Volume 37, No. 2 March/April 2016 ISSN 0143-2044
	Cryopreservation on a cryo-plate of Arundina graminifolia protocorms, dehydrated with silica gel and drying beads Luis B. Cordova II and Kanchit Thammasiri	68-76
	Melatonin-loaded alginate beads improve cryopreservation of Yam (Dioscorea alata and D. cayenensis) Esther E. Uchendu and E. R. Joachim Keller	77-87
	The revascularization and follicular survival of Mouse ovarian grafts treated with FSH during cryopreservation by vitrification Hong Zhang, Yanzhou Yang. Wenye Ma, Hao Wu, Xiaomin Zheng, Changchun Hei, Miao Sun, Wenzhi Ma, Huiming Ma, Qing Chang, Hongyan Wang, Yufang Cai, Yan Xie, Chengjun Zhao, Xiuying Pei and Yanrong Wang	88-102
	Effect of Cell Membrane Permeability Protein on Porcine oocyte vitrification Van Hanh Nguyen, Viet Linh Nguyen, Nghia Son Hoang and Trung Kien Do	103-109
	Cardiac mitochondrial oxidative capacity is partly preserved after cryopreservation with dimethyl sulfoxide Alain Meyer, Anne-Laure Charles, François Singh, Joffrey Zoll, Samy Talha, Irina Enache, Anne Charloux, Marie-Eve Isner-Horobeti and Bernard Geny	110-114
	Effect of melatonin supplementation on cryopreserved sperm quality in Mouse Xue-jiao Chen, Yue Zhang, Gong-xue Jia, Qing-gang Meng, Thomas D. Bunch, Guo-shi Liu, Shi-en Zhu and Guang-bin Zhou	115-122
	Comparative effects of slow freezing and vitrification on cryosurvival of spermatozoa obtained from West African Dwarf Goat bucks Daramola J.O. and Adekunle E.O.	123-128
	Both death receptor and mitochondria mediated apoptotic pathways participated the occurrence of apoptosis in Porcine vitrified MII stage oocytes Jianjun Dai, Yingfang Niu, Caifeng Wu, Shushan Zhang and Defu Zhang	129-136
	Dog sperm cryopreservation using one step dilution with glycerol-free Tris extender Md. Ataur Rahman, Sang-Hyun Park and Il-JeoungYu	137-141

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CryoLetters 37 (2), 68-76 (2016)
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CRYOPRESERVATION ON A CRYO-PLATE OF Arundina graminifolia PROTOCORMS, DEHYDRATED WITH SILICA GEL AND DRYING BEADS

Luis B. Cordova II and Kanchit Thammasiri*

Department of Plant Science, Faculty of Science, Mahidol University, Rama VI Road, Phayathai, Bangkok 10400, Thailand.
*Corresponding author email: kanchitthammasiri@gmail.com

Abstract

BACKGROUND: There are various methods for the cryopreservation of plant material, with each biological specimen potentially requiring protocol optimization to maximize success. OBJECTIVE: The aim of this study is to compare droplet-vitrification, encapsulation-dehydration, and the cryo-plate method for cryopreservation of protocorms of the orchid Arundina graminifolia, using silica gel and drying beads as the desiccation materials. MATERIALS AND METHODS: The cryo-plate method included preculture of protocorms, developed from seeds, placed on aluminium cryo-plates and embedded in alginate gel. Cryo-plates were surface dried using sterile filter paper, placed in Petri dishes containing 50 g silica gel or 30 g drying beads in a laminar air-flow cabinet. Specimens on cryo-plates were dehydrated to 25% moisture content, placed into 2 mL cryotubes and plunged directly into liquid nitrogen for 1 d. RESULTS: For cryopreservation, the cryo-plate method, involving dehydration with 30 g drying beads gave the highest regrowth (77%), followed by the encapsulation-dehydration method with 30 g drying beads (64% regrowth) and the droplet-vitrification method, following exposure to PVS2 solution for 20 min (33% regrowth). CONCLUSIONS: Regrowth of cryopreserved protocorms using the cryo-plate method was rapid with the highest survival and regrowth.

