322 EFFECT OF SODIUM NITROPRUSSIDE ON BUFFALO SPERM CAPACITATION IN VITRO

2007 ◽  
Vol 19 (1) ◽  
pp. 276 ◽  
Author(s):  
L. Boccia ◽  
L. Attanasio ◽  
A. De Rosa ◽  
G. Pellerano ◽  
R. Di Palo ◽  
...  
Keyword(s):  
Sodium Nitroprusside ◽  
Acrosome Reaction ◽  
Production Efficiency ◽  
Control Group ◽  
Sperm Capacitation ◽  
Promoting Effect ◽  
Vitro Fertilization ◽  
Domestic Species ◽  
Frozen Semen

The overall in vitro embryo production efficiency in buffalo is hampered by the poor fertilization rate. It is known that the quality of the frozen semen may affect fertilization efficiency. However, it is not possible to rule out that the process of capacitation, required by spermatozoa to acquire the fertilizing ability, is impaired in the in vitro fertilization (IVF) system. Although several agents have been proven to induce sperm capacitation in vitro, heparin treatment is still the most efficient method in most of the domestic species. There is evidence that capacitation is part of an oxidative process and that nitric oxide (NO) acts as a capacitation inducer in human (Herrero et al. 1999 Biol. Reprod. 61, 575–581) and bovine (Rodriguez et al. 2005 Anim. Reprod. Sci. 85, 231–242) spermatozoa. The aim of the present study was to evaluate whether sodium nitroprusside (SNP), a well-known generator of NO in vitro, improves buffalo sperm capacitation in vitro. Frozen–thawed sperm from a bull previously tested for IVF were treated by swim-up in order to select only the motile population. Spermatozoa were incubated in the presence of 0.01 mM heparin (control group) for 1 h (n = 266), 2 h (n = 270), and 3 h (n = 306), and in the presence of 10 �M SNP for 1 h (n = 302), 2 h (n = 286), and 3 h (n = 260). The concentration of SNP was chosen on the basis of a preliminary dose-response trial (0.1 �M, 1 �M, and 10 �M). Following incubation with these agents, sperm were exposed for 15 min to 60 �g mL-1 of lysophosphatidylcholine, an agent known to induce acrosome reaction only on capacitated spermatozoa. Trypan blue was used first to differentiate live from dead spermatozoa and the dried smears were then fixed in 37% formaldehyde and stained with Giemsa for acrosome evaluation by microscopic examination. The proportion of acrosome-reacted spermatozoa in each group was used to assess the efficiency of capacitation under different incubation conditions. Differences between groups were analyzed by chi-squared test. No dead spermatozoa were found in all groups. Following 1-h sperm treatment with either heparin or SNP, the proportion of acrosome-reacted spermatozoa was similar (35.3% vs. 28.5%, respectively). However, extending the incubation time to 2 h, SNP significantly (P < 0.01) increased the incidence of acrosome reaction compared to heparin (60.1% vs. 44.1%, respectively). Analogously, when the sperm treatment was prolonged to 3 h, SNP gave a significantly (P < 0.01) higher percentage of acrosome reaction compared to the control (68.8% vs. 36.6%, respectively). In conclusion, sperm treatment with SNP for either 2 or 3 h significant improved the efficiency of buffalo sperm capacitation in vitro compared with heparin, that is, the capacitating agent currently used in the IVF system. The promoting effect of SNP indirectly indicates that NO acts as a capacitation inducer in buffalo spermatozoa. Finally, these results suggest the need to evaluate the effect of SNP on the fertilizing capability of buffalo spermatozoa in vitro.

scholarly journals 239 CAPACITATION OF BUFFALO SPERMATOZOA IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 270 ◽  
Author(s):  
L. Boccia ◽  
A. De Rosa ◽  
L. Attanasio ◽  
R. Di Palo ◽  
L. Zicarelli ◽  
...  
Keyword(s):  
Acrosome Reaction ◽  
Technical Assistance ◽  
Critical Role ◽  
Male Gamete ◽  
Sperm Capacitation ◽  
Cleavage Rate ◽  
Domestic Species ◽  
Frozen Semen

