Study on fish embryo responses to the treatment of cryoprotective chemicals using impedance spectroscopy

Investigations using electrical impedance spectroscopy to measure the responses of fish embryos to the cryoprotective chemicals, methanol and dimethyl sulphoxide (DMSO), were carried out. Zebrafish (Danio rerio) embryos were used as a model to study the newly proposed technique. The normalised permittivity and conductivity changes of the embryos were measured continuously over a 20-min period in a customised embryo-holding chamber. The normalised permittivity and conductivity spectra were obtained during embryo exposure to different concentrations of methanol (1.0, 2.0 and 3.0 M) and DMSO (0.5, 1.0 and 2.0 M) solutions. The results showed significant permittivity and conductivity changes after embryo exposure to methanol and DMSO at the optimum embryo loading level (six embryos). Embryos in different concentrations of methanol and DMSO also resulted in quantitative responses shown in the normalised permittivity and conductivity spectra. The results demonstrated that fish embryo membrane permeability to cryoprotective chemicals could be monitored in real-time. The measurement of permittivity at a lower frequency range (10–10³ Hz) and conductivity at a higher frequency range (10⁴–10⁶ Hz) during fish embryo exposure to cryoprotective chemicals using impedance spectroscopy can be used as a new tool for the fast screening of most effective cryoprotective chemicals. The results from the present study also demonstrated the possibility of quantifying the level of cryoprotective chemicals penetrating the fish embryos.     

Development of a new rapid measurement technique for fish embryo membrane permeability studies using impedance spectroscopy

Information on fish embryo membrane permeability is vital in their cryopreservation. Whilst conventional volumetric measurement based assessment methods have been widely used in fish embryo membrane permeability studies, they are lengthy and reduce the capacity for multi-embryo measurement during an experimental run. A new rapid 'real-time' measurement technique is required to determine membrane permeability during cryoprotectant treatment. In this study, zebrafish (Danio rerio) embryo membrane permeability to cryoprotectants was investigated using impedance spectroscopy. An embryo holding cell, capable of holding up to 10 zebrafish embryos was built incorporating the original system electrods for measuring the impedance spectra. The holding cell was tested with deionised water and a series of KCl solutions with known conductance values to confirm the performance of the modified system. Untreated intact embryos were then tested to optimise the loading capacity and sensitivity of the system. To study the impedance changes of zebrafish embryos during cryoprotectant exposure, three, six or nine embryos at 50% epiboly stage were loaded into the holding cell in egg water, which was then removed and replaced by 0.5, 1.0, 2.0 or 3M methanol or dimethyl sulfoxide (DMSO). The impedance changes of the loaded embryos in different cryoprotectant solutions were monitored over 30 min at 22 degrees C, immediately following embryo exposure to cryoprotectants, at the frequency range of 10-10(6)Hz. The impedance changes of the embryos in egg water were used as controls. Results from this study showed that the optimum embryo loading level was six embryos per cell for each experimental run. The optimum frequency was identified at 10(3.14) or 1,380 Hz which provided good sensitivity and reproducibility. Significant impedance changes were detected after embryos were exposed to different concentrations of cryoprotectants. The results agreed well with those obtained from conventional volumetric based studies.     

Early steps of cryopreservation of day one honeybee (Apis mellifera) embryos treated with low-frequency sonophoresis

Honeybees, major providers of pollination, are endangered in many areas. Embryo cryopreservation may be a very useful tool to maintain their genetic diversity. However, it is complex in insects, because embryos are chill sensitive and are surrounded by two protectant membranes, the chorion and vitelline. These membranes prevent penetration of cryoprotectant in the embryos. This study aimed to test different conditions of embryo preparation before cryopreservation, including low-frequency sonophoresis, a physical method of permeabilization, and passages through cryoprotectant solutions. Apis mellifera ligustica embryos were collected in artificial cell plugs 7.5 h after queens had been caged, in two different seasons (winter, spring) and were then incubated in vitro overnight (16.5 h). Embryos were individually sonicated and then incubated in three cryoprotectant baths (B1 = 10%, B2 = 20% and B3 = 40% of cryoprotectant) and quenched in liquid nitrogen. Artificial cell plugs and in vitro incubation device were efficient in producing future embryos hatching. Embryos stained ruby red with rhodamine B after sonophoresis treatment indicated that low-frequency ultrasound had permeabilized embryos. According to the treatment, different significant hatching rates were obtained after sonophoresis (up to 25%). After three cryoprotectant incubations, best hatching rates were obtained after 10 min in B1 and B2, and 40 s in B3. These results show that sonophoresis is an efficient tool to permeabilize the chorion and vitelline membrane of the day one honeybee embryo allowing a hatching rate of more than 20%. They also show that the season is an important variability factor.     

