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Volume 42, No. 1 January/February 2021
ISSN 0143-2044
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Editorial: Coming into PERSPECTIVE (PDF)
Hugh W Pritchard
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ii-iv
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PERSPECTIVE: Encapsulation nanotechnology in sperm cryopreservation: systems preparation methods and antioxidants enhanced delivery (PDF)
Lamia Taouzinet, Sofiane Fatmi, Malika Lahiani-Skiba,
Mohamed Skiba and Mokrane Iguer-Ouada
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1-12
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Post-rewarming
developmental competence of in vitro produced buffalo (Bubalis bubalis) embryos vitrified using the solid surface technique
Sandeep Rahangdale, Dharmendra Kumar, Ajit Pratap Singh,
Lalit Mohan Jeena and Bikash Chandra Sarkhel
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13-18
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Vitrification
and ultra-rapid laser warming of yeast Saccharomyces cerevisiae
Estefania Paredes
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19-24
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Cryopreservation of orchid pollinia using the V cryo-plate method
N. Jitsopakul, P. Homchan and K. Thammasiri
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25-32
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Effect
of cryopreservation on semen quality parameters in relation to lipid peroxidation and antioxidant profile in Indian buffalo
Amarjeet Bisla, Rupali Rautela, Vinay Yadav, Athanas Alex Ngou,
Abhishek Kumar, S K Ghosh, Sadhan Bag and N Srivastava
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33-38
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Dimethylacetamide in rooster semen cryopreservation
Norton Luis Souza Gatti, Carine Dahl Corcini, Jorge Squeif Filho,
Sara Lorandi Soares, Andréia Nobre Anciuti,
Rafael Mielke Barbosa, Nathália Wacholz Knabah,
Amauri Telles Tavares, Denise Calisto Bongalhardo
and Antonio Sergio Varela Junior
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39-43
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Cryopreservation
of dog spermatozoa using essential and non-essential amino acids solutions in an egg yolk-free polyvinyl alcohol extender
Nabeel Abdelbagi Hamad Talha, Yubyeol Jeon and Il-Jeoung Yu
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44-52
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Damage of
granusola and stroma cells exposed to mouse ovarian tissue cryopreservation: potential mechanism of ovarian injury
Jian-Min Zhang, Hong-Xia Wang and Xi-Lan Lu
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53-58
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CryoLetters 42 (1), 1-12 (2020)
© CryoLetters, editor@cryoletters.org
PERSPECTIVE:
ENCAPSULATION NANOTECHNOLOGY IN SPERM CRYOPRESERVATION:
SYSTEMS PREPARATION METHODS AND ANTIOXIDANTS ENHANCED DELIVERY
Lamia TAOUZINET*1,2, Sofiane FATMI1,2,3, Malika LAHIANI-SKIBA3,
Mohamed SKIBA3 and Mokrane IGUER-OUADA2
1Technology Pharmaceutical Laboratory, Department of Processes Engineering,
Faculty of Technology, Université de Bejaia, 06000 Bejaia, Algeria.
2Associated Laboratory in Marine Ecosystems and Aquaculture, Faculty of Nature
and Life Sciences, Université de Bejaia, 06000 Bejaia, Algeria.
3Technology Pharmaceutical and Bio pharmaceutics Laboratory, UFR Medicine and
Pharmacy, Rouen University, 22 Blvd. Gambetta, 76183, Rouen, France.
*Corresponding author's details: taouzinetl93.univ@gmail.com (E-mail); 0000-0001-8992-1591 (ORCID)
Abstract
Sperm cryopreservation promotes the storage and transport of germplasm for its use in
artificial insemination (AI) and other advanced reproductive technologies. However, sperm cryopreservation causes several stresses including thermal shock, osmotic
damage, and ice crystal formation, thereby reducing sperm quality. Supplementing cryoprotectant media with antioxidants has been reported to be positive in different
species. It has been widely suggested to combine antioxidants with nanotechnology, to maximize therapeutic activity and to minimize undesirable side effects. In this
review, we discuss the role of different antioxidants in sperm cryopreservation and their improved therapeutic effect through their formulation using nanotechnology. In addition,
we report the different nano-systems preparation methods present in literature. Whilst the use of nanotechnology in animal production is still in its infancy, encouraging
results from nutrition, biocidal, remedial, and reproductive studies are driving further investigations.
