72 COMPARISON OF CRYOPRESERVATION METHODS: SLOW COOLING VERSUS RAPID COOLING BASED ON SPERM VIABILITY OF SPERMATOZOA OBTAINED FROM THE CAUDA EPIDIDYMIS OF ALPACA (VICUGNA PACOS)

2015 ◽  
Vol 27 (1) ◽  
pp. 129
Author(s):  
E. Mellisho ◽  
M. Moina
Keyword(s):  
Vital Staining ◽  
Freezing Process ◽  
Sperm Viability ◽  
Solid Carbon Dioxide ◽  
Slow Cooling ◽  
Rapid Cooling ◽  
Significance Level ◽  
Progressive Motility ◽  
Environment Temperature ◽  
Hypoosmotic Swelling Test

In alpacas, the male has a low reproductive performance due to the small size of the testes, extended period of ejaculation, and low quality of semen. This work had an objective to evaluate 2 methods of cryopreservation on sperm viability of spermatozoa obtained from the cauda epididymis of male alpacas. Testes from 19 male alpacas (>3 years old) were obtained from the slaughterhouse of Huancavelica and transported to the laboratory in isothermal conditions within 3 h of slaughter. The spermatozoa were obtained by slicing the head of the epididymis, diluting in tris-yolk-glycerol at environment temperature, and then refrigerating for 2.5 h at 5°C. The freezing process was carried out by 2 methods, slow cooling and rapid cooling, the results for percentage of progressive motility, vital staining (eosin nigrosin staining), and hypoosmotic swelling test for each method were evaluated. Cryopreservation of spermatozoa by slow cooling was using 0.25-mL straws immediately after the addition of the extenders and sealed with polyvinyl alcohol. The freezing procedure consisted of placing a metallic rack in a polystyrene foam box of 25 × 20 × 15 cm (length × width × height) and pouring LN (–196°C) within the box, keeping the level of LN below the surface of the metallic rack by 6 cm. The straws were placed onto the metallic rack exposing them to the vapors of the LN and closing the box hermetically by 10 min to freeze and then store by immersion in LN. The cryopreservation of spermatozoa by rapid cooling was carried out in pellets of 0.25 mL immediately finishing the addition of the extenders a final concentration of 60 × 106 sperm mL–1. The freezing process consisted of placing the suspension of spermatozoa in holes made on the surface of a block of solid carbon dioxide (dry ice, –79°C) with a micropipette placing aliquots of 0.25 cc quickly and successively, trying to not let the time between the first pellet formed and the last exceed a minute, and then stored by immersion in LN. Semen was thawed at 37°C in hot bath for 1 to 2 min for pellets and 30 s for straws. Percentage of progressive motility, vital staining, and hypoosmotic swelling test were analysed statistically using ANOVA at a significance level of P < 0.05 using the statistical program SAS® 8.0 (Statistic Analysis System, SAS Institute Inc., Cary, NC, USA). There were statistical differences between the 2 methods slow cooling and rapid cooling for percentage of progressive motility (21.7%a v. 36.2%b), vital staining (30.4%a v. 39.7%b), and hypoosmotic swelling test (21.6a v. 19.0a) for the epididymis spermatozoa. We conclude according to the viability parameters for frozen-thawed spermatozoa that the method of rapid cooling (pellets) is a good alternative for cryopreserved spermatozoa from male alpaca epididymis.

scholarly journals Viability of Najawa carp (Cyprinus carpio L.) sperm at 4°C temperature storage

2020 ◽  
Vol 147 ◽  
pp. 01015
Author(s):  
Dimas Fendy Pradana ◽  
Ignatius Hardaningsih ◽  
Dini Wahyu Kartika Sari
Keyword(s):  
Low Temperature ◽  
Cyprinus Carpio ◽  
Sperm Viability ◽  
Low Temperature Storage ◽  
Progressive Motility ◽  
C Storage ◽  
Randomized Design ◽  
Significant Difference ◽  
Carp Cyprinus Carpio ◽  
Temperature Storage

