Volume 38, No. 3 May/June 2017 ISSN 0143-2044
	Defined combinations of cryomedia and thawing extenders influence the viable x-y boar sperm ratio in vitro Wasamon Korchunjit, Kampon Kaeoket, Yindee Kitiyanant, Jane Taylor and Tuempong Wongtawan	160-165
	Follicle development in grafted mouse ovaries after vitrification processes under static magnetic field Vida Kazemein Jasemi, Firooz Samadi, Hussein Eimani, Saeed Hasani, Rouhollah Fathi and Abdolhossein Shahverdi	166-177
	Cryopreservation of waigieu seaperch (Psammoperca waigiensis) sperm Minh Hoang Le and Hung Quoc Pham	178-186
	Antioxidant effect of xanthan gum on ram sperm after freezing and thawing Gustavo D.A. Gastal, Estela F. Silva, Bruna Mion, Antonio S. Varela Junior, Carlos E. Rosa, Carine D. Corcini, Rafael G. Mondadori, Arnaldo D. Vieira Ivan Bianchi and Thomaz Lucia Jr.	187-193
	Basic fibroblast growth factor improved angiogenesis of vitrified human ovarian tissues after in vitro culture and xenotransplantation Beijia Kang, Yan Wang, Long Zhang and Shangwei Li	194-201
	Long term conservation at -80ºC of Pinus radiata embryogenic cell lines: recovery, maturation and germination Itziar A. Montalbán and Paloma Moncaleán	202-209
	Effects of washing protocols on cryosurvival of spermatozoa from West African dwarf goat bucks Daramola J.O. and Adekunle E.O.	210-215
	The cold hardiness of Phrynocephalus erythrurus, the lizard living at highest altitude in the world Xiangtao Li, Yan Wang, Songsong Lu, Mei Li, Shengkang Men, Yucheng Bai, Xiaolong Tang and Qiang Chen	216-227
	Exogenous catalase and pyruvate dehydrogenase improve survival and regeneration and affect oxidative stress in cryopreserved Dendrobium nobile protocorm-like bodies Wei Di, Mengxue Jia, Jin Xu, Bingling Li and Yan Liu	228-238
	Determination of an optimal membrane-permeable cryoprotectant addition and dilution protocol for water buffalo spermatozoa Sundus Mehmood, Hussain Ahmed, Syed Aftab Hussain Shah, Feroza Hamid Wattoo, Shamim Akhtar and Syed Murtaza Hassan Andrabi	239-249
	Hop powdery mildew (Podosphaera macularis) spore cryopreservation Sugae Wada and Barbara M. Reed	250-256

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CryoLetters 38 (3), 160-164 (2017)
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DEFINED COMBINATIONS OF CRYOMEDIA AND THAWING EXTENDERS INFLUENCE THE VIABLE X-Y BOAR SPERM RATIO IN VITRO

Wasamon Korchunjit¹, Kampon Kaeoket^2,3, Yindee Kitiyanant^4,5,
Jane Taylor⁶ and Tuempong Wongtawan^1,7,*

¹Laboratory of Cellular Biomedicine and Veterinary Medicine, Faculty of Veterinary Science, Mahidol University, Salaya campus, Phuttamonthon, Nakhon Pathom, 73170, Thailand.
²Semen Laboratory, Faculty of Veterinary Science, Mahidol University, Salaya, Phuttamonthon, Nakhon Pathom, 73170, Thailand.
³Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, Salaya, Phuttamonthon, Nakhon Pathom, 73170, Thailand.
⁴Department of Anatomy, Faculty of Science, Mahidol University, Phayathai, Ratchatewi, Bangkok, 10400, Thailand.
⁵Reproductive and Stem Cell Biology Research Group, Institute of Molecular Biosciences, Mahidol University, Salaya, Puttamonthon, Nakhon Pathom, 73170, Thailand.
⁶College of Medicine and Veterinary Medicine, Centre for Regenerative Medicine, the University of Edinburgh, 49 Little France Crescent, Edinburgh EH16 4SB, UK.
⁷Department of Preclinic and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, Salaya, Phuttamonthon, Nakhon Pathom, 73170, Thailand.
*Corresponding author email: tuempong.wan@mahidol.edu

