Abstracts: CryoLetters 39 (5), 2018

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



Volume 39, No. 5 September/October 2018

ISSN 0143-2044



Effect of liquid nitrogen flushing of extender on seminal antioxidant profile of murrah buffalo during cryopreservation
B Balamurugan, SK Ghosh, SA Lone, JK Prasad, GK Das,
R Katiyar, Abdul Rahman Mustapha, Ajay Kumar
and MR Verma




Protective effect of vitrified-warmed media with clove bud (Syzygium aromaticum) extract on mouse oocytes and resultant blastocysts
Soghra Bahmanpour, Azizollah Bakhtari
and Beheshteh Abouhamzeh




Evaluation of fertilizing ability of frozen equine sperm using a bovine zona pellucida binding assay
Hariany Seabra Martins, Olindo Assis Martins Filho,
Márcio Sobreira Silva Araujo, Nelson Rodrigo Martins
and Monique de Albuquerque Lagares




Supplementation of vitrification medium with vanadium: evaluation of histological change and follicle growth
Kobra Zaleh Samangani and Mehri Azadbakht




Evaluation of apoptotic markers and tissue histology indicate a slight advantage of slow freezing method over vitrification for sheep ovarian tissues
Samane Adib, Mojtaba Rezazadeh Valojerdi and
Mehdi Alikhani




Successful cryopreservation of Vitis shoot tips: novel pre-treatment combinations applied to nine species
Gayle M. Volk, Ashley N. Shepherd and Remi Bonnart




A smart box with adjustable cooling rate for cryopreservation
Kaixuan Zhu, S. M. Chapal Hossain, Li Yufang, Yuan Fuquan,
Peng Hu and Gang Zhao




Pre-freezing treatment with butylated hydroxytoluene and cholesterol-loaded methyl-β-cyclodextrin improves quality of cryopreserved boar semen
Santosh Kumar Baishya, Ranjan Kumar Biswas,
Kadirvel Govindasamy, Bharat Chandra Deka, Sudip Sinha
and Mahak Singh






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CryoLetters 39 (5), 279-287 (2018)
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Balamurugan B1, SK Ghosh1, SA Lone2*, JK Prasad1, GK Das1,
R Katiyar1, Abdul Rahman Mustapha3, Ajay Kumar4
and MR Verma4

1Division of Animal Reproduction, ICAR-Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, 243122, India.
2Artificial Breeding Research Center (ABRC), Animal Reproduction, Gynecology and Obstetrics, ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, India.
3Department of Theriogenology, Faculty of Veterinary Medicine, University of maiduguri, Borno  State, Nigeria.
4Biochemistry Division, ICAR-Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, 243122, India.
4Division of Livestock Economics and Statistics, ICAR-Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, 243122, India.
*Corresponding author email address:


BACKGROUND: The dissolved oxygen in the extender may act as a source for the production of reactive oxygen species that may lead to reduced seminal antioxidant profile which in turn may be responsible for impaired frozen thawed sperm quality and fertility. OBJECTIVE: To study the effect of adding liquid nitrogen into the extender on semen freezability and seminal antioxidant profile in buffalo. MATERIALS AND METHODS: Semen extender was prepared freshly and divided into two sub extenders namely, Extender I: control (non deoxygenated) and Extender II: partially deoxygenated by using LN2 flushing). The estimation of dissolved oxygen (DO) level was done in both extenders. Semen samples with mass motility of ≥ 3+ and individual progressive motility of 70% and above, collected from murrah buffalo bulls were utilized for the present study.  Each semen sample was split into two group’s viz., group I: diluted with extender I and group II: diluted with extender II up to 60×106 sperm/mL. The diluted semen samples were packed into French mini straws (0.25 mL), sealed with polyvinyl alcohol powder, kept for 3 h at 5ºC for equilibration and then kept in automatic programmable freezer until temperature of straws reached -145°C followed by plunging into liquid nitrogen (-196°C). The evaluation of semen samples was carried out for various seminal attributes (sperm motility, live sperm count, acrosomal integrity, and hypo-osmotic swelling (HOS) response) and antioxidant profile (superoxide dismutase (SOD), glutathione peroxidase (GPx) and total antioxidant capacity (TAC)) at pre freeze and post thaw stage. RESULTS: Sperm motility, live sperm count, acrosomal integrity, HOS response were significantly (P<0.05) higher in group II as compared to group I. The average seminal SOD, GPx and TAC levels were significantly (P<0.05) higher in group II as compared to group I at pre freeze and post thaw stage. CONCLUSION: It is concluded that partial deoxygenation of the extender prior to its addition to semen enhances sperm quality in terms of sperm motility, live sperm count, acrosomal integrity, and hypo-osmotic swelling (HOS) response and also improves seminal antioxidant profile (superoxide dismutase (SOD), glutathione peroxidase (GPx) and total antioxidant capacity (TAC).

