Abstracts: CryoLetters 39 (2), 2018

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



Volume 39, No. 2 March/April 2018

ISSN 0143-2044



Vitrification has an effect like culture on gene expression and histone modification in mouse embryos
Seyedeh Saeideh Sahraei, Maryam Shahhoseini and
Bahar Movaghar




The complementary effect of cholesterol and vitamin e preloaded in cyclodextrins on frozen bovine semen: motility parameters, membrane integrity and lipid peroxidation
A Khellouf, K. Benhenia, S. Fatmi and M. Iguer-Ouada




Association between DMSO and sugars in the sperm cryopreservation of Pacu
Diego Martins Pires, Carine Dahl Corcini,
Alessandra Cardoso da Silva, Stela Mari Menegello Gheller,
Fernanda Alves Pereira, Jéssica Ribeiro Pereira,
Juan Ramon Esquivel Muelbert, Rodrigo Dessesards Jardim,
Juan Ramon Esquivel Garcia and Antonio Sergio Varela Junior




Effects of antifreeze protein supplementation on the development of porcine morulae stored at hypothermic temperatures
Thanh-Van Nguyen, Fuminori Tanihara, Maki Hirata,
Takayuki Hirano, Katsutoshi Nishio, Lanh Thi Kim Do,
Thanh Van Nguyen, Masahiro Nii and Takeshige Otoi




An experimental and numerical study on nodular gel phantom during cryotherapy
Chandrika Kumari, A. Kumar, S.K. Sarangi and
A. Thirugnanam




Learning aspects in implementing a vitrification program using mouse blastocysts
E.J. Hoekman, K.E. Tucker, C.A.M. Jansen and C.G. Hilders




Supplementation of rosmarinic acid has reduced oxidative stress on bull spermatozoa following the freeze thawing process
Deniz Yeni, Muhammed Enes İnanç, Fatih Avdatek,
Pürhan Barbaros Tuncer, Beste Çil, Ruhi Türkmen and
Umut Taşdemir






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CryoLetters 39 (2), 102-112 (2018)
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Seyedeh Saeideh Sahraei1, Maryam Shahhoseini2 and Bahar Movaghar1*

1Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
2Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Phone number: +982122339901 Fax: +982122339916
*Corresponding author email:


OBJECTIVE: This paper reports results from studies on modifications of histone marks in transcriptional regulatory regions of H19, Igf2 and Mest genes in vitrified-warmed and cultured mouse embryos.MATERIALS AND METHODS: Non vitrified and vitrified-warmed 8-cell embryos were cultured in Ham's F10+10% BSA into blastocyst stage in two experimental groups. Gene expression level was assessed by quantitative PCR and histone modifications were evaluated by ChIP assay method. RESULTS: The expression levels of the selected genes in vitrified embryos were found to be markedly reduced compared to the control group. Significant increase in H3K9me2 as a gene silencer was seen in the regulatory regions of all genes in vitrified group. Furthermore, H3K4me3 and H3K9ac decreased in some regulatory regions but not in all of them. Additionally, H3K9me2 in regulatory regions of Igf2 and H19 significantly increased in group I, whereas, there was no change in Mest compared to the control group. CONCLUSIONS: Vitrification and culture condition affects gene down regulation of Igf2 and H19. Although changes in each histone mark are not always the same as gene expression, the total pattern and sum of their changes are in line with gene down regulation upon vitrification and culture.

