Abstracts: CryoLetters 40 (2), 2019

CryoLetters is a bimonthly, international journal for low temperature science and technology



Volume 40, No. 2 March/April 2019

ISSN 0143-2044



Application of pectin from Aloe arborescens Mill. to the cryopreservation of human leukocyte cell suspensions
O.O. Zaitseva, V.V. Golovchenko, M.I. Sergushkina and
А.N. Khudyakov, T.V. Polezhaeva




Effect of L-tryptophan on sperm quality of Tigris scraper (Capoeta umbla) (Pisces: Cyprinidae) after cryopreservation
Filiz Kutluyer, Önder Aksu and Mehmet Kocabaş




The impact of cryopreservation-thawing conditions on umbilical cord blood quality and transplantation outcomes
Shaoqing Wu, Jieying Wu, Jinsong Chen, Yan Lu, Yan Li,
Xuewei Tang, Xunsha Sun, Guie Xie and Can Liao




Effect of tris-egg yolk, soya milk, and liposome-based extenders on Sahiwal (Bos indicus) sperm quality during pre- and post-cryopreservation stages
Ajeet Singh, Mukesh Bhakat, Tushar Kumar Mohanty,
Santu Mondal, S. K. Yadav, Pradeep Kumar, Raj Kumar,
Abdul RahimR. Sinha and Nadeem Shah




Modifications to a Vitis shoot tip cryopreservation procedure: effect of shoot tip size and use of cryoplates
Jean Carlos Bettoni, Remi Bonnart, Ashley N. Shepherd,
Aike Anneliese Kretzschmar and Gayle M. Volk




Cryopreservation of endangered wild species, Aster altaicus var. uchiyamae Kitam, using droplet-vitrification procedure
Chung-Ho Choi, Elena Popova, Hyoeun Lee, Sang-Un Park,
Jajung Ku, Jung-Hwa Kang and Haeng-Hoon Kim




Using Rapid I method for vitrification of bovine oocytes
Patrycja Mrowiec, Agnieszka Nowak, Joanna Kochan
and Wiesława Młodawska




Optimization of the protocol for cryopreservation of Arabian stallion spermatozoa: effects of centrifugation, semen extenders and cryoprotectants
A.M. Ghallab, Mostafa M. Abou-Ahmed, Aya M. Fadl, Diya A. El-Badry, Abdallah M. Shahat and Adel R. Moawad







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CryoLetters 40 (2), 71-76 (2019)
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O.O. Zaitseva*, V.V. Golovchenko, M.I. Sergushkina, А.N. Khudyakov,
T.V. Polezhaeva

Physiology Institute at the Komi Scientific Center of the Russian Academy of Sciences, Komi Republic, Syktyvkar, Russian Federation.
* For correspondence:


BACKGROUND: The molecular weight (MW) of pectic polysaccharide АА3 equal to 150 kDa and its structure suggest that it has a non-permeating protector action. OBJECTIVE: To study the ability of low methylesterified galacturonan АА3, extracted from Aloe arborescens Mill., to influence the cryoprotective properties of the refrigerant in the cryopreservation of leukocytes (-20°С, -80°С). METHODS: Pectic polysaccharide АА3 was extracted from fresh leaves using a 0.7% (w/w) solution of ammonium oxalate. The cryoscopic method was used to study its ability in different concentrations (0.1-1% w/v) in a mixture with glycerol to change the osmolarity and freezing temperature of the venous blood. Venous blood was protected with a comprehensive cryopreservative solution (3.5 % glycerol + 0.1 % pectic polysaccharide АА3) and stored at -20°C and -80°C for 1 and 7 days. RESULT: The cryoprotective properties of glycerol solution (3.5%) containing AA3 at 0.1 to 1.0% led to a decrease in the osmolarity of venous blood and accelerated the process of water freezing. So, these solutions act as nucleators. A solution of AA3 (0.1%) and glycerol (3.5%) improves the morphological preservation of granulocytes, resistance of cell membranes to eosin and phagocytic activity of neutrophils after -20°C storage for 7 days. CONCLUSIONS: The addition to glycerol solution (3.5%) of pectic polysaccharide АА3 (0.1%) may facilitate the formation of a three-dimensional network of intermolecular hydrogen bonds between the functional groups of pectin (mainly carboxyl) and hydroxyl groups of polyhydric alcohol. Better linkage of cellular water molecules in the biological specimen may improve cryoprotection.

Keywords: AA3, crystallization temperature, leukocytes, galacturonic acid, degree of etherification, cells viability.




