Evaluation of the function of fresh and frozen - thawed sex-sorted and non-sorted stallion spermatozoa using a heterologous oocyte binding assay

2010 ◽  
Vol 22 (4) ◽  
pp. 710 ◽  
Author(s):  
J. R. Clulow ◽  
G. Evans ◽  
W. M. C. Maxwell ◽  
L. H. A. Morris
Keyword(s):  
Zona Pellucida ◽  
Binding Assay ◽  
Binding Ability ◽  
Treatment Groups ◽  
Functional Integrity ◽  
Sperm Binding ◽  
Heterologous Sperm ◽  
Bovine Oocytes ◽  
Better Than

The aim of the present study was to evaluate the potential oocyte binding ability and functional integrity of fresh or frozen–thawed, sex-sorted or non-sorted stallion spermatozoa. In the absence of effective IVF procedures in the horse, a heterologous sperm-binding assay was used as an indicator of fertilising capacity to assess differences in the ability of stallion spermatozoa to bind to bovine oocytes. The functional integrity of four treatment groups was assessed: (1) fresh non-sorted spermatozoa; (2) fresh sex-sorted spermatozoa; (3) frozen–thawed non-sorted spermatozoa; and (4) frozen–thawed sex-sorted spermatozoa. Spermatozoa found in association with the zona pellucida of the bovine oocytes were deemed ‘attached’ or ‘bound’ depending on their characterisation as either acrosome intact or acrosome reacted, respectively. Significantly less frozen–thawed spermatozoa were found attached to the oocytes compared with fresh spermatozoa. No significant differences were identified between the number of attached sex-sorted and non-sorted frozen–thawed spermatozoa. However, significantly more sex-sorted than non-sorted fresh spermatozoa were found attached to the oocytes after 1 h coincubation, although after 3 h coincubation this difference was no longer apparent. In conclusion, sex-sorted fresh and frozen–thawed stallion spermatozoa are functionally capable of attaching and binding to bovine oocytes in vitro. Furthermore, fresh sex-sorted spermatozoa attach better than non-sorted spermatozoa, suggesting that they have a more advanced capacitation-like status.

scholarly journals Fertilization of Pronase-Treated Mouse Ova in Vitro

1981 ◽  
Vol 34 (2) ◽  
pp. 245 ◽  
Author(s):  
P Quinn ◽  
JD Stanger
Keyword(s):  
Zona Pellucida ◽  
Binding Sites ◽  
Relative Effectiveness ◽  
Fertilization Rate ◽  
Enzyme Treatment ◽  
Perivitelline Space ◽  
Sperm Binding ◽  
Mechanical Removal ◽  
Better Than

In order to obtain consistently a large number of zona-free mouse ova for studies of sperm-egg interactions, a study was made of the relative effectiveness of removing the zona pellucida from ova by mechanical or enzymatic treatments. Ova exposed to pronase before mechanical removal of the zona pellucida in medium devoid of pronase had similar fertilization rates in vitro compared with ova mechanically denuded in the absence of pronase. Ova with pronase-weakened zonae were easier to denude and survived the mechanical manipulations better than the ova denuded by vigorous aspiration in narrow-bore pipettes. However, exposure to pronase did significantly lower the incidence of polyspermy in the naked ova, indicating that some of the enzyme may have diffused across the perivitelline space and damaged sperm-binding sites on the vitelline plasma membrane. The enzyme treatment also reduced the fertilization rate of zona-intact ova.

Differences between antigenic determinants of pig and cat zona pellucida proteins

Reproduction ◽  
2000 ◽  
pp. 15-23 ◽  
Author(s):  
K Jewgenow ◽  
M Rohleder ◽  
I Wegner
Keyword(s):  
In Vitro Fertilization ◽  
Zona Pellucida ◽  
Antigenic Determinants ◽  
Vitro Fertilization ◽  
Contraceptive Vaccine ◽  
Sperm Binding ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Dose Dependent