Keywords: Extender; Deoxygenation; Sperm; Antioxidant




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CryoLetters 39 (5), 288-297 (2018)
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Soghra Bahmanpour1,2, Azizollah Bakhtari2
and Beheshteh Abouhamzeh3*

1Department of Anatomy, Shiraz University of Medical Sciences, Shiraz, Iran
2Department of Reproductive Biology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
3Department of Anatomical Sciences, Faculty of Medicine AJA University of Medical Sciences, Tehran, Iran
*Corresponding author email:


BACKGROUND: Although oocyte vitrification has become an essential part of infertility treatments, the formation of reactive oxygen species can reduce the quality of oocytes. OBJECTIVE: Protective effects of vitamin E or clove bud extract in the vitrification and warming media of vitrified mature oocytes were evaluated on blastocysts derived from the warmed oocytes. MATERIALS AND METHODS: O ocytes were vitrified-warmed with vitamin E (0, 50, 100, 200 and 400 mM) or hydroethanolic extract of clove bud (0, 5, 10, 20 and 40 µg/ml) in vitrification and warming media, and resultant blastocysts were evaluated. RESULTS: Mid concentrations of these antioxidants (10 and 20 µg/ml of clove bud extract and 100 and 200 µM of vitamin E) improved the blastocyst formation (P<0.05). The expression of catalase (P<0.01) and superoxide dismutase-1 (Sod1) genes, as well as the inner cell mass number (P<0.05), were significantly less in the blastocyst of the control (untreated) vitrified oocytes, however these levels were restored to normal by clove bud extract. In both antioxidant groups Bcl2l1 gene expression was promoted in comparison to the controls (P<0.05). CONCLUSION: Clove bud extract, with antioxidant and anti-apoptotic properties, may serve to prevent or reduce oxidative stress condition of oocyte vitrification.

Keywords: Syzygium aromaticum, α-tocopherol, antioxidant, apoptosis, vitrification




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CryoLetters 39 (5), 298-305 (2018)
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Evaluation of Fertilizing ability of frozen equine sperm using a bovine zona pellucida binding assay

Hariany Seabra Martins1, Olindo Assis Martins Filho2,
Márcio Sobreira Silva Araujo2, Nelson Rodrigo Martins3
and Monique de Albuquerque Lagares1*

1Departamento de Clinica e Cirurgia Veterinárias, Escola de Veterinária, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, CEP: 31270-901, Belo Horizonte, MG, Brazil
2Laboratório de Biomarcadores de Diagnóstico e Monitoração, Centro de Pesquisas René Rachou - Fiocruz, Belo Horizonte, MG, Brazil.
3Departamento de Medicina Preventiva da Escola de Veterinária da Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
*Corresponding author email:


BACKGROUND: Frozen equine semen has lower fertility compared to cooled semen. Due to the difficulty to obtain equine oocytes, a heterologous zona pellucida binding assay (ZBA) is an alternative method to predict the fertilizing capability of equine frozen sperm. The rate of capacitated and hyperactivated sperm according to their motility characteristics were analyzed with a Computer Assisted Sperm Analyzer. We believe this report describes for the first time the in vitro hyperactivation induction and the heterologous ZBA to predict the fertilizing ability of frozen equine sperm. OBJECTIVE: This work aimed at developing an assay to evaluate the fertilizing ability of frozen equine sperm using a bovine ZBA with the use of an in vitro capacitation and hyperactivation media with procaine and calcium ionophore A23187, respectively. MATERIALS AND METHODS: The sperm motility characteristics, intact and acrosome reacted sperm rates, and number of stallion sperm bound to the bovine ZP were calculated. RESULTS: The procaine group showed a hyperactivation motility pattern, although it improved ZP sperm binding similarly to the capacitation group. CONCLUSION: The capacitation medium improved the IVF capability of frozen equine sperm, allowing the highest possibility of sperm-oocyte interaction.

