Volume 41, No. 5 September/October 2020 ISSN 0143-2044
	Remembering Dirk K Hincha	ii
	PERSPECTIVE: Possible protective mechanisms of coenzyme Q10 action on spermatozoa during cryopreservation or cooled-stored condition (PDF) Michael Osei Appiah, Bismark Asante-Badu, Jing Zhao, Hongyu Liu, Jun Wang and Wenfa Lu	246-256
	Incubation with cholesterol loaded cyclodextrin and subsequent dilution in partially deoxygenated extender improves the freezability of crossbred bull sperm Rahul Katiyar, Subrata Kumar Ghosh, Jai Kishan Prasad, Abhishek Kumar, Lhendup Bhutia, Vinod Gupta and Rupali Rautela	257-266
	The potential of cryotherapy to remove sugarcane mosaic virus from sugarcane (Saccharum spp. hybrids) shoot tips M.T. González-Arnao, M. Banasiak, S.J. Snyman and Sershen	267-271
	Cryopreservation of collared peccary (Pecari tajacu  Linnaeus, 1758) somatic cells is improved by sucrose and high concentrations of fetal bovine serum Gabriela Pereira de Oliveira Lira, Alana Azevedo Borges, Matheus Barbosa do Nascimento, Leonardo Vitorino Costa de Aquino, Moacir Franco de Oliveira, Alexandre Rodrigues Silva and Alexsandra Fernandes Pereira	272-280
	Storage behavior and cryopreservation of Citrus cavaleriei, an endangered, cold-resistant species of northeast India with exceptionally large seeds S. K. Malik, Ravish Choudhary, Sukhdeep Kaur, Rekha Chaudhury and Hugh W. Pritchard	281-290
	Detection of the metastable ice phase during water crystallization Kosuke Kondo, Kenji Hara, Osaka Keiichi, Shuji Abe and Kazuhito Kajiwara	291-296
	Oxidative stress-induced changes in fertility status of cryopreserved sperm: a diagnosis based on sperm chromatin dispersion assay Archana Kumar and T.B. Sridharan	297-302
	A microfluidic approach for cryoprotectant screening: preliminary validation with human red blood cells Qianqian Hu, Lingxiao Shen, Xiaojie Guo, Xiaoyu Hu, Zhifeng Liu and Gang Zhao	303-307

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CryoLetters 41 (5), 246-256 (2020)
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PERSPECTIVE:
POSSIBLE PROTECTIVE MECHANISMS OF COENZYME Q10 ACTION ON SPERMATOZOA DURING CRYOPRESERVATION OR COOLED-STORED CONDITION

Michael Osei Appiah^{1,2,3 #}, Bismark Asante-Badu^4,#, Jing Zhao^1,2,3,
 Hongyu Liu^1,2,3, Jun Wang^1,2,3,* and Wenfa Lu^1,2,3,*

¹Joint Laboratory of Modern Agricultural Technology International Cooperation,
²Key Laboratory of Animal Production, Product Quality, and Security,
³College of Animal Science and Technology,
⁴College of Resources and Environmental Science,
Jilin Agricultural University, Changchun, 130118, P.R. China.
^# These authors contributed equally to this work.
* Corresponding authors' E-mails: Jun Wang, E-mail: moa4short@outlook.com

Abstract

Artificial insemination (AI) with frozen or cooled-stored semen plays a key role in the widespread distribution of germplasm of elite livestock resources and the protection of endangered species. Cryopreservation provides long-term preservation of sperm and also encourages a greater exchange of genetic material between distant populations. However, freezing has some detrimental effects on sperm, including premature induction of acrosome response, reduced sperm motility, reduced viability, and impaired sperm DNA integrity and fertility. The transition of the membrane phase occurs when the sperm cools down, and lipid accumulation damages the micro-domain, thereby impairing membrane functions, leaving a gap between the gel and the liquid membrane region. Coenzyme Q10 (CoQ10) is a vital lipophilic molecule found in all respiratory eukaryotic cells, including spermatozoa. When such a lipophilic antioxidant is added to the sperm, it can directly diffuse into the polyunsaturated lipid chain present in the plasma membrane, thereby affecting the structure and function of the sperm by generating energy and preventing reactive oxygen. Coenzyme Q10 treatment of sperm from various species improves sperm quality during cryopreservation and cooled-stored condition. It is, however, unclear how this antioxidant affects sperm to improve survival during freezing or cooled-stored condition. Thus, this review highlights the potential protective mechanisms of coenzyme Q10 action during the sperm freezing process.

