Open Pulled Straw Research Articles

Closed pulled straw vitrification of in vitro–produced and in vivo–produced bovine embryos

The objective of this study was to evaluate the efficiency of the closed pulled straw (CPS) method for cryopreserving in vitro–produced and in vivo–produced bovine ( Bos taurus) embryos. Based on the open pulled straw (OPS) protocol, the top end of a CPS was closed by tweezers (heated in a flame) to prevent the cryoprotectant medium containing embryos from contacting the liquid nitrogen. Bovine in vitro or in vivo morulae and early blastocyst embryos were frozen by slow cryopreservation, OPS vitrification, or CPS vitrification. Morphology of postthawed embryos was evaluated, and normal embryos were used for successive culture for 72 h. There were no significant differences between OPS and CPS freezing groups in postthawed in vitro–produced embryos with respect to rates of morphologically normal embryos (mean ± SD, 87.9 ± 5.2% vs. 85.4 ± 4.9%), survival at 24 h (58.0 ± 6.8% vs. 56.3 ± 4.4%), and survival at 72 h (35.2 ± 6.0% vs. 34.9 ± 6.7%). However, both OPS and CPS vitrification resulted in higher postthaw rates of morphologically normal embryo and survival at 24 and 72 h than those of the slow-freezing method (P < 0.05). Similar results were obtained for in vivo–derived embryos. We concluded that CPS vitrification was a feasible method to cryopreserve both in vitro–derived and in vivo–derived bovine embryos. This method not only eliminated the risk of embryo contamination by preventing contact with liquid nitrogen but also retained the advantages of the OPS vitrification method.

Effect of container, vitrification volume and warming solution on cryosurvival of in vitro-produced bovine embryos

The aim of the present research was to develop a low cost and easy to perform vitrification method for in vitro-produced cattle embryos. Effect of container material was evaluated (plastic straw compared to glass capillary, experiment 1), two volume sample (1 compared to 0.5 μL, experiment 2) and warming solution composition medium (Tissue Culture Medium 199 (TCM-199) compared to phosphate buffered saline (PBS), experiment 3) as modifications of the open pulled straw (OPS) system in order to reduce embryo damage caused by exposure to cold. In all experiments, day 7 and expanded blastocysts of cattle were exposed to the vitrification solution 1 for 3 min and 30 s in solution 2. After this, embryos were placed in a droplet and loaded in a narrow end container, and immediately submerged into liquid nitrogen. For warming, vitrified embryos were plunged into warming solution 1 for 3 min, and transferred into warming solution 2 for 1 min. Fresh embryos kept in culture were used as control group. Hatching rates were recorded in all cases at day 13. In experiment 1 there was no significant effect of container material on hatching rates. Postwarming survival rate of vitrified embryos was lower than control (27.5% plastic straws, 18.9% glass capillary and 80.5% control, P < 0.05). In experiment 2, there was no significant effect of volume in hatching rates (58.3% 1 μL, 61.3% 0.5 μL and 80.5% control, P < 0.05). In experiment 3, the composition of the holding medium of warming solution influenced hatching rates (84.1% TCM-199, 74.8% PBS and 91.1% control P < 0.05). These data suggest that neither glass capillaries nor reduced sample volume could improve hatching rates after vitrification–warming with open pulled straw (OPS) procedure, and that PBS can replace TCM-199 in warming solutions, but lesser hatching rates should be expected.

In vitro penetration of swine oocytes by homologous spermatozoa: Distinct systems for gamete's co-incubation and oocyte's cryopreservation

