45 EFFECT OF VITRIFICATION ON KIDDING OF CAPRINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 136
Author(s):  
M. M. Toishibekov ◽  
H. Blackburn ◽  
G. A. Valieva ◽  
S. M. Askarov ◽  
B. B. Molzhigitov
Keyword(s):  
Survival Rate ◽  
Ethylene Glycol ◽  
Statistical Difference ◽  
Developmental Competence ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Heat Transmission ◽  
Bovine Semen ◽  
Fresh Frozen ◽  
Fresh Embryos

This work evaluated different methods: vitrification (V) and super-cooling ultra-rapid vitrification (SCURV). The goat morulae were cryopreserved into the High Security Vitrification (HSV) Kit (Cryo Bio System, Paris, France). The vitrification method was applied according to the method described by Vajta et al. (1998). Both treatments used a vitrification solution [VS; 20% (3.6 mol L–1) ethylene glycol (EG), 20% (2.4 mol L–1) dimethyl sulfoxide (Me2SO), and 0.5 mol L–1 of sucrose in Dulbecco's PBS (DPBS) with 10% BSA in both methods]. In our experiment, we used the Vit-Master™ apparatus (MTG GmbH, Bruckberg, Germany). The supercooled LN facilitates heat transmission between LN and the cryosolution interface and this is efficient for bovine semen and blastocyst cryoconservation (Arav et al. 2002). By surgical flushing of 30 superstimulated (1200 IU of Folligon, Intervet International, Boxmeer, the Netherlands) goats, 137 transferable morulae were harvested; 41 morulae were transferred fresh to synchronized recipients (control) and the others were cryopreserved by V (n = 47) or SCURV (n = 49), respectively thawed or warmed, and transferred to recipients. Embryos were vitrified using the HSV Kit. They were first incubated in 50% VS for 2 min and then transferred for 30 s into 100% VS. Each embryo was loaded by HSV Kit, which was immediately submerged into and stored in LN. Warming was done by placing the narrow end of the straw into DPBS + 0.25 M sucrose for 5 min. Embryos were then transferred into DPBS + 0.125 M sucrose for 3 min and finally to DPBS until transfer. The SCURV morulae were then exposed to 50 and 100% VS at 37°C for 2 min and 30 s, respectively. Embryos after saturation in VS were transferred by HSV Kit and using negative pressure of LN in the chamber for freezing with the VIT-Master. Thawing vitrified embryos was accomplished by placing the vitrified embryos in solutions of sucrose 0.25 and 0.125 M, with exposures of 2 and 3 min, accordingly. After thawing embryos, only good-quality embryos were transferred. The kidding rate following transfer of fresh, frozen-thawed vitrification, and SCURV methods were 25, 17, and 19 kids, respectively. No statistical difference was found for the percentage of does kidding following transfer of thawed embryos after vitrification (36.2 ± 4.4%a) and SCURV methods (38.7 ± 6.5%b). The survival rate following transfer of fresh embryos (60.9 ± 5.3c) was higher and in line with previous findings using VS. Differences were statistically significant (ac, bc: P < 0.05). Importantly, our data suggest that the SCURV method can be used for cryopreservation of goat morulae as the vitrification method. Although further work on the developmental competence of embryos cryopreserved with the SCURV method are needed, these data suggest that with SCURV, a faster freeze rate and lower level of cryoprotectants is able to minimize ice crystal formation and should be further evaluated as a routine mechanism for cryopreserving goat embryos.

44 SUCCESSFUL KIDDING AFTER ULTRARAPID VITRIFICATION OF GOAT EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 129
Author(s):  
Y. Toishibekov ◽  
M. Yermekova
Keyword(s):  
Survival Rate ◽  
Liquid Nitrogen ◽  
Statistical Difference ◽  
Developmental Competence ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Heat Transmission ◽  
Bovine Semen ◽  
Fresh Frozen ◽  
Fresh Embryos