Abstract

BACKGROUND: It is believed that plasma membrane X- and Y-chromosome bearing sperm are different; therefore the freezing and thawing process may affect X- and Y-sperm differently. OBJECTIVE: The objective of this study was to investigate the effect of cryomedia and thawing extenders on the survival of X and Y-sperm.  MATERIALS AND METHODS: Three different cryomedia and thawing extenders were compared. Viable motile sperm were separated using a swim-up technique. Real-time PCR was used to identify the sperm type. RESULTS: Using CryoA for freezing and Beltsville-Thawing-Solution (BTS) as the thawing extender yielded significantly higher numbers of viable motile Y sperm (64%) than control (48%) (P<0.01). Conversely, semen freezing with CryoC and thawing with Androstar^®Plus gave a significantly lower number of viable motile Y sperm (32%) than control (51%).  CONCLUSION: Our results revealed that defined combinations of cryomedia and thawing extenders significantly altered the survival ratio of frozen-thawed X-Y sperm in vitro, which has potential implications for artificial insemination.

Keywords: sperm, cryomedia, sexual selection, thawing extenders

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CryoLetters 38 (3), 166-177 (2017)
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FOLLICLE DEVELOPMENT IN GRAFTED MOUSE OVARIES AFTER VITRIFICATION PROCESSES UNDER STATIC MAGNETIC FIELD

Vida Kazemein Jasemi¹, Firooz Samadi¹, Hussein Eimani^2*,3,
Saeed Hasani¹, Rouhollah Fathi² and Abdolhossein Shahverdi²

¹Department of Animal and Poultry Physiology, Faculty of Animal Science, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Golestan, Iran.
²Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
³Department of Anatomy, Faculty of Medicine, Baqiyatallah University of Medical Sciences,Tehran, Iran.
*Correspondence Address: P.O.Box: 19395-4644, Embryology Department, Royan Institute for Reproductive Biomedicine Research Center, ACECR, Tehran, Iran 
*Corresponding author email: eimanih@royaninstitute.org

Abstract

BACKGROUND: Ovarian cryopreservation has emerged as an important method of fertility preservation. Magnetic field enhanced cryopreservation has been considered in recent times as a promising type of ovarian cryopreservation but the effectiveness of the process is still not clear. OBJECTIVE: The aim of the present study was to investigate the effect of applying 1-mT SMF (static magnetic field) on the vitrification of ovarian tissue and the follow-up investigation of the morphology and functions of vitrified- warmed ovarian tissue after transplantation. MATERIALS AND METHODS: Ovaries of 6-8 week-old female mice from the Naval Medical Research Institute (NMRI) were exposed of the static magnetic field during different steps of the vitrification process. Immunohistological studies were performed on the ovaries. RESULTS: The mean percentage of damaged primordial follicles was lowest in control group and the group with ovaries exposed to magnetic field during the equilibration step. The latter group also had the highest percentage of intact primordial follicles after transplantation. CONCLUSION: Exposure of mice ovaries to static magnetic field during first step of vitrification process (the equilibration step) resulted in greater resistance against injury.

Keywords: apoptosis, mouse, ovarian vitrification, static magnetic field

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CryoLetters 38 (3), 178-186 (2017)
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CRYOPRESERVATION OF WAIGIEU SEAPERCH (PSAMMOPERCA WAIGIENSIS) SPERM

Minh Hoang Le* and Hung Quoc Pham

Institute of Aquaculture, Nha Trang University, Nha Trang City, Vietnam.
*Corresponding author eamielhoanglm@ntu.edu.vn