In vitro penetration (IVP) of swine oocytes by homologous spermatozoa was evaluated in two experiments using four boars as semen donors. In experiment 1, the IVP rate and the number of penetrating spermatozoa (PSP) were compared using three co-incubation systems for vitrified oocytes and fresh sperm: (1) 35 mL petri dishes in a CO 2 incubator, (2) 35 mL petri dishes in bags (submarine system) and (3) glass flasks partially submerged in water bath with the same gas mixture used for the bag system. Mean PSP was 8.2 ± 10.1 and the IVP rate was 90.5%. The PSP differed across all systems ( P = 0.0006): 15.5 ± 0.5 for flasks, 6.3 ± 0.4 for CO 2, and 3.9 ± 0.4 for bags. The IVP rate for flasks (95.0%) was greater ( P = 0.01) than for CO 2 and bags (90.8% and 85.0%, respectively), but it did not differ between flasks and CO 2 for three boars ( P > 0.05). In experiment 2, co-incubation was done as described for glass flasks in experiment 1. The IVP rate and PSP were compared for cryopreserved oocytes: either vitrified in open pulled straws (OPS), or frozen in cryotubes. Mean PSP was 5.4 ± 6.5 and IVP rate was 89.6%. Both PSP and IVP rate were greater ( P < 0.0001) for oocytes frozen in cryotubes (7.0 ± 0.3% and 95.8%, respectively) than those frozen in OPS (3.7 ± 0.3% and 83.4%, respectively), with no differences found for three boars ( P > 0.05). In summary, successful IVP of swine oocytes by homologous spermatozoa can be achieved using gametes incubated in glass flasks and oocytes frozen in cryotubes.

Viability of OPS Vitrified Sheep Embryos After Direct Transfer

The aim of this study was to evaluate the viability in the effect of open pulled straw (OPS) vitrification procedure of sheep embryos after direct transference. Embryos were produced in vivo and cryopreserved in slow freezing or OPS vitrification. The survival rates of cryopreserved embryos were compared to non-frozen standard pattern. In a first set of experiments, embryos at morula and blastocyst stages were dived in ethylene glycol (1.5 M) and frozen in an automatic freezer. After being thawed, they were directly or indirectly transferred to ewes recipient. A second group of embryos were drawn into OPS and plunged into liquid nitrogen after being exposed at room temperature for 1 min and 45 s in 10% EG plus 10% dimethyl sulphoxide (DMSO), then again for 30 s in 20% EG + 20% DMSO + 0.5 M sucrose. After being warmed, embryos were also directly transferred using a French mini straw as the catheter for the transplantation process or after in vitro dilution of cryoprotectants (two-step-process). No significant difference was observed among fresh, frozen or vitrified embryos on pregnancy rate (50.0%, 38.6% and 55.8%). However, when we evaluated only the direct transference, the pregnancy rate of OPS vitrified embryos was higher than that of frozen embryos (57.1% vs 34.8%) (p = 0.07). In addition, vitrified morulae had a higher pregnancy rate than the one with frozen embryos (64.0% vs 38.9%) (p = 0.07). Finally, our results indicate that OPS vitrification technique in association with direct transference improves the viability of sheep embryos with potential applications to field conditions.

Fertility rates following the transfer of ovine embryos cryopreserved using three protocols

The aim of this study was to compare the efficiency of three cryopreservation techniques namely, controlled slow freezing, conventional vitrification and open pulled straw (OPS) vitrification, for the cryopreservation of in vivo produced Portuguese Black Merino ovine embryos. Fresh (control group) and cryopreserved embryos (3 methods) were transferred by laparoscopy to recipient ewes (2 embryos/recipient). Embryo survival rates were assessed and pregnancy diagnosis was performed by serum progesterone concentration on day 18 and by ultrasonography on day 45, and at lambing. Successful pregnancy and embryo survival rates did not differ between the fresh (73.3 and 50.0%, respectively) and cryopreserved (58.3 and 38.5%, respectively) embryos. No differences in lambing and embryo survival rates were observed between the three cryopreservation methods used (68.4 and 44.7%; 50.0 and 33.3%; 54.5 and 36.3% for controlled slow freezing, vitrification and OPS, respectively). Pregnancy rate based on serum progesterone concentration on day 18 was higher than the pregnancy diagnosed at 45 days following transfer for the cryopreserved embryos. It could be concluded that all three cryopreservation techniques were equally efficient in preserving and propagating genetic material in Portuguese Black Merino sheep.

