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.     

Vitrification devices affect structural and molecular status of in vitro matured ovine oocytes

We evaluated the effect of three different cryodevices on membrane integrity, tubulin polymerization, maturation promoting factor (MPF) activity and developmental competence of in vitro matured (IVM) ovine oocytes. IVM oocytes were exposed during 3 min to 7.5% DMSO and 7.5% ethylene glycol (EG) in TCM199 and 25 sec to 0.5 M sucrose, 16.5% DMSO and 16.5% EG, loaded in open pulled straws (OPS), cryoloops (CL) or cryotops (CT) and immersed into liquid nitrogen. Untreated (CTR) or exposed to vitrification solutions but not cryopreserved (EXP) oocytes were used as controls. After warming, double fluorescent staining evidenced a lower membrane integrity in vitrified groups compared to the controls (P < 0.01). After in vitro fertilization and culture OPS and CL groups evidenced a lower cleavage rate than CT and controls (P < 0.01) while blastocysts were obtained only in CL and EXP, at a lower rate than CTR (P < 0.01). All vitrified groups showed alterations in spindle conformation, which were partially recovered in OPS and CT groups. MPF activity was lower in treated compared to CTR and CT showed the lowest value (P < 0.01). After 2 hr culture MPF activity was restored in all groups except CT. Parthenogenetic activation was higher in treated compared to CTR and CT evidenced the highest value. Our results indicate that cryodevice influences not only the ability to survive cryopreservation but is also associated with molecular alterations which affect developmental competence.     

In vitro fertilization of ovine oocytes vitrified by solid surface vitrification at germinal vesicle stage

Cryopreservation of immature oocytes at germinal vesicle (GV) stage would provide a readily available source of oocytes for use in research and allow experiments to be performed irrespective of seasonality or other constraints. This study was designed to evaluate the recovery, viability, maturation status, fertilization events and subsequent development of ovine oocytes vitrified at GV stage using solid surface vitrification (SSV). Cumulus oocyte complexes (COCs) obtained from mature ewes were randomly divided into three groups (1) SSV (oocytes were vitrified using SSV), (2) EXP (oocytes were exposed to vitrification and warming solutions without vitrification) or (3) Untreated (control). Following vitrification and warming, viable oocytes were matured in vitro for 24h. After that, nuclear maturation was evaluated using orcein staining. Matured oocytes were fertilized and cultured in vitro for 7days. Following SSV, 75.7% 143/189 oocytes were recovered. Of those oocytes recovered 74.8%, 107/143 were morphologically normal (viable). Frequencies of in vitro maturation were significantly (P<0.01) decreased in SSV and EXP groups as compared to control. In vitro fertilization rates were significantly (P<0.01) decreased in SSV (39.3%) group as compared to EXP (56.4%) and control (64.7%) groups. Cleavage at 48h post insemination (pi) and development to the blastocyst stage on day 7 pi were significantly (P<0.001) decreased in SSV oocytes as compared to EXP and control groups. In conclusion, immature ovine oocytes vitrified using SSV as a simple and rapid procedure can survive and subsequently be matured, fertilized and cultured in vitro up to the blastocyst stage, although the frequency of development is low.     

Improved development of ovine matured oocyte following solid surface vitrification (SSV): effect of cumulus cells and cytoskeleton stabilizer

