Non-vitrified Control Research Articles

CRIOTOLERANCIA DE EMBRIONES BOVINOS CULTIVADOS INDIVIDUALMENTE EN UN MEDIO SUPLEMENTADO CON L-CARNITINA

<p><strong>Background:</strong> L-carnitine is a lipid metabolism enhancer and a potent antioxidant that prevents oxidative damage and improves cryotolerance of bovine embryos. <strong>Objective:</strong> To determine the effect of L-carnitine during oocyte maturation on developmental competence and cryotolerance of single bovine embryo cultured. <strong>Methodology:</strong> Embryos were produced in vitro using abattoir-derived cumulus-oocyte complexes (COCs). In experiment 1, two individual maturation, fertilization and culture systems were used in 24-well plates with 20 µL drops of medium covered with mineral oil and 96-well plates with 30 µL drops of medium. In experiment 2, oocytes were randomly distributed into two groups and single matured in 96-well plates in medium supplemented or not with 0.6mg/mL L-carnitine. On day 7 post fertilization, blastocysts were vitrified on solid surface in Fiberplug. Non-vitrified blastocysts were used as control. Embryonic survival after devitrification was determined by blastocysts re-expansion and hatching rate at 24 and 48 hours of post-devitrification culture. Total cell number and apoptotic rate by TUNEL-DAPI staining were used as quality and cryotolerance indicator. In both cases, cleavage and blastocyst rates were evaluated at 48 hours and 7 days post fertilization, respectively. <strong>Results:</strong> No significant differences were found for embryonic development between single culture systems. There was no effect of L-carnitine supplementation during maturation on embryo development, but embryo survival had increased (P < 0.05) at 24- and 48-hours post devitrification. <strong>Implications:</strong> Treatment with L-carnitine had increased (P < 0.05) post-thaw re-expansion rates (86.8 ± 3.0 vs 70.0 ± 4.4) and it was similar to non-vitrified control (89.7 ± 2.6). Mean cell number and apoptotic cell index, were similar for all treatment groups. <strong>Conclusion:</strong> L-carnitine supplementation during maturation, does not improve division rate and subsequent development of single cultured embryos, however increases cryotolerance post devitrification.</p>

Vitrification of porcine immature oocytes and zygotes results in different levels of DNA damage which reflects developmental competence to the blastocyst stage.

The present study investigated the effects of vitrification of porcine oocytes either at the immature Germinal Vesicle (GV) stage before in vitro maturation (GV-stage oocytes) or at the pronuclear stage after in vitro maturation and fertilization (zygotes) on DNA integrity in relevance with their subsequent embryo development. Vitrification at the GV stage but not at the pronuclear stage significantly increased the abundance of double-strand breaks (DSBs) in the DNA measured by the relative fluorescence after γH2AX immunostaining. Treatment of GV-stage oocytes with cryoprotectant agents alone had no effect on DSB levels. When oocytes were vitrified at the GV stage and subjected to in vitro maturation and fertilization (Day 0) and embryo culture, significantly increased DSB levels were detected in subsequent cleavage-stage embryos which were associated with low cell numbers on Day 2, the upregulation of the RAD51 gene at the 4-8 cell stage (measured by RT-qPCR) and reduced developmental ability to the blastocyst stage when compared with the non-vitrified control. However, total cell numbers and percentages of apoptotic cells (measured by TUNEL) in resultant blastocysts were not different from those of the non-vitrified control. On the other hand, vitrification of zygotes had no effect on DSB levels and the expression of DNA-repair genes in resultant embryos, and their development did not differ from that of the non-vitrified control. These results indicate that during vitrification GV-stage oocytes are more susceptible to DNA damages than zygotes, which affects their subsequent development to the blastocyst stage.

Vitrification within a nanoliter volume: oocyte and embryo cryopreservation within a 3D photopolymerized device

