25 A preliminary study of the effects of breed and nanowater as extender diluent on ram semen characteristics post-thawing

2020 ◽  
Vol 32 (2) ◽  
pp. 138
Author(s):  
M. Murawski ◽  
J. Szymanowicz ◽  
T. Schwarz ◽  
P. Bartlewski
Keyword(s):  
Sperm Morphology ◽  
Egg Yolk ◽  
Plasma Generator ◽  
Deionized Water ◽  
Ex Situ ◽  
Survival Times ◽  
Becton Dickinson ◽  
Significant Difference ◽  
Semen Extender ◽  
Difference Test

This study examined the effect of genotype and extender diluent on the characteristics of frozen-thawed ram semen. Twenty ejaculates collected from 10 rams aged 4-12 years (2 Polish Lowland (PON), 4 synthetic line BCP (Berrichon du Cher×Charolais×PON/Polish Merino), and 4 SCP (Suffolk×Charolais×PON/Polish Merino) in mid-breeding season were divided into six equal portions each and frozen in semen extender prepared by mixing commercial Triladyl extender (MiniTub GmbH) with deionized water (Aqua Purificata, Prolab) or nanowater (NW; deionized water declusterized in a cold plasma generator for 15, 30, 45, 60, or 90 minutes (15’, 30’, 45’, 60’, or 90’, respectively); Nanotechnology Systems) and Gallus domesticus egg yolk (1:3:1 vol/vol/vol). All semen samples were evaluated for progressive motility (Sperm Class Analyzer) and sperm morphology (Nikon Eclipse 80i microscope, Nikon Corp.). In addition, the ex situ survival time at 37°C and extender content of ALT (alanine transferase), ALP (alkaline phosphatase), and aspartate aminotransferase were measured, and the numbers of apoptotic, necrotic, and live spermatozoa were determined post-thawing with flow cytometry (BD Accuri™ C6 Plus, Becton Dickinson). Data were analysed by two-way analysis of variance and least significant difference test (SigmaPlot, Systat Software Inc.). The PON exceeded (P<0.05) SCP rams in the occurrence of mid-piece and double tail defects using NW30’ and NW45’ extenders. The proportion of spermatozoa with proximal droplets was greater (P<0.05) in PON than in SCP or BCP rams with NW15’ and NW30’ extenders, respectively, but the percentage of sperm with detached heads was greater (P<0.05) in BCP and SCP compared with PON using NW15’ extender. Overall, spermatozoa in PON rams’ semen had survival times greater by 25-50min (P<0.05) compared with those for semen from BCP and SCP synthetic lines. The ALT levels were significantly elevated in SCP compared with PON and BCP rams only in NW90’ extender. The numbers of necrotic spermatozoa were greater (P<0.05) in SCP and BCP compared with PON rams, and the numbers of apoptotic spermatozoa were greater (P<0.05) in BCP than in SCP rams with NW45’, NW60’, and NW90’ extenders. Live spermatozoa were more prevalent (P<0.05) in PON than in BCP rams in NW45’ extender post-thawing. To summarise, semen from PON rams had higher survival times and fewer spermatozoa with detached heads but more mid-piece defects and sperm with double tails post-thawing compared with BCP and SCP breeds. The number of necrotic cells was greater in semen from BCP and SCP rams compared with PON. It can be concluded that breed-related differences in post-thaw sperm morphology were apparent in extenders containing NW15-60’, whereas the variations in cell necrosis/apoptosis were primarily seen with the use of NW45-90’ extenders.

39 Nanowater enhances cryoprotective effects of glycerol during ram semen freezing

2020 ◽  
Vol 32 (2) ◽  
pp. 145
Author(s):  
J. Szymanowicz ◽  
M. Murawski ◽  
T. Schwarz ◽  
P. Bartlewski
Keyword(s):  
Alkaline Phosphatase ◽  
Survival Time ◽  
Semen Quality ◽  
Sperm Concentration ◽  
Egg Yolk ◽  
Plasma Generator ◽  
Ex Situ ◽  
Live Cells ◽  
Water Control ◽  
Becton Dickinson