The objective of the present study was to examine the effects of cumulus cells, cytochalasin B (CB), and taxol on the development of ovine matured oocyte following solid surface vitrification (SSV). In experiment 1, effects of cumulus cells during the vitrification were examined. Survival rates after warming were not different between ovine mature oocytes with cumulus cells and without cumulus cells. After in vitro fertilization, rates of embryonic cleavage and development to blastocyst were not different between these two groups. In experiment 2, the effects of cytochalasin B (CB) on vitrification of ovine matured oocytes were examined. The rates of survived ovine matured oocytes were not significantly different among the treatment with 0, 2.5, 5.0, 7.5 and 10.0 microg/mL CB. After in vitro fertilization, the rate of cleavage was not different between the five treatment groups. However, vitrified oocytes treated with 7.5 or 10.0 microg/mL CB resulted in a higher (8.1+/-4.6% and 7.8+/-2.4% respectively, P<0.05) blastocyst development rate than those of oocytes treated with lower CB concentrations. In Experiment 3, the effects of taxol on vitrification of ovine matured oocytes were examined. The rate of survived oocytes was not significantly different among the taxol treatment group with 0, 0.5, 1.0, and 5.0 microM taxol. After in vitro fertilization, the rates of embryos that reached cleavage were not different between the four treatment groups. However, vitrified oocytes treated with 0.5 microM taxol resulted in a higher blastocyst (10.1%+/-6.3, P<0.05) development rate compared to other treatment groups. In conclusion, no effect of cumulus cells on vitrification of ovine matured oocytes was detected in this study. Pretreatment of ovine matured oocytes with cytoskeletal inhibitor cytochalasin B or taxol have a positive effect and helps to reduce the damage induced by vitrification and is a potential way to improve the development of vitrified/warmed ovine matured oocytes.     

Delay on the in vitro kinetic development of prepubertal ovine embryos

In the present study we characterize the developmental potential of prepubertal and adult ovine oocytes, analyzing the developmental speed to two-cell and blastocyst stages and its relationship with hatching from the zona pellucida, development after vitrification and the number and allocation of inner mass and trophoblastic cells. Prepubertal and adult ovine oocytes were matured and fertilized in vitro and first cleavage rates at 22, 26 and 32 h were recorded. Cleaved oocytes were cultured and blastocyst production was assessed at 6-9 days post-fertilization (dpf). Blastocysts from the two sources obtained on different days were divided into two groups: the first was vitrified, warmed and cultured in vitro to evaluate re-expansion of the blastocoelic cavity; blastocysts of the second were cultured separately to allow for hatching and count of trophoblastic and inner mass cells of hatched blastocysts by differential staining. We observed a significantly lower rate (P < 0.01) of cleaved prepubertal oocytes at 22 and 26 h after fertilization while it was higher (P<0.01) at 32 h than in the adult ones. Adult blastocyst production was significantly lower (P < 0.01) in prepubertal than in adult groups and began on the seventh dpf, later (P < 0.01) than in the adult group, where they appeared on the sixth dpf. Prepubertal blastocysts hatched at a lower rate than the adult ones (P < 0.01) and in both experimental groups faster blastocysts showed a higher (P < 0.01) hatching rate. Similarly, prepubertal derived blastocysts showed lower viability after vitrification (P < 0.01) compared to the adult counterparts, and in particular slower embryos had reduced viability after vitrification compared to the fastest (P < 0.01). Cell number was not different between blastocysts of both groups obtained at 6 and 7 dpf, which were higher (P < 0.01) than those obtained at 8 and 9 dpf. The ICM/trophoblast cell ratio was similar in 6- and 7-day obtained blastocyst and increased (P < 0.01) in those obtained 1 or 2 days later. These findings show that differences in kinetic development between prepubertal and adult derived embryos reflect differences in developmental capacity of the oocytes from which they derive and could be indicative of embryo quality.     

Vitrification of pre-pubertal ovine cumulus-oocyte complexes: effect of cytochalasin B pre-treatment