This work evaluated methods for goat morulae cryopreservation by using cryoloop: vitrification (V) and super-cooling ultra-rapid vitrification (SCURV). The vitrification method was applied according to the method described by Vajta et al. (1998 Mol. Reprod. Dev. 51, 53–58). Both treatments used a vitrification solution [VS: 20% (3.6 mol L−1), ethylene glycol (EG), 20% (2.4 mol L−1) dimethylsulfoxide (Me2SO)] 0.5 mol L−1 sucrose in DPBS with 10% BSA in both methods. In our experiment, we used the Vit-Master™ (MTG, Bruckberg, Germany). The super-cooled LN facilitates heat transmission between LN and the cryosolution interface and this is efficient for bovine semen and blastocyst cryoconservation (Arav et al. 2002 Mol. Cell. Endocrinol. 187, 77–81). By surgical flushing 25 super stimulated goats, 127 transferable morulae were harvested; 39 morulae were transferred fresh to synchronized recipients (control) and the others were cryopreserved by V (n = 46) or SCURV (n = 42), respectively thawed or warmed, and transferred to recipients. Embryos were vitrified using the cryoloop. They were first incubated in 50% VS for 2 min and then transferred for 30 s into 100% VS. Each embryo was loaded by cryoloop, which was immediately submerged into and stored in liquid nitrogen. Warming was done by placing the narrow end of the cryoloop into DPBS + 0.25 M sucrose for 5 min. Embryos were then transferred into DPBS + 0.125 M sucrose for 3 min and finally to DPBS until transfer. The SCURV morulae were then exposed to 50% and 100% VS at 37°C for 2 min and 30 s, respectively. Embryos after saturation in VS were transferred by cryoloop and using negative pressure of liquid nitrogen in the chamber for freezing with the VIT-Master. Thawing vitrify embryos was accomplished by placing the vitrified embryos in solutions of sucrose 0.25 M and 0.125 M with 2- and 3-min exposures accordingly. After thawing, embryos were transferred. Statistical analysis was done using Student’s test. The kidding rate following transfer of fresh, frozen-thawed vitrification, and SCURV methods were 22, 16, and 16 kids, respectively. No statistical difference was found for the percentage of does kidding following transfer thawed after vitrification (34.7 ± 4.5%a), and SCURV methods (38.1 ± 5.9%b). The survival rate following transfer of fresh embryos (56.4 ± 4.9c) was higher and in line with previous findings using VS. Differences were statistically significant (ac, bc P < 0.05). Importantly, our data suggested that the SCURV method can be used for cryopreservation of goat morulae and has similar success to the vitrification method. While further work on the developmental competence of embryos cryopreserved with the SCURV method is needed, we hypothesise that SCURV, with a faster freeze rate and potentially a lower level of cryoprotectants, may be able to minimize ice crystal formation; SCURV should be further evaluated as a routine mechanism for cryopreserving goat embryos.

120 SUCCESSFUL LAMBING AFTER SUPER-COOLING ULTRARAPID VITRIFICATION SHEEP MORULAE

2010 ◽  
Vol 22 (1) ◽  
pp. 218
Author(s):  
Y. M. Toishibekov ◽  
H. D. Blackburn
Keyword(s):  
Ethylene Glycol ◽  
Liquid Nitrogen ◽  
Toxic Elements ◽  
Developmental Competence ◽  
Alternative Methods ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Heat Transmission ◽  
Bovine Semen ◽  
Loop Volume