It was suggested that unique physicochemical properties of nanowater (NW, water declustered in the cold plasma generator), such as a low dielectric constant and high diffusivity, might ameliorate ram semen freezing by improving bioavailability of extender constituents. This study was undertaken to evaluate the effects of NW added to glycerol-containing semen extenders on the characteristics of frozen-thawed ram semen. A total of 18 ejaculates collected from 6 Olkuska breed rams during the breeding season were divided into six equal portions. The ejaculates were frozen in the modified fructose-skimmed milk-egg yolk Kareta extender containing 3 or 7% glycerol and diluted in deionized water (control extenders; Aqua Purificata, Prolab; C3% and C7%, respectively) or NW (declustered for 15min (NW15’) or 30min (NW30’); Nanotechnology Systems) to a final concentration of 800×106 spermatozoa/mL. They were placed in 0.25-mL plastic straws and frozen in liquid nitrogen. The two declusterization times were chosen on the basis of previous laboratory tests and fertility trials yielding the best results in terms of semen quality post-thawing and pregnancy rates after AI. All semen samples were evaluated for sperm concentration, progressive motility (Sperm Class Analyzer), and morphological defect rates (Nikon Eclipse 80i microscope, Nikon Corp.). In addition, the ex situ survival time at 37°C and extender content of alanine transferase, alkaline phosphatase, and aspartate aminotransferase were measured, along with the proportions of apoptotic, necrotic, and live spermatozoa determined post-thawing with flow cytometry (BD Accuri™ C6 Plus, Becton Dickinson) at 0 and 1h post-thawing. Data were analysed by one-way analysis of variance and Holm-Sidak method using SigmaPlot statistical software (Systat Software Inc.). The proportion of spermatozoa with mid-piece defects was significantly (P<0.05) reduced in NW15’-3% compared with C3% (1.3±0.3% vs. 4.0±06%; mean±s.e.m.). The proportion of necrotic spermatozoa 1h after thawing was greater (P<0.05) in C7% compared with NW30’-7% (20.7±0.4% vs. 17.6±1.0%), whereas the proportions of live cells detected immediately (0h) and 1h after thawing were greater (P<0.05) in NW30’-7% than in C30’ (0 h: 54.0±0.6% vs. 50.4±1.2%; 1 h: 54.4±1.2% vs. 59.2±0.4%, respectively). The mean survival time of spermatozoa was greater (P<0.05) in extenders dissolved in NW30’compared with their respective controls (NW30’-3% vs. C3%: 215±4min vs. 189±6 min; and NW30’-7% vs. C7%: 242±8min vs. 217±4min, respectively). Alkaline phosphatase concentrations in extenders prepared with NW30’ were lower (P<0.05) compared with the control groups (NW30’-3% vs. C3%: 3105±231IU vs. 4490±458 IU; and NW30’-7% vs. C7%: 2516±128IU vs. 3956±263IU, respectively). Our results indicate that NW significantly improves the effects of glycerol on ram sperm viability post-thawing, with the reduction in sperm necrosis/overall enhancement of sperm survivability being greater with 7% compared with 3% of glycerol in semen extender.

scholarly journals Effect of the Administration of PGF2α Analogue to Extended Boar Semen on Sperm Motility, Morphology and Kinematic Parameters

2017 ◽  
Vol 40 (2) ◽  
pp. 125-129
Author(s):  
Mihail Chervenkov ◽  
Teodora Ivanova ◽  
Paulina Taushanova ◽  
Rossen Stefanov ◽  
Boyko Georgiev
Keyword(s):  
Sperm Motility ◽  
Sperm Morphology ◽  
Prostaglandin F2α ◽  
Limited Data ◽  
Kinematic Parameters ◽  
Significant Difference ◽  
Myometrial Contractility ◽  
Boar Semen ◽  
Semen Extender ◽  
Motility Parameters

AbstractThe addition of prostaglandin F2α (PGF2α) to boar semen prior to insemination improves the conception and farrowing rates in sows. It is accepted that this is due to increased myometrial contractility, which improves the spermatozoa movement. However, there are limited data about the effect of the exogenous PGF2α analogs on sperm motility parameters and morphology. The aim of the current study was to define if there are changes in motility, morphology and kinematic parameters of spermatozoa on 1st and 24th hour after addition of PGF2α analogue to extended boar semen. A total of 18 ejaculates, obtained from clinically healthy boars were diluted 1:3 in semen extender, and each of them was separate into four aliquots, 50 ml each. PGF2α was added to 3 of them in concentrations of 6, 12 and 25 μg/ml, and the fourth served as untreated control. The motility, kinematic parameters and morphology of spermatozoa were evaluated on 1st and 24th hours after addition of PGF2α. There was no significant difference in sperm morphology, total and progressive motility between the untreated and treated groups. There was however a significant decrease in the rapid velocity and some of the kinematic parameters (VCL, VSL and VAP) in the group treated with 25 μg/ml compared to the control at the 1st hour after PGF2α treatment, which (except for the rapid velocity) persisted to the 24th hour. The results indicate that addition of Oestrophan (Bioveta, CZ) to the extended boar semen did not improve the sperm motility, morphology and kinematic parameters of the spermatozoa.

