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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. Author contributions
  9. References

Three experiments were designed to test a solid-surface vitrification system for bovine in vitro-produced embryos and to develop a simple method of in-straw dilution after warming, which can be potentially used for direct transfer in the field. Experiment 1 evaluated embryo survival rates (i.e. re-expansion and hatching) after vitrification and warming in three different solutions: VS1 (20% ethylene glycol (EG) + 20% propanediol (PROH) + 0.25 m trehalose (Tr)), VS2 (20% EG + 1M Tr) or VS3 (30% EG + 0.75 m Tr). Re-expansion and hatching rates were higher (p < 0.05) for embryos vitrified in VS3 (72.2 ± 1.9 and 58.2 ± 0.8) than VS1 (64.4 ± 0.9 and 37.2 ± 2.5) or VS2 (68.5 ± 1.5 and 49.6 ± 1.0; p < 0.05). Experiment 2 was designed to compare two methods of vitrification: glass micropipettes or solid surface, using the VS1 or VS3 solutions. No significant differences were detected between the two methods; but re-expansion and hatching rates were higher (p < 0.05) with VS3 (73.5 ± 3.1 and 47.1 ± 2.1) than VS1 (63.3 ± 3.3 and 39.7 ± 2.8). In experiment 3, embryos were vitrified by solid surface in VS1 or VS3 solutions and cryoprotectants were diluted in-straw after warming in a TCM 199, 0.25 m sucrose solution or holding media. Survival rates of embryos vitrified in VS3 did not differ between those exposed to 0.25 m sucrose (74.7 ± 1.3 and 57.2 ± 2.2) or holding (77.3 ± 1.4 and 58.0 ± 2.5) medium after warming; however, survival rates of embryos vitrified in VS1 were higher (p < 0.05) in those exposed to 0.25 m sucrose (67.7 ± 2.3 and 47.0 ± 1.7) than holding medium (54.5 ± 1.0 and 27.7 ± 3.1). In conclusion, solid-surface vitrification using simplified EG-based solutions and in-straw dilution with holding media may be a practical alternative for cryopreservation and direct transfer of in vitro-produced bovine embryos.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. Author contributions
  9. References

Since Rall and Fahy (1985) successfully vitrified mammalian embryos, the vitrification method has become the main cryopreservation technique used by several laboratories for in vitro-produced (IVP) bovine embryos; especially those in South America (Viana et al. 2010). Several studies have shown that conventional cryopreservation procedures, such as controlled slow-freezing, have lower survival rates with in vitro produced than in vivo-produced bovine embryos (Dinnyes and Nedambale 2009; Nicacio et al. 2011; Rodriguez Villamil et al. 2012). Vitrification allows the cryopreservation of embryos through fast cooling and warming rates, preventing intracellular ice crystal formation (Yu et al. 2010). It is necessary to combine three important factors to consistently achieve a glass-like solidification: high cooling rates, high viscosity of the cryopreservation medium and minimum volume (Saragusty and Arav 2011). In an attempt to achieve a stable vitreous state, most vitrification solutions require the use of high concentrations of permeating and non-permeating cryoprotectants (>4 m) than those used for slow-freezing (1–2 m); however, these high concentrations of cryoprotectants may be cytotoxic to the embryos if they are exposed for a prolonged period of time (Kasai and Mukaida 2004). For this reason, solutions are composed of the least toxic cryoprotectants or combination of different cryoprotectants to prevent the osmotic and toxic effects that affect the blastomeres (Liebermann et al. 2003; Kasai and Mukaida 2004). Among the different permeating cryoprotectants, ethylene glycol (EG) is one of the most commonly used, due to its high permeability (Dochi et al. 2006). Furthermore, vitrification solutions based on EG alone or EG combined with other permeating and non-permeating cryoprotectants have been shown to be effective for the cryopreservation of IVP bovine embryos (Saha et al. 1996; Vajta et al. 1998; Campos-Chillon et al. 2006; Vieira et al. 2007; Inaba et al. 2011).