The aim of this study was to evaluate the effect of cytochalasin B (CCB) pre-treatment before vitrification on ability of immature oocytes from lamb ovaries to progress until metaphase II (MII) stage after vitrification/warming procedure. Cumulus-oocyte complexes (COCs) were obtained from ovaries of lambs, from 80 to 90 days old, collected from a local slaughterhouse. Before vitrification, COCs were randomly distributed in two experimental groups corresponding to the incubation with or without 7.5 microg/ml CCB for 30 min. In order to study cryoprotectant and CCB pre-treatment toxicity (toxicity test), oocytes were exposed to cryoprotectants, with or without CCB pre-treatment, but without plunging into N2 liquid. Vitrification solution was composed by 4.48 M EG plus 3.50 M DMSO supplemented with 0.25 M sucrose. Two-step addition was performed. After vitrification or toxicity test, COCs were matured in bicarbonate-buffered TCM 199 containing 10% foetal calf serum and 10 ng/ml epidermal growth factor. A sample of COCs was directly in vitro matured (control group). Rates of MII oocytes of toxicity groups both, with or without CCB pre-treatment were lower than control group (41.1-50.0 versus 79.9, respectively; P<0.05). After vitrification, a lower number of oocytes progressed to MII stage in comparison with non-vitrification groups (P<0.05). In vitrified groups both with or without CCB pre-treatment 8.0 and 12.7%, respectively, of immature oocytes reached MII stage by the end of in vitro maturation culture. No effect of CCB was observed, either in the toxicity or vitrified groups. In conclusion, no effect of CCB pre-treatment before vitrification was detected in this study with immature oocytes of pre-pubertal sheep. More studies are needed in order to increase ovine oocyte survival after vitrification.     

Effect of macromolecules in solutions for vitrification of mature bovine oocytes

This study was designed to evaluate vitrification procedures for in vitro matured bovine oocytes for efficient blastocyst production after warming, IVF and culture. A second goal was to replace serum as the macromolecular component of the vitrification solution, without compromising efficacy. The first experiment compared two containers, open pulled straws (OPS) versus cryoloops, and two vitrification protocols: short equilibration (H-TCM-199+10% EG+10% DMSO+20% FCS for 30s, followed by H-TCM-199+20% EG+20% DMSO+20% FCS+0.48M galactose for 20s) versus long equilibration (H-TCM-199+3% EG+20% FCS for 10min, followed by H-TCM-199+31% EG+20% FCS+1M galactose for 20s). Subsequent experiments used only cryoloops and the short equilibration protocol to evaluate the effect of replacing FCS with defined macromolecules (BSA, Ficoll, PVP, and PVA) in vitrification solutions. Cryoloops were superior to OPS for vitrification of oocytes as determined by blastocyst production (P<0.05). The short and long vitrification protocols gave similar results. The presence of macromolecules in vitrification solutions for bovine oocytes was necessary for acceptable post-warming developmental capacity; 20% FCS, 1% and 2% BSA, 6% and 18% Ficoll, 6% and 20% PVP, 1% PVA, and the combinations of 18% Ficoll+1% BSA, and 6% PVP+1% BSA provided similar protection during vitrification of oocytes; development ranged from 14.8% to 23.0% blastocysts/oocyte, which was not different (P>0.05) from non-vitrified controls (26.9-34.0% blastocysts/oocyte). Too much (6%) and too little (0.3%) BSA, and 0.3% PVA for vitrification resulted in lower blastocyst production (P<0.05) relative to unvitrified oocytes.     

Pig oocyte vitrification by cryotop method: effects on viability, spindle and chromosome configuration and in vitro fertilization