Despite many efforts, the control of reproduction in feral cat populations is still a problem in urban regions around the world. Immunocontraception is a promising approach; thus the present study examined the suitability of the widely used pig zona pellucida proteins (pZP) for contraception in feral domestic cats. Purified zona pellucida proteins obtained from pig and cat ovaries were used to produce highly specific antisera in rabbits. Antibodies against pZP raised in rabbits or lions were not effective inhibitors of either in vitro sperm binding (cat spermatozoa to cat oocytes) or in vitro fertilization in cats, whereas antibodies against feline zona pellucida proteins (fZP) raised in rabbits showed a dose-dependent inhibition of in vitro fertilization. Immunoelectrophoresis, ELISA and immunohistology of ovaries confirmed these results, showing crossreactivity of anti-fZP sera to fZP and to a lesser extent to pZP, but no interaction of anti-pZP sera with fZP. It is concluded that cat and pig zonae pellucidae express a very small number of shared antigenic determinants, making the use of pZP vaccine in cats questionable. A contraceptive vaccine based on feline zona pellucida determinants will be a better choice for the control of reproduction in feral cats if immunogenity can be achieved.

scholarly journals DIFFERENCE BETWEEN ANTIMICROBIAL EFFECTS OF PAPACARIE AND PAPAIN ON STREPTOCOCCUS MUTANS IN VITRO

2019 ◽  
pp. 79-83
Author(s):  
TITTY SULIANTI ◽  
NILAKESUMA DJAUHARI ◽  
BAMBANG NURSASONGKO
Keyword(s):  
Inhibitory Concentration ◽  
Minimum Bactericidal Concentration ◽  
Zone Of Inhibition ◽  
Antimicrobial Effects ◽  
Treatment Groups ◽  
Group Control ◽  
Caries Removal ◽  
And Diffusion ◽  
Better Than

Objective: The aim is to compare the antimicrobial effects of papain and Papacarie with dilution and diffusion tests.Methods: There were two treatment groups and one Group control. The treatment group received papain and Papacarie, and the control groupreceived chlorhexidine, in five liquids with different concentrations of 0.5%, 0.25%, 0.125%, 0.0625%, and 0.03%. The dilution and diffusion testswere used to determine the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and zone of inhibition for eachtreatment material.Results: MICs of papain and Papacarie were 12.5%, indicating that at a concentration of 12.5%, the material can inhibit the growth of Streptococcusmutans. Papain does not have an MBC value but the Papacarie has an MBC at 25%, which indicating that at a concentration of 25%, Papacarie hasbactericidal effects on S. mutans. The zone of inhibition of papain was lower than Papacarie.Conclusion: Based on chemomechanical caries removal materials, the antimicrobial effects of Papacarie were better than those of papain.

scholarly journals The activity of extracts chara vulgaris against promastigotes of leishmania tropica.

2018 ◽  
Vol 12 (2) ◽  
pp. 66-72
Author(s):  
Ban Hussein Ali ◽  
Thaer A. Saleh ◽  
Mohammed M. Al-Halbosiy
Keyword(s):  
Major Health Problem ◽  
Organic Extract ◽  
Major Health ◽  
Treatment Groups ◽  
Chara Vulgaris ◽  
Incubation Periods ◽  
Potential Activity ◽  
Diphenyl Tetrazolium Bromide ◽  
Better Than

            Leishmaniasis is a widespread parasitic disease caused by Leishmania parasite, this disease considers as a major health problem worldwide. The available therapy is unsatisfactory expensive with a cytotoxic side effects. Studies of marine algae as a source of pharmacological active compounds have increased worldwide.  This study was aimed to investigate the effect of a type of green algae (Chara vulgaris) on promastigotes of L. tropica, by using. various concentrations (500, 250, 125, 62.5, 31.25, 15.6 µg/mL) in vitro by MTT assay [3-(4.5-dimethylthiazol-2-yl)- 2.5-diphenyl tetrazolium bromide)], to investigate its effect on the proliferation of promastigotes, by three incubation periods (24, 48, 72 hr.) The results showed a significant (p< 0.05)   decrease in survived of promastigotes in treatment groups with concentrations that ranged between 15 to 500 μg/ ml.  This study revealed a major growth inhibition effect of the organic extract of C. vulgaris against L. tropica promastigotes, and the extract of ethyl acetate showed potential activity is better than the aqueous extract.

