scholarly journals 255 PRONUCLEUS FORMATION IN RAT ZONA-FREE OOCYTES CO-CULTURED WITH HOMOLOGOUS POST-THAW SPERMATOZOA

2005 ◽  
Vol 17 (2) ◽  
pp. 277
Author(s):  
Y. Seita ◽  
Y. Okuda ◽  
A. Takizawa ◽  
N. Hirahara ◽  
M. Koichi ◽  
...  
Keyword(s):  
Cooling Rate ◽  
Sperm Motility ◽  
Phase Contrast ◽  
Room Temperature ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Student’S T ◽  
Percentage Data ◽  
Student’S T Test

The aim of the present study was to develop an IVF system with frozen/thawed rat spermatozoa. We examined the effect of cooling rate to 5.0°C on post-thaw sperm motility and membrane integrity, and also investigated the ability of post-thaw spermatozoa to form pronuclei. Under room temperature, epididymal spermatozoa of Wistar rats were collected in 2.0 mL of egg yolk medium containing 8.0% (w/v) lactose monohydrate and 0.7% (v/v) Equex Stem. Samples were loaded into 0.25-mL straws and cooled to 5.0°C in the chamber of a programmed freezer. For cryopreservation, the samples were exposed to liquid nitrogen (LN) vapor for 10 min and then plunged into LN. Straws were thawed in a 37.0°C water bath for 10 s. Ovulated oocytes were collected and the zona pellucidae were removed with 0.1% pronase. One-hundred μL of thawed samples were put into a droplet of 400 μL R1ECM and pre-incubated for 1 h. R1ECM solution was added to the droplet to adjust to 0.5–1.5 × 106 sperm mL−1. The zona-free oocytes were then transferred into the droplet and co-cultured for 10 h. Oocytes were observed for pronuclei formation by means of an inverted phase contrast microscope. In Experiment I, the influence of sperm cooling rate to 5.0°C on sperm motility and membrane integrity was evaluated. Portions of samples were cooled at 54.0°C/min, 0.9°C/min, 0.5°C/min, and 0.3°C/min. The remainders were then frozen. The non-cooled samples were designated as controls. In Experiment II, we examined whether post-thaw spermatozoa have the ability to form pronuclei in vitro or not. All percentage data were arc-sine transformed and then analyzed by the Student's t-test. In Experiment I, the membrane integrity between the spermatozoa cooled at 0.5°C/min and the non-cooled spermatozoa was not different (38.1% vs. 37.2%; P > 0.05), but the integrity of these was higher than in spermatozoa cooled directly at 54.0°C/min (38.1% vs. 25.3%; P < 0.05). After culture for 1 h, the motility of spermatozoa cooled at 0.5°C/min was higher than that of those cooled at 54.0°C/min (61.3% vs. 53.3%; P < 0.05). At 2 h post-thaw the motility of spermatozoa cooled at 0.5°C/min was higher than that of spermatozoa cooled at 54.0°C/min and at 0.9°C/min (11.0% vs. 4.5%, 4.9%; P < 0.05). The membrane integrity of post-thaw spermatozoa cooled at 0.5°C/min was also higher compared to that of spermatozoa cooled at 54.0°C/min (22.5% vs. 8.4%; P < 0.01). In Experiment II, 28 (26.2%) of 107 oocytes had pronuclei when the post-thaw spermatozoa cooled at 0.5°C/min were used. The results indicated that the frozen/thawed spermatozoa cooled to 5.0°C at 0.5°C/min showed higher sperm motility and membrane integrity, and that spermatozoa can form pronuclei in homologous zona-free oocytes in vitro. Although in the rat sperm damage occurred during cooling to 5.0°C, and sperm motility and membrane integrity were also decreased by the cold shock, it is possible to decrease the damage by cooling slowly to 5.0°C at 0.5°C/min.

scholarly journals Effect of split ejaculation and seminal extenders on longevity of donkey semen preserved at 5° C