PurposeVitrification permits long-term banking of oocytes and embryos. It is a technically challenging procedure requiring direct handling and movement of cells between potentially cytotoxic cryoprotectant solutions. Variation in adherence to timing, and ability to trace cells during the procedure, affects survival post-warming. We hypothesized that minimizing direct handling will simplify the procedure and improve traceability. To address this, we present a novel photopolymerized device that houses the sample during vitrification.MethodsThe fabricated device consisted of two components: the Pod and Garage. Single mouse oocytes or embryos were housed in a Pod, with multiple Pods docked into a Garage. The suitability of the device for cryogenic application was assessed by repeated vitrification and warming cycles. Oocytes or early blastocyst-stage embryos were vitrified either using standard practice or within Pods and a Garage and compared to non-vitrified control groups. Post-warming, we assessed survival rate, oocyte developmental potential (fertilization and subsequent development) and metabolism (autofluorescence).ResultsVitrification within the device occurred within ~ 3 nL of cryoprotectant: this volume being ~ 1000-fold lower than standard vitrification. Compared to standard practice, vitrification and warming within our device showed no differences in viability, developmental competency, or metabolism for oocytes and embryos. The device housed the sample during processing, which improved traceability and minimized handling. Interestingly, vitrification-warming itself, altered oocyte and embryo metabolism.ConclusionThe Pod and Garage system minimized the volume of cryoprotectant at vitrification—by ~ 1000-fold—improved traceability and reduced direct handling of the sample. This is a major step in simplifying the procedure.

High survival of bovine mature oocytes after nylon mesh vitrification, as assessed by intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is an alternative technique to in vitro fertilization (IVF) for producing transferable blastocysts, especially in combination with cryopreserved oocytes, when the IVF system does not work sufficiently. The present study was conducted to directly compare the efficacy of producing bovine blastocysts by ICSI and IVF from vitrified-warmed and fresh oocytes. Denuded oocytes with a detectable first polar body were vitrified-warmed using a nylon mesh device. In the non-vitrified control group, blastocyst yields 8 days after IVF and ICSI were 32.0 and 26.8%, respectively. Oocyte vitrification and subsequent IVF resulted in an impaired blastocyst yield (15.0%); however, such a loss of efficacy due to vitrification was not observed in the ICSI group (blastocyst yield, 25.2%). The alignment of cortical granules beneath the oolemma was comparable between the fresh control and vitrified-warmed oocytes. Here, we report the high survival of vitrified-warmed bovine oocytes, as assessed by ICSI.

Solid surface vitrification of goat testicular cell suspension enriched for spermatogonial stem cells

This study reports solid surface vitrification (SSV) of goat testicular cell suspensions (TCS) enriched for spermatogonial stem cells (SSCs). The TCS was isolated from pre-pubertal goat testis by enzymatic digestion, enriched for SSCs by filtration and differential plating, and were vitrified-warmed by SSV. The study showed that SSV could successfully vitrify goat TCS although the percentage of live cells in the vitrified-warmed group was lower (74.8 ± 4.1%) than in non-vitrified control (80.6 ± 6.27%). The vitrified-warmed TCS formed putative SSC colonies upon their in vitro culture, but the colony size of vitrified-warmed cells (24.3 ± 1.8 μm) was smaller than those of non-vitrified warmed cells (58.4 ± 2.5 μm). Mitochondrial activity (0.40 vs. 0.38 A U.), population doubling time (33.45 ± 1.25 h vs. 31.86 ± 1.90 h), and the cell proliferation rate (0.72 ± 0.10 vs. 0.75 ± 0.11 per day) of total cells (including putative SSCs and other somatic cells) did not differ (p > 0.05) between control and SSV vitrified-warmed groups. However, during in vitro culture for 96 h, vitrified-warmed cells showed significantly lower (0.75 vs. 1.33 A U.; p < 0.05) mitochondrial activity than non-vitrified controls. The DCFDA assay showed that ROS activity was significantly (p < 0.05) higher in vitrified-warmed cells (52.8 ± 4.1 A U) than non-vitrified control cells (32.8 ± 2.1 AU). In conclusion, our results suggest that SSC-enriched goat TCS could be successfully cryopreserved by SSV. However, ROS-induced damages to cell cytoplasmic components reduce their cellular proliferation and require further improvement in the protocol. To the best of our knowledge, this study is the first report on the SSV of SSC-enriched goat TCS.

26 Effect of different cryoprotectant concentrations on vitrification of invitro-matured bovine oocytes in paper containers

