scholarly journals Protective Effect of Chlorogenic Acid on Human Sperm: In Vitro Studies and Frozen—Thawed Protocol

Antioxidants â—½  
2021 â—½  
Vol 10 (5) â—½  
pp. 744
Author(s):  
Daria Noto â—½  
Giulia Collodel â—½  
Daniela Cerretani â—½  
Cinzia Signorini â—½  
Laura Gambera â—½  
...  
Keyword(s):  
Chlorogenic Acid â—½  
Sperm Motility â—½  
Plasma Membranes â—½  
Culture Media â—½  
Human Sperm â—½  
Dna Integrity â—½  
H2o2 Treatment â—½  
Transmission Electron â—½  
The Media

The study evaluated the chlorogenic acid (CGA) antioxidant potential on oxidative stress (OS) induced in vitro in human spermatozoa and during cryopreservation procedure. Swim-up selected spermatozoa were treated with 100 µM CGA, 100 µM H2O2 to induce lipid peroxidation (LPO), and with both compounds and the effects on mitochondrial membrane potential (MMP) by JC-1, DNA integrity by acridine orange (AO), and sperm ultrastructure by transmission electron microscopy (TEM), were evaluated. CGA antioxidant activity was assessed by measuring malondialdehyde (MDA) and F2-isoprostanes (F2-IsoPs) in the media. The CGA protective activity and the immunolocalization of Phospho-AMPKα (Thr172) were explored in frozen-thawed sperm. CGA was not toxic for sperm motility, DNA integrity and MMP. The increase in MDA (p < 0.05) and F2-IsoPs (p < 0.001), DNA damage (p < 0.01) and low MMP (p < 0.01) levels after H2O2 treatment were reduced in presence of CGA as well as the percentage of broken plasma membranes (p < 0.01) and altered acrosomes (p < 0.01) detected by TEM. Treated frozen-thawed spermatozoa showed increased sperm motility (p < 0.01), DNA integrity (p < 0.01), MMP (p < 0.01), reduced MDA (p < 0.01) and increased sperm percentage with Phospho-AMPKα labelling in the head (p < 0.001). CGA can be used to supplement culture media during semen handling and cryopreservation where OS is exacerbated.

Omeprazole does not alter human sperm motility, viability or DNA integrity in vitro

Andrologia â—½  
10.1111/and.13260 â—½  
2019 â—½  
Vol 51 (6) â—½  
pp. e13260 â—½  
Author(s):  
Saleem Ali Banihani â—½  
Alayna'‐almarddyah Abdullah Al‐khawalde
Keyword(s):  
Sperm Motility â—½  
Human Sperm â—½  
Dna Integrity

scholarly journals Effect of Microsphaeropsis sp. Strain P130A on Germination and Production of Sclerotia of Rhizoctonia solani and Interaction Between the Antagonist and the Pathogen

Phytopathology â—½  
2001 â—½  
Vol 91 (8) â—½  
pp. 782-791 â—½  
Author(s):  
Odile Carisse â—½  
Souad El Bassam â—½  
Nicole Benhamou
Keyword(s):  
Rhizoctonia Solani â—½  
Plasma Membranes â—½  
Culture Media â—½  
Electron Microscopic â—½  
Transmission Electron â—½  
Incubation Periods â—½  
Percent Germination â—½  
Wall Appositions â—½  
Potential Antagonist