Studies on membrane permeability of zebrafish (Danio rerio) oocytes in the presence of different cryoprotectants

Investigation into fish oocyte membrane permeability is essential for developing successful protocols for their cryopreservation. The aim of the present work was to study the permeability of the zebrafish (Danio rerio) oocyte membrane to water and cryoprotectants before cryopreservation protocol design. The study was conducted on stage III and stage V zebrafish oocytes. Volumetric changes of stage III oocytes in different concentrations of sucrose were measured after 20 min exposure at 22 degrees C and the osmotically inactive volume of the oocytes (Vb) was determined using the Boyle-van't Hoff relationship. Volumetric changes of oocytes during exposure to different cryoprotectant solutions were also measured. Oocytes were exposed to 2 M dimethyl sulphoxide (DMSO), propylene glycol (PG), and methanol for 40 min at 22 degrees C. Stage III oocytes were also exposed to 2 M DMSO at 0 degrees C. Oocyte images were captured on an Olympus BX51 cryomicroscope using Linkham software for image recording. Scion Image was used for image analysis and diameter measurement. The experimental data were fitted to a two-parameter model using Berkeley Madonna 8.0.1 software. Hydraulic conductivity (L(p)) and solute (cryoprotectant) permeability (Ps) were estimated using the model. The osmotically inactive volume of stage III zebrafish oocytes was found to be 69.5%. The mean values+/-SE of Lp were found to be 0.169+/-0.02 and 0.196+/-0.01 microm/min/atm in the presence of DMSO and PG, respectively, at 22 degrees C, assuming an internal isosmotic value for the oocyte of 272 mOsm. The Ps values were 0.000948+/-0.00015 and 0.000933+/-0.00005 cm/min for DMSO and PG, respectively. It was also shown that the membrane permeability of stage III oocytes decreased significantly with temperature. No significant changes in cell volume during methanol treatment were observed. Fish oocyte membrane permeability parameters are reported here for the first time. The Lp and Ps values obtained for stage III zebrafish oocytes are generally lower than those obtained from successfully cryopreserved mammalian oocytes and higher than those obtained with fish embryos and sea urchin eggs. It was not possible to estimate membrane permeability parameters for stage V oocytes using the methods employed in this study because stage V oocytes experienced the separation of outer oolemma membrane from inner vitelline during exposure to cryoprotectants.     

Effects of methanol and developmental arrest on chilling injury in zebrafish (Danio rerio) embryos

Stage-dependent chilling sensitivity has been reported for many species of fish embryos. Most of these studies reveal that developmental stages beyond 50% epiboly are less sensitive to chilling, but the chilling sensitivity accelerates rapidly at subzero temperatures. In this study, the effects of methanol and developmental arrest on chilling injury were studied using zebrafish (Danio rerio) embryos at 64-cell, 50% epiboly, 6-somite, prim-6 and long-bud stages. Embryos were exposed to methanol or anoxic conditions before they were cooled to 0 or -5 degrees C with slow (1 degrees C/min), medium (30 degrees C/min) or fast ( approximately 300 degrees C/min) cooling rates and were held at these temperatures for different time periods. Embryo survival was evaluated in terms of the percentage of treated embryos with normal developmental appearance after 3-day culture. Experiments on the effect of methanol on chilling sensitivity of the embryos showed that the addition of methanol to embryo medium increased embryo survival significantly at all developmental stages and under all cooling conditions. Higher concentration of methanol treatment generally improved embryo survival when embryos were cooled at a fast cooling rate of 300 degrees C/min. Experiments on the effect of developmental arrest on chilling sensitivity of embryos showed that embryos at 50% epiboly and prim-6 stages underwent developmental arrest almost immediately after 15 min oxygen deprivation. After 4h in anoxia, the survival rates of the embryos were not significantly different from their respective aerobic controls. Anoxia and developmental arrest had no effect on the chilling sensitivity of zebrafish embryos.     