A great challenge for successful oocyte vitrification is the development of a low-cytotoxic cryoprotectant solution in a safe device allowing ultra-rapid cooling. This study compared different concentrations of cryoprotectants for bovine IVM-oocyte vitrification in a safe paper container device on oocyte survival and cleavage rates. Abattoir ovaries were obtained and cumulus–oocyte complexes (COCs) were recovered by aspirating follicles of 3 to 6mm in diameter. A total of 470 COCs with homogeneous cytoplasm oocytes, surrounded by several layers of cumulus cells were selected, in 5 replicates. Groups of ∼50 COCs were matured in 500µL of semi-defined IVM medium for 22h at 38.8°C in a humidified atmosphere with 5% CO2. After IVM, COCs were allocated to 1 of 3 groups of 20 to 30 COCs, differing only in final concentration of cryoprotectants. A nonvitrified control group (CG) was also tested, totalling 4 groups. Before vitrification, each group was transferred to 500µL of TCM-199 HEPES with 20% fetal bovine serum (FBS) (Base medium, BM) for 5min at 34°C, and COCs were partially denuded by gentle pipetting. Vitrification followed a 3-step protocol at room temperature and groups of 4 to 5 COCs were transferred to BM solution drops containing (1) 5% ethylene glycol (EG) + 5% dimethyl sulfoxide (DMSO) for 30 s; (2) 10% EG + 10% DMSO + 0.25M sucrose for 30 s; and (3) vitrification solution (VS), according to each group: high (HG), 20% EG + 20% DMSO + 0.5M sucrose; medium (MG), 15% EG + 15% DMSO + 0.5M sucrose; or low (LG), 10% EG + 10% DMSO + 0.5M sucrose for 30s. Afterwards, COCs were loaded in &amp;lt;1µL of solution and placed in a homemade paper container device, and immediately plunged in liquid nitrogen. Warming was performed placing the paper container in 3mL of 1M sucrose in BM for 2min. After warming, a 3-step protocol was conducted and COCs were transferred to (1) 500µL of 0.5M sucrose in BM for 2 min; (2) 500µL of 0.25M sucrose for 2 min; (3) 500µL of BM for 2min. Then, COCs from each group were transferred to 250µL of semi-defined IVF medium. Motile sperm were recovered by Percoll washing from one bull and added to IVF medium (Day 0) at final concentration of 106 sperm mL−1 for 18h. At Day 1, all presumptive zygotes were cultured in 25µL of SOF medium with 5% FBS under mineral oil at 38.8°C with 5% CO2 and 5% O2. Normal data were subjected to ANOVA and post hoc Tukey test. Cleavage rate was recorded at Day 2 after IVF. Oocyte survival rate was similar (P&amp;gt;0.05) among vitrified groups (HG, 80%; MG, 86%; LG,87%). Cleavage rate differed (P&amp;lt;0.05) in all vitrified groups compared with control (CG, 82%; HG, 10%; MG, 16%; LG, 16%). Although no difference (P&amp;gt;0.05) was observed among vitrified groups, MG and LG showed a slightly increased oocyte survival and cleavage rates compared with HG. In conclusion, the use of either medium or low concentrations of cryoprotectants may be a less toxic alternative for vitrification of IVM bovine oocytes on paper device. This research was funded by CAPES/COFECUB (#88881.142966/2017-01).

Mitochondrial function, blastocyst development and live foals born after ICSI of immature vitrified/warmed equine oocytes matured with or without melatonin

Oocyte vitrification is considered experimental in the horse with only three live foals reported. The oxidative conditions induced by vitrification could in part explain the poor results and melatonin, a powerful antioxidant, could stimulate ROS metabolization and restore mitochondrial function in these oocytes. Our objective was to determine the oxidative status of vitrified equine oocytes and to analyze the effect of melatonin on mitochondrial-specific ROS (mROS), oocyte maturation, ICSI embryo development and viability. Immature, abattoir-derived oocytes were held for 15 h and vitrified in a final concentration of 20% EG, 20% DMSO and 0.65 M trehalose. In Experiment 1, overall ROS was determined by DCHF-DA; vitrification increased ROS production compared to non-vitrified controls (1.29 ± 0.22 vs 0.74 ± 0.25 a. u.; P = 0.0156). In Experiment 2, mROS was analyzed by MitoSOX™ in vitrified/warmed oocytes matured with (+) or without (−) supplementation of 10−9 M melatonin; mROS decreased in vitrified and non-vitrified oocytes matured in presence of melatonin (P < 0.05). In Experiment 3, we assessed the effect of melatonin supplementation on oocyte maturation, embryo development after ICSI, and viability by pregnancy establishment. Melatonin did not improve oocyte maturation, cleavage or blastocyst rate of non-vitrified oocytes. However, vitrified melatonin (+) oocytes reached similar cleavage (61, 75 and 77%, respectively) and blastocyst rate (15, 29 and 26%, respectively) than non-vitrified, melatonin (+) and (−) oocytes. Vitrified, melatonin (−) oocytes had lower cleavage (46%) and blastocyst rate (9%) compared to non-vitrified groups (P < 0.05), but no significant differences were observed when compared to vitrified melatonin (+). Although the lack of available recipients precluded the transfer of every blastocyst produced in our study, transferred embryos from non-vitrified oocytes resulted in 50 and 83% pregnancy rates while embryos from vitrified oocytes resulted in 17 and 33% pregnancy rates, from melatonin (+) and (−) treatments respectively. Two healthy foals, one colt from melatonin (+) and one filly from melatonin (−) treatment, were born from vitrified/warmed oocytes. Gestation lengths (considering day 0 = day of ICSI) were 338 days for the colt and 329 days for the filly, respectively. Our work showed for the first time that in the horse, as in other species, intracellular reactive oxygen species are increased by the process of vitrification. Melatonin was useful in reducing mitochondrial-related ROS and improving ICSI embryo development, although the lower pregnancy rate in presence of melatonin should be further analyzed in future studies. To our knowledge this is the first report of melatonin supplementation to an in vitro embryo culture system and its use to improve embryo developmental competence of vitrified oocytes following ICSI.