Microsphaeropsis sp. strain P130A was evaluated for the control of tuber-borne inoculum of Rhizoctonia solani based on the viability of sclerotia produced in vitro and on both the viability and production of tuber-borne sclerotia. The interactions between the antagonist and the pathogen, as well as the effect of the toxins produced by the antagonist on mycelial growth of R. solani were studied using transmission electron microscopy. On sclerotia produced in vitro, for all incubation periods (1 to 42 days), Microsphaeropsis sp. significantly reduced germination. Percent germination of sclerotia treated with Microsphaeropsis sp. decreased with increasing incubation period from an average of 82.0% after 1 day to stabilize at an average of 5.8% after 35 days. Similarly, percent germination of tuber-borne sclerotia was significantly lower when tubers were treated with Microsphaeropsis sp. Both 2% formaldehyde and Microsphaeropsis sp. treatments significantly reduced sclerotia germination to approximately 10% after 42 days of incubation at 4°C. Furthermore, on tubers treated with the antagonist, the number of sclerotia per square centimeter decreased from 1.6 to 0.5 during the 8 months of storage at 4°C, whereas an increase from 1.2 to 7.8 sclerotia per square centimeter was observed on untreated tubers. Microsphaeropsis sp. (strain P130A) colonized hyphae of R. solani within 4 days after contact on culture media. Transmission electron microscopic observations showed that the antagonist induced a rupture of the pathogen plasma membrane and that a chitin-enriched matrix was deposited at sites of potential antagonist penetration. Host penetration was not associated with pathogen cell wall alterations, which occurred at the time of progress of the antagonist in the pathogen cytoplasm. In the presence of a crude extract of Microsphaeropsis sp., cells of R. solani showed cytoplasm disorganization and breakdown of plasma membranes. Antibiosis and mycoparasitism were involved in the antagonism of R. solani by Microsphaeropsis sp., but the sequence by which these events occur, as well as the significance of wall appositions produced by R. solani, is yet to be established.

scholarly journals Cytotoxic Effects of Benzene Metabolites on Human Sperm Function: An In Vitro Study

ISRN Toxicology â—½  
10.1155/2013/397524 â—½  
2013 â—½  
Vol 2013 â—½  
pp. 1-6 â—½  
Author(s):  
Priyanka Mandani â—½  
Ketki Desai â—½  
Hyacinth Highland
Keyword(s):  
Sperm Motility â—½  
Nuclear Dna â—½  
In Vitro Study â—½  
Human Sperm â—½  
Dna Integrity â—½  
Sperm Function â—½  
Cytotoxic Effects â—½  
Membrane Function â—½  
Atp Production

In recent years, individuals are rampantly exposed to vapours of benzene, through paint, plastic, petroleum industries, fuel exhaust, and tobacco smoke. Hence the present investigation was directed towards determining the effect of benzene metabolites, namely, phenol-hydroquinone and catechol, on the motility, viability, and nuclear integrity of the human spermatozoa. From the results obtained it was clear that exposure to phenol-hydroquinone caused a significant decline in both, sperm motility and viability. Exposure to a phenol-hydroquinone (Phase I) microenvironment may therefore inhibit metabolically active enzymes, thus impeding ATP production, and in turn lowers sperm motility and viability. In addition, the present study also revealed that both metabolites of benzene caused significant denaturation of sperm nuclear DNA. Hence, exposure to phenol-hydroquinone in vitro could have resulted in generation of free radicals and altered membrane function, which is reflected by a decline in the motility, viability, and loss of sperm nuclear DNA integrity. In Phase II, the exposure of human sperm in vitro to varied concentrations of catechol caused only insignificant changes in sperm motility and viability as compared to those observed on exposure to phenol-hydroquinone. Hence, exposure to catechol appeared to have less toxic effects than those of phenol-hydroquinone.

scholarly journals Testing Strategies of the In Vitro Micronucleus Assay for the Genotoxicity Assessment of Nanomaterials in BEAS-2B Cells

Nanomaterials â—½  
2021 â—½  
Vol 11 (8) â—½  
pp. 1929
Author(s):  
Tereza Cervena â—½  
Andrea Rossnerova â—½  
Tana Zavodna â—½  
Jitka Sikorova â—½  
Kristyna Vrbova â—½  
...  
Keyword(s):  
Cytochalasin B â—½  
Culture Media â—½  
Methodological Approach â—½  
Micronucleus Assay â—½  
Toxicity Testing â—½  
In Vitro Toxicity â—½  
Testing Strategies â—½  
Transmission Electron â—½  
The Impact