Japanese flounder (Paralichthys olivaceus) embryos are difficult to cryopreserve by vitrification

The first successful cryopreservation of fish embryos was reported in the Japanese flounder by vitrification [Chen and Tian, Theriogenology, 63, 1207-1219, 2005]. Since very high concentrations of cryoprotectants are needed for vitrification and fish embryos have a large volume, Japanese flounder embryos must have low sensitivity to cryoprotectant toxicity and high permeability to water and cryoprotectants. So, we investigated the sensitivity and the permeability of Japanese flounder embryos. In addition, we assessed the survival of flounder embryos after vitrification with solutions containing methanol and propylene glycol, following Chen and Tian's report. The embryos were relatively insensitive to the toxicity of individual cryoprotectants at lower concentrations, especially methanol and propylene glycol as their report. Although their permeability to water and cryoprotectants could not be measured from volume changes in cryoprotectant solutions, the embryos appeared to be permeable to methanol but less permeable to DMSO, ethylene glycol, and propylene glycol. Although vitrification solutions containing methanol and propylene glycol, which were used in Chen and Tian's report, were toxic to embryos, a small proportion of embryos did survived. However, when vitrified with the vitrification solutions, no embryos survived after warming. The embryos became opaque during cooling with liquid nitrogen, indicating the formation of intracellular ice during cooling. When embryos had been kept in vitrification solutions for 60 min after being treated with the vitrification solution, some remained transparent during cooling, but became opaque during warming. This suggests that dehydration and/or permeation by cryoprotectants were insufficient for vitrification of the embryos even after they had been over-treated with the vitrification solutions. Thus, Chen and Tian's cryopreservation method lacks general application to Japanese flounder embryos.     

Differential scanning calorimetry studies of intraembryonic freezing and cryoprotectant penetration in zebrafish (Danio rerio) embryos

Nucleation temperatures of intraembryonic water and cryoprotectant penetration in zebrafish embryos were studied using differential scanning calorimetry. The effects of embryo developmental stage, dechorionation, partial removal of yolk, cooling rate, and cryoprotectant treatment on the temperatures of intraembryonic freezing were investigated. Embryo stages were found to have a significant effect on the nucleation temperatures of intact embryos. Freeze onset temperatures of -11.9 +/- 1.5, -15.6 +/- 0.3, and -20.5 +/- 0.1 degrees C were obtained for intact embryos at 6-somite, prim-6, and high-pec stages, respectively. After dechorionation, the freeze onset temperatures of intraembryonic water shifted to significantly lower temperatures, being -23.5 +/- 0.8, -18.7 +/- 0.7, -24.9 +/- 0.8 degrees C for 6-somite, prim-6, and high-pec stages, respectively. Yolk-reduced high-pec stage embryos showed significantly lower nucleation temperatures with an average onset at -27.9 +/- 0.4 degrees C. The effect of cryoprotectant treatment on the nucleation temperatures of intraembryonic water varies among different embryo stages and different cryoprotectants. Thirty-minute treatment with 2 M methanol significantly decreased the nucleation temperatures of dechorionated 6-somite embryos whilst no temperature decrease was observed for prim-6 or yolk-reduced high-pec embryos. Thirty-minute exposure to 1 M propylene glycol did not significantly affect the nucleation temperatures of dechorionated 6-somite, prim-6, or yolk-reduced high-pec embryos. In order to increase the permeability of embryos to cryoprotectants, the yolk sacs of dechorionated embryos at 6-somite or prim-6 embryos were punctured with a sharp micro-needle before exposure to cryoprotectants. The punctured prim-6 embryos showed significantly lower temperatures of intraembryonic freezing after 30 min of exposure to 2 M methanol following the multi-punctures. The nucleation temperatures of punctured 6-somite or prim-6 embryos were also decreased significantly after exposure to 1 M propylene glycol for 30 min. These results suggested that in intact embryos, intraembryonic freezing appeared to be seeded by the external ice in the perivitelline fluid and that in dechorionated embryos (in the absence of external water) intraembryonic freezing was more likely a consequence of heterogeneous nucleation. Methanol was demonstrated to show a limited degree of penetration into prim-6 stage embryos, but it did not penetrate later-stage embryos such as prim-6 and yolk-reduced high-pec. No propylene glycol permeation was observed for embryos at all stages. However, multi-punctures of yolk resulted in the permeation of both cryoprotectants into prim-6 embryos and propylene glycol permeation into 6-somite embryos. These findings may have important implications in overcoming the problem associated with the low membrane permeability of zebrafish embryos to cryoprotectants.     