Keywords: Cryopreservation, droplet-vitrification, encapsulation-dehydration, cryo-plate, orchid, protocorm, Arundina graminifolia

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CryoLetters 37 (2), 77-87 (2016)
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MELATONIN-LOADED ALGINATE BEADS IMPROVE  CRYOPRESERVATION OF YAM (DIOSCOREA ALATA AND D. CAYENENSIS)

Esther E. Uchendu^1* and E. R. Joachim Keller²

¹Department of Agronomy, Faculty of Agriculture and Forestry, University of Ibadan, Ibadan, Nigeria.
²Leibniz Institute for Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Stadt Seeland OT Gatersleben, Germany.
*Corresponding author email: ee.uchendu@ui.edu.ng

Abstract

BACKGROUND: The cryopreservation of yam is constrained with many challenges. OBJECTIVE: This study tested the effects of melatonin on shoot tips of D. alata and D. cayenensis accessions exposed to water and liquid nitrogen (LN) stresses. MATERIALS AND METHODS: Sucrose pretreatment (0.3 M) was applied for 48 h before cryopreservation. Shoot tips were encapsulated in beads loaded with 0.75 M sucrose, with and without melatonin and desiccated over sterile dry silica gel for 0 - 9 h. RESULTS: The beads moisture content declined from 100% to ~ 13% after 9 h. The 3 h desiccation period without melatonin produced a significantly higher regeneration compared to 6 h and 9 h. Shoot tips with melatonin had significantly higher regeneration after 3 - 6 h desiccation compared to 9 h and the regeneration of all accessions after 6 h was >80%. Regeneration following 6 h desiccation and LN was significantly greater for melatonin-treated shoot tips compared to non-treated ones. CONCLUSION: The results indicate that melatonin significantly increased regeneration from 15% to 35%.

Keywords: antioxidant, Dioscorea spp., desiccation, encapsulation-dehydration, melatonin, oxidative stress, and plant tissue culture

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CryoLetters 37 (2), 88-102 (2016)
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THE REVASCULARIZATION AND FOLLICULAR SURVIVAL OF MOUSE OVARIAN GRAFTS TREATED WITH FSH DURING CRYOPRESERVATION BY VITRIFICATION

Hong Zhang#¹,   Yanzhou Yang#¹,   Wenye Ma¹,   Hao Wu²,
Xiaomin Zheng¹,  Changchun Hei¹, Miao Sun¹, Wenzhi Ma¹,
Huiming Ma¹,  Qing Chang¹, Hongyan Wang¹, Yufang Cai¹ ,
Yan Xie¹, Chengjun Zhao¹, Xiuying Pei*¹ and Yanrong Wang*¹

¹Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Key Laboratory of Reproduction and Genetics in Ningxia; Department of Histology and Embryology, Ningxia Medical University; Tissue Organ Bank & Tissue Engineering Centre, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, 75004, P.R. China.
²Department of Orthopedics, The People No.3 Hospital of Anyang, Henan
*Corresponding author email: Prof. Wang Yanrong and Pei Xiuying: 4083304@163.com; peixiuying@163.com; nxykdyang@163.com
#Zhang Hong and Yang Yanzhou equally contributed to this work.

Abstract

BACKGROUND: Ovarian cryopreservation by vitrification is a very effective pathway for the preservation of female fertility during radiotherapy and chemotherapy. However, damage of follicles was triggered by cryo-injure during the process of ovarian vitrification and ischemia/reperfusion during the process of ovarian transplantation. Appropriate FSH play important roles in anti-apoptosis and neoangiogenesis during ovarian follicle development. OBJECTIVE: Therefore, the purpose of this study was to investigate the effect of FSH on the revascularization and follicular survival of vitrified-warmed ovarian grafts. MATERIALS AND TMETHODS: Four-week-old C57BL/6J mice with diestrus were used and the ovaries were randomized into the following three groups: fresh control group (FCG), vitrified/warmed group (VCG) and vitrified/warmed group treated with 0.3 IU/mL FSH (FSH-VG) during ovarian vitrification. After warming, the ovaries of the three groups were allotransplanted into the renal capsule of receptor mice. Assessment of follicular quantity was performed by histological analysis. The angiogenesis factors, CD31 and MMP-2, and cell survival factors, PCNA, EdU and survivin were examined by immunohistochemistry and western blot analysis. Angiogenesis was detected by vascular perfusion with the fluorescent dye 2MD-FITC-Dextran.  RESULTS: The expression of CD31and MMP-2 were not significantly different in either VCG or FSH-VG compared with FCG, but when the ovaries were transplanted 48 hours later, the expression levels of CD31 and MMP-2 were lower for VCG than FCG (P < 0.05) and FSH-VG was not significantly different from FCG. Before transplantation, the expression levels of PCNA and survivin were lower for VCG and FSH-VG than FCG (p < 0.05), but FSH-VG was higher than VCG (p < 0.05). After 48 h of ovarian transplantation, the expression of survivin was lower for VCG than FCG (P < 0.05), but FSH-VG was not significantly different from FCG. In addition, these data were further supported by the results from detecting the 2MD-FITC-Dextran and EdU.  CONCLUSION: Taken together, supplementation with 0.3 IU/mL FSH during ovarian cryopreservation by vitrification increased the revascularization and follicular survival for mouse ovarian grafts through the up-regulated expression of angiogenesis and ovarian survival factors.