OPS vitrification of mouse immature oocytes before or after meiosis: the effect on cumulus cells maintenance and subsequent development

Cryopreservation can cause cumulus cell damage around the immature oocytes, which may result in poor subsequent development. To evaluate the effect of the meiosis stage on the cumulus cell cryoinjury and determine the suitable stage for cryopreservation in immature oocytes, mouse oocytes at germinal vesicle (GV) and germinal vesicle breakdown (GVBD) stages were vitrified using open pulled straw (OPS) method. Cumulus cells damage was scored immediately after thawing by double-fluorescent staining. The survival rate of the oocytes was evaluated and the subsequent development of oocytes was assessed through in vitro culture (IVC) and in vitro fertilization (IVF) separately. After vitrification, a higher proportion of cumulus cells of GV oocytes were damaged than those of GVBD and untreated control groups. The survival rate of vitrified GVBD oocytes (94.1%) was significantly higher (p < 0.05) than that of GV oocytes (85.4%). Oocytes vitrified at GVBD stage (55.7%) showed similar cleavage rate compared to those at GV stage (49.2%), but significantly higher (p < 0.05) blastocyst rate (40.9% vs. 27.4%). These results demonstrate that oocytes at GVBD stage remain better cumulus membrane integrity and developmental ability during vitrification than those at GV stage, indicating they are more suitable for immature oocytes cryopreservation in mice.

Effect of addition of hyaluronan to embryo culture medium on survival of bovine embryos in vitro following vitrification and establishment of pregnancy after transfer to recipients

Two experiments were conducted to determine whether addition of hyaluronan to culture medium could improve survival of bovine embryos after vitrification or following embryo transfer. In Experiment 1, embryos were produced in vitro and cultured for 7 days in modified synthetic oviductal fluid (SOF) containing one of four concentrations of hyaluronan (0, 0.1, 0.5, or 1 mg/mL), with or without 4 mg/mL of bovine serum albumin (BSA). On Day 7 after insemination, blastocysts and expanded blastocysts were vitrified using open-pulled straws. At a concentration of 1 mg/mL, hyaluronan increased ( P < 0.05) the percentage of oocytes that were blastocysts and re-expansion rate at 24 h after warming. At 0.5 mg/mL, hyaluronan tended ( P < 0.10) to increase re-expansion rate at 48 h after warming and increased ( P < 0.05) embryo hatching rate at 24 and 72 h. Treatment with BSA caused a slight reduction in cleavage rate ( P < 0.05), but only for cultures containing hyaluronan (BSA × hyaluronan, P = 0.10), an increase in the percentage of oocytes that became blastocysts ( P < 0.001), and a reduction in re-expansion rates ( P < 0.001) and hatching rates ( P < 0.05 or P < 0.01) at all times examined. In Experiment 2, embryos were produced in vitro and cultured in modified SOF containing 4 mg/mL BSA, with or without 1 mg/mL hyaluronan. At 159–162 h after insemination, grade 1 morula, blastocysts and expanded blastocysts were harvested for embryo transfer. Harvested embryos were transferred individually to lactating Holstein recipients with a palpable corpus luteum on Day 7 after presumptive ovulation. There was an interaction ( P < 0.05) between hyaluronan and embryo stage on pregnancy rate. Recipients that received morula and blastocyst stage embryos treated with hyaluronan had a higher pregnancy rate than recipients that received control embryos of the same stage. There was no effect of hyaluronan on pregnancy rates of recipients that received expanded blastocysts. In conclusion, addition of hyaluronan to embryo culture enhanced blastocyst yield, improved survival following vitrification, and enhanced the post-transfer survival of fresh morula and blastocyst stage embryos.