Keywords: vitrification, histone modification, imprinting gene, blastocyst embryo




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CryoLetters 39 (2), 113-120 (2018)
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    A Khellouf1*, K. Benhenia2,3, S. Fatmi4 and M. Iguer-Ouada1

1Associated Laboratory in Marine Ecosystems and Aquaculture, Department of Biological Sciences of the Environment, Faculty of Nature and Life Sciences, Abderrahmane-Mira-University, Route de Targua Ouzemmour, 06000 Bejaia, Algeria
2High National Veterinary School, 16270, Street Issad Abbes, Oued Smar, Algiers, Algeria
3National Center for Biotechnology Research (CRBt), Ali Mendjli Nouvelle Ville UV 03 BP E73, Constantine, Algeria
4Pharmaceutical Laboratory, Department of Engineering Processes, Abderrahmane-Mira-University, Route de Targua Ouzemmour, 06000 Bejaia, Algeria
Corresponding author email:


BACKGROUND: During cryopreservation sperm cells suffer from two major deleterious impacts: oxidative stress and cold shock.  OBJECTIVE: To investigate in bovine species the benefit of cholesterol and vitamin E, both loaded in cyclodextrins, as a double protection against oxidative stress and cold shock. MATERIALS AND METHODS: Semen was collected from nine mature bulls using an artificial vagina and each ejaculate was split into four equal aliquots. The control aliquot was diluted with Fraction A (Tris+citric acid+fructose+penicillin) without further supplementation; the treated samples were diluted in Fraction A supplemented with cholesterol-loaded cyclodextrins (CD-CHL), vitamin E-loaded cyclodextrins (CD-Vit E) or CD-CHL in association with CD-Vit E (CD-CHL-VitE). After incubation at 22°C for 15 min and addition of Fraction B (Fraction A+egg yolk+glycerol), all aliquots were frozen in 0.25 ml straws. Straws were then thawed individually at 37°C for 30 seconds in a water bath and immediately analyzed for motility, using Computer Aided Semen Analysis (CASA), membrane integrity and oxidative stress status. RESULTS: The results showed that samples treated with CD-CHL and CD-Vit E were protected against the deleterious impact of freezing thawing process.  However, the optimal protection was observed when the two complexes CD-CHL and CD-Vit E were simultaneously used. All analysed semen parameters including motility, membrane integrity and oxidative stress status were significantly improved in CD-CHL-Vit E compared to all other treatments. CONCLUSION: C holesterol and vitamin E, both preloaded in cyclodextrins to increase their solubility, appeared as a powerful protection in cryopreserved bovine semen to fight simultaneously cold shock and oxidative stress.

Keywords: Bull sperm; Cryopreservation; Cholesterol; Cyclodextrins; vitamin E; oxidative stress




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CryoLetters 39 (2), 121-130 (2018)
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Diego Martins Pires1, Carine Dahl Corcini1,
Alessandra Cardoso da Silva1, Stela Mari Menegello Gheller1,
Fernanda Alves Pereira2, Jéssica Ribeiro Pereira2,
Juan Ramon Esquivel Muelbert3, Rodrigo Dessesards Jardim2,
Juan Ramon Esquivel Garcia4 and Antonio Sergio Varela Junior2*

1Pós Graduação em Veterinária, Universidade Federal de Pelotas (UFPEL), Pelotas, RS, Brasil.;
2Instituto de Ciências Biológicas, Universidade Federal do Rio Grande (FURG), Rio Grande, RS, Brasil.;
3Piscicultura Panamá, Paulo Lopes, SC, Brasil. 4Universidade do Sul de Santa Catarina (Unisul), Florianópolis, SC, Brasil.
*Corresponding author email:


BACKGROUND: The cryopreservation protocol that has been developed exclusively for the preservation of the sperm of the species different. OBJECTIVE: this study was to evaluate the effect of the association of 10% DMSO with trehalose, raffinose, sucrose and lactose concentrations on the sperm cells of Piaractus mesopotamicus. MATERIALS AND METHODS: Sperms were collected from the animals through abdominal massage. The samples were diluted in the Beltsville Thawing Solution without different concentrations of other sugars (test conditions). Sixty days after the cryopreservation, cell movement analysis was performed using CASA. RESULTS: The results revealed that the parameters for total motility and motility period were superior when 100mM raffinose (P<0.05). The lateral displacement of the head was observed to be improved was 100mM lactose, 150mM sucrose and 150mM raffinose (P<0.05) as compared to treatment wherein lactose (0mM) was omitted. CONCLUSION: the results of our study indicated that the ideal parameters for cryopreservation, were obtained when the cryopreservation fluid contained 100mM raffinose in association with DMSO.

