Preservation Of Genetic Resources Research Articles

Beyond botany to genetic resource preservation: the S.P. Vander Kloet Vaccinium collections1This article is part of a Special Issue entitled “A tribute to Sam Vander Kloet FLS: Pure and applied research from blueberries to heathland ecology”.

Sam P. Vander Kloet, botanist, traveled the world examining and obtaining specimens to redefine infrageneric taxonomic units within Vaccinium L., family Ericaceae. Besides his botanical treatises, his legacy includes herbarium voucher specimens and ex situ genetic resource collections including a seed bank and living plant collections at the Agriculture and Agri-Food Canada Research Centre, Kentville, Nova Scotia, Canada; the K.C. Irving Environmental Science Centre and Harriet Irving Botanical Gardens, Acadia University, Wolfville, Nova Scotia, Canada; the Canadian Clonal Genebank, Harrow, Ontario, Canada; and the US Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository, Corvallis, Oregon, United States. Sam P. Vander Kloet’s collections include representatives of wild Ericaceae with special emphasis on collections of North American and subtropical endemic Vaccinium species. These reference collections are significant and represent a lifetime of dedicated research. Representatives of his heritage collections have now been deposited not only in American genebanks (in Canada and the United States) but also in the World Genebank in Svalbard, Norway, for long term conservation and future evaluation of Vaccinium for the service of humanity. The bequest of his wild collected germplasm will continue to be available to facilitate utilization of an extended Vaccinium gene pool for development and breeding throughout the world.

Preservation and management of the genetic resources of Arabica coffee (Coffea arabica L): A challenge for Ethiopia

Les forets tropicales humides d’altitude du sud-ouest du plateau ethiopien constituent l’habitat naturel et le centre primaire de diversite de Coffea arabica L. Si la deforestation est un phenomene ancien en Ethiopie, elle s’est accentuee recemment sous l’action de nombreux facteurs dont les principaux sont la croissance demographique et la mise en œuvre de politiques publiques qui entrent en conflit avec les droits d’usage des communautes locales. Apres un bref historique et un etat des lieux des initiatives de conservation des ressources genetiques des cafeiers d’Ethiopie dans des collections ex situ, nous developpons plus particulierement les concepts qui sont mis en œuvre par des projets de recherche et de developpement impliques dans la conservation in situ des cafeiers dans les forets et les jardins (reserve de biosphere, gestion participative des forets, certification du cafe, indications geographiques). Ces projets visent a concilier les objectifs de conservation de la biodiversite, de developpement economique et de preservation des cultures et des savoir-faire paysans locaux.

Genetic diversity in commercial wineries: effects of the farming system and vinification management on wine yeasts

Analysis of the diversity and distribution of wine yeasts isolated from organically and conventionally grown grapes, and during the subsequent fermentation with or without starter cultures in six different commercial wineries. PCR-RFLP screening of isolates revealed the involvement of ten different species. Saccharomyces cerevisiae, scarcely isolated from grapes, was the dominant species during the latter phases of fermentation, identifying 108 different genotypes by means of SSR analysis. Species and strains' diversity and presence were strongly influenced by the farming system used to grow the grapes and the system of vinification. Organic farming management was more beneficial in terms of diversity and abundance than the conventional one. Induced fermentation generated a great replacement of native yeasts. Although winery-resident yeasts resulted to be predominant in the process, some noncommercial strains originally in the vineyard were found in final stages of the fermentation, confirming that autochthonous strains of S. cerevisiae are capable to conduct the fermentation process up to its end. The study of natural yeast communities from commercial vineyards and wineries is an important step towards the preservation of native genetic resources. Our results have special relevance because it is the first time that the real situation of the yeast ecology of alcoholic fermentation in commercial wineries belonging to the relevant wine-producing Appellation of Origin 'Vinos de Madrid' is shown.

Genetic variability of bottle gourd Lagenaria siceraria (Mol.) Standley and its morphological characterization by multivariate analysis

A wide range of bottle gourd Lagenaria siceraria (Mol.) Standley germplasm was collected from different parts of the world. Genetic resource preservation and determination of genetic variability was carried out as a foundation for future breeding work. The germplasm diversity collected in Serbia is a result of its adaptation to diverse ecological conditions and farmers? selection in accordance with their preference and ethnobotanical utilization. The broad intraspecific variation of the plant, fruit and seed morphology is a direct result of the research carried out. Principal component analysis (PCA) of L. siceraria with 13 quantitative traits showed continuous variation among accessions, primarily due to fruit and seed size and shape. The evident reduction in trait variation is a direct result of the preference for ornamental use that favored certain shapes and sizes of the fruit, which has not significantly changed over the centuries.