Keywords: antioxidants; nano-systems; preparation methods; sperm cryopreservation.
Download the paper: ENCAPSULATION NANOTECHNOLOGY IN SPERM CRYOPRESERVATION: SYSTEMS PREPARATION METHODS AND ANTIOXIDANTS ENHANCED DELIVERY (PDF)
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CryoLetters 42 (1), 13-18 (2020)
© CryoLetters, editor@cryoletters.org
POST-REWARMING DEVELOPMENTAL COMPETENCE OF IN VITRO PRODUCED BUFFALO (Bubalis bubalis) EMBRYOS VITRIFIED USING THE SOLID SURFACE
TECHNIQUE
Sandeep Rahangdale, Dharmendra Kumar, Ajit Pratap Singh,
Lalit Mohan Jeena and Bikash Chandra Sarkhel*
Animal Biotechnology Centre, Nanaji Deshmukh Veterinary Science University, Jabalpur, Madhya Pradesh, India 482004
* Corresponding author's E-mail: sarkhelbc@gmail.com
Abstract
BACKGROUND: Vitrification is an ultra-rapid freezing technique for germplasm
preservation under high salt concentration with very short exposure time. OBJECTIVE: To assess the post-thawed developmental potential of in vitro-produced buffalo
embryos vitrified by solid surface technique using different concentrations of cryoprotectants. MATERIALS AND METHODS: The slaughterhouse derived oocytes
were in vitro matured and fertilized with epididymal sperm. IVF embryos at the morula stage were vitrified under two protocols; (i) Protocol-1: ethylene glycol (35%) (ii)
Protocol-2: ethylene glycol (15%) and dimethyl sulfoxide (15%). The vitrified-thawed embryos were in vitro cultured up to the blastocyst stage. RESULTS: Post-thawed
development of embryos vitrified under Protocol-1 was significantly higher in terms of compact morula formation as compared to Protocol-2. However, blastocyst
developmental rates were not significantly different between the two protocols. The developmental rates of the non-vitrified control were significantly higher than embryos vitrified by either protocols.
CONCLUSION: The process of cryopreservation, under both protocols, significantly affected the developmental potential of pre-implant
embryos as compared to fresh embryos. Hence the nature and concentrations of cryoprotectants needs to be optimized for efficient, viable embryonic development.
Keywords: buffalo; cryoprotectant; embryo; in vitro fertilization; vitrification.
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CryoLetters 42 (1), 19-24 (2020)
© CryoLetters, editor@cryoletters.org
VITRIFICATION AND ULTRA-RAPID LASER WARMING OF YEAST Saccharomyces cerevisiae
Estefania Paredes
Centro de Investigación Mariña, Universidade de Vigo, Laboratorio de Ecoloxia
Costeira (ECOCOST), Departamento de Ecoloxia e Bioloxia Animal, Vigo, Spain.
Author's E-mail: eparedes@uvigo.es
Abstract
BACKGROUND: As fundamental model organisms, yeasts have been used for the
study and understanding of cryopreservation and freezing damage mechanisms, in particular Saccharomyces cerevisiae. OBJECTIVE: As cryopreservation success
requires optimization of the cooling and warming rates, the objective was to test how ultra-rapid warming could improve yeast cell cryopreservation. MATERIALS AND METHODS: S. cerevisiae cells were exposed to concentrations of vitrification solutions
containing a combination of permeating and non-permeating cryoprotectants (EAFS) and to a simple 1 M sucrose solution prior to vitrification (cooling rate 69,000°C min-1).
Cells were then warmed ultra-rapidly with a laser (warming rate 107°C min-1). RESULTS: When using a vitrification solution (0.33xEAFS) survival was 80 ± 16%.