scholarly journals LÍQUIDO FOLICULAR EQÜINO NA MATURAÇÃO IN VITRO DE OÓCITOS BOVINOS

2004 ◽  
Vol 9 (1) ◽  
Author(s):  
M.G.L. PINTO ◽  
M.I.B. RUBIN ◽  
C.A.M. SILVA ◽  
T.F. HILGERT ◽  
M.F. SÁ FILHO ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Mineral Oil ◽  
Control Group ◽  
Sodium Pyruvate ◽  
Vitro Fertilization ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Bovine Oocytes

O desenvolvimento embrionário de oócitos bovinos maturados in vitro (MIV) foi avaliado em meio suplementado com líquido folicular eqüino (Lfe). Foram distribuídos 1045 oócitos em 11 repetições formando três grupos tratamentos (T1, T2, T3) e um controle (C). O meio de maturação utilizado foi o TCM-199 acrescido de piruvato de sódio, hormônio folículo estimulante recombinante (rFSHh) e hormônio luteinizante equino (LHe). Suplementou-se esse meio com 10% de soro de égua em estro para o grupo controle e para T1, T2 e T3, o meio foi suplementado com 5, 10, e 20% de LFe, respectivamente. Os oócitos foram maturados in vitro (MIV) por 24h. A fecundação in vitro (FIV) foi realizada em meio Talp-Fert. A MIV e a FIV foram realizadas em estufa a 39ºC com 5% de CO2 em ar e umidade saturada. Os zigotos foram cultivados em meio SOFaaci, sob óleo mineral no interior de bolsas plásticas gaseificadas. As taxas de clivagem e de blastocistos foram observadas diariamente (D), e em D7, foram superiores (P0,05) às do grupo controle. Em D9, a taxa de blastocistos do T2 foi superior (P0,05). O LFe, na concentração de 10% pode ser utilizado, em substituição ao soro de égua em estro para suplementar o meio de MIV de oócitos bovinos. Equine follicular fluid on in vitro maturation of bovine oocytes Abstract Embryo development of bovine oocytes was evaluated using maturation medium supplemented with equine follicular fluid (eFF). One thousand and forty five (1045) oocytes were distributed in 11 replications forming three treatment groups (T1, T2 e T3) and one Control (C). TCM-199 added with sodium pyruvate, rFSHh and LHe was used as maturation medium. This medium was supplemented with 10% estrous mare serum for Control group, and 5, 10, and 20% eFF, respectively, for T1, T2 e T3 groups. In vitro maturation (IVM) of all groups was performed during 24h. In vitro fertilization (IVF) was performed in TALP-FERT medium. IVM and IVF were carried out in an incubator at 39ºC with 5% CO2 in air and saturated humidity. Zygotes were cultured in SOFaaci medium, under mineral oil in gasified bags. Cleavage and blastocyst rates were daily observed (D), and at D7, were higher (P0.05) for those from control group. At D9, blastocyst rate of T2 was higher (P0.05). The eFF, at a 10% concentration, can replace the use of estrous mare serum to supplement the IVM medium of bovine oocytes.

87 EFFECT OF CRYOPROTECTANT CONCENTRATION IN THE VITRIFICATION SOLUTION ON THE ZONA PELLUCIDA HARDENING AND SPERMATOZOA PENETRATION OF BOVINE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 149 ◽  
Author(s):  
A. Quiñones Martorello ◽  
G. Rios ◽  
A. Cano ◽  
R. H. Alberio
Keyword(s):  
Zona Pellucida ◽  
Calcium Influx ◽  
Oocyte Activation ◽  
High Concentration ◽  
Hoechst 33342 ◽  
Polar Bodies ◽  
Dmso Concentration ◽  
Sperm Penetration ◽  
Bovine Oocytes