2000 ◽  
Vol 52 (4) ◽  
pp. 372-378 ◽  
Author(s):  
S.L.V. Mello ◽  
M. Henry ◽  
M.C. Souza ◽  
S.M.P. Oliveira
Keyword(s):  
Phase Contrast ◽  
Room Temperature ◽  
Sperm Morphology ◽  
Sperm Concentration ◽  
Egg Yolk ◽  
The Rich ◽  
Student’S T ◽  
The One ◽  
Artificial Vagina ◽  
Student’S T Test

The aim of this study was to evaluate the longevity of donkey sperm comparing the rich seminal fraction and the whole semen in two extenders, Kenney and modified Baken extenders. Semen of five donkeys were collected through an open-end artificial vagina once a week for five consecutive weeks. The two first jets (rich fraction) of semen were collected separately from the rest of the ejaculate. Whole semen samples were obtained mixing proportionally part of the rich with part of the poor seminal fractions. Seminal samples were immediately diluted 1:1 in each extender and maintained at room temperature during sperm concentration analysis. Samples were further diluted to rich 50×10(6) sperm per ml, cooled in a refrigerator at the initial rate of -0.6° C/min and preserved at 5° C. Total motility (TM), progressive motility (PM) and sperm vigor (V) were examined after final dilution and cooling, and every 24 hours up to the decrease of total motility under 10%. Sperm morphology was evaluated using a phase contrast microscope directly after dilution, on days 3, 6 and 9 post collection. It was used a 2×2 factorial design in a randomised bloc experiment, and means were compared by Student’s t test. Longevity did not vary between the rich seminal fraction and the whole semen for both extenders used. TM, PM, V and sperm morphology were better preserved in the extender with egg yolk (modified Baken extender) than in the one with skimmed milk (Kenney) in both seminal fractions.

77 ADDITION OF CHOLESTEROL IN FRESH GOAT SPERM IMPROVES CRYOSURVIVAL RATES

2013 ◽  
Vol 25 (1) ◽  
pp. 186
Author(s):  
B. G. Silva ◽  
E. A. Moraes ◽  
C. S. Oliveira ◽  
W. D. Ferrari Junior ◽  
W. C. G. Matos ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Phase Contrast ◽  
Room Temperature ◽  
Petri Dish ◽  
Egg Yolk ◽  
Irreversible Damage ◽  
Hypoosmotic Swelling Test ◽  
Contrast Microscopy ◽  
Working Solution

Cryopreservation causes irreversible damage to goat sperm membranes, measured by a loss of motile and functional normal cells, compared with fresh sperm. The objective of this study was to determine if the addition of cholesterol-loaded cyclodextrin (CLC) to goat semen improved sperm cryosurvival. The CLC was prepared as described by Purdy and Graham (2004 Cryobiology 48, 36–45) with some modifications: 200 mg of cholesterol were dissolved in 1 mL of chloroform and 1 g of methyl-beta-cyclodextrin was dissolved in 2 mL of methanol. A 0.45-mL aliquot of the cholesterol solution was added to the cyclodextrin solution, after which the mixture was poured into a glass Petri dish and the solvents allowed to evaporate on a warm plate for 24 h. The resulting crystals were removed from the dish and stored at 22°C. A working solution of the CLC was prepared by adding 50 mg of CLC to 1 mL TALP at 37°C. Thirty ejaculates from 5 bucks were collected, diluted 1 : 1 in Tris diluent, divided into 7 equal aliquots, and centrifuged at 800g for 10 min. The sperm pellets were resuspended in Tris diluent, to which 0, 0.75, 1.5, 3.0, 4.5, 6.0, or 7.5 mg of CLC/120 million sperm were added. All treatments were incubated for 15 min at room temperature and then cooled to 4°C over 2 h. The samples were then diluted with Tris-egg-yolk diluent containing 2% glycerol, and the sperm were packaged into 0.5-mL straws, frozen in static liquid-nitrogen vapour for 20 min, and plunged into liquid nitrogen. Straws were thawed in 37°C water for 30 s, extended in Tris, and analyzed using optic microscopy. To test thermal resistance, after thawing, 0.5 mL of semen from each treatment were placed in 1.5-mL Eppendorf tubes in a water bath at 37°C for 3 h. At 0, 60, 120, and 180 min, subsamples were evaluated for sperm progressive motility. A hyposmotic test was also conducted by adding 10 µL of sperm to 2 mL of each solution and incubating them for 1 h/37°C. Sequentially, 20 µL of sperm was diluted in hypoosmotic solution (150 mOsm), and the samples were evaluated using phase-contrast microscopy. A total of 100 spermatozoa were counted in at least 5 different fields, and sperm tails were classified as either noncoiled or coiled. Data were analyzed using ANOVA, and treatment means were separated, using the SNK test at 5% probability. The sperm motility (50.4, 33.8, and 22.5%) was significantly higher for sperm treated with 0.75 mg of cholesterol after 0, 60, and 120 min of incubation after thawing, when compared with other treatments. No treatment differences in the hypoosmotic swelling test were observed. The addition of 0.75 mg of cholesterol to fresh goat semen improved sperm motility after cryopreservation for up to 3 h. Supported by FACEPE and CAPES.