105 COMPARISON BETWEEN TWO EXTENDERS FOR CRYOPRESERVATION OF BACTRIAN CAMEL SEMEN

2006 ◽  
Vol 18 (2) ◽  
pp. 161
Author(s):  
A. Niasari-Naslaji ◽  
S. Mosaferi ◽  
A. A. Gharahdaghi ◽  
A. Abarghani ◽  
A. Ghanbari ◽  
...  
Keyword(s):  
Citric Acid ◽  
Liquid Nitrogen ◽  
Egg Yolk ◽  
Deionized Water ◽  
Bactrian Camel ◽  
Equilibration Time ◽  
Persian Language ◽  
Significant Difference ◽  
Arcsine Transformation ◽  
Artificial Vagina

A Tris-based extender (SHOTOR diluent) has been developed for preserving Bactrian camel semen at 4�C (Niasari-Naslaji et al. 2005 Reprod. Fertil. Dev. 17, 198 (abstr.)). The present study investigated the possibility of utilizing the SHOTOR diluent for the cryopreservation of Bactrian camel semen. A modified bovine artificial vagina (Masaferi et al. 2005 Theriogeology 63, 92-101) was used to collect semen from three fertile bulls. The viscosity of the semen was reduced mechanically (Mosateri et al. 2005) and the homogenized semen was divided equally into two parts. Each part was sequentially diluted with either IMV buffers (Green buffer: first extender; White buffer: second extender; IMV, France) or SHOTOR diluents (without glycerol: first extender; with 12% glycerol: second extender). SHOTOR diluent consists of 2.6 g TIS, 1.35 g citric acid, 1.2 g glucose, and 0.9 g fructose in 100 mL of deionized water, with an osmolality of 330 mOsm/kg and pH of 6.9. All extenders had 20% egg yolk and antibiotics. The semen was diluted at the ratio of 1:1 with the first extender. The diluted semen was then cooled within 2 h to 4�C. At this temperature, the second extender was added at the same volume as the diluted semen in three steps with an equal volume, 10 min apart. After a 30-min equilibration time, beginning after addition of the last fraction of the second extender, the diluted semen was loaded into 0.5-mL straws at a concentration of 50 � 106 sperm per straw. The straws were maintained for 20 min at 4 cm above the liquid nitrogen surface, after which they were plunged into liquid nitrogen. The semen was thawed at 40�C water bath for 20 s. Progressive forward motility of spermatozoa was assessed at the time of dilution and immediately after thawing of the semen. The experiment was replicated four times. Data were analyzed using GLM procedure in SAS/STAT after arcsine transformation. At the time of dilution, there was no significant difference in progressive forward motility of spermatozoa between IMV buffers (51.8%) and SHOTOR diluent (61%; P > 0.05). However, after thawing, there was a significant decrease in progressive forward motility of spermatozoa in IMV buffers (4.2%) compared to SHOTOR diluent (29.9%, P < 0.05). In conclusion, in this experiment, SHOTOR diluent was more efficient for cryopreserving Bactrian camel semen than IMV extender. Shotor means camel in the Persian language.

scholarly journals Daya Hidup Spermatozoa Epididimis Sapi Persilangan yang Dipreservasi dengan Air Kelapa Muda pada Suhu 5oC (VIABILITY OF EPIDIDYMAL SPERMATOZOA CROSSBREED CATTLE PRESERVED WITH COCONUT WATER AT 5oC)

2018 ◽  
Vol 18 (4) ◽  
pp. 571
Author(s):  
Muhammad Rizal ◽  
Muhammad Riyadhi ◽  
Bambang Irawan ◽  
Anis Wahdi ◽  
Habibah Habibah ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Saline Solution ◽  
Egg Yolk ◽  
Coconut Water ◽  
Randomized Design ◽  
Significant Difference ◽  
Epididymal Spermatozoa ◽  
Completely Randomized Design ◽  
Difference Test

