108 NEGLIGIBLE LEVEL OF DNA DAMAGE IN BOVINE OOCYTES VITRIFIED USING MINIMUM VOLUME METHODS

2006 ◽  
Vol 18 (2) ◽  
pp. 162
Author(s):  
K. Papis ◽  
E. Stachowiak ◽  
M. Kruszewski ◽  
T. Iwanenko ◽  
T. Bartlomiejczyk
Keyword(s):  
Comet Assay ◽  
Dna Fragmentation ◽  
Sucrose Solution ◽  
Objective Assessment ◽  
Significant Proportion ◽  
Positive Control ◽  
Negative Control ◽  
Dna Integrity ◽  
Minimum Volume ◽  
Bovine Oocytes

A relatively high number of bovine cryopreserved oocytes analyzed by the comet assay (Men et al. 2003 Mol. Reprod. Dev. 64, 245) showed compromised DNA integrity. The DNA fragmentation (comet tails) was found in 29% of slow cooled oocytes, in 20% of oocytes vitrified in straws and in 24% of oocytes vitrified in open pulled straws (OPS). Present study used the comet assay to compare the DNA status of 151 in vitro matured bovine oocytes vitrified in straws, in OPS or in droplets. It was assumed that the droplet method (Papis et al. 2000 Theriogenology 54, 651), which has gentle pre-equilibration prior to vitrification, would offer better protection of DNA. OPS vitrification was performed using a solution consisting of 20% DMSO, 20% ethylene glycol (EG), and 0.5 M sucrose. For in-straw and in-droplet vitrification, VS14 (5.5 M EG and 1.0 M sucrose) solution was used. In these two methods pre-equilibration in 3% EG solution for 15 min was applied. Fresh oocytes exposed to 0.5 mM of hydrogen peroxide for 5 min served as the positive control. Fresh M II oocytes served as the negative control. The comet assay was performed according to the procedure of Men et al. (2003) with some modifications aimed at enhancing the sensitivity of the method. The zona pellucida was removed using 0.5% pronase solution, followed by placing of the oocytes in droplets of low-melting agarose on slides. These were subjected to overnight treatment in lysis buffer, followed by 40 min of DNA unfolding and 30 min electrophoresis. Following air drying, the slides were stained with DAPI fluorochrome and photographed. The pictures were saved as anonymous consecutive files to enable objective assessment. Of 119 vitrified oocytes, 112 (94%) were evaluated. The remainder were lost or displayed atypical pictures. The comets could not be analyzed with the Comet v.3.0 software, possibly due to the large size of each oocyte. Six main classes of comet tails were distinguished ranging from no tail (class 0) to heavy and long tail (class 5). Positive control oocytes displayed class 4 (36%) or 5 (64%) tails. Negative control oocytes formed class 0 (18%) to class 3 (47%) comet tails. The oocytes vitrified using minimum volume methods fell within the same range, with 80% and 76% of oocytes vitrified in droplets and OPS, respectively, forming class 1 or 2 tails. One OPS vitrified oocyte (2.2%) expressed a class 5 tail. A small but significant proportion of oocytes vitrified in straws (15.4%, P d 0.05, ANOVA) formed class 4 tails typical of positive control oocytes. In conclusion, in spite of pre-equilibration, a significant proportion of oocytes vitrified in straws contained detectable levels of DNA fragmentation, due probably to the lower cooling rate. The minimum volume protocols (the droplet and OPS methods) caused virtually no damage as assessed by the DNA comet assay. Results presented here differ from those reported previously. Reasons for differences remain to be established.

scholarly journals AEDES AEGYPTI SURVIVORSHIP ON SALT TOLERANT CALIFORNIA LANDSCAPE PLANTS

2021 ◽  
Vol 66 (1) ◽  
pp. 7-10
Author(s):  
Christopher S. Bibbs
Keyword(s):  
Aedes Aegypti ◽  
Plant Species ◽  
Sucrose Solution ◽  
Water Solution ◽  
The United States ◽  
Positive Control ◽  
Negative Control ◽  
Arid Environments ◽  
Salt Cedar ◽  
Riparian Plants