SCIENTIFIC AND TECHNOLOGICAL INFORMATION AND DATA SOURCE IN INDONESIA A BRIEF OVERVIEW

There are a total of 581 special libraries and information sources unevenly distributed in 65 cities in Indonesia. The majority are located in Java, with the highest concentration in Jakarta. A large proportion of these libraries are affiliated to educational institutions/ universities, with an interest in sciences in general, with a holding of 1,000 - 10,000 book volumes; only a small number of them have computer facilities. To optimize the services of these libraries and information centers, a National Scientific Documentation and Information Network System had been set up with four centers as focal points respectively in the fields of humanities, science & technology, biology & agricultural sciences, and health & medical sciences. No less than 14 institutions can be considered as data centers on various fields of science and technology. Some of them which deal with biological resources have worked together in a network under the coordination of the National Commission for the Preservation of Plant and Animal Genetic Resources. Fourteen of these tutions had been identified also as microbial collections.

44 REACTIVE OXYGEN SPECIES IN VITRIFIED BOVINE IN VITRO-MATURED OOCYTES

Vitrification of in vitro-matured oocytes has important applications in fertility preservation and management of genetic resources. However, despite the increasing interest, the efficiency of oocyte vitrification needs to be improved. It was demonstrated that under stressful conditions of cryopreserving pig oocytes accumulate reactive oxygen species (ROS; Gupta et al. 2010 Fertility and Sterility 93, 2602–2607). Reactive oxygen species are known to exert harmful effects such as mitochondrial damage, ATP (ATP) depletion, altered calcium oscillation during fertilization and consequently their developmental ability may be compromised (Takahashi et al. 2003 Mol. Reprod. Dev. 66, 143–152). The aim of the present study was to evaluate whether the exposure to cryoprotectants and vitrification procedure affect ROS production in bovine in vitro-matured oocytes. Abattoir-derived bovine (n = 360, over 6 replicates) cumulus oocyte complexes (COCs), were in vitro-matured. COCs were mechanically stripped of their cumulus cells by gentle pipetting, washed and divided into 3 groups: control (C; i.e. fresh non treated oocytes), toxicity (T) and vitrification (V) groups. In group V, oocytes were exposed to 10% ethylene glycol (EG) + 10% DMSO for 3 min, then to 20% EG + 20% DMSO and 0.5 M sucrose, loaded on cryotops and plunged into liquid nitrogen within 25 s. Oocytes were warmed into a 1.25 M sucrose solution for 1 min and then to decreasing concentrations of sucrose (0.625 M, 0.42 M and 0.31 M) for 30 s each. In group T, oocytes were simply exposed to the vitrification and warming solutions. ROS determination was carried out by a spectrofluorometer at 495 nm excitation and 525 nm emission. Frozen oocytes were thawed and incubated in 500 μL of TRIS-HCl 40 mM, pH 7.0 in the presence of 5 μmol L–1 of 2′,7′-dichlorfluorescein-diacetate, for 20 min at 37°C into a shaker. After incubation, the extraction was obtained by a syringe and the samples were centrifuged at 3000 rpm for 10 min at 4°C. Data were expressed as arbitrary ROS units per oocyte per min (U) and analysed by ANOVA. The results of this study showed that in bovine oocytes ROS levels tend to increase in the T and V groups compared to group C (76.0 ± 6.4, 249.9 ± 87.3 and 147.6 ± 42.6 in C, T and V groups, respectively). However, there were no statistical differences among groups and this was mainly due to the high variability recorded in both treated groups. In conclusion, these results suggest that both exposure to cryoprotectants and vitrification of in vitro-matured oocytes may influence ROS generation. However, the high variability recorded among replicates recommends further investigations.

DO WE PRESERVE UNIQUE APPLE GERMPLASM?