When using only a non-permeating solute (1 M sucrose) for cryoprotection, the results were slightly lower, viz. 61 ± 26 %. CONCLUSION: These results add information to
the study of the effect of numerous cooling and warming rates for baker´s yeast cryopreservation and provide further examples of the application of vitrification and
ultra-fast laser warming. Ultra-rapid warming seems to be applicable to a wide range of cells and tissues from diverse species.
Keywords: laser-warming; recrystallization; Saccharomyces cerevisiae; vitrification; yeast.
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CryoLetters 42 (1), 25-32 (2020)
© CryoLetters, editor@cryoletters.org
CRYOPRESERVATION OF ORCHID POLLINIA USING THE V CRYO-PLATE METHOD
N. Jitsopakul1*, P. Homchan2 and K. Thammasiri3
1 Department of Plant Science, Textile and Design, Faculty of Agriculture and
Technology, Rajamangala University of Technology Isan, Surin Campus, Surin 32000, Thailand.
2 Department of Science and Mathematics, Faculty of Agriculture and Technology,
Rajamangala University of Technology Isan, Surin Campus, Surin 32000, Thailand.
3 Department of Plant Science, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
*Corresponding author's E-mail: njitsopakul@hotmail.com
Abstract
BACKGROUND: Preserving pollinia viability and fertility for pollination at specific times
of the year is very important in orchid breeding. One possible method is cryopreservation using aluminium cryo-plate which is increasingly used for the long-term storage of plant genetic resources.
OBJECTIVE: To determine a V cryo-plate method for the cryopreservation of orchid pollinia and apply it to some Thai orchid species for breeding. MATERIALS AND METHODS: Pollinia of Rhynchostylis
gigantea (L.) Ridl. were collected from completely open flowers in the morning and then were placed on aluminium cryo-plates embedded in alginate gel, then immersed
in loading solution containing 2 M glycerol and 0.4 M sucrose for 15 min at room temperature (29°C), and then dehydrated with PVS2 solution for 0-60 min at 29°C. The
cryo-plates with pollinia were directly plunged into liquid nitrogen for 40 min, and rapidly warmed in 1 M sucrose for 15 min. The cryopreserved and non-cryopreserved
pollinia were used for hand-pollinating flowers of the same species for producing hybrids. RESULTS: The viability of non-cryopreserved and cryopreserved pollinia
dehydrated with PVS2 was 100%. The highest capsule set after pollinating flowers with cryopreserved pollinia dehydrated with PVS2 for 40 min was also up to 100%. The protocol for cryopreservation of R. gigantea (L.) Ridl pollinia using V cryo-plate method
was successfully applied for the cryopreservation of nine Thai orchid species. The exposure time to PVS2 affected the pollinia viability (range 40-100 %; average 93%)
and capsule set (range 20-100 %; average 78%) of the nine species. The successful capsule set and seed production after pollination with cryopreserved pollinia in all
orchid hybrids were observed. Seed germinated into protocorms and developed to plantlets cultured on modified Vacin and Went (1949) agar medium. CONCLUSION:
Cryopreservation of freshly collected orchid pollinia using V cryo-plate method is an efficient tool for the long-term storage of plant germplasm and for orchid breeding.
Keywords: aluminium cryo-plate; capsule; fertility; orchid; pollinia; protocorm.
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CryoLetters 42 (1), 33-38 (2020)
© CryoLetters, editor@cryoletters.org
EFFECT OF CRYOPRESERVATION ON SEMEN QUALITY PARAMETERS IN RELATION TO LIPID PEROXIDATION AND ANTIOXIDANT PROFILE IN INDIAN BUFFALO
Amarjeet Bisla1*, Rupali Rautela1, Vinay Yadav2, Athanas Alex Ngou1,
Abhishek Kumar1, S K Ghosh1, Sadhan Bag3 and N Srivastava1
1 Division of Animal Reproduction, ICAR-Indian Veterinary Research Institute, Uttar Pradesh, India.
2 Department of Veterinary Gynaecology and Obstetrics, Lala Lajpat Rai University of
Veterinary and Animal Sciences, Haryana, India.