Natural honey acts as a nonpermeating cryoprotectant for promoting bovine oocyte vitrification.

Sugars are commonly supplemented into vitrification solution to dehydrate cells in order to reduce the formation of fatal intracellular ice crystals. Natural honey is a mixture of 25 sugars (mainly fructose and glucose) that have different biological and pharmacological benefits. The present study was designed to determine if honey can be used as a nonpermeating cryoprotectant in vitrification of bovine oocytes. In the first experiment, denuded-MII oocytes were exposed to 0.25, 0.5, 1.0, 1.5 or 2.0 M of honey or sucrose. Natural honey and sucrose caused similar ooplasm dehydration. A significant relationship existed between time and ooplasm volume change (P < 0.05), during dehydration and rehydration phases, in both honey and sucrose solutions. In the second experiment, the immature cumulus-oocyte complexes (COCs) were vitrified in an EG/DMSO-based vitrification solution containing honey (0.5, 1 or 1.5 M) or sucrose (0.5 M) as a gold standard. The vitrified-warmed COCs were matured in vitro and evaluated for nuclear maturation. The maturation (MII) rate was greater in nonvitrified control (81%) than vitrified groups (54%, P < 0.05). In the third experiment, COCs were either remained nonvitrified (control) or vitrified in 1.0 M honey or 0.5 M sucrose, followed by IVM, IVF and IVC (for 9 days). Cleavage rate was greater in control (74%) than in vitrified groups (47%, P < 0.05), without significant difference between sugars. Blastocyst rate was 34, 13 and 3% in control, honey and sucrose groups respectively (P < 0.05). In conclusion, natural honey acted as a nonpermeating cryoprotectant in vitrification solution and improved the embryonic development in vitrified bovine COCs.

Effect of microfluidic sperm sorting on equine ICSI blastocyst rate

Oocyte vitrification is considered experimental in the horse with only three live foals reported. The oxidative conditions induced by vitrification could in part explain the poor results and melatonin, a powerful antioxidant, could stimulate ROS metabolization and restore mitochondrial function in these oocytes. Our objective was to determine the oxidative status of vitrified equine oocytes and to analyze the effect of melatonin on mitochondrial-specific ROS (mROS), oocyte maturation, ICSI embryo development and viability. Immature, abattoir-derived oocytes were held for 15 h and vitrified in a final concentration of 20% EG, 20% DMSO and 0.65 M trehalose. In Experiment 1, overall ROS was determined by DCHF-DA; vitrification increased ROS production compared to non-vitrified controls (1.29 ± 0.22 vs 0.74 ± 0.25 a. u.; P = 0.0156). In Experiment 2, mROS was analyzed by MitoSOX™ in vitrified/warmed oocytes matured with (+) or without (−) supplementation of 10−9 M melatonin; mROS decreased in vitrified and non-vitrified oocytes matured in presence of melatonin (P < 0.05). In Experiment 3, we assessed the effect of melatonin supplementation on oocyte maturation, embryo development after ICSI, and viability by pregnancy establishment. Melatonin did not improve oocyte maturation, cleavage or blastocyst rate of non-vitrified oocytes. However, vitrified melatonin (+) oocytes reached similar cleavage (61, 75 and 77%, respectively) and blastocyst rate (15, 29 and 26%, respectively) than non-vitrified, melatonin (+) and (−) oocytes. Vitrified, melatonin (−) oocytes had lower cleavage (46%) and blastocyst rate (9%) compared to non-vitrified groups (P < 0.05), but no significant differences were observed when compared to vitrified melatonin (+). Although the lack of available recipients precluded the transfer of every blastocyst produced in our study, transferred embryos from non-vitrified oocytes resulted in 50 and 83% pregnancy rates while embryos from vitrified oocytes resulted in 17 and 33% pregnancy rates, from melatonin (+) and (−) treatments respectively. Two healthy foals, one colt from melatonin (+) and one filly from melatonin (−) treatment, were born from vitrified/warmed oocytes. Gestation lengths (considering day 0 = day of ICSI) were 338 days for the colt and 329 days for the filly, respectively. Our work showed for the first time that in the horse, as in other species, intracellular reactive oxygen species are increased by the process of vitrification. Melatonin was useful in reducing mitochondrial-related ROS and improving ICSI embryo development, although the lower pregnancy rate in presence of melatonin should be further analyzed in future studies. To our knowledge this is the first report of melatonin supplementation to an in vitro embryo culture system and its use to improve embryo developmental competence of vitrified oocytes following ICSI.