Efficiency of osmotic and chemical treatments to improve the permeation of the cryoprotectant dimethyl sulfoxide to Japanese whiting (Sillago japonica) embryos

Insufficient cryoprotectant permeation is one of the major obstacles for successful fish embryo cryopreservation. The purpose of this study was to test the effectiveness of osmotic and chemical treatments to enhance cryoprotectant uptake by fish embryos. Japanese whiting Sillago japonica embryos at the somites and tail elongation stages were treated with hyperosmotic sugar solutions (1 M trehalose and sucrose) for 2-6 min, or a permeating agent (2-6 mg/mL pronase) for 30-120 min, and then impregnated with 10-15% DMSO in artificial sea water or aqueous solutions containing inorganic salts (0.125-0.25 M MgCl₂ and CaCl₂). The viability of the embryos after the treatments was estimated from hatching rates and the internal DMSO concentration was measured by HPLC. Treatment with trehalose for 3 min prior to impregnation with DMSO enhanced the uptake of the cryoprotectant by 45% without significantly affecting embryo viability, whereas pronase had no noticeable effect on cryoprotectant permeation. Incorporation of DMSO into the embryos was enhanced by 143-170% in the presence of 0.25 M MgCl₂ and 0.125 M CaCl₂ compared to sea water. A combination of treatments with trehalose and MgCl₂ was even more effective in promoting DMSO permeation (191% compared to untreated embryos). Tail elongation embryos were less tolerant of the treatments, but had higher DMSO impregnation. In conclusion, the use of trehalose (as dehydrating agent) and MgCl₂/CaCl₂ (as a vehicle during impregnation) greatly promoted cryoprotectant uptake and may be a promising aid for the successful cryopreservation of fish embryos.     

Effect of different cryoprotectants and vitrificant solutions on the hatching rate of turbot embryos (Scophthalmus maximus)

Vitrification could provide a promising tool for the cryopreservation of fish embryos. However, in order to achieve a vitrifiable medium, a high concentration of permeable cryoprotectants must be employed, and the incorporation of high molecular weight compounds should also be considered. The toxicity of these permeable and non-permeable agents has to be assessed, particularly when high concentrations are required. In the present study, permeable and non-permeable cryoprotectant toxicity was determined in turbot embryos at two development stages (F stage-tail bud and G stage-tail bud free). Embryos treated with pronase (2mg/ml, 10 min at 22 degrees C) were incubated in dimethyl sulfoxide (Me2SO), methanol (Meth.) or ethylene glycol (EG) in concentrations ranging from 0.5 to 6M for periods of 10 or 30 min, and in 5, 10, and 15% polyvinylpyrrolidone (PVP), 10, 15, and 20% sucrose or 0.1, 1, and 2% X-1000 for 2 min. The embryos were then washed well and incubated in seawater until hatching. The toxicity of permeable cryoprotectants increased with concentration and exposure time. There were no significant differences between permeable cryoprotectants. However, embryos tolerated higher concentrations of Me2SO than other cryoprotectants. Exposure to permeable cryoprotectants did not affect the hatching rate except at G stage with X-1000 treatment and 20% sucrose. Taking into account the cryoprotectant toxicity and the vitrification ability of cryoprotectant mixtures, three vitrification solutions (V1, V2, and V3), and one protocol for stepwise incorporation were designed. The tested solutions contained 5M Me2SO+2M Meth+1M EG plus 5% PVP, 10% sucrose or 2% X-1000. The hatching rate of embryos that had been exposed to the the vitrification solutions was analyzed and no significant differences were noticed compared with the controls. Our results demonstrate that turbot embryos can be subject to this cryoprotectant protocol without deleterious effect on the hatching rate.     