Keywords: coenzyme Q10; cryopreservation; cooled-stored; mechanism; spermatozoa

Download the paper: POSSIBLE PROTECTIVE MECHANISMS OF COENZYME Q10 ACTION ON SPERMATOZOA DURING CRYOPRESERVATION OR COOLED-STORED CONDITION (PDF)

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CryoLetters 41 (5), 257-266 (2020)
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INCUBATION WITH CHOLESTEROL LOADED CYCLODEXTRIN AND SUBSEQUENT DILUTION IN PARTIALLY DEOXYGENATED EXTENDER IMPROVES THE FREEZABILITY OF CROSSBRED BULL SPERM

Rahul Katiyar^*, Subrata kumar Ghosh, Jai Kishan Prasad,
Abhishek Kumar, Lhendup Bhutia, Vinod Gupta and Rupali Rautela

Division of Animal Reproduction, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, Uttar Pradesh, India.
*Corresponding author E-mail: rahul.katiyarvet@gmail.com

Abstract

BACKGROUND: The cryopreservation process induces osmotic stress, membrane changes and production of reactive oxygen species resulting in damage to the spermatozoa. Together, the presence of oxygen in the extender aggravates the oxidative stress that further reduces the cryosurvival rate of sperm cells. OBJECTIVE: To study the combined effect of cholesterol loaded cyclodextrin (CLC) and partial deoxygenation on post-thaw semen quality in crossbred bulls. MATERIALS AND METHODS: A total of 18 ejaculates from three crossbred bulls with ≥3+ mass motility and ≥70% individual progressive motility were utilized for the study. Each semen sample was divided into four groups: Group I (containing extender without partial deoxygenation or CLC addition); Group II (extender containing 3 mg CLC/120X10⁶ spermatozoa); Group III (extender containing 3 mg CLC/120X10⁶ spermatozoa and 4 ppm dissolved oxygen (DO) level); Group IV (extender containing 3 mg CLC/120X10⁶ spermatozoa and 6 ppm DO level). The samples in each group were finally extended to have 80×10⁶ progressive motile sperm/mL of extender, filled and sealed in French mini straws (0.25 mL) and frozen following equilibration. The effect of CLC addition and partial deoxygenation was assessed at fresh (post-dilution), pre-freeze and post-thaw stages by evaluating various variables [sperm motility, viability, hypo-osmotic swelling (HOS) response, acrosomal integrity, capacitation status and mitochondrial membrane potential (MMP)]. RESULTS: The sperm population was significantly more positive for motility, viability, HOS response, acrosome intactness, high MMP and had less capacitation-like changes in groups supplemented with CLC and partially deoxygenation. However, the positive effect was most pronounced in the group in extender with CLC+4 ppm DO. CONCLUSION: Partial deoxygenation of extender, and CLC addition in combination, could be part of a rationale for improving post-thaw semen quality in cross-bred bulls.

Keywords: cyclodextrin, dissolved oxygen, oxidative stress, bulls.

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CryoLetters 41 (5), 267-271 (2020)
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THE POTENTIAL OF CRYOTHERAPY TO REMOVE SUGARCANE MOSAIC VIRUS FROM SUGARCANE (Saccharum spp. HYBRIDS) SHOOT TIPS

M.T. González-Arnao¹, M. Banasiak², S.J. Snyman^2,3* and Sershen⁴

¹Facultad de Ciencias Químicas, Universidad Veracruzana, Orizaba, Veracruz, Mexico.
2South African Sugarcane Research Institute, Private Bag X02, Mount Edgecombe, 4300, South Africa.
³University of KwaZulu-Natal, Westville Campus, Private Bag X54001, Durban, 4000, South Africa.
⁴Department for Biodiversity and Conservation Biology, University of the Western Cape, Private Bag X17, Bellville, 7535, South Africa.
*Corresponding author E-mail: sandy.snyman@sugar.org.za