Effect of ovum pick-up medium on blastocyst rate

In this study, we compared two staining protocols assessing the nuclear chromatin stage of equine oocytes after vitrification using permeable and nonpermeable cryoprotectants. Slaughterhouse-derived oocytes (n = 155) were obtained from a total of 32 mares and in vitro matured in M199 medium for 42 hours at 38.5°C in 5% CO2. In the first experiment, two concentrations of Hoechst 33342 (HO) were tested (10 μg/mL; P1 and 2.5 μg/mL; P2) combined with 50 μg/mL of propidium iodide as staining protocols to evaluate the visibility of matured oocytes (n = 44). In the second experiment, 111 oocytes were evaluated using the staining protocol P2, before (C, control) and after vitrification following a two-step conventional protocol with (15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose; V1) or without (1 M sucrose; V2) using permeable cryoprotectants. Our results showed that P2 provided a higher percentage of oocytes with outstanding visibility of the nuclear chromatin stage (52.17%; P < .05) in comparison with P1 (19.04%). In the second experiment, no cryoprotectant-free vitrified oocytes reached the metaphase II maturation stage. This result was significantly lower (P < .05) than conventional vitrification (15.38%) and both lower in comparison with the nonvitrified control group (42.11%). In conclusion, permeable cryoprotectant-free vitrification of equine oocytes obtained poor results and therefore cannot be considered an alternative to vitrification using permeable cryoprotectants. In addition, a staining protocol with a low concentration of HO is recommended to evaluate the nuclear chromatin stage of equine oocytes after in vitro maturation.

Exogenous inorganic ions, partial dehydration, and high rewarming temperatures improve peach palm (Bactris gasipaes Kunth) embryogenic cluster post-vitrification regrowth

Previous studies on the vitrification of peach palm (Bactris gasipaes Kunth) embryogenic cluster highlighted PVS3 as essential for survival, but toxic nonetheless. The objective of this work was to identify methods to improve embryogenic cluster survival through either modification to PVS3 or other changes to the established protocol. PVS3 reduced to 80% strength was less toxic to non-vitrified controls and showed no significant difference in vitrified embryogenic cluster at any incubation time (60, 120, 180, or 240 min) compared to full strength PVS3. The addition of 0.1 M KCl, MgCl2, MgSO4, or K2H(PO4) to 80% PVS3 increased vitrified embryogenic cluster regrowth. D2O had no significant effect on embryogenic cluster regrowth when used as a solvent for PVS3 compared to H2O. Larger embryogenic cluster showed greater regrowth than smaller ones. Regrowth increased with rising rewarming temperature up to 70 °C, the highest tested temperature. The duration of rewarming between 1, 2, or 3 min had no overall significant effect, suggesting that the most critical events in rewarming occur within the first minute. The most effective strips used for droplet vitrification were made from aluminum or silver, which were significantly more effective than polystyrene and expanded polystyrene. Droplet vitrification was initially more effective than conventional cryovial immersion vitrification at 60 min incubation in PVS3, but not significantly different at 120, 180 or 240 min incubations. Partial dehydration for 1 h in a laminar flow hood significantly increased regrowth, but longer dehydration times decreased regrowth when combines with longer PVS3 incubation times. Inorganic ions in diluted PVS3 reduced damage to embryogenic clusters compared to PVS3. Partial dehydration was an effective pre-treatment. Regrowth improved with rising rewarming temperatures up to the maximum tested.