scholarly journals Effects of apple and orange juices on quality of refrigerated goat semen

2018 ◽  
Vol 63 (1) ◽  
pp. 53-65
Author(s):  
Ezekiel Adekunle ◽  
James Daramola ◽  
Olusiji Sowande ◽  
John Abiona ◽  
Monsuru Abioja
Keyword(s):  
Sperm Motility ◽  
Orange Juice ◽  
Apple Juice ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Fruit Juices ◽  
Sperm Viability ◽  
In Vitro Storage

This study investigated the effects of apple and orange juices on quality of refrigerated spermatozoa of goat bucks. Semen samples from WAD goat bucks were diluted with Tris-egg yolk extenders each supplemented with apple and orange juices at 0, 2.5, 5, 7.5 and 10/100 ml of diluents. The diluted semen samples were assessed for sperm viability and malondialdehyde (MDA) concentration after in vitro storage for 240 hours at 5oC. The ability to maintain sperm motility was higher in the extenders with 7.5% orange juice followed by 10% apple juice compared to other treatments (P<0.05). The extenders supplemented with 2.5%, 5% and 7.5% apple juice, and 5% orange juice had higher intact acrosome compared to other treatments and the control (P<0.05). The 10% orange juice had higher percentage membrane integrity compared to other treatments. Consistent and reduced (P<0.05) MDA levels were observed in the extenders supplemented with fruit juices and lower MDA was observed in the extenders supplemented with 10% apple juice compared to other treatments and the control (P<0.05). The findings reveal that additions of the fruit juices to semen extenders to maintain the viability of refrigerated spermatozoa were best at concentrations of 10 ml/100 ml of apple juice and 7.5 ml/100 ml of orange juice.

Freezability of Andalusian donkey (Equus asinus) spermatozoa: effect of extenders and permeating cryoprotectants

2016 ◽  
Vol 28 (12) ◽  
pp. 1990 ◽  
Author(s):  
D. Acha ◽  
M. Hidalgo ◽  
I. Ortiz ◽  
M. J. Gálvez ◽  
J. J. Carrasco ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Equus Asinus ◽  
Sperm Membrane ◽  
Freeze Test ◽  
Pooled Samples

The aim of this study was to compare the effect of two semen extenders and four permeating cryoprotectants on post-thaw sperm quality of Andalusian donkeys. First, 32 ejaculates were pooled, split and frozen in either Gent B or INRA 96 with egg yolk and glycerol. Second, 12 pooled semen samples were simultaneously frozen in Gent B (glycerol) or Gent A containing ethylene glycol (EG; 1 or 1.5%) or dimethyl sulfoxide (DMSO; 1.5 or 2%). Finally, nine pooled samples were simultaneously cryopreserved in Gent A containing 1% EG (as control), dimethylformamide (DMFA; 1 or 2.5%) or a combination of 1% EG and 1.5% DMFA. Gent B yielded a higher (P < 0.01) post-thaw sperm motility than modified INRA96. EG 1% increased the sperm membrane integrity (P < 0.001), whereas DMSO affected sperm motility and membrane integrity (P < 0.001). DMFA 2.5% yielded higher (P < 0.001) values for sperm motility and membrane integrity. We concluded that Gent B improves in vitro post-thaw sperm quality of donkey spermatozoa, but the replacement of glycerol with 1% EG or 2.5% DMFA increased sperm protection against cryodamage. The use of DMSO for freezing donkey semen was unsuccessful and a toxic effect is suspected. These extenders should be included in the pre-freeze test for each donkey.

