Open Pulled Straw Research Articles

Vitrification of pig embryos dysregulates the microRNA transcriptome profile

This study examined how the vitrification of pig blastocysts using either the superfine open pulled straw (SOPS) or Cryotop method affects the expression profile of embryonic microRNA (miRNA) transcriptomes, as well as its relation to changes in the expression of target genes (TGs). Surgically collected pig blastocysts were vitrified using either the SOPS method (n = 60; 4–6 embryos/device) or the Cryotop system (n = 60; 20 embryos/device). Embryos were cultured in vitro for 24 h after warming. Fresh blastocysts (n = 60) cultured for 24 h served as controls. After in vitro culture, five pools of eight viable blastocysts from each group were prepared for miRNA expression analysis based on a microarray approach. Then, biological interpretation of miRNAs profiles and integrative analysis of miRNA and mRNA transcriptome data were performed. Survival after 24 h of in vitro culture was similar (>96 %) for both the vitrification systems and the control group (100 %). Compared with the controls, the SOPS-vitrified blastocysts had 94 (one upregulated and 93 downregulated) differentially expressed (DE) miRNAs, and the Cryotop-vitrified blastocysts had 174 DE miRNAs (one upregulated and 173 downregulated). One DE miRNA (miR-503) in the SOPS group and three DE miRNAs (miR-7139–3p, miR-214 and miR-885–3p) in the Cryotop group were annotated for Sus scrofa. The integrative analysis showed that 27 and 61 DE TGs were regulated by the DE miRNAs in blastocysts vitrified with the SOPS and Cryotop systems, respectively. The TGs enriched one pathway (the TGF-β signaling pathway) for the SOPS system and four pathways (HIF-1, Notch, ascorbate and aldarate metabolism and glycosphingolipid biosynthesis-ganglio series) for the Cryotop system. In summary, vitrification via the SOPS and Cryotop systems dysregulates miRNAs, with slight differences between methods. The altered miRNAs identified in this study were related mainly to cell proliferation, apoptosis, and the response to cell stress. Further studies are needed to clarify the consequences of dysregulation of miRNAs involved in the TGF-β (SOPS-vitrified blastocyst) and Notch (Cryotop-vitrified blastocyst) signaling pathways, particularly if they can affect embryonic development.

Effects of different cryopreservation methods on canine isolated preantral follicles

The aim of the present study was to compare the survival and developmental rate of canine isolated preantral follicles (PAFs) after cryopreservation with different methods (closed vs open vitrification). Follicles were isolated from ovaries randomly divided into three groups: fresh control, OPS (open pulled straw) vitrified and cryotube (CT) vitrified. Post-thaw viability of follicles and oocytes was assessed. Fresh and vitrified/thawed PAFs were cultured in 20 µl drops of FSH-supplemented medium for 10 days. Follicular growth, survival rate, estradiol production and ovulation rate were examined. CT method resulted in lower rate of live cells (58.7%) and oocytes (38.8%) than that of fresh ones (83.6% and 64%, respectively) and OPS (80.3% and 79.3%, respectively). Survival rate was similar to fresh follicles in OPS group (98.5% and 95.4%, respectively), while CT decreased the survival to 81.2%. Fresh follicles showed continuous growth, while CT follicles stopped to increase their size after 2 day. In the OPS vitrified follicles, this halting occurred between Day5 and Day10. Fresh follicles showed the highest estradiol production (range: 26.9 – 266.2 pg/ml). Comparing the two vitrified groups, lower estradiol concentration range was measured in the CT group (7.8–48.7 pg/ml vs. 15.4–89.6 pg/ml). Ovulation rate in each group was lowest in the OPS group (1.7% vs 7% and 8.9% in fesh and CT, respectively). Our data show that OPS vitrification provides superior survival rate, in vitro growth and hormonal production to CT. To our knowledge, these are the first results on comparing different cryopreservation protocols on canine isolated preantral follicles.

Effects of L-carnitine and cryodevices on the vitrification and developmental competence of invitro fertilized buffalo oocytes