Keywords: Ovarian vitrified cryopreservation, Heterotopic transplantation, Revascularization, Follicular survival

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CryoLetters 37 (2), 103-109 (2016)
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EFFECT OF CELL MEMBRANE PERMEABILITY PROTEIN ON PORCINE OOCYTE VITRIFICATION

Van Hanh Nguyen¹*, Viet Linh Nguyen¹, Nghia Son Hoang²
and Trung Kien Do³

¹Laboratory of Embryo Biotechnology, Institute of Biotechnology, Vietnam Academy of Science and Technology, Hanoi
²Department of Animal Biotechnology, Institute of Tropical Biology, Vietnam Academy of Science and Technology, Ho Chi Minh City; ³Fisheries College, Tuson District, Bacninh Province, Vietnam
*Corresponding author email: nvhanh@ibt.ac.vn

Abstract

BACKGROUND: The discovery of proteins with inherent cell membrane-translocating activity will expand our ability to study and manipulate various intracellular processes in living systems. OBJECTIVE: We investigated the effect of TAT-EGFP (trans-activator of transcription-enhanced green fluorescent protein) intra-cellular delivery on the survival and development of mature porcine oocytes after cryopreservasion.  MATERIALS AND METHODS: Cumulus-oocyte complexes (COCs) collected from follicles 3 to 6 mm in diameter in abattoir-derived oocytesries of prepubertal gilts were on vitro matured (IVM). After IVM, the oocytes were used for TAT-EGFP delivery test and cryopreservation with and without TAT-EGFP supplementation. Oocyte viability was assayed by staining with fluorescein diacetate. Live oocytes were parthened and cultured in vitro, to assess their ability to be activated and to therefore develop. RESULTS: The results show that the TAT-EGFP was well delivered into the nuclear of the Hela cell and oocytes also. In the medium toxic test, the proportion of viable oocytes in seven groups showed no significance. In vitrification experiments, the viability of oocytes in group supplemented with TAT-EGFP was significantly higher than that in the without TAT-EGFP group and the control groups (27.7%, 90.4%, and 100%, respectively). Among the three groups, the developmental abilities of oocytes in the supplement TAT-EGFP, EGFP and Control groups revealed that the vitrified group had a significantly reduced ability to undergo first cleavage (34.4%, 63.3%, and 69.0%, respectively). CONCLUSION: the supplement of TAT-EGFP protein into vitrification medium does not affect the viability of the oocytes whereas it improved the viability and developmental potential of oocytes after it was vitrified.

Keywords: cell membrane permeability protein, porcine oocytes, vitrification, cryopreservation

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CryoLetters 37 (2), 110-114 (2016)
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CARDIAC MITOCHONDRIAL OXIDATIVE CAPACITY IS PARTLY PRESERVED AFTER CRYOPRESERVATION WITH DIMETHYL SULFOXIDE

Alain Meyer^1,2*, Anne-Laure Charles¹, François Singh^1,2,
Joffrey Zoll^1,2, Samy Talha^1,2, Irina Enache^1,2, Anne Charloux^1,2,
Marie-Eve Isner-Horobeti^1,3 and Bernard Geny^1,2

¹Fédération de Médecine Translationnelle, Université de Strasbourg;
²Service de Physiologie et d'Explorations Fonctionnelles, Pôle de Pathologie Thoracique Hôpitaux Universitaires de Strasbourg;
³Service de Médecine Physique et de Réadaptation, Institut Universitaire Clémenceau et Hôpitaux Universitaires de Strasbourg, 67000 Cedex, France.
^*Corresponding author email: Alain Meyer (Bernard.Geny@chru-strasbourg.fr)