Several methods and devices using minimum volume have been created with the objective of increasing cooling rates and enhancing the probability of vitrification (Saragusty and Arav 2011); however, most of these techniques require a laboratory for the warming and dilution of cryoprotectants before the embryos can be transferred to recipients in the field. The open-pulled straw (OPS) technique is one of the most commonly used microvolume techniques (Saragusty and Arav 2011), and optimal survival rates have been reported after in-straw warming and cryoprotectant dilution (Vajta et al. 1999; Yang et al. 2007). However, volume variations created by handmade devices such as the OPS may alter cooling and warming rates, reducing embryo survival (Vajta et al. 1999; Vieira et al. 2007). Furthermore, the in-straw dilution method for this type of device requires highly skilled operators to handle the devices during warming, making these methods difficult to apply in the field by embryo-transfer practitioners (Vieira et al. 2007; Inaba et al. 2011). The solid-surface vitrification procedure is a method that allows embryos to be vitrified using a metal solid surface cooled in liquid nitrogen (LN2; −196°C), without contact to LN2 itself (Dinnyes et al. 2000). With this method, it is possible to use vitrification solutions with lower cryoprotectant concentrations, using higher cooling rates (>20 000°C/min) and minimum standard volumes (0.6 μL) (Lindemans et al. 2004; Peachey et al. 2004; Dinnyes and Nedambale 2009). Unfortunately, laboratory conditions are still necessary for the warming and dilution procedures before the embryos can be transferred into recipients. Three experiments were designed to test a solid-surface vitrification system for bovine IVP embryos and to develop a simple method of in-straw dilution after warming that can be potentially used for direct transfer in the field. Experiment 1 was designed to evaluate the survival rates after vitrification of embryos exposed to three different cryoprotectant solutions. Experiment 2 was designed to compare two methods of vitrification: glass micropipettes (GMP) or solid-surface, whereas experiment 3 evaluated survival rates after warming and in-straw dilution of cryoprotectants using a 0.25 m sucrose solution or holding medium.

Material and Methods

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. Author contributions
  9. References

Except where otherwise indicated, all chemicals and water were obtained from Sigma Chemical Co. (St. Louis, MO, USA).

In vitro embryo production

Bovine ovaries from cross-bred beef cows were obtained at the slaughterhouse and kept at 37°C during transport to the laboratory. Cumulus–oocyte complexes (COC) were aspirated from follicles 2–8 mm in diameter and then washed in tissue culture medium 199 (TCM-199) and transferred to 4-well plates containing 500 μL of maturation medium per well (30–50 COCs per well) and matured for approximately 24 h at 38.8°C in an atmosphere of saturated humidity and 5% CO2. The maturation medium consisted of TCM-199 supplemented with 10% (v/v) foetal bovine serum (Natocor®, Natocor, Cordoba, Argentina), 0.2 mm sodium pyruvate, 35 mg/ml of porcine FSH (Folltropin-V, Bioniche Animal Health, Belleville, Canada) and antibiotics. After in vitro maturation, a straw of frozen semen from an Angus bull was thawed for 1 min at 37°C. Sperm were prepared for fertilization with a Percoll gradient system (90–45%). COCs were fertilized with a final concentration of 1 × 106 spz/ml in 500 μL droplets (30–50 COCs/droplet) covered with mineral oil into modified TALP fertilization medium. After 20 h of in vitro fertilization, the COCs were vortexed to remove the cumulus cells and excess sperm and washed once in synthetic oviductal fluid with essential amino acids medium (SOFaa) supplemented with foetal bovine serum (FBS; Natocor®, Argentina). Then, oocytes were transferred into SOFaa culture medium drops (500 μL) under mineral oil in a controlled atmosphere (5% CO2, 5% O2 and 90% N2) at 38.8°C. Cleavage rates were observed on day 2 (day 0 = day of fertilization), and embryo development rates were observed on day 7 of the culture period. Only Grade 1 blastocysts (stage 6) were selected for the experiments.