Cryotolerance of Bovine Blastocysts is Affected by Oocyte Maturation in Media Containing Palmitic or Stearic Acid

In this study, non-esterified fatty acids (NEFAs) were added during in vitro maturation at concentrations measured previously in follicular fluid (FF) of high-producing dairy cows in a negative energy status to evaluate their subsequent effect on the embryos cryotolerance. Oocytes were matured for 24 h in serum-free media with or without (negative control) the addition of NEFAs dissolved in ethanol or ethanol alone (positive control). Matured oocytes were fertilized and cultured for 7 days in synthetic oviduct fluid medium supplemented with 5% FCS. Embryos that had at least reached the blastocyst stage were vitrified by open pulled straw (OPS) vitrification. Addition of palmitic (C16 : 0) or stearic acid (C18 : 0) during oocyte maturation had significant negative effects on embryo cryotolerance, whereas ethanol or oleic acid (C18 : 1) had no effect. These in vitro results suggest that high NEFA concentrations in FF during a period of negative energy balance in high-yielding dairy cows can have carry-over effects on embryo quality.

Vitrification of Small Volume of Normal Human Sperms: Use of Open Pulled Straw Carrier

Vitrification of Small Volume of Normal Human Sperms: Use of Open Pulled Straw Carrier

69 A HOMEMADE N2 SUPERCOOLING DEVICE (NITROCOOLER) ENHANCES VIABILITY AFTER VITRIFICATION OF BOVINE OOCYTES BUT NOT OF IN VITRO-PRODUCED EMBRYOS

Increasing the cooling rate is a common strategy to enhance vitrification efficiency of bovine oocytes and in vitro-produced (IVP) embryos. Under vacuum conditions, liquid N2 (LN2) temperature decreases, increasing the cooling rate during the vitrification procedure. However, commercially available brands of equipment to supercool nitrogen are expensive. The aim of this study was to verify the effectiveness of a low-cost homemade nitrogen supercooling apparatus (Nitrocooler) for vitrification of bovine oocytes and IVP embryos. The device consists of a vacuum pump coupled to a close-tight-lidded flask with a styrofoam cup filled with 300 mL of LN2. After 5 to 7 min of vacuum pumping, LN2 goes through the slushing phenomenon, turning solid. After the lid is opened, the N2 turns liquid again, but in a stable, supercooled physical state lasting for approximately 10 min, boiling off when objects are plunged into it. Nitrocooler was tested for vitrified oocytes (Experiment I), vitrified oocytes in different containers (Experiment II), and embryos in 2 different vitrification solutions (Experiment III). In Experiment I (Ciência Rural, 2006 36, 1501–1506), immature (IM, n = 172) or in vitro-matured (IVM, n = 174) oocytes were exposed to 10% ethylene glycol (EG) + 10% DMSO for 30 s, followed by 20% EG + 20% DMSO + 0.5 m sucrose for 20 s, loaded in open-pulled straws (OPS), and plunged into LN2 or Nitrocooler-pumped LN2. For both IM (8.8%) and IVM (10.6%) oocytes, Nitrocooler-pumped LN2 increased blastocyst rates (Bonferroni, P &lt; 0.05). In Experiment II, 1454 oocytes were vitrified using Nitrocooler after loading in different containers: stainless steel straws (SSS), beveled-tip straws (BTS), open-OPS, and glass-pulled straws (GPS). Greater blastocyst rates were obtained in the SSS (10.2%) and the GPS (8.0%) treatments, both being greater than the BTS (6.1%) and OPS (6.1%) containers (Tukey, P &lt; 0.05). In Experiment III (Acta Sci. Vet. 2006 34, 77–82), 195 IVP bovine blastocysts were vitrified with or without Nitrocooler under 2 different cryoprotectant solutions: 10% EG + 10% DMSO or 10% EG + 10% propylene glycol (PROP). Hatching rates using Nitrocooler for EG + DMSO (50.2%) or EG + PROP (54.0%) were similar (ANOVA, P &gt; 0.05) to normal atmosphere rates using EG + DMSO (50.1%) or EG + PROP (56.0%). Under these experimental conditions, our results suggest that the greater cooling rates obtained favored an increase in oocyte viability after vitrification, whereas no such beneficial effects were detected for vitrified IVP embryos. In conclusion, Nitrocooler was proven effective as a low-cost device to supercool LN2, increasing viability after vitrification of bovine oocytes, but not of IVP embryos. Table 1.Effect of Nitrocooler apparatus on cryopreservation of bovine oocytes and embryos

