine immature oocytes. Bovine cumulus oocyte complexes were randomly allocated into three groups: (1) controls, (2) liquid nitrogen vitrification, and (3) liquid helium vitrification. Oocytes were vitrified and then warmed, the percentage of morphologically normal oocytes in liquid helium group (89.0%) was significantly higher (P < 0.05) than that of the liquid nitrogen group (81.1%). When the vitrified–thawed oocytes were matured in vitro for 24 h, the maturation rate in liquid helium group (50.6%) was higher (P < 0.05) than liquid nitrogen group (42.6%). Oocytes of liquid helium vitrification had higher cleavage and blastocyst rates (41.1% and 10.0%) than that of liquid nitrogen vitrification (33.0% and 4.5%; P < 0.05) after in vitro fertilization. Moreover, the expression of GDF9 (growth/differentiation factor-9), BAX (apoptosis factor) and ZAR1 (zygote arrest 1) was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) when the vitrified–thawed oocytes were matured 24 h. The expression of these genes was altered after vitrification. Expression of GDF9 and BAX in the liquid helium vitrification group was not significantly different from that of the control, however there were significant differences between the liquid nitrogen vitrification group and control. In conclusion, it was feasible to use liquid helium for vitrifying bovine immature oocytes. There existed an association between the compromised developmental competence and the altered expression levels of these genes for the vitrified oocytes.