Experiment 1

In experiment, 1320 expanded blastocysts (IETS codes 6 and 7, Robertson and Nelson 2010) were randomly allocated into one of four treatment groups, in 6 replicates. Embryos in the control group (n = 76) remained in culture until hatching and were not vitrified or exposed to any vitrification solution or vitrified. The remaining 244 embryos were randomly distributed to be exposed to the following solutions: VS1: 10% ethylene glycol (EG) + 10% propanediol (PROH) + 0.25 m trehalose (Tr) for 1 min and 20% EG + 20% PROH + 0.25 m Tr for 30 s; VS2: 10% EG + 0.25 m Tr for 1 min and 20% EG + 1M Tr for 30 s or VS3: 15% EG + 0.25 m Tr for 1 min and 30% EG + 0.75 m Tr for 30 s. All the solutions were based in buffered TCM-199 medium, supplemented with 0.4% of BSA. After exposure, embryos were vitrified using the GMP procedure. The GMP procedure is a modification of the OPS technology originally described by Vajta et al. (1998), replacing the plastic straws by glass capillary tubes that are heated and pulled manually until their outer diameter reaches approximately 0.6 mm (Kong et al. 2000). Briefly, embryos were loaded by capillarity, placing the narrowest end of glass micropipettes into 2–4 μL cryoprotectant droplets and then plunged into liquid nitrogen (LN2). Embryo warming and cryoprotectant dilution were performed by direct plunging of the GMP and unloading of the embryos into 300 μL droplets of TCM-199 supplemented with 0.25 m sucrose at 37°C for 5 min. Embryos were then washed in holding media (Vigro Holding Plus, Bioniche Canada Inc. Belleville, ON, Canada) and placed into SOFaa medium droplets (200 μL) for in vitro culture. Re-expansion and hatching rates were evaluated at 24 h and 72 h of in vitro culture.

Experiment 2

Grade 1 blastocysts (IETS codes 6 and 7; n = 594) were randomly allocated to 5 groups, in 10 replicates. Embryos in the control group (n = 95) remained in culture until hatching and were not vitrified. The remaining 499 embryos were exposed to two of the three vitrification solutions used in experiment 1 (VS1 and VS3) and then further subdivided to be vitrified by GMP or solid-surface vitrification. The solid-surface vitrification procedure used was a modification of that described by Dinnyes et al. (2000), using a commercial vitrification system (CVM®, Cryologic, Vic., Australia; Peachey et al. 2004). This method allows embryos to be vitrified using a metal solid surface cooled at −196°C, without contact to LN2. Briefly, a 0.6 μL droplet of the vitrification solution containing an embryo was placed in a hook attached to a straw plug (called Fyberplugs™; Cryologic, Mulgrave, Australia) using a pipette and immediately exposed to the solid metal surface by touching the drop containing the embryo to the metal surface cooled at −196°C (Lindemans et al. 2004). Then the Fyberplugs were inserted into short plastic straw that were also cooled to −196°C by contact with metal block as described by Lindemans et al. (2004). After at least 1 week storage in LN2 tanks, vitrified embryos were warmed by firstly twisting off the Fyberplugs from the short plastic straws inside LN2, to release pressure before removal from the short plastic straws, and immediate plunging of the drop containing the embryo in 0.25 m sucrose solution that was pre-warmed at 39°C. After 5 min into the sucrose solution at 39°C, embryos were placed in SOFaa medium and cultured for 72 h as described in 'Experiment 3'.

Experiment 3

Grade 1 blastocysts (IETS codes 6 and 7; n = 572) were selected and randomly allocated into five groups in five replicates. As in previous experiments, embryos in the control group (n = 100) remained in culture until hatching and were not vitrified. The remaining 472 embryos were exposed to two different vitrification solutions (VS1 or VS3) and vitrified by the solid-surface system as described in 'Statistical analysis', except that instead of using the hemi-straw provided by the manufacturer, Fyberplugs were inserted into 0.5-ml plastic straws that contained 200 μL of either of the two diluting solutions: holding medium (Vigro®; Bioniche Animal Health) or TCM-199 with 0.25M sucrose (Fig. 1). The volume of the diluting solutions was calculated to provide a 5-mm air space between the Fyberplug and the solution, to avoid direct contact with the vitrified embryos. The straws containing the diluting solution were frozen in LN2 and kept in the metal block at −196°C until the embryos were vitrified, and the Fyberplugs were inserted into the straws and then plunged in LN2. After at least 1 week storage in LN2, the straws containing the diluting solutions and vitrified embryos were warmed by immersion in a 39°C water bath until the diluting solution was clear (approximately 15–30 s) and then the diluting solution and the drop containing the embryo in the vitrification solution were immediately mixed by gently agitating the straw as shown in Fig. 2. After mixing, the straws with the embryos were maintained on a thermal plate at 37°C for 5 min, simulating the transfer period. Then, embryos were placed in SOFaa culture medium for 72 h as in experiment 1.

image

Figure 1. In-straw dilution used in experiment 3. (a) Fyberplug with vitrification solution drop (b) 0.5-ml straw containing 200 μl of warming solution (c) Fyberplug plunged into the straw with warming solution

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image

Figure 2. In-straw dilution procedure used in Experiment 3. (a) The Fyberplug was twisted off inside liquid nitrogen to release pressure, and then the straw was exposed to air for 5 s (b) the straw was plunged into water bath (37°C) for 30 s (c) The diluting solution into and the drop containing the embryo were mixed by gentle agitation (d) 0.5-ml straw containing embryo for direct transfer

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Statistical analysis

Re-expansion and hatching rates were first transformed by square root and then analysed by anova, to evaluate the main effects of treatments and their interactions. The protected least significant difference (LSD) test was used for subsequent multiple comparisons when anova revealed a significant main effect or interaction (p < 0.05).