The aim of this work was to establish alternative methods for sheep morulae cryopreservation by using vitrification by open pulled straw (OPS) methods and super-cooling ultra-rapid vitrification (SCURV). Both treatments used a vitrification solution (VS) of 20% (3.6 mol L-1) ethylene glycol (EG), 20% (2.4 mol L-1) dimethylsulfoxide (DMSO), 0.5 mol L-1 sucrose in DPBS with 10% BSA in both methods. In our experiment we used the Vit-Master™ (MTG, Germany). The super-cooled LN facilitates heat transmission between LN and the cryosolution interface, and this is efficient for bovine semen and blastocyst cryoconservation (Arav et al. 2002). By surgical flushing of 24 super stimulated ewes 121 transferrable morulae were harvested; 30 morulae were transferred fresh to synchronised recipients and the others were cryopreserved by OPS (n = 49) or SCURV (n = 42), respectively thawed or warmed, and transferred to recipients. Embryos were vitrified using the OPS method. They were first incubated in 50% VS for 2 min and then transferred for 30 s into 100% VS. Each embryo was loaded by touching a 1-μL drop with the straw, which was immediately submerged into and stored in liquid nitrogen. Warming was done by placing the narrow end of the straw into DPBS + 0.25M sucrose for 5 min. Embryos were then transferred into DPBS + 0.125 M sucrose for 3 min and finally to DPBS until transfer. The SCURV morulae were then exposed to 50 and 100% VS at 37°C for 2 min and 30 s, respectively. Embryos after saturation VS have been transferred by on a surface of a nylon loop (volume 20 μL, diameter 0.5 mm) and using negative pressure temperature of liquid nitrogen in the chamber for freezing with the VIT-Master. Thawing vitrified embryos was accomplished by placing the vitrified embryos in solutions of sucrose 0.25 and 0.125 with expositions of 2 and 3 min, accordingly. After embryos were thawed, only good quality embryos were transferred. Importantly, our data suggest that by using the SCURV method, the toxic elements contained in the cryopreservation solution can be reduced while maintaining a similar ability to produce viable morulae for implantation as the OPS method. Although further work on the developmental competence of embryos cryopreserved with the SCURV method are needed, these data suggest that the faster freeze rate and lower levels of cryoprotectants of SCURV are able to minimize ice crystal formation and should be further evaluated as a routine mechanism for cryopreserving sheep morulae. Table 1.Effect vitrification and ultra-rapid super-cooling vitrification on the viability and lambing of sheep morulae

155 EFFECT OF VITRIFICATION OF MORULAE ON PREGNANCY RATE IN GOAT

2015 ◽  
Vol 27 (1) ◽  
pp. 168
Author(s):  
M. M. Toishibekov ◽  
G. A. Valieva ◽  
S. M. Askarov
Keyword(s):  
Liquid Nitrogen ◽  
Statistical Difference ◽  
Developmental Competence ◽  
Alternative Methods ◽  
Heat Transmission ◽  
Group V ◽  
Bovine Semen ◽  
Student’S T ◽  
Student’S T Test ◽  
Fresh Embryos

This work evaluated alternative methods for goat morulae cryopreservation by using the High Security Vitrification Kit (Cryobiosystem): vitrification (V) and super-cooling ultra-rapid vitrification (SCURV). Vitrification was applied according to the method described by Vajta et al. (1998). Both treatments used a vitrification solution (VS) containing 20% ethylene glycol (EG), 20% dimethylsulfoxide (Me2SO), 0.5 mol L–1 sucrose in DPBS with 10% BSA. In our experiment we used the Vit-Master™ (MTG, Germany). Super-cooled liquid nitrogen (LN) facilitates heat transmission between LN and the cryosolution interface suggested to be beneficial for bovine semen and blastocyst cryoconservation. By surgical flushing of 30 super-stimulated goats, 137 transferable morulae were harvested; 41 morulae were transferred fresh to synchronized recipients (control) and the others were cryopreserved by V (n = 47) or SCURV (n = 49), respectively thawed, and transferred to recipients. Embryos were vitrified using the HSV Kit. They were first incubated in 50% VS for 2 min and then transferred for 30 s into 100% VS followed by vitrification (group V). Accordingly, morula of SCURV group were exposed to 50% VS for 2 min and to 100% VS for 30 s at 37°C. Thereafter, embryos were transferred into the VIT-Master for freezing with liquid nitrogen using negative pressure. Thawing of vitrified embryos was accomplished by placing the vitrified embryos in solutions of 0.25 M sucrose for 2 min and 0.125 M sucrose for 3 min, respectively. After thawing only survived embryos were transferred. Statistical analyses were performed with Student's t-test. After transfer of fresh or frozen-thawed embryos of V and SCURV groups, 25, 17, and 19 kids were born. No statistical difference was found for the percentage of viability of thawed embryos after vitrification (36.2 ± 4.4%), and SCURV methods (38.7 ± 6.5%). The survival of fresh embryos, however, was significantly higher (60.9 ± 5.3%). Differences were statistically significant (P < 0.05). Importantly, our data suggest that the SCURV method can be used for cryopresevation of goat morulae. Nevertheless, further work regarding the developmental competence of embryos cryopreserved with the SCURV method is needed.