In the current, study the effect of the addition of L-carnitine (LC) in in vitro maturation (IVM) medium for buffalo oocytes and different cryo-devices on developmental competence. They were matured in IVM medium supplemented with 0.0, 0.3, 0.6 and 1.2 mg/mL of LC and vitrified by using either straw (ST), open pulled-straw (OPS), solid surface vitrification (SSV). The effects of LC during invitro fertilization and invitro culture on the developmental potential were examined. ST showed a higher recovery rate when using 0.6 mg/ml; viability rate (VR) when using 0.3mg/ml and lower zona pellucida (ZP) and cytoplasmic abnormalities when using 1.2 mg/ml LC (P<0.05). OPS showed a higher recovery and viability rates when using 0.3 mg (P<0.05). SSD showed a higher recovery and VR when using 0.6 mg/ml LC (P<0.05). Maturation and fertilization rates were increased when using 0.3 mg LC and OPS (P<0.05). Cleavage was increased in 0.3 (OPS) and 0.6 mg LC (SSD). The blastocyst rate was increased in 0.3 (OPS) and 0.6 mg/ml (SSD) (P< 0.05). In conclusion, 0.3 mg/ml LC and OPS gives a higher recovery and viability rates but 0.6 mg/ml LC and of both SSD and ST gives a higher recovery and viability rates.

Recent Advancements in Vitrification Cryodevices for Gamete and Gonadal Tissue

Cryopreservation of gametes and gonadal tissue is nowadays primarily accomplished through vitrification. Variables such as cooling rate, viscosity and volume of vitrification solution are critical in gamete vitrification. In addition, sample size and stepwise exposure are also crucial for gonadal tissue vitrification. Recently a class of cryodevices has been developed to reduce the volume of vitrification solution so as to achieve higher cooling rates. Vitrification devices are classified as "open" or "closed" depending on whether the medium comes into direct contact with liquid nitrogen during the process. Examples of the open cryodevices for gamete vitrification are Cryotop, Cryolock, open pulled straw (OPS), etc., and closed devices are Vitrisafe, CryoTip, and high security vitrification kit. Similarly, for tissue vitrification open cryodevices used are needles, cryovials and closed devices used are Cryotissue, ovarian tissue cryosystem, etc. Among all the gamete cryodevices, Cryotop is unique and the best-selling micro-volume storage device. Use of this device has resulted in the highest number of babies born after embryo or oocyte vitrification. Another novel device, Kitasato vitrification system, is a vitrification solution absorber, which is similar to Cryotop but differs in one way, as it possesses a porous membrane that absorbs extra solution from the gamete. This review provides an update on the recent use of cryodevices for gamete and gonadal tissue vitrification.

Impact of Presence or Absence of Trehalose during Vitrification on Viability and Development of Vitrified/Warmed Immature Dromedary Camel Oocytes

Vitrification of immature oocytes at the germinal vesicle (GV) stage is important to preserve female gametes. The standard formula for vitrification solutions has long been a debate. Herein, we investigated the effect of the presence or absence of trehalose in vitrification solution on viability, in vitro maturation (IVM) rates, and development of vitrified/warmed immature dromedary camel oocytes. Cumulus oocyte complexes (COCs) obtained at slaughter from the ovaries of mature she-camels were randomly allocated into three groups; namely, control group, oocytes were directly subjected to IVM without vitrification, vitrification solution 1 (VS1) group, oocytes were vitrified in a solution composed of 25% ethylene glycol (EG) plus 25% dimethyl sulfoxide (DMSO) + 0.5 M trehalose; and vitrification solution 2 (VS2) group, oocytes were vitrified in a solution composed of 25% EG plus 25% DMSO. Vitrification of COCs was conducted by open pulled straws (OPS) method. Following vitrification and warming, morphologically viable oocytes were matured in vitro for 36 h. COCs were then fertilized and cultured in vitro for 7 days. The percentage of viable oocytes was significantly higher (P 0.05) in VS2 than VS1 group (80.0% vs. 63.3%, respectively). Nuclear maturation, cleavage (48 h post-insemination; pi), and blastocyst rates (7 days pi) were significantly higher (P camel oocytes in trehalose-free solution (VS2) was more advantageous than that in trehalose supplemented media since it did not reduce viability and development.

Melatonin promotes in vitro maturation of vitrified-warmed mouse GV oocytes potentially by modulating MAD2 protein expression of SAC component through MTRs