Keywords: Pacu; Sugars; Flow cytometry; Kinetics




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CryoLetters 39 (2), 131-136 (2018)
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EFfects of antifreeze protein supplementation on the development of porcine morulae stored at Hypothermic temperatures

Thanh-Van Nguyen1, Fuminori Tanihara1, Maki Hirata1,
Takayuki Hirano1, Katsutoshi Nishio1, Lanh Thi Kim Do1,2,
Thanh Van Nguyen2, Masahiro Nii3 and Takeshige Otoi1

1Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan
2Faculty of Veterinary Science, Vietnam National University of Agriculture, Vietnam
3Tokushima Prefectural Livestock Research Institute, Tokushima, Japan
*Corresponding author email:


BACKGROUND: Short-term storage is valuable method to reuse manipulated embryos. OBJECTIVE: The present study evaluated the effects of antifreeze protein (AFP) supplementation on the quality and development of in vitro-produced porcine morulae after short-term storage (24 h). MATERIALS AND METHODS: The morulae were stored with various concentrations of AFP type III for 24 h at 5, 15 and 25C. RESULTS: Supplementation of AFP type III (1.0 µg/ml) improved the developmental competence of embryos stored at 25°C. The proportions of DNA-fragmented nuclei in the blastocysts did not differ between the embryos stored at 25°C and the control embryos without storage treatment. However, the developmental competence of embryos stored at hypothermic temperatures decreased relative to that of the control embryos. CONCLUSION: Supplementation of AFP type III (1.0 µg/ml) maintained the quality of embryos stored at 25°C, but did not have beneficial effects on the development of embryos stored at hypothermic temperatures.

Keywords: antifreeze proteins, chilling, embryo, porcine, quality




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CryoLetters 39 (2), 137-146 (2018)
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Chandrika Kumari1, A. Kumar2, S.K. Sarangi3 and A. Thirugnanam1*

1Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha-769008, India.
2Department of Mechanical Engineering, Indian Institute of Technology, Varanasi (BHU), Uttar Pradesh-221005, India.
3Department of Mechanical Engineering, National Institute of Technology, Rourkela, Odisha-769008, India.
*Corresponding author email: (A.Thirugnanam)


BACKGROUND: Cryogen spray cooling is an effective method to treat nodular basal cell carcinoma. OBJECTIVE: To study the effect of spraying distance (z = 27 mm, 18 mm and 9 mm) on a nodular gel phantom and to validate the numerical model. MATERIALS AND METHODS: Liquid nitrogen is sprayed on agarose gel phantom using 0.8 mm nozzle diameter. A two-dimensional Pennes equation with phase change is solved on the axisymmetric non-orthogonal grid. RESULTS: The lethal front is obtained at 4 mm from the gel surface, irrespective of spraying distance and the same is validated with numerical results. The maximum necrotic volume and lethal front achieved by -50°C and -25°C isotherms seem to have a lower dependence on the spraying distance as opposed to those corresponding to the 0ºC isotherm. In contrast, the initiation of these parameters is highly dependent on the nozzle to gel height. CONCLUSION: The numerical study presents good agreement with experimental data. The decrease in spraying distance leads to higher rate of maximum ablation volume during freezing, but final ablation volume remained approximately similar.

