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ABSTRACT ARCHIVE |
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CryoLetters is a bimonthly, international journal for low temperature science and technology |
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CryoLetters 40 (6), 312-321 (2019) DETERMINATION OF MEDIUM CONDITION EFFECTIVE TO CRYOPRESERVATION OF PRIMARY SPERMATOGONIAL STEM CELLS DERIVED FROM PORCINE NEONATAL TESTES Min Hee Park1, Ji Eun Park1, Min Seong Kim1, Jung Im Yun2, 1 Dept. Animal Life Science, Kangwon National University, Chuncheon 24341, Republic of Korea Abstract BACKGROUND: The superior genetic resources of breeding pigs have been preserved for use through freezing the sperm or semen. However, because there is no way to collect their sperm or semen after depletion, the generation of sperm via the differentiation of porcine spermatogonial stem cells (SSCs) can be an alternative. To date, there have been no reports of techniques customized to in-vitro culture and differentiation into sperm in porcine SSCs. Accordingly, it is important to preserve porcine SSCs with outstanding genetic backgrounds until these technologies are developed. Unfortunately, a protocol for the long-term preservation of porcine SSCs has yet to be reported. OBJECTIVE: We tried to develop a cryopreservation medium to preserve the characteristics of undifferentiated porcine SSCs for long-term cryopreservation. MATERIALS AND METHODS: SSCs retrieved from porcine testes were freeze-cryopreserved in StemPro-34 medium supplemented with various concentrations of fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), and trehalose; then, after 7 days, the viability and alkaline phosphatase (AP) activity was measured in thawed porcine SSCs. Additionally, we investigated the use of hypotaurine and/or glutathione as antioxidants in the optimized freezing medium for maintaining the viability and AP activity of porcine SSCs during the freezing-cryopreservation-thawing process. RESULTS: Porcine SSCs frozen-cryopreserved-thawed in StemPro-34 medium supplemented with 10% (v/v) FBS, 10% (v/v) DMSO, 200 mM trehalose, 5 mM hypotaurine, and 5 mM glutathione showed the highest viability and AP activity. CONCLUSION: We optimized a cryopreservation medium that inhibits the loss of viability and the increases differentiation post-thawing of the frozen porcine SSCs. Keywords: cryopreservation, medium, pigs, spermatogonial stem cells, undifferentiation.
CryoLetters 40 (6), 322-332 (2019) EFFECT OF CRYOPRESERVATION ON PLANT GROWTH, BULB CHARACTERISTICS, AND VIRUS REDUCTION OF GARLIC (Allium sativum L.) Xiao-Xue Liu1, Si-Wei Mou2 and Zhi-Hui Cheng1* 1 College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, People’s Republic of China. Abstract BACKGROUND: Cryopreservation is a promising plant germplasm preservation technique that provides genetically stable plantlets. OBJECTIVE: To investigate whether the cryopreservation protocol developed for garlic compromises the performance of the cryopreserved plants, compared to garlic plants grown in the field. MATERIALS AND METHODS: The field performance of cryopreserved garlic plantlets under in vivo conditions was compared with garlic derived from the field. We assessed both net photosynthetic rate, bulb characteristics and the efficiency of cryotherapy-induced virus eradication. RESULTS: The superiority of the morphological traits of cryopreserved garlic increased gradually with the growth of the garlic plant. Cryopreservation also improved the net photosynthetic rate, bulb diameter (by 13.4-18.2%), bulb weight (by 37.9-50.5%), and clove number per bulb (by 25.7-63.6%). The highest virus elimination rate due to cryopreservation was 75.0% for onion yellow dwarf virus (OYDV). CONCLUSION: These findings suggest cryopreservation as a promising method for conservation and improvement of garlic. Keywords: cryopreserved garlic, morphological trait, net photosynthetic rate, bulb characteristics, virus elimination rate.