Aedes aegypti has expanded its range in the United States to include various arid and desert geographies, with notable introduction into various parts of California. Because resources are limited in arid environments, it is currently an important topic to understand how Ae. aegypti interacts with its surrounding environment for survival and proliferation. Three common plant species in peridomestic landscape, i.e., salt cedar (Tamarix aphylla), arrow weed (Pluchea sericea) and four wing saltbush (Atriplex canescens), were collected for survival bioassays to understand how Ae. aegypti is persisting in arid, chaparral landscapes in California, USA. These three plant-species along with a 10% sucrose solution (positive control) and reverse osmosis water solution (negative control-) were added to cages of Ae. aegypti to assess their survival at 24h, 48h, and 96h. It was found, in comparison with the negative control and four wing salt bush, that arrow weed and to a lesser extent salt cedar, promoted survival of Ae. aegypti in the first 24h. After the first day, only arrow weed significantly supported mosquito survival out to 96h as compared to the controls. Arrow weed and salt cedar are both riparian plants producing some nectaries which could be energy resources provided through stem sap or nectar to Ae. aegypti amidst peridomestic chaparral in California.

99 EFFECT OF CYSTEAMINE ON SURVIVAL OF BOVINE AND OVINE OOCYTES VITRIFIED USING THE MINIMUM VOLUME COOLING (MVC) CRYOTOP METHOD

2006 ◽  
Vol 18 (2) ◽  
pp. 158 ◽  
Author(s):  
J. Kelly ◽  
D. Kleemann ◽  
M. Kuwayama ◽  
S. Walker
Keyword(s):  
Ethylene Glycol ◽  
Sucrose Solution ◽  
Comparable Result ◽  
Individual Treatment ◽  
Minimum Volume ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Blastocyst Rate ◽  
Main Effects ◽  
Bovine Oocytes

The addition of cysteamine to maturation (IVM) media increases glutathione (GSH) synthesis in bovine oocytes and improves embryo development and quality (de Matos et al. 1995 Mol. Reprod. Dev. 42, 432–436). This study assesses the effect of adding cysteamine to IVM media on the survival of bovine and ovine oocytes following vitrification using the MVC cryotop method (Kuwayama and Kato 2000 J. Assist. Reprod. Genet. 17, 477 abst.). Abattoir-sourced bovine and ovine cumulus–oocyte complexes (COC) were matured in IVM media with or without 100 μM cysteamine for 24 h. After maturation, the COC were partially denuded and a proportion vitrified. Oocytes were equilibrated with 10% ethylene glycol (EG) and 10% dimethyl sulfoxide (DMSO) for 30 s and then exposed to 20% EG, 20% DMSO, 0.5 M sucrose, and 20% FCS for 15 s. Oocytes were loaded onto a MVC plate (Cryotop, Kitazato Supply, Tokyo), and plunged into liquid nitrogen. After 5 days, oocytes were thawed directly into 1.25 M sucrose solution at 38.5°C, followed by stepwise dilution of the cryoprotectants. Ova were subsequently fertilized (Day 0) and cultured in modified SOF. Oocyte survival was assessed by cleavage and development to Day 8 compared with the development of fresh oocytes. Main effects, interactions and individual treatment differences were tested using procedure CATMOD in SAS. Cleavage rate was higher (P < 0.001) for fresh oocytes than for vitrified oocytes and it increased (P < 0.05) only for fresh ovine oocytes when cysteamine was added to maturation media. Blastocyst development was influenced by a significant (P < 0.001) interaction between species and whether or not oocytes were vitrified. This interaction occurred because cysteamine improved blastocyst rate in fresh ovine and vitrified bovine oocytes but not in other treatments. These results show that bovine oocytes (38.3% blastocyst rate) can be vitrified successfully when maturation occurs in the presence of cysteamine; however, a comparable result did not occur in ovine oocytes (10.6% blastocyst rate) despite >70% cleavage. Table

scholarly journals Low cost and comprehensive pork detection in processed food products with a different food matrix