Three experiments were designed to evaluate the effects of vitrification using Cryotop method on MII porcine oocyte viability, chromosomes configuration, meiotic spindle morphology and in vitro fertilization; to do this, in vitro matured oocytes were subjected to the cryoprotectant treatment excluding the plunging into liquid nitrogen, the whole vitrification/warming/rehydration procedure or no treatment (control). In experiment 1 viable oocytes were not reduced by either cryoprotectants or vitrification when they were evaluated immediately after warming and cryoprotectant dilution. However, after a 2 h incubation, the survival rate significantly decreased (P<0.05). In experiment 2 cryoprotectant exposure significantly (P<0.05) influenced spindle morphology even if chromosome organization did not vary, while vitrification significantly (P<0.05) increased oocytes with damaged spindles and chromosomes displaced from the metaphase plate. No significant improvements in these parameters were observed after 2 h of incubation but, on the contrary, the rate of oocytes with normal chromosome configuration was reduced. In experiment 3 significant differences among the three groups in the fertilization rate but not in the percentages of monospermy fertilization were recorded; in addition, exposure to cryoprotectants and vitrification significantly (P<0.05) increased degenerated oocyte rate. Overall, these findings confirm that porcine oocytes at MII stage are very sensitive to vitrification, which reduces the rate of viable oocytes and alters microtubule organization, thus impairing fertilization; in addition, incubation of oocytes for 2 h after devitrification seems to be detrimental rather than ameliorative. Further improvements of the current protocol will be necessary in order to optimize the Cryotop method for vitrifying pig matured oocytes.     

Developmental capacity of Antarctic minke whale ( Balaenoptera bonaerensis ) vitrified oocytes following in vitro maturation, and parthenogenetic activation or intracytoplasmic sperm injection

The present study investigated the effects of the sexual maturity of oocyte donors on in vitro maturation (IVM) and the parthenogenetic developmental capacity of fresh minke whale oocytes. The effects of cytochalasin B (CB) pretreatment and two types of cryoprotectant solutions (ethylene glycol (EG) or ethylene glycol and dimethylsulfoxide (EG + DMSO)) on the in vitro maturation of vitrified immature whale oocytes were compared, and the developmental capacity of vitrified immature whale oocytes following IVM and intracytoplasmic sperm injection examined (ICSI). The maturation rate did not differ significantly with sexual maturity (adult, 60.9%; prepubertal, 53.1%), but the parthenogenetic activation rate of oocytes from adult donors (76.7%) was significantly higher (p < 0.05) than that of oocytes from prepubertal donors (46.4%). The maturation rates after vitrification and warming were not significantly different between the EG (22.2%) and EG + DMSO groups (30.2%), or between the CB-treated (30.4%) and non-CB-treated groups (27.3%). These results indicate that parthenogenetic activation of in vitro matured oocytes from adult minke whales was superior to that from prepubertal whales, but that the developmental capacity of the whale oocytes after parthenogenetic activation or ICSI was still low. The present study also showed that CB treatment before vitrification and two kinds of cryoprotectants did not improve the IVM rate following the vitrification of immature whale oocytes.     

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.     

Embryo development and structural analysis of in vitro matured bovine oocytes vitrified in flexipet denuding pipettes

The aim of this study was to compare the effectiveness of two different vitrification carrier systems for oocyte cryopreservation. In vitro matured (IVM) bovine oocytes were vitrified in open pulled straws (OPS) or flexipet denuding pipettes (FDP), and the effects of cryopreservation determined on the cytoskeletal components and developmental capacity of the oocytes. Three experimental groups were established according to whether the oocytes were vitrified in OPS (OPS group), FDP (FDP group) or left untreated (CTR group). Twenty two hours after the onset of maturation, sub-groups of 2–4 oocytes were pre-equilibrated in 1 mL of Hepes-TCM 199 with 20% fetal calf serum (FCS) (HM), 10% dimethyl sulfoxide (DMSO) and 10% ethylene glycol (EG) for 30 s. The oocytes were then transferred to a 20-μL drop of HM containing 20% DMSO, 20% EG and 0.5 M of sucrose, which was used to load the OPS or FDP before their immersion in liquid nitrogen (LN₂). Oocytes were thawed by plunging the OPS or FDP into 0.25 M sucrose in HM, and then placed for 5 min each in 0.15 and 0 M sucrose in HM. After warming, spindle configuration, chromosome distribution and embryo development were assessed. Frozen–thawed semen was used for fertilization. Zygotes were denuded at 22 h post-insemination, and cultured in SOF medium for 9 days at 38.5 °C in a 5% CO₂, 5% O₂ and 90% N₂ atmosphere. All experiments were performed using both cow and calf oocytes to establish sensitivity differences. After in vitro fertilization and culture, oocytes in the FDP group showed a lower cleavage rate than those in the OPS or control groups (P < 0.05), while blastocysts were only obtained in the OPS group, at a lower rate than controls. After warming, double fluorescent staining revealed higher rates of spindle and chromosome abnormalities in the FDP group compared to the OPS group (P < 0.05). No differences between cow and calf oocytes were observed in the different experiments. Our results indicate that the carrier system affects the capacity of IVM oocytes to survive cryopreservation. Unexpectedly, the flexipet denuding pipette failed to improve results and high rates of clustered chromatin and abnormal spindles were observed in calf and cow oocytes vitrified by the FDP method. In conclusion, the use of the flexipet denuding pipette modifies the cytoskeletal components and compromises the developmental capacity of in vitro matured calf and cow oocytes.     

