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Abstracts: CryoLetters 43 (2), 2022



Volume 43, No. 2 March/April 2022

ISSN 0143-2044



PERSPECTIVE: Systemic and local hypothermia in the context of cell regeneration (PDF)
Basheer Abdullah Marzoog




Evaluation of cold tolerance of seven walnut varieties
Rui Zhang, Bing Liu, Guo Xin, Xiayi Zhang, Jianhong Li
and Yanxiu Wang




Whole-body cryotherapy enhances the expression of heat shock protein 70 and related hormones
Nobuhiko Eda, Kazuhiro Shimizu, Ai Takemura, Emi Narumi, Mariko Nakamura, Masako Hoshikawa and Michiko Dohi




The evaluation of rosemary (Rosmarinus officinalis) leaf extract inclusion in freezing medium on quality parameters of buffalo bull spermatozoa
Hussain Ahmed, Sarwat Jahan, Israr Alam, Farhad Ullah
and Muhammad Umar Ijaz




Effect of synthetic cholesterol (SyntheChol®) supplementation in an egg yolk-free extender on dog sperm cryopreservation
Nabeel Abdelbagi Hamad Talha, Yubyeol Jeon
and Il-Jeoung Yu




Sulfated polysaccharides from seaweed as a supplement to Prochilodus brevis sperm freezing medium
Renata Vieira do Nascimento,
Priscila Silva de Almeida-Monteiro, Vanessa Alves Pereira, Thais Maia Torres, Larissa Teixeira Nunes, Yara Silvino Sales, Bruna Farias Brito, Francisco Hiago Gadelha Moreira,
Ianna Wivianne Fernandes Araújo,
José Ariévilo Gurgel Rodrigues, Assis Rubens Montenegro and Carminda Sandra Brito Salmito-Vanderley




Relationship between toxicity of cryoprotectants, osmotic and oxidative stresses in Awassi ram sperm
Ömer Varişli, Faruk Bozkaya, Nurettin Aydilek
and Abdullah Taşkin






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CryoLetters 43 (2), 66-73 (2022)
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Basheer Abdullah Marzoog

National Research Mordovia State University, Bolshevitskaya Street 31, Saransk, Mordovia Republic.
Author’s E-mail: and ORCID: 0000-0001-5507-2413.


Local and systemic cooling is an inducer of cell proliferation. Cell proliferation and transdifferentiation or stem cells differentiation involves microenvironment regulation such as temperature. Mild hypothermia downregulates the production of pro-inflammatory cytokines and reduces the immune response against pathogens. In addition, mild tissue cooling improves endothelial cell function. Endothelial cells are involved in angiogenesis during regeneration strategies; therefore, their death is catastrophic and affects regeneration, but not cell proliferation. The potential mechanism underlying the effects of local or systemic hypothermia on cell regeneration has not yet been elucidated. Hypothermia reduces the production of reactive oxygen species and organelle activity. Hypothermia therapeutic effects depends on the targeted organ, exposure duration, and hypothermia degree. Therefore, determining these factors may enhance the usage of hypothermia more effectively in regenerative medicine. The paper introduces the hypothermia role in paracrine/endocrine cell secretion, reception, and the immune state after local and systemic hypothermia application.

Keywords: cryotherapy; hypothermia; regeneration: immune cells; cytokine; proliferation; differentiation.





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CryoLetters 43 (2), 74-82 (2022)
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Rui Zhang1, Bing Liu1, Guo Xin2, Xiayi Zhang1, Jianhong Li3
and Yanxiu Wang1*

1 College of Horticulture, Gansu Agricultural University, Lanzhou, Gansu, P.R. China.
2 Longnan Economic Forest Research Institute, Longnan, Gansu, P.R. China.
3 Forestry Science and Technology Extension Station of Gansu Province, Lanzhou, Gansu, P.R. China.
*Corresponding author's E-mail:


BACKGROUND: Winter injury often threatens phenology and walnut survival in late spring and winter in northern areas of China. OBJECTIVE: This study aimed at evaluating seven walnut varieties which have superior economic traits. MATERIALS AND METHODS: Mature functional leaves were observed by paraffin section and the related anatomical structure indices were measured. The stem segments were placed at six temperatures [4 (control), -10, -15, -20, -25, -30°C] for low temperature stress treatment, and the relevant indices were measured. RESULTS: The thickness of palisade tissue (PT), sponge tissue (ST) and the PT/ST of ‘Wen185’ was significantly higher than in ‘Longboxiang 2’, ‘Longboxiang 3’ and ‘Suizhuang’. However, the leaf thickness (LT) of ‘Longboxiang 3’ was markedly lower than that of other varieties. With the decrease in temperature, relative electrolyte leakage (REL), proline (Pro) and soluble sugar content (SS) of all varieties increased, while superoxide dismutase (SOD) and peroxidase (POD) activities showed a rise and fall trend. According to the LT50, the cold tolerance of seven walnut varieties was ranked from high to low, viz. ‘Yuanlin’ > ‘Wen185’ > ‘Xiangling’ > ‘Suizhuang’ > ‘Qingxiang’ > ‘Longboxiang 2’ > ‘Longboxiang 3’. CONCLUSION: The variety ‘Yuanlin’ can adapt to low temperature and should be widely promoted.

Keywords: anatomic structure; antioxidant enzyme activity; cold tolerance; electrolyte leakage; osmotic substances.



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CryoLetters 43 (2), 83-90 (2022)
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Nobuhiko Eda1,2*, Kazuhiro Shimizu2, Ai Takemura2, Emi Narumi2, Mariko Nakamura2, Masako Hoshikawa2 and Michiko Dohi2

1Department of Fundamental Education, Dokkyo Medical University, Shimotsugagun, Tochigi, Japan;
2Japan Institute of Sports Sciences, Kita-Ku, Tokyo, Japan.
* Corresponding author's E-mail:


BACKGROUND: Whole-body cryotherapy (WBC) is used as a conditioning method for athletes. However, the scientific evidence for its effects is still insufficient. OBJECTIVE: To elucidate the effects of transient WBC on the expression of heat shock protein (HSP) 70 and the secretion of related hormones in humans. MATERIALS AND METHODS: The participants in this study were six healthy adult men. WBC was performed for 3 min in a booth at a temperature in the range of -150 to -120°C, and measurements were taken immediately before (Pre), immediately after (Post), and 60 min after WBC (Post60). For measurement of core body temperature (gastrointestinal temperature), participants ingested a capsule-type wireless temperature sensor. The body surface temperature was measured using a noncontact thermometer, and measurements were taken at four sites on the body surface (chest, abdomen, front of the thigh, and front of the lower thigh). Leukocyte count, lactate dehydrogenase, creatine kinase, hemoglobin, hematocrit, adrenaline, noradrenaline, cortisol, adrenocorticotropic hormone (ACTH), erythropoietin, and HSP70 in the collected blood were measured. RESULTS: The results showed a decrease in body surface temperature and an increase in noradrenaline and ACTH immediately after WBC. In addition, the core body temperature decreased 60 min after WBC, accompanied by an increase in HSP70 expression. CONCLUSION: WBC may increase HSP70 expression via noradrenaline and ACTH. The results of this study suggest the usefulness of WBC in triggering protein synthesis and the maintenance of immune function after training

Keywords: conditioning; core temperature; cryostimulation; heat shock protein; hormone; recovery.




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CryoLetters 43 (2), 91-98 (2022)
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Hussain Ahmed1,2*, Sarwat Jahan2, Israr Alam1, Farhad Ullah3 and Muhammad Umar Ijaz4

1Department of Zoology, University of Buner, Khyber Pakhtunkhwa (KP), Pakistan.
2Reproductive Physiology Laboratory, Department of Animal Sciences, Quaid-i-Azam University (QAU), Islamabad, Pakistan.
3Department of Zoology, Islamia College University, Peshawar, KP, Pakistan.
4Department of Zoology, Wildlife and Fisheries, University of Agriculture, Faisalabad, Pakistan.
*Corresponding author's E-mail:


BACKGROUND: The discrepancy between the endogenous antioxidants concentrations and free radicals results in oxidative stress and cellular injury. OBJECTIVE: To appraise the usefulness of Rosemarinus officinalis (RO) aqueous extract in protecting buffalo spermatozoa during freezing / thawing process. MATERIALS AND METHODS: Qualifying ejaculates from four well-restrained bulls were evaluated initially and then diluted in a freezing medium supplemented with RO-0.00, RO-0.50%, RO-1.00%, RO-2.00%, and RO-4.00%, cooled to 4ºC in 2 h, equilibrated for 4 h at 4ºC, packed in straws, and cryopreserved, and thawed at 37ºC for 30 s followed by evaluation. RESULTS: We found that freezing medium supplemented with RO-2.00% improves progressive motility (%) compared to the control. Similarly, a lower rate of apoptosis-like changes (%) was recorded with RO-4.00% than the control, RO-0.50% and RO-1.00%. This response was accompanied by an increment in viable spermatozoa. Semen samples supplemented with RO-2.00% and RO-4.00% displayed higher TAC (total antioxidant capacity, µM/L) and ATP (nmol/million) content than the control. In addition, semen samples supplemented with RO-2.00% displayed lower concentrations of ROS (reactive oxygen species, 104 RLU/20 min/25 million) than the control and RO-0.05%. Also LPO (lipid peroxidation, µM/L) with RO-2.00% and RO-4.00% was lower than the control. CONCLUSION: The inclusion of rosemary aqueous extract ameliorates motility features, structural and functional parameters, viability, TAC and ATP content of bull sperm. Conversely, the inclusion of rosemary aqueous extract alleviates apoptosis-like changes, ROS and LPO in comparison to the control. Further studies are required to determine the mechanism of action of rosemary aqueous extract in ameliorating semen quality and fertility of buffalo spermatozoa.

Keywords: CASA; cryopreservation; fertility; plasmalemma; ROS; semen quality.




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CryoLetters 43 (2), 99-109 (2022)
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Nabeel Abdelbagi Hamad Talha1,2, Yubyeol Jeon1 and Il-Jeoung Yu1*

1Laboratory of Theriogenology and Reproductive Biotechnology, College of Veterinary Medicine and Bio-safety Research Institute, Jeonbuk National University, Iksan 54596, Republic of Korea.
2Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, Sudan University of Science and Technology, Khartoum 11111, Sudan.
*Corresponding author's E-mail:


BACKGROUND: SyntheChol® is a new synthetic, non-animal-derived cholesterol that is easily dissolved in ethanol, ready to use, and behaves in a similar way as natural cholesterol. Therefore, it could be used as a substitute of natural cholesterol in dog sperm freezing extender. OBJECTIVE: To evaluate the effect of supplementing an egg yolk-free (EY-free) extender with synthetic cholesterol (SyntheChol®) on cryopreserved dog sperm. MATERIALS AND METHODS: Spermatozoa (1 × 108 sperm/mL) were suspended in EY-free extender supplemented with 0% (control), 0.25, 0.5, 1, 2, 4, or 6% SyntheChol® (Extender 1), cooled at 4 °C for 1 h, and diluted (1:1, v/v) with Extender 1 containing 1 M glycerol. The spermatozoa were then cooled to 4 °C for 30 min. Sperm-containing straws were frozen using LN2 vapor. Sperm motility (computer-assisted sperm analysis, CASA), sperm membrane integrity (SYBR-14 and PI staining), and acrosome integrity (FITC-PSA) were evaluated after thawing. Thereafter, optimal concentrations were determined (0.25, 0.5, 1, or 2%) and used to evaluate reactive oxygen species (ROS) generation, apoptosis, and the gene expression of motility-related sperm mitochondria-associated cysteine-rich protein, apoptosis-related B-cell lymphoma 2 (BCL2), and BCL2-associated X protein (BAX) in cryopreserved sperm. RESULTS: Sperm progressive motility, membrane integrity, and acrosome integrity were markedly greater in the SyntheChol®-supplemented groups (0.25, 0.5, 1, or 2%) than in the control group. Only BAX expression was significantly reduced in the SyntheChol® groups (0.25, 1, or 2%) compared with the control group. However, there were no significant effects on the ROS generation or apoptosis index. CONCLUSION: SyntheChol® (0.25, 1, or 2%) proved to be effective in reducing the BAX gene expression level and improving sperm progressive motility, and membrane and acrosome integrity.

Keywords: dog spermatozoa, cryopreservation, gene expression, SyntheChol®.




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CryoLetters 43 (2), 110-119 (2022)
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Renata Vieira do Nascimento1*, Priscila Silva de Almeida-Monteiro1, Vanessa Alves Pereira1, Thais Maia Torres1, Larissa Teixeira Nunes1, Yara Silvino Sales2, Bruna Farias Brito1,
Francisco Hiago Gadelha Moreira1,
Ianna Wivianne Fernandes Araújo3, José Ariévilo Gurgel Rodrigues3, Assis Rubens Montenegro1
and Carminda Sandra Brito Salmito-Vanderley1