Abstract

BACKGROUND: Virus-free sugarcane is difficult to achieve due to the multiple vegetative propagation cycles employed commercially. In vitro culture using small (1 mm) meristematic shoot tips has eliminated viruses but survival is low with small explants. OBJECTIVE: Droplet- Vitrification (D-V) and V-Cryoplate protocols were investigated for the elimination of Sugarcane mosaic virus (SCMV) from large (c. 3 mm) in vitro-derived shoot tips. MATERIALS AND METHODS: Shoot tips excised from NCo376 and N19 cultivars were exposed to both cryogenic procedures. Virus indexing by RT-qPCR was performed 16 weeks after recovery. RESULTS: Explants exposed to cryo-treatments that recovered and multiplied was 30-92%, while at least 90% of control explants regenerated. No virus was detected in multiplied shoots from either cultivar after D-V and liquid nitrogen immersion. In NCo376, virus was eliminated after D-V without cooling. CONCLUSION: The preliminary findings suggest that cryotherapy and/or osmotherapy are viable options for SCMV removal from infected plants.

Keywords: cryopreservation, osmotherapy, qRT-PCR detection, shoot tip size.

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CryoLetters 41 (5), 272-280 (2020)
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CRYOPRESERVATION OF COLLARED PECCARY (Pecari tajacu LINNAEUS, 1758) SOMATIC CELLS IS IMPROVED BY SUCROSE AND HIGH CONCENTRATIONS OF FETAL BOVINE SERUM

Gabriela Pereira de Oliveira Lira¹, Alana Azevedo Borges¹,
Matheus Barbosa do Nascimento¹,
Leonardo Vitorino Costa de Aquino¹, Moacir Franco de Oliveira²,
Alexandre Rodrigues Silva³ and Alexsandra Fernandes Pereira¹*

¹Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoro, RN, Brazil.
²Laboratory of Applied Animal Morphophysiology, UFERSA, Mossoro, RN, Brazil.
³Laboratory of Animal Germplasm Conservation, UFERSA, Mossoro, RN, Brazil
*Corresponding author E-mail: alexsandra.pereira@ufersa.edu.br

Abstract

BACKGROUND: The formation of somatic cell banks is affected by, amongst other factors, the cryoprotectant solution used. The selection of an effective solution, therefore, is a primary parameter. OBJECTIVE: We optimized the cryoprotectant used for collared peccary somatic cell cryopreservation. MATERIALS AND METHODS: We categorized cells into different groups based on their cryopreservation and evaluated the morphology, viability, proliferative activity, metabolism, and oxidative stress. One group was cryopreserved in 10% DMSO with 10% fetal bovine serum (DMSO-10FBS), and another with 50% FBS (DMSO-50FBS). The cryopreservation of both groups included the presence of 0.2 M sucrose (DMSO-SUC-10FBS and DMSO-SUC-50FBS). Non-cryopreserved cells and cells cryopreserved with 10% DMSO (DMSO) supplemented with 0.2 M sucrose (DMSO-SUC) were used as controls. RESULTS: There was no difference observed in morphology or viability among the groups. Proliferative activity was reduced in DMSO-10FBS when compared to controls. Although cryopreservation reduced metabolism, no difference was observed among solutions. A lower level of reactive oxygen species was observed in cells of DMSO-SUC-50FBS when compared to other cryoprotectants. Only cells of DMSO-SUC-50FBS had mitochondrial potential similar to non- cryopreserved cells. CONCLUSION: 10% DMSO supplemented with 50% FBS and 0.2 M SUC was observed to be the most efficient cryoprotectant for preserving collared peccary somatic cells.

Keywords: Peccaries, extracellular cryoprotectants, slow freezing, cryobanking.