2018 ◽  
Vol 23 (1) ◽  
pp. 21
Author(s):  
Fenny Aulia Sugiana ◽  
Henni Widyowati ◽  
Muhammad Ali Warisman ◽  
Suryani Suryani ◽  
Desriani Desriani
Keyword(s):  
18S Rrna ◽  
Low Cost ◽  
Meat Products ◽  
Positive Control ◽  
Negative Control ◽  
Dna Integrity ◽  
Processed Meat ◽  
Duplex Pcr ◽  
Processed Food ◽  
Food Matrix

The adulteration of processed beef-based meat products with pork is a sensitive issue in Indonesia. In this study, we developed a detection method for the low cost identification of pork in processed meat products. We used the cost-efficient Taq DNA polymerase, DreamTaq Green PCR master mix (2x), and duplex PCR method to recognize pork simultaneously with 18S rRNA detection. A positive control containing a pork gene inserted into pGEM®-T easy was prepared, along with a negative control. The results of the duplex PCR were used to assess its specificity, detection limit, and its ability to recognize pork in processed meat products with a different food matrix. 18S rRNA detection was for confirming DNA integrity of DNA extracted from the processed food, while the positive control confirmed that the reagents were working well and the negative control confirmed a non-contamination problem. Following this, the duplex PCR was optimized and the optimum concentration primer for duplex PCR detection was found to be 3 µm for pork and 0.2 µm for 18S rRNA. As little as 3.125 ng of the DNA template could be used to detect whether a sample contained pork. Duplex PCR is a simple, fast, sensitive, specific, and low cost method of detecting pork in processed meat products.

scholarly journals Pre-Incubation with Kisspeptin Improves the Adverse Effects of Freeze-Thawed Human Ejaculated Sperms

2020 ◽  
Author(s):  
Tayebeh Kermani ◽  
Seyede Fatemeh Hossein ◽  
Tahereh Talaei-Khozani ◽  
Elham Aliabadi
Keyword(s):  
Sperm Quality ◽  
Fluorescein Isothiocyanate ◽  
Positive Control ◽  
Negative Control ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Dna Integrity ◽  
Freezing And Thawing ◽  
Freeze Thaw ◽  
Beneficial Effects ◽  
Thaw Cycle

Abstract Objectives: Sperm cryopreservation reduces sperm quality. Kisspeptin (KP), as an antioxidant, has beneficial effects on the sperm functions. Therefore, the present study was conducted to use kisspeptin in order to mitigate detrimental effects of the sperm freeze-thawing process and compare it with Glutathione (GSH), as positive control. Materials and methods: 30 normal semen samples, prepared by swim-up procedure, were divided into 3 aliquots: negative control with no treatment; positive control receiving 1mM GSH; and experimental aliquot treated with 10 µM KP for 30 min. All aliquots were cryopreserved, and then thawed after 48 hr. Sperm motility was assessed according to WHO guidelines. Acrosomal reaction and capacitation were evaluated by Fluorescein Isothiocyanate-Conjugated Peanut Agglutinin (FITC-PNA), Wheat Germ Agglutinin (WGA), and Concanavalin A (ConA); percentage of positive cells was evaluated by flow cytometry. Sperm DNA quality was evaluated by Acridine Orange (AO), Aniline Blue (AB), Chromomycin A3 (CMA3), and TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) staining methods. Statistical analyses were performed using ANOVA and LSD.Results: Results of the study showed that, KP supplementation improved motility and led to an increase in the percentage of capacitated and acrosome-intact sperms compared to both controls. Freeze-thaw procedure damaged the DNA integrity severely, and KP pre-treatment significantly reduced frequency of apoptotic sperms as well as those with histone-protamine substitution impairment. Conclusion: Pre-exposure of the sperms to KP can protect the sperm quality including motility and DNA integrity against detrimental influence of freeze–thaw cycle. Therefore, it can be considered as a good pre-additive substance to control the sperm quality during freezing and thawing procedure.