In many European countries, preservation of genetic resources in apple is directed mainly, or even exclusively, to so-called heirloom cultivars, i.e., old and presumably indigenous cultivars. However, there is very little information about the extent to which such collections contain a unique and desirable set of genes as opposed to just a random sample of genes from the global population of apple germplasm. Molecular markers, especially Simple Sequence Repeats (SSR), are commonly used for quantification of the amount of diversity in national collections of various plant crops. Provided that the same SSR primers are used and that allele sizing is standardized, data obtained in different studies can be pooled, thus allowing us to estimate not only diversity but also the degree of differentiation among e.g., apple collections preserved in different national gene banks. We have now used primer pairs from a 'standard set', defined by the European Community for Plant Genetic Resources working group on molecular markers, to analyse a total of 144 apple cultivars preserved in Sweden, Finland and Ireland, as well as some other European and non-European cultivars. Preliminary SSR data indicate that the Swedish, Finnish and Irish heirloom cultivars are very diverse. The groups are, however, only slightly differentiated from one another, with the other European cultivars being quite evenly distributed among the cultivars from the three national collections.

Enhance Beef Cattle Improvement by Embryo Biotechnologies

Embryo biotechnology has become one of the prominent high businesses worldwide. This technology has evolved through three major changes, that is, traditional embryo transfer (in vivo embryo production by donor superovulation), in vitro embryo production by ovum pick up with in vitro fertilization and notably current cloning technique by somatic cell nuclear transfer and transgenic animal production. Embryo biotechnology has widely been used in dairy and beef cattle industry and commercial bovine embryo transfer has become a large international business. Currently, many developed biotechnologies during the period from early oocyte stage to pre-implantation embryos can be used to create new animal breeds and accelerate genetic progression. Based on recent advances in embryo biotechnologies and authors current studies, this review will focus on a description of the application of this technology to beef cattle improvement and discuss how to use this technology to accelerate beef cattle breeding and production. The main topics of this presentation include the following: (i) how to increase calf production numbers from gametes including sperm and oocyte; (ii) multiple ovulation and embryo transfer breeding schemes; (iii) in vitro fertilization and intracytoplasm sperm injection in bovine; (iv) pronuclear development and transgenic animals; (v) sex selection from sperm and embryos; (vi) cloning and androgenesis; (vii) blastocyst development and embryonic stem cells; (viii) preservation of beef cattle genetic resources; and (ix) conclusions.

Priorities for genetic resource collection and preservation of wild gymnosperms in Yunnan: an analysis based on the “3E” principle

Priorities for genetic resource collection and preservation of wild gymnosperms in Yunnan: an analysis based on the “3E” principle

In vitro Germination and Pollen Conservation of Some Musa Species

The yield ofbanana and plantain which is an important food is affected especially by black leaf streak disease. Pollen from resistant wild types is used by conventional breeding to create new hybrids resistant to black leaf streak disease. A strategy of pollen genetic resource preservation and storage is necessary for this breeding. The main objective of this study was to optimize the conditions for in vitro germination, conservation and storage of pollen from three ban ana diploids (Musa acuminata). Pollen grains used in this study were collected from Njombe and Ekona banana collections. The basal media used for in vitro culture were th ose of Heslop-Harrison and Brewbaker and Kwack. Different temperature and pH values tested allowed for the evaluation of germination rates. Pollen were desiccated and stored in a freezer ( -20°C) and in a refrigera tor ( + 10°C). The results obtained showed that the basal medium of Heslop-Harrison supplemented with 5% sucrose allowed to obtain optimum pollen germination rate. The optimum temperature was 30°C and the optimum pH values were 6.5 for Calcutta and 6.8 for M53 and Zebrina. The pollen grains of the three varieties can be conserved freshly in the refrigerator at+ 10°C for at !east two months. These results are very important for ban ana and plantain breeding programs because the conditions of pollen storage for the three varieties are weil established and permit genetic material sharing and diffusion between different ban ana research centers. (Resume d'auteur)

Complete mitochondrial genome of the black-headed snake Sibynophis collaris (Squamata, Serpentes, Colubridae)

The black-headed snake Sibynophis collaris (Reptilia, Squamata, Colubridae) is a least concern species in the world. Two universal and two specific polymerase chain reaction (PCR) primers were used for long PCRs to amplify the whole mitochondrial genome of S. collaris. The products were subjected to do sequencing reactions. The complete genome is 17,163 bp in size, containing 37 genes coding for 13 proteins, 2 rRNAs, 22 tRNAs, and 2 control regions (CRI and CRII). The results could play an important role in the preservation of genetic resources for helping conservation of the endangered species.