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CryoLetters 40 (2), 77-82 (2019)
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Filiz Kutluyer1*, Önder Aksu1 and Mehmet Kocabaş2

1 Munzur University, Fisheries Faculty, 62000, Tunceli, Turkey.
2 Karadeniz Technical University Faculty of Forestry, Department of Wildlife Ecology and Management 61080, Trabzon, Turkey.
3 Munzur University, Engineering Faculty, Department of Food Engineering, 62000, Tunceli, Turkey.
*Corresponding author email:


BACKGROUND: The aromatic amino acid L-tryptophan is a versatile molecule and needed for the biosynthesis of proteins. Due to metabolic functions, it has been widely used in research and clinical trials. OBJECTIVE: The present study aimed to test the usefulness of L-tryptophan as an extender of Tigris scraper (Capoeta umbla) sperm during cryopreservation. MATERIALS AND METHODS: Semen samples were diluted with a ratio of 1:3 (sperm: extender) and base extenders were supplemented with L-tryptophan (0 mM, 0.5 mM, 1 mM, 2 mM and 4 mM). Sperm motility and duration of post-thawed samples were assessed after the cryopreservation process. RESULTS: Our results revealed that motility rate and duration of sperm increased when the cryomedia was supplemented with L-tryptophan. On the other hand, an increase in the concentration of L-tryptophan in the extender caused a significant decrease (p<0.05) in the motility rate of Tigris scraper (C. umbla) sperm. The ideal results were obtained at 1 mM L-tryptophan; however, no motile sperm were shown in any samples when a concentration of 2 mM was used. CONCLUSION: The current study shows the importance of conducting further studies related to long-term storage and reproduction management.

Keywords: Tigris scraper, Capoeta umbla, L-tryptophan, sperm cryopreservation.




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CryoLetters 40 (2),83-93 (2019)
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Shaoqing Wu, Jieying Wu, Jinsong Chen, Yan Lu, Yan Li,
Xuewei Tang, Xunsha Sun, Guie Xie* and Can Liao*

Guangzhou Cord Blood Bank, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, China.
*For correspondence: and


BACKGROUND: Cord blood units (CBUs) go through the cryopreservation-thawing process for storage before use in transplantation. The differences in the cryopreservation-thawing process affect the quality of CBUs. The effects of the cryopreservation-thawing process on the final outcomes of CBU transplantation has not been defined well. OBJECTIVE: To study the impact of differences in the cryopreservation-thawing process on the quality of CBUs and the final clinical outcomes of transplantation. MATERIALS AND METHODS: The differences in cryopreservation-thawing conditions were analyzed to determine their effect on the quality and clinical outcomes of transplanted CBUs. CBUs were detected using the same reagents, instruments and methods for minimizing experimental errors.  RESULTS: The differences in the cryopreservation-thawing process did not change cell survival, TNCC recovery (CD34+ and CFUs), the implantation rates and recovery time of neutrophils/100-day platelets.  CONCLUSION: The present study shows that the differences in the cryopreservation-thawing conditions do not influence the quality and transplantation outcomes of CBUs.

Keywords: cord blood, cryopreservation, clinical transplantation




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CryoLetters 40 (2), 94-102 (2019)
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Ajeet Singh1, Mukesh Bhakat1*, Tushar Kumar Mohanty1,
Santu Mondal1, S. K. Yadav1, Pradeep Kumar2, Raj Kumar1,
Abdul Rahim1 R. Sinha1 and Nadeem Shah.

1 Artificial Breeding Research Centre, ICAR-National Dairy Research Institute, Karnal-132001 (Haryana) India.
2 Division of Dairy Microbiology, ICAR-National Dairy Research Institute-132001.
 *Corresponding author’s e-mail:


BACKGROUND: Even though there are plenty of semen cryopreservation extenders available, their adoption is limited. Although normal tris-based egg yolk (EYC) extender is widely used, it leads to compromised post-thaw sperm quality. OBJECTIVE: To find a standard semen extender, six different semen extenders were validated. METHODS: In a split study, six aliquots of zebu cattle fresh semen ejaculate were cryopreserved in extenders containing egg yolk obtained from hen which was reared either in 1) normal, 2) omega-3 enriched, and 3) herbal enriched diet supplementation, and egg yolk free extenders such as 4) soya lecithin, 5) Bioxcell and 6) Optixcell. RESULT: Significantly poor sperm quality and kinematics were observed in extender containing herbal egg yolk. However, omega-3 enriched egg yolk extender was on par with EYC. Among all extenders, soya lecithin and bioxcell have shown better sperm quality. Sperm motility was significantly higher in semen extended in liposome-based extender Optixcell. CONCULSION: Optixcell can be considered as a standard extender for cattle semen cryopreservation to maintain adequate sperm quality required for artificial insemination.