Abstract

BACKGROUND: The cryopreservation protocols have been applied for sperm prevervation of many fish species, but have not been developed for Waigieu seaperch Psammoperca waigiensis, an important aquaculture species in Vietnam. OBJECTIVE: The objectives of this study were to find the best cryoprotectant, extender, freezing method and dilution ratio for sperm cryopreservation of Waigieu seaperch. MATERIALS AND METHODS: An effective protocol was established by comparing different cryodiluents created by mixing various cryoprotectants and extenders. The different freezing methods and dilution ratios were also used in an effective protocol. The motility (MOT), straight-linear velocity (VSL) and fertility rates of post-thawed sperm were comparable to that of fresh sperm. RESULTS: The results indicated that at ratio of 1:3 in cryodiluent contained ASP (artificial seminal plasma) as extender supplement 10% DMSO as cryoprotectant and cryopreserved with the freezing method as two steps, reached the best MOT and VSL of post-thawed sperm. The fertilization rate and hatching rate of the post-thawed sperm cryopreserved for 1 week, 1 month, or 1 year in liquid nitrogen (66.93 ± 0.93% and 44.16 ± 1.47%, 65.40 ± 1.11% and 43.88 ± 1.54%, or 65.13 ± 1.31% and 43.24 ± 1.41%) were similar to that of fresh sperm (68.67 ± 1.27% and 45.12 ± 1.92%). CONCLUSION: Using cryodiluent contained the ASP as extender and 10% DMSO as cryoprotectant to semen at the ratio of 1:3 (v/v) in the freezing method as two steps (-76°C for 5 minutes and -196°C) is an effective protocol for cryopreservation, especially hatching success of egg fertilized by post-thawed sperm of Waigieu seaperch.

Keywords: Waigieu seaperch, sperm cryopreservation, protocol, cryodiluent, extender, cryoprotectant

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CryoLetters 38 (3), 187-193 (2017)
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ANTIOXIDANT EFFECT OF XANTHAN GUM ON RAM SPERM AFTER FREEZING AND THAWING

Gustavo D.A. Gastal^1,2, Estela F. Silva^1,3, Bruna Mion^1,2,
Antonio S. Varela Junior^1,5, Carlos E. Rosa⁵, Carine D. Corcini^1,2,
Rafael G. Mondadori^1,4, Arnaldo D. Vieira^1,2, Ivan Bianchi^1,2 and
Thomaz Lucia Jr.^1,2*  

¹ReproPel,²Faculdade de Veterinária,³Centro de Biotecnologia, ⁴Instituto de Biologia, Universidade Federal de Pelotas, 96010-900, Pelotas-RS, Brazil
⁵Instituto de Ciências Biológicas, Universidade Federal do Rio Grande, 96201-900, Rio Grande-RS, Brazil
*Corresponding author email: thomaz@pq.cnpq.br

Abstract

BACKGROUND: Xanthan gum is used as thickener in media to preserve food products, having cryoprotectant and antioxidant properties that may be relevant for sperm cryopreservation. OBJECTIVE: To evaluate the effects of adding xanthan gum to freezing extenders on post-thawing quality and oxidant activity of ram sperm. METHODS: Ejaculates from seven rams extended TRIS-egg yolk-glycerol were split in three treatments including xanthan gum (0.15%; 0.20%; and 0.25%) and a control with no xanthan gum. RESULTS: After thawing, motility and production of reactive oxygen species (ROS) with 0.20% and 0.25% xanthan gum were lower than for the control (P < 0.05), but mitochondrial functionality and integrity of membrane, acrosome and DNA did not differ (P > 0.05). Xanthan gum at 0.20% and 0.25% may be an efficient antioxidant for frozen-thawed ram sperm, due to the reduction in ROS production.

Keywords: biopolymer; freezing; reactive oxygen species, ram sperm

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CryoLetters 38 (3), 194-201 (2017)
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BASIC FIBROBLAST GROWTH FACTOR IMPROVED ANGIOGENESIS OF VITRIFIED HUMAN OVARIAN TISSUES AFTER IN VITRO CULTURE AND XENOTRANSPLANTATION

Beijia Kang, Yan Wang, Long Zhang and Shangwei Li*

Reproductive Medical Center of West China 2nd University Hospital, Sichuan University, Chengdu, Sichuan, China.
*Corresponding author email: lswivfo1@126.com

Abstract

BACKGROUND: Basic fibroblast growth factor is a potent angiogenic factor. OBJECTIVE: To study the concentration and in vitro culture time of bFGF that maximize the angiogenesis for transplanted human ovarian tissues.  MATERIALS AND METHODS: Vitrified and rewarmed human ovarian tissues were cultured in vitro with bFGF (0, 25, 50, 100, 150 and 200 ng/ml) for different periods (1 h, 2 d, 5 d and 7 d) before transplantation. The effect of bFGF on follicle survival was studied by evaluating the pregraft group, control group (no bFGF) and bFGF-treated group. CD34, Ki-67 and AC-3 immuno-histochemical (IHC) staining and histological analysis was used to evaluate angiogenesis, proliferration, apoptosis and follicular quantity.  RESULTS: Treatments with 100 and 150 ng/ml bFGF improved the angiogenesis for grafted human ovarian tissues after in vitro culture for 2 days. The proliferation and survival of follicles were significantly increased.  CONCLUSION: bFGF improved the quality of vitrified-warmed human ovarian tissues after transplantation.