Keywords: cryopreservation, stallion, spermatozoa, semen, hyperactivation, capacitation




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CryoLetters 39 (5), 306-312 (2018)
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Kobra Zaleh Samangani and Mehri Azadbakht*

Department of Biology, Faculty of Basic Sciences, Razi University, Kermanshah, Iran.
*Corresponding author email:


BACKGROUND: Cryopreservation of ovary is an effective option for treatment of infertility and much effort has been made to improve the ovarian cryopreservation. OBJECTIVE: The study was to investigate the effect of vanadium in the vitrification medium on histology, viability rate and follicle growth. MATERIALS AND METHODS: Mouse ovaries were vitrified in the presence of 0, 10, 100 and 250 μM vanadium (assigned as Group V0, V1, V2 and V3). After thawing, the ovaries were fixed for histological studies, mechanically isolated follicles were cultured, and then viability and growth rate of follicles were assessed. RESULTS: Vanadium supplementation to the vitrification medium significantly increased the number of morphologically normal follicles. In vitro growth and viability rate of follicles was higher in the V2 than V0 group (P < 0.05). CONCLUSION: Vanadium can improve in vitro growth and viability rate of isolated follicles from the vitrified ovaries.

Keywords: Ovary, Vitrification, Vanadium, Follicle growth




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CryoLetters 39 (5), 313-321 (2018)
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Samane Adib1, Mojtaba Rezazadeh Valojerdi1, 2* and Mehdi Alikhani3

1Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran;
2Department of Embryology, Reproductive Biomedicine Research Center and 3Department of Molecular Systems Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
*Corresponding author email: and


BACKGROUND: An efficient cryopreservation method is very important for preserving the fertility of sheep ovarian tissues. OBJECTIVE: To compare the slow freezing method and vitrification for cryopreservation of sheep ovarian tissues. METHODS: Dissected cortex fragments from ten sheep ovaries were used for the comparative study. Cryopreserved and control tissues were cultured for 24h and then evaluated according to follicular morphology and apoptotic assessment. RESULTS: In both slow freezing and vitrification methods, normal follicles were reduced when compared to the non-frozen control group. There were significantly more abnormal follicles with vitrification than with slow freezing method (P < 0.05). However, there were no significant differences with regard to apoptotic gene expression or the percentage of Caspase3 positive follicles among cryopreserved and control groups. CONCLUSION: A slight advantage of the slow freezing method was observed over vitrification for cryopreservation of sheep ovarian tissue fragments.

Keywords: ovarian tissue; slow freezing; vitrification; apoptotic marker




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CryoLetters 39 (5), 322-330 (2018)
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Gayle M. Volk*, Ashley N. Shepherd and Remi Bonnart

USDA-ARS National Center for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, CO 80521
*Corresponding author email:


BACKGROUND: Secure back-up of Vitis genetic resource collections requires cryopreservation methods that give long-term survival of clonal germplasm having diverse genetic backgrounds. OBJECTIVE: This work sought to increase survival of Vitis shoot tips exposed to liquid nitrogen using combinations of pretreatments and cryoprotection procedures. The new procedure should give high survival of shoot tips from a wide range of Vitis species. MATERIALS AND METHODS: In vitro plants from nine Vitis species were used as source material for nodal sections. Shoot tips were then excised from nodal sections that were grown on medium containing benzyladenine, salicylic acid, glutathione, and ascorbic acid. The shoot tips were treated with loading solution, and then half-strength PVS2 for 30 minutes, prior to full-strength PVS2 treatments for between 60 and 90 minutes prior to liquid nitrogen (LN) exposure. RESULTS: Shoot tip regrowth levels were highest 90 minutes in PVS2+LN and ranged from 24-43% and averaged 35±2% across the nine Vitis species. CONCLUSION: The pretreatment, cryopreservation, and recovery methods yielded successful regrowth for multiple Vitis species using a droplet-vitrification procedure.