Results

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. Author contributions
  9. References

Experiment 1

Re-expansion rates were significantly higher (p < 0.01) for embryos vitrified in VS3 than those vitrified in VS1; re-expansion rates in embryos vitrified in VS2 were intermediate and not different from those vitrified in VS1 and VS2 (Table 1). Furthermore, hatching rates differed (p < 0.04) among the three groups and were lowest in VS1 and highest in VS3 (Table 1). Hatching rates in the control group were significantly higher than those in the three vitrification groups (p < 0.05).

Table 1. Re-expansion and hatching rates of IVP bovine embryos vitrified to three vitrification solutions
SolutionsNo. embryosRe-expanded embryos (%)Hatched embryos (%)
  1. Means within the same column with different superscript (upper case) are significantly different, p < 0.05.

  2. a

    VS1: 10% EG + 10% PROH for 1 min followed by 20% EG + 20% PROH + 0.25 m Tr for 30 s.

  3. b

    VS2 = 10% EG +0.25 m Tr for 1 min followed by 20% EG +1 m Tr for 30 s.

  4. c

    VS3 = 15% EG + 0.25 m Tr for 1 min followed by VS3 = 30% EG + 0.75 m Tr for 30 s.

VS1a8164.4 ± 0.9B37.2 ± 2.5D
VS2b8168.5 ± 1.5AB49.6 ± 1.0C
VS3c8272.2 ± 1.9A58.2 ± 0.8B
Control7674.4 ± 2.5A

Experiment 2

There were no interactions between vitrification methods and vitrification solutions with regard tore-expansion and hatching rates. Furthermore, there were no differences in re-expansion and hatching rates between both vitrification methods (Table 2); however, embryos vitrified with the VS3 solution had higher (p < 0.05) re-expansion and hatching rates than those vitrified with the VS1 solution (Table 3). As in previous studies, hatching rates in the control group were higher (p < 0.05) than those in the vitrified groups.

Table 2. Re-expansion and hatching rates of embryos vitrified using glass micropipettes (GMP) or solid surface (CVM)
Vitrification methodsNo. embryosRe-expanded embryos (%)Hatched embryos (%)
  1. Means within the same column with different superscript are significantly different, p < 0.05.

Control9577.0 ± 1.3a
GMP25067.8 ± 4.4a40.8 ± 3.4b
CVM24969.0 ± 2.0a46.0 ± 1.3b
Table 3. Re-expansion and hatching rates of vitrified embryos using two different cryoprotectant solutions
Vitrification solutionsNo. embryosRe-expanded embryos (%)Hatched embryos (%)
  1. Means within the same column with different superscript (upper case) are significantly different, p < 0.05.

  2. a

    VS1: 10% EG + 10% PROH for 1 min followed by 20% EG + 20% PROH+ 0.25 m Tr for 30 s.

  3. b

    VS3 = 15% EG + 0.25 m Tr for 1 min followed by VS3 = 30% EG + 0.75 m Tr for 30 s.

Control9577.0 ± 1.3A
VS1a25063.3 ± 3.3B39.7 ± 2.8C
VS3b24973.5 ± 3.1A47.1 ± 2.1B

Experiment 3

As in previous experiments, embryos vitrified in VS3 had higher (p < 0.05) re-expansion and hatching rates (179/235, 76% and 135/235, 57%, respectively) than those vitrified in VS1 (146/237, 61% and 88/237, 37%, respectively). However, there was an interaction (p < 0.05) between vitrification and diluting solutions. Although re-expansion and hatching rates of embryos vitrified in VS3 did not differ between embryos exposed to 0.25 m sucrose or holding medium after warming, re-expansion and hatching rates were higher (p < 0.05) in embryos vitrified in VS1 that were exposed to 0.25 m sucrose after warming than those exposed to holding medium (Table 4).