Effect of Different Combinations of Cryoprotectants onIn VitroMaturation of Immature Buffalo (Bubalus bubalis) Oocytes Vitrified by Straw and Open-Pulled Straw Methods

This study was designed to evaluate effects of different combinations of cryoprotectants on the ability of vitrified immature buffalo oocytes to undergo in vitro maturation. Straw and open-pulled straw (OPS) methods for vitrification of oocytes at the germinal vesicle stage also were compared. The immature oocytes were harvested from ovaries of slaughtered animals and were divided into three groups: (i) untreated (control); (ii) exposed to cryoprotectant agents (CPAs); or (iii) cryopreserved by straw and OPS vitrification methods. The vitrification solution (VS) consisted of 6 m ethylene glycol (EG) as the standard, control vitrification treatment, and this was compared with 3 m EG + 3 m dimethyl sulfoxide (DMSO), 3 m EG + 3 m glycerol, and 3 m DMSO + 3 m glycerol. Cryoprotectants were added in two steps, with the first step concentration half that of the second (and final) step concentration. After warming, oocyte samples were matured by standard methods and then fixed and stained for nuclear evaluation. Rates of MII oocytes exposed to CPAs without vitrification were lower (54.3 +/- 1.9% in EG, 47.5 +/- 3.4% in EG + DMSO, 36.8 +/- 1.2% in EG + glycerol and 29.9 +/- 1.0% in DMSO + glycerol; p < 0.05) than for the control group (79.8 +/- 1.3%). For all treatments in each vitrification experiment, results were nearly identical for straws and OPS, so all results presented are the average of these two containers. The percentages of oocytes reaching telophase-I or metaphase-II stages were lower in oocytes cryopreserved using all treatments when compared with control. However, among the vitrified oocytes, the highest maturation rate was seen in oocytes vitrified in EG + DMSO (41.5 +/- 0.6%). Oocytes cryopreserved in all groups with glycerol had an overall low maturation rate 19.0 +/- 0.6% for EG + glycerol and 17.0 +/- 1.1% for DMSO + glycerol. We conclude that the function of oocytes was severely affected by both vitrification and exposure to cryoprotectants without vitrification; the best combination of cryoprotectants was EG + DMSO for vitrification of immature buffalo oocytes using either straw or OPS methods.

Comparison of the Level(s) of DNA Damage Using Comet Assay in Bovine Oocytes Subjected to Selected Vitrification Methods

It was suggested that the cryodamage to oocytes' DNA has been responsible for the compromised developmental competence of cryopreserved oocytes. Vitrification of bovine oocytes affected not only cellular components, but also nuclear material. A significant rate of DNA fragmentation was found in bovine frozen or vitrified oocytes analysed by Comet assay regardless of cryopreservation method. Our method of vitrification using droplet system after gentle pre-equilibration treatment is one of the most effective cryopreservation methods employed for bovine oocytes so far, making it possible to develop 30% blastocyst stage embryos. In this study, the extent of DNA damage in bovine oocytes vitrified using three vitrification methods (droplet system, Open Pulled Straw and traditional vitrification in 0.25 ml insemination straws) was compared using Comet assay. Vitrification in droplet system and Open Pull Straws vitrification did not result in detectable cryoinjuries of DNA of bovine oocytes. On the contrary, DNA fragmentation was found in four of 26 oocytes vitrified in 0.25 ml straws (15.4%, p <or= 0.05 in comparison with the other vitrification methods).