The evaluation of the frequency of micronuclei (MN) is a broadly utilised approach in in vitro toxicity testing. Nevertheless, the specific properties of nanomaterials (NMs) give rise to concerns regarding the optimal methodological variants of the MN assay. In bronchial epithelial cells (BEAS-2B), we tested the genotoxicity of five types of NMs (TiO2: NM101, NM103; SiO2: NM200; Ag: NM300K, NM302) using four variants of MN protocols, differing in the time of exposure and the application of cytochalasin-B combined with the simultaneous and delayed co-treatment with NMs. Using transmission electron microscopy, we evaluated the impact of cytochalasin-B on the transport of NMs into the cells. To assess the behaviour of NMs in a culture media for individual testing conditions, we used dynamic light scattering measurement. The presence of NMs in the cells, their intracellular aggregation and dispersion properties were comparable when tests with or without cytochalasin-B were performed. The genotoxic potential of various TiO2 and Ag particles differed (NM101 < NM103 and NM302 < NM300K, respectively). The application of cytochalasin-B tended to increase the percentage of aberrant cells. In conclusion, the comparison of the testing strategies revealed that the level of DNA damage induced by NMs is affected by the selected methodological approach. This fact should be considered in the interpretation of the results of genotoxicity tests.

Biochemical markers for pregnancy in the spent culture medium of in vitro produced bovine embryos

2021 â—½  
Author(s):  
Gabriela de Oliveira Fernandes â—½  
Marcella Pecora Milazzotto â—½  
Andrei Antonioni Guedes Fidelis â—½  
Taynan Stonoga Kawamoto â—½  
Ligiane de Oliveira Leme â—½  
...  
Keyword(s):  
Mass Spectrometry â—½  
Biochemical Markers â—½  
Culture Medium â—½  
Culture Media â—½  
Ionization Mass â—½  
Metabolic Profiles â—½  
Bovine Embryos â—½  
The Media

Abstract The present study aimed to identify biomarkers to assess the quality of in vitro produced (IVP) bovine embryos in the culture media. IVP embryos on Day (D) 5 of development were transferred to individual drops, where they were maintained for the last 48 h of culture. Thereafter, the medium was collected and the embryos were transferred to the recipients. After pregnancy diagnosis, the media were grouped into the pregnant and nonpregnant groups. The metabolic profiles of the media were analyzed via electrospray ionization mass spectrometry, and the concentrations of pyruvate, lactate, and glutamate were assessed using fluorimetry. The spectrometric profile revealed that the media from embryos from the pregnant group presented a higher signal intensity compared to that of the nonpregnant group; the ions 156.13 Da [M + H]+, 444.33 Da [M + H]+, and 305.97 Da [M + H]+ were identified as biomarkers. Spent culture medium from expanded blastocysts (Bx) that established pregnancy had a greater concentration of pyruvate (p = 0.0174) and lesser concentration of lactate (p = 0.042) than spent culture medium from Bx that did not establish pregnancy. Moreover, pyruvate in the culture media of Bx can predict pregnancy with 90.9% sensitivity and 75% specificity. In conclusion, we identified markers in the culture media that helped in assessing the most viable IVP embryos with a greater potential to establish pregnancy.

scholarly journals Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Essential Oils from AgNPs and AuNPs Elicited Lavandula angustifolia In Vitro Cultures

Molecules â—½  
2019 â—½  
Vol 24 (3) â—½  
pp. 606 â—½  
Author(s):  
Aneta Wesołowska â—½  
Paula Jadczak â—½  
Danuta Kulpa â—½  
Włodzimierz Przewodowski
Keyword(s):  
Molecular Weight â—½  
Silver Nanoparticles â—½  
Essential Oils â—½  
Culture Media â—½  
In Vitro Cultures â—½  
Gas Chromatography Mass Spectrometry â—½  
Lavandula Angustifolia â—½  
Gold And Silver Nanoparticles â—½  
The Media