Previous studies have shown that melatonin (MT) can ameliorate vitrification-inflicted damage in mouse germinal vesicle (GV) oocytes, however, the key mechanistic basis of this improvement still remains poorly understood. This study was conducted to investigate whether MT can improve in vitro developmental potential of vitrified-warmed GV oocytes through its receptors. The fresh oocytes were randomly divided into four groups: untreated (control group, F), vitrified by open-pulled straw method (vitrification group, V), vitrification group with 100 nmol/L MT supplementation (vitrification + MT group, VM), and with 100 nmol/L MT plus 100 nmol/L luzindole administration (vitrification + MT + luzindole group, VML) or with 50 nmol/L ramelteon addition (vitrification + ramelteon group; VR). After warming, oocytes were cultured in vitro, and MT receptors (MTRs), MAD2 (mitotic arrest deficient 2), Securin and CyclinB1 protein levels and spindle morphology were evaluated. The ratio of oocytes developed to the metaphase I (MI) and metaphase II (MII) stages was also assessed. The results showed that after vitrification-warming, the in vitro maturation rate of GV oocytes was significantly lower compared to the control (F) group. Vitrification also significantly impaired the spindle morphology, decreased the protein level of MTRs and Securin, and decreased MAD2 levels in MI oocytes. However, when MT or ramelteon (MTRs agonist) were added (group wise) to warming and maturation media, the maturation rate of GV oocytes was significantly increased, the normal proportion of the spindle morphology increased, and the expression level of MAD2 increased in their resulting MI oocytes compared to the vitrification group. However, following addition of both MT and ramelteon, the maturation rate of GV oocyte showed no significant difference between VML and vitrification groups. The spindle morphology and MAD2 levels in MI oocytes were comparable to the vitrification group but differed significantly from the VM group. Taken together, finding of the present study shows that MT (100 nmol/L) can ameliorate the in vitro maturation of vitrified-warmed mouse GV oocytes, potentially by improving the spindle morphology, modulating MAD2 protein level and promoting the development of MI stage oocytes through MTRs.

Ultrastructural changes in immature ovine cumulus-oocyte complexes vitrified in conventional and open pulled straws

This study aimed to evaluate the effect of different cryodevices [conventional straws (CS) and open pulled straws (OPS)] of vitrification on viability, morphology and ultrastructure of ovine immature oocytes. Ovaries of slaughtered ewes were transported from a local abattoir to our laboratory in 0.9 % normal saline at 25–30 °C. The visible follicles (1–6 mm in diameter) were sliced with a microblade and their cumulus-oocyte complexes (COCs) were released in Dulbecco’s phosphate buffer saline. Upon examination under a stereomicroscope, good quality oocytes (n = 266) were selected and randomly assigned into three groups: 1) Fresh oocytes (control, n = 78); 2) Oocytes vitrified using CS (n = 95) and 3) Oocytes vitrified using OPS (n = 93). The gross morphology of COCs was evaluated under a stereomicroscope whilst their viability was evaluated in Trypan blue stained smear. Moreover, glutaraldehyde-fixed COCs (n = 45) were processed for transmission electron microscopy. It was observed that the proportion of recovered normal ovine oocytes after vitrification in OPS (89.24 %) was significantly higher (P < 0.05) than those vitrified in CS (73.68 %). Meanwhile, the proportion of abnormal oocytes was greater (16.84 %) in CS than OPS (8.60 %). The proportion of viable ooplasm and viable cumulus oocytes was significantly (P < 0.05) higher in the fresh group (88.88 %) than OPS (69.11 %) and CS (54.54 %) groups with significant (P < 0.05) difference between the two vitrification groups. Notably, ultrastructural alterations of vesicles and lipid droplets in fresh oocytes were similar with OPS and lower than CS vitrified-warmed oocytes without significant difference between the two vitrified groups. In conclusion, OPS vitrified-warmed oocytes have less cryoinjuries and higher survivability in comparison with CS vitrified-warmed oocytes, although ovine immature oocytes were more vulnerable to cryopreservation by vitrification.

28 Evaluation invitro of two protocols of vitrification from alpaca (Vicugna pacos) embryos

The reproductive efficiency of South American camelids as the alpaca is low, with a few number of animals having a good genetic characteristic. The transfer of cryopreserved embryos has great potential to disseminate valuable genetic, but the suitable protocol for such cryopreservation still needs to be developed. In this study, two protocols of vitrification of alpaca embryos were tested. Day 6.5 post-mating, embryos (n=66) were recovered from 14 female alpacas through a non-surgical technique and classified according to the characteristics of old world camelids reported by Skidmore et al. 2004 (Reprod. Fertil. Dev. 16, 605–609). Only quality 1 and 2 embryos were used for the study. They were placed together in 50-µL drops of holding medium for 30min and transferred to a 100-µL drop of equilibration solution 1, consisting of 7.5% (v/v) ethylene glycol (EG) + 0.25M sucrose. After 1min, embryos were transferred to equilibration solution 2, consisting of 15% (v/v) EG + 0.5M sucrose. After 2min, embryos were transferred into 2 consecutive drops of vitrification solutions A [SA: 30% (v/v) EG + 1M sucrose] for 20s each, then in 2 other drops of vitrification solution B [SB: 30% (v/v) EG + 3% glycerol + 1M sucrose] for 20s each. Thereafter, embryos were quickly loaded into open pulled straws (OPS) in a volume of 10µL and then plunged into liquid nitrogen. For warming, the OPS were held in air for 5s and subsequently thawed at 37°C for 50s. Straws were emptied into 1mL of prewarmed holding medium solution (HMS1) containing 1M sucrose for wash and the thawed blastocysts were transferred into a second 1mL of prewarmed HMS1. After 5min incubation at 37°C, the blastocysts were transferred into 1mL of warmed Holding medium solution 2 (HMS2) containing 0.5M sucrose maintained at room temperature (∼24°C) for evaluation. Data were analysed by the Chi-squared test. Post-thaw embryo expansion results were 81.3% and 58.8% for SA and SB (P&amp;lt;0.05), respectively. Post-thaw embryo quality (1 and 2) were found at 62.5% and 29.1% with SA and SB, respectively (P&amp;lt;0.05). In conclusion, the vitrification of alpaca embryos with the ethylene glycol:sucrose solution results in better post-thaw outcomes than the ethylene glycol:sucrose:glycerol. Further experiments with embryo transfer are needed. This research was funded by FONDECYT project no. 149-2017.