Keywords: Bioxcell, Optixcell, Sahiwal bull, semen quality parameters, soya lecithin,Tris-egg yolk extender.




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CryoLetters 40 (2), 103-112 (2019)
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Jean Carlos Bettoni123*, Remi Bonnart3, Ashley N. Shepherd3,
Aike Anneliese Kretzschmar1, Gayle M. Volk3*

1Santa Catarina State University (UDESC), Lages, SC, 88520000, Brazil
2CAPES Foundation, Ministry of Education of Brazil, Brasília, DF, 70040020, Brazil
3USDA-ARS National Laboratory for Genetic Resources Preservation, Fort Collins, CO 80521, USA
*Corresponding author email:;


BACKGROUND: Cryopreservation has been considered a preferred method for the long-term storage of plant germplasm, especially to efficiently conserve and maintain the genetic integrity of genebank materials. Droplet-vitrification (DV) procedures have been developed to cryopreserve Vitis shoot tips from in vitro-grown plants. OBJECTIVE: This research focused on optimizing shoot tip sizes for DV and the feasibility of using cryo-plates for Vitis cryopreservation. MATERIALS AND METHODS: Uniform shoot tips were obtained from nodal sections cultured from in vitro-grown stock plants of Vitis aestivalis and Vitis jacquemontii (PI 135726). Shoot tips were precultured for 3 days on medium containing 0.3 M sucrose, salicylic acid, glutathione (reduced form), and ascorbic acid. They were cryopreserved using either DV on aluminum foil strips or by placement in calcium alginate gel in the wells of aluminium cryo-plates (V cryo-plate method). Shoot tips were then treated with loading solution followed by PVS2 treatment prior to liquid nitrogen (LN) exposure. Shoot tips were warmed in unloading solution and placed on recovery medium. The effect of extraction or non-extraction of the cryopreserved shoot tips from alginate beads was also tested. RESULTS: The highest regrowth levels of cryopreserved shoot tips were obtained using 1 mm shoot tips and a PVS2 exposure for 90 min at 0C with the DV method on aluminum foil strips or by using 30 min of PVS2 at 22C using V cryo-plates. CONCLUSION: Shoot tip size is an important factor in the cryopreservability of Vitis shoot tips; 1 mm shoot tips were the most successful for the DV cryopreservation method that was tested. In addition, the V cryo-plate cryopreservation technique described herein can be easily executed and results in high regrowth levels (≥70%) with quality plants obtained from cryo-exposed shoot tips, making it a practical and promising Vitis cryopreservation methodology.

Keywords: cryo-plate, vitrification, genebanking, PVS2




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CryoLetters 40 (2), 113-122 (2019)
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Cryopreservation of ENDANGERED WILD SPECIES Aster altaicus var. uchiyamae Kitam, Using Droplet-vitrification Procedure

Chung-Ho Choi1, Elena Popova2, Hyoeun Lee3, Sang-Un Park4,
Jajung Ku5, Jung-Hwa Kang6, Haeng-Hoon Kim3*

1Gyeonggi-do Forestry Environment Research Center, Osan-si, 52319, Korea
2National Cell Culture Collection, Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia.
3Dept. of Well-being Resources, Sunchon National University, Suncheon, 57922, Korea.
4Div. Plant Science and Resources, Chungnam National University, Daejeon 34134, Korea.
5Forest Policy Division, Korea Forest Service, 189 Cheongsa-ro, Daejeon 35208, Korea
6Hantaek Botanical Garden Foundation, 2 Hantaek-ro, Yongin-si, 17183, Korea.


BACKGROUND: Aster altaicus var. uchiyamae Kitam is an endemic and endangered species in urgent need of a comprehensive conservation strategy. OBJECTIVE: To develop an efficient cryopreservation protocol using in vitro shoot tips to complement traditional conservation approaches in case seeds are not available or insufficient for conservation programs. METHODS: Shoot tips of in vitro plants were cryopreserved using a droplet-vitrification method following improvement of pre-culture, osmoprotection, vitrification solution (VS), unloading and post-culture treatments. The starting protocol included step-wise pre-culture with 10% and 17.5% sucrose for 55 h and 17 h, respectively, followed by osmoprotection with C4-35% (17.5% glycerol + 17.5% sucrose) for 30 min, and cryoprotection with B5-80% (40% glycerol + 40% sucrose) for 60 min. RESULTS: Shoot tips of A. altaicus were found to be moderately sensitive to the osmotic stress. Pre-culture and osmoprotection were not critical for the regeneration of cryopreserved explants when either of these treatments was applied.  Osmoprotection with C4-35% on ice for 60 min followed by cryoprotection with A3-80%, a modified and diluted PVS2, on ice for 60 min resulted in the highest (65.3%) regeneration of cryopreserved shoot tips. Among alternative VSs tested, A3-80% and B5-80% were superior to PVS2 and PVS3 used under the same conditions. Step-wise recovery of shoot tips on ammonium-free medium followed by GA3-containing medium and medium without growth regulators were critical for the normal regeneration of both VS-treated and cryopreserved shoot tips. CONCLUSIONS: Cryopreservation of in vitro shoot tips using droplet-vitrification was developed as a complementary conservation approach for A. altaicus. Adjustment of the composition of regrowth media depending on recovery stage was important for the regeneration of healthy plants from cryopreserved shoot tips.