The objectives of this study were to evaluate the sperm viability of Najawa carp (Cyprinus carpio L.) in cryopreservation pre-conditions at 4°C. The design used in this study was Complete Randomized Design with 4 treatments, BSS as a control, 10% DMSO, 0,2 M Sucrose, and 5% DMSO + 0,1 M Sucrose; each consist of three replications. The parameters observed were progressive motility of fresh sperm, diluted sperm before low temperature storage, and 2 hours; 3 hours; 4 hours; 5 hours; one day; one week; a month after 4°C storage. The data were analyzed by ANOVA. The data showed that there was no significant difference between treatment (P>0.05). The best viability was 40.56% of sperm motility which survive for one week, it was achieved by 5% DMSO + 0,1 M Sucrose.

Viability of ovarian tissue after freezing

1957 ◽  
Vol 147 (929) ◽  
pp. 520-528 ◽  
Keyword(s):  
Systematic Study ◽  
Normal Saline ◽  
Ovarian Tissue ◽  
Cumulus Cells ◽  
Rapid Freezing ◽  
Freezing And Thawing ◽  
Slow Cooling ◽  
Rapid Cooling ◽  
Fertilized Egg ◽  
Cells Cultured

The fertilized egg of the rabbit, obtained from the Fallopian tube, is very sensitive to freezing and thawing, even after treatment with glycerol (Smith 1953). By contrast, the cumulus cells of the follicular granulosa which adhere to the egg after ovulation are much more resistant, and a few survive without any special precautions being taken (Smith 1949). A systematic study of the viability of the cumulus cells cultured after freezing and thawing by various methods (Smith 1953) gave the following results: (1) A majority of the cells survived when suspended in homologous serum, cooled slowly to — 79° C and thawed rapidly at + 40° C. Very few survived rapid freezing. (2) Addition of 15 % glycerol to the suspending medium improved survival after slow cooling but not after rapid cooling. (3) Cells suspended in normal saline failed to survive either slow or rapid cooling. Addition of glycerol promoted some survival after slow cooling. These experiments emphasized the need for slow cooling, already demonstrated with bull spermatozoa, and the superiority of serum over saline as a suspending medium. The addition of glycerol to the medium, though conducive to survival after freezing and thawing, was not as necessary as with spermatozoa.

scholarly journals Effect of fruit-juice on spermatozoa viability and lipid peroxidation of Red Sokoto bucks during liquid storage

2021 ◽  
Vol 41 (1) ◽  
pp. 16-27
Author(s):  
J. O. Daramola ◽  
T. A. Sorongbe ◽  
O. M. Onagbesan ◽  
A. V. Jegede ◽  
A. O. Ladokun ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Cell Damage ◽  
Egg Yolk ◽  
Protective Effects ◽  
Sperm Viability ◽  
Progressive Motility ◽  
Liquid Storage ◽  
Watermelon Juice ◽  
And Control

Antioxidants are linked with sperm viability because of their protective effects against cell damage during preservation. In order to enhance the life span of refrigerated buck semen, this study was carried out to determine the effect of fruit-rich antioxidants on spermatozoa viability and lipid peroxidation (LPO) of buck semen during liquid storage. Pooled semen from five Red Sokoto bucks was diluted with Tris-egg yolk based extender and supplemented each with juices from pawpaw tomato and watermelon at 0, 2.5, 5, 7.5 and 10/ 100 ml respectively. Following dilution, the semen samples were assessed subjectively after in vitro storage at 5°C for 24, 48, 72 and 96 hours as regards sperm motility, abnormalities, and acrosome status using a phase-contrast microscope. The concentration of malondialdehyde (MDA) as indices of lipid peroxidation (LPO) in the stored semen was measured in thiobarbituric acid reactive substances (TBARS) at 24, 48, 72 and 96 hours. The results showed highest progressive motility in watermelon juice at 2.5% (P<0.05) during the first 24 hours of storage while the lowest progressive motility was recorded at various levels of pawpaw juice (P<0.05). After 48 hours of storage, extender supplemented with watermelon and tomato juices had better progressive motility compared to control except 7.5% and 10%% of tomato juice (P<0.05). Irrespective of level of juice in the extender, the percentage of intact acrosome was similar among the various juices and control. The results showed that spermatozoa extended with watermelon juice had the lowest (P<0.05) percentage abnormality compared to other extenders at 24, 48, 72 and 96 hours of storage. Higher (P<0.05) percent spermatozoa abnormality compared to other fruit juices and control was observed at 72 and 96 hours of storage in spermatozoa extended with pawpaw juice. Significant reductions of MDA concentrations were achieved by addition of fruit-rich antioxidants to Tris-egg yolk based extender during the first 72 hours and the reduction was much pronounced in extender supplemented with pawpaw juice compared to control (P<0.05). The findings reveal that fruit-rich antioxidants from watermelon and tomato have protective ability to maintain sperm viability and to reduce concentration MDA of buck semen during liquid storage.