The efficiency of in vitro embryo production (IVEP) in buffalo is hampered by the poor cleavage rate. The quality of the frozen semen may affect fertilization efficiency, due to damages of the male gamete that occur following cryopreservation. However, it is not possible to rule out that the process of capacitation, required by spermatozoa to acquire the fertilizing ability, is impaired in the IVF system. Although several agents have been proven to induce sperm capacitation in vitro, heparin is still the most efficient method in most of the domestic species. The aim of the study was to evaluate the efficiency of buffalo estrus serum (BES) and follicular fluid (FF) to induce buffalo sperm capacitation in vitro, indirectly assessed by estimating the capability of spermatozoa to acrosome react. Frozen semen from a bull previously tested for IVF, thawed at 37°C for 40 s in water, was treated by swim-up in order to select only the motile population. Spermatozoa (n = 1546) were assessed immediately after swim-up separation, to evaluate the incidence of acrosomal loss in nontreated cells (time 0). The remaining spermatozoa were incubated in the presence of 0.01 mM heparin (control; n = 3531), 20% BES (n = 2442) and 20% FF (n = 1419), the latter recovered from a pool of dominant follicles, for 1, 2, and 3 hours. Sperm was then exposed for 15 min to 60 μg mL−1 of lysophosphatidylcholine, an agent known to induce acrosome reaction only on capacitated spermatozoa. Trypan blue was used to differentiate live from dead spermatozoa, and the dried smears were then fixed in 37% formaldehyde and stained with Giemsa for acrosome evaluation by microscopic examination. The proportion of acrosome-reacted spermatozoa in each group was used to assess the efficiency of capacitation under different incubation conditions. Differences between groups were analyzed by χ2. No dead spermatozoa were found in all groups. Acrosomal loss was observed in only 15.3% of the sperm population at time 0; it may be accounted for either by damages preceding cell death or by freezing-induced capacitation. No differences were found between incubation times within each treatment group. Interestingly, sperm treatment with both BES and FF resulted in a significantly higher incidence of acrosome reaction compared with heparin (84.3, 94.5 vs. 50.1%, respectively; P < 0.001), the capacitating agent currently used in the IVF system, and, in particular, FF showed the highest percentage of acrosome reaction at all incubation times, even when compared with BES (P < 0.01). It is likely that factors derived by BES and FF, present in the oviduct environment around fertilization, play a critical role in processing the male gamete in vivo. These preliminary results show the possibility of significantly improving the efficiency of sperm capacitation in vitro in buffalo species with BES and FF and strongly suggest investigating the effects of these factors also on the fertilizing capability of buffalo spermatozoa. The authors thank to Dr. O. Paciello for his technical assistance.

50 Effects of Cathepsin B Inhibitor E64 on the Survival Rate of Cryopreserved Semen from Korean Brindled Bulls

2018 ◽  
Vol 30 (1) ◽  
pp. 164
Author(s):  
S. W. Kim ◽  
M.-S. Kim ◽  
C.-L. Kim ◽  
I. S. Jeon
Keyword(s):  
Cathepsin B ◽  
Room Temperature ◽  
Egg Yolk ◽  
Coat Colour ◽  
Control Group ◽  
Vitro Fertilization ◽  
Frozen Semen ◽  
The Difference ◽  
The Individual

Korean brindled cattle have distinctive coat colour and are regarded as rare cattle in the Korean peninsula. To preserve the line as a genetic resource for diversity in cattle, semen cryopreservation has been used for selection of individuals for breeding between local AI centers. Nevertheless, the survival and viability of frozen semen from Korean brindled bulls is not uniform due to the difference between the individual bulls or experimental techniques. In this study, E64, a cathepsin B inhibitor, was used at final concentrations of 0.05, 0.5, and 1 µM to test the viability of frozen semen. To prepare frozen semen, a triladyl-egg yolk diluent with 6.4% glycerol was used in a two-step freezing method with 4 different bulls with 3 repeats. A total of 12 ejaculates was diluted to a concentration of 50 to 100 × 106 mL−1 at room temperature, and slowly cooled from room temperature to 5°C in 2 to 3 h. The cooled semen was diluted 1:1 with a secondary diluent containing E64 to prepare the experimental group. After loading, 0.5-mL straws were immersed into liquid nitrogen after 10 min exposure at 5 cm above the nitrogen using a styrofoam box. The viability of spermatozoa after thawing at 37°C for 40 s was analysed by Student’s t-test. The rate of surviving sperm in the 1 µM E64 group (82 ± 4.3%) was significantly higher than that of the control group (71.2 ± 2.0%; P < 0.05). However, the 0.05 and 0.5 µM E64 treatment groups lead to similar rates (77.5 ± 2.0% and 75.0 ± 5.0%, respectively; P > 0.05). Based on these results, it is expected that E64 could be used for the improvement of productivity of frozen semen; further results on in vitro fertilization and development are ongoing.

