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CryoLetters is a bimonthly, international journal for low temperature science and technology |
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CryoLetters 42 (2), 59-66 (2020) PERSPECTIVE: Andrea Svoradová1*, Lenka Kuželová2, Jaromír Vašíček1,3, 1 National Agricultural and Food Centre, Research Institute for Animal Production Nitra, Hlohovecká 2, 951 41 Lužianky, Slovak Republic. Abstract Unsuccessful rooster fertility following cryopreservation may be linked to specific changes in spermatozoa quality, which can be determined using various methods. These determinations also facilitate the design of improved freezing and thawing processes. Here, we update the current state of methodologies available for the assessment of rooster semen quality after cryopreservation. Computer-assisted sperm analyses (CASA) is one of the main systems used to analyse motion parameters of spermatozoa (total motility, progressive motility and motion parameters). Moreover, fluorescent techniques and flow cytometry can improve the assessment of various aspects of semen quality (viability, acrosome status, mitochondrial potential, lipid peroxidation, DNA damage, lipid peroxidation and cell debris removal) using specific fluorescent markers such as ethidium bromide, Yo-Pro-1, Annexin V, propidium iodide, SYBR-14, PNA, JC-1, BODIPY, acridine orange and DRAQ5. Transmission electron microscopy also yields valuable information on spermatozoa ultrastructure. The application of these techniques to rooster spermatozoa is reviewed in relation to specific freezing techniques, the effects of cryoprotective agents (CPAs) and extenders, and the determination of spermatozoa quality after cryopreservation. Keywords: cryopreservation; quality evaluation; rooster; semen. Download the paper: ROOSTER SPERMATOZOA CRYOPRESERVATION AND QUALITY ASSESSMENT (PDF)
CryoLetters 42 (2), 67-72 (2020) EFFECT OF MOUSE OVARIAN VITRIFICATION ON PROMOTER METHYLATION OF INHBA AND INHBB IN GRANULOSA CELLS OF FOLLICLES Maryam Damavandi1, Parisa Farrokh*1,2 and Saeed Zavareh1,2 1School of Biology, Damghan University, Damghan, Iran 36716-41167 Abstract BACKGROUND: Cryopreservation can induce cellular, genomic, and epigenetic abnormalities. OBJECTIVE: To analyse the impact of ovarian vitrification on follicular development and its epigenetic effect on promoter methylation of Inhba and Inhbb in granulosa cells. MATERIALS AND METHODS: Mouse ovaries were divided into control, toxicity, and vitrified groups. The growth and development of follicles were examined. After in vitro culture of follicles, DNA was extracted from isolated granulosa cells and treated with sodium bisulfite. The promoter methylation of Inhba and Inhbb was analyzed by direct PCR sequencing. RESULTS: Vitrification reduced the growth of follicles; however, antral cavity formation was not influenced negatively. Vitrification reduced the percentage of 5-methylcytosine in the Inhba promoter, while CpG sites in the promoter of Inhbb remained unmethylated. CONCLUSION: Vitrification had adverse effects on follicle growth and the epigenetics of granulosa cells. The results of the current study show that vitrification methods of ovary need more improvement. Keywords: activin; DNA methylation; granulosa cells; inhibin; vitrification.
CryoLetters 42 (2), 73-80 (2020) IN VITRO ASSESSMENT OF TRIS EGG YOLK AND SOYBEAN LECITHIN BASED EXTENDERS FOR CRYOPRESERVATION OF CROSSBRED RAM SEMEN Arjuma Khatun1, M.R.Fazili2, A. A. Malik1, R.A. Shah3, H.M. Khan4, 1Division of Animal Reproduction, Gynaecology and Obstetrics, Abstract BACKGROUND: The replacement of egg yolk with alternative plant-derived soybean lecithin is gaining interest in both animal and human sperm cryopreservation owing to biosecurity issues with egg yolk based extenders. OBJECTIVE: To evaluate the comparative effect of egg yolk and soyabean lecithin based extenders on the quality of cryopreserved crossbred ram semen. METHODS: Pooled ejaculates (total ejaculates = 36) were divided into two aliquots and extended with Tris egg yolk extender (Tris extender) and soybean lecithin based commercial extender (Ovixcell) RESULTS: Among the two extenders, Ovixcell showed better sperm quality both at the pre-freeze (Sperm motility) and post-thaw stages. Lower malondialdehyde (MDA) level (nmol/mL) was observed in Ovixcell as compared to Tris extender. Both sperm quality and MDA level decreased significantly (P<0.05) from pre-freeze to post-thaw in both the extenders. CONCLUSION: The findings of the present study indicate that Ovixcell is a comparable alternative to Tris extender for the cryopreservation of crossbred ram semen. Keywords: cryopreservation; IVF; ram; semen; soybean lecithin; Tris egg yolk.