Birth of a Live Cria After Transfer of a Vitrified-Warmed Alpaca (Vicugna pacos) Preimplantation Embryo.

The alpaca (Vicugna pacos) is an important species for the production of fiber and food. Genetic improvement programs for alpacas have been hindered, however, by the lack of field-practical techniques for artificial insemination and embryo transfer. In particular, successful techniques for the cryopreservation of alpaca preimplantation embryos have not been reported previously. The objective of this study was to develop a field-practical and efficacious technique for cryopreservation of alpaca preimplantation embryos using a modification of a vitrification protocol originally devised for horses and adapted for dromedary camels. Four naturally cycling non-superovulated Huacaya females serving as embryo donors were mated to males of proven fertility. Donors received 30 μg of gonadorelin at the time of breeding, and embryos were non-surgically recovered 7 days after mating. Recovered embryos (n = 4) were placed individually through a series of three vitrification solutions at 20°C (VS1: 1.4 M glycerol; VS2: 1.4 M glycerol + 3.6 M ethylene glycol; VS3: 3.4 M glycerol + 4.6 M ethylene glycol) before loading into an open-pulled straw (OPS) and plunging directly into liquid nitrogen for storage. At warming, each individual embryo was sequentially placed through warming solutions (WS1: 0.5 M galactose at 37°C; WS2: 0.25 M galactose at 20°C), and warmed embryos were incubated at 37°C in 5% CO2 in humidified air for 20–22 h in 1 ml Syngro® holding medium supplemented with 10% (v/v) alpaca serum to perform an initial in vitro assessment of post-warming viability. Embryos whose diameter increased during culture (n = 2) were transferred individually into synchronous recipients, whereas embryos that did not grow (n = 2) were transferred together into a single recipient to perform an in vivo assessment of post-warming viability. Initial pregnancy detection was performed ultrasonographically 29 days post-transfer when fetal heartbeat could be detected, and one of three recipients was pregnant (25% embryo survival rate). On November 13, 2019, the one pregnant recipient delivered what is believed to be the world's first cria produced from a vitrified-warmed alpaca embryo.

The effect of artificial insemination prior to transfer of a limited number of vitrified and warmed porcine embryos by open pulled straw (OPS) method on their survival ability for farrowing.

Embryo transfer (ET) of 20 porcine expanded blastocysts (ExBs) vitrified and warmed (VW) by open pulled straw (OPS) to a recipient allows stable piglet production. The efficiency of artificial insemination (AI) prior to ET of 10 VW ExBs for piglet production was investigated. For one trial, 10-15 VW ExBs from single donor were assigned, 10 were used for ET and the remains were assessed for their in vitro viability. In the non-AI/ET group, 10 were transferred to each of five recipients. As AI/ET group, 10 were transferred to each of five recipients after AI. In AI/non-ET group, only AI was performed to seven gilts. In the non-AI/ET group, the pregnancy rate was 40%, but none of them farrowed. In the AI/ET group, all recipients produced piglets. Four (80.0%) delivered piglets from transferred VW ExBs. The survival rate of VW ExBs to term was 20.0% (10/50). In the AI/non-ET group, six of the seven gilts farrowed. There was no difference in in vitro viability between the non-AI/ET and AI/ ET groups (62.5% and 68.3%, respectively). AI prior to ET can be an appropriate way to maintain pregnancy and assist the development of a low number of VW ExBs to term.