Molecular characterization of the Latvian apple (Malus) genetic resource collection based on SSR markers and scab resistance gene Vf analysis

Apple cultivars are an integral part of the Latvian State Program for Preservation and Sustainable Use of Genetic Resources for Food and Agriculture. Latvian apple (n = 109) cultivars of local origin, nominated as National Plant Genetic Resources, were analyzed with eight simple sequence repeat (SSR) markers recommended by the European Cooperative Programme for Plant Genetic Resources (ECPGR) Malus/Pyrus working group and a marker for scab resistance gene (Vf). All SSR loci exhibited a high level of polymorphism – 12 to 39 alleles, 18.75 in average, with high observed heterozygosity (Ho) ranging from 0.64 to 0.89 and a mean of 0.78. The gene diversity (polymorphism information content value) varied from 0.79 to 0.90, with an average of 0.86. All cultivars could be distinguished with the tested set of SSR loci, with a high frequency of rare (38%) or unique (26%) alleles. For the Vf gene, all three possible genotypes (VfVf, Vfvf and vfvf) were detected for 1, 6 and 102 varieties, respectively. The analyzed germplasm showed high genetic diversity, particularly landraces, while the modern cultivars clustered mainly into two groups. Cluster analysis did not reveal a clear pattern with well-defined groups, but confirmed some relationships based on known or putative pedigrees, as well as suggesting the possible parentage of some cultivars.

Phylogenetic relationships of the Hucul horse from Romania inferred from mitochondrial D-loop variation

The existence of the Hucul horse on Romanian territory has been documented from the very distant past; today Hucul is a unique breed that is part of the FAO Program for the Preservation of Animal Genetic Resources. We compared Hucul with several primitive European and Asiatic breeds in order to elucidate the origin of these horses. We analyzed a 683-bp mitochondrial DNA (mtDNA) D-loop fragment in a population of Hucul horses and compared the polymorphic sites with sequences from other primitive breeds, including Exmoor, Icelandic Pony, Sorraia, Przewalski Horse, Mongolian Wild Horse, Konik, and Shetland Pony, as well as with Arabian, Akhal Teke and Caspian Pony. The sequences were truncated to 247 bp to accommodate short sequence data for the other species. Eighty horses were analyzed; 35 polymorphic sites representing 33 haplotypes were observed. The mean percentage of polymorphic sites was 14.2% for this mtDNA fragment. A neighbor-joining phylogenetic tree was constructed based on Kimura two-parameter distances and the Network 3.111 software was used for phylogenetic analysis. The Hucul horse was classified separately from all other primitive breeds. It is possible that the Hucul horse is not part of the pony class, as it segregated apart from all primitive pony breeds. We found multiple origins in the maternal lineage of domestic horse breeds and demonstrated the uniqueness of the Hucul breed; its origins remain unclear.

Identification of Germline Chimeric Chickens Produced by Transfer of Primordial Germ Cells Using a Hinai-dori-specific Microsatellite Marker

The production of germline chimeric chickens by transfer of primordial germ cells (PGCs) is an effective technique for the preservation and regeneration of genetic resources in chickens. To date, the most widely used method to identify germline chimeric chickens is a testcross using differences in plumage color between the donor and recipient breeds. However, this method is time consuming and laborious. Therefore, simple high-precision molecular techniques for the identification of germline chimeric chickens need to be developed. In this study, we verified the practicability of using a Hinai-dori-specific microsatellite marker that was previously developed to identify germline chimeric chickens. A Hinai-dori-specific microsatellite marker ABR0633 can distinguish the Hinai-dori breed from White Leghorn (WL); thus, these two breeds were used as donor and recipient embryos, respectively, in this study. PGCs obtained from embryonic gonads of Hinai-dori were micro-injected into either the subgerminal cavity or dorsal aorta of WL recipient embryos to produce germline chimeric chickens. A portion of both left and right testes was removed from the manipulated male chicks and was used for genotyping to verify the possibility of identifying germline chimeric chickens. Semen was then obtained from matured manipulated male chickens and was used for genotyping. Simultaneously, these chickens were crossed with Hinai-dori females by artificial insemination to produce offspring. No donor-derived Hinai-dori allele was detected in the testicular tissues from the manipulated male chicks. However, donor-derived Hinai-dori allele was detected in the semen from two manipulated male chickens (2/2) that were produced by microinjection of Hinai-dori PGCs into the dorsal aorta. In the progeny tests, Hinai-dori PGC-derived offspring were obtained from these two chickens. Moreover, only the donor-derived Hinai-dori allele was detected in the offspring that were judged as the Hinai-dori breed by plumage color. Therefore, the offspring were confirmed as the Hinai-dori breed both phenotypically and genotypically. We conclude that a Hinai-dori-specific microsatellite marker is suitable to identify germline chimeric chickens and the use of this method could reduce the time and labor needed for testcrosses.