The aim of this study was to determine how the addition of gold and silver nanoparticles to culture media affects the composition of essential oils extracted from Lavandula angustifolia propagated on MS media with the addition of 10 and 50 mg·dm−3 of gold (24.2 ± 2.4 nm) and silver (27.5 ± 4.8 nm) nanocolloids. The oil extracted from the lavender tissues propagated on the medium with 10 mg·dm−3 AgNPs (silver nanoparticles) differed the most with respect to the control; oil-10 compounds were not found at all, and 13 others were detected which were not present in the control oil. The addition of AuNPs (gold nanoparticles) and AgNPs to the media resulted in a decrease of lower molecular weight compounds (e.g., α- and β-pinene, camphene, δ-3-carene, p-cymene, 1,8-cineole, trans-pinocarveol, camphoriborneol), which were replaced by those of a higher molecular weight (τ- and α-cadinol 9-cedranone, cadalene, α-bisabolol, cis-14-nor-muurol-5-en-4-one, (E,E)-farnesol).

284 COMPARATIVE FERTILITY OF FRESHLY-COLLECTED VERSUS FROZEN - THAWED SPERMATOZOA FOR IN VITRO FERTILIZATION IN THE FISHING CAT (PRIONAILURUS VIVERRINUS)

2006 â—½  
Vol 18 (2) â—½  
pp. 249
Author(s):  
G. Magarey â—½  
J. Herrick â—½  
K. Thiangtum â—½  
W. Tunwattana â—½  
W. Swanson
Keyword(s):  
Sperm Motility â—½  
Culture Medium â—½  
Culture Media â—½  
Egg Yolk â—½  
Ovarian Follicles â—½  
Blastocyst Stage â—½  
Motile Sperm â—½  
Vitro Fertilization â—½  
International Transport

Wild populations of fishing cats (Prionailurus viverrinus) in Southeast Asia are in decline, primarily due to habitat loss. Because the fishing cat population in North American zoos is small (n = 69) and inbred (F = 0.17) with relatively low genetic variation (86%), infusion of new founder genes from Asia is a conservation priority. Importation of cryopreserved semen for use with IVF and ET may offer one alternative to the international transport of living animals. In this study, our objectives were to (1) compare motility longevity of fresh vs. frozen-thawed fishing cat spermatozoa in two culture media, (2) evaluate ovarian responses to exogenous gonadotropins, and (3) assess development of IVF embryos produced with fresh vs. frozen-thawed spermatozoa. Raw semen was collected via electroejaculation from male fishing cats (n = 4), divided into groups, and washed. Two sperm pellets were resuspended in either Ham's F10 medium (HF10; with 5% FBS) or our feline optimized culture medium (FOCM; with 0.4% BSA); another pellet was diluted in TEST egg yolk, cooled to 5�C over 3 h, glycerated (4%), and cryopreserved in straws over LN2 vapor. Frozen sperm samples were thawed, washed, and diluted in either HF10 or FOCM. Fresh and frozen-thawed sperm motility (percent motile, rate of forward progress) in each medium (10 � 106 motile sperm/mL) was assessed (at 0, 1, 3, and 6 h) in microdrops under oil during culture (38�C; 6% CO2 in air). Female fishing cats (n = 10) were treated with exogenous gonadotropins (150 IU eCG, 100 IU hCG, 85-h interval) and ovarian follicles were aspirated laparoscopically. Recovered oocytes were inseminated with fresh (2 � 105 motile sperm/mL) or frozen-thawed (5 � 105 motile sperm/mL) spermatozoa in FOCM microdrops; resulting embryos were either cryopreserved or cultured in FOCM (with 5% FBS added at 72 h post-insemination) for 7 days. Sperm motility over time did not differ (P > 0.05) between media for either fresh or frozen-thawed samples; however, across media, frozen-thawed sperm motility was lower (P < 0.05) and declined faster (P < 0.05) compared to fresh spermatozoa. Females produced an average (�SEM) of 9.8 � 2.9 mature ovarian follicles, allowing recovery of 7.3 � 2.6 high-quality oocytes per female. Oocyte cleavage percentage at 42 h p.i. was lower (P < 0.05) with frozen-thawed spermatozoa (38%, 11/29) compared to freshly collected spermatozoa (68%, 17/25). Overall, 35% (6/17) of cultured embryos developed to blastocysts with no difference (P > 0.05) between embryos produced with frozen-thawed (4/11) vs. fresh (2/6) spermatozoa. Although fishing cat sperm motility and fertility appear compromised after cryopreservation, our results demonstrate the ability of frozen-thawed spermatozoa to produce IVF embryos that are capable of developing to blastocyst stage in vitro. This work was supported by (NIH RR015388).