scholarly journals Cryopreservation in Coffea canephora Pierre seeds: Slow and fast cooling

2018 ◽  
Vol 42 (6) ◽  
pp. 588-597
Author(s):  
Stefânia Vilas Boas Coelho ◽  
Sttela Dellyzete Veiga Franco da Rosa ◽  
Tatiana Botelho Fantazzini ◽  
Júlia Lima Baute ◽  
Luciano Coutinho Silva
Keyword(s):  
Water Content ◽  
Liquid Nitrogen ◽  
Coffea Canephora ◽  
Slow Cooling ◽  
Rapid Cooling ◽  
Promising Alternative ◽  
Biochemical Alterations ◽  
Physiological Quality ◽  
Fast Cooling

ABSTRACT Coffee is one of the main agricultural commodities in the country, and it is important to conservation of plant material for breeding programs. Cryopreservation is a promising alternative for preserving in the long-term the germplasm of species considered recalcitrant. However, studies should be performed to achieve maximum survival of seedlings after immersion in liquid nitrogen. The objective of this work was to find a cryopreservation protocol for storing seeds of Coffea canephora, studying two methods of cryopreservation, slow and fast cooling. Seeds were subjected to drying in silica gel up to the water content of 0.25 g g-1. In the first experiment, dried seeds were subjected to treatments of slow cooling at speeds of -1 ºC min-1,-3 ºC min-1 and -5 ºC min-1 until the end temperatures of -40 ºC, -50 ºC and -60 ºC, by means of a bio freezer and subsequently immersed in liquid nitrogen. In the second experiment, the best result was selected of the first experiment and compared with the rapid cooling, in which dried seeds, with 0.25 g g-1 of water content, were immersed directly into liquid nitrogen. Physiological and biochemical alterations occurring in the seeds after cryopreservation were evaluated. Coffea canephora seeds respond better to cryopreservation by rapid cooling, when compared to slow cooling. Drying, one of the cryopreservation steps does not affect the viability of Coffea canephora Pierre seeds, when these seeds are dried to 0.25 g g-1 of water content. Catalase and esterase enzymes are good biochemical markers for cryopreserved coffee seeds and their activity is greater in larger seed physiological quality.

256 EFFECT OF SPERM SELECTION ON THE RATE OF IN VITRO FERTILIZATION IN ALPACA (VICUGNA PACOS)

2015 ◽  
Vol 27 (1) ◽  
pp. 217
Author(s):  
E. Mellisho ◽  
V. Rivas ◽  
J. Ruiz ◽  
G. Mamani
Keyword(s):  
Extended Period ◽  
Cumulus Cells ◽  
Sperm Selection ◽  
Cleavage Rate ◽  
Slow Cooling ◽  
Vitro Fertilization ◽  
Progressive Motility ◽  
Frozen Semen ◽  
Washing Method