Stability of the cytoskeleton of matured buffalo oocytes pretreated with cytochalasin B prior to vitrification

Stabilizing the cytoskeleton system during vitrification can improve the post-thaw survival and development of vitrified oocytes. The cytoskeleton stabilizer cytochalasin B (CB) has been used in cryopreservation to improve the developmental competence of vitrified oocytes. To assess the effect of pretreating matured buffalo oocytes with CB before vitrification, we applied 0, 4, 8, or 12 μg/mL CB for 30 min. The optimum concentration of CB treatment (8 μg/mL for 30 min) was then used to evaluate the distribution of microtubules and microfilaments, the expression of the cytoskeleton proteins actin and tubulin, and the developmental potential of matured oocytes that were vitrified-warmed by the Cryotop method. Western blotting demonstrated that vitrification significantly decreased tubulin expression, but that the decrease was attenuated for oocytes pretreated with 8 μg/mL CB before vitrification. After warming and intracytoplasmic sperm injection, oocytes that were pretreated with 8 μg/mL CB before vitrification yielded significantly higher 8-cell and blastocyst rates than those that were vitrified without CB pretreatment. The values for the vitrified groups in all experiments were significantly lower (P < 0.01) than those of the control groups. In conclusion, pretreatment with 8 μg/mL CB for 30 min significantly improves the cytoskeletal structure, expression of tubulin, and development capacity of vitrified matured buffalo oocytes.     

Vitrification solution containing DMSO and EG can induce parthenogenetic activation of in vitro matured ovine oocytes and decrease sperm penetration

This study was designed to examine the reduced incidence of normal fertilization in vitrified ovine oocytes. After in vitro maturation for 24 h, the oocytes were randomly allocated into three groups: (1) untreated (control), (2) exposed to vitrification solution (VS) without being plunged into liquid nitrogen (toxicity), or (3) vitrified by open-pulled straw method (vitrification). In experiment 1, the treated and control oocytes were matured for another 2 h, and the oocytes were then in vitro fertilized for 12 h to examine sperm penetration. The percentage of monospermy in toxicity group (29.3%) and vitrification group (28.2%) dramatically decreased compared to the control group (45.0%) (P<0.05). To find the mechanism that the VS decreased the monospermy, some treated and control oocytes were used to test the distribution of CG and the resistance of zona pellucida (ZP) to 0.1% pronase E immediately (IVM 24 h), after another 2 h of maturation (IVM 26 h), and after 12 h of in vitro fertilization (IVF 12 h) respectively. Others were used to examine female pronucleus formation after 12 h of culture in fertilization medium with the absence of sperm. The results showed that the percentage of CG completely release in the oocytes (IVM 24 and 26 h) of toxicity group (41.2% and 39.9%) and vitrification group (41.7% and 51.7%) was significantly higher than that of control group (7.1% and 18.4%) (P<0.05). The ZP digestion duration in the oocytes (IVM 26 h) of the toxicity group (435.6 s) and vitrification group (422.3 s) was longer than that of control group (381.6 s) (P<0.05). The percentage of female pronucleus formation in toxicity group (58.7%) and vitrification group (63.9%) was higher than that (8.2%) of control group (P<0.05). The data above demonstrated that the VS containing DMSO and EG could parthenogenetically activate in vitro matured ovine oocytes, resulting in ZP hardening and decreased sperm penetration.     