CONSERVATION AND VALORISATION OF APRICOT LOCAL VARIETIES

The genetic resources preservation is justified by the necessity of protecting the varieties in danger of extinction. In Southern Italy, a high number of old ecotypes of apricot are present. The aim of the research was to valorise the bio-agronomic and qualitative characteristics of the ecotypes individualized and harvested at optimum maturity during the summer 2004. Characteristics of fruit, stone and pulp were measured. The observed ecotypes showed significant differences in the morphological and qualitative characteristics of fruits and a wide range in ripening time for the market.

Germination rates of perilla (Perilla frutescens (L.) Britton) mericarps stored at 4°C for 1–20 years

Periodic update of stored seeds is indispensable for sustainable use and preservation of plant genetic resources, and performance of this task requires precise data on the relationship between storage period and the germination rate of seeds after long-term storage. In this study, germination rates of pure strains of perilla (Perilla frutescens (L.) Britton var. crispa) were investigated for three strains by using the mericarps stored for 1-20 years at 4 degrees C under dry conditions. The germination rates were fairly good (65-70%) for 5-8 years after harvest, but dropped to almost 0% for mericarps stored more than 9 years. Mericarps that did not germinate were analyzed for viability by using 2,3,5-triphenyltetrazolium chloride. For old mericarps (stored more than 9 years), the germination rates and seed viability did not correlate. Further study on days required for germination of mericarps harvested each year was derived from our observation record, suggesting that germination rates and longevity of mericarp life were increasingly influenced by individual difference as the storage period lengthened.

ACCUMULATION, PRESERVATION AND INVESTIGATION OF LITHUANIAN ORNAMENTAL PLANTS IN THE BOTANICAL GARDEN OF VILNIUS UNIVERSITY

At present genetic resources collections in the Floriculture Department consists of 900 flower taxa developed by Lithuanian plant breeders. Lithuanian flower breeders released a lot of new cultivars of Crocus L., Dahlia Cav., Gladiolus L., Hemerocallis L., Iris L., Lilium L., Paeonia L., Primula L., Tulipa L. and etc. The aim of this research is to study and evaluate ornamental properties of flower cultivars released by the Lithuanian breeders. The investigations, descriptions and evaluations of morphological, bioecological and ornamental properties of gladiolus (Gladiolus L.) and peony (Paeonia L.) cultivars were carried out in the period of 1998-2008 according to the requirements of the International Union for the Protection of New Varieties of Plants (UPOV) and methodologies used in neighbouring countries. Having collected, accumulated, researched and evaluated the flowers created by Lithuanian breeders, in the future it will be possible to select the most valuable cultivars (genetic resources) and to make a system of the effective preservation and rational usage of the genetic resources. In general, Lithuanian cultivars are originals adapted to the local climate conditions, and it is urgent to conserve, investigate and foster it as a part of the land culture. The most distinguishing and attractive (unbelievable form, perfect display of blooms in the spike, colour harmony) Lithuanian gladiolus cultivars, investigated in the Botanical Garden of Vilnius University, are the following ones: 'Fiji' (author A. Lukosevicius), 'Nu, Gromov, Pogodi!' (P. Ciplijauskas), 'Laimute' (P. Ciplijauskas), 'Merkurijus' (A. Lukosevicius), 'Onute-3' (P. Balcikonis), 'Paparcio Ziedas' (P. Ciplijauskas), 'Saules Takas' (P. Balcikonis), 'Snieguole' (P. Ciplijauskas), 'Solveiga' (A. Lukosevicius), and 'Spalvingas Sapnas' (J.A. Liutkevicius). Forty-four cultivars of paeony created by Lithuanian plant breeders (O. Skeiviene and E. and J. Tarvidai) are confirmed by Orders of Minister of Environmental as National Plant Genetic Resources. These cultivars will be preserved in future.