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CryoLetters 41 (5), 281-290 (2020)
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STORAGE BEHAVIOR AND CRYOPRESERVATION OF Citrus cavaleriei, AN ENDANGERED, COLD-RESISTANT SPECIES OF NORTHEAST INDIA WITH EXCEPTIONALLY LARGE SEEDS

S. K. Malik¹*, Ravish Choudhary², Sukhdeep Kaur¹, Rekha Chaudhury¹ and Hugh W. Pritchard³

¹Tissue Culture and Cryopreservation Unit, ICAR-National Bureau of Plant Genetic Resources, New Delhi-110012, India
²Division of Seed Science and Technology, ICAR-Indian Agricultural Research Institute, New Delhi-110012
³Royal Botanic Gardens, Kew, Wakehurst, Ardingly, West Sussex RH17 6TN, UK
*Corresponding author E-mail: skm1909@gmail.com

Abstract

BACKGROUND: Ichang Papeda (Citrus cavaleriei H.Lév. ex Cavalerie) is a wild and endangered species of NE India that requires urgent preservation of its genetic resources. OBJECTIVE: To characterize the storage physiology of the seeds and to cryopreserve the embryo and embryonic axis (EA). MATERIALS AND METHODS: The morphology and storage physiology of the seeds were determined, and the cryopreservation of embryos and EA attempted using various techniques (viz., air desiccation-freezing, vitrification and encapsulation-dehydration). RESULTS: Weighing up to 4 g, seeds of Citrus cavaleriei are the largest known in the genus. Based on estimates using the seed coat ratio - seed mass (SCR- SM) model there was a very high probability of seed desiccation intolerance, which was validated physiologically; seeds lost vigour on drying below 30% moisture content (MC) and no seeds germinating after drying to <12 % MC. Embryos and EAs could be air dried to 25- 30% MC and cryopreserved with c. 50% survival. In contrast, EA optimally exposed to PVS2 (20 min) or encapsulated, sucrose pretreated (0.5 M, 24 h) and dehydrated (6 h) had c. 40% survival after cryopreservation. CONCLUSION: Citrus cavaleriei produces large, recalcitrant seeds that can, nevertheless, be cryopreserved as embryos or isolated EA after air drying to c. 25-30% MC; encapsulation-dehydration and vitrification provide alternative options for the cryopreservation of EA.

Keywords: seed coat ratio-seed mass (SCR-SM) model, recalcitrant seed, crop wild relative.

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CryoLetters 41 (5), 291-296 (2020)
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DETECTION OF THE METASTABLE ICE PHASE DURING WATER CRYSTALLIZATION

Kosuke Kondo¹, Kenji Hara², Osaka Keiichi³, Shuji Abe¹
and Kazuhito Kajiwara¹*

¹Graduate School of Bionics, Computer and Media Science, Bionics Program, Tokyo University of Technology, Hachioji, Tokyo, Japan.
²Graduate School of Engineering, Sustainable Engineering Program, Tokyo University of Technology, Hachioji, Tokyo, Japan.
³Industrial Application Division, Japan Synchrotron Radiation Research Institute (JASRI), Mikazuki-cho, Sayou-gun, Hyogo, Japan.
^{* Corresponding author E-mail:} kajiwara@stf.teu.ac.jp

Abstract

BACKGROUND: Under atmospheric pressure, the identifiable phases of ice crystals are hexagonal (stable) and cubic (metastable). OBJECTIVE: This study aimed to test the hypothesis that water crystallizes into the cubic phase at the beginning and then changes to the hexagonal phase. MATERIALS AND METHODS: Aqueous solutions of 40% (w/w) and 50% (w/w) glucose, and 40% (w/w) ammonium hydrogen sulfate, as well as emulsified water, were investigated. RESULTS: The cubic-to-hexagonal ice phase transition was detected in 40% (w/w) glucose solution within a 1 s integration interval, whereas the cubic ice formed in 50% (w/w) glucose solution did not transition to the hexagonal phase. The cubic phase was also confirmed in the 40% (w/w) ammonium hydrogen sulfate solution, but not in emulsified water. CONCLUSION: The cubic-to-hexagonal ice phase transition was detected in three aqueous solutions tested upon freezing. It was not possible to clearly capture the transition process in emulsified water under the study condition.

