scholarly journals Inhibitors of zinc-dependent metalloproteases hinder sperm passage through the cumulus oophorus during porcine fertilization in vitro

Reproduction ◽  
2012 ◽  
Vol 144 (6) ◽  
pp. 687-697 ◽  
Author(s):  
J Beek ◽  
H Nauwynck ◽  
D Maes ◽  
A Van Soom
Keyword(s):  
Sperm Quality ◽  
Membrane Integrity ◽  
Inhibitory Effect ◽  
Fertilization Rate ◽  
Quality Parameters ◽  
Fertilization In Vitro ◽  
Cumulus Oophorus ◽  
Sperm Penetration

In this study, we report for the first time on a possible contribution of metalloproteases in sperm passage through the cumulus matrix in pigs. The presence of 20 μM 1,10-phenanthroline (1,10-PHEN), inhibitor of zinc-dependent metalloproteases, strongly inhibited the degree of sperm penetration in cumulus-intact (CI), but not in cumulus-free (CF), porcine oocytes during IVF. The inhibitory effect of 1,10-PHEN was due to the chelation of metal ions as a non-chelating analog (1,7-PHEN) did not affect IVF rates. Furthermore, incubation with 1,10-PHEN did not affect sperm binding to the zona pellucida nor sperm motility, membrane integrity, or acrosomal status. These findings led to the assumption that 1,10-PHEN interacts with a sperm- or cumulus-derived metalloprotease. Metalloproteases are key players in physiological processes involving degradation or remodeling of extracellular matrix. In vivo, their proteolytic activity is regulated by tissue inhibitors of metalloproteases (TIMP1–TIMP4). We tested the effect of TIMP3 on fertilization parameters after porcine IVF. Similar to 1,10-PHEN, TIMP3 inhibited total fertilization rate of CI but not CF oocytes and did not influence sperm quality parameters. Although the inhibitory effect was stronger in CI oocytes, TIMP3 also reduced the degree of sperm penetration in CF oocytes, suggesting the involvement of a metalloprotease in a subsequent step during fertilization. In conclusion, our results indicate the involvement of TIMP3-sensitive, zinc-dependent metalloprotease activity in sperm passage through the cumulus oophorus in pigs. The results should provide the basis for further biochemical research toward the localization and identification of the metalloprotease involved.

scholarly journals The Re-Addition of Seminal Plasma after Thawing Does Not Improve Buck Sperm Quality Parameters

Animals ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 3452
Author(s):  
Uchechi Linda Ohaneje ◽  
Uchebuchi Ike Osuagwuh ◽  
Manuel Alvarez-Rodríguez ◽  
Iván Yánez-Ortiz ◽  
Abigail Tabarez ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Quality Parameters ◽  
Mitochondrial Activity ◽  
Dna Integrity ◽  
Vitro Fertilization

In order to achieve a higher post-thaw buck sperm quality, an approach in the thawing protocol of cryopreserved sperm doses under in vitro capacitation conditions mimicking the in vivo female environment was studied. Therefore, functional and kinetic characteristics of buck thawed sperm from males of different ages, the season of collection, and melatonin implanted males in the non-breeding season were assessed after 3 h of incubation in an in vitro fertilization (IVF) media with 20% of buck seminal plasma (SP). Previously, fresh ejaculates were collected via artificial vagina from eight males of the Cabra Blanca de Rasquera breed during two consecutive years in breeding and non-breeding periods. Prior to semen collection in non-breeding seasons, males were split into two groups: one group was implanted with melatonin, while the other was not. In each group, semen samples were pooled, centrifuged, and diluted in an extender containing 15% powdered egg yolk and 5% glycerol before freezing. After thawing, sperm were washed and incubated in three different media: (a) control media (modified phosphate-buffered saline (PBS), (b) IVF commercial media, and (c) IVF media + 20% SP. Sperm motility was evaluated by CASA, while plasma and acrosome membrane integrity, mitochondria activity, and DNA fragmentation were analysed by flow cytometer at 0 h and after 3 h incubation. A significant reduction in motility, mitochondrial activity, plasma, and acrosome membrane integrity were observed after incubation in the presence of SP, although similar to that observed in IVF media alone. DNA integrity was not affected under in vitro capacitation conditions, regardless of SP addition. In conclusion, the addition of SP failed to improve post-thaw buck sperm quality under in vitro conditions irrespective of male age, the season of collection, and melatonin implant.