294 THE EFFECT OF PLASMIN ON THE IN VITRO FERTILIZATION ABILITY OF PORCINE GAMETES

2006 ◽  
Vol 18 (2) ◽  
pp. 254
Author(s):  
H.-H. Rhee ◽  
S.-J. Sa ◽  
H.-T. Cheong ◽  
B.-K. Yang ◽  
C.-K. Park
Keyword(s):  
In Vitro Fertilization ◽  
Zona Pellucida ◽  
Acrosome Reaction ◽  
Proteolytic Enzymes ◽  
Extracellular Proteins ◽  
Control Group ◽  
Sperm Viability ◽  
Vitro Fertilization ◽  
Sperm Binding

Plasminogen activators (PAs) are specific proteolytic enzymes that convert the inactive proenzyme plasminogen to plasmin. The plasmin formed is a nonspecific, potent protease that cleaves blood fibrin clots and several other extracellular proteins. The purposes of the present study were (1) to assess the effect of plamin on sperm viability and acrosome reaction (AR), (2) to examine the effect of plasmin on zona pellucida (ZP) solubility and the binding of sperm to ZP, and (3) to evaluate the effect of plasmin on fertilization responses, including penetration and incidence of polyspermy during in vitro fertilization in the pig. Ejaculated semen was collected from three mature Duroc boars by artificial vagina. The same three boars were used for all experiments. The oocyte maturation medium used was North Carolina State University-23 (NCSU-23) medium supplemented with 10% (v/v) porcine follicular fluid (pFF), 0.6 mM cysteine, 10 IU/mL human chorionic gonadotropin (hCG; Sigma-Aldrich Corporation, St. Louis, MO, USA), and 10 IU/mL pregnant mare's serum gonadotropin (PMSG; Sigma). Porcine spermatozoa, which were washed in Dulbecco PBS (Sigma), were resuspended and incubated in fertilization medium (mTBM) containing 0, 0.1, 1.0, 10.0, or 100.0 ng/mL plasmin (Sigma). Data were analyzed by ANOVA and Duncan's multiple-range test using the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). The present study suggests that sperm viability was not affected by plasmin treatment. Also, addition of plasmin in doses ranging between 0.1 and 100.0 ng/mL for 2, 4, or 6 h to washed boar spermatozoa resulted in enhancement of acrosome reaction (AR), compared with untreated cells. Concentrations of 0 and 0.1 ng/mL plasmin (83 � 15 and 95 � 18 sperm/oocyte, respectively) had no effect on sperm binding, whereas 1.0 (123 � 21 sperm/oocyte), 10.0 (124 � 16 sperm/oocyte), and 100 ng/mL (124 � 15 sperm/oocyte) plasmin increased (P < 0.05) sperm binding, compared with the control. The zona pellucida solubility (zona digestion time) was significantly (P < 0.05) lower in medium with 1.0 (123 � 24 s), 10.0 (99 � 15 s), or 100.0 ng/mL (95 � 19 s) plasmin, compared with control (176 � 27 s). When porcine oocytes and spermatozoa were co-incubated in various concentrations of plasmin for 6 h, the penetration rate was significantly (P < 0.05) higher in medium with 1.0 ng/mL plasmin (77.5 � 3.1%), compared with control. However, there were no significant differences in the polyspermic rates and mean numbers of sperm (MNS)/oocyte among the groups treated with plasmin and the control group. We found that addition of plasmin to fertilization medium increases the percentage of acrosome-reacted spermatozoa and the sperm-binding ability of the pig ZP. These results suggest that plasmin may play a role in events related to fertilization in the pig.