Keywords: cryopreservation, angiogenesis, bFGF, human ovary

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CryoLetters 38 (3), 202-209 (2017)
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LONG TERM CONSERVATION AT -80ºC OF PINUS RADIATA EMBRYOGENIC CELL LINES: RECOVERY, MATURATION AND GERMINATION

Itziar A. Montalbán and Paloma Moncaleán^*

Neiker-Tecnalia, Campus Agroalimentario de Arkaute Apdo. 46, 01080 Vitoria-Gasteiz, Spain
*Corresponding author email: paloma.moncalean@gmail.com

Abstract

BACKGROUND: Pinus radiata is an economically important conifer, and somatic embryogenesis is being currently used for its propagation. But the embryogenic competence of cultures decreases with culture age. To cope with this, cryopreservation protocols have been developed lately for different Pinus species. Although cryopreservation reduces the costs associated with embryogenic cultures maintenance, the initial investment and the maintenance of cryotanks are expensive when dealing with somatic embryogenesis basic research issues. OBJECTIVE: To study the feasibility of storing embryogenic cell lines at -80ºC for over a year. MATERIALS AND METHODS: The feasibility of the conservation method was assessed in terms of recovery, maturation and germination rates. RESULTS: The recovery rates were up to 77%, and maturation and germination rates were 86% and 83%, respectively. CONCLUSION: The work described here is a simple and low-cost protocol that enables successful conservation of embryogenic cell lines for over a year.

Keywords: clonal forestry, conifer, in vitro , Pinus radiata, plant regeneration, somatic embryo, somatic embryogenesis

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CryoLetters 38 (3), 210-215 (2017)
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EFFECTS OF WASHING PROTOCOLS ON CRYOSURVIVAL OF SPERMATOZOA FROM WEST AFRICAN DWARF GOAT BUCKS

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

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

Abstract

BACKGROUND: Sperm washing in some species helps remove the detrimental effect of enzymes secreted from the bulbourethral gland, but such an effect has not been evaluated for cryopreserved sperm obtained from West African Dwarf (WAD) goat bucks. OBJECTIVE: This study evaluated the effects of washing protocols on the viability parameters of spermatozoa obtained from WAD goat bucks prior to cryopreservation. MATERIALS AND METHODS: Semen samples collected from WAD goat bucks with the aid of artificial vagina were subjected to washing protocols by centrifuging once, twice and thrice while the control group was not washed. Following washing semen samples were diluted in Tris-based extenders, cryopreserved for 30 days in liquid nitrogen. RESULTS: Sperm motility, acrosome integrity, live spermatozoa and arginase activity were higher, and malondialdehyde (MDA) concentration was reduced in semen washed prior to cryopreservation compared to the control. CONCLUSION: Semen washing improved the quality.

Keywords: artificial insemination, goat, cryopreservation, oxidative stress, sperm viability

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CryoLetters 38 (3), 216-227 (2017)
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THE COLD HARDINESS OF PHRYNOCEPHALUS ERYTHRURUS, THE LIZARD LIVING AT HIGHEST ALTITUDE IN THE WORLD

Xiangtao Li, Yan Wang, Songsong Lu, Mei Li, Shengkang Men,
Yucheng Bai, Xiaolong Tang and Qiang Chen*

School of Life Sciences, Lanzhou University, 222 Tian Shui South Road, Lanzhou, 730000, PR China
*Corresponding author e-mail: chenq@lzu.edu.cn (Q. Chen)

Abstract

BACKGROUND: Phrynocephalus erythrurus living at Qinghai-Tibet Plateau, is believed to be the highest lizard in the world, but we know little about how these lizards cope with very low temperatures in winter. OBJECTIVE: The aim of this study was to find the difference of the lizards before and after cold acclimatization. MATERIALS AND METHODS: In this study the limit of supercooling and inoculative freezing, the concentration of four organic osmolytes, and the activity of lactate dehydrogenase in the plasma were measured in samples shortly after capture and in other samples after 7~8 weeks of acclimatization at 2~4C. RESULTS: Animals acquired an ability to undergo deeper supercooling and inoculative freezing through the course of acclimatization. We find no regular changes of the four organic osmolytes after the acclimatization. CONCLUSION: We think that this species of lizard is partly freeze-tolerant and conclude that it uses supercooling to survive in winter.