Abstract

BACKGROUND: Cardiac muscle cryopreservation is a challenge for both diagnostic procedure requiring viable tissues and therapeutic advance in regenerative medicine. Mitochondria are targets of both direct and indirect damages, secondary to congelation per se and/or to cryoprotectant's toxic effects, which participate to diminution of viability and/or functioning of cells after freezing. At the cardiac muscle level, only one study had investigated mitochondrial respiration after cryopreservation. OBJECTIVE: To determine the effect of cryopreservation on mitochondrial respiration of cardiac muscle. MATERIALS AND TMETHODS: We recorded mitochondrial respiration through complexes I, II, III and IV along with mitochondrial coupling in fresh and cryopreserved rat left ventricles samples and assessed difference of the means, correlation and agreement between the measures in all samples. RESULTS: Mitochondrial respiration was partly maintained up to 70% in cryopreserved samples whatever the substrate. A significant correlation was observed between fresh and cryopreserved samples (r = 0.71, p<0.0001). However, mitochondrial coupling significantly decreased after cryopreservation (- 1.44 ± 0.15; p <0.005) suggesting that mitochondrial intactness was not totally preserved by cryopreservation. Further, the fluctuations around the mean difference were wide (-14.06, +5.08 µmol/min/g), increasing with respiration rates (p <0.0001). CONCLUSION: Thus, fresh samples extemporaneous analysis should be preferred when available despite the fact that cryopreservation using DMSO partly protect cardiac mitochondrial respiration and coupling. These data support the interest to further refine cryopreservation methods.

Keywords: heart, muscle, mitochondria, oxygraphy, cryopreservation, DMSO

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CryoLetters 37 (2), 115-122 (2016)
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EFFECT OF MELATONIN SUPPLEMENTATION ON CRYOPRESERVED SPERM QUALITY IN MOUSE

Xue-jiao Chen ¹, Yue Zhang ¹, Gong-xue Jia ², Qing-gang Meng ^3,*,
Thomas D. Bunch ³, Guo-shi Liu², Shi-en Zhu² and Guang-bin Zhou ^1,*

¹Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu.
²College of Animal Science and Technology, China Agricultural University, Beijing, China.
³Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, Utah, USA.
^* Corresponding author email: q.meng@aggiemail.usu.edu or zguangbin@sicau.edu.cn

Abstract

BACKGROUND: Antioxidants protect spermatozoa against cell damage during cryopreservation.  OBJECTIVE: To investigate whether melatonin supplement in the extender may improve the quality of cryopreserved mouse sperm. METHODS: Kunming mice sperm frozen in extender R₁₈S₃ (18% (w/v) rafӿnose and 3% (w/v) skim milk) supplemented with melatonin were thawed and evaluated.  RESULTS: Mouse spermatozoa were cryopreserved in the freezing extender R₁₈S₃ that contained melatonin at 0, 0.125, 0.25 and 0.5 mg/mL melatonin. The extender without melatonin supplement was associated with increased formation of reactive oxygen species (ROS) and decreased sperm motility. Melatonin supplement at 0.125 mg/mL significantly increased the progressive motility of sperm in comparison to other melatonin concentration or control. The percentage of thawed viable sperm with ROS was lower in the melatonin-treated groups than in untreated group. Melatonin supplement also increased antiapoptotic gene Bcl-xl expression in the thawed sperm.  CONCLUSION: Supplement of 0.125 mg/mL melatonin could reduce oxidative damage and apoptosis.

Keywords: Mouse spermatozoa, cryopreservation, melatonin, ROS, apoptotic genes

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CryoLetters 37 (2), 123-128 (2016)
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COMPARATIVE EFFECTS OF SLOW FREEZING AND VITRIFICATION ON CRYOSURVIVAL OF SPERMATOZOA OBTAINED FROM WEST AFRICAN DWARF GOAT BUCKS

Daramola J.O.^* and Adekunle, E.O.

Department of Animal Physiology, Federal University of Agriculture Abeokuta, P.M.B 2240, Ogun State, Nigeria.
Corresponding author email: daramolajames2003@yahoo.com

Abstract

BACKGROUND: Slow freezing and vitrification are used to improve the viability of spermatozoa from various species but comparative effects of these cryoprotocols have never been evaluated for spermatozoa obtained from West African Dwarf (WAD) goat bucks. OBJECTIVE: This study evaluated the comparative effects of slow freezing and vitrification on the viability of spermatozoa of WAD goat bucks. MATERIALS AND METHODS: Semen samples collected with the aid of artificial vagina were allocated to slow freezing and vitrification protocols and cryopreserved for 30 days in liquid nitrogen.  RESULTS: Consistent higher (P<0.05) sperm motility, acrosome integrity, membrane integrity, live sperm, acrosome reaction and capacitation in slow freezing compared to vitrification. Abnormal sperm cells and malondialdehyde (MDA) concentrations reduced (P<0.05) in slow freezing compared to vitrification. Higher (P<0.05) arginase activity was observed in slow freezing compared to vitrification. CONCLUSION: The findings indicated that spermatozoa obtained from WAD goat bucks were better preserved in slow freezing compared to vitrification.