The Effect of Different Vitrification and Staining Protocols on the Visibility of the Nuclear Maturation Stage of Equine Oocytes

In this study, we compared two staining protocols assessing the nuclear chromatin stage of equine oocytes after vitrification using permeable and nonpermeable cryoprotectants. Slaughterhouse-derived oocytes (n = 155) were obtained from a total of 32 mares and in vitro matured in M199 medium for 42 hours at 38.5°C in 5% CO2. In the first experiment, two concentrations of Hoechst 33342 (HO) were tested (10 μg/mL; P1 and 2.5 μg/mL; P2) combined with 50 μg/mL of propidium iodide as staining protocols to evaluate the visibility of matured oocytes (n = 44). In the second experiment, 111 oocytes were evaluated using the staining protocol P2, before (C, control) and after vitrification following a two-step conventional protocol with (15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose; V1) or without (1 M sucrose; V2) using permeable cryoprotectants. Our results showed that P2 provided a higher percentage of oocytes with outstanding visibility of the nuclear chromatin stage (52.17%; P < .05) in comparison with P1 (19.04%). In the second experiment, no cryoprotectant-free vitrified oocytes reached the metaphase II maturation stage. This result was significantly lower (P < .05) than conventional vitrification (15.38%) and both lower in comparison with the nonvitrified control group (42.11%). In conclusion, permeable cryoprotectant-free vitrification of equine oocytes obtained poor results and therefore cannot be considered an alternative to vitrification using permeable cryoprotectants. In addition, a staining protocol with a low concentration of HO is recommended to evaluate the nuclear chromatin stage of equine oocytes after in vitro maturation.

Addition of cholesterol loaded cyclodextrin prior to GV-phase vitrification improves the quality of mature porcine oocytes in vitro

The purpose of this study was to determine whether the mitochondrial membrane potential, pro-apoptotic gene expression, and ubiquitylation status of zona pellucida proteins (ZP1, ZP2, and ZP3) of vitrified GV-stage mature oocytes could be protected by treatment with cholesterol-loaded methyl-β-cyclodextrin (CLC) prior to vitrification. Porcine GV oocytes were treated with CLC prior to the vitrification process, and the effects on the mitochondrial membrane potential and ZP ubiquitylation status were determined by JC-1 single staining and western blot assays. We found that porcine GV-stage oocytes were treated with CLC at different concentrations (0.5, 5, and 10 mg/mL) prior to vitrification improved in vitro maturation of these oocytes (P < 0.05). The mitochondrial membrane potential of matured oocyte without vitrification or treated with 5 mg/mL CLC vitrification treatment was higher than that of the 0 mg/mL CLC group and other treatment groups (vitrified) (P < 0.05). The expression of Caspase 3, Caspase 8, and Caspase 9 genes in the high concentration CLC treatment groups (5 and 10 mg/mL) was significantly lower than that in the 0 (vitrified) mg/mL CLC group (P < 0.05). ZPs protein and ZP3 protein ubiquitylation were also higher in the non-vitrified controls, 5 and 10 mg/mL CLC-treated oocytes than in the 0 (vitrified) and 0.5 mg/mL vitrified groups (P < 0.05). Whereas the sperm–oocyte binding capacity was improved in the CLC treatment groups (P < 0.05) but the embryonic development rate was not improved. In conclusion, pretreatment with CLC can improve the survival rate and maturation rate of oocytes and protect their mitochondria and zona pellucida of porcine oocytes from cryodamage during the vitrification process.

Cryopreservation of murine testicular Leydig cells by modified solid surface vitrification with supplementation of antioxidants

Reports on the vitrification of somatic cells are scarce. Here, we show that Leydig cells (murine cell line TM3) could be successfully vitrified by both open vitrification [plastic straw (PS) and plastic vials (PV)] and closed ultravitrification [microdrop (MD) and solid surface vitrification (SSV)], after protocol optimization. However, open ultravitrification resulted in better post-warming viability than closed systems of vitrification with highest success obtained in modified SSV (84.8 ± 1.86%; p < 0.05). Leydig cells vitrified-warmed by modified SSV also showed superior (p < 0.05) cell growth, mitochondrial activity and cytoplasmic esterase enzyme activity, than MD, PS and PV, respectively. It was also observed that vitrified-warmed cells had higher level of ROS activity than non-vitrified control cells (41.6 ± 4.0 vs. 16.7 ± 1.0; p < 0.05). Treatment of cells with glutathione (GSH) or 2-mercaptoethanol (2-ME) (0, 10, 50, 100 μM) significantly (p < 0.05) reduced the ROS activity but had no significant (p > 0.05) effect on post-warm viability. Nevertheless, antioxidant-treated cells had improved mitochondrial activity, cytoplasmic esterase activity and cell growth during in vitro culture (p < 0.05). In conclusion, our results suggest that modified SSV offers a viable method for vitrifying single cell suspension of Leydig cells. To the best of our knowledge, this is the first report on cryopreservation of Leydig cells by vitrification.