Germinal stage vitrification is superior to MII stage vitrification in prepubertal mouse oocytes

This study investigated if in vitro maturation (IVM) before or after vitrification would be more successful for prepubertal oocytes. To mimic prepubertal conditions in an experimental setup, oocytes were collected from healthy 14, 21 and 28day old Swiss albino mice. The germinal vesicle (GV) stage oocytes and in vitro matured MII oocytes were subjected to vitrification-warming. Both structural (meiotic spindle morphology, mitochondrial integrity, cortical granules) and functional (sperm zona binding, fertilization) characteristics were assessed in oocytes after warming. This study demonstrated that IVM was more detrimental to prepubertal oocytes than to young adults. Further, vitrification of the IVM oocytes resulted in an increase in the number of abnormal meiotic spindles, a change in the cortical distribution pattern, a reduction in sperm zona binding and the fertilization rate. Importantly, oocyte integrity was better when prepubertal oocytes were vitrified before, rather than after, IVM. The above observations support GV stage vitrification for prepubertal oocytes requiring fertility preservation. Understanding the mechanisms behind the differing outcomes for oocytes from immature females will help in refining current protocol, thereby retaining the oocytes' maximum structural and functional integrity Further investigation is necessary to determine whether human prepubertal oocytes also behave in a similar way. It is to be noted here, with great emphasis, that a major limitation of this study is that the oocytes’ abilities were tested only until fertilisation, as a consequence of which the study cannot reveal the developmental potentials of the embryos beyond fertilisation.     

Effects of cryodevice type and donors' sexual maturity on vitrification of minke whale (Balaenoptera bonaerensis) oocytes at germinal vesicle stage

Germinal-vesicle-stage oocytes enclosed with compact cumulus cell layers (COCs) were recovered from adult or prepubertal minke whale ovaries, and were vitrified in a solution containing 15% ethylene glycol, 15% DMSO and 0.5 M sucrose using either a Cryotop or an open-pulled straw (OPS) as the cryodevice. The post-warm COCs with normal morphology were cultured for 40 h in a 390 mosmol in vitro maturation medium, and oocytes extruding the first polar body were considered to be matured. The proportion of morphologically normal COCs after vitrification and warming was higher when the COCs were cryopreserved by Cryotop (adult origin, 88.4%; prepubertal origin, 80.8%) compared with the OPS (adult origin, 67.7%; prepubertal origin, 64.2%). The oocyte maturation rate was higher in the adult/Cryotop group (29.1%) compared with those of the prepubertal/Cryotop group (14.4%), the adult/OPS group (14.3%) and the prepubertal/OPS group (10.6%). These results indicate that the Cryotop is a better device than the OPS for vitrification of immature oocytes from adult minke whales.     

Viability, maturation and embryo development in vitro of immature porcine and ovine oocytes vitrified in different devices

This study was designed to evaluate the effects of vitrification on immature porcine and ovine oocytes, collected at a slaughterhouse, by performing vitrification in devices with different volumes. Viability was evaluated both before and after vitrification and maturation. Immediately after warming, the percentage of viable pig oocytes was 81% regardless the type of device, while in the control (after oocyte selection) was 95%. The viability of matured pig oocytes after warming, vitrified in beveled edge open straws (BES) was 6%, in small-open-pulled-straw (SOPS) was 17% and in cryotop was 4%, while the viability of the control group was 86%. The viability and maturation results were similar with all devices. Embryo development (ED) was observed in fresh porcine oocytes with 15% 2-8 cell embryos, 7% morulae and 3% blastocysts, and non-embryo cleavage was observed in warmed oocytes. The viability of sheep oocytes immediately after warming averaged 90% in all devices, while that of the control (after oocyte selection) averaged 95%. The viability of warmed oocytes after maturation was: BES 21%, SOPS 30%, cryotop 21% and control group 86%; while maturation values were 11, 21, 34 and 70%, respectively. After vitrification, the highest ED was achieved with ovine oocytes vitrified in SOPS, with 17% morulae development and it was the only device in which blastocysts developed. A direct relationship was observed between viability and actin filament integrity in both species.     

Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on spindle and chromosome configuration and embryo development

Two experiments were designed to assess the effectiveness of cryopreserving bovine MII oocytes using cryotops as the carrier system for vitrification. In the first experiment, we examined the developmental competence of oocytes after: (i) vitrification in open-pulled straws (OPS method); or (ii) vitrification in <0.1 μl medium droplet on the surface of a specially constructed fine polypropylene strip attached to a plastic handle (Cryotop method). In the second experiment, warmed oocytes that had been vitrified in OPS or cryotops were fixed to analyze spindle and chromosome configuration. In all experiments both cow and calf oocytes were used. Significantly different fertilization rates were observed between the vitrification groups: 31.5% and 20.2% for the cow and calf oocytes vitrified in OPS, respectively, versus 46.1% and 46.4% for the oocytes vitrified using cryotops. After in vitro fertilization, 3.8% of the calf oocytes and 5.3% of the cow oocytes developed to the blastocyst stage. All blastocysts from vitrified oocytes resulted from the Cryotop method. A significantly lower percentage of the OPS-vitrified calf oocytes showed a normal spindle configuration (37.8%) compared to control fresh oocytes (69.9%), while normal spindle and chromosome configurations were observed in a significantly higher proportion of the cryotop-vitrified calf oocytes (60.2%). For the cow oocytes, 60.6% in the OPS group and 60.3% in the Cryotop group exhibited a normal morphology after warming. These findings suggest the cryotop system is a more efficient carrier for vitrification than OPS for the cryopreservation of bovine oocytes.     

Calcium concentration in vitrification medium affects the developmental competence of in vitro matured ovine oocytes

The present study was designed to determine whether different calcium concentrations in the vitrification solutions could improve the developmental competence of in vitro matured ovine oocytes after cryopreservation. In vitro matured oocytes were vitrified with 16.5% ethylene glycol (EG) + 16.5% dimethylsulfoxide (DMSO) vitrification media. The base media contain different calcium concentrations, so that five experimental groups were obtained: TCM/FCS (TCM 199 + 20% fetal calf serum (FCS), [Ca²⁺] 9.9 mg/dl); PBS/FCS (Dulbecco Phosphate Buffered Saline (PBS) + 20% FCS, [Ca²⁺] 4.4 mg/dl); PBS^{CaMg free}/FCS (PBS without Ca²⁺ and Mg²⁺ + 20% FCS [Ca²⁺] 2.2 mg/dl); PBS/BSA (PBS + 0.4% bovine serum albumin (BSA), [Ca²⁺] 3.2 mg/dl) and PBS^{CaMg free}/BSA (PBS without Ca²⁺ and Mg²⁺ +0.4% BSA, [Ca²⁺] 0.4 mg/dl). After warming, the oocytes from the five experimental groups were assessed for survival, spontaneous parthenogenetic activation and developmental capacity via in vitro fertilization. Oocyte survival after vitrification procedures was better preserved in group PBS^{CaMg free}/FCS compared to the others (P < 0.05). In addition, a positive correlation was found between calcium concentration in vitrification solutions and spontaneous parthenogenetic activation (correlation index 0,82; P < 0.001). Development of vitrified oocytes was significantly affected by vitrification media composition (P < 0.01). In particular, oocytes from group PBS^{CaMg free}/FCS led to higher cleavage rates and blastocyst rate compared to the others. Our data showed that lowering calcium concentration in the vitrification medium improves the blastocyst rate of vitrified ovine oocytes, probably reducing the effect of EG + DMSO during vitrification. On the contrary, the replacement of FCS with BSA dramatically reduces the developmental potential of these oocytes.     