scholarly journals Validation of the Turkey Semen Cryopreservation by Evaluating the Effect of Two Diluents and the Inseminating Doses

Animals ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1329
Author(s):  
Michele Di Iorio ◽  
Giusy Rusco ◽  
Roberta Iampietro ◽  
Lucia Maiuro ◽  
Achille Schiavone ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Sperm Concentration ◽  
Membrane Integrity ◽  
In Vivo Test ◽  
Sperm Analysis ◽  
Fertilizing Ability ◽  
Vitro Analysis ◽  
Hatching Rates

This study was designed to test the fertilizing ability of cryopreserved turkey semen, and here, two experiments were performed: an in vitro analysis to assess the effects of Tselutin and Lake diluents and an in vivo test to determine the fertility and hatching rates by also studying the feat of three insemination doses (250, 400 and 600 × 106 sperm/hen). Pooled semen samples were diluted with Tselutin or Lake extender which contained 20% of dimethylsulfoxide and 1 mM of Ficoll at final sperm concentration of 3 × 109 sperm/mL. Thereafter, semen was packaged into straws and frozen on liquid nitrogen. The post-thaw sperm quality was evaluated considering motility (computer-aided sperm analysis—CASA system) and membrane integrity (flow cytometry). Significantly higher values of progressive motility and some kinetic parameters in semen frozen with Lake were found. When we compared the extenders in vivo, no significant effects were detected, whilst sperm concentration significantly affected both fertility and hatching rates, with the best results obtained with the sperm concentration of 400 × 106 sperm/hen. From the results obtained, it emerged that the extender type only affected sperm motility characteristics, not the fertilizing ability of frozen-thawed semen, while inseminating dose markedly affected fertility and hatching rates.

scholarly journals Expression and putative function of fibronectin and its receptor (integrin α5β1) in male and female gametes during bovine fertilization in vitro

Reproduction ◽  
2009 ◽  
Vol 138 (3) ◽  
pp. 471-482 ◽  
Author(s):  
Mirjan Thys ◽  
Hans Nauwynck ◽  
Dominiek Maes ◽  
Maarten Hoogewijs ◽  
Dries Vercauteren ◽  
...  
Keyword(s):  
Sperm Cell ◽  
Acrosome Reaction ◽  
Male Gamete ◽  
Inhibitory Effect ◽  
Fertilization In Vitro ◽  
Integrin Receptor ◽  
Male And Female ◽  
Sperm Penetration ◽  
Bovine Spermatozoa

Fibronectin (Fn) is a 440 kDa glycoprotein assumed to participate in sperm–egg interaction in human. Recently, it has been demonstrated that Fn – when present during bovine IVF – strongly inhibits sperm penetration. The present study was conducted firstly to evaluate the expression of Fn and its integrin receptor (α5β1) on male and female bovine gametes using indirect immunofluorescence and secondly, to determine the function of Fn during bovine IVF. Endogenous Fn was detected underneath the zona pellucida (ZP) and integrin α5 on the oolemma of cumulus-denuded oocytes. Bovine spermatozoa displayed integrin α5 at their equatorial segment after acrosome reaction. We established that the main inhibitory effect of exogenously supplemented Fn was located at the sperm–oolemma binding, with a (concurrent) effect on fusion, and this can probably be attributed to the binding of Fn to spermatozoa at the equatorial segment, as shown by means of Alexa Fluor 488-conjugated Fn. Combining these results, the inhibitory effect of exogenously supplemented Fn seemed to be exerted on the male gamete by binding to the exposed integrin α5β1 receptor after acrosome reaction. The presence of endogenous Fn underneath the ZP together with integrin α5 expression on oolemma and acrosome-reacted (AR) sperm cell surface suggests a ‘velcro’ interaction between the endogenous Fn ligand and corresponding receptors on both (AR) sperm cell and oolemma, initiating sperm–egg binding.

scholarly journals The Antibacterial and Antioxidant Roles of Buckwheat Honey (BH) in Liquid Preservation of Boar Semen

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Qun Lan ◽  
Yingyu Xie ◽  
Jiahua Pan ◽  
Qiaohui Chen ◽  
Tianfang Xiao ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Antioxidant Properties ◽  
Membrane Integrity ◽  
Storage Period ◽  
Antibiotic Use ◽  
Quality Parameters ◽  
Progressive Motility ◽  
Liquid Storage ◽  
Boar Semen