Vitrification of early-stage bovine and equine embryos

The objectives of this study were to: (1) determine an optimal method and stage of development for vitrification of bovine zygotes or early embryos; and (2) use the optimal procedure for bovine embryos to establish equine pregnancies after vitrification and warming of early embryos. Initially, bovine embryos produced by in-vitro fertilization (IVF) were frozen and vitrified in 0.25 mL straws with minimal success. A subsequent experiment was done using two vitrification methods and super open pulled straws (OPS) with 1- or 8-cell bovine embryos. In Method 1 (EG-O), embryos were exposed to 1.5 M ethylene glycol (EG) for 5 min, 7 M ethylene glycol and 0.6 M galactose for 30 s, loaded in an OPS, and plunged into liquid nitrogen. In Method 2 (EG-DMSO), embryos were exposed to 1.1 M ethylene glycol and 1.1 M dimethyl sulfoxide (DMSO) for 3 min, 2.5 M ethylene glycol, 2.5 M DMSO and 0.5 M galactose for 30 s, and loaded and plunged as for EG-O. Cryoprotectants were removed after warming in three steps. One- and eight-cell bovine embryos were cultured for 7 and 4.5 d, respectively, after warming, and control embryos were cultured without vitrification. Cleavage rates of 1-cell embryos were similar ( P > 0.05) for vitrified and control embryos, although the blastocyst rates for EG-O and control embryos were similar and higher ( P < 0.05) than for EG-DMSO. The blastocyst rate of 8-cell embryos was higher ( P < 0.05) for EG-O than EG-DMSO. Therefore, EG-O was used to cryopreserve equine embryos. Equine oocytes were obtained from preovulatory follicles. After ICSI, injected oocytes were cultured for 1–3 d. Two- to eight-cell embryos were vitrified, warmed and transferred into recipient's oviducts. The pregnancy rate on Day 20 was 62% (5/8) for equine embryos after vitrification and warming. In summary, a successful method was established for vitrification of early-stage bovine embryos, and this method was used to establish equine pregnancies after vitrification and warming of 2- to 8-cell embryos produced by ICSI.

Association between HSP70 Genotypes and Oocytes Development on In vitro Maturation/Fertilization in Pig

This study was performed to clarify whether the variation of stress related heat shock protein 70 (HSP70) (GenBank X68213) gene was associated with the nuclear morphological change of in vitro maturation and in vitro capacitation in oocytes of pig ovaries obtained at the slaughterhouse. The nucleic acid substitution of C to G at the 483rd position was found out in HSP70 K1 (290- 512) from X68213. The ovaries were categorized into CC, CG, and GG genotypes using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (BsiHKA I). After the second in vitro maturation of immature fresh oocytes, the relation of nuclear morphological change in oocytes with the genotype of HSP70 K1 gene was such that the MII ratios of the genotype GG and CG (46.93% and 42.20%, respectively) were significantly higher than that of the CC genotype (10.71%) (p<0.05). With respect to in vitro maturation of frozen-thawed oocytes by an open pulled straw (OPS) method, the percentage of oocytes matured to MII stage of the CG genotype showed a higher trend than CC and GG genotypes. After the in vitro maturation of immature fresh oocytes and frozen-thawed oocytes by the OPS method, the relation of the pronuclei change in oocytes matured in vitro with HSP70 genotype was assessed, and the result showed that the enlarged sperm heads (ESH) of matured fresh oocytes and frozen-thawed oocytes were 80.0% and 60.0% in the CC genotype, respectively. The CC genotype group had a significantly higher rate of ESH than the CG and the GG genotype group (p<0.05). The ratios of polyspermic invasion were not different among HSP70 of the three genotypes. It was considered that the rate of in vitro maturation of fertilized oocytes was expected to differ according to genotype of the stress related gene. (Key Words : Fertilization, Maturation, Porcine Oocytes, HSP70 Genotypes)