1Fish Reproduction Biotechnology Laboratory, Postgraduate Program in Veterinary Sciences, State University of Ceará, Fortaleza, Ceará, Brazil.
2Fish Reproduction Biotechnology Laboratory, State University of Ceará, Fortaleza, Ceará, Brazil.
3Federal University of Ceará, Fortaleza, Ceará, Brazil.
4Polymers and Material Innovation Laboratory, Federal University of Ceará, Fortaleza, Ceará, Brazil.
*Corresponding author's E-mail:


BACKGROUND: Using sulfated polysaccharides (SP) in fish sperm freezing medium promotes cell maintenance. OBJECTIVE: To evaluate the effect of different SP concentrations, extracted from two seaweeds (Gracilaria domingensis and Ulva fasciata), as a supplement to the sperm freezing medium of Prochilodus brevis. MATERIALS AND METHODS: Five semen pools were diluted in a solution composed of 5% glucose, 10% dimethyl sulfoxide (DMSO) and different SP concentrations (0, 0.5, 1.0, 1.5, 2.0, 2.5 or 3.0 mg/mL). The samples were cryopreserved and, after 7 days, rewarmed and analyzed for morphology, plasma membrane integrity, DNA integrity, mitochondrial activity and sperm kinetics [total motility, progressive motility, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN), and wobble (WOB)]. RESULTS: There was no interaction between seaweed and SP concentrations. Similar effects were observed with SP extracted from the two seaweeds, regardless of concentration. When comparing the SP concentrations, regardless of the seaweed, 1.0 mg/mL SP showed better results for VCL and VSL. For VAP and WOB, 1.0 mg/mL SP showed better results, but differed from 3.0 mg/mL. LIN followed the same pattern, but differed from SP at 2.5 and 3.0 mg/mL. For progressive motility, 1.0 mg/mL G. domingensis showed superior results compared to the control. For mitochondrial activity, G. domingensis was superior to U. fasciata, regardless of concentration. The lowest concentrations (0.5 and 1.0 mg/mL) showed the best results, regardless of the seaweed. However, the control was superior to all treatments tested. CONCLUSION: G. domingensis SP at the lowest concentrations might be a potential supplement to the P. brevis freezing medium.

Keywords: antioxidants; Brazilian bocachico; cryopreservation; oxidative stress; sperm kinetics.




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CryoLetters 43 (2), 120-128 (2022)
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Ömer Varişli1, Faruk Bozkaya2, Nurettin Aydilek3 and Abdullah Taşkin4

1Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Kırıkkale University, Kırıkkale, Turkey.
2Department of Genetics, Faculty of Veterinary Medicine, Harran University, Şanlıurfa, Turkey.
3Department of Physiology, Faculty of Veterinary Medicine, Dicle University, Diyarbakır, Turkey.
4Faculty of Health Sciences, Harran University, Şanlıurfa, Turkey.
*Corresponding author's E-mail:


BACKGROUND: The relationship between the toxicity of cryoprotectants and their osmotic and/or oxidative stresses remains to be further investigated. OBJECT?VE: To investigate the toxic effects of different cryoprotectants and osmotic stress on Awassi ram sperm and to determine the relationship between oxidative and antioxidative status of the sperm. MATERIALS AND METHODS: Pooled sperm samples were exposed to sucrose solutions of different concentrations (75 to 900 mOsm) and isosmotic condition (290-325 mOsm) was re-established by adding HEPES buffered Tyrode’s lactate. Sperm samples were mixed with 0.5, 1.0 and 1.5 M of glycerol, methanol, 2-methoxyethanol, dimethylacetamide or 1,2-propanediol for 5 min and returned to isosmotic condition. Sperm samples were exposed to cryoprotectants at 4°C for 2 hours and isosmotic conditions were re-established. Motility, viability, acrosome integrity and oxidative or antioxidative parameters were determined. RESULTS: Treatment with hypo- or hyperosmotic sucrose solution reduced motility and viability without affecting acrosome integrity. The addition and removal of glycerol and dimethylacetamide (1.0 or 1.5 M) decreased sperm motility, while cryoprotectants had no effect on viability except for 1.5 M glycerol. Chilling significantly reduced the motility and viability of the sperm, but not the acrosome integrity. Rapid addition or removal of cryoprotectants also did not affect the acrosome integrity. Cryoprotectants changed only the ceruloplasmin level, while there were significant post-chilling differences in lipid hydroperoxide, paraoxonase and ceruloplasmin levels. CONCLUSION: Cryoprotectants without other additives have limited protection and glycerol can be toxic to spermatozoa. The oxidative stress plays a role in cryoprotectant toxicity and chilling stress.

Keywords: cryoprotectants; oxidative stress; ram; toxicity; sperm.

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