CryoLetters 42 (2), 81-86 (2020) EVALUATION OF THE FREEZABILITY OF THE BOVINE EPIDIDYMIS TAIL SPERM WITH THE ADDITION OF ANTIOXIDANTS Gabriela Passamani da Cruz1, Ana Paula Zanfrilli dos Santos1, 1Paranaense University, Umuarama/PR, Brazil. Abstract BACKGROUND: The cryopreservation and recovery of epididymis tail sperm is an important biotechnology dependent on the composition of the freezing medium. OBJETIVE: To evaluate the effect of melatonin, added to commercial freezing medium extender, on the kinetics and viability of bovine epididymis tail sperm. MATERIAL AND METHODS: Five routines were performed, each consisting of eight epididymis and the structures were sliced onto a glass plate containing a commercial diluting medium for Botubov®. The samples were divided into four groups, with 80 x 106 spermatozoa per mL. Group 1: samples diluted in Botubov®. Group 2: samples centrifuged (600 g, 10 min), and the pellet re-suspended in Botubov®. Group 3, samples diluted in Botubov® containing 100 pM melatonin. Group 4: samples centrifuged (600 g, 10 min) and the pellet resuspended in Botubov® with 100 pM melatonin. The samples were transferred to 0.5 mL straws at 40 x 106 viable spermatozoa, stabilized at 5º C for 4 h, transferred to liquid nitrogen vapour for 20 min, dipped in liquid nitrogen and stored in a cryogenic cylinder. After thawing (46ºC, 15 s), sperm kinetics and viability parameters were evaluated. RESULTS: There was no difference in the parameters of total motility (MT, %), progressive motility (MP, %), progressive linear velocity (VSL, µm/s), curvilinear velocity (VCL, µm/s), linearity (LIN, %), spermatozoa with rapid movement (RAP, %) and level of intact plasma membranes and acrosome (IPMA, %) among the groups studied. However, a difference was observed between the routines performed. CONCLUSION: The protocol for freezing bovine epididymis tail sperm is applicable; however, there is an influence of the epididymis used, for the best efficacy of this biotechnology. Keywords: bovine epididymis; freezing; melatonin; spermatic parameters.
CryoLetters 42 (2), 87-95 (2020) IMPACTS OF SPECIFIC CRYOPROTECTANTS ON SPERM FREEZING AND RELATIONSHIPS BETWEEN CRYODAMAGE AND OXIDATION STRESS PARAMETERS IN AWASSI RAM SPERM Ömer Varışlı1*, Serkan Erat2, Faruk Bozkaya3, Nurettin Aydilek4 1 Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Kırıkkale University, Kırıkkale, Turkey Abstract BACKGROUND: The role of oxidative stress during cryoprotectant treatment has received little attention. OBJECTİVE: To assess the effects of different cryoprotectants and discover relationships between cryodamage and oxidative stress parameters on Awassi ram sperm. MATERIALS AND METHODS: The sperm samples diluted with Salamon's tris-citrate (TRIS) containing 20% centrifuged egg yolk and 0.5, 1.0 or 1.5 M Glycerol (Gly), methanol (M), 2-methoxyethanol (2-ME), dimethylacetamide (DMA) and 1.2 propanediol (PR). After 2 h of equilibration at +4 ºC, the sperm samples were frozen in liquid nitrogen vapour and stored. RESULTS: The best post-thaw motility (43.3%, 41.7%) of sperm was achieved when protected with 0.5 and 1.0 M glycerol. Arylesterase and ceruloplasmin parameters were significantly different after equilibration, whereas sulfhydryl groups were significantly different after freezing in their respective groups (P< 0.05). CONCLUSION: The increased use of glycerol caused greater loss of motility. The role of oxidative stress in freezing was also found to be limited. Keywords: Awassi; cryopreservation; cryoprotectants; oxidative stress; ram; sperm.