Vitrification of in vitro matured oocytes of Mangalica and Large White pigs

The breeding of Mangalica, a native pig breed in Hungary, had been started in 1833, but this pig breed almost became extinct in Hungary in the past decades. In 1991, the number of sows was only 200. Although in these days the existing Mangalica population consists of more than 6000 animals representing different colour variations, the preservation of this traditional pig breed is still very important. Vitrification is a potential tool for the preservation of gametes and embryos of these animals. The aim of this study was to investigate the effects of vitrification on the developmental competence of Mangalica (M) and Large White (LW) oocytes following fertilisation. The oocytes were vitrified by the Open Pulled Straw (OPS) method using different concentrations of ethylene glycol and dimethyl sulphoxide as cryoprotectants. After rehydration the oocytes underwent in vitro fertilisation; the resultant zygotes were then cultured in vitro for four days to assess embryonic development. In the first experiment, in vitro maturation of M and LW oocytes was compared. No significant difference was observed in the nuclear maturation rate of LW and M oocytes. In the second experiment, the sensitivity of oocytes to vitrification was examined by evaluating oocyte morphology after thawing. A higher percentage of LW oocytes showed normal morphology compared to M oocytes, indicating that Mangalica oocytes are more sensitive to cryoprotectants than Large White oocytes. After warming and in vitro fertilisation, more than 50% of the oocytes started embryonic development and by the end of the incubation period morula stage embryos had developed in both groups. The results show that the OPS vitrification technique is well suited to preserve Mangalica oocytes and from these oocytes morula embryos can be produced.

Vitrification of immature bubaline cumulus oocyte complexes by the open-pulled straw and conventional straw methods and their subsequent in vitro fertilization

The &lt;i&gt;in vitro&lt;/i&gt; maturation (IVM), fertilization (IVF) and morphological changes in buffalo cumulus oocyte complexes (COCs) cryopreserved by ultra rapid freezing using conventional (CON) and open-pulled straw (OPS) methods were tested. COCs were cryopreserved using a vitrification solution comprising of DPBS + 0.5M sucrose + 0.4% BSA and two concentrations (4.5 or 5.5M) of each cryoprotectant ethylene glycol (EG) and dimethylsulfoxide (DMSO) and cryopreserved by either CON or OPS method. Vitrified COCs were stored in LN&lt;sub&gt;2&lt;/sub&gt; for seven days and then thawed, and morphologically normal COCs were used for IVM (&lt;i&gt;n&lt;/i&gt; = 864) and IVF (&lt;i&gt;n&lt;/i&gt; = 933) in two separate experiments to record (1) morphological damage of COCs due to vitrification, (2) nuclear maturation 24 h after culture (nine replicates) and (3) fertilization 24 h after insemination (10 replicates). The COCs were matured &lt;i&gt;in vitro&lt;/i&gt; in TCM-199 medium using hormone supplements and fertilized using TALP-BSA. Freshly collected COCs were separately used for IVM (&lt;i&gt;n&lt;/i&gt; = 110) and IVF (&lt;i&gt;n&lt;/i&gt; = 130) and kept as control. The arcsin transformed data of the proportion of COCs matured or fertilized was compared by DNMR test. The highest proportion of morphologically normal COCs were seen in 5.5M EG with CON method (94.5%) and the lowest were seen in 4.5M DMSO with OPS method (82.4%). At the end of Experiment 1, it was revealed that IVM in all vitrification groups was significantly lower (&lt;i&gt;P&lt;/i&gt; &lt; 0.05) compared to control (66.4%). Amongst the various vitrification treatments the highest IVM was seen in 5.5M EG with OPS method (39.2%) and the lowest in 4.5M DMSO with CON method (19.3%). Comparison of both concentrations of EG and DMSO showed that the proportion of COCs attaining metaphase-II (M-II) increased with increasing concentration of both the cryoprotectants. However, at equal concentration of EG and DMSO the proportion of COCs attaining M-II were significantly higher in OPS method compared to CON method. In Experiment 2, a significantly higher (&lt;i&gt;P&lt;/i&gt; &lt; 0.05) IVF was seen for fresh COCs (45.4%) compared to vitrified COCs. Amongst the vitrification treatments the highest fertilization was seen for 5.5M EG with the OPS method (33.6 %) and the lowest for the 4.5M DMSO with CON method (15.17%). A dose dependant increase in the proportion of oocytes fertilized was seen with increasing concentration of both EG and DMSO [CON: 4.5M (15.2%), 5.5M (25.6%), OPS: 4.5M (21.3%) and 5.5M (27.5%)] in both CON and OPS methods. Comparison of the two cryoprotectants revealed that EG was better compared to DMSO. At equal concentrations of EG or DMSO a significantly higher (&lt;i&gt;P&lt;/i&gt; &lt; 0.05) proportion of fertilized oocytes were seen in OPS method compared to the CON method. It was concluded that developmental capacity of vitrified buffalo COCs could be improved by using OPS in comparison to conventional straws.

