Abstracts: CryoLetters 38 (5), 2017

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



Volume 38, No. 5 September/October 2017

ISSN 0143-2044



Timings of major glaciations, inherent selection schemes and the origins of cold, hardy animal phyla
Robyn Beckett and Jonathon Stone




Effects of cryopreservation on ultrastructural morphology of white shrimp (Litopenaeus Vannamei) sperm
Yongzhen Zhao, Chunling Yang, Xiuli Chen, Min Peng,
Xiaohan Chen and Digang Zeng




A simple method for cryopreservation of shoot tips of Arabidopsis genotypes
Johanna Stock, Angelika Senula, Manuela Nagel,
Hans-Peter Mock and E.R. Joachim Keller




Effect of tert-butyl hydroquinone on bull semen cryopreservation
Sayed Mohammad Hadi Hussaini, Mahdi Zhandi,
Ahamd Zare Shahneh, Mohsen Sharafi,
Ardeshir Nejati-Javaremi, Alireza Yousefi, Mojtaba Emamverdi,
Mohamed Ahmed Mohamed Mahmoud Shehab-El-Deen
and Felipe Martinez-Pastor




Effect of cryolipolysis on abdominal adiposity of women
Patricia Froes Meyer, Anne Caroline Guarines Furtado,
Sayonara Fatima Tomaz Morais,
Luis Gonzaga de Araujo Neto,
Rodrigo Marcel Valentim da Silva, Melyssa Lima Medeiros
and Claudete Arruda Maciel de Queiroz




(Cryo)conservation of Ajania pacifica (Nakai) Bremer et Humphries shoot tips via encapsulation-dehydration technique
Dariusz Kulus and Agnieszka Abratowska




Effect of developmental stage of embryos at freezing on live birth outcomes after frozen embryo transfer
Xiao jian Zhang, Qun Lv, Li hua Min, Xue hua Cao
and Xiao jie Li




The parthenogenetic development of porcine in vitro matured oocytes vitrified before or after electric activation
Guoquan Wu, Decai Xiang, Bin Zhang, Qionghua Hong
and Guobo Quan




An in vitro assessment of the value of sucrose supplementation to warming media for the vitrification of bovine embryos
Van Huong Do, Simon Walton, Sally Catt
and Andrew W. Taylor-Robinson






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CryoLetters 38 (5), 347-356 (2017)
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Robyn Beckett* and Jonathon Stone

Department of Biology, McMaster University, 1280 Main St W, Hamilton Canada L8S 4L8
*Corresponding author email:


BACKGROUND: Knowing the origin dates of specific phyla may help in understanding the intricate relationships between environments and their biota. Past extreme environments would have challenged biota and led to the evolution of phyla that were and possibly still are able to withstand extreme conditions.  OBJECTIVE: We test the hypothesis that major glaciation events imposed strong selection schemes, ultimately leading to the origins of cold hardy phyla. METHODS: We identified dates of past major glaciation events, cold hardy phyla and their origin dates and synthesized these data in a phylogenetic context. RESULTS: Origin dates of cold hardy phyla do not correspond with major glaciation events, falsifying the hypothesis.  CONCLUSION: An alternative hypothesis is proposed in which varying degrees of cold hardiness evolved at other taxonomic levels within these phyla.

Keywords: Adaptation, cladogram, hypothesis testing, Snowball Earth




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CryoLetters 38 (5), 357-363 (2017)
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Yongzhen Zhao1, Chunling Yang1, Xiuli Chen, Min Peng,
Xiaohan Chen2 and Digang Zeng2

Guangxi Key Laboratory of Aquatic Genetic Breeding and Healthy Aquaculture, Guangxi Academy of Fisheries Sciences, Nanning, China
The first two authors contributed equally to this work.
*Corresponding author email:


BACKGROUND: The structural integrity of a spermatozoon is very important for the processes of fertilization and embryo development. OBJECTIVE: To provide valuable data for developing better cryopreservation techniques for shrimp sperm.  MATERIALS AND METHODS: Using scanning electron microscopy and transmission electron microscopy, we examined the morphological alteration of Litopenaeus vannamei sperm after cryopreservation with different concentrations of dimethylsulfoxide (DMSO). RESULTS: We found that the damaged post-thaw sperm presented either vesiculated acrosomal contents, wrinkled membranes, perforated membranes, and loss of the acrosomal spike. The seriously damaged sperm showed missing acrosomal spikes, deformed nuclei, burst nuclear membranes, and vacuolated nuclei. In addition, we found that the post-thaw sperm stored with 5% DMSO had the highest viability rate and lowest DNA damage coefficient by eosin-nigrosin staining and comet assay. CONCLUSION: Our results suggested that cryopreservation has deleterious effects on ultrastructural morphology of L. vannamei sperm, especially on acrosomal spikes and membranes.