Keywords: Phrynocephalus erythrurus; acclimatization; supercooling; inoculative freezing; organic osmolytes; plasma LDH

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CryoLetters 38 (3), 228-238 (2017)
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EXOGENOUS CATALASE AND PYRUVATE DEHYDROGENASE IMPROVE SURVIVAL AND REGENERATION AND AFFECT OXIDATIVE STRESS IN CRYOPRESERVED DENDROBIUM NOBILE PROTOCORM-LIKE BODIES

Wei Di^1#, Mengxue Jia^1#, Jin Xu², Bingling Li¹ and Yan Liu^1*

¹Beijing Laboratory of Urban and Rural Ecological Environment, Beijing Key Laboratory of Ornamental Plants Germplasm Innovation & Molecular Breeding, National Engineering Research Center for Floriculture, College of Landscape Architecture, Beijing Forestry University, Beijing 100083, People's Republic of China
²College of Architecture & Urban Planning, Guangzhou University, Guangzhou 510006, PR China
^*Corresponding author email: yanwopaper@yahoo.com
^#contributed equally to this work

Abstract

BACKGROUND: Reactive oxygen species (ROS)-induced oxidative damage is responsible for viability loss in plant tissues following cryopreservation. Antioxidants may improve viability by preventing or repairing the injury. OBJECTIVE: This work aimed at studying the effect of catalase (CAT) and pyruvate dehydrogenase (PDH), which are involved in ROS metabolism and are differentially expressed during pollen cryopreservation, for cryopreservation of Dendrobium nobile Lindl. 'Hamana Lake Dream' protocorm-like bodies (PLBs).  MATERIALS AND METHODS: Different concentrations of exogenous CAT or PDH were added at the loading, PVS2 treatment, unloading steps during vitrification-cryopreservation of PLBs. Their survival and regeneration were evaluated and correlated with physiological oxidative indexes. RESULTS: PLB survival increased significantly when CAT and PDH were added separately to the unloading solution at a suitable concentration. CAT at 400 U·ml^-1 increased PLB survival and regeneration by 33.5 and 14.6% respectively. It had no impact on the production of superoxide anion radical (·O₂^-) and on superoxide dismutase (SOD) activity, but it reduced the hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents and enhanced ascorbic acid (AsA) and endogenous CAT levels compared to PLBs cryopreserved using the standard vitrification protocol (CK1). PDH at 0.1 U·ml^-1 significantly improved PLB survival (by 2.5%), but it had no marked effect on regeneration compared to the CK1 group. It induced the same variations in ·O₂^-, AsA and endogenous CAT levels that were observed following CAT addition. However, PDH did not affect the H₂O₂ and MDA content but significantly increased SOD activity.  CONCLUSION: These results indicate that the addition of 400 U·ml^-1 CAT and 0.1 U·ml^-1 PDH at the unloading step increased survival of cryopreserved PLBs and that this improvement was associated with scavenging of H₂O₂ and the repair of oxidative damage. Exogenous CAT also significantly improved PLB regeneration after cryopreservation, while PDH had no obvious effect. The effect of exogenous CAT on PLB survival and regeneration was stronger than that of PDH, which may be due to the increased SOD activity by PDH addition.