In the present study, we hypothesized that buckwheat honey (BH) should be regarded as a potential alternative to antibacterial and antioxidant agent in liquid storage of boar semen. To this end, boar semen was firstly studied for in vitro dose tolerability to BH by measuring sperm progressive motility. The optimum progressive motility of boar spermatozoa was observed in extender with 0.5% and 0.6% BH addition. Afterward, sperm quality parameters, bacterial profile and composition, total antioxidant (T-AOC), catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) levels of control, BH supplementation, antibiotics supplementation, and incorporated supplementation were compared during liquid storage period, to further investigate antibacterial and antioxidant properties of BH. The results showed that BH supplementation significantly improved sperm motility, acrosome integrity, plasma membrane integrity, inhibited opportunistic bacterial growth, and altered microbial compositions at the end of preservation. Additionally, T-AOC, SOD, and CAT levels were significantly higher in the BH supplementation group than those in the control and antibiotic supplementation group, whereas MDA level exhibited opposite change pattern. Importantly, BH addition to the extender was able to exert a synergistic effect in combination of antibiotic use. Our findings suggested that the appropriate concentrations (0.5% and 0.6%) of BH were added to the extender could act antibacterial and antioxidant roles in liquid preservation of boar semen.

scholarly journals Comparing the Effect of Different Antibiotics in Frozen-Thawed Ram Sperm: Is It Possible to Avoid Their Addition?

2021 ◽  
Vol 8 ◽  
Author(s):  
Luis Anel-Lopez ◽  
Marta F. Riesco ◽  
Rafael Montes-Garrido ◽  
Marta Neila-Montero ◽  
Juan C. Boixo ◽  
...  
Keyword(s):  
Bacterial Growth ◽  
Sperm Quality ◽  
Inhibitory Effect ◽  
Contamination Control ◽  
Health Agencies ◽  
Different Temperatures ◽  
Species Specific ◽  
Ram Sperm

It is crucial to perform a deep study about the most extensively used antibiotics in sperm extenders. Most of the protocols and concentrations used in ram are direct extrapolations from other species. It is important to establish species-specific antibiotic treatments to optimize their use and if it is possible to reduce the quantity. Previews studies have assessed some aspects of sperm quality in vitro, but this study aimed to go further and assess the effect of three different antibiotic treatments, which are the most extensively used, not only in sperm quality or assessing the inhibitory effect on bacterial growth but also assessing these important parameters of productivity such as fertility, prolificacy, fecundity, and sex-ratio during a freeze-thaw process. Gentamicyn (G) treatment showed the worst results, not only concerning sperm quality but also in the reproductive trials exhibiting a toxical effect at the experiment concentration, and being the most powerful inhibiting bacterial growth. For its part, Lincomicyn-spectinomycin (LS) showed similar results inhibiting bacterial growth but it did not show a detrimental effect either in sperm quality or in reproductive parameters. Penicillin-streptomycin (PS) showed good results in the sperm quality and in the reproductive in vivo trials, but it showed a very poor effect inhibiting bacterial growth probably due to some kind of antibiotic resistance. According to our results, there is not a significant positive relationship between the higher bacterial inhibitory activity of LS and PS samples, and the sperm quality respect Control samples (without antibiotics). In the case of G, which exhibited the most effective as antibacterial, we observed a toxic effect on sperm quality that could be translated on productivity parameters. Our results suggest that the bacterial contamination control in frozen-thawed semen may be possible without the use of antibiotics, although the effects of longer periods of cooling storage and different temperatures of storage need to be further investigated for animal semen. At this point, a reflection about a drastic reduction in the use of antibiotic treatments in sperm cryopreservation is mandatory, since freezing conditions could keep sperm doses contamination within the levels recommended by regulatory health agencies.

scholarly journals Effect of L-carnitine on sperm quality during liquid storage of boar semen

2020 ◽  
Vol 33 (11) ◽  
pp. 1763-1769
Author(s):  
Kang Yang ◽  
Na Wang ◽  
Hai-Tao Guo ◽  
Jing-Ran Wang ◽  
Huan-Huan Sun ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Quality Parameters ◽  
Atp Content ◽  
Developmental Potential ◽  
Positive Effects ◽  
Liquid Storage ◽  
Boar Sperm