7 Pregnancy from a vitrified-warmed alpaca pre-implantation embryo

The alpaca (Vicugna pacos) is a ruminant livestock species in the South American camelid family. There are more than 9 million South American camelids globally that make important contributions to the livelihoods of rural farmers through conversion of low quality roughages to high quality food and fibre. Reproductive biotechnologies for alpacas are not well developed compared with those for other ruminant livestock species. In particular, embryo cryopreservation technologies are lacking. The objective of this study was to evaluate under field conditions a vitrification protocol originally developed for old world camels that we adapted for use in alpacas. Potential donors were evaluated for follicular development using a 7.5-MHz ultrasound probe. Hembras (sexually mature female alpacas) with ovarian follicles 7-10mm in diameter were behaviour tested to determine sexual receptivity, and receptive females were naturally mated to a proven herd sire. At the time of breeding, non-superovulated donors (n=4) received 30μg gonadorelin. Embryos were nonsurgically collected 7 days after breeding and handled at 20°C. Diameter of harvested embryos (n=4 quality grade 1 hatched expanded blastocysts) was measured using an eyepiece reticle. All recovered embryos were placed individually into 0.5-mL drops of vitrification solution (VS1: 1.4M glycerol) for 5min, 0.5-mL drops of VS2 (1.4 M glycerol + 3.6M ethylene glycol) for 5min, 0.05-mL drops of VS3 (3.4 M glycerol + 4.6M ethylene glycol) for 20s, and 0.05-mL drops of VS3 for 20s while loading into open-pulled straws (OPS). Each OPS was plunged directly into liquid nitrogen for storage for 29 days. At warming, each OPS was submerged into a 1-mL drop of warming solution 1 (WS1: 0.5M galactose) for 1min followed by 1min in WS2 (0.25 M galactose) for 5min before being incubated at 37°C in 5% CO2 in humidified air for 21h in 1mL of Syngro holding medium supplemented with 10% (vol/vol) alpaca serum. Embryos that grew during culture (n=2) were transferred individually into synchronous recipients, and embryos that did not appear to grow (n=2) were transferred together as a pair. Prior to embryo transfer, potential recipients were evaluated ultrasonographically as described previously. Hembras with ovarian follicles 7-10mm in diameter were behaviour tested, and sexually receptive females received 30μg gonadorelin 6 days before embryo transfer. Final selection of recipients (n=3) was based on presence of a corpus luteum and nonreceptive behaviour to a herd sire 24h before transfer. Pregnancy was detected ultrasonographically, and fetal heartbeat was detected 29 days post-transfer in one of the three recipients. Ultrasound at 177 days post-transfer revealed that the pregnancy, generated from a 400μm×375μm vitrified-warmed embryo that had grown in culture, was still ongoing. If this pregnancy results in the birth of a live cria (newborn alpaca), it would represent-to the best of our knowledge-the world's first cria born from a cryopreserved alpaca pre-implantation embryo. It would also demonstrate the potential utility of this protocol under field conditions.

50 Effect of colony-stimulating factor 2 on competence of bovine blastocysts to survive vitrification

Colony-stimulating factor 2 (CSF2) is an important maternal regulator of embryonic development. Treatment of invitro-produced embryos with CSF2 from Days 5 to 7 after insemination increased competence of embryos to establish pregnancy after transfer. One action of CSF2 is to protect embryos from stress. Treatment with CSF2 reduced heat-shocked induced apoptosis in morulae and regulated expression of multiple genes involved in cellular stress responses in the blastocyst. Here we tested whether CSF2 would promote survival of embryos to another stress: cryodamage caused by vitrification. Ovaries were recovered from an abattoir and used to harvest cumulus-oocyte complexes (COCs). Groups of 10 COCs were matured for 20-22h. After maturation, COCs were fertilised with 1×106mL−1 isolate-purified spermatozoa from a pool of frozen-thawed semen from 3 bulls. Fertilization proceeded for 20-22h. Cumulus cells were removed from putative zygotes using 1000UmL−1 hyaluronidase by agitation and putative zygotes were cultured in groups of 30 in 50-µL drops of synthetic oviducal fluid-bovine embryo 2 (SOF-BE2) medium until Day 7 after insemination. Drops of cultured embryos were randomly assigned to receive either 10ngmL−1 recombinant bovine CSF2 or vehicle [90% (vol/vol) SOF-BE2 and 10% (vol/vol) Dulbecco's phosphate-buffered saline containing 1mgmL−1 bovine serum albumin) on Day 5. Blastocyst-stage embryos were selected for vitrification at Day 7. Blastocysts were vitrified in open-pulled straws and rewarmed according to procedures of Vajta et al. (1996 Theriogenology 45, 683-689). After warming, embryos were cultured for 72h in groups of 5-12 in SOF-BE2 containing 10% fetal bovine serum and 50 µM dithiothreitol. Blastocysts were assessed for re-expansion (defined as restoration of a reconstituted blastocoele) and hatching (defined as partial or completion exit from the zona pellucida). A total of 11 replicates were performed using 1745 zygotes treated with CSF2 and 1690 zygotes treated with vehicle. The number of blastocysts vitrified were 280 for CSF2 and 289 for vehicle. Data on percent presumptive zygotes becoming blastocysts and on blastocyst re-expansion and hatching after thawing were calculated for each replicate and analysed by analysis of variance using the GLIMMIX procedure of SAS (SAS Institute Inc.). The percentage of putative zygotes that became blastocysts was not affected by treatment (21.5±1% for CSF2 vs. 20.9±1% for vehicle). Overall, CSF2 increased (P=0.021) the percentage of blastocysts that re-expanded (77.8 + 0.05% vs. 73.3 + 0.05%), but there was no effect of CSF2 on the percentage of blastocysts that underwent hatching (39.3 + 0.06% vs. 37.4 + 0.06%). In conclusion, CSF2 increased blastocyst survival following vitrification. Further work will be performed to evaluate whether CSF2 also increases survival of vitrified embryos after embryo transfer. Support was provided by NIH R01 HD088352.