Aktivitas Antifungi Air Perasan Bawang Merah (Allium ascalonicum L.) Terhadap Candida albicans Secara In Vitro

2017 ◽  
Vol 9 (2) ◽  
pp. 71
Author(s):  
Nurhasanah Nurhasanah ◽  
Fauzia Andrini ◽  
Yulis Hamidy
Keyword(s):  
Candida Albicans ◽  
Antifungal Activity ◽  
Allium Sativum ◽  
Fungicidal Activity ◽  
Positive Control ◽  
Negative Control ◽  
Randomized Design ◽  
Significant Difference ◽  
Completely Randomized Design

Shallot (Allium ascalonicum L.) has been known as traditional medicine. Shallot which has same genus with garlic(Allium sativum L.) contains allicin that is also found in garlic and has been suspected has fungicidal activity toCandida albicans. It is supported by several researches. Therefore, shallot is suspected has antifungal activity too.The aim of this research was to know antifungal activity of shallot’s water extortion againsts Candida albicans invitro. This was a laboratory experimental research which used completely randomized design, with diffusion method.Shallot’s water extortion was devided into three concentrations, there were 50%, 100% and 200%. Ketoconazole 2%was positive control and aquadest was negative control. The result of this research based on analysis of varians(Anova), there was significant difference between several treatments and was confirmed with Duncan New MultipleRange Test (DNMRT) p<0,05, there was significant difference between 100% shallot’s water extortion with othertreatments, but there was no significant difference between 50% shallot’s water extortion with 200% shallot’s. Theconclusion was shallot’s water extortion had antifungal activity againsts Candida albicans with the best concentration100%, but it was lower than ketoconazole 2%.

The Comparison of Ostecyte in the Pressure area and Tension area on Tooth Movement Because of Hyperbaric Oxygen Therapy

DENTA ◽  
2018 ◽  
Vol 12 (1) ◽  
pp. 34
Author(s):  
Arya Barahmanta ◽  
Muhammad Faizal Winaris ◽  
Pambudi Raharjo
Keyword(s):  
Hyperbaric Oxygen ◽  
Oxygen Therapy ◽  
Tooth Movement ◽  
Bone Remodelling ◽  
Orthodontic Tooth Movement ◽  
Negative Control Group ◽  
Positive Control ◽  
Negative Control ◽  
Control Group ◽  
Pressure Area

<p><strong><em>Background:</em></strong><em> Orthodontic tooth movement is a </em><em>interaction prosess</em><em> of resorption and deposition of bone remodeling. Orthodontic tooth movement by mechanical strength causes changes in alveolar bone. Osteocyte is an essential cell to respond bone remodelling. Hyperbaric Oxygen Therapy affects production of osteocyte because it can release Reactive Oxygen Species (ROS) and Nitrid Oxide (NO).  <strong>Purpose: </strong>To determine the difference number  of osteocyte in pressure and tension area during tooth movement by adjuvant of Hyperbaric Oxygen 2,4 ATA during 7 days starting on day 8 to day 14. <strong>Materials and Methods</strong>: This research used Completery Randomized Control Group Post Test Only Design. 36 cavia cobaya (male)  were divided into 3 groups randomly : the negative control groups, positive control group, and treatment group. Preparat staining used Hematoxylin Eosin (HE) and calculated on microscop 1000x with 20 field of view. Data analyses used one way ANOVA and LSD test then compared each area by using paired T test. <strong>Result:</strong> The data showed that the treatment group (P=10,67) tension area has the highest number of osteocyte than  negative control group (K-=3,67), positive control (K+=7,42). In the pressure area showed that negative control group (K-=5,