3 Division of Veterinary Physiology and Climatology, ICAR-Indian Veterinary Research Institute, Uttar Pradesh, India.
*Corresponding author's E-mail: amarjeetbislav@gmail.com
Abstract
BACKGROUND: Lipid peroxidation (LPO) due to oxidative stress leads to structural and functional changes in spermatozoa. OBJECTIVE:
To evaluate any association of various seminal characteristics at the pre- and post-cryopreservation stages with LPO and total antioxidant capacity (TAC) in Murrah buffalo semen samples.
MATERIALS AND METHODS: Sixty-five ejaculates from seven bulls were processed for cryopreservation in liquid nitrogen. RESULTS: Only 31 (47.7%) samples were found
satisfactory for inclusion in the further artificial insemination. A strong negative correlation was observed between LPO and individual progressive motility, TAC,
viability, plasma membrane integrity as well as acrosome integrity of fresh spermatozoa. At the post-thaw stage, post-thaw motility, viability, plasma membrane
integrity and acrosome integrity had strong positive correlation with TAC. CONCLUSION: The effort to minimize LPO and enhance TAC shall play a pivotal role
in improving buffalo semen quality upon cryopreservation.
Keywords: antioxidant capacity; buffalo semen; cryopreservation; lipid peroxidation.
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CryoLetters 42 (1), 39-43 (2020)
© CryoLetters, editor@cryoletters.org
DIMETHYLACETAMIDE IN ROOSTER SEMEN CRYOPRESERVATION
Norton Luis Souza Gatti1, Carine Dahl Corcini2,* , Jorge Squeif Filho1,
Sara Lorandi Soares3, Andréia Nobre Anciuti1,
Rafael Mielke Barbosa1, Nathália Wacholz Knabah1,
Amauri Telles Tavares4, Denise Calisto Bongalhardo5
and Antonio Sergio Varela Junior6
1 Pós-Graduação em Veterinária, Faculdade de Veterinária;
2 Departamento de Patologia Animal, Laboratório de Reprodução Animal, Faculdade
de Veterinária;
3 Pós-Graduação em Biotecnologia, Centro de desenvolvimento tecnológico CDTEC/Biotecnologia;
4 Instituto de Biologia, Departamento de Fisiologia e Farmacologia, Universidade
Federal de Pelotas;
5 Reprodução Animal Comparada, Instituto de Ciência Biológicas, Universidade Federal de Rio Grande, Rio Grande, Brasil.
*Corresponding author's E-mail: antoniovarela@furg.br
Abstract
BACKGROUND: Sperm cryopreservation of cockerels is a major challenge, and so far
there is no adequate information to enable commercial use of frozen semen. OBJECTIVE: To test the toxicity of dimethylacetamide (DMA). MATERIALS AND METHODS:
DMA was added at 3%, 6%, 9% and 12% to the freezing diluent, and maintained for equilibration with the semen sample for 1 min, 3 min, 5 min, 7 min and
9 min prior to freezing. Thawed semen was evaluated for kinetic characteristics by computer-assisted semen analysis (CASA) and for structural and functional properties
by flow cytometry (plasma membrane rupture, mitochondrial functionality and plasma membrane functionality). RESULTS and CONCLUSION: The addition of 6% DMA for
3-min equilibration resulted in the highest total and progressive motility, 42.0% and 36.9%, respectively. The point of intersection between a good protection and low
plasma membrane rupture was obtained with the addition of 6% of DMA for 3-min equilibration with the rooster semen.
Keywords: amides; cryopreservation; rooster sperm.
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CryoLetters 42 (1), 44-52 (2020)
© CryoLetters, editor@cryoletters.org
CRYOPRESERVATION OF DOG SPERMATOZOA USING ESSENTIAL AND NON-ESSENTIAL AMINO ACIDS SOLUTIONS IN AN EGG YOLK-FREE POLYVINYL
ALCOHOL EXTENDER
Nabeel Abdelbagi Hamad Talha1,2, Yubyeol Jeon1 and Il-Jeoung Yu1, *
1 Laboratory of Theriogenology and Reproductive Biotechnology, College of Veterinary
Medicine and Bio-safety Research Institute, Jeonbuk National University, Iksan, Republic of Korea.