Faster, cheaper, defined and efficient vitrification for immature porcine oocytes through modification of exposure time, macromolecule source and temperature

Vitrification reduces the developmental competence of porcine immature oocytes. We investigated the effects of modifying various factors on the viability and development of oocytes after vitrification. These factors included: 1) exposure to the vitrification solution, 2) macromolecule addition (bovine serum albumin (BSA) or polyvinyl pyrrolidone (PVP)), 3) treatment with cytochalasin B, 4) equilibration temperature, and 5) vitrification method (microdrop or Cryotop). Oocytes were equilibrated and vitrified using medium containing ethylene glycol and propylene glycol. After warming, oocytes were subjected to in vitro maturation, stimulated parthenogenetically, and cultured in vitro. Survival rate, nuclear maturation, cleavage, development to the blastocyst stage and their quality were compared between the vitrified groups and the non-vitrified control group. It was found that 1) exposure to the vitrification solution for longer than 30 s was detrimental to embryo development; 2) replacement of BSA with PVP improved embryo development; 3) cytochalasin B treatment reduced the survival rates, but did not affect the blastocyst development rates, 4) equilibration at room temperature (25 °C) was the most beneficial, and 5) the microdrop method improved survival rates. With these adjustments, we were able to establish a simplified and defined cryopreservation system for porcine immature oocytes with improved efficacy.

Developmental Potential and Apoptosis Incidence of In Vitro Produced Buffalo Embryos Vitrified by Solid Surface Technique

Under the present study the post-thawed developmental potential and apoptotic incidence of in vitro-produced (IVP) buffalo embryos vitrified by solid surface technique were assessed. The abattoir derived oocytes were in vitro matured in maturation media and fertilized with capacitated epididymal sperm. The fertilized embryos were cultured in commercial Research vitro cleave media for in vitro embryo development. Embryos at morula stages were vitrified using vitrification media composed of 35% Ethylene glycol. The vitrified morulae were thawed and allowed to grow in culture media similar to non-vitrified control embryos up to the blastocyst stage. The post-thawed developmental competence of vitrified embryos was recorded to be significantly lower in terms of compact morula (57±1.22% vs 81±1.87%), blastocyst (32±1.22% vs 58±1.22%) and blastomere count (129.0±1.22 vs 159.2±1.31) as compared to non-vitrified control. Furthermore, Annexin-V and PI staining dye were used for staining the embryos for apoptotic study under confocal microscope. The apoptotic study of embryos observed by confocal microscopy revealed that rate of apoptosis was significantly higher in vitrified group as compared to control (53.33±2.13% vs 26.67±3.43%). Our study concluded that the vitrification increased the rate of apoptosis which significantly affected the development potential of vitrified-thawed embryos.

Effect of antioxidant on in-vitro maturation of vitrified bovine oocytes

The present study was undertaken to investigate the effect of -tocopherol on in-vitro maturation of vitrified bovine oocytes. The cumulus-oocyte-complexes (COCs) recovered from follicles (3–5 mm diameter) by aspiration and slicing techniques were equally divided into 5 (five) groups consisting 66 COCs in each. The vitrified bovine oocytes were sub-grouped into control and treatment group. The vitrified- and non-vitrified- control group of oocytes were in-vitro matured in TCM-199 media without supplementation of vitamin E. In three vitrified treatment groups, the oocytes were in-vitro cultured in TCM-199 media supplemented with vitamin E (-tocopherol) @ 50 μM, 100 μM and 200 μM, respectively. The mean percentage of cumulus cell expansion and polar body formation in non-vitrified control and vitrified control groups were 85.18±2.57 and 65.38±1.83, and 51.67±1.94 and 30.26±0.16, respectively. The rate of cumulus cell expansion and polar body formation in the oocytes of three vitrified treatment groups media supplemented with vitamin E @ 50 μM, 100 μM and 200 μM were 56.39±3.49 and 34.62±1.83, 69.95±3.20 and 56.23±1.61, and 54.44±4.73 and 31.62±4.50% respectively. Mean percentage of both cumulus cell expansion and polar body formation in the non-vitrified control group were significantly higher than that in the vitrified control group. In the treatment group supplemented with vitamin E @ 100 μM, both cumulus cells expansion and polar body formation were significantly higher than that in media supplemented with 50 μM and 200 μM vitamin E as well as in vitrified control group.