Development of in vitro tests to predict fertility of bulls

2005 ◽  
Vol 85 (1) ◽  
pp. 47-52 ◽  
Author(s):  
G. Giritharan ◽  
N. Ramakrishnappa ◽  
A. Balendran ◽  
K. M. Cheng ◽  
R. Rajamahendran
Keyword(s):  
Acrosome Reaction ◽  
Return Rate ◽  
In Vitro Tests ◽  
In Vitro Test ◽  
Motile Sperm ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Frozen Semen ◽  
Vitro Test

The overall objective was to develop an in vitro test to predict fertility of bulls in the field. We investigated the bull effect on in vitro embryo production, zona binding and acrosome reaction, and the correlation of this effect to field fertility meas ured by 60–90 d non-return rate. Frozen semen from three separate ejaculates of eight unrelated young bulls, obtained from an artificial insemination (AI) center, was used. On thawing, ejaculates from each bull were pooled, motile sperm were selected and (a) subjected to immunofluorescent assay at 0 and 4 h of incubation in capacitation medium to assess acrosome status, (b) used in an in vitro fertilization assay system to assess cleavage and blastocyst production rates, and (c) sperm-zona binding assay was carried out to determine the number of sperm bound to the zona pellucida of mature oocytes. Percentage of pre-freeze motile sperm (PrFM) and non-return rate data were obtained from the AI center. PrFM, percentage of acrosome reacted sperm at 0 h (AR1), increase in percentage of acrosome reacted sperm after 4 h (InAR) and sperm-zona binding rates (ZB) differed (P < 0.05) among sperm samples obtained from different young bulls. Significant correlations (P < 0.05) were observed between PrFM and AR1 (r = -0.31), InAR (r = 0.36), and ZB (r = 0.32). AR1 was negatively correlated to ZB (r = -0.27) and cleavage rate (r = -0.20), InAR was positively correlated with ZB (r = 0.31) and cleavage rate (r = 0.26). None of the in vitro tests was correlated with non-return rate. These findings indicate that along with pre-freeze motility, a combination of in vitro tests including the percentage of spontaneously acrosome reacted sperm at thawing, might be useful in predicting bull field fertility. Such a combination of assays, however, has yet to be determined. Key words: Field fertility, acrosome reaction, zona binding, IVF, fertility assay

Effect of culture, incubation and acrosome reaction of fresh and frozen-thawed ram spermatozoa for in vitro fertilization and intracytoplasmic sperm injection

1997 ◽  
Vol 9 (7) ◽  
pp. 665 ◽  
Author(s):  
M. C. Gómez ◽  
J. W. Catt ◽  
L. Gillan ◽  
G. Evans ◽  
W. M. C. Maxwell
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Acrosome Reaction ◽  
Vitro Fertilization ◽  
Fertilization Rates ◽  
Frozen Semen ◽  
Acrosome Integrity ◽  
And Control ◽  
Fresh Semen ◽  
Percoll Centrifugation

This study evaluated different sperm treatments for fertilization of sheep oocytes by intracytoplasmic sperm injection (ICSI) and in vitrofertilization (IVF). In Experiment 1, fresh and frozen semen was separated by Percoll centrifugation and incubated at 30°C or 39°C in HSOF or BSOF medium for 1 h before use for IVF or ICSI. For IVF, oocytes were inseminated and incubated with sperm for 30 min, 4 h and 19 h. Sperm were assessed for acrosome integrity after Percoll centrifugation and 1 h incubation, and those used for IVF were assessed after each period of exposure to the oocytes. Fertilization rates after ICSI were higher for fresh than for frozen-thawed sperm and were highest 19 h after IVF with fresh or frozen-thawed sperm in the presence of HSOF at 30°C. In Experiment 2, fresh semen was separated by Percoll centrifugation and incubated for 5 h in HSOF, and the acrosome reaction was induced with lysophosphatidylcholine. Acrosome integrity was then assessed. Fertilization rates after ICSI were similar for acrosome-reacted and control spermatozoa. These results suggest that induction of the acrosome reaction in spermatozoa before ICSI is unnecessary, whereas a capacitating treatment of spermatozoa is required before IVF.