Keywords: cryopreservation, grape, shoot tips, Vitis, vitrification




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CryoLetters 39 (5), 331-335 (2018)
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Kaixuan Zhu1, #, S. M. Chapal Hossain1, #, Li Yufang1, Yuan Fuquan1,
Peng Hu2,* and Gang Zhao1,*

1Department of Electronic Science and Technology; 2Department of Thermal Science and Energy Engineering, University of Science and Technology of China, Hefei, China.
# These authors contributed equally.
*Corresponding author email: or


BACKGROUND: The box-in-box (BIB) has recently been developed as a reliable low-cost device with a passively controlled cooling rate. However, the typical box-in-box design is only applicable to –80°C freezer. The transfer process to liquid nitrogen may affect the quality of the sample.  OBJECTIVE: To develop a smart cooling BIB that can be immersed directly into liquid nitrogen. MATERIALS AND METHODS: Different thickness and materials of the box were used to alter the cooling rate. Experiments were carried out to measure the cooling rate and modeling was performed to verify experimental results. RESULTS: The average cooling rate of the solid cassette with a wall thickness of 10 mm was 7°C/min. The average cooling rate of the hollow boxes was about 2.5°C/min and 1.5°C/min for polystyrene box and aerogel box, respectively. CONCLUSION: The cooling rate of the internal sample can be controlled by controlling the thickness of the box as well as by changing the filling material of the hollow box for cooling by immersion into liquid nitrogen.

Keywords: Cooling rate, box-in-box, heat transfer model, cryopreservation




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CryoLetters 39 (5), 336-344 (2018)
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Santosh Kumar Baishya1*, Ranjan Kumar Biswas2,
Kadirvel Govindasamy1, 3,, Bharat Chandra Deka2, Sudip Sinha2
and Mahak Singh1 ,3, 4

1Krishi Vigyan Kendra, ICAR Research Complex for NEH Region Nagaland Centre, Longsachung, Nagaland;
2Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary Science, Assam Agricultural University, Khanapara, Assam;
3Division of Livestock Production, ICAR Research Complex for NEH Region, Umiam, Meghalaya;
4ICAR Research Complex for NEH Region Nagaland Centre, Jharnapani, Dimapur, Nagaland, India.
*Corresponding author email:


BACKGROUND: Pre-freezing treatment of boar sperm with additives improves the quality of post-thaw sperms. OBJECTIVES: The study aimed to determine the efficacy of butylated hydroxy-toluene (BHT) and cholesterol-loaded methyl-β-cyclodextrin (CLC) for the improvement of the frozen-thawed boar sperm quality. METHODS: Split samples of 30 ejaculates from six boars were cryopreserved in lactose-egg yolk-glycerol extender containing BHT (0.2 mM), CLC (5 mg/ 200-240 x 106 sperm) or BHT (0.2 mM) plus CLC (5 mg per 200-240106 sperm). Semen samples were evaluated for motility, membrane integrity, acrosomal status, mitochondrial membrane potential (MMP), DNA integrity and lipid peroxidation after equilibration and after freezing. RESULTS: The addition of BHT and CLC into the extender significantly improved (P<0.05) plasma membrane integrity and decreased (P<0.05) lipid peroxidation after freezing. Post-thaw motility and live intact acrosome were significantly (P<0.05) higher in the extenders with BHT or BHT plus CLC. The post-thaw MMP of viable spermatozoa and DNA integrity were not affected. BHT plus CLC showed a significant (P < 0.05) improvement on motility as compared to BHT and CLC alone. CONCLUSION: Treatment of boar spermatozoa with BHT and CLC improved post-thaw sperm quality.

Keywords: Butylated hydroxytoluene, boar semen, cholesterol-loaded methyl-β-cyclodextrin, cryopreservation, sperm quality

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