Keywords: Freezing, goat, oxidative stress, sperm viability

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CryoLetters 37 (2), 129-136 (2016)
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BOTH DEATH RECEPTOR AND MITOCHONDRIA MEDIATED APOPTOTIC PATHWAYS PARTICIPATED THE OCCURRENCE OF APOPTOSIS IN PORCINE VITRIFIED MII STAGE OOCYTES

Jianjun Dai^1,2, Yingfang Niu^1,2, Caifeng Wu^1,2, Shushan Zhang^1,2
and Defu Zhang^1,2*

¹Institute of Animal Science and Veterinary Science, Shanghai Academy of Agricultural Sciences, Shanghai, 201106, China
²Division of Animal Genetic Engineering, Shanghai Municipal Key Laboratory of Agri-Genetics and Breeding, Shanghai, 201106, China.
*Corresponding author email: zhangdefuzdf@163.com

Abstract

BACKGROUND: Oocytes vitrification is widely used for cryopreservation of female genetic resources. OBJECTIVE: In order to illuminate the apoptotic pathways of porcine MII stage oocytes after vitrification. MATERIALS AND METHODS: This study used in situ fluorescence staining and RT-PCR to detect the expression levels of some key molecules from death receptor and mitochondria mediated apoptotic pathways. RESULTS: (1) Early stage apoptosis were detected in both PI staining survival oocytes and PI staining dead oocytes. (2) The fluorescence intensity of caspase 8, caspase 9, caspase 3 and pan caspase from vitrified oocytes were 32.03, 16.56, 16.70 and 8.43 respectively, which were much higher than those from fresh oocytes (4.02, 4.83, 4.23 and 3.08, P < 0.05). (3) Not only the genes from death receptor mediated apoptotic pathway, but also from mitochondrial mediated apoptotic pathway were changed greatly. CONCLUSION: The death of porcine vitrified oocytes could be induced by apoptosis, both death receptor and mitochondria mediated apoptotic pathways participated the occurrence of apoptosis in porcine vitrified MII stage oocytes.

Keywords: pig; oocytes; vitrification; caspase activity; apoptotic pathway

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CryoLetters 37 (2), 137-141 (2016)
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DOG SPERM CRYOPRESERVATION USING ONE STEP DILUTION WITH GLYCEROL-FREE TRIS EXTENDER

Md. Ataur Rahman, Sang-Hyun Park and Il-JeoungYu^*

Laboratory of Theriogenology and Reproductive Biotechnologies, College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Iksan 54596, Republic of Korea.
Corresponding author email: iyu@jbnu.ac.kr

Abstract

OBJECTIVE: The present study was to investigate a freezing method using one step-dilution with glycerol-free TRIS extender containing 172.2 mM glucose (GFTG). MATERIALS AND METHODS: The sperm pellet from selected ejaculates was resuspended in GFTG at 1×10⁸ cells/mL. The semen was cooled for 10, 30, 50 or 70 min in GFTG at 4°C and was frozen in LN₂ vapor or in deep freezer (-80°C, DF) for 20 min before plunge into LN₂. Post-thaw sperm characteristics were examined. The phosphatidylserine (PS) translocation (Annexin V-FITC) and DNA integrity (TUNEL assay) were assessed using flow cytometry. RESULTS: Progressive motility and viability were significantly higher in 50 and 70 min groups than the other groups (P<0.05). PS translocation index was significantly lower in spermatozoa cooled for 50 or 70 min compared to 10 min (P<0.05). Freezing methods using LN₂ vapor showed higher progressive motility than DF method (P<0.05), while viability and DNA fragmentation were not different between two freezing methods. CONCLUSION: Cryopreservation of canine sperm cooled for 50 or 70 min following one step dilution in GFTG yields more viable sperm with lower PS translocation and freezing method using LN₂ vapor is more effective on progressive motility.

Keywords: canine sperm, cryopreservation, DNA integrity, glycerol-free TRIS, PS translocation

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