Objective: This study was conducted to investigate the effect of L-carnitine on the pig semen characteristics during storage.Methods: Spermatozoa samples were examined for spermatozoa quality and then randomly divided into 5 groups: 0 (control), 12.5, 25, 50, and 100 mM L-carnitine. Sperm motility, plasma membrane integrity and antioxidant parameters (total reactive oxygen species, total antioxidant capacity, and malondialdehyde) were evaluated after 0, 3, 5, and 10 day cooledstorage at 17°C. Moreover, ATP content, mitochondria activity as well as sperm-binding and in vitro fertilizing ability of preserved boar sperm were also investigated.Results: Supplementation with 50 mM L-carnitine could effectively maintain boar sperm quality parameters such as sperm motility and membrane integrity. Besides, we found that L-carnitine had positive effects on boar sperm quality mainly through improving antioxidant capacities and enhancing ATP content and mitochondria activity. Interestingly, by assessing the effect of L-carnitine on sperm fertility and developmental potential, we discovered that the extender containing L-carnitine could improve sperm quality and increase the number of sperms bounding to zona pellucida, without improving in vitro fertility and development potential.Conclusion: These findings suggested that the proper addition of L-carnitine to the semen extender improved boar sperm quality during liquid storage at 17°C.

Efficacy of tumor treatment field (TTF) therapy alone and in combination with chemotherapyin pancreatic cancer preclinical models.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e14616-e14616
Author(s):  
Moshe Giladi ◽  
Rosa S. Schneiderman ◽  
Yaara Porat ◽  
Aviran Itzhaki ◽  
Daniel Mordechovich ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Pancreatic Adenocarcinoma ◽  
Cell Lines ◽  
Treatment Modality ◽  
Membrane Integrity ◽  
Inhibitory Effect ◽  
Pancreatic Tumors ◽  
In Vivo Models

e14616 Background: TTF therapy is a novel, non-invasive treatment modality for solid tumors and was recently approved by the FDA for recurrent glioblastoma. It utilizes alternating electric fields to inhibit tumor growth, by mitotic spindle disruption and destruction of plasma membrane integrity during cytokinesis. TTF inhibits the growth of many solid tumor cell lines in vitro and in vivo. The optimal treatment for pancreas cancer remains elusive, thus we sought to evaluate the efficacy of TTF in pre-clinical pancreatic cancer models. Methods: Cultures of hamster and human pancreatic adenocarcinoma cell lines (PC1-0 and AsPC-1, respectively) were treated with TTF (frequencies ranging from 75 to 300 kHz), using two pairs of perpendicularly oriented insulated transducer arrays. Once determining optimal frequency, TTF was combined with chemotherapy (gemcitabine or 5-Fluorouracil, 5-FU). Hamsters bearing syngeneic, orthotopic pancreatic tumors were treated with either TTF alone or in combination with gemcitabine or 5-FU. Results: TTF treatment had significant inhibitory effect on proliferation of pancreatic cancer cultures. The maximal inhibitory effect for PC1-0 and ASPC-1 was observed when TTF frequencies of 100 and 150 kHz were applied (respectively). The application of TTF to cultures treated with either gemcitabine or 5-FU resulted in an additive inhibitory effect. In-vivo, TTF therapy, either alone or in combination with chemotherapy, resulted in a significant decrease in tumor weight and volume. Compared to chemotherapy alone, TTF increased tumor response to both gemcitabine and 5-FU. Histological analysis demonstrated higher mitotic index in TTF-treated tumors, consistent with the mitotic arrest previously shown in TTF treated cultures. Conclusions: TTF therapy demonstrated efficacy in pancreatic adenocarcinoma in both in vitro and in vivo models. These results support the evaluation of this novel treatment modality in combination with standard chemotherapy in pancreatic cancer patients. A pilot study is in development to test the clinical benefit of combined TTF and gemcitabine in patients with advanced pancreatic adenocarcinoma.

