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.

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.

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.

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

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.

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).

97 BOER GOAT OFFSPRING BORN FOLLOWING TRANSFER OF CRYOPRESERVED EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 207
Author(s):  
K. C. Lehloenya ◽  
J. P. C. Greyling
Keyword(s):  
Survival Rate ◽  
Pregnancy Rate ◽  
Genetic Material ◽  
Embryo Survival ◽  
Boer Goat ◽  
Frozen Embryos ◽  
Group 2 ◽  
The University ◽  
Fresh Embryos ◽  
Cryopreserved Embryos

Cryopreservation of embryos is an important technique in the whole MOET program, which could help improve the transportation of genetic material across South Africa and globally. This trial evaluated the survival rate of goat embryos following transfer with cryopreserved Boer goat embryos. Twenty-seven multiparous Boer goat recipients were synchronized with CIDR for 16 days and injected with 300 IU of eCG at CIDR withdrawal. The recipients were allocated into 3 groups (n = 9). Group 1 received fresh embryos; Group 2 received slow frozen embryos; and Group 3 received vitrified embryos. Expanded blastocysts used were surgically collected from donors superovulated with pFSH on 7 following AI. Two blastocysts were transferred laparoscopically to the uterine horn ipsilateral to functional CL. A pregnancy rate of 85.7% (6) was obtained following the transfer of fresh embryos and tended to be better than in the does receiving slow frozen and vitrified embryos, (n = 4; 50.0% and n = 3; 37.5% does pregnant, respectively) with no significant differences. The kidding rate of the recipient does declined to 57.0% (4) and 25.0% (2) for fresh and slow frozen groups, respectively. The embryo survival rate of 35.7% (n = 5) for fresh, 25.0% (n = 4) for conventional slow freezing and 31.3% (n = 5) for vitrification was obtained and was not affected by the number of CL present on the respective ovaries at the time of embryo transfer. Although the pregnancy rate following the transfer of fresh embryos was satisfactory, the embryo survival rate following the transfer of fresh or cryopreserved embryos tended to be lower. The authors acknowledge the University of the Free State for financial and facility support and National Research Foundation (Thuthuka) for financial support for conducting this trial.

scholarly journals Crystal-plane-dependent effects of antifreeze glycoprotein impurity for ice growth dynamics

2019 ◽  
Vol 377 (2146) ◽  
pp. 20180393 ◽  
Author(s):  
Yoshinori Furukawa ◽  
Ken Nagashima ◽  
Shunichi Nakatsubo ◽  
Salvador Zepeda ◽  
Ken-ichiro Murata ◽  
...  
Keyword(s):  
Crystal Growth ◽  
Growth Rates ◽  
Space Station ◽  
Supercooled Water ◽  
Normal Growth ◽  
Crystal Plane ◽  
Impurity Effect ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Antifreeze Glycoprotein

An impurity effect on ice crystal growth in supercooled water is an important subject in relation to ice crystal formation in various conditions in the Earth's cryosphere regions. In this review, we consider antifreeze glycoprotein molecules as an impurity. These molecules are well known as functional molecules for controlling ice crystal growth by their adsorption on growing ice/water interfaces. Experiments on free growth of ice crystals in supercooled water containing an antifreeze protein were conducted on the ground and in the International Space Station, and the normal growth rates for the main crystallographic faces of ice, namely, basal and prismatic faces, were precisely measured as functions of growth conditions and time. The crystal-plane-dependent functions of AFGP molecules for ice crystal growth were clearly shown. Based on the magnitude relationship for normal growth rates among basal, prismatic and pyramidal faces, we explain the formation of a dodecahedral external shape of an ice crystal in relation to the key principle governing the growth of polyhedral crystals. Finally, we emphasize that the crystal-plane dependence of the function of antifreeze proteins on ice crystal growth relates to the freezing prevention of living organisms in sub-zero temperature conditions. This article is part of the theme issue ‘The physics and chemistry of ice: scaffolding across scales, from the viability of life to the formation of planets’.

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