99 DIFFERENTIAL GENE REGULATION OF STEROIDOGENIC TRANSCRIPTS AND ESTRADIOL PRODUCTION FOLLOWING IN VITRO PIG EMBRYO ELONGATION IN ALGINATE HYDROGEL THREE-DIMENSIONAL MATRIX

10.1071/rdv24n1ab99 â—½  
2012 â—½  
Vol 24 (1) â—½  
pp. 162
Author(s):  
J. R. Miles â—½  
C. N. Sargus â—½  
S. A. Plautz â—½  
J. L. Vallet â—½  
A. K. Pannier
Keyword(s):  
Equal Opportunity â—½  
Culture Media â—½  
Morphological Changes â—½  
Three Dimensional â—½  
Differential Gene Regulation â—½  
Alginate Hydrogels â—½  
The Media â—½  
And Control

Between Day 10 and 12 of gestation, the pig embryo elongates from a sphere to a long thin, filament. During this time, the embryo increases the production of oestrogen via an increase in steroidogenic transcripts, which is critical for maternal recognition of pregnancy. To date, attempts to elongate porcine embryos in vitro have been unsuccessful. Therefore, the objective of this study was to utilise alginate hydrogels to establish a culture system that promotes in vitro embryo elongation with a corresponding increase in steroidogenic transcripts and oestradiol production. In 3 replicate collections, White crossbred gilts (n = 15) were bred at Day 0 of the oestrous cycle. At Day 9 of gestation, reproductive tracts were collected and flushed with RPMI-1640 containing antibiotics. Embryos were recovered, grouped according to size and washed with RPMI-1640 containing antibiotics and 10% fetal bovine serum (FBS). Embryos were randomly assigned to be encapsulated using a double encapsulation technique (0.7% sodium alginate and 1.5% calcium chloride solution) or used as controls. Encapsulated and control embryos were cultured for 96 h in CO2 -pretreated RPMI-1640 containing antibiotics and 10% FBS at 38°C, 5% CO2 in air and 100% humidity. Every 24 h, the embryos were imaged and half of the media was replaced. The removed media was stored at –20°C and used to assess oestradiol levels by radioimmunoassay. At the end of culture, a subset of encapsulated and control embryos were snap frozen and used to assess the expression level of steroidogenic transcripts (STAR, CYP11 and CYP19) using quantitative PCR. All data were analysed using general linear model (GLM) procedures for ANOVA. Cell survival, assessed by blastocyst fragmentation and confirmed by live/dead staining in representative embryos, was greater (P = 0.01) for encapsulated embryos (60.1 ± 4.8%) compared with controls (33.3 ± 4.8%). Of encapsulated embryos, 27% had some morphological change (minor flattening and tubal formation) and 14% had significant morphological changes (considerable flattening and tubal formation elongating through the gel), consistent with in vivo embryo elongation. In contrast, the control embryos had no morphological changes observed and remained spherical during culture. The expression levels of STAR, CYP11 and CYP19 were significantly (P < 0.05) greater in encapsulated embryos compared with control embryos. Furthermore, a significant (P < 0.01) time-dependent increase in oestradiol levels in the culture media of encapsulated embryos was identified compared with controls and culture media alone. These results illustrate that cultured pig embryos encapsulated in alginate hydrogels undergo limited morphological changes with increased expression of steroidogenic transcripts and oestrogen production. †USDA is an equal opportunity provider and employer.

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