Keywords: water crystallization, cubic ice, hexagonal ice, cubic to hexagonal phase transition.

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CryoLetters 41 (5), 297-302 (2020)
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OXIDATIVE STRESS-INDUCED CHANGES IN FERTILITY STATUS OF CRYOPRESERVED SPERM: A DIAGNOSIS BASED ON SPERM CHROMATIN DISPERSION ASSAY

Archana Kumar and T.B. Sridharan*

Department of Biotechnology, School of BioSciences and Technology, VIT, Vellore, Tamil Nadu, India
*Corresponding author E-mail: tbsridharan@vit.ac.in; archanatnau@gmail.com

Abstract

BACKGROUND: Cryopreservation introduces iatrogenic damage to sperm cells due to excess production of reactive oxygen species (ROS) that can damage sperm macromolecules and alter the physiochemical properties of sperm cells. These altered properties can affect the biological potential of sperm cell towards fertility. OBJECTIVE: The study was designed to assess the role of oxidative stress in sperm DNA damage upon cryopreservation. MATERIALS AND METHODS: Semen samples (160) were classified into fertile and infertile on the basis of Computer Assisted Semen Analysis (CASA), and cryopreserved. Thawed samples were analyzed for 8OHdG marker, sperm chromatin dispersion (SCD)-based DNA fragmentation index (SCD-DFI) and ROS levels. Receiver Operating Characteristics (ROC) was performed to find the specificity and sensitivity of SCD-DFI in assessing the sperm DNA integrity. Principle component analysis (PCA) was performed to group semen parameters. RESULTS: SCD-DFI significantly correlates with 8OHdG in infertile samples (r=0.73, p<0.0001) and moderately in fertile samples (r=0.52, p<0.001). Among SCD-DFI, ROS and 8OHdG values, SCD-DFI possess a higher area under the curve (0.83) in ROC, suggesting its potential in representing sperm DNA damage. PCA analysis extracted SCD-DFI and sperm progressive motility as two independent parameters, differentiating fertile and infertile cryopreserved semen. CONCLUSION: The study proves that SCD can potentially represent ROS-induced sperm DNA damage in infertile semen upon cryopreservation.

Keywords: cryopreservation, male infertility, receiver operating characteristics, sperm DNA damage.

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CryoLetters 41 (5), 303-307 (2020)
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A MICROFLUIDIC APPROACH FOR CRYOPROTECTANT SCREENING: PRELIMINARY VALIDATION WITH HUMAN RED BLOOD CELLS

Qianqian Hu¹, Lingxiao Shen¹, Xiaojie Guo², Xiaoyu Hu²,
Zhifeng Liu³* and Gang Zhao¹*

¹Department of Electronic Science and Technology, University of Science and Technology of China, Hefei, P.R. China.
²Hefei Blood Center, Hefei, P.R. China.
³Department of Thermal Sciences and Energy Engineering, University of Science and Technology of China, Hefei, P.R. China.
*Corresponding author E-mail: zhaog@ustc.edu.cn(GZ); lzf123@ustc.edu.cn(ZL).

Abstract

BACKGROUND: The application of microfluidics has overcome a series of long-term existing bottlenecks in the field of cryobiology. However, there is still no investigation regarding the on- chip rapid screening of cryoprotectant concentration optimization for the integration of the entire freeze, thaw and cell counting process. OBJECTIVE: To establish an on-chip approach for rapid cryoprotectant screening and concentration optimization. MATERIALS AND METHODS: Rapid freezing of red blood cells with glycerol and dimethyl sulfoxide was used to validate the proposed on-chip method. RESULTS: The optimal cryoprotectant and its concentration can be determined with the described method, without the need of additional samplings and assays. The optimal concentration for glycerol and dimethyl sulfoxide used in the validation experiments was 10% (w/v). CONCLUSION: The microfluidic approach can be used for rapid cryoprotectant screening and concentration optimization.

Keywords: microfluidics; cryopreservation; cryoprotectant screening.

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