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 (LN 2). 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% CO 2, 5% O 2 and 90% N 2 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.

One-step dilution of open-pulled-straw (OPS)-vitrified mouse blastocysts in sucrose-free medium

Embryos vitrified by the open-pulled-straw (OPS) method are only briefly exposed to cryoprotectants and not fully equilibrated with the cryoprotectant. That being the case, conceivably the post-thawing de- and rehydration processes may be omitted. This would render thawing and dilution in a single step and direct transfer to recipients possible without the need for a microscope and other laboratory equipment. Morphologically intact mouse blastocysts from superovulated 5- to 8-week-old virgin female NMRI mice were vitrified according to a protocol [6] slightly modified from the classical OPS-procedure of Vajta et al. [29] consisting of exposure to 10% dimethyl-sulfoxide (Me 2SO) + 10% ethylene glycol (EG) for 1 min, followed by 20% Me 2SO + 20% EG for 20 s before loading into straws that are plunged into liquid nitrogen. In Group 1, 75 blastocysts were exposed to the standard thawing and dilution regimen involving exposure to three solutions of decreasing sucrose content (Control). In Groups 2, 3 and 4, 75 blastocysts each were transferred, in a single step, to medium at 37 °C containing 0.66, 0.33 or 0 M sucrose, respectively. After 48 h of in vitro culture the proportion of hatched blastocysts was determined. In Group 1, this proportion amounted to 82.7%, in Groups 2, 3 and 4 to 76.0%, 73.3% and 78.7%, respectively ( P > 0.05). To examine their potential to continue development in vivo, OPS-vitrified blastocysts thawed according to the regimens of Groups 1 and 4 were transferred to recipients (10 embryos/recipient). In Group 1, 9/10 recipients got pregnant with 4.7 ± 0.6 (mean ± SEM) fetuses, in Group 4, 8/10 recipients with 5.0 ± 0.5 fetuses. The overall embryo survival rate per group was 42% for Group 1 and 40% for Group 4. All fetuses were normally developed and viable and there were no significant differences between groups ( P > 0.05). It may be concluded that warming and transfer of OPS-vitrified mouse embryos in a single step in medium devoid of sucrose is feasible, which is tantamount to a substantial simplification of embryo transfer operations.

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.

Open-pulled straw (OPS) vitrification of in vitro fertilised mouse embryos at various stages

The present study was designed to investigate the cryotolerance of in vitro fertilised (IVF) mouse embryos at various preimplantation developmental stages. IVF mouse embryos were vitrified by the open-pulled straw (OPS) method. After warming, embryos were morphologically evaluated and assessed by their development to blastocysts, hatched blastocysts or term. The results showed that a high proportion (93.3-100.0%) of vitrified embryos at all developmental stages were morphologically normal after recovery. The developmental rate of vitrified 1-cell embryos to blastocyst (40.0%) or hatched blastocyst (32.7%) or term (9.3%) was significantly lower than that from other stages (P < 0.05). Vitrified embryos from 2-cell to early blastocyst stage showed similar blastocyst (71.8-89.5%) and hatched blastocyst rates (61.1-69.6%) and could develop to term without a significant loss of survival compared with those of fresh embryos (P > 0.05). Vitrified 2-cell embryos showed the highest survival rate in vivo (50.6%, 88/174), compared with that from other stages (9.3-30.5%, P < 0.05). The data demonstrate that the OPS method is suitable for the cryopreservation of IVF mouse embryos from 2-cell stage to early blastocyst stage without a significant loss of survival. Embryos at the 2-cell stage had the best tolerance for cryopreservation in the present study.