57 THE EFFECT OF VITRIFICATION FOR SHEEP EMBRYO VIABILITY

2015 ◽  
Vol 27 (1) ◽  
pp. 121
Author(s):  
G. A. Valieva ◽  
M. M. Toishibekov ◽  
S. M. Askarov ◽  
B. B. Molzhigitov
Keyword(s):  
Developmental Competence ◽  
Embryo Cryopreservation ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Embryo Viability ◽  
Fresh Frozen ◽  
Loop Volume ◽  
Student’S T ◽  
Phosphate Buffered Saline ◽  
Student’S T Test

This work evaluated different methods for sheep embryo cryopreservation by vitrification (V) and super-cooling ultra-rapid vitrification (SCURV). The vitrification method was applied according to the method described by Vajta et al. Both treatments used a vitrification solution (VS) containing 20% ethylene glycol, 20% dimethylsulfoxide (Me2SO), 0.5 mol L–1 sucrose in Dulbecco's phosphate buffered saline (DPBS) with 10% BSA. The super-cooled LN facilitates heat transmission between LN and the cryosolution interface, and this is efficient for bovine semen and blastocyst cryoconservation (Arav et al. 2002). By surgical flushing 25 super-stimulated ewes, 109 transferable morulae were harvested; 35 morulae were transferred fresh to synchronized recipients (control) and the others were cryopreserved by V (n = 36) or SCURV (n = 38), respectively, thawed or warmed, and transferred to recipients. Embryos were vitrified using the HSV Kit. They were first incubated in 50% VS for 2 min and then transferred for 30 s into 100% VS. Each embryo was loaded by HSV Kit, which was immediately submerged into and stored in LN. Warming was done by placing the narrow end of the straw into DPBS + 0.25 M sucrose for 5 min. Embryos were then transferred into DPBS + 0.125 M sucrose for 3 min and finally to DPBS until transfer. The SCURV morulae were then exposed to 50 and 100% VS at 37°C for 2 min and 30 s, respectively. Embryos after saturation in VS were transferred on a surface of a nylon loop (volume 20 μL, diameter 0.5 mm) and using negative pressure of LN in the chamber for freezing with the VIT-Master. Thawing vitrified embryos was accomplished by placing the vitrified embryos in solutions of sucrose 0.25 M and 0.125 M with expositions 2 and 3 min accordingly. After thawing embryos, only good-quality embryos were transferred. Statistical analyses were performed with Student's t-test. The lambing rate following transfer of fresh, frozen-thawed vitrification and SCURV methods were 18, 12, 14 lambs accordingly. No statistical difference was found for the percentage of does lambing following transfer thawed after vitrification (33.4 ± 5.2a%) and SCURV methods (36.8 ± 6.3b%). The survival rate following transfer of fresh embryos (51.4 ± 4.8c) was higher and in line with previous findings using VS. Differences were statistically significant (ac,bc P < 0.05). Importantly, our data suggest that the HSV Kit can be used to produce viable morulae for implantation as the SCURV, and to as vitrification method. Although further work on the developmental competence of embryos cryopreserved with the SCURV method are needed, these data suggest that with SCURV a faster freeze rate and lower level of cryoprotectants is able to minimize ice crystal formation and should be further evaluated as a routine mechanism for cryopreserving sheep embryos.

96 RECOVERY RATES OF BOVINE IVP EMBRYOS CRYOPRESERVED BY SLOW FREEZING AND VITRIFICATION (OPS AND SSV) METHODS

2010 ◽  
Vol 22 (1) ◽  
pp. 207
Author(s):  
L. P. Landim Junior ◽  
L. T. S. Yamazaki ◽  
O. Watanabe ◽  
E. C. D. Benzi ◽  
D. P. Corneglian ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Slow Freezing ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Gas Atmosphere ◽  
Atmosphere Control ◽  
New Methodologies ◽  
Embryo Stage ◽  
Conventional Systems