In the murine model, it has been shown that the high concentration of cryoprotectants required for vitrification can activate the oocytes through a process mediated by calcium influx. This activation induces the zona pellucida (ZP) hardening and affects the sperm penetration. This study aimed to evaluate the effect of exposure of bovine oocytes to the vitrification solutions (VS1 and VS2) in calcium-free medium with 3 concentrations of etilenglycol (EG) and dimetylsulfoxide (DMSO) on the oocyte activation. Cumulus oocyte complexes (COC) were matured in vitro (22 h), partially denuded through pipetting in medium with hyaluronidase, and subject to four treatments: T1, untreated (control); T2, exposed to 20% EG+0% DMSO (VS1) and then 40% EG+0% DMSO (VS2); T3, 10% EG+10% DMSO (VS1) and then 20% EG+20% DMSO (VS2); and T4, 0% EG+20% DMSO (VS1) and then 0% EG+40% DMSO (VS2). The contact with each VS was 3 min and 30 s, respectively. After this, the COC were matured up to 24 h. In Expt. 1, COC were denuded and placed in a solution of pronase E in PBS (1 mg mL–1) to determine the number of oocytes with ZP digested after 9 min of exposure to the enzyme. In Expt. 2, COC were fertilized in TALP medium with 50 mg mL–1 heparin and 1 million mL–1 sperm. After 12 h, COC were denuded and stained with bisbenzimide (Hoechst 33342) and examined under epi-fluorescence. The number of oocytes indicating spermatic penetration was determined by presence of intact sperm heads, spermatic pro-nucleus, or 2 polar bodies. Data were analysed by the PROC GENMOD (SAS; see Table 1). In Expt. 1, there were no differences in the percentage of oocytes without ZP after pronase treatment in groups T1, T2, and T3. The T4 group had the lowest percentage of digestion, and T3 was not different from T4. In Expt. 2 there were no differences in the percentage of sperm penetration between T2, T3, and T4. All treatments had lower values than T1. In conclusion, bovine oocytes undergo hardening of the ZP when put in contact with the cryoprotectants, and this effect was significantly increased with the use of DMSO. Moreover, there was a decrease in sperm penetration in all treated groups, indicating that the natural blocking of polyspermy depends not only on the hardening of the ZP, but another process that could act at the plasma membrane. It is possible that cryoprotectants, regardless of their concentration, may trigger this early block through a mechanism that would be independent of calcium. Table 1.Effect of EG and DMSO concentration in the VS on the ZP hardening and sperm penetration of bovine oocytes exposed to these solutions Acknowledgment: the National Research Agency through the grant PICT 2007/1205.

Ultrastructural Imaging Analysis of the Zona Pellucida Surface in Bovine Oocytes

2019 ◽  
Vol 25 (4) ◽  
pp. 1032-1036 ◽  
Author(s):  
Francisco Báez ◽  
Álvaro A. Camargo ◽  
Gustavo D.A. Gastal
Keyword(s):  
Zona Pellucida ◽  
Ultrastructural Change ◽  
Early Embryo ◽  
Imaging Analysis ◽  
Mature Group ◽  
Immature Group ◽  
Imagej Software ◽  
Bovine Oocytes ◽  
Objective Methodology

AbstractThe aims of the present study were to: (i) evaluate the ultrastructural differences in the zona pellucida (ZP) surface between immature and mature bovine oocytes, and (ii) describe a new objective technique to measure the pores in the outer ZP. Intact cumulus–oocyte complexes (COCs) obtained from a local abattoir were immediately fixed (immature group) or submitted to in vitro maturation (IVM) at 38.5 °C for 24 h in a humidified atmosphere of 5% CO2 in air (mature group). Oocytes from both groups were morphologically evaluated via Scanning Electron Microscopy (SEM) and the images were processed in the Fiji/ImageJ software using a new objective methodology through the Trainable Weka Segmentation plugin. The average number of pores in ZP was greater (p < 0.05) in the mature group than the immature group. However, the size and circularity of pores in ZP did not differ (p > 0.05) between groups. In conclusion, it has been shown that the number of pores highlighted the main ultrastructural change in the morphology of the ZP surface of bovine oocytes during the IVM process. We have described an objective method that can be used to evaluate ultrastructural modifications of the ZP surface during oocyte maturation and early embryo development.

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