2 Department of Veterinary Medicine and Surgery, College of Veterinary Medicine,
Sudan University of Science and Technology, Khartoum, Sudan.
*Corresponding author's E-mail: iyu@jbnu.ac.kr
Abstract
BACKGROUND: Amino acids (AAs) have been indicated to have cryoprotective and
antioxidative effects on sperm freezing using egg yolk (EY)-based extender. However, EY-based extender is difficult to be standardized for the effect of amino acids because
the EY composition varies with the animal's diet. OBJECTIVE: To test the effect of AAs in EY-free polyvinyl alcohol (EY-free PVA) extender and develop a chemically
defined extender for dog sperm cryopreservation. MATERIALS AND METHODS: In the first experiment (E1), dog spermatozoa (1 x 108 sperms/mL) were frozen with EY-free
PVA extender without AAs or supplemented with essential (EAAs, 50 x: 1, 2, 4 %) or non-essential amino acids (NEAAs, 100 x: 1, 2, 4 %). In the second experiment (E2),
spermatozoa were frozen with EY-free PVA extender supplemented with 0, 0.5, 1 or 2 % of an EAA-NEAA mixture. Motility, viability and acrosome integrity were evaluated
after thawing in E1 and E2. In the third experiment (E3), spermatozoa were frozen using an extender supplemented with 2 % EAAs, 2 % NEAAs or a 0.5 % EAA-NEAA
mixture. Reactive oxygen species (ROS) and phosphatidylserine (PS) translocation were assessed. Expression of genes for motility-related sperm
mitochondrial-associated cysteine-rich protein (SMCP), apoptosis-related B-cell lymphoma 2 (BCL2) and BCL2 associated X protein (BAX) was measured. RESULTS:
Addition of EAAs, NEAAs or an EAA-NEAA mixture to EY-free PVA extender significantly increased sperm motility without affecting viability. Only 1 % NEAAs
significantly increased the acrosome membrane. EAA-NEAA mixture (0.5 %) significantly increased SMCP, BCL2 and BAX expression compared to the control
group without significant effect on PS translocation or ROS. CONCLUSION: EAAs and NEAAs addition in EY-free PVA extender improved sperm motility, with limited effect
on acrosome integrity and gene expression of SMCP, BCL2 and BAX during dog sperm cryopreservation.
Keywords: amino acids; cryopreservation; dog spermatozoa; gene expression.
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CryoLetters 42 (1), 53-58 (2020)
© CryoLetters, editor@cryoletters.org
DAMAGE OF GRANUSOLA AND STROMA CELLS EXPOSED TO MOUSE OVARIAN TISSUE CRYOPRESERVATION: POTENTIAL MECHANISM OF OVARIAN INJURY
Jian-Min Zhang1*, Hong-Xia Wang2 and Xi-Lan Lu2
1Weifang Nursing Vocational College, Weifang, Shandong, P.R. China.
2Department of Reproductive Medicine, Jinan Central Hospital & Cheeloo College of
Medicine, Shandong University, Jinan, Shandong, P.R. China.
*Corresponding author's E-mail: jmzwfhl@163.com
Abstract
BACKGROUND: Several obstacles must be overcome before ovarian tissue cryopreservation can be used as a standard procedure.
OBJECTIVE: To carry out a morphologic and functional study of the effect of cryopreservation on mouse follicles and stroma cells. MATERIALS AND METHODS:
Female mice were divided into three groups (control, fresh graft and cryopreserved graft). Ultrastructural features of follicles
and stroma cells were evaluated using transmission electron microscopy. After autologous transplantation, micro-vessel densities of grafts were examined. RESULTS:
Vacuoles in granulosa cells and stromal cells are significantly greater than that of oocytes. The microvessel density of fresh grafts is significantly higher than that in frozen-thawed grafts.
CONCLUSION: Granusola and stroma cells, rather than oocytes, are vulnerable to cryoinjury. Injuries to granulosa cells and stromal cells
could be the critical part of ovarian damage caused by cryopreservation.
Keywords: cryopreservation; granusola cell; oocyte; stroma cell.
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