297 BIRTH OF LIVE RATS THROUGH IN VITRO FERTILIZATION USING CRYOPRESERVED SPERMATOZOA: SPERM FERTILITY IMPROVED BY FREEZING AT - 150°C

2006 ◽  
Vol 18 (2) ◽  
pp. 256
Author(s):  
Y. Seita ◽  
Y. Okuda ◽  
A. Takizawa ◽  
S. Hisamatu ◽  
T. Inomata ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
In Vitro Fertilization ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Plasma Membrane Integrity ◽  
Freezing Temperatures

We previously reported that damages to spermatozoa by cold shock can be avoided by cooling slowly at 0.5�C/min to 5.0�C (Seita et al. 2005 Reprod. Fertil. Dev. 17, 277-278). The objective of the present study was to develop an in vitro fertilization (IVF) system with frozen-thawed rat spermatozoa for more efficient reproduction of live offspring. We examined the effect of freezing temperatures (cooling 5.0�C to pre-plunging) on post-thaw sperm motility, plasma membrane integrity, and fertility in vitro. Epididymal spermatozoa of Wistar rats were collected in 2.0 mL of freezing medium containing 23% (v/v) egg yolk, 8.0% (w/v) lactose monohydrate, and 0.7% (v/v) Equex STM (Nova Animal Sales, Inc., Scituate, MA, USA) at room temperature. Samples were loaded into 0.25-mL straws and cooled to 5.0�C at 0.5�C/min in a programmable freezer. Next, the samples were exposed to liquid nitrogen (LN) vapor at various freezing temperatures (-120�C, -150�C or -180�C) above the LN level for 15 min and then plunged into LN. Straws were thawed in a 37�C water bath for 10 min. The thawed samples were diluted to 0.5-1.5 � 106 sperm/mL in a droplet of 200 �L of R1ECM and then pre-incubated for 5 h. Ovulated oocytes were introduced into the droplet and co-cultured for 10 h. The oocytes were denuded, fixed, and/or examined for two pronuclei (2PN) formation microscopically. The denuded oocytes, which were fertilized with spermatozoa frozen at -150�C and were microscopically confirmed to have 2PN formation, were transferred to pseudo-pregnant recipient females. IVF was also performed by the same method using fresh spermatozoa as the control. Differences in the sperm motility and plasma membrane integrity were analyzed by ANOVA, and the IVF data were analyzed by chi-square test. At 2 h after thawing the motility of spermatozoa frozen at -150�C was significantly higher than that of spermatozoa frozen at -180�C (19.8% and 11.1%; P < 0.05), although the sperm plasma membrane integrity was not significantly different among different freezing temperatures, -120�C, -150�C, and -180�C (18.2%, 23.5%, and 17.9%; P > 0.05). The percentage of oocytes with 2PN was not significantly different between the -150�C frozen and the control (fresh spermatozoa) groups [59% (131/221) and 62% (155/251); P > 0.05], although that of frozen spermatozoa at -120�C and -180�C [20% (38/188) and 23% (35/153)] were significantly lower than that of frozen spermatozoa at -150�C (P < 0.05). A total of 168 putative fertilized zygotes with 2PN were transferred to eleven recipients, and 87 live young were born. In conclusion, our results indicated that post-thaw motility of cryopreserved rat spermatozoa was improved by using a suitable cooling protocol, and the IVF system used in the present study would effectively produce offspring from the cryopreserved epididymal rat spermatozoa. To our knowledge, this procedure is the first successful production of live offspring from cryopreserved rat spermatozoa through in vitro fertilization.

scholarly journals Ram and Goat Semen Immunosexed and Diluted in Powdered Coconut Water-Based Preservation Medium (ACP101/102c).