New Approaches for Studying the Permeability of Fish Embryos: Toward Successful Cryopreservation

This paper describes some new approaches for understanding the permeability of teleost embryos. The dechorionated zebrafish (Brachydanio rerio) was used as a model for basic studies of water and cryoprotectant permeability. These embryos are composed of two compartments, a large yolk (surrounded by the yolk syncytial layer) and differentiating blastoderm cells. Cellular water was distributed unequally in each compartment. Measurements indicated that the total water in the embryo was 74%, while the total water in the yolk was 42%, and total water in the blastoderm was 82%. The internal isosmotic value for the zebrafish embryo is unknown. However, for one-compartment modeling studies of membrane permeability, the mean Lp (±SEM) values were 0.022 ± 0.002 to 0.049 ± 0.008 μm × min⁻¹atm⁻¹at 40 mOsm (assuming this was one possible internal isosmotic value for the entire embryo) and 0.040 ± 0.004 to 0.1 ± 0.017 μm × min⁻¹atm⁻¹at 300 mOsm (assuming this was another possible internal isosmotic value for the entire embryo). When three- and six-somite embryos were placed in 1.5 and 2.0Mcryoprotectants (dimethyl sulfoxide and propylene glycol), osmometric measurements of volume changes indicated no cryoprotectant permeation. However, similar measurements with methanol revealed a small volume decrease (ca. 8%) and recovery (ca. 5%) for six-somite embryos in a 2.0Msolution. Magnetic resonance (MR) images of the spatial distribution of three cryoprotectants (dimethyl sulfoxide, propylene glycol, and methanol) demonstrated that only methanol permeated the entire embryo within 15 min. The other cryoprotectants exhibited little or no permeation into the yolk over 2.5 h. The results from MR spectroscopy and cryoprotectant microinjections into the yolk suggested that the yolk syncytial layer plays the critical limiting role for cryoprotectant permeation throughout the embryo.     

Freezing mammalian embryos: A review of the techniques

This article reviews the literature on freezing mammalian oocytes and embryos, with emphasis on embryos of domestic animals. Mammalian embryos must be stored in a quiescent state to retain viability for long periods. This has been accomplished by freezing and storing the embryos at ?196°C. To freeze embryos, a cryoprotectant like dimethyl sulfoxide (DMSO) or glycerol was required, slow cooling (0.1 to 2.0°C/min) and warming (1 to 50°C/min) rates were used, enucleation or seeding the freezing medium was a necessity, and stepwise addition and removal of the cryoprotectant at room temperature seemed to be beneficial. Using the above parameters embryos have been frozen and stored at ?196°C for several years and upon thawing and transfer to a suitable recipient, viable offspring have developed. Initially embryo viability was low after freezing-thawing, but with refinement of freezing-thawing techniques has increased sufficiently so that freezing embryos is no longer a laboratory technique, but is applicable to field use.     