CryoLetters 40 (6), 333-340 (2019) OCCURRENCE OF LATENT BACTERIA DURING CRYOPRESERVATION OF LONG-TERM IN VITRO CULTURES OF COLTSFOOT, Tussilago farfara Melanie Hambeck1, Angelika Senula2 and Andrea Kodym1,3* 1 Department of Pharmacognosy, University of Vienna, 1090 Vienna, Austria. Abstract BACKGROUND: Long-term in vitro cultures of Tussilago farfara (L.), a traditional medicinal plant in Austria, had been stored at 14°C for over 20 years. The cultures were vigorous and showed no visual signs of bacterial presence. The transfer from growth regulator-free culture medium to medium containing kinetin and the increase of temperature from 14°C to 25°C for fast propagation led to the emergence of latent bacteria in all twelve accessions studied. OBJECTIVE: To investigate latent infections occurring during the development of a cryopreservation protocol of genetically interesting material using droplet-vitrification. MATERIALS AND METHODS: Two protocols for droplet-vitrification were tested using plant vitrification solutions (PVS) 2 and 3. The bacteria were isolated and identified using 16S rDNA analysis. Next, non-cryopreserved in vitro plantlets were acclimatized and transferred to the glasshouse. After 6 weeks, shoot tips were harvested from the pot plants, surface-sterilized and initiated into culture. Further, newly acquired achenes of Tussilago were surface-sterilized and germinated in vitro and seedlings checked for bacteria. RESULTS: The bacteria from the long-term cultures were isolated and identified as Luteibacter. Regeneration after cryopreservation using PVS3 was successful despite the continuing presence of Luteibacter. Luteibacter could no longer be detected in the newly-initiated in vitro material in subsequent tests and it was also not detected in the seedlings. CONCLUSION: Luteibacter withstood the cryopreservation procedure. Re-initiation of infected material may be an efficient alternative to antibiotic treatment to manage bacteria in micropropagation systems. Keywords: endophytic-free cultures, contamination, infection, sequencing, antibiotics
CryoLetters 40 (6), 341-3461 (2019) THE EFFICACY OF LASER ZONA THINNING ON THE OUTCOMES OF VITRIFIED HUMAN EMBRYO TRANSFERS IS RELATED TO FERTILIZATION METHOD AND NOT VITRIFICATION PER SE Jie Zhao 1*, Zhengyuan Huang 2*, Fang Liu 1, Xiujuan Chen 1# 1 Reproductive Center, Department of Obstetrics and Gynecology, the Affiliated Hospital of Inner Mongolia Medical University, Hohhot; Abstract BACKGROUND: Embryo vitrification induces zona hardening, which can further influence the pregnancy outcome after vitrified human embryo transfer. However, the factors in fertilization or cryopreservation that contribute to zona hardening and influence the efficacy of laser zona thinning (LZT) is not well studied. OBJECTIVE: To investigate whether fertilization methods and vitrification affect the efficacy of LZT treatment. MATERIALS AND METHODS: The present study is a retrospective analysis of embryo transfers. The process involves two fertilization methods before embryo vitrification. LZT is used to deal with zona hardening. The pregnancy outcomes of 1274 embryo transfer cycles were reviewed statistically. RESULTS: LZT treatment on vitrified embryos fertilized with intracytoplasmic sperm injection (ICSI) resulted in a significantly lower pregnancy rate and live birth rate compared to that by in vitro fertilization (IVF) (43.0% vs 54.3%, P=0.023; 34.4% vs 45.7%, P=0.021). LZT treatment on vitrified embryos fertilized with ICSI had a negative impact on pregnancy rate and live birth rate. CONCLUSION: The efficacy of LZT treatment is related to the fertilization method rather than vitrification. LZT treatment for zona hardening of the vitrified embryos fertilized with ICSI leads to a lower pregnancy rate and live birth rate. Keywords: embryo, intracytoplasmic sperm injection, laser zona thinning, vitrification.
CryoLetters 40 (6), 347-351 (2019) A NOVEL RECOMBINANT EEL POUT (Macrozoarces americans) TYPE III ANTIFREEZE PROTEIN IMPROVES CRYOSURVIVAL OF BUFFALO SPERM S. Qadeer1, M. A. Khan2, M. S. Ansari3, B. A. Rakha4, R. Ejaz5, 1 Department of Biological Sciences, University of Sargodha-Mianwali Campus-42200, Pakistan Abstract BACKGROUND: The quality of cryopreserved buffalo semen is low due to high susceptibility of sperm membranes to cold shock. OBJECTIVE: The present study was designed to investigate the effect of recombinant type-III antifreeze protein from the eel pout Macrozoarces americanus (rAFPIII) on freezability of buffalo semen. MATERIALS AND METHODS: Semen was collected from three buffalo bulls for three weeks (replicates). Qualified ejaculates (N=18) were split into four aliquots and diluted in Tris-citric acid extender containing 0.0, 0.1, 1 and 10 µg mL-1 of rAFPIII. Semen was cooled to 4 °C, evaluated for sperm motility and PMI, cryopreserved and assessed for post-thaw quality. RESULTS: Supplementation of the extender with rAFPIII didn’t affect motility and PMI of chilled semen. Post-thaw sperm motility and PMI were higher in extender supplemented with rAFPIII (10µg mL-1) compared to control. Sperm viability and acrosome integrity remained the same. CONCLUSION: Addition of rAFPIII in extender improved motility and PMI of cryopreserved buffalo semen. Keywords: antifreeze proteins, extender, buffalo, semen, cryopreservation.