scholarly journals Cysteamine, glutathione and ionomycin treatments improve in vitro fertilization of prepubertal goat oocytes

Zygote ◽  
2004 ◽  
Vol 12 (4) ◽  
pp. 277-284 ◽  
Author(s):  
Aixa Urdaneta ◽  
Ana Raquel Jiménez ◽  
María-Teresa Paramio ◽  
Dolors Izquierdo
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Control Group ◽  
Sperm Capacitation ◽  
Vitro Fertilization ◽  
Maturation Medium

The aim of this study was to improve in vitro embryo development of prepubertal goat oocytes by studying the effect of adding cysteamine to in vitro maturation medium, glutathione (GSH) to in vitro fertilization medium and ionomycin to the sperm capacitation medium. In experiment 1, we analysed the effect of 1 mM GSH added to fertilization medium of oocytes matured with 400 μM cysteamine. The control group were oocytes without cysteamine and GSH. In experiment 2, oocytes matured and fertilized in the presence of 400 μM cysteamine and 1 mM GSH, respectively, were inseminated with spermatozoa treated with ionomycin or heparin. In experiment 1, the percentages of total and normal fertilized oocytes were significantly higher for oocytes supplemented with cysteamine and GSH (40.26% and 30.20%, respectively) than for oocytes from the control group (16.66%, and 10.61%, respectively). The percentage of total embryos obtained after 7 days of culture was significantly higher in the group supplemented with cysteamine and GSH (30.62%) than in the control group (8.09%) . In experiment 2, percentages of total and normal fertilized oocytes were significantly higher for the group of spermatozoa capacitated with ionomycin (52.21% and 37.17%, respectively) than with heparin (38.62% and 28.35%, respectively). After 7 days of culture, total embryo rate was significantly higher in the group of sperm capacitated with ionomycin (44.91%) than with heparin (38.69%) . However, the percentage of embryos developed to the blastocyst stage was not affected by any of the treatments studied.

Levels interleukine-4 and interleukin-17 (IL-4, IL-17) of blood in patients with habitual recurrent pregnancy loss, which occurred in a loop in vitro fertilization

2016 ◽  
pp. 137-139
Author(s):  
K.P. Golovatyuk ◽  
Keyword(s):  
In Vitro Fertilization ◽  
Conditioned Medium ◽  
Mononuclear Cells ◽  
Recurrent Miscarriage ◽  
Interleukin 4 ◽  
Control Group ◽  
Interleukin 17 ◽  
Vitro Fertilization ◽  
Blood Mononuclear Cells

The objective: was to investigate the levels of cytokines IL-4 and IL-17 in serum and conditioned medium cultures of blood mononuclear cells (MNC) and evaluation association between their products and miscarriage, which occurred in IVF cycles. Patients and methods. We observed 240 patients with recurrent miscarriage, came in IVF cycles, and 100 apparently healthy fertile women in the control group. The concentrations of IL-4 and IL-17 in serum and conditioned medium of MNC cultures were determined. Results. The levels of IL-4 in the serum and conditioned medium in spontaneous and stimulated mitogen secretion was not significantly different from those in the control group, whereas IL-17 levels were higher than those in the control group serum, in conditioned media of stimulated and non-stimulated MNCs. Conclusion. Disregulation of activity of circulating blood mononuclear cells in women with recurrent miscarriage that followed IVF, is accompanied by increased secretion of IL-17 and almost constant production of IL-4 on the back of high stimulation index of production of these cytokines. Key words: in vitro fertilization, miscarriage, interleukin-4, interleukin-17, serum stimulated and non-stimulated mononuclear blood.

scholarly journals IN VITRO FERTILITY TEST OF HUMAN SPERMATOZOA MEMBRANE PROTEIN FERTILIN BETA ANTIBODY IN MICE (Mus musculus Balb/c) AS IMMUNOCONTRACEPTIVE CANDIDATE