Combined Inhibitory Effect against Postharvest Storage Rots and Their Effects on Postharvest Quality Parameters in Cherry Tomatoes by Cassia Oil and Calcium Chloride

2013 ◽  
Vol 76 (11) ◽  
pp. 1873-1878 ◽  
Author(s):  
WU FENG ◽  
XIAODONG ZHENG ◽  
JIAPING CHEN
Keyword(s):  
Calcium Chloride ◽  
Enzyme Activities ◽  
Inhibitory Effect ◽  
Quality Parameters ◽  
Significant Inhibition ◽  
Postharvest Quality ◽  
Related Enzyme ◽  
Cherry Tomatoes

The inhibitory effect of cassia oil alone or in combination with calcium chloride (CaCl2) against Alternaria alternata in vitro and in vivo was assessed on cherry tomatoes. The results demonstrated that concentrations of CaCl2 ranging from 0.25 to 3%enhanced the inhibitory effects of 200 μl of cassia oil per liter on the growth of A. alternata in vitro. The combination of 0.25% CaCl2 and 500 μl of cassia oil per liter showed a significant inhibition effect on decay development in both wounded artificially infected and unwounded naturally infected fruit. Importantly, these treatments did not reduce the overall quality of tomatoes. Defense-related enzyme activities were also evaluated. The results indicated that cassia oil alone or in combination with CaCl2 significantly enhanced defense-related enzyme activities, such as peroxidase and polyphenol oxidase. Together, these data suggest that the combination of cassia oil and CaCl2 may be an efficient method to limit cherry tomato decay caused by fungi.

309 DIFFERENCES IN PRONUCLEUS FORMATION RATES BETWEEN BULLS IN RESPONSE TO GAMMA IRRADIATION OF FROZEN - THAWED SEMEN

2007 ◽  
Vol 19 (1) ◽  
pp. 270
Author(s):  
M. M. Petersen ◽  
G. B. Boe-Hansen ◽  
A. Birck ◽  
B. Avery ◽  
M. Jensen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Gamma Irradiation ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Fertilization Rate ◽  
High Dose ◽  
Exact Test ◽  
Bovine Spermatozoa ◽  
Gamma Irradiated

Gamma irradiation induces DNA damage to mature bovine spermatozoa but does not affect motility, membrane integrity, or function (Fatehi et al. 2006 J. Androl. 27, 176–188), making it a useful model to evaluate the effect of fertilization with DNA-damaged spermatozoa. The objective of the present study was to analyze the fertilizing capacity of frozen–thawed gamma-irradiated sperm from 2 bulls: a bull with a high sperm quality and fertilization rate (bull A), and a bull with a low sperm quality and fertilization rate (bull B). To ensure that DNA damage was induced, frozen–thawed semen was exposed to a high dose of irradiation (90 Gy). Cumulus–oocyte complexes were obtained from abattoir ovaries and were in vitro-matured (IVM) using standard procedures (23 h in DMEM with 5% serum and eCG/hCG). For each treatment group, 3 to 5 straws of semen from the same ejaculate were used. After thawing, semen from the 2 bulls was either irradiated or held at room temperature before 1:1 dilution in sperm-TALP. During irradiation, semen was kept in the straws. The irradiated and nonirradiated sperm were used for IVF (23 h in IVF-TALP) within 1 h of thawing. IVM and IVF were carried out at 38.5�C in 5% CO2 in air. After IVF, presumptive zygotes were whole mount-fixed and 24 h later were stained with 1% aceto-orcein followed by determination of fertilization status. Fertilization was considered normal if 2 pronuclei (PN) were present. A total of 4 replicates were performed and 286 zygotes analyzed. No difference was detected between replicates, and the results were pooled. Fisher's exact test was used to determine effect of treatment. More zygotes had 2 PN after fertilization with semen from bull A (47/67 = 70%), compared with that from bull B (16/71 = 23%; P < 0.0001) using nonirradiated sperm. Irradiation of sperm significantly increased the fraction of zygotes with 2 PN from bull A (61/70 = 87%; P = 0.03), but decreased the 2 PN fractions in zygotes fertilized with sperm from bull B (2/75 = 3%; P = 0.0002). Ideally, to avoid straw variation, semen should have been pooled and divided into groups before irradiation. The variation between straws might explain the higher fertilization rates in bull A using irradiated sperm compared with nonirradiated sperm. In conclusion, there appear to be differences in fertilizing ability between bulls after irradiation of frozen–thawed sperm. This could be due to suboptimal DNA packaging, which made sperm from bull B more susceptible to radiation-induced damage. The potential irradiation-induced increase in DNA fragmentation in sperm from bull B compared with that from bull A might delay or prevent the formation of the 2 PN. Further studies are needed to investigate differences in fertilization and early embryonic development using sperm with intact or damaged DNA.

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