CryoLetters 40 (6), 352-356 (2019) OVIDUCTAL PROTEINS EFFECT IN ROOSTER SPERMATIC CRYOPRESERVATION Ricardo Camarillo1, Irma Jiménez2, Adrián Guzmán3, Ana M Rosales3, Metropolitan Autonomous University, Mexico: Abstract BACKGROUND: Cryopreservation induces spermatic cryo capacitation, which can decrease thawed sperm fertilizing capability. OBJECTIVE: To evaluate the effect of uterus-vaginal union protein factors to inhibit sperm cryo capacitation and mantain viability and fertilizing capability of rooster spermatozoa. MATERIALS AND METHODS: Rooster spermatozoa was cryopreserved using Lake extender supplemented with different hen's uterus-vaginal junction protein concentrations, to determine spermatic viability, sperm physiological condition and fertilizing capability in vivo. RESULTS: It was possible to induce spermatic decapacitation in vitro, inhibiting cryo capacitation and allowing fertility results comparable to those obtained with fresh semen. CONCLUSION: Uterus-vaginal protein extracts induce spermatic decapacitation in vitro. Keywords: poultry, reproduction, cryopreservation.
CryoLetters 40 (6), 357-366 (2019) RESPONSES OF THE BUDS OF THREE SOUTH AFRICAN SWEET POTATO (Ipomoea batatas) ACCESSIONS TO DIFFERENT CRYOPROTECTANTS Machoene Tshidi Manamela 1, 2* and David John Mycock1 1 University of Witwatersrand, School of Animal, Plant and Environmental Sciences, Private Bag 3, Wits, 2050, South Africa. Abstract BACKGROUND: Plant Vitrification Solution 2 (PVS2) was reported to be a successful cryoprotectant solution in regenerating plantlets from sweet potato shoot tips after cryostorage. However, this was not the case for the South African sweet potato accessions. OBJECTIVE: This study assessed the responses of buds of sweet potato to different concentrations of the components of PVS2 both individually and in combination. MATERIALS AND METHODS: The percentage material that regenerated into plantlets was recorded against the total that was evaluated. RESULTS: Regeneration of buds after treatment with 5% dimethyl sulphoxide was significantly better (p<0.001) than 10-15% dimethyl sulphoxide while exposure to ethylene glycol concentrations of 5-10% were better than 15% for all but one accession. Significant difference (p<0.001) was observed in treatment with 30-40% glycerol. Combination of the components at 5% (PVS5%) was better than 10-15% dimethyl sulphoxide and ethylene glycol and 20% glycerol. CONCLUSION: It is recommended that PVS5% be used for further development of the cryopreservation protocol for the South African accessions. Keywords: dimethyl sulphoxide, ethylene glycol, glycerol, plant vitrification solution, axillary buds, sweet potato.
CryoLetters 40 (6), 367-373 (2019) PRODUCTION OF HANDMADE OPEN PULLED STRAW (OPS) USING DIGITAL HEATING GUN FOR THE VITRIFICATION PROCESS Sung Woo Kim 1, †,*, M. M. R. Chowdhury1, 2, †, Yeoung-Gyu Ko 1, 1 Animal Genetic Resources Research Center, National Institute of Animal Science, Rural Development Administration (RDA), Jeonbuk, Republic of Korea Abstract BACKGROUND: Vitrification is the most popular technique for the cryopreservation of oocytes and embryos, replacing slow freezing methods. MATERIALS AND METHODS: This study evaluated the efficient manufacturing methods of handmade open pulled straw (OPS) with a digital heating gun that could be proposed for vitrification. RESULTS: Production efficiency of OPS using 0.5 mL straw was detected at 0, 66.1, 90.5 and 85.7% for 1~2 s and 9.5, 33.3, 47.6 and 23.8% for 2~3 s of heating time at 250, 350, 400 and 450°C respectively. The production rate of OPS using 0.25 mL straw was perceived at 33.3, 76.2, 83.3, 95.2 and 57.6 % for 1.5~2.5 s time with the optimized heat setting at 330, 340, 350, 360 and 370°C respectively. The desired inner diameter (200~300 μm) of OPS could be varied according to the gamete size, embryo developmental stages or cell lines of different species. Based on our data, the production efficiency of OPS using 0.25-mL straw were increased beyond using a 0.5-mL straw. CONCLUSION: Handmade OPSs could be efficiently produced with a digital heating gun to generate a vitrification device for freezing gametes, embryos and cell lines. Keywords: digital heating gun, open pulled straw, embryo, vitrification. |
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For Abstracts published from meetings, such as SLTB meetings, go to the
relevant Volume Year of the journal (above). For Full text Free Access Content (from 2000 onwards) go to CryoLetters at Ingenta and look for the blue symbol. |
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