Melatonin Improves In Vitro Development of Vitrified-Warmed Mouse Germinal Vesicle Oocytes Potentially via Modulation of Spindle Assembly Checkpoint-Related Genes.

The present study aimed to investigate the effect of melatonin (MT) supplementation on in vitro maturation of vitrified mouse germinal vesicle (GV) oocytes. The fresh oocytes were randomly divided into three groups: untreated (control), or vitrified by open-pulled straw method without (vitrification group) or with MT supplementation (vitrification + MT group). After warming, oocytes were cultured in vitro, then the reactive oxygen species (ROS) and glutathione (GSH) levels, mitochondrial membrane potential, ATP levels, spindle morphology, mRNA expression of spindle assembly checkpoint (SAC)-related genes (Mps1, BubR1, Mad1, Mad2), and their subsequent developmental potential in vitro were evaluated. The results showed that vitrification/warming procedures significantly decreased the percentage of GV oocytes developed to metaphase II (MII) stage, the mitochondrial membrane potential, ATP content, and GSH levels, remarkably increased the ROS levels, and significantly impaired the spindle morphology. The expressions of SAC-related genes were also altered in vitrified oocytes. However, when 10−7 mol/L MT was administered during the whole length of the experiment, the percentage of GV oocytes matured to MII stage was significantly increased, and the other indicators were also significantly improved and almost recovered to the normal levels relative to the control. Thus, we speculate that MT might regulate the mitochondrial membrane potential, ATP content, ROS, GSH, and expression of SAC-related genes, potentially increasing the in vitro maturation of vitrified-warmed mouse GV oocytes.

Melatonin Improves Parthenogenetic Development of Vitrified⁻Warmed Mouse Oocytes Potentially by Promoting G1/S Cell Cycle Progression.

This study aimed to investigate the effect of melatonin on the cell cycle of parthenogenetic embryos derived from vitrified mouse metaphase II (MII) oocytes. Fresh oocytes were randomly allocated into three groups: untreated (control), or vitrified by the open-pulled straw method without (Vitrification group) or with melatonin (MT) supplementation (Vitrification + MT group). After warming, oocytes were parthenogenetically activated and cultured in vitro, then the percentage of embryos in the G1/S phase, the levels of reactive oxygen species (ROS) and glutathione (GSH), and the mRNA expression of cell cycle-related genes (P53, P21 and E2F1) in zygotes and their subsequent developmental potential in vitro were evaluated. The results showed that the vitrification/warming procedures significantly decreased the frequency of the S phase, markedly increased ROS and GSH levels and the expression of P53 and P21 genes, and decreased E2F1 expression in zygotes at the G1 stage and their subsequent development into 2-cell and blastocyst stage embryos. However, when 10−9 mol/L MT was administered for the whole duration of the experiment, the frequency of the S phase in zygotes was significantly increased, while the other indicators were also significantly improved and almost recovered to the normal levels shown in the control. Thus, MT might promote G1-to-S progression via regulation of ROS, GSH and cell cycle-related genes, potentially increasing the parthenogenetic development ability of vitrified–warmed mouse oocytes.

AANAT transgenic sheep generated via OPS vitrified-microinjected pronuclear embryos and reproduction efficiency of the transgenic offspring.