In current commercial bovine in vitro embryo production (IVP) systems, the majority of costs are related to acquisition and preparation of recipient cows, which must be proportional to the produced embryos. Nevertheless, most of the time this relation is not obtained, and one option is the cryopreservation of extra embryos. Unfortunately, there is a large difference in the pregnancy rates of fresh and cryopreserved IVP embryos or the rates of recovery of frozen-thawed embryos according to the technique used for cryopreservation. The aim of this work was to compare recovery taxes (re-expansion and eclosion) of bovine IVP embryos produced following conventional systems, without any specific media or supply for cryopreservation, simulating one condition of extra produced embryos. The COC obtained from abattoir ovaries were matured (TCM-199, supplemented with FCS, LH, FSH, E2, pyruvate, and antibiotic) for 24 h and fertilized (Fert-TALP supplemented with BSA, PHE, and heparin) for 18 to 22 h (Day 0) in vitro. At Day 1, presumptive zygotes were transferred to development media (SOFaa supplemented with BSA and FCS), and at Day 7, grade I embryos were submitted to 3 different cryopreservation methods: slow freezing (ethylene glycol 1.5 M from 6°C to -35°C, 1°C min-1) and vitrification (DMSO, ethylene glycol, and sucrose) by OPS or SSV After the cryopreservation process, all embryos were specifically thawed according to their method and re-cultured in SOFaa for 48 h when the re-expansion and eclosion taxes were evaluated (number of viable embryos after the cryopreservation process). According to results, the taxes of viable embryos cryopreserved by vitrification methods were better than slow freezing, except expanded blastocysts cryopreserved by slow freezing, disproving literature data that show more ability for ice crystal formation in this embryo stage than others due to liquid storage. When the total of embryos is considered in the different methods (n = 1464), the vitrification method was superior to the others, but when new methodologies could be applied aimed at less lipid storage in structures (oocytes and embryos) by gas atmosphere control or total-defined cultured media, better rates can most likely be obtained with the OPS method as SSV or slow freezing. Table 1.Viable embryo rates after different cryopreservation methods WTA-Watanabe Applied Technology.

scholarly journals 46 VITRIFICATION OF IMMATURE AND MATURE BOVINE OOCYTES

2017 ◽  
Vol 29 (1) ◽  
pp. 130
Author(s):  
P. T. Hardin ◽  
F. A. Diaz ◽  
B. A. Foster ◽  
E. J. Gutierrez ◽  
K. R. Bondioli
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Maturation Stage ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Exact Test ◽  
Immature Oocytes ◽  
Commercial Source ◽  
Bovine Oocytes

While vitrification has become a valuable system used in oocyte and embryo preservation, there is still much to be learned in optimizing this protocol. Both mature and immature oocytes can be vitrified but each presents challenging aspects. Mature oocytes have microfilaments that are not yet developed in immature oocytes, which are fragile and may be disrupted by ice crystal formation during freezing. Further, currently many different cryoprotectants are used in different concentrations, most being combinations of dimethyl sulfoxide (DMSO), glycerol, and ethylene glycol. This study aimed to determine if vitrification solutions composed of ethylene glycol and either dimethyl sulfoxide or glycerol resulted in more-competent post-thaw oocytes, and to determine if maturation stage affected optimal vitrification solution. As validation of the IVF protocol, fresh mature oocytes from a commercial source were fertilized and proportion, with pronuclei formation 48 h post-IVF was recorded. Two experiments evaluated 2 cryoprotectant solutions by analysing post-vitrification and thaw competence of in vitro-fertilized oocytes to form pronuclei. Oocytes in both studies were exposed to 2 sequential vitrification solutions containing 10% DMSO or glycerol, 10% ethylene glycol and 0.5 M sucrose, and then 20% DMSO/glycerol and ethylene glycol and 0.5 M sucrose, before vitrification on cryolocks. In the first study, immature bovine oocytes (n = 200) were vitrified. Following thawing and IVM, they were analysed for pronuclei formation, with 8.49% and 0% fertilization following vitrification in DMSO and glycerol, respectively (P < 0.01). In the second study, mature oocytes were vitrified (n = 200), thawed, and fertilized using the same methods as in study 1. In total, 12.62% and 3.4% of the mature oocytes were successfully fertilized following vitrification in DMSO and glycerol, respectively (P < 0.05). Fisher’s exact test was used for all statistics in both studies. These results suggest that DMSO in combination with ethylene glycol may be superior to glycerol for vitrification of both immature and mature bovine oocytes.