Keywords: ammonium nitrate, Asteraceae family, post-culture media, shoot tips, vitrification solution.




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CryoLetters 40 (2), 123-128 (2019)
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Patrycja Mrowiec1, Agnieszka Nowak1*, Joanna Kochan1
and Wiesława Młodawska1

1University of Agriculture in Krakow, Faculty of Animal Sciences, Institute of Veterinary Sciences, al. Mickiewicza 21, 30-120 Krakow, Poland
*For correspondence:


BACKGROUND: Vitrification is commonly used for cryopreservation of gametes. OBJECTIVE: The study aimed at evaluating viability and developmental competence of bovine oocytes vitrified by Rapid-I method. MATERIALS AND METHODS: Oocytes after collection (group 1) and IVM (group 2) were vitrified using medium containing 18% Ficoll, 40% ethylene glycol, 0.3 M sucrose. To evaluate viability, oocytes after warming were stained with fluorescein diacetate and ethidium bromide. In experiment 2, oocytes after IVM and vitrification were activated by 0.5 µM ionomycin in TCM 199 combined with 2 mM 6-DMAP in TCM 199 with 10% fetal bovine serum. RESULTS: Survival rate in group 1 was 58%, and 88% in the control. In group 2, 63% viable oocytes were found, compared to 82% in the control group. After parthenogenetic activation 27.2% morulas were observed. This percentage was lower than in the non-vitrified group (31%). CONCLUSION: Maturity stage of bovine oocytes has no effect on their survival after vitrification.

Keywords: bovine, oocyte, Rapid I, vitrification




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CryoLetters 40 (2), 129-138 (2019)
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A.M. Ghallab1, Mostafa M. Abou-Ahmed1, Aya M. Fadl1,
Diya A. El-Badry2, Abdallah M. Shahat1 and Adel R. Moawad1*

1 Department of Theriogenology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt.
2 Department of Artificial Insemination and Embryo Transfer, Animal Reproduction Research Institute, Al Aharam, Giza, Egypt
* Corresponding author’ email:


BACKGROUND: Cryopreservation of Arabian stallion semen is important in order to improve the function and fertility of frozen/thawed semen in this breed. OBJECTIVE: To investigate the effects of centrifugation, type of semen extenders, and type of cryoprotectants on the quality of frozen/thawed Arabian stallion spermatozoa. MATERIALS AND METHODS: Semen samples collected from four adult Arabian stallions (one ejaculate per week for 10 consecutive weeks) were either processed directly without centrifugation (no centrifugation; NC) or subjected to centrifugation on the gel-free fraction. Centrifugation protocols were divided into six categories; 600 x g for 10 min (C1), 600 x g for 15 min (C2), 900 x g for 10 min (C3), 900 x g for 15 min (C4), 1200 x g for 10 min (C5), or 1200 x g for 15 min (C6) (Experiment 1). Two semen extenders, INRA-82 and modified Kenney's were compared (Experiment 2). Three different cryoprotectants, [namely 5% glycerol, 5% dimethylformamide (DMF) and 2.5% glycerol] plus 2.5% DMF were used (Experiment 3). Following freezing and thawing, motility, viability, plasma membrane integrity, acrosome status and viability index were evaluated. RESULTS: Centrifugation at 600 x g for 15 min before cryopreservation increased (P< 0.05) sperm motility, viability, membrane integrity and percentage of spermatozoa with intact acrosome compared to other centrifugation protocols. Dilution of Arabian stallion semen with INRA-82 before cryopreservation improved (P< 0.05) sperm quality after freezing and thawing compared to modified Kenney's extender. Supplementation of semen diluent INRA-82 with 5% DMF improved (P< 0.05) the quality of frozen/thawed Arabian stallion spermatozoa compared to 5% glycerol. CONCLUSION: These findings suggest that optimized conditions such as centrifugation, types of semen extenders and cryoprotectants play an important role in processing Arabian stallion spermatozoa for cryopreservation.

Keywords: Arabian stallion, centrifugation, cryopreservation, extender, semen.

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