Keywords: Litopenaeus vannamei, Sperm, Cryopreservation, Ultrastructural morphology




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CryoLetters 38 (5), 364-371 (2017)
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Johanna Stock*, Angelika Senula, Manuela Nagel,
Hans-Peter Mock and E.R. Joachim Keller

Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstraße 3, D-06466 Seeland OT Gatersleben, Germany.
*Corresponding author email:


BACKGROUND: The use of the model plant Arabidopsis could be a valuable tool to elucidate the basic mechanisms involved in plant cryopreservation. OBJECTIVE: A simple and powerful protocol, independent of Arabidopsis genotypes, was established using a PVS2 protocol.  MATERIALS AND METHODS: Two PVS2 (a and b), one PVS3 droplet-vitrification and one DMSO droplet-freezing protocol were tested with alternating temperatures during the growing phase of donor plants. RESULTS: PVS2 protocols, including cold acclimation of donor plants, resulted in highest recovery. The PVS2a protocol was successfully applied to a collection of different Arabidopsis genotypes with an average recovery of 94%. In addition, Differential Scanning Calorimetry confirmed the occurrence of glass transitions in the PVS3 and PVS2 protocols.  CONCLUSION: The PVS2a protocol is suitable to screen the large collection of Arabidopsis mutants and transgenic lines with the aim to identify cellular functions associated with cryopreservation tolerance.

Keywords: Arabidopsis thaliana, cryopreservation, DSC, PVS2, shoot tips




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CryoLetters 38 (5), 372-378 (2017)
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Sayed Mohammad Hadi Hussaini1, Mahdi Zhandi1*,
Ahamd Zare Shahneh1, Mohsen Sharafi2,3, Ardeshir Nejati-Javaremi1,
Alireza Yousefi1, Mojtaba Emamverdi1,
Mohamed Ahmed Mohamed Mahmoud Shehab-El-Deen4
and Felipe Martinez-Pastor5,6

1Department of Animal Science, College of Agriculture and Natural Resources, University of Tehran,Karaj, Iran
2Department of Poultry Sciences, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran
3Department of Embryology at Reproduction Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACER, Tehran, Iran
4Department of Animal Production, Faculty of Agriculture, Suez Canal University, Ismailia, Egypt
5 NDEGSAL, University of León, 24071 León, Spain
6 Department of Molecular Biology, University of León, 24071 León, Spain
*Corresponding author email:


BACKGROUND: Many studies have been shown that freezing induced oxidative stress has detrimental effect on post-thaw sperm quality. OBJECTIVE : This study was conducted to investigate the effect of tert-butyl hydroquinone (tBHQ) on bull semen crtopreservation. MATERIALS AND METHODS: In this study, four different levels of tBHQ [Optidyl® containing zero (T0), 2.5 (T2.5), 5 (T5), and 7.5 µM (T7.5) tBHQ] was used to study the effect of tBHQ on freezability of bull semen. On each collection day, four ejaculates were collected (a total of 24 ejaculates from four bulls), pooled and divided to four equal parts. Each part was diluted with one of the above-mentioned extenders and frozen. After thawing, sperm motility, plasma membrane functionality and integrity, apoptosis status and mitochondrial activity were assessed. RESULTS: The results show that total sperm motility was significantly higher in T5 compared to other groups. The value of VSL was significantly lower in T5 compared to T0. Also, T5 resulted in lower LIN and STR versus T0 and T2.5 groups. All extenders containing tBHQ resulted in a significantly higher percentage of sperm with functional membrane compared to T0 groups. Finally, Apoptosis related parameters and mitochondrial activity were not significantly difference between the groups. CONCLUSION: adding 5 µM tBHQ to the bull semen extender can be beneficial for post-thaw sperm quality. Also, in vivo or in vitro fertility test is recommended to test fertilizing ability of tBHQ exposed sperm.

Keywords:  antioxidant, bovine, freezing, sperm




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CryoLetters 38 (5), 379-386 (2017)
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Patricia Froes Meyer*, Anne Caroline Guarines Furtado,
Sayonara Fatima Tomaz Morais, Luis Gonzaga de Araujo Neto,
Rodrigo Marcel Valentim da Silva, Melyssa Lima Medeiros
and Claudete Arruda Maciel de Queiroz.

Potiguar University, Department of physiotherapy, Natal, Brazil.
Patricia Froes Meyer, Rua Maxaranguape 550 apto 2603 Tirol, Natal, RN, Brasil – CEP: 59020160.
*Corresponding author email:


BACKGROUND: The cryolipolysis is on the spotlight as a non-invasive method which reduces fat layer thickness with no damage to surrounding tissues. OBJECTIVE: This study aims to verify the effectiveness of cryolipolysis in the reduction of localized adiposity in women. MATERIALS AND METHODS: This is an experimental study, without a control group, with pre and post treatment evaluation through a single application on the lower abdominal area. Setting: Research conducted in the period from July to December 2015 at the University Potiguar. Participants: A group of 15 women, age between 25-50 years. The cryolipolysis was used in the following parameters: temperature (-7ºC); suction power (30 kPa), and application time (60 min). Measurements: After the cryolipolysis was performed, a follow-up of 2 months was conducted to verify the changes related to weight, body circumference, fat layer thickness, which were evaluated by ultrasonography and photogrammetry.  RESULTS: From data analysis, the reductions observed on perimeter (p=0.03) and ultrasonography (p=0.03) showed significant results, considering p<0.05. As of body weight results (p=0.57), the average value varied during the study; however, at the end of the research, no significant weight increase or decrease was reported, as it is known that this method does not interfere with this variable. Additionally, quantitative data were satisfactory. The photogrammetry analysis showed that cryolipolysis positively affected subjects’ results. CONCLUSION: A change in body contouring, especially in individuals with lower body mass, reinforces the idea that the parameters must be  suitable for individual needs.

Keywords: Adipose tissue, adiposity, photogrammetry, cryolipolysis




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CryoLetters 38 (5), 387-398 (2017)
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Dariusz Kulus1 and Agnieszka Abratowska2

1Laboratory of Biotechnology – Department of Ornamental Plants and Vegetable Crops, UTP University of Science and Technology in Bydgoszcz, Bernardyńska 6-8, 85-029 Bydgoszcz, Poland
2Laboratory of Ecotoxicology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland


BACKGROUND: Ajania pacifica, a novelty on the horticultural market, is valued as an ornamental and a medicinal plant. Breeding programs led to the creation of numerous cultivars. Therefore, it is important to develop species storage methods. OBJECTIVE: The aim of this study has been to develop an encapsulation-based cryopreservation protocol of Ajania pacifica shoot tips. MATERIALS AND METHODS: Shoot tips of ajania ‘Bengo‘ were precultured on media with different sucrose and ABA concentrations, encapsulated and dehydrated for various periods and stored in liquid nitrogen. After thawing the explants were inoculated on different recovery media. RESULTS: A higher (9%) sucrose concentration and addition of ABA (15 µM) during preculture, followed by 4-hour desiccation (41% moisture content), as well as the application of cytokinins (1.11 µM BA or 1.16 µM KIN) in the post-thawing recovery medium were necessary to provide a high survival rate of ajania ‘Bengo’ shoot tips. CONCLUSION: Despite good survival of shoot tips of ajania following cryopreservation procedure, the stimulation of their further growth is a problem.

Keywords: Abscisic acid, cryostorage, desiccation, sucrose, synthetic seeds, TEM




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CryoLetters 38 (5), 399-406 (2017)
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Effect of developmental stage of embryos at freezing on live birth outcomes after frozen embryo transfer

Xiao jian Zhang*, Qun Lv, Li hua Min, Xue hua Cao and Xiao jie Li

Department of assisted reproductive medical center, Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital, No.32 West Second Section First Ring Road, Chengdu, Sichuan, China
*Corresponding author email:


PURPOSE: Our objective was to evaluate the birth outcomes of FET from different developmental stage of embryo at freezing (Day 3, Day 5 and Day 6).
METHODS: All vitrified-warmed day 3 (D3) and day 5/6 (D5, D6) embryo transfers during the time period from January 2013 until December 2015 were included in the study. The Birthweight, Low birthweight (LBW), sexual distinction, gestation and for singleton births and twins conceived by FET were compared retrospectively between three cryopreservation strategies utilizing either D3, D5 or D6 embryo freezing. All embryos were vitrified by Dimethylsulfoxide (DMSO), Ethylene glycol (EG) and Sucrose used as Cryoprotectants and using Cryoleaf as the receptacles. RESULTS: A total of 993 infants from 771 women. The length of embryos storage was 3-38 months, with a median of 8 months. For singleton, birthweight from D5 and D6 blastocysts transfers were signiӿcantly heavier than for D3 cleavage-stage embryos transfers (P =0.0065 P =0.0006). For twins, birthweight from D6 blastocysts transfers were significantly heavier than for D5 blastocysts transfers (P =0.0044), and children born after D3 were at a signigicantly increased risk of being born a LBW. CONCLUSIONS: Birthweights from FET are influenced by developmental stage of embryos at freezing, which were lower for cleavage-stage embryos transfer than blastocysts transfer after FET in singletons. The birth rate of LBW infants was higher in the twins. The study was small so there may be other factors than cryopreservation which affected outcomes. A higher sample size or a multi-centre prospective randomized design could be used in future studies to corroborate the current findings.

Keywords: Freezing, Embryo transfer, Cleavage-stage embryos, Blastocysts, Live Birth




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CryoLetters 38 (5), 407-413 (2017)
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Guoquan Wu, Decai Xiang, Bin Zhang, Qionghua Hong
and Guobo Quan*

Yunnan Provincial Engineering Laboratory of Animal Genetic Resource Conservation and Germplasm Enhancement, Yunnan Animal Science and Veterinary Institute, Kunming, Yunnan, 650224, People’s Republic of China.
*Corresponding author email:


BACKGROUND: The highly efficient production of parthenogenetic embryos from vitrified porcine in vitro matured oocytes has become essential for biotechnology and biomedicine research.  OBJECTIVE: To investigate the survival and parthenogenetic development of oocytes vitrified before or after electric activation. MATERIALS AND METHODS: The vitrified oocytes were parthenogenetically activated at 0, 0.5, 1, 2 and 4 h post warming (h.p.w.), and fresh oocytes were vitrified at 0, 0.5, 1, 2 and 4 h post electric activation (h.p.a.).  RESULTS: In comparison with non-vitrified oocytes, the rates of survival and activation of oocytes vitrified at 0.5, 1, 2 and 4 h.p.a. were similar, but the parameters in other vitrified groups significantly decreased. Parthenogenetic development in vitrified 0.5, 1, 2 and 4 h.p.a. groups was also significantly higher than that in other vitrified groups. Moreover, the total cell numbers of blastocysts were similar among all groups. CONCLUSION: Porcine oocytes vitrified at 0.5-4 h h.p.a. showed acceptable survival and pronuclear formation, and a higher blastocyst yield could be obtained from these oocytes.

Keywords: Porcine oocytes, vitrification, survival, parthenogenetic development




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CryoLetters 38 (5), 414-418 (2017)
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    Van Huong Do1,2, Simon Walton3, Sally Catt4
    and Andrew W. Taylor-Robinson1*

1School of Health, Medical and Applied Sciences, Central Queensland University, Rockhampton, Australia.
 2 National Key Laboratory of Animal Cell Technology, National Institute of Animal Sciences, Hanoi, Vietnam.
 3 Australian Reproductive Technologies, Mt Chalmers, Australia.
4 Education Program in Reproduction and Development, Department of Obstetrics and Gynaecology, Monash University, Clayton, Australia.
*Corresponding author email:


BACKGROUND: In order to thaw slow-cooled bovine embryos it is standard practice to draw out permeating cryoprotectants by passing embryos through successively decreasing osmotic solutions. However, recently it has been suggested that sucrose may not be needed in the warming media.  OBJECTIVE: The aim of this experiment was to compare the effect of warming media prepared with or without the inclusion of sucrose on the survival and hatching capacity of vitrified in vitro-derived bovine embryos. MATERIALS AND METHODS: Expanded blastocysts were produced in vitro and vitrified. Vitrified embryos were warmed either successively through 0.5, 0.3 and 0.2 M sucrose solutions (‘stepwise’), or by placing directly into the blastocyst solution without the addition of sucrose (‘direct’). A total of 93 expanded blastocysts were assigned randomly to two treatment groups, respectively. RESULTS: The re-expansion rates of vitrified embryos warmed after 24h in vitro culture were similar between the two groups (46/46, 100%; 46/47, 97.9%). From those vitrified embryos that expanded at 24 h there was also no significant difference in hatching rates after 48 h in vitro culture (42/46, 91.3%; 40/46, 87.0%).  CONCLUSION: The findings indicate that stepwise warming through sucrose solutions is not required for continued embryo development. Hence, a more time-efficient warming method for vitrified embryos may be followed when conducting cattle embryo transfers.

Keywords: cattle; in vitro-derived embryo; vitrification; freezing; thawing; sucrose.

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