Defined media for vitrification,warming, and rehydration: effects on post-thaw protein synthesis and viability of in vitro derived ovine embryos

The purpose of this study was to assess the viability (rates of re-expanding and hatching in vitro), of in vitro derived ovine blastocysts using vitrification and warming/rehydration media containing fetal calf serum (20% FCS) or polyvinyl alcohol (0.1% PVA), and the incorporation of labelled methionine in protein synthesised during the first 4h after cryopreservation. In experiment 1, after 60 h culture in TCM-199 supplemented with 10% FCS, the hatching rates of blastocysts that had been vitrified, warmed, and rehydrated in media containing only PVA (p/p) were significantly (P<0.05) lower than those vitrified in medium containing PVA with warming and rehydration in medium containing FCS (p/s). Blastocysts that were vitrified in medium containing FCS and warmed and rehydrated in medium with PVA (s/p) had hatching rates that were significantly lower (P<0.01) than those vitrified, warmed, and rehydrated in media with only FCS (s/s). After warming, the number of dead cells in the p/p group was significantly (P<0.05) lower than in all other groups. In experiment 2, the [35S]methionine uptake by embryonic cells of the s/p group was significantly (P<0.01) higher than in other groups. The incorporation of labelled methionine into newly synthesised proteins was significantly lower in the p/p group (P<0.01) than in all other groups. No differences in the newly synthesised proteins were observed between groups. In conclusion, these results suggest that it is possible to replace serum with defined macromolecules in vitrification and warming/rehydration media for in vitro derived ovine blastocysts but this leads to a decrease in viability and a reduction in protein synthesis after warming.     

Microtubule and microfilament organization in immature, in vitro matured and in vitro fertilized prepubertal goat oocytes

The aim of our study was to analyse the cytoskeletal organization of prepubertal goat oocytes. Microtubule and microfilament organization during in vitro maturation of prepubertal and adult goat oocytes and presumptive zygotes of in vitro matured-in vitro fertilized (IVM-IVF) prepubertal goat oocytes were analysed. Oocytes were matured in M-199 with hormones and serum and inseminated with frozen-thawed sermatozoa. Oocytes and presumptive zygotes were treated with anti-alpha-tubulin antibody and fluorescein isothiocyanate (FITC)-labelled goat anti-mouse antibody to stain the microtubules. Microfilaments were localized by means of phalloidin 5 microg/ml conjugated with fluorescein isothiocyanate (FITC-phalloidin). DNA was stained with propidium iodide. Stained oocytes were observed under a confocal laser scanning microscope. At the germinal vesicle nuclear stage, microfilaments were distributed at the cortex of the oocytes. After in vitro maturation, 91.7% of metaphase II (MII) oocytes from adult goats displayed microfilaments in the cortex and within the polar body and were characterized by the presence of a microfilament thickening at the cortical region over the meiotic spindle. In prepubertal goat MII oocytes only 5.7% of oocytes displayed microfilaments at the cortex and within the polar body. After insemination, most of the zygotes displayed microfilaments distributed at the cortex. An undefined microtubular network was observed in adult and prepubertal goat oocytes at the germinal vesicle stage. After in vitro maturation, 100% of MII oocytes from adult goats displayed microtubules on the meiotic spindle and within the polar body. This pattern of distribution was observed in 71.6% of prepubertal goat oocytes. Undefined microtubule networks were present in most of the zygotes analysed. In conclusion, cytoskeletal differences were found between prepubertal and adult goat MII oocytes. Furthermore, most of the zygotes from IVM-IVF prepubertal goat oocytes displayed cytoskeletal anomalies.