Table 4. Re-expansion and hatching rates of embryos vitrified using two solutions and that were diluted in-straw after warming in 0.25 m sucrose or holding media
Vitrification solutionWarming solutionNo. embryosRe-expansion (%)Hatching (%)
  1. Means within the same column with different superscript (upper case) are significantly different, p < 0.05.

  2. a

    VS1: 10% EG + 10% PROH for 1 min followed by 20% EG + 20% PROH + 0.25 m Tr for 30 s.

  3. b

    VS3 = 15% EG + 0.25 m Tr for 1 min followed by VS3 = 30% EG + 0.75 m Tr for 30 s.

VS1aSucrose12067.7 ± 2.3B47.0 ± 1.7C
Holding11754.5 ± 1.0C27.7 ± 3.1D
VS3bSucrose11774.7 ± 1.3A57.2 ± 2.2B
Holding11877.3 ± 1.4A58.0 ± 2.5B
Control10076.1 ± 1.9A

Discussion

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. Author contributions
  9. References

Although vitrification has been one of the most widely used techniques for embryo cryopreservation of IVP embryos for many years, it has not been widely used commercially due to variable results and the difficulty of the warming and diluting methodologies needed for embryo transfer in the field (Campos-Chillon et al. 2006). The present study revealed that the use of a simplified EG-based solution in combination with the solid-surface vitrification methodology allowed the in-straw dilution of vitrified bovine embryos successfully and may be a feasible method to use in the field, diminishing the chances of manipulation errors during the warming and diluting process prior to embryo transfer.

Vitrification is a physical process that solidifies a solution at low temperatures not by crystallization but by extreme elevation in viscosity during cooling (Rall and Fahy 1985). To attempt a stable vitreous state, it is necessary to combine high cooling rates and small volumes of highly concentrated cryoprotectant solutions (Yavin and Arav 2007). It is known that EG is one of the intracellular cryoprotectants with less toxicity (Dochi et al. 2006), and it is the main component of most of the cryopreservation solutions (Saha et al. 1996; Vajta et al. 1998; Campos-Chillon et al. 2006; Vieira et al. 2007). However, it has been shown that the glass-forming capacity of EG alone is weaker than when it is combined with other cryoprotectants such as PROH (Baudot and Odagescu 2004; Fahy et al. 2004). However, in experiment 1, embryos vitrified in a simple solution using 30% EG and the extracellular cryoprotectant Tr showed higher survival rates than the EG-PROH-Tr composed solution. Although the combination EG and PROH could potentially reduce the toxicity of the high EG concentration (Fahy et al. 2004), the addition of a non-permeable cryoprotectant that remains in the extracellular area such as Tr may have been beneficial because it may have decreased the osmotic effects and reduced the formation of intracellular ice (Liebermann et al. 2003; Kasai and Mukaida 2004). Furthermore, it has been shown that Tr enhances the capacity of EG to form a stable vitreous state while reducing the osmotic stress of the embryos during the vitrification and warming processes (Saha et al. 1996; Liebermann et al. 2003; Lawson et al. 2011). Conversely, the use of a lower concentration of EG (i.e. 20%) as the unique permeable cryoprotectant resulted in poor survival rates, that may have been the consequence of using a less stable solution increasing the possibilities of intracellular crystallization to form (Mazur et al. 2007).

In experiment 2, two different vitrification systems were compared. As we mentioned, high cooling rates are also necessary to obtain a stable vitreous state (Yavin and Arav 2007). Furthermore, the thermal conductivity of the containers and the surface to volume ratio of the medium surrounding the embryos are critical (He et al. 2008; Rodriguez et al. 2010). Several cooling devices have been used to achieve high cooling rates for vitrification, such as the open-pulled straw (Vajta et al. 1998), cryoloops (Lane et al. 1999), hemi-straw (Vanderzwalden et al. 2000), gel-loading tips (Tominaga and Hanada 2001), fine diameter plastic (Cremades et al. 2004) and cryotop that were reported to result in cooling rates approximately 20 000°C/min (Kuwayama et al. 2005). It is known that higher cooling rates are easier to achieve using containers with a greater heat transfer than plastic, such as glass or quartz (Cho et al. 2002; He et al. 2008), or using a container-less system (Lane et al. 1999; Dinnyes et al. 2000), which may result in an improvement of embryo survival rates after vitrification (Cho et al. 2002; Lindemans et al. 2004; Rodriguez Villamil et al. 2012). Our results have not shown significant differences in hatching rates of embryos vitrified in the GMP or a container-less system such as the solid-surface system. These findings are in agreement with the results observed by other authors that also compared different microvolume containers without finding significant differences among the devices (Cho et al. 2002; Lindemans et al. 2004; Rios et al. 2010; Yu et al. 2010). Although both methods evaluated in the current study have not shown significant differences on survival rates, the solid-surface system has the commercial advantage that the embryos are not directly in contact with LN2, decreasing the contamination risks during vitrification or storage (Bielanski and Lalonde 2009; Beebe et al. 2011). Therefore, we decided to develop an in-straw dilution system for the solid-surface vitrification in experiment 3.