In alpacas, improvement of reproductive efficiency of male camelids is limited by the small size of the testes, extended period of ejaculation, and low quality of semen. This study was designed to determine the effect of 2 sperm preparation treatments before IVF on the cleavage rate. The sperm was obtained by slicing the head of the epididymis of slaughtered male alpacas (n = 8), diluting in Tris-yolk-glycerol, and freezing with the slow-cooling method. Frozen semen straws per each male were thawed in a water bath at 37°C for 15 s and evaluated for percentage of progressive motility (32 ± 8.6%) and concentration (66.5 ± 24 × 106 sperm mL–1) post-thawing. Sperm selection by the swim-up method was performed by centrifugation at 1077 × g for 5 min with washing sperm medium eliminating the supernatant; sperm were settled in inclined tube with fertilization medium (without capacitating agent) for 60 min, after which 100 μL from the surface was recovered for use in IVF. The washing method consisted in repeated washing (twice) of sperm in washing sperm medium and fertilization medium by centrifugation at 1077 × g for 5 and 3 min, respectively, and recovery of 50 μL from the bottom of the tube for use in IVF. Sperm selected by swim-up or washing methods had similar characteristics of progressive motility (18 and 23%); however, the concentration was higher for the washing v. swim-up method (52 v. 14 × 106 sperm mL–1, respectively). Cumulus-oocyte complexes (COC) were recovered from 278 ovaries of alpacas killed at abattoirs and classified (Grade 1 and 2) for in vitro maturation (38.5°C at 5% CO2 in air for 27 h in 50 μL of 10 COC per drop). A total of 839 oocytes cultured for 27 h in maturation medium were partially stripped out of cumulus cells by gentle aspiration with a pipette. Sperm suspensions in Fert TALP medium (5 μL) from each treatment group were added to each fertilization drop with 10 oocytes per drop of 45 μL obtaining a final concentration of 10 × 106 sperm mL–1 and cultivated for 72 h until their evaluation. The data for the 13 repetitions of the rate of cleavage (2 to 8 cells) were converted to angular values (angle = arcsin √%) with the object of normalizing the distribution of the data; the analysis of variance was performed (complete randomised design with sub-sampling, P < 0.05) using SAS® version 8.0 for Windows. The rate of cleavage (cell division) did not show statistical differences (P = 0.67) for the swim-up method (37%; 155/421) v. washing method (35%; 147/418). The methods of sperm selection (swim-up and washing) did not affect the rate of IVF.

163 EVALUATION OF VIABILITY OF BULL SEMEN COLLECTED BY ELECTRO-EJACULATION USING COMMERCIAL SEMEN EXTENDER AND 2 CULTURE MEDIA AT CONTROLLED ROOM TEMPERATURE

2017 ◽  
Vol 29 (1) ◽  
pp. 190
Author(s):  
A. M. Raseona ◽  
O. A. Ajao ◽  
L. D. Nethengwe ◽  
L. R. Madzhie ◽  
T. L. Nedambale ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Reproductive Technologies ◽  
Sperm Viability ◽  
Progressive Motility ◽  
Bull Semen ◽  
Significant Difference ◽  
Semen Extender ◽  
Motility Rate

Preservation of semen is an important process to ensure that semen quality is sufficient for assisted reproductive technologies. The aim of this study was to evaluate the viability of bull semen collected by electro-ejaculation using commercial semen extender and 2 modified culture media stored at controlled RT (24°C) for 72 h. Two Nguni bulls were used for semen collection; after collection, the semen was evaluated macroscopically for volume, pH, and colour, and microscopically for sperm motility, viability, and morphology. Uncontaminated semen samples with progressive motility >70% and morphological defects <20% were pooled after collection before being aliquoted into 3 extenders, namely Triladyl, modified Ham’s F10, and TCM-199 culture media, at a dilution ratio of 1:4 and then stored at controlled RT (24°C). Sperm motility rate was analysed using the computer-aided sperm analyser after 0, 24, 48, and 72 h of storage. Sperm morphology and viability was performed after staining the sperm cells with spermac and nigrosine-eosin stain, respectively. The study was replicated 4 times and data were analysed using ANOVA. Triladyl had a higher sperm viability rate (41.3%) and total motility rate (96.3%) for 72 h (P < 0.01) compared with the 2 modified culture media, Ham’s F10 (26.5 and 86.8%) and TCM-199 (25.0 and 86.7%), respectively. However, Ham’s F10 had higher progressive motility rate (37.8%) as compared with the other extenders, TCM-199 (31.7%) and Triladyl (23.4). There was no significant difference (P > 0.05), in viability rate between Ham’s F10 (26.5%) and TCM-199 (25.0%). No significant difference (P > 0.05) in straight line velocity was observed for the three extenders. Furthermore, no significant difference was observed in total sperm abnormalities, except for reacted acrosomes and absent tails (P > 0.05), between the 2 Nguni bulls. Nguni semen can be preserved in Triladyl or modified Ham’s F10 and TCM-199 culture media stored at 24°C and stay viable for 72 h. Triladyl proved to be the best suitable extender of the 3 extenders, showing higher sperm viability and total motility rate as compared with Ham’s F10 and TCM-199 modified culture media.