Vitrification, not cryoprotectant exposure, alters the expression of developmentally important genes in in vitro produced porcine blastocysts

The vitrification of embryos is common practice in advanced livestock breeding programs and in human fertility clinics. Recent studies have revealed that vitrification results in aberrant expression of a number of stress related genes. However, few studies have examined the effect that vitrification has on developmentally important genes, and none have been conducted in porcine embryos. The aim of this study was to determine the effects that different vitrification procedures and cryoprotectant combinations have on the expression of imprinted genes in in vitro produced (IVP) porcine blastocysts. The transcript levels of insulin-like growth factor 2 (IGF2) were lower in all groups of vitrified blastocysts compared to that in non-vitrified control blastocysts (P < 0.05). Expression levels of IGF2 and IGF2 receptor (IGF2R) in blastocysts that had been exposed to cryoprotectants without being vitrified were similar to that in non-vitrified control blastocysts (P > 0.05). Furthermore, blastocysts vitrified using ethylene glycol and propanediol combined, and those vitrified in a closed device, had IGF2R transcript levels similar to that in non-vitrified control blastocysts (P > 0.05). In conclusion, vitrification, but not exposure to cryoprotectants, caused aberrant expression of the imprinted genes IGF2 and IGF2R. Vitrification protocols that incorporated propanediol or a closed device were found to be least disruptive of gene expression in IVP porcine blastocysts.

An improved vitrification protocol for equine immature oocytes, resulting in a first live foal.

The success rate for vitrification of immature equine oocytes is low. Although vitrified-warmed oocytes are able to mature, further embryonic development appears to be compromised. The aim of this study was to compare two vitrification protocols, and to examine the effect of the number of layers of cumulus cells surrounding the oocyte during vitrification of immature equine oocytes. Experimental invitro and invivo trials. Immature equine oocytes were vitrified after a short exposure to high concentrations of cryoprotective agents (CPAs), or a long exposure to lower concentrations of CPAs. In Experiment 1, the maturation of oocytes surrounded by multiple layers of cumulus cells (CC oocytes) and oocytes surrounded by only corona radiata (CR oocytes) was investigated. In Experiment 2, spindle configuration was determined for CR oocytes vitrified using the two vitrification protocols. In Experiment 3, further embryonic development was studied after fertilisation and culture. Embryo transfer was performed in a standard manner. Similar nuclear maturation rates were observed for CR oocytes vitrified using the long exposure and nonvitrified controls. Furthermore, a lower maturation rate was obtained for CC oocytes vitrified with the short exposure compared to control CR oocytes (P=0.001). Both vitrification protocols resulted in significantly higher rates of aberrant spindle configuration than the control groups (P<0.05). Blastocyst development only occurred in CR oocytes vitrified using the short vitrification protocol, and even though blastocyst rates were significantly lower than in the control group (P<0.001), transfer of five embryos resulted in one healthy foal. The relatively low number of equine oocytes and embryo transfer procedures performed. For vitrification of immature equine oocytes, the use of 1) CR oocytes, 2) a high concentration of CPAs, and 3) a short exposure time may be key factors for maintaining developmental competence.

Embryonic survival, development and cryoinjury of repeatedly vitrified mouse preimplantation embryos

ObjectiveThe aim of this study is to investigate the embryonic survival, development, and expressions of cryoinjury- or antioxidant-related genes in once, twice, or three-time vitrified mouse preimplantation embryos. Study desisgn: Six hundred 8-cell stage embryos were obtained from 60 female mice and randomly assigned to control and three experimental groups. Embryos were vitrified by indirect methods The developmental outcomes such as survival rate, blastocyst-forming rate, and the percentage of hatching/hatched blastocyst were assessed. The cell numbers of hatching/hatched blastocyst were counted after nuclear staining. From hatching/hatched blastocysts, the mRNA expressions for Cirbp, Casp3, Sod1, Gpx3, and Cat were quantified by real-time quantitative RT-PCR. ResultsIn once, twice, or three-time vitrified mouse 8-cell stage embryos, survival rates, blastocyst-forming rates, the percentages of hatching/hatched blastocyst, and the cell counts were all similar when compared with non-vitrified control group. The mRNA expression levels of Cirbp, Casp3, Sod1, Gpx3 and Cat were not affected. ConclusionRepeatedly vitrified mouse 8-cell stage embryos well developed up to blastocyst stage without cryoinjury and without decrease of antioxidant-related genes.