Most warming protocols for vitrified embryos involve diluting embryos in decreasing concentrations of sucrose to counterbalance the swelling caused by water moving into the cells more quickly than the permeable cryoprotectant can move out, affecting embryo viability (Kasai and Mukaida 2004; Guignot et al. 2006). Our results, suggest that a non-permeating cryoprotectant like sucrose is necessary when a combination of two permeating cryoprotectants like EG and PROH are used with the vitrification technique used in the present study. However, when embryos were vitrified in EG as the only permeating cryoprotectant, there were no significant differences in survival rates between the use of sucrose or holding medium as warming solutions. These results were similar to those reported by other authors (Saha et al. 1996; Campos-Chillon et al. 2006), which also suggested that using or not using sucrose during warming of vitrification solutions is irrelevant when vitrification solutions are composed with solutions containing EG as the only intracellular cryoprotectant. Furthermore, vitrified embryos diluted in-straw have higher survival rates when embryos were cryopreserved in simplified solutions with one permeable cryoprotectant (Pugh et al. 2000; Taniguchi et al. 2007). In vitro assessment of the in-straw dilution system demonstrated that it is a feasible method for direct transfer, reducing the chances of manipulation errors and variations during the warming process. Several studies have shown that rapid warming is equally important to rapid cooling for the survival of vitrified embryos (Liebermann et al. 2003). Although the use of a 0.5-ml straw that is plunged into a 39°C water bath for 30 s may possibly result is a slower warming and dilution process than plunging directly the vitrified embryos into the warming solution, the survival rates obtained in the present study suggest that warming and dilution were not long enough to significantly affect embryo viability. Other studies have shown similar hatching rates with in-straw dilution for devices with minimum volume such as OPS (Vajta et al. 1999), GMP (Vieira et al. 2007) or Cryotop (Inaba et al. 2011), but these devices required an involved method of warming and dilution, such as the introduction of the vitrification devices into 0.25-ml plastic straws containing the warming solutions. In these warming procedures, the operator must manipulate the device between warming and dilution that may result in temperature variations. Another problem related to this methodology is the time required to perform the warming and dilution, which may slow down the work when high numbers of embryos are transferred in the field. Both problems can be avoided with the system developed in the present study because straws can be warmed, gently agitated and then transferred directly into a recipient; just as it is now commonly done with in vivo embryos frozen in EG. The only caveat is the requirement of a 0.5-ml transfer gun to perform this technique, but this can be easily solved by using an equine-disposable embryo-transfer device (Squires et al. 2003) or just an insemination device for 0.5-ml straws. Further experiments are necessary to confirm these results and test this methodology by obtaining adequate pregnancy rates in the field.

In conclusion, solid-surface vitrification using simplified EG-based solutions and in-straw dilution with holding media may be a practical option for cryopreservation and direct transfer of IVP bovine embryos.

Conflict of interest

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. Author contributions
  9. References

None of the authors have any conflict of interest to declare.

Author contributions

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. Author contributions
  9. References