The objective of this research was to examine the effectiveness of coconut water with various egg yolk concentrations in maintaining the viability of epididymal spermatozoa of crossbreed cattle preserved at 5oC. Five testis with epididymides of crossbreed cattle were obtained from slaughterhouse. Epididymal spermatozoa was collected by the combination of slicing, flushing and tissues pressure methods of cauda epididymides with saline solution (0.9% NaCl). Collected-spermatozoa was equally divided in volume into four tubes and diluted with lactose extender containing 20% egg yolk (control), 90% coconut water + 10% egg yolk (CWEY10), 85% coconut water + 15% egg yolk (CWEY15), and 80% coconut water + 20% egg yolk (CWEY20), repectively. Diluted-spermatozoa was stored in refrigerator at 5oC. Quality of dilutedspermatozoa including percentages of motile spermatozoa (MS), live spermatozoa (LS), spermatozoa with intact plasma membrane (IPM) were evaluated every day during four days of storage. Data were analyzed by using completely randomized design with four treatments and five replicates. Means were compared with significant difference test at 0.05 significant level. Results of this study showed that mean of spermatozoa concentration, percentage of MS, percentage of LS, percentage of spermatozoa abnormal, and percentage of IPM of crossbreed cattle fresh epididymal spermatozoa were 1,414 million cell/ml, 72%, 85%, 9%, and 90%, respectively. At day-4 of the storage, percentages of MS, LS, and IPM of control (43, 52.2, 59.2%) and CWEY20 (42, 52, 59%) were significantly (P<0.05) higher than CWEY10 (33, 45.4, 52.8%) and CWEY15 (37, 50, 54.6%). In conclusion, lactose and CWEY20 extenders could be maintaining the quality of epidydimal spermatozoa of crossbreed cattle for three days preservation at 5oC and two days for CWEY10 and CWEY15.

scholarly journals Cryopreservation of spermatozoa of indigenous stinging catfish, Heteropneustes fossilis (Bloch) for ex-situ conservation

2020 ◽  
Vol 32 (1) ◽  
pp. 11-22
Author(s):  
MD. RAFIQUL ISLAM SARDER ◽  
MD. ABUL KALAM AZAD ◽  
K.M. SHAHRIAR NAZRUL ◽  
MOHAMMAD RASHED
Keyword(s):  
Ex Situ Conservation ◽  
Egg Yolk ◽  
Ringer Solution ◽  
The Other ◽  
Ex Situ ◽  
Nacl Solution ◽  
Heteropneustes Fossilis ◽  
Significant Difference ◽  
Cryopreserved Sperm ◽  
Cryopreservation Protocol

An experiment was conducted to develop cryopreservation protocol for spermatozoa of stinging catfish, Heteropneustes fossilis and to use the cryopreserved sperm in its breeding trials. The activation of sperm motility at various concentrations of NaCl solution was tested and complete activation and inhibition of sperm were obtained at 0.4% and 0.9% to 1% NaCl solution respectively. In toxicity test, sperms were incubated with DMSO, methanol and ethanol at 5, 10, and 15% concentrations where DMSO and methanol produced better motility at 5 and 10% concentration with Alsever’s solution and egg-yolk citrate at 5 and- 10 min incubation period. Three extenders- Alsever’s solution, egg-yolk citrate and Ginsburg Fish Ringer solution and three cryoprotectants- DMSO, methanol and ethanol were used for cryopreservation of sperm, and among the diluents, Alsever’s solution with 10% DMSO showed best performance producing 77.50±3.22% post-thaw motility. On the other hand, egg-yolk citrate and Ginsburg Fish Ringer solution along with 10% DMSO produced 63.75±2.39% and 62.50±3.22% post-thaw motility, respectively. Sperm preserved with Alsever’s solution plus 10% DMSO produced 52.5±3.34% and 38.0±2.39%fertilization and hatching, and those preserved with Alsever’s solution plus 10% methanol produced 46.9±3.11% and 32.7±2.70% fertilization and hatching respectively. The fry produced using cryopreserved and fresh sperm grew well and no significant difference (p>0.05) was found between two groups.