scholarly journals Sodium bicarbonate and HEPES as buffer components for cooling the semen of pony stallions

2018 ◽  
Vol 39 (2) ◽  
pp. 631
Author(s):  
Janislene Mach Trentin ◽  
Luiz Augusto Machado Centeno ◽  
Taison De Souza Balestrin ◽  
Thainá Minela ◽  
Guilherme Machado Zanatta ◽  
...  
Keyword(s):  
Sodium Bicarbonate ◽  
Artificial Insemination ◽  
Membrane Integrity ◽  
Milk Powder ◽  
Skim Milk ◽  
Skim Milk Powder ◽  
Mitochondrial Activity ◽  
Sperm Viability ◽  
Progressive Motility ◽  
Before And After

The composition of semen diluents can modify its viability during cooling. The buffering effects of HEPES and sodium bicarbonate were evaluated considering the pH and sperm viability. The semen of seven adult Brazilian ponies was evaluated before and after cooling at 5oC for 24 h and 48 h. A non-buffered skim milk powder extender (C) and the same extender buffered with sodium bicarbonate (SB) and HEPES (H) were used. After dilution, semen (three ejaculates/pony) was centrifuged and the seminal plasma discarded. Sperm was then diluted with SB, H or C and its concentration adjusted to 50 x 106 sptz/mL. Progressive motility evaluated after dilution showed similar results with all extenders (71.42% (SB), 74.28% (H), and 74.52% (C)). Sperm motility was evaluated 24 h and 48 h after cooling for SB (44.76% and 25.23%), H (51.42% and 38.09%) and C (54.05% and 41.66%, respectively). Plasma membrane integrity was similar after exposure to the three extenders (62.71% (SB), 68.76% (H), and 69.23% (C)). Mitochondrial activity was higher in SB immediately after dilution (SB= 1.05nm, H= 0.81nm, C= 0.79nm), and after 24 h (0.83nm (SB), 0.73nm (H) and 0.64nm (C)). After 48 h, the mitochondrial activity decreased to 0.72nm (SB), 0.69nm (H), and 0.63nm (C) (P > 0.05). The pH, osmolarity and pH after 48 h of cooling of the diluted semen were higher in SB (8; 382; 7.9), intermediate in H (7.5; 362; 7.32) and lower in C (7.16; 350; 7.07). Lipid peroxidation and its induction were similar in all groups. Data were analyzed by analysis of variance (ANOVA), and Duncan’s test was used to evaluate the significant differences (P < 0.05). Sodium bicarbonate reduced the progressive motility and increased the semen pH. The extender C was considered more appropriate for immediate use in artificial insemination. The non-buffered and HEPES-buffered extenders were considered appropriate for the cooling of equine semen for 48 h at 5°C.