Paula Rodriguez contributed with all the research, acquisition interpretation data and to draft the paper. Felipe Ongaratto contributed on the acquisition and analysis of data. Mariana Fernandez contributed with the analysis and interpretation data, and Gabriel Bo helped in the experimental design, interpretation of data and drafting and reviewing this manuscript.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. Author contributions
  9. References
  • Baudot A, Odagescu V, 2004: Thermal properties of ethylene glycol aqueous solutions. Cryobiology 48, 283294.
  • Beebe LFS, Bouwmana EG, McIlfatrick SM, Nottle MB, 2011: Piglets produced from in vivo blastocysts vitrified using the Cryologic Vitrification Method (solid surface vitrification) and a sealed storage container. Theriogenology 75, 14531458.
  • Bielanski A, Lalonde A, 2009: Effect of cryopreservation by slow cooling and vitrification on viral contamination of IVF embryos experimentally exposed to bovine viral diarrhea virus and bovine herpesvirus-1. Theriogenology 72, 919925.
  • Campos-Chillon LF, Walker DJ, de la Torre-Sanchez JF, Seidel GE, 2006: In vitro assessment of a direct transfer vitrification procedure for bovine embryos. Theriogenology 65, 12001214.
  • Cho SK, Cjo SG, Bae IH, Park CS, Kong IK, 2002: Improvement in post-thaw viability of in vitro-produced bovine blastocysts vitrified by glass micropipette (GMP). Anim Reprod Sci 73, 151158.
  • Cremades N, Sousa M, Silva J, Viana P, Sousa S, Oliveira C, Teixeira da Silva J, Barros A, 2004: Experimental vitrification of human compacted morulae and early blastocysts using fine diameter plastic micropipettes. Human Reprod 19, 300305.
  • Dinnyes A, Nedambale TL, 2009: Cryopreservation of manipulated embryos: tackling the double jeopardy. Reprod Fertil Dev 21, 4559.
  • Dinnyes A, Dai Y, Jiang S, Yang X, 2000: High development rates of vitrified bovine oocytes following parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. Biol Reprod 63, 513518.
  • Dochi O, Imai K, Matoba S, Miyamura M, Hamano S, Koyama H, 2006: Essential methods of freezing embryos for application in animal reproduction management. J Reprod Dev 52, 6570.
  • Fahy GM, Wowk B, Wu J, Paynter S, 2004: Improved vitrification solutions based on the predictability of vitrification solution toxicity. Theriogenology 48, 2235.
  • Guignot F, Bouttier A, Baril G, Salvetti P, Pignon P, Beckers JF, Touzé JL, Cognié J, Traldi AS, Cognié Y, Mermillod P, 2006: Improved vitrification method allowing direct transfer of goat embryos. Theriogenology 66, 10041011.
  • He X, Park EYH, Fowler A, Yarmush ML, Toner M, 2008: Vitrification by ultra-fast cooling at a low concentration of cryoprotectants in a quartz micro-capillary: a study using murine embryonic stem cells. Cryobiology 56, 223232.
  • Inaba Y, Aikawa Y, Hirai T, Hashiyada Y, Yamanouchi T, Misumi K, Othake M, Somfai T, Kobayashi S, Saito N, Matoba S, Konishi K, Imai K, 2011: In-Straw cryoprotectant dilution for bovine embryos vitrified using cryotop. J Reprod Dev 57, 437443.
  • Kasai M, Mukaida T, 2004: Cryopreservation of animal and human embryos by vitrification. Reprod Biomed Online 9, 164170.
  • Kong IK, Lee SI, Cho SG, Cho SK, Park CS, 2000: Comparison of open pulled Straw (OPS) vs glass micropipette (GMP) vitrification in mouse blastocysts. Theriogenology 53, 18171826.
  • Kuwayama M, Vajta G, Leda S, Kato O, 2005: Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reprod Biomed Online 11, 608614.
  • Lane M, Bavister BD, Lyons EA, Forest KT, 1999: Containerless vitrification of mammalian oocytes and embryos. Nat Biotechnol 17, 12341236.
  • Lawson A, Ahmad H, Sambanis A, 2011: Cytotoxicity effects of cryoprotectants as single-component and cocktail vitrification solutions. Cryobiology 62, 115122.
  • Liebermann J, Dietl J, Vanderzwalmen P, Tucker M, 2003: Recent developments in human oocyte, embryo and blastocyst vitrification: where are we now? Reprod Biomed Online 7, 623633.
  • Lindemans W, Sangalli L, Kick A, Earl CR, Fry RC, 2004: Vitrification of bovine embryos using the CLV method. Reprod Fertil Dev 16, 174