2017 ◽  
Vol 52 (3) ◽  
pp. 209
Author(s):  
Reny I’tishom ◽  
Doddy M Soebadi ◽  
Aucky Hinting ◽  
Hamdani Lunardhi ◽  
Rina Yudiwati
Keyword(s):  
In Vitro Fertilization ◽  
Membrane Protein ◽  
Mus Musculus ◽  
Human Spermatozoa ◽  
Control Group ◽  
Reproductive Process ◽  
Vitro Fertilization ◽  
Antibody Induction

One of the materials as potential candidates immunocontraception material is spermatozoa. Fertilin beta is spermatozoa membrane protein and is found only in mature spermatozoa and ejaculate, which serves as an adhesion molecule. Spermatozoa membrane protein that is used as an ingredient immunocontraception candidate, must have specific criteria that the specificity of spermatozoa, the role of antigen in the fertilization process, which includes the formation of immunogenicity sufficient antibody response has the potential to block fertilization. Antibodies against spermatozoa affect the stages before fertilization of the reproductive process and can hinder the development of the embryo after fertilization. Until now very little research data spermatozoa membrane protein as an ingredient immunocontraception are up to the test of experimental animals. The research objective is to prove the role of the resulting antibody induction of antibodies fertilin beta protein in the membrane of human spermatozoa induce agglutination and reduce motility thus reducing the number of in vitro fertilization. Research conducted at the IVF Laboratory, Department of Biology of Medicine, Faculty of Medicine, University of Airlangga. This research includes: Test the potential of antibody protein beta fertilin membrane of human spermatozoa and inhibit the role of antibodies in vitro fertilization in mice (Mus musculus Balb/c). In vitro studies have resulted in fertilization figure of 25% is smaller than the number that is equal to control fertilization of 58.7%, whereas previously the spermatozoa were incubated first with a beta membrane protein antibody fertilin human spermatozoa. While the percentage of inhibition of sperm to fertilize an oocyte by 33.75%. Potential imunokontraseptif considered effective if it decreased significantly (P <0.05) than the numbers fertilization in the treatment group compared with the control group. This shows fertilin beta membrane protein antibody has the ability to inhibit human spermatozoa to fertilize oocytes that reduce the number of fertilization.

scholarly journals The effectiveness of collaborative infertility counseling (CIC) on pregnancy outcome in infertile women undergoing in vitro fertilization: A randomized controlled trial

2019 ◽  
Author(s):  
Mahboobeh Rasoulzadeh Bidgoli ◽  
robab latifnejad roudsari ◽  
ali montazeri
Keyword(s):  
In Vitro Fertilization ◽  
Pregnancy Rate ◽  
Treatment Success ◽  
Intervention Group ◽  
Control Group ◽  
Vitro Fertilization ◽  
Infertile Women ◽  
Significant Difference ◽  
And Control

Abstract Background: Infertility is an emotional tension which influences the whole aspects of relationships in infertile couples. A main objective of infertility treatments is elevation of pregnancy rate. The present study aimed to examine the effect of collaborative counseling on pregnancy rate in infertile women, undergoing in vitro fertilization in Mashhad, Iran. Methods: In this clinical trial, 60 women with primary infertility were selected from an infertility research center and were randomly allocated into intervention (n=29) and control (n=31) groups. The intervention group received individual counseling, based on the collaborative reproductive healthcare model with collaboration of a midwife, a gynecologist and a clinical psychologist in five sessions during a two-month period. The control group received routine care. Positive pregnancy test was considered as a criterion of treatment success at the end of the study. Data were analyzed using statistical tests including independent samples t-test. Results: There was no significant difference in pregnancy rate between intervention and control groups (P = 0.298). Also, there were no significant differences in follicle and embryo numbers between two groups. However, a significant difference was observed between two groups in terms of oocyte numbers where the intervention group had more oocyte (P = 0.014). Conclusion: Overall the findings indicated that the collaborative infertility counseling did not improve treatment success in infertile women undergoing in vitro fertilization

Sign in / Sign up

Export Citation Format

Share Document