Clinicopathological Features, Prognostic Factors, Survival Trends, and Treatment of Malignant Ovarian Germ Cell Tumors: A SEER Database Analysis

2021 ◽  
Vol 44 (4) ◽  
pp. 145-152
Author(s):  
Hualei Guo ◽  
Hao Chen ◽  
Wenhui Wang ◽  
Lingna Chen
Keyword(s):  
Prognostic Factors ◽  
Germ Cell ◽  
Proportional Hazards ◽  
Germ Cell Tumors ◽  
Cox Proportional Hazards ◽  
Low Grade ◽  
Seer Database ◽  
Survival Times ◽  
Survival Curves ◽  
Significant Difference

Objective: The aim of this study was to investigate the clinicopathological prognostic factors of malignant ovarian germ cell tumors (MOGCT) and evaluate the survival trends of MOGCT by histotype. Methods: We extracted data on 1,963 MOGCT cases diagnosed between 2000 and 2014 from the Surveillance, Epidemiology, and End Results (SEER) database and the histological classification of MOGCT, including 5 categories: dysgerminoma, embryonal carcinoma (EC), yolk sac tumor, malignant teratoma, and mixed germ cell tumor. We examined overall and disease-specific survival of the 5 histological types. Kaplan-Meier and Cox proportional hazards regression models were used to estimate survival curves and prognostic factors. We also estimated survival curves of MOGCT according to different treatments. Results: There was a significant difference in prognosis among different histological classifications. Age, histotype, grade, SEER stage, and surgery were independent prognostic factors for survival of patients with MOGCT. For all histotypes, 1-, 3-, and 5-year survival rate estimates were >85%, except for EC, which had the worst outcomes at 1 year (55.6%), 3 years (44.4%), and 5 years (33.3%). In the distant SEER stage, both chemotherapy and surgery were associated with improved survival outcomes compared with surgery- and chemotherapy-only groups. Conclusions: Dysgerminoma patients had the most favorable outcomes, whereas EC patients had the worst survival. A young age, low grade, and surgery were all significant predictors for improved survival. In contrast, a distant SEER stage was a risk factor for poor survival. Chemotherapy combined with surgery contributed to longer survival times of patients with MOGCT in the distant SEER stage.

Osmotic and ionic effects of NaCl on germination, early seedling growth, and ion content of Atriplex halimus (Chenopodiaceae)

2002 ◽  
Vol 80 (3) ◽  
pp. 297-304 ◽  
Author(s):  
Mohammed Bajji ◽  
Jean-Marie Kinet ◽  
Stanley Lutts
Keyword(s):  
Calcium Concentration ◽  
Deionized Water ◽  
Ion Content ◽  
Short Term ◽  
Atriplex Halimus ◽  
Early Seedling Growth ◽  
Significant Difference ◽  
Early Seedling ◽  
Ionic Effects ◽  
Higher Doses

The effects of salt and osmotic stresses on the germination processes in seeds of the perennial halophyte species Atriplex halimus L. were compared using iso-osmotic concentrations of NaCl and mannitol. The lowest stress intensity delayed germination, while higher doses of NaCl and mannitol reduced final germination percentages. No significant difference occurred between the effects of these solutes on germination percentages or seedling dry weights. At an external osmotic potential of –0.7 MPa, however, the water content of mannitol-treated seedlings was reduced compared to that of seedlings that developed from NaCl-exposed seeds. The K, Mg, and Pi content decreased in seedlings that developed from mannitol-treated seeds while calcium concentration was strongly reduced in those arising from NaCl-treated seeds. Inhibited seeds were able to germinate at levels similar to those of the control after rinsing in deionized water and imbibition in control conditions. Seedlings produced from NaCl pre-treated seeds had a lower Ca and a higher Na content than control seedlings. The effect of salinity on the germination phase of development is mainly due to its osmotic component, and inhibition of germination is reversible. Both salt and osmotic stresses may have an impact on the mobilization of minerals from the seeds to the young seedling, but this effect does not have any consequence on growth processes analysed on a short-term basis.Key words: Atriplex halimus, halophyte, osmotic stress, recovery of seed germination, salinity.

scholarly journals In situ acoustic analysis of two twentieth-century heritage carillons

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Scott Allan Orr
Keyword(s):  
Twentieth Century ◽  
Acoustic Analysis ◽  
Mechanical Action ◽  
Ex Situ ◽  
Physical Acoustics ◽  
Acoustic Characteristics ◽  
Dynamic Level ◽  
Significant Difference ◽  
And Function