  • Mazur P, Pinn IL, Kleinhans FW, 2007: The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at that temperature. Cryobiology 54, 223233.
  • Nicacio AC, Simões R, de Paula-Lopes FF, de Barros FR, Peres MA, Assumpção ME, Visintin JÁ, 2011: Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos. Zygote 16, 16.
  • Peachey B, Hartwich K, Cockrem K, Marsh A, Pugh A, Van Wagtendonk A, Lindemans W, 2004: Assessment of viability of in vitro produced bovine embryos following vitrification by CVM or slow freezing with ethylene glycol and triple transfer. Reprod Fertil Dev 17, 199.
  • Pugh PA, Tervit HR, Niemann H, 2000: Effects of vitrification medium composition on the survival of bovine in vitro produced embryos, following in straw-dilution, in vitro and in vivo following transfer. Anim Reprod Sci 58, 922.
  • Rall WF, Fahy GM, 1985: Ice-free cryopreservation of mouse embryos at -196°C by vitrification. Nature 313, 573575.
  • Rios GL, Mucci NC, Kaiser GG, Alberio RH, 2010: Effect of container, vitrification volume and warming solution on cryosurvival of in vitro-produced bovine embryos. Anim Reprod Sci 118, 1924.
  • Robertson RE, Nelson I, 2010: Chapter 9 Certification and identification of embryos. In: 4th Edition (Stringfellow DA, Givens MD (eds), Manual of the International Embryo Transfer Society (IETS). International Embryo Transfer Society, Inc, Champaign, IL, USA, pp. 86105.
  • Rodriguez Villamil P, Lozano D, Oviedo JM, Ongaratto FL, Bó GA, 2012: Developmental rates of in vivo and in vitro produced bovine embryos cryopreserved in ethylene glycol based solutions by slow freezing or solid surface vitrification. Anim Reprod 9, 8692.
  • Rodriguez P, Ongaratto FL, Silva DS, Rodrigues BA, Rodrigues JL, 2010: Survival of vitrified mouse blastocysts loaded into glass micro-capillaries. Rev Col de cienc Pec 23, 2834.
  • Saha S, Otoi T, Takagi M, Boediono A, Sumantri C, Suzuki T, 1996: Normal calves obtained after direct transfer of vitrified bovine embryos using ethylene glycol, trehalose, and polyvinylpyrrolidone. Cryobiology 33, 291299.
  • Saragusty J, Arav A, 2011: Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification. Reproduction 141, 119.
  • Squires EL, Carnevale EM, McCue PM, Bruemmer JE, 2003: Embryo technologies in horse. Theriogenology 59, 151170.
  • Taniguchi M, Ikeda A, Arikawa E, Wongsrikeao Agung B, Naoi H, Nagai T, Otoi T, 2007: Effect of cryoprotectant composition on in vitro viability of in vitro fertilized and cloned bovine embryos following vitrification in-straw dilution. J Reprod Dev 53, 963969.
  • Tominaga K, Hanada Y, 2001: Gel-loading tip as container for vitrification of in vitro produced bovine embryos. J Reprod Dev 47, 267273.
  • Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, 1998: Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 51, 5358.
  • Vajta G, Murphy CN, Machaty Z, Prather RS, Greve T, Callesen H, 1999: In-straw dilution of bovine blastocyst after vitrification with the open pull straw method. Vet Rec 144, 180181.
  • Vanderzwalden P, Bertin G, Debauche CH, Standaart V, Schoysman E, 2000: In vitro survival of metaphase II oocytes (MII) and blastocyst after vitrification in an hemi-straw (HS) system. Fertil Steril 74, 215216.
  • Viana JHM, Siqueira LGB, Palhão MP, Camargo LSA, 2010: Use of in vitro fertilization technique in the last decade and its effect on Brazilian embryo industry and animal production. Acta Sci Vet 38, 277831.
  • Vieira AD, Forell F, Feltrin C, Rodrigues JL, 2007: In-straw cryoprotectant dilution of IVP bovine blastocysts vitrified in hand-pulled glass micropipettes. Anim Reprod Sci 99, 377383.
  • Yang QE, Hou YP, Zhou GB, Yang ZQ, Zhu SE, 2007: Stepwise In-straw dilution and direct transfer using open pulled straw (OPS) in the mouse: a potential model for field manipulation of vitrified embryos. J Reprod Dev 53, 211218.
  • Yavin S, Arav A, 2007: Measurement of essential physical properties of vitrification solutions. Theriogenology 67, 8189.
  • Yu XL, Deng W, Liu FJ, Li YH, Li XX, Zhang YL, Zan LS, 2010: Closed pulled straw vitrification of in vitro–produced and in vivo–produced bovine embryos. Theriogenology 73, 474479.