Keywords: orchid; CAT; PDH; vitrification; oxidative damage

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CryoLetters 38 (3), 239-249 (2017)
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DETERMINATION OF AN OPTIMAL MEMBRANE-PERMEABLE CRYOPROTECTANT ADDITION AND DILUTION PROTOCOL FOR WATER BUFFALO SPERMATOZOA

Sundus Mehmood^1,2, Hussain Ahmed¹_, Syed Aftab Hussain Shah¹_,
Feroza Hamid Wattoo², Shamim Akhtar¹ and
Syed Murtaza Hassan Andrabi^1*

¹Animal Reproduction Laboratory, Animal Sciences Institute, National Agricultural Research Center, Islamabad 45500, Pakistan
²Department of Biochemistry, Faculty of Sciences PMAS Arid Agriculture University Rawalpindi, Pakistan
*Corresponding author email: andrabi123@yahoo.com

Abstract

BACKGROUND: There is a possibility to reduce the toxicity of glycerol and osmotic stress of Me₂SO/DMSO by lowering their concentrations in freezing extenders. OBJECTIVE: To investigate the effect of glycerol and dimethyl sulfoxide (Me₂SO/DMSO) in tris-citric acid based extender on post- thaw quality of buffalo (Bubalus bubalis) bull spermatozoa. MATERIALS AND METHODS: Semen was collected from five adult buffalo bulls with artificial vagina. Five aliquots of semen per bull were separated for dilution with the treatment extenders. The first aliquot was diluted at 37°C with 6% glycerol (T1). The second aliquot was diluted at 37°C with extenders containing 4.5% glycerol and 1.5% DMSO (T2). The third aliquot was diluted with extenders containing 4.5% glycerol at 37°C and 1.5% DMSO at 4°С (T3). The fourth aliquot was diluted with extenders containing 1.5% DMSO at 37°C and 4.5% glycerol at 4°С (T4). The fifth aliquot was diluted with extender containing 2.5% DMSO at 37 as well as at 4°C (T5). The final concentration of spermatozoa was 50×10⁶/ml in all the treatment groups. Semen was cooled from 37 to 4°C in 2 h and equilibration was done at 4 °C for 4 h. Later on, packing of cooled semen was undertaken in 0.54 ml French straws and frozen in a programmable cell freezer. RESULTS: At post thawing, treatment groups T1 and T2 yielded significant (P<0.05) outcome for CASA parameters, longevity, acrosomal integrity, plasma membrane integrity, mitochondrial transmembrane potential and DNA integrity. CONCLUSION: We concluded that by decreasing glycerol concentration (4.5%) and combining it with Me₂SO/DMSO (1.5%) at 37°C (T2) in tris-citric acid based extender provided similar results to those observed when glycerol (6 %) alone is used at 37°C (T1) for improving the post-thaw quality of buffalo bull spermatozoa.

Keywords: glycerol; Me₂SO/DMSO; longevity; cryopreservation; sperm; buffalo

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CryoLetters 38 (3), 250-256 (2017)
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HOP POWDERY MILDEW (PODOSPHAERA MACULARIS) SPORE CRYOPRESERVATION

Sugae Wada¹ and Barbara M. Reed^2*

¹Department of Horticulture, Oregon State University 4017 Agriculture and Life Science Bldg. Corvallis, OR 97331-7304
²USDA-ARS, Retired, National Clonal Germplasm Repository and Department of Horticulture, Oregon State University, Corvallis, OR 97331-7304
*Corresponding author e-mail: Reedba@onid.oregonstate.edu

Abstract

BACKGROUND: Hop powdery mildew (HPM), Podosphaera macularis, an important disease organism for hops, is an obligate parasite, requiring constant culture on living plant tissue for strain maintenance. OBJECTIVE: This study determined the parameters required to successfully cryopreserve HPM spores for the first time and reduce the need for constant culture.  MATERIALS AND METHODS: Spores of an Oregon HPM strain, OSU C-100 were desiccated over silica gel for 2-10 h to determine the spore moisture content (MC). Regrowth of the hyphae before and after drying and liquid nitrogen exposure was determined on glass slides and leaf discs of several susceptible hop cultivars. A second mixture of strains was later tested with the protocol. RESULTS: Desiccation to an optimal 2-3% MC produced hyphal growth on slides and infection of leaf discs. The OSU C-100 HPM spore strain required 8-10 h desiccation to reach 2-3% MC while the mixed strains required 6-8 h due to slightly different MC when collected. CONCLUSION: HPM strains should be placed in cryovials, dried to 2-3% MC over silica gel, cryopreserved by direct immersion in liquid nitrogen. They can be rewarmed for 1 min each in 45^◦C and 20^◦C water and the viability tested on isolated leaf discs.

Keywords: fungus, Humulus, Leaf discs, moisture content, Podosphaera, vitrification

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