40Ar/39Ar and K–Ar age constraints on shear zone evolution, southern Taltson magmatic zone, northeastern Alberta

1995 ◽  
Vol 32 (3) ◽  
pp. 281-291 ◽  
Author(s):  
H. E. Plint ◽  
M. R. McDonough
Keyword(s):  
Cooling Rate ◽  
Shear Zone ◽  
High Grade ◽  
Slow Cooling ◽  
Rapid Cooling ◽  
Deformational History ◽  
The Mean ◽  
Exhumation Rates ◽  
Northeastern Alberta ◽  
Age Constraints

New 40Ar/39Ar analyses of hornblende, muscovite, biotite, and K-feldspar constrain the timing of deformation and cooling of the southern Taltson magmatic zone, which underwent lower granulite to upper amphibolite grade deformation, in part synchronous with voluminous 1.99–1.92 Ga magmatism. New data are combined with existing K–Ar dates into a regional cooling framework to provide thermotemporal constraints on the deformational history. 40Ar/39Ar hornblende ages of ca. 1900 Ma are interpreted to record relatively rapid cooling following ductile thrusting on the Andrew Lake shear zone, and younger anatectic magmatism. These data, with published K–Ar and U–Pb data, support relatively rapid cooling of the Taltson magmatic zone from monazite closure temperature of 725 °C at ca. 1930 Ma to 525 °C at ca. 1900 Ma. Cooling rate estimates are about 7 °C/Ma, which suggests moderate exhumation rates during the high-grade part of the deformational history. A muscovite 40Ar/39Ar plateau age of 1803 ± 11 Ma is consistent with the mean muscovite K–Ar age of 1792 Ma, indicating regional cooling through about 350 °C at ca. 1800 Ma. 40Ar/39Ar ages from magmatic biotite of 1856 and 1799 Ma also suggest slow cooling during greenschist grade deformation, which can be no older than ca. 1860 Ma. A K-feldspar 40Ar/39Ar age of 1681 Ma provides a lower limit for the time of greenschist grade deformation. Cooling rate estimates during amphibolite to greenschist grade deformation are 1.75–2.25 °C/Ma.

Stainless Steels Sintered Form the Mixture of Prealloyed Stainless Steel and Alloying Element Powders

2011 ◽  
Vol 672 ◽  
pp. 165-170 ◽  
Author(s):  
Zbigniew Brytan ◽  
Marco Actis Grande ◽  
Mario Rosso ◽  
Róbert Bidulský ◽  
L.A. Dobrzański
Keyword(s):  
Mechanical Properties ◽  
Impact Energy ◽  
Stainless Steels ◽  
Charpy Impact ◽  
Charpy Impact Test ◽  
Duplex Stainless Steels ◽  
Vacuum Furnace ◽  
Slow Cooling ◽  
Rapid Cooling ◽  
Plastic Properties

The aim of the presented paper is to describe the sintered duplex stainless steels manufactured in sinter-hardening process and their structural and mechanical properties. Duplex stainless steels were obtained through powder metallurgy starting from austenitic 316L or ferritic 410L prealloyed base powders by controlled addition of alloying elements powder. Prepared mixes were compacted at 700MPa and sintered in a vacuum furnace with argon backfilling at temperature of 1240°C for 1h. After sintering different cooling cycles were applied: rapid cooling (6°C/s) using nitrogen under pressure and slow cooling (0.1°C/s) with furnace in argon atmosphere. Produced sintered duplex stainless steels were studied by scanning and optical microscopy and EDS chemical analysis of microstructure components as well as X-ray analysis. Mechanical properties were studied through tensile and three-point bending tests and Charpy impact test. It was demonstrated that austenitic-ferritic microstructures with regular arrangement of both phases and absence of precipitates can be obtained with properly designed powder mix composition as well as sintering cycle with rapid cooling rate. Produced sintered duplex steels show good mechanical properties which depend on austenite/ferrite ratio in the microstructure and elements partitioning (Cr/Ni) between phases. The optimal mechanical properties were obtained for compositions based on ferritic 410L powder where the balanced distribution of α and γ is present and the tensile strength can reach value about 500MPa with 16% of elongation and impact energy about 120J. The precipitations of hard intermetallic σ-FeCr phase take place when sintering with slow cooling cycle what cause substantial decrease of plastic properties, including reduce of elongation to 7% and in particular decrease of impact energy to 68 J.

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