BackgroundThe open pulled straw (OPS) vitrification method has been successfully applied in mouse, pig, and goat embryos as well as in buffalo oocytes, but it has not yet been applied to the microinjected embryos. This study examined the effects of OPS vitrification on embryo development and the reproductive capacity of the transgenic offspring in order to establish a method for preservation of microinjected embryos.MethodsOvine pronuclear embryos were microinjected with the exogenous aralkylamine N-acetyltransferase gene (AANAT), frozen by the OPS method, and subsequently thawed for embryo transplantation. Pregnancy rate, lambing rate, survival rate, average birth weight and transgenic positive rate as well as reproduction efficiency and hormone level of the transgenic offspring were investigated to analyze the effect of OPS vitrification on microinjectd pronuclear embryos.ResultsNo significant differences were observed in the birth rate, lamb survival rate and transgenic positive rate between the frozen and non-frozen AANAT-microinjected pronuclear embryos. The average birth weight of the frozen embryos offspring was greater than that of the non-frozen embryos. Importantly, the transgenic offspring that overexpressed the AANAT gene showed improved ovulation efficiency and lambing rate by regulating their hormone levels.ConclusionsThe OPS vitrification approach may be a valuable method in microinjected- embryo transfer technology, which could reserve embryos and result in fewer unnecessary animal sacrifices. In addition, the AANAT+ transgenic offspring exhibited improved reproductive capacity on account of regulation effect of melatonin on reproductive hormone. These data may provide available references for human-assisted reproduction.

Technical Note: Transfer of caprine blastocysts vitrified by the open pulled straw (OPS) or the solid surface procedure and warmed in sucrose-free medium

The present paper outlines ways of non-surgical collection and semi-laparoscopic transfer of caprine embryos. Two different ways of embryo cryo-preservation by way of vitrification are described; the open pulled straw (OPS) procedure, known to be well suited and the solid surface procedure called for in situations where contact between embryos and non-sterile liquid nitrogen is to be avoided. Based on 13 transfers of OPS-vitrified and 9 transfers of solid surface-vitrified blastocysts (2 blastocysts/recipient) it was shown that either procedure is applicable (54% vs. 56% pregnancy- and 39% vs. 44% kidding rate). Furthermore the experiment showed that warming of vitrified embryos may be accomplished by one-step procedure (88% transferable post-warming embryos), opening up the possibility to transfer vitrified embryos under field conditions.

Production of blastocysts following in vitro maturation and fertilization of dromedary camel oocytes vitrified at the germinal vesicle stage.

Cryopreservation of oocytes would serve as an alternative to overcome the limited availability of dromedary camel oocytes and facilitate improvements in IVP techniques in this species. Our goal was to develop a protocol for the vitrification of camel oocytes at the germinal vesicle (GV) stage using different cryoprotectant combinations: 20% EG and 20% DMSO (VS1), 25% EG plus 25% DMSO (VS2) or 25% EG and 25% glycerol (VS3) and various cryo-carriers; straws or open pulled-straw (OPS) or solid surface vitrification (SSV); and Cryotop. Viable oocytes were cultured in vitro for 30 h. Matured oocytes were fertilized with epididymal spermatozoa and then cultured in vitro in modified KSOMaa medium for 7 days. Survival and nuclear maturation rates were significantly lower (P ≤ 0.05) in oocytes exposed to VS3 (44.8% and 34.0%, respectively) than those exposed to VS1 (68.2% and 48.0%, respectively) and VS2 (79.3% and 56.9%, respectively). Although recovery rates were significantly lower (P ≤ 0.05) in SSV and Cryotop vitrified oocytes (66.9% to 71.1%) than those vitrified by straws with VS1 or VS2 solutions (86.3% to 91.0%), survival rates were higher in the SSV and Cryotop groups (90.7% to 94.8%) than in the straw and OPS groups (68.2% to 86.5%). Among vitrified groups, maturation and fertilization rates were the highest in the Cryotop-VS2 group (51.8% and 39.2%, respectively). These values were comparable to those seen in the controls (59.2% and 44.6%, respectively). Cleavage (22.5% to 27.9%), morula (13.2% to 14.5%), and blastocyst (6.4% to 8.5%) rates were significantly higher (P ≤ 0.05) in SSV and Cryotop groups than in straws. No significant differences were observed in these parameters between the Cryotop and control groups. We report for the first time that dromedary oocytes vitrified at the GV-stage have the ability to be matured, fertilized and subsequently develop in vitro to produce blastocysts at frequencies comparable to those obtained using fresh oocytes.