Methanol as a cryoprotectant for equine embryos

Equine embryos (n=43) were recovered nonsurgically 7-8 days after ovulation and randomly assigned to be cryopreserved in one of two cryoprotectants: 48% (15M) methanol (n=22) or 10% (136 M) glycerol (n=21). Embryos (300-1000 microm) were measured at five intervals after exposure to glycerol (0, 2, 5, 10 and 15 min) or methanol (0, 15, 35, 75 and 10 min) to determine changes (%) in diameter over time (+/-S.D.). Embryos were loaded into 0.25-ml plastic straws, sealed, placed in a programmable cell freezer and cooled from room temperature (22 degrees C) to -6 degrees C. Straws were then seeded, held at -6 degrees C for 10 min and then cooled to -33 degrees C before being plunged into liquid nitrogen. Two or three embryos within a treatment group were thawed and assigned to be either cultured for 12 h prior to transfer or immediately nonsurgically transferred to a single mare. Embryo diameter decreased in all embryos upon initial exposure to cryoprotectant. Embryos in methanol shrank and recovered slightly to 76+/-8 % of their original diameter; however, embryos in glycerol continued to shrink, reaching 57+/-6 % of their original diameter prior to cryopreservation. Survival rates of embryos through Day 16 of pregnancy were 38 and 23%, respectively (P>0.05) for embryos cryopreserved in the presence of glycerol or methanol. There was no difference in pregnancy rates of mares receiving embryos that were cultured prior to transfer or not cultured (P>0.05). Preliminary experiments indicated that 48% methanol was not toxic to fresh equine embryos but methanol provided no advantage over glycerol as a cryoprotectant for equine blastocysts.     

The effect of internal and external cryoprotectants on zebrafish (Danio rerio) embryos

The study investigated the effects of internal (DMSO, 1,2-propanediol, glycerol, ethylene glycol, methanol, N,N-dimethylacetamide) and external cryoprotectants (glucose, sucrose) on the viability and on morphometric parameters of zebrafish embryos. From the tested internal cryoprotectants, DMSO had the lowest toxicity, followed by 1,2-propanediol and glycerol. The external cryoprotectants were less toxic then the internal ones. Early ontogenetic stages were more sensible to cryoprotectant exposure than advanced stages. Two-step incubation procedures in increasing concentrations of internal and external cryoprotectants were superior to multiple-step exposure procedures. All tested vitrification solutions exceeded the tolerance limit of embryos. The tolerance of zebrafish embryos to cryoprotectants was highly variable in a concentration range causing approximately 50% embryo mortality. The width of the perivitelline space showed significant morphometrical changes due to cryoprotectant exposure. In the germinative tissue non-significant changes occurred. The yolk did not change morphometrically after exposure to internal cryoprotectants and showed no sign of dehydration after exposure to external cryoprotectants. Based on these results the study comes to the following conclusions: as yolk dehydration was impossible and as vitrification solutions were over the tolerance limit it seems unlikely that successful vitrification of zebrafish embryos can be achieved. Under these considerations slow freezing methods would be a better option as lower cryoprotectant concentrations can be used and embryos can be dehydrated during freezing.     

Successful direct transfer of vitrified sheep embryos

The use of a simple cryopreservation method, adapted to direct transfer of thawed embryos may help to reduce the costs of embryo transfer in sheep and increase the use of this technique genetic improvement of this species. Two experiments were made to test a vitrification method that is easy to apply in field conditions. All embryos were collected at Day 7 of the estrous cycle of FSH-stimulated donor ewes and were assessed morphologically, washed in modified PBS and incubated for 5 min in 10% glycerol, for 5 min in 10% glycerol and 20% ethylene glycol and were transferred into the vitrification solution (25% glycerol and 25% ethylene glycol). All solutions were based on mPBS. Embryos were loaded in straws (1 cm central part, the remaining parts being filled with 0.8 M galactose in mPBS) and plunged into liquid N2 within 30 sec of contact with the vitrification solution. The straws were thawed (10 sec at 20 degrees C) and the embryos were either transferred directly or after 5 min of incubation in the content of the straw (followed by washing in PBS) into the uterus of a recipient ewe. In Trial 1, the pregnancy rates at term (72 vs. 72%) as well as the embryo survival rates (60 vs 50% respectively) were not different between fresh (n = 48 embryos) and vitrified (n = 50) embryos. In a second trial no difference was observed between vitrified embryos transferred after in vitro removal of the cryoprotectant (n = 86 embryos) or directly after thawing (n = 72) both in terms of lambing rate (67 vs. 75%, respectively) and embryo survival rate (lambs born/embryos transferred; 49 vs. 53%). This method of sheep embryo cryopreservation provided high pregnancy and embryo survival, even after direct transfer of the embryos.     