2021 ◽  
Vol 49 ◽  
Author(s):  
Bruna Farias Brito ◽  
Bárbara Mara Bandeira Santos ◽  
Leonardo Alves Rodrigues Cabral ◽  
Luiz Carlos Pinheiro Maia ◽  
Natanael Aguiar Braga Negreiros ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Specific Protein ◽  
Coconut Water ◽  
In Vivo Studies

Background: Sperm sexing aims to separate sperm populations in carriers of the “X” or “Y” chromosome. Currently, flow cytometry is a technique that allows greater accuracy; however, it causes structural changes in sperm, reduces viability, and has a high cost. As a result, other methods have been researched, including immunosexing, which uses monoclonal antibodies to detect sex-specific surface antigens. Thus, the objective of this study was to evaluate the immunosexing technique using a monoclonal antibody against sex-specific protein (HY) in the conservation of ram and goat semen in ACP101/102c.Materials, Methods & Results: Ejaculates from 5 rams and 5 goats were collected with the aid of an artificial vagina; they were evaluated and submitted to the immunosexing protocol, according to the manufacturer's recommendations, using the Monoclonal Antibody Kit specific for mammalian sperm with “Y” chromosomes (HY; HY Biotechnology, Rio de Janeiro, RJ, Brazil). After sexing, the supernatant was resuspended in the cryopreservation diluent: ACP ram (ACP101/102c + 20% egg yolk + 7% glycerol) and ACP goat (ACP101/102c + 2.5% egg yolk + 7% glycerol), packaged in 0.25 mL straws, refrigerated at 4°C, stabilized for 30 min, frozen in liquid nitrogen vapor (-60°C) for 15 min, immersed in liquid nitrogen, and stored in cryogenic cylinders. The samples were evaluated in natura (T1), after immunosexing (T2) and after thawing (T3) for sperm motility subjectively using conventional microscopy (40x). Plasma membrane integrity (IMP) and sperm cell morphology were evaluated by the smear staining technique using eosin-nigrosine dye, and the percentages of healthy and morphologically defect spermatozoa were determined. In the evaluation of ram semen regarding sperm motility and IMP, no statistically significant differences were observed between treatments after sexing in the evaluation of absolute data (P > 0.05), with the difference being observed only between T1 and T2, and T3 (P < 0.05). Regarding the relative percentage and sperm morphology, no statistically significant differences were observed (P > 0.05). Regarding the evaluation of goat semen samples, the motility parameters were consistent with the technique submitted; however, the IMP data did not appear as expected, requiring further evaluation for a better assessment of the technique for this species.Discussion: The data obtained from ram semen submitted to the immunosexing protocol, regarding the absolute evaluation of motility and IMP, demonstrated that the non-sexed semen (T1) was superior to the sexed treatments (T2 and T3); however, it is noteworthy that freezing started with approximately 50% of the cells, since the immunosexing technique results in a loss of viability of approximately 50% of the sperm, which corresponds to the ratio of sperm carrying the X chromosome. In addition, when the data in this study were transformed into relative values, no statistical differences were observed, indicating that the immunosexing protocol, as well as the freezing protocol, did not significantly affect the quality of ram sperm cells. In relation to the immunosexing of goat semen, future studies should be conducted in vitro to define a more appropriate protocol for the species and, in addition, in vivo studies should be performed to prove the quality of the technique. It was concluded that the immunosexing process using a monoclonal antibody against sex-specific protein (HY) associated with the use of powdered coconut water diluent (ACP101/102c) in the cryopreservation of semen proved to be efficient in the in vitro evaluation of ovine species.