Expression of CD9 and CD81 in bovine germinal vesicle oocytes after vitrification followed by in vitro maturation

The present study aimed to investigate the effect of vitrification on the expression of fertilization related genes (CD9 and CD81) and DNA methyl transferases (DNMT1 and DNMT3b) in bovine germinal vesicle (GV) oocytes and their resulting metaphase Ⅱ (MⅡ) stages after in vitro maturation culture. GV oocytes were vitrified using the open-pulled straw method; after warming, they were cultured in vitro. The vitrified-warmed GV oocytes and more developed MII oocytes were used to calculate the maturation rates (first polar body extrusion under a stereomicroscopy), and to detect mRNA expression (qRT-PCR). Fresh GV oocytes and their in vitro-derived MII oocytes served as controls. The results showed that both the maturation rate (54.23% vs. 42.93%) and the relative abundance of CD9 mRNA decreased significantly (p < 0.05) in bovine GV oocytes after vitrification, but the expression of CD81 and DNMT3b increased significantly. After in vitro maturation of vitrified GV oocytes, the resulting MII oocytes showed lower (p < 0.05) mRNA expression of genes (CD9, CD81, DNMT1 and DNMT3b) when compared to the control group (MII oocytes). Altogether, vitrification decreased the maturation rate of bovine GV oocytes and changed the expression of fertilization related genes and DNA methyl transferases during in vitro maturation.

Comparison of The Effects of Vitrification on Gene Expression of Mature Mouse Oocytes Using Cryotop and Open Pulled Straw.

BackgroundOocyte cryopreservation is an essential part of the assisted reproductive technology (ART), which was recently introduced into clinical practice. This study aimed to evaluate the effects of two vitrification systems-Cryotop and Open Pulled Straw (OPS)-on mature oocytes gene expressions.Materials and MethodsIn this experimental study, the survival rate of metaphase II (MII) mouse oocytes were assessed after cryopreservation by vitrification via i. OPS or ii. Cryotop. Then we compared the fertilization rate of oocytes produced via these two methods. In the second experiment, we determined the effects of the two vitrification methods on the expression of Hspa1a, mn-Sod, and ß-actin genes in vitrified-warmed oocytes. Denuded MII oocytes were vitrified in two concentrations of vitrification solution (VS1 and VS2) by Cryotop and straw. We then compared the results using the two vitrification methods with fresh control oocytes.Resultsmn-Sod expression increased in the vitrified-warmed group both in OPS and Cryotop compared with the con- trols. We only detected Hspa1a in VS1 and control groups using Cryotop. The survival rate of the oocytes was 91.2% (VS1) and 89.2% (VS2) in the Cryotop groups (P=0.902) and 85.5% (VS1) and 83.6% (VS2) in the OPS groups (P=0.905). There were no significant differences between the Cryotop and the OPS groups (P=0.927). The survival rate in the Cryotop or the OPS groups was, nevertheless, significantly lower than the control group (P<0.001). The fertilization rates of the oocytes were 39% (VS1) and 34% (VS2) in the Cryotop groups (P=0.902) and 29 %( VS1) and 19.7% (VS2) in the OPS groups (P=0.413). The fertilization rates were achieved without significant differences among the Cryotop and OPS groups (P=0.755).ConclusionOur results indicated that Cryotop vitrification increases both cooling and warming rates, but both Cryo- top and OPS techniques have the same effect on the mouse oocytes after vitrification.

Dynamic changes in the global transcriptome of bovine germinal vesicle oocytes after vitrification followed by in vitro maturation.

This study was conducted to investigate the effect of vitrification on the dynamics of the global transcriptome in bovine germinal vesicle (GV) oocytes and their in vitro-derived metaphase II (MII) oocytes. The GV oocytes were vitrified using the open-pulled straw method. After warming, GV oocytes and the resulting MII-stage oocytes were cultured in vitro for 2h and 24h respectively and were then collected. The fresh GV oocytes and their in vitro-derived MII oocytes were used as controls. Then, each pool (fresh GV, n=3; vitrified GV, n=4; fresh MII, n=1 and MII derived from vitrified GV, n=2) from the different stages was used for mRNA transcriptome sequencing. The results showed that the in vitro maturation rates of GV oocytes were significantly decreased (32.36% vs 53.14%) after vitrification. Bovine GV oocyte vitrification leads to 12 significantly upregulated and 19 downregulated genes. After culturing in vitro, the vitrification-derived MII oocytes showed 47 significantly upregulated and six downregulated genes when compared with those from fresh GV oocytes. Based on molecular function-gene ontology terms analysis and the Kyoto encyclopaedia of genes (KEGG) pathway database, the differentially expressed genes were associated with the pathways of cell differentiation and mitosis, transcription regulation, regulation of actin cytoskeleton, apoptosis and so on, which potentially result in the lower in vitro development of GV bovine oocytes.