Suitability of cryoprotectants and impregnation protocols for embryos of Japanese whiting Sillago japonica

The purpose of this study was to examine the suitability of cryoprotectant agent (CPA) impregnation protocols for the embryos of Japanese whiting (Sillago japonica), a small-sized, easy-to-rear, and prolific marine fish which may constitute a suitable experimental material for the development of cryopreservation methods for fish embryos. Our immediate goals were to assess the toxicity and permeability of various CPAs to whiting embryos of different developmental stages. Exposure of gastrula, somites, tail elongation, and pre-hatching embryos to 10%, 15%, and 20% solutions of propylene glycol (PG), methanol (MeOH), dimethyl sulfoxide (Me2SO), dimethylformamide (DFA), ethylene glycol (EG), and glycerol (Gly) in artificial sea water (ASW; 33 psu) for 20 min revealed that CPA toxicity for whiting embryos increased in the order of PG相似文献   

A simple vitrification technique for sheep and goat embryo cryopreservation

The aim of this study was to evaluate pregnancy and embryo survival rate of vitrified in vivo produced Merino sheep and Criolla goat (morulae and blastocysts) embryos, using the plastic tips of micropipettes, as containers (Cryo-tips). The embryos were exposed, at room temperature, to two successive equilibration solutions for a period of 5 min and then to a vitrification solution (VS) for 30 s. Then embryos were then loaded in 1 μl VS, into a plastic micropipette tip, and plunged into liquid nitrogen. On thawing, the embryos were warmed (37 °C) and placed into cryoprotectant dilutions (three-step-process). In the ovine, the morula and blastocyst pregnancy rates (47.1% vs 50%) and embryo survival rates (41.2% vs 50%) recorded were similar for both embryonic stages. Unlike the sheep, no pregnancies were recorded in goat vitrified/thawed morulae embryos, following transfer. However, in contrast, goats receiving blastocysts recorded high rates of pregnancy and embryo survival (64% and 64%, respectively). This technique allows for easy handling of cryopreserved embryos, is simple and efficient in both ovine embryo stages and also for goat vitrified blastocysts. The technique has definite potential application.     

Cryopreservation of sheep embryos using ethylene glycol

The objective of the study was to evaluate the use of ethylene glycol (EG) for cryopreservation of sheep embryos. A 2 × 2 factorial treatment arrangement examining one-step vs. two-step cryoprotectant addition and removal was used. The one-step cryoprotectant addition involved placement of embryos directly into 1.5 mol EG, whereas the two-step addition utilized an intermediate 10 min exposure to 0.75 mol EG. Similarly, the one-step cryoprotectant removal involved direct placement of thawed embryos into 1.0 mol sucrose, and the two-step procedure included a 10 min exposure to 0.25 mol sucrose before placement in 1.0 mol sucrose. A total of 185 frozen-thawed embryos was placed into in vitro culture for 96 h to determine viability. No differences were observed between cryoprotectant addition or removal techniques, and overall survival was 69%. To validate the results obtained in vitro, a limited number of embryo transfers was performed. Four ewes receiving a total of 11 frozen-thawed embryos produced eight lambs (73% survival) which compared favorably with 74% survival obtained by transferring 19 non-cryopreserved embryos to eight recipients. It is concluded that one-step addition of 1.5 mol ethylene glycol followed by one-step removal in a 1.0 mol sucrose gradient is an appropriate technique for cryopreservation of sheep embryos.     