CryoLetters 42 (2), 96-105 (2020) CRYO-RADIOFREQUENCY STIMULATES BROWNING OF ADIPOCYTES IN MICE Marina Chaves de Oliveira1,#, Amanda Carla Clemente de Oliveira1,#, 1Department of Nutrition, Nursing School, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. Abstract BACKGROUND: Local fat accumulation is a health risk and this has raised interest in the development of aesthetic treatments, such as cryo-radiofrequency (CRF). OBJECTIVE: To evaluate the consequences of CRF in adipose tissue remodeling in a model system. MATERIALS AND METHODS: Lean and high-fat diet-induced obese mice were assessed 7 days after one CRF application; and lean mice were assessed 0, 3, 6 and 12 h after one application of CRF. Assessments included histology, DNA degradation, gene expression, ELISA of cytokines, serum analysis and neutrophil presence. RESULTS: Unchanged fat mass was found 7 days after CRF in obese and lean mice. However, lean mice showed smaller adipocyte size with areas resembling a browning process. TNF levels, apoptotic cells, and UCP-1 expression increased 7 days after CRF in inguinal adipose tissue of lean mice. Although no differences were found in fat mass, adipocyte size decreased just after CRF and this changed was maintained until 12 h, with cells resembling beige adipocytes. CONCLUSION: We suggest that CRF therapy is capable of inducing thermogenic adipocytes in lean mice. Keywords: adipose tissue; browning; cryo-radiofrequency (CRF); obesity.
CryoLetters 42 (2), 106-110 (2020) EXPOSURE OF Teramnus labialis (L.F.) SPRENG SEEDS TO LIQUID NITROGEN DOES NOT AFFECT NUTRITIONAL STATUS OF FIELD GROWN ADULT PLANTS Yanier Acosta1, 2 , Lianny Pérez3, Doris Escalante2, 1Faculty of Agricultural Sciences; Abstract BACKGROUND: Teramnus labialis (L.f.) Spreng is a potentially important legume species, and can be used as an animal feed and to enhance soil physicochemical characteristics. Despite the biological and agricultural importance, the low availability of seeds, their small size and the low percentage germination limit their large-scale use by farmers. We previously reported a method to cryopreserve seeds of T. labialis which also allowed for the breaking of seed dormancy. OBJECTIVE: The current study reports on the nutritional status of 5 month old field grown plants regenerated from cryostored and control seeds. MATERIALS AND METHODS: Biomass (fresh and dry mass of leaves and stems) and contents of ash, neutral detergent fiber, acid detergent fiber, lignin, cellulose, crude protein, P, Ca, Mg and K were measured. RESULTS: Seeds germinated and emerged faster following immersion in liquid nitrogen (LN) which was supported by quantitative evaluations of fresh and dry weights per m2. However, the ratio of leaf:stem mass were not altered by seed exposure to LN. CONCLUSION: The results showed that exposure of seeds to cryogenic temperatures did not alter the nutritional composition of regenerated plants. Keywords: animal feed; crops; cryopreservation; legumes; nitrogen fixation; seed dormancy.
CryoLetters 42 (2), 111-119 (2020) EFFECT OF MITO-TEMPO INCORPORATED SEMEN EXTENDER ON PHYSICO-MORPHOLOGICAL ATTRIBUTES AND FUNCTIONAL MEMBRANE INTEGRITY OF FROZEN THAWED BUFFALO SPERMATOZOA Abhishek Kumar1*, Subrata Kumar Ghosh1*, Rahul Katiyar1, 1Division of Animal Reproduction, Abstract BACKGROUND: Sperm mitochondria are the major site of reactive oxygen species (ROS) production and excess production during freezing-thawing process inflicts oxidative damages to spermatozoa. Buffalo spermatozoa are more prone to oxidative damage due to inherently more polyunsaturated fatty acids and low cholesterol to phospholipids ratio in the plasma membrane. A mitochondrial targeted antioxidant, Mito-TEMPO was used in this study. OBJECTIVE: To study the effect of Mito-TEMPO incorporated semen extender on the post-thaw semen quality in buffalo. MATERIALS AND METHODS: A total of 18 ejaculates from three murrah buffalo bulls with ≥70% individual progressive motility were utilized for the study. Each semen sample was equally divided and extended with five groups: Group I (Control, without Mito-TEMPO addition); Group II (10 µM Mito-TEMPO); Group III (50 µM Mito-TEMPO); Group IV (100 µM Mito-TEMPO); Group V (500 µM Mito-TEMPO) to have 80×106 progressive motile sperm/mL of extender, filled and sealed in French mini straws (0.25 mL) and frozen following equilibration. The effect of Mito-TEMPO was assessed at fresh/post-dilution and post-thaw stages by evaluating physico-morphological attributes and functional membrane integrity such as hypo-osmotic swelling test (HOST). RESULTS: Initial progressive motility, viability, acrosomal integrity and HOS response was significantly (p<0.05) improved and sperm abnormality was significantly (p<0.05) reduced in extended semen with Mito-TEMPO (50 µM) compared to control at post-thaw stage, although improvement was also observed at 10 and 100 µM in post-thaw samples. CONCLUSION: Mito-TEMPO incorporated semen extender at 50 µM concentration, could be part of a rationale for improving post-thaw semen quality in buffalo. Keywords: buffalo; Mito-TEMPO; oxidative stress; semen cryopreservation. |
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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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