Parameters of Successful Tissue Freezing, Studied After Substitution at – 50°C With 70% Glycol

1967 ◽  
Vol 25 ◽  
pp. 26-27
Author(s):  
Daniel C. Pease
Keyword(s):  
Ethylene Glycol ◽  
Direct Contact ◽  
Eutectic Mixture ◽  
Mechanical Deformation ◽  
Freezing Process ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Fresh Tissue ◽  
Subzero Temperatures ◽  
Macromolecular Systems

Much of the water of fresh tissue can be successfully substituted with an eutectic mixture of ethylene glycol (70%) at -50°C since this removes sufficient water to stabilize macromolecular systems. Dehydration then can be completed at subzero temperatures if it is so desired. The virtue of the method is its consistent reliability. It is also possible to add 5%. glutaraldehyde to the substitution medium and so fix the tissue as it is being dehydrated. It would appear that this obviates any gross specimen damage during dehydration. Thus it makes possible an accurate assessment of damage due to gross ice crystal formation during the freezing process itself.Kidney and pancreatic tissues in particular have been studied after subjecting them to different sorts of freezing in an effort to determine the simplest and most reliable method that is fully effective. It appears that preparations rapidly frozen in a supercooled 70%. ethylene glycol bath at dry ice temperature (-77°C) regularly are morphologically as well preserved as comparable specimens frozen at -160°C in a Freon 22 bath, or as specimens frozen as rapidly as possible at -196°C by direct contact with a massive polished brass block kept immersed in liquid nitrogen until immediately before use. Indeed, the -77°C glycol bath was superior to the -160°C Freon 22 bath in that there was never any problem with gaseous films forming protective envelopes, and it was superior to pressing specimens against the -196°C block in that there was no mechanical deformation of the tissue. The effect iveness of 70% glycol at -77°C as a quenching bath has been further demonstrated by studying liver, cartilage and brain.

High-pressure freezing and freeze substitution of plant tissue

1992 ◽  
Vol 50 (1) ◽  
pp. 748-749
Author(s):  
William P. Sharp ◽  
Robert W. Roberson
Keyword(s):  
High Pressure ◽  
Cell Structure ◽  
Leaf Tissue ◽  
Freeze Substitution ◽  
Crystal Formation ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Cell Components ◽  
Cold Metal ◽  
Brome Grass

The aim of ultrastructural investigation is to analyze cell architecture and relate a functional role(s) to cell components. It is known that aqueous chemical fixation requires seconds to minutes to penetrate and stabilize cell structure which may result in structural artifacts. The use of ultralow temperatures to fix and prepare specimens, however, leads to a much improved preservation of the cell’s living state. A critical limitation of conventional cryofixation methods (i.e., propane-jet freezing, cold-metal slamming, plunge-freezing) is that only a 10 to 40 μm thick surface layer of cells can be frozen without distorting ice crystal formation. This problem can be allayed by freezing samples under about 2100 bar of hydrostatic pressure which suppresses the formation of ice nuclei and their rate of growth. Thus, 0.6 mm thick samples with a total volume of 1 mm3 can be frozen without ice crystal damage. The purpose of this study is to describe the cellular details and identify potential artifacts in root tissue of barley (Hordeum vulgari L.) and leaf tissue of brome grass (Bromus mollis L.) fixed and prepared by high-pressure freezing (HPF) and freeze substitution (FS) techniques.

The “KIMWIPE” Effect in Ultra-Thin Cryosections

1985 ◽  
Vol 43 ◽  
pp. 458-459
Author(s):  
I. Taylor ◽  
P. Ingram ◽  
J.R. Sommer
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Muscle Fibers ◽  
Ice Crystals ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Thin Sections ◽  
Skeletal Muscle Fibers ◽  
Freeze Dried ◽  
Ice Crystal Formation

In studying quick-frozen single intact skeletal muscle fibers for structural and microchemical alterations that occur milliseconds, and fractions thereof, after electrical stimulation, we have developed a method to compare, directly, ice crystal formation in freeze-substituted thin sections adjacent to all, and beneath the last, freeze-dried cryosections. We have observed images in the cryosections that to our knowledge have not been published heretofore (Figs.1-4). The main features are that isolated, sometimes large regions of the sections appear hazy and have much less contrast than adjacent regions. Sometimes within the hazy regions there are smaller areas that appear crinkled and have much more contrast. We have also observed that while the hazy areas remain still, the regions of higher contrast visibly contract in the beam, often causing tears in the sections that are clearly not caused by ice crystals (Fig.3, arrows).

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