Altering fish embryos with aquaporin-3: an essential step toward successful cryopreservation

Fish populations are globally threatened by overharvesting and habitat degradation. The ability to bank fish embryos by cryopreservation could be crucial for preserving species diversity, for aquaculture (allowing circannual fish farming), and for managing fish models used in human biomedical research. However, no nonmammalian embryo has ever been successfully cryopreserved. For fish, low membrane permeability prevents cryoprotectants from entering the yolk to prevent cryodamage. Here, we present evidence of a membrane mechanism hindering cryopreservation of fish and propose a novel solution to this obstacle. Zebrafish (Danio rerio) embryos have rectifying membranes that allow water to leave but not to reenter readily. This feature may be an evolutionary trait that allows freshwater embryos to grow in hypoosmotic environments without osmoregulatory organs. However, this trait may also prevent successful fish embryo cryopreservation because both water and cryoprotectants must move into and out of cells. As a solution, we injected zebrafish embryos with mRNA for the aquaporin-3 water channel protein and demonstrated increased membrane permeability to water and to a cryoprotectant. Modeling indicates that sufficient cryoprotectant enters aquaporin-3-expressing zebrafish embryos to allow cryopreservation.     

Development of OPS vitrified pig blastocysts: effects of size of the collected blastocysts, cryoprotectant concentration used for vitrification and number of blastocysts transferred

Unhatched blastocysts from Large White hyperprolific gilts (n=103) were identified, measured and vitrified using the Open Pulled Straw (OPS) technique to evaluate the effects of the collected blastocyst size and cryoprotectant concentrations used for vitrification, and the number of embryos transferred per recipient. Vitrified/warmed blastocyst viability was estimated in vitro, as the percentage of embryos developing after 72h, and in vivo, on pregnancy Day 30. In the in vitro study, we compared the use of three cryoprotectant concentrations (16.5, 18, or 20% DMSO+16.5, 18, or 20% EG+0.4M sucrose). Survival rates differed significantly between the control (98.3%) and the three cryoprotectant concentrations (67, 62.3, and 57%, respectively). Blastocyst size at vitrification determined the further in vitro development of embryos (26% survival for blastocysts 126-144microm versus 100% for blastocysts >199microm). For the in vivo study, blastocysts were vitrified using cryoprotectant concentrations of 16.5 or 18% DMSO+EG and transferred surgically in groups of 20 or 30 per recipient (n=40). Recipients were slaughtered on pregnancy D30. No significant differences were detected in gestation rates (50-70%) and embryo survival rates (14.7-25%), although survival was higher (P=0.0003) when 20 blastocysts were transferred compared to 30 (24.7% versus 15.5%). Our findings indicate that best results, in terms of subsequent in vivo embryo survival, were achieved after transferring 20 embryos at the blastocyst or expanded blastocyst stage, previously vitrified using cryoprotectant concentrations of 16.5 or 18%.     

Preliminary studies on the vitrification of red sea bream (Pagrus major) embryos

The objective was to identify an appropriate cryoprotectant and protocol for vitrification of red sea bream (Pagrus major) embryos. The toxicity of five single-agent cryoprotectants, dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycerol (GLY), and methyl alcohol (MeOH), as well as nine cryoprotectant mixtures, were investigated by comparing post-thaw hatching rates. Two vitrifying protocols, a straw method and a solid surface vitrification method (copper floating over liquid nitrogen), were evaluated on the basis of post-thaw embryo morphology. Exposure to single-agent cryoprotectants (10% concentration for 15 min) was not toxic to embryos, whereas for higher concentrations (20 and 30%) and a longer duration of exposure (30 min), DMSO and PG were better tolerated than the other cryoprotectants. Among nine cryoprotectant mixtures, the combination of 20% DMSO+10% PG+10% MeOH had the lowest toxicity after exposure for 10 min or 15 min. High percentages of morphologically intact embryos, 50.6+/-16.7% (mean+/-S.D.) and 77.8+/-15.5%, were achieved by the straw vitrifying method (20.5% DMSO+15.5% acetamide+10% PG, thawing at 43 degrees C and washing in 0.5M sucrose solution for 5 min) and by the solid surface vitrification method (40% GLY, thawing at 22 degrees C and washing in 0.5M sucrose solution for 5 min). After thawing, morphological changes in the degenerated embryos included shrunken yolks and ruptured chorions. Furthermore, thawed embryos that were morphologically intact did not consistently survive incubation.