AbstractCarillons are a diverse and global form of musical and civic heritage: musical instruments comprised of a series of 23 or more bells, typically hung in a tower-like structure, tuned chromatically and played from a touch-sensitive manual and pedal console using an elaborate mechanical action. Carillon bells have a distinct series of musical overtones which should be accurately tuned to one another and with other bells they sound alongside. Although these overtones have been previously studied ex situ, this study assesses the acoustic characteristics of two early-twentieth century carillons in Toronto, Canada as a combination of structure, bells, and mechanical action. Thus, the instrument and its context are considered holistically, more accurately reflecting the musical sensitivity of a carillonist. Spectral analysis of audio samples of each bell at different musical dynamic levels enabled the analysis of the acoustic qualities of the bells and the mechanical action of the instruments. The tuning of bells in the instruments varied; most importantly, there was a significant difference between the audial intensity of the bell tones produced by the instruments, demonstrating the importance of the mechanical action as part of the ‘carillon system’. This was represented with a resistive power-law model, that represents the sensitivity of intensity to carillonist musical dynamic level. A discussion of the implications for artistic and heritage practice follows. Understanding the in situ physical acoustics of the carillon as a holistic instrument in its context informs performers, arrangers, and composers of how they can best embrace the instrument’s unique qualities to improve artistic pursuits and support the appreciation of carillons as heritage instruments and function as civic voices.

Surface culture of Steinernema sp. on two solid media and their pathogenicity against Galleria mellonella

2021 ◽  
Vol 95 ◽  
Author(s):  
C.I. Cortés-Martínez ◽  
A.I. Rodríguez-Hernández ◽  
M.R. López-Cuellar ◽  
N. Chavarría-Hernández
Keyword(s):  
Galleria Mellonella ◽  
Egg Yolk ◽  
Infective Juvenile ◽  
Surface Culture ◽  
Solid Media ◽  
Significant Difference ◽  
Bacterial Lawn ◽  
The Mean

Abstract The use of native entomopathogenic nematodes as biocontrol agents is a strategy to decrease the environmental impact of insecticides and achieve sustainable agriculture crops. In this study, the effect of the surface culture of Steinernema sp. JAP1 over two solid media at 23–27°C on infective juvenile (IJ) production and pathogenicity against Galleria mellonella larvae were investigated. First, the bacterial lawn on the surface of the media with egg yolk (P2) or chicken liver (Cl) were incubated in darkness at 30°C for 48 and 72 h, and 100 surface-sterilized IJs were added. Four harvests were conducted within the next 35 days and the mean accumulated production was superior on Cl (210 × 103 IJs) than on P2 (135 × 103 IJs), but the productivity decreased up to 10% when the incubation time of the bacterial lawn was of 72 h. The mean pathogenicity of in vitro- and in vivo-produced IJs were of 47–64% and 31%, respectively. It is worth noting that none of the two solid media had a statistically significant difference in IJ pathogenicity. Considering that the maximum multiplication factor of IJs on solid media was 2108 and that the pathogenicity against G. mellonella was outstanding, Steinernema sp. has a good potential for in vitro mass production.

scholarly journals In vitro quantitative evaluation of marginal microleakage in class II restorations confected with a glass ionomer cement and two composite resins

2001 ◽  
Vol 15 (4) ◽  
pp. 277-282 ◽  
Author(s):  
Maria Fernanda Borro BIJELLA ◽  
Maria Francisca Thereza Borro BIJELLA ◽  
Salete Moura Bonifácio da SILVA
Keyword(s):  
Glass Ionomer Cement ◽  
Class Ii ◽  
Deionized Water ◽  
Composite Resins ◽  
Glass Ionomer ◽  
Dye Penetration ◽  
Significant Difference ◽  
Distal Direction ◽  
Ionomer Cement

This study evaluated, in vitro, marginal microleakage in class II restorations confected with the glass ionomer cement Vitremer and with the composite resins Ariston pHc and P-60. The aims of the study were to assess the effect of thermocycling on those materials and to evaluate two methods utilized in the analysis of dye penetration. Sixty premolars divided in three groups were utilized; the teeth had proximal cavities whose cervical walls were located 1 mm below the cementoenamel junction. Half of the test specimens from each group underwent thermocycling; the other half remained in deionized water, at 37ºC. The specimens were immersed, for 24 hours, in a basic 0.5% fuchsin solution at 37ºC. For the analysis of microleakage, the specimens were sectioned in a mesio-distal direction, and the observation was carried out with the software Imagetools. The results were evaluated through the 2-way ANOVA and through the Tukey’s test. All groups presented marginal microleakage. The smallest values were obtained with Vitremer, followed by those obtained with the composite resins P-60 and Ariston pHc. There was no statistically significant difference caused by thermocycling, and the method of maximum infiltration was the best for detecting the extension of microleakage.

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