Keywords: cryospray, gel phantom, lethal front, ablation volume, basal cell carcinoma




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CryoLetters 39 (2), 147-155 (2018)
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E.J. Hoekman1,2, K.E. Tucker2, C.A.M. Jansen2 and C.G. Hilders1,2

1Dept. of Gynecology, Leiden University Medical Center, P/O Box 9600, 2300RC Leiden, The Netherlands
2IVF Dept., Reinier de Graaf Group, P/O Box 998, 2275 CX Voorburg, The Netherlands
*Corresponding author email:


BACKGROUND: Oocyte vitrification is important for fertility preservation.  There are debates, however, surrounding the components of the procedure itself.  OBJECTIVE: before starting a vitrification program, we decided to (1) determine if blastocysts derived from previously frozen mouse embryos would be suitable to practice vitrification and (2) to analyze the factors that contributed to improving our "learning curve". MATERIALS AND METHODS: 58 expanded blastocysts cultured from commercially available frozen 1-cell mouse embryos (B6D2F1 x B6C3F1) were used. Embryos were vitrified, during 2 separate attempts, in a closed device (Cryopette) and vitrified and warmed using Ethylene Glycol-cryoprotectant vitrification kit (Vitrification Attempt 1 and 2 – VA1 and 2; Warming Attempt 1 and 2 – WA1 and 2). Differences in attempts by more focusing on the reaction of the embryo itself. The survival rate (SR) of embryos was evaluated immediately after warming (T0), then rinsed and cultured for 24 hours in an HTF-like medium (Cleavage Medium – Sage; Cooper Surgical; U.S.A.).  Additional evaluations were done at 2 and 24 hours of culture (T2 and T24, respectively). RESULTS: The combined SR for both attempts were 70.6, 60.3 and 34.4% at T0, T2 and T24, respectively. Embryo loss was significantly higher in WA1 compared to WA2 (30.8 versus 10.1%; p=.05). The SR at T0 after VA1 and VA2 were similar (90.9 versus 80.9%) and the SR after VA1+WA1 versus VA1+WA2 was 55.6 vs. 76.9% at T2 and 22.2 vs. 61.5% at T24, respectively (NS).  CONCLUSION: Commercially frozen mouse embryos can provide essential information for initiating a Vitrification Program applicable to human embryos and oocytes.  Practice is critical, particularly Warming, which can provide the confidence needed to adapt and optimize these protocols before vitrifying limited human tissues or gametes.

Keywords: Vitrification, Fertility preservation, frozen mouse embryos, learning curve




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CryoLetters 39 (2), 156-165 (2018)
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Deniz Yeni1, Muhammed Enes İnanç2, Fatih Avdatek1,
Pürhan Barbaros Tuncer3, Beste Çil4, Ruhi Türkmen5
and Umut Taşdemir6

1Afyon Kocatepe University, Faculty of Veterinary Medicine, Department of Reproduction and Artificial Insemination, Afyon, Turkey
2Mehmet Akif University, Faculty of Veterinary Medicine, Department of Reproduction and Artificial Insemination, Burdur, Turkey
3Mersin University, Technical Sciences Vocational School, Mersin, Turkey.
4Ankara University, Faculty of Veterinary Medicine, Department of Reproduction and Artificial Insemination, Ankara, Turkey.
5Afyon Kocatepe University, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Afyon, Turkey
6*Aksaray University, Technical Sciences Vocational School, TR-68100, Aksaray, Turkey
Phone: +90 535 6654375
*Corresponding author email:


BACKGROUND: Cryopreservation has a side effect on the motility, chromatin integrity and viability of sperm cells. OBJECTIVE: The present study investigated the effects of supplementation with rosmarinic acid (RA) Tris extender on sperm quality parameters, plasma and acrosome membrane damage, antioxidant enzyme activity and chromatin integrity following the freeze thawing process on bull spermatozoa. MATERIALS AND METHODS: Ejaculates were split into five aliquots and diluted to a final concentration of 15x106 spermatozoa/ml with the Tris extender containing RA (25, 50, 100 and 200 µg/ml) and (control) and then frozen at a controlled rate. RESULT: Treatments did not give better results on the percentages of sperm progressive, total motility and sperm motion characters (P>0.05); however, RA25 and RA50 exhibited favourable chromatin integrity. In conclusion, RA25 and RA50 increased total antioxidant activity. As a consequence, the amount of MDA and chromatin damage were reduced in sperm cells.

Keywords: Antioxidant, bull semen cryopreservation, oxidative stress, rosmarinic acid

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