Efficient system for preservation and regeneration of genetic resources in chicken: concurrent storage of primordial germ cells and live animals from early embryos of a rare indigenous fowl (Gifujidori)

The unique accessibility of chicken primordial germ cells (PGCs) during early development provides the opportunity to combine the reproduction of live animals with genetic conservation. Male and female Gifujidori fowl (GJ) PGCs were collected from the blood of early embryos, and cryopreserved in liquid nitrogen for >6 months until transfer. Manipulated GJ embryos were cultured until hatching; fertility tests indicated that they had normal reproductive abilities. Embryos from two lines of White Leghorn (24HS, ST) were used as recipients for chimera production following blood removal. The concentration of PGCs in the early embryonic blood of 24HS was significantly higher than in ST (P < 0.05). Frozen-thawed GJ PGCs were microinjected into the bloodstream of same-sex recipients. Offspring originating from GJ PGCs in ST recipients were obtained with a higher efficiency than those originating from GJ PGCs in 24HS recipients (23.3% v. 3.1%). Additionally, GJ progeny were successfully regenerated by crossing germline chimeras of the ST group. In conclusion, the cryogenic preservation of PGCs from early chicken embryos was combined with the conservation of live animals.

Die Deutsche Genbank Obst, ein dezentrales Netzwerk zur nachhaltigen Erhaltung genetischer Ressourcen bei Obst

The preservation of fruit genetic resources is the basis for ensuring a sustainable fruit production in Germany. The preservation of fruit cultivars has a long-lasting tradition in Germany, which reaches back to the early decades of the 20 th century. Since that time, a multitude of cultivars of different fruit crop species are preserved in public and private germplasm collections. However, the loss of individual cultivars can not be excluded using such form of preservation. The German Fruit Genebank has been recently established to minimize the risk of losing fruit genetic resources. The German Fruit Genebank is a decentral network, which is aimed on the coordination of different germplasm collections in Germany. The work will be organized in species specific networks (e.g. apple network, strawberry network etc.). Preservation guidelines for each fruit species will be developed within the species specific networks. The first three networks for strawberry, cherries, and apple recently have taken up their activities. The establishment of networks for further fruit crops has been scheduled.

94 VITRIFICATION OF WHOLE OVARIES IN YOUNG RATS

Cryopreservation of reproductive organs is an important technology for preservation of genetic resources of experimental, domestic, and wild animals. In addition, cryopreservation of the ovary could be applied to restore the fertility of young women diagnosed with cancer because it could not only provide future fertility, but could also decrease the emotional consequences of cancer therapy for women afflicted with such devastating diseases. Cryopreservation of whole ovary particularly would enable such females to be pregnant by natural mating after transplantation. The aim of the present study was to evaluate the possible of vitrification for young rat ovaries as a possible model for human ovaries using a Cryotop method. Whole ovaries were collected from 10-day-old female rats (Brown-Norway × Wistar) and then washed several times in PB1 medium containing 20% (v/v) fetal calf serum (FCS). The ovaries were submerged in equilibration solution [10% (v/v) dimethyl sulfoxide (DMSO), 10% (v/v) ethylene glycol (EG), and 20% (v/v) FCS in PB1] for 1, 3, 5 or 10 min and then vitrified in vitrification solution (20% DMSO, 20% EG, and 20% FCS in PB1) for 5 min, being plunged into liquid nitrogen on Cryotops. Vitrified ovaries were later warmed in 37°C in PB1 containing 1 M sucrose and 20% FCS for 5 min and then PB1 containing 0.5 M sucrose and 20% FCS for 5 min. Eight ovaries were embedded in paraffin and cut into 4 μm slices. The slices were stained by hematoxylin-eosin (HE) and integrity of nuclei and cytoplasm in the follicles was histologically evaluated. In each group, 3 ovaries were used for the staining of live/dead (Invitrogen LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells, #L3224). Follicles were separated from the ovary with enzyme and then classified into 2 categories by staining. Follicles possessing more than 50% of green-stained and red-stained granulosa cells were divided as live and dead, respectively. Data were analyzed by Tukey’s test. Results of the HE staining showed that integrity of nuclei and cytoplasm in ovaries equilibrated for 10 min (11%) was lower than those for 1 min (25%), 3min (42%), and 5 min (30%). As for evaluation by live/dead staining, most of follicles in ovaries equilibrated for 10 min (25%) were stained with red (dead). In vitrified ovaries equilibrated for 1 min, 3 min, and 5 min (18%, 16%, and 18%, respectively), there were no significant differences in red-stained follicles. These results suggest that optimal equilibration time is 3 to 5 min for vitrification of whole ovaries of 10-day-old rats. Using this equilibration time, we are now trying to evaluate the capability of vitrified/warmed ovaries after transplantation to recipient rats. The work was supported in part by Grant-in-Aid for Scientific Research from JSPS (KAKENHI) (21789253) to J. I. This work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan through a Grant-in-Aid for Matching Fund Subsidy for Private Universities to J.I. and N.K.