Kriopreservasi Semen Kambing Boer dengan Konsentrasi Pengencer Nira Aren dan Gliserol Berbeda

2019 ◽  
Vol 6 (1) ◽  
pp. 1
Author(s):  
Muhammad Riyadhi ◽  
Anis Wahdi ◽  
Muhammad Rizal
Keyword(s):  
Sperm Motility ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Boer Goat ◽  
Palm Juice ◽  
Sperm Membrane ◽  
Live Sperm

ABSTRAK                                                                        Penelitian bertujuan untuk mengetahui efektivitas nira aren sebagai pengencer alternatif dalam proses pembekuan (kriopreservasi) semen kambing boer.Kriopreservasi semen kambing boer menggunakan pengencer tris-gliserol-kuning telur (P1 73-7-20%), nira aren-gliseol-kuning telur(masing-masing P2 74-6-20%, P3 73-7-20%, dan P4 72-8-20%) dan andromed (P5 tanpa mengandung kuning telur dan gliserol). Parameter evaluasi meliputi motilitas, viabilitas, dan membrane plasma utuh setelah pengenceran, ekuilibrasi dan thawing.  Evaluasi motilitas pasca thawing menunjukkan P5 52% berbeda nyata (P<0.05) dengan P1 42%, selanjutnya P5 dan P1 berbeda sangat nyata (P<0.05) dengan P2 8%, P3 6% dan P4 12%.  Viabilitas pasca thawing menunjukkan P5 65,4% tidak berbeda nyata (P>0,05) dengan P1 61,8%, akan tetapi P5 dan P1 berbeda sangat nyata (P<0.05) dengan P2 26,2%, P3 29,8%, dan P4 34%.  Membran plasma utuh (MPU) pasca thawing menunjukkan P5 66,2% tidak berbeda nyata (P>0,05) dengan P1 65,4%, akan tetapi keduanya berbeda sangat nyata (P<0.05) dengan P2 39%, P3 38%, dan P4 36,2%.  Disimpulkan kriopreservasi semen kambing boer dengan pengencer nira aren dan gliserol pada konsentrasi berbeda belum dapat dipergunakan sebagai sumber bibit berdasarkan standar nasional Indonesia.Kata Kunci : Kambing boer, semen, nira arenABSTRACTThe experiment was conducted to determine the effect of sugar palm juice as alternative extender for cryopreservation process of boer semen.Tris-glycerol-egg yolk (P1 73-7-20%), Sugar palm juice-glyserol-egg yolk (P2 74-6-20%, P373-7-20%, dan P4 72-8-20%), and andromed (P5) used as a extender  in the cryopreservation process of boer semen.  Sperm motility (%), live sperm (%) and sperm membrane integrity (%) were recorded after diluted, equilibration and freeze-thawing.  Result of post thawing motility showed that P5 52% was significantly different (P <0.05) with P1 42%, then P5 and P1 were significantly different (P <0.05) with P2 8%, P3 6% and P4 12%. Viability after thawing showed P5 65.4% was not significantly different (P> 0.05) with P1 61.8%, but P5 and P1 significantly different (P <0.05) with P2 26.2%, P3 29.8 %, and P4 34%. Spermmembrane integrity post-thawing showed P5 66.2% was not significantly different (P> 0.05) with P1 65.4%, but both were very significantly different (P <0.05) with P2 39%, P3 38% and P4 36.2%. Conclusions, sugar palm juice-glycerol-egg yolk with differentconcentrationsineffectively as an alternative extenderin cryopreservation of boer semen.Keywords: boer goat, semen, sugar palm juice

scholarly journals 209FUNCTIONAL ASSESSMENT OF WHITE RHINOCEROS CERATHOTERIUM SIMUM EPIDIDYMAL SPERMATOZOA BEFORE AND AFTER CRYOPRESERVATION

2004 ◽  
Vol 16 (2) ◽  
pp. 226 ◽  
Author(s):  
F. Martinez-Pastor ◽  
F. Olivier ◽  
T. Spies ◽  
L. Anel ◽  
P. Bartels
Keyword(s):  
Plasma Membrane ◽  
Assisted Reproduction ◽  
Phase Contrast ◽  
Egg Yolk ◽  
Phase Contrast Microscopy ◽  
White Rhinoceros ◽  
Contrast Microscopy ◽  
Before And After ◽  
Epididymal Spermatozoa

Biological Resource Banks represent a potentially valuable tool for species conservation. It is, however, necessary to understand the species-specific cryopreservation process and its consequences for spermatozoa to aid in the development of assisted reproduction as a future conservation tool. The aim of this study was to assess the in vitro functionality of white rhinoceros Cerathoterium simum epididymal spermatozoa both before and after cryopreservation. Testes from a harvested white rhino bull were removed and transported at 5°C to the laboratory within 4h. The cauda epididymis was dissected out and flushed with 2mL of Tris-citrate egg yolk extender (fraction A, Biladyl, Minitüb, Germany). A 0.1mL aliquot was removed for analysis and the balance (9mL; 2mL fraction A+7mL sperm sample) mixed with an additional 27.2mL of Tris-citrate egg yolk with glycerol (fraction B, Bidadyl). The extended sample was allowed to cool to 4°C over a 6-h period before an additional 29.2mL of cooled fraction B were added (final sperm concentration=150×106mL−1). Sperm samples were loaded into 0.25-mL straws and frozen over LN2 vapor (4cm for 20min) for later assessment. Sperm straws were thawed by placing the straws in water at 37°C for 30s. Pre-freeze and post-thaw evaluations were carried out in the same manner. Media used included: HEPES for washing (20mM HEPES, 355mM sucrose, 10mM glucose, 2.5mM KOH) and HEPES saline (197mM NaCl, instead of sucrose). An aliquot was diluted with HEPES (washing) and centrifuged for 5min at 600×g; the pellet was resuspended in HEPES saline. Sperm motility (total motility %, TM;; and progressive motility %, PM) was assessed using phase contrast microscopy (×200; 37°C). Sperm plasma membrane status was assessed using the fluorescent dye, propidium iodide (50ngmL−1 in HEPES saline;; 10min, RT). Percentage of cells with plasma membranes intact (unstained;; PMI) was recorded. Mitochondrial status was assessed with the fluorescent dye, JC-1 (7.5μM in HEPES saline;; 30min, 37°C). The % of cells with an orange-stained midpiece was recorded (active mitochondria;; MIT). Resilience to hypoosmotic shock (HOS test) was assessed by diluting a sample in 100mOsm/kg HEPES saline (1:20; 15min, RT). An aliquot was stained with PI to assess plasma membrane status (HOSPMI), and the rest was fixed with formaldehyde, and % coiled tails (positive endosmosis;; HOST) was estimated using phase contrast microscopy (×400). Evaluations of PMI, MIT and HOSPMI were performed using fluorescence microscopy (×400, 450–490nm excitation filter). The results indicated that quality was good pre-freezing (TM: 60%; PMI: 86%; MIT: 100%), except for a PM value of 15%. After thawing, although there was a drop in TM (30%), there was no decrease in PM (20%). Our in vitro functional assessment indicated a loss of quality between the pre-freeze and post-thaw evaluations, but PMI and MIT maintained their pre-thaw levels (60% and 72%, respectively). The HOS test, which indicates plasma membrane integrity, decreased from the pre-freeze level (91%) to a post-thaw value of 70%. HOSTPMI was 72% pre-freeze, and decreased to 54% post-thaw. In conclusion, epididymal spermatozoa from the white rhino may retain its functionality after cryopreservation in a commerically available cryo-extender (Bidadyl). The use of assisted reproduction techniques could someday play a role in the management and conservation of the white rhinoceros and related species.

scholarly journals Cooling of equine semen at 16°C for 36 hours with addition of different glutathione concentrations

2015 ◽  
Vol 36 (6) ◽  
pp. 3699
Author(s):  
Rodrigo Arruda de Oliveira ◽  
Marco Antônio De Oliveira Viu ◽  
Maria Lúcia Gambarini
Keyword(s):  
Lipid Peroxidation ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Membrane Lipid ◽  
Sperm Cells ◽  
Sperm Viability ◽  
Membrane Lipid Peroxidation ◽  
Acrosomal Membrane ◽  
In Vitro Effects

Handling equine semen during the refrigeration process reduces sperm viability, and consequently causes membrane lipid peroxidation, among other challenges. The present study aimed to evaluate the in vitro effects of glutathione (control, 1. 0, 1. 5, and 2. 5 mM) on equine semen in a refrigeration protocol of 16ºC for 36 hours. The following variables were evaluated after 0, 12, 24, and 36 hours refrigeration: total sperm motility, vigor, viability, and plasma and acrosomal membrane integrity. Motility was higher with 2. 5mM of glutathione (57. 8 ± 7. 3) after 12 hours of refrigeration compared to the control (53. 2 ± 8. 3) (P < 0. 05). After 36 hours of refrigeration, motility was higher with 1. 5 mM (43. 4 ± 12. 7) and 2. 5mM glutathione (45. 5 ± 6. 2), than it was with 1mM glutathione (38. 2 ± 9) and the control (35. 5 ± 18. 4) (P < 0. 05), respectively. Vigor was highest with 1. 5mM glutathione (3. 7 ± 0. 3) after 36 hours compared to the control (3. 2 ± 1. 1), (P < 0. 05). Viability differed between control and 1mM treatments (79. 5 ± 1. 8) only after 24 hours (75. 5 ± 9. 7) (P < 0. 05). Throughout the investigation, no significant differences were noted in plasma and acrosomal membrane integrity (P > 0. 05). The 1. 5 and 2. 5mM glutathione levels were more efficient in protecting sperm cells and yielded higher total motility values after 36 hours of refrigeration.

scholarly journals Inhibition of Demineralization at Restoration Margins of Z100 and Tetric EvoCeram Bulk Fill in Dentin and Enamel

2019 ◽  
Vol 6 (2) ◽  
pp. 36
Author(s):  
Cristina Leon-Pineda ◽  
Kevin Donly
Keyword(s):  
Light Microscopy ◽  
Polarized Light ◽  
In Vitro Study ◽  
The Other ◽  
Solution Ph ◽  
Class V ◽  
The Mean ◽  
Student’S T ◽  
Student’S T Test

Recurrent caries is still considered the main reason restorations need to be replaced. There are different materials available now that promise to reduce the possibility of recurrent caries by releasing fluoride and inhibiting restoration marginal caries. The purpose of this in vitro study was to evaluate the demineralization inhibition potential of a non-fluoride-releasing resin (Z100TM 3M, St. Paul, MN, USA) and a glass containing resin-based composite (Tetric EvoCeram Bulk Fill, Ivoclar/Vivadent AG, Schaan, Liechtenstein), which contains fluoride. Class V preparations were placed on 22 premolars; the gingival margin was below the cementoenamel junction and the occlusal margin was placed above the cemento-enamel junction. Ten teeth were randomly selected to be restored with Z100 while the other 10 were restored with Tetric EvoCeram Bulk Fill. Both groups were restored following manufacturer’s instructions. All teeth had an acid resistant varnish placed within one millimeter of the preparation margins. Both groups were placed in artificial caries challenge solution (pH 4.4). At the end of the 4 days; 100 µm buccolingual sections were obtained for each tooth; these were photographed under polarized light microscopy and the demineralized areas adjacent to the restorations were measured and quantified. The mean (±S.D.) area (µm2) of demineralization from the occlusal margin (enamel) and dentin margin were: Z100 2781.889 ± 1045.213; 3960.455 ± 705.964 and for Tetric EvoCeram Bulk Fill 1541.545 ± 1167.027; 3027.600 ± 512.078. Student’s t-test indicated that there was significantly less enamel and dentin demineralization adjacent to Tetric EvoCeram Bulk Fill compared to Z100; there was significantly less demineralization in enamel compared to dentin in both Tetric EvoCeral Bulk Fill and Z100. Tetric EvoCeram Bulk Fill performed better inhibiting demineralization at restoration margins when compared to Z100 and provided better demineralization inhibition in enamel than cementum/dentin.

Sign in / Sign up

Export Citation Format

Share Document