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Abstracts: CryoLetters 40 (4), 2019



Volume 40, No. 5 September/October 2019

ISSN 0143-2044



Abstracts: Society for Low Temperature Biology Annual Meeting 2018
6-7 September 2018, Crop Research Institute
(Výzkumný ústav rostlinné výroby, v.v.i.), Czech Republic




Effect of extender supplemented with boron on post-thaw motility, viability, DNA damage and fertilization ability of cryopreserved brown trout (Salmo trutta macrostigma) spermatozoa
Yusuf Bozkurt, İlker Yavaş, Aziz Gül, Mustafa Numan Bucak,
Deniz Yeni and Fatih Avdatek




Cold-hardiness of the common spadefoot Pelobates fuscus  (Pelobatidae, Anura, Amphibia)
Daniil I. Berman, Nina A. Bulakhova,
Ekaterina N. Meshcheryakova, Mikhail V. Yermokhin and
Vasily G. Tabachishin




Cryopreservation of axillary buds of Cannabis sativa L. by V-cryoplate droplet-vitrification: the critical role of sucrose preculture
Hemant Lata, Esther Uchendu, Suman Chandra,
Chandrani G. Majumdar, Ikhlas A. Khan and
Mahmoud A. ElSohly




The addition of açaí palm fruit extract in the cryopreservation diluent of bull's semen with low performance on freezing
Janaina Barros Luz, Kaliandra Souza Alves,
André Maciel Crespilho, Rafael Mezzomo,
Cláudia Siqueira Caldas,
Paulo Castelo Branco de Gouveia Filho,
José Carlos Ferreira-Silva, Marcos Antonio Lemos Oliveira and
Luis Rennan Sampaio Oliveira




Development of cryopreservation protocol for Vanilla siamensis: an endangered orchid species in Thailand
Vinan Vince Chaipanich, David L. Roberts, Sureerat Yenchon,
Sompong Te-chato and Minoo Divakaran





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CryoLetters 40 (5), 275-283 (2019)
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Yusuf Bozkurt1*, İlker Yavaş2, Aziz Gül3, Mustafa Numan Bucak4,
Deniz Yeni5, Fatih Avdatek5

1 İskenderun Technical University, Faculty of Marine Sciences and Technology, Department of Aquaculture, İskenderun, Hatay, Turkey.
2 Mustafa Kemal University, Faculty of Veterinary Medicine,  Department of Reproduction and Artificial Insemination, Antakya, Hatay, Turkey.
3 Mustafa Kemal University, Faculty of Agriculture, Department of Animal Sciences, Antakya, Hatay, Turkey.
4 Selcuk University, Faculty of Veterinary Medicine, Department of Reproduction and Artificial Insemination, Konya, Turkey.
5 Afyon Kocatepe University, Faculty of Veterinary Medicine, Department of Reproduction and Artificial Insemination, Afyonkarahisar, Turkey.
*Corresponding author:


BACKGROUND: Boron has been considered as an essential nutrient for decreasing lipid peroxidation and improving antioxidant mechanism in different animal species. On the other hand, its effect on quality or DNA damage following cryopreservation process in fish sperm is still unclear. OBJECTIVE: Experiments were designed to analyse the effect of an ionic based extender supplemented with boron on post-thawed motility, viability, fertility and DNA integrity of cryopreserved brown trout (Salmo trutta macrostigma) sperm. MATERIALS AND METHODS: Sperm samples were cryopreserved with the ionic extender containing different boron concentrations (0.1, 0.2, 0.3 and 0.4 mM) using a controlled freezer at two different freezing rates (FR-I: 10°C min-1 from +4 °C to -40°C and FR-II: 15°C min-1 from +4°C to -40°C). Sperm motility, viability, fertilization, eyeing and DNA fragmentations were determined in post-thawed samples. RESULTS: Freezing rate-I provided significantly higher fertilization and eyeing rates compared to freezing rate-II (p<0.05). Higher post-thaw motility (62.8±1.4%) and fertilization (75.2±0.9%) rates were obtained with the 0.4 mM boron concentration at freezing rate-I. CONCLUSION: Supplementation of the extender with boron increased fertilization and eyeing rates and also decreased DNA damages at both freezing rates. 

Keywords: brown trout, boron, sperm, cryopreservation, motility, DNA damage



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CryoLetters 40 (5), 284-290 (2019)
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Daniil I. Berman1, Nina A. Bulakhova1,2*, Ekaterina N. Meshcheryakova1,
Mikhail V. Yermokhin3 and Vasily G. Tabachishin4

1 Institute of Biological Problems of the North, Far Eastern Branch, Russian Academy of Sciences, Magadan, 685000, Russia.
2 Research Institute of Biology and Biophysics, Tomsk State University, Tomsk, 634050, Russia.
3 Saratov State University, Saratov, 410012, Russia.
4 Saratov branch of A. N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Saratov, 410028, Russia.
*Corresponding author:


BACKGROUND: Pelobates fuscus is an anuran amphibian that overwinters on land deep in soil, including regions with low sub-zero temperatures. It is not yet known if such behaviour can be explained by low cold-hardiness of the species. OBJECTIVE: To measure cold-hardiness of P. fuscus and compare the results with data about its location in winter in nature. METHODS: Lower lethal temperatures and supercooling points (SCP) were measured in specimens collected near the Saratov city in south-eastern European Russia. RESULTS: SCP of P. fuscus range from -1.7 to -2.7°C. All individuals withstood exposure of 1°С for 20 days; but freezing is lethal to animals: one individual out of five withstood exposure of -1°С for 3 days, and -2°С is lethal to all animals. The condition of supercooling is unstable. Freezing at -2°C is lethal to the animals. CONCLUSION: The lower tolerable temperature is probably about 0°С. Low cold hardiness determines overwintering deep in substrates, which allowed the species to disperse over a large geographical range, which also includes cold regions with little snow in winter.

Keywords: Pelobates fuscus, cold-hardiness, SCP, lower lethal temperatures, overwintering, Saratov Oblast region.



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CryoLetters 40 (5), 291-298 (2019)
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Hemant Lata1*, Esther Uchendu1,2, Suman Chandra1,
Chandrani G. Majumdar1, Ikhlas A. Khan1,3 and Mahmoud A. ElSohly1,4

1 National Center for Natural Products Research, School of Pharmacy, University of Mississippi, MS 38677, USA.
2 Department of Agronomy, University of Ibadan, Ibadan, Nigeria.
3 Department of Biomolecular Sciences, School of Pharmacy, University of Mississippi, MS 38677, USA.
4 Department of Pharmaceutics and Drug Delivery, School of Pharmacy, University of Mississippi, University, MS 38677, USA.
*Corresponding author:


BACKGROUND: Cryopreservation is the only method allowing the safe and cost-effective long-term conservation of important germplasm. Recent use of the cryo-plate system has proven beneficial in further simplifying the cryopreservation protocols. OBJECTIVE: Developing an efficient protocol for the cryopreservation of axillary buds of Cannabis sativa elite cultivars (MX and V1-20) by the V-cryoplate droplet-vitrification technique. MATERIALS AND METHODS: Stem segments (~5 cm in length) with mature axillary buds collected from indoor-grown plants were surface sterilized and then either precultured on MS basal medium with 0.1 M sucrose (1st step preculture) for 72 h or non-precultured. All mature axillary buds (~1 mm) were aseptically excised from stem segments and precultured for an additional 48 h on MS basal medium with sucrose (0.3 M) and 5% DMSO prior to cryopreservation (2nd step preculture). Biomass samples of fully mature mother plants and regrown cryopreserved plants were analyzed for Δ9-THC and CBD content using gas chromatography-flame ionization detector (GC/FID). RESULTS: The survival and regrowth rates of cryopreserved axillary buds of cultivar MX following this two-step preculture were 45% and 42% respectively, while those of cultivar V1-20 were 47% and 44% respectively. A direct preculture of axillary buds (2nd step preculture) on high sucrose (0.3M sucrose) significantly decreased both the survival and regrowth levels of axillary buds of cultivar MX (5% and 3% respectively) as well as those of cultivar V1-20 (20% and 17% respectively). Δ9-THC and CBD content of mother plants and regrown cryopreserved plants were found to be highly comparable to each other. CONCLUSION: The resulting plants after cryopreservation appeared normal without any callus formation or morphogenetic variation. On maturity, mother plants and re-grown cryopreserved plants were comparable in terms of Δ9-THC and CBD content. This report provides an efficient protocol for cryopreservation of axillary buds of Cannabis sativa cultivars which may be applicable to other important cultivars, plant parts and other related medicinal plants.

Keywords: Conservation, cryoprotectant, liquid nitrogen, micropropagation, (Δ9--tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD)



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CryoLetters 40 (5), 299-304 (2019)
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Janaina Barros Luz1*, Kaliandra Souza Alves1, André Maciel Crespilho2,
Rafael Mezzomo1, Cláudia Siqueira Caldas1,
Paulo Castelo Branco de Gouveia Filho3, José Carlos Ferreira-Silva3,
Marcos Antonio Lemos Oliveira3, Luis Rennan Sampaio Oliveira1

1 Laboratory of Biotechnology in Animal Reproduction of Carajás, Federal Rural University of Amazonia, Parauapebas-PA, Brazil.
2 Reproduction Laboratory, University of Santo Amaro, São Paulo-SP, Brazil.
3 Laboratory of Reproductive Biotechniques, Federal Rural University of Pernambuco, Recife-PE, Brazil.
*Corresponding author:


BACKGROUND: Semen freezing is of great importance for animal production because it allows the use and the rapid diffusion of the genetic material from economically important animals. OBJECTIVE: To evaluate the effect of açaí (Euterpe oleracea; Arecaceae family) extract addition to the semen cryopreservation diluent on the morphology, sperm motility parameters, and plasma membrane integrity of spermatozoa. MATERIALS AND METHODS: The ejaculates, obtained from five bulls with low performance on semen freezing, were fractionated and distributed according to the experimental group. The control samples did not have açaí extract added, whereas to the treated groups were added 5, 10, 15 or 20 mg ml-1 of açaí extract into the semen diluent. The sperm morphology was evaluated with a formalin-saline-buffered solution. The plasma membrane integrity was evaluated by the epifluorescent test, while the cellular kinetics was assessed by an automated analysis of the spermatic movement. RESULTS: The sperm defects showed a linearly decreasing effect (P < 0.05) with the addition of different concentrations of açaí extract. The plasma membrane integrity was higher (P < 0.05) after the açaí addition to the cryopreservation diluent. There was no significant effect (P > 0.05) of the açaí extract on the kinetics of spermatozoa. CONCLUSION: The addition of açaí extract to the cryopreservation diluent provided better preservation of the structural integrity of the sperm plasma membrane in the bull's semen with low tolerance to the cryopreservation process.

Keywords: bull, freezing, Euterpe oleracea Martius, plasma membrane.



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CryoLetters 40 (5), 305-311 (2019)
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Vinan Vince Chaipanich1, David L. Roberts1, Sureerat Yenchon2,3*,
Sompong Te-chato2,3 and Minoo Divakaran4

1 Durrell Institute of Conservation and Ecology, School of Anthropology and Conservation, Faculty of Social Sciences, University of Kent, Kent, United Kingdom.
2 Department of Plant Science, Faculty of Natural Resources, Prince of Songkla University, Hat Yai, Songkhla, Thailand.
3 Centre of Excellent in Agricultural and Natural Resources Biotechnology (CoE-ANRB phase II) Faculty of Natural Resources, Prince of Songkla University, Hat Yai, Songkhla, Thailand.
4 Department of Botany, Providence Women's College, Kozhikode, Kerala, India.
*Corresponding author:


BACKGROUND: Vanilla siamensis is listed in Appendix-II of Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) as an endangered species in Thailand. OBJECTIVE: To develop an optimum cryopreservation protocol for V. siamensis. MATERIALS AND METHODS: Protocorms were precultured on solid ½ MS medium with 0.5 M sucrose for 0-7 d. For encapsulation-dehydration, encapsulated protocorms (beads) were dehydrated for 0-6 h. In the case of encapsulation-vitrification, the beads were loaded with a plant vitrification solution 2 (PVS2) at 0 ˚C for 0-90 min. RESULTS: Protocorms precultured for 3 d gave the highest post-cryopreservation survival of 17%. Dehydration of the encapsulated protocorm beads for 4 h gave the highest survival of 33% and a regrowth of 25%. Protocorms subjected to the encapsulation-vitrification method did not survive at all. CONCLUSION: Protocorms precultured with 0.5 M sucrose for 3 d, encapsulated with 3% sodium alginate and dehydrated to a moisture content c. 14% before plunging into LN was a suitable method for the successful cryopreservation of V. siamensis protocorms.

Keywords: Protocorm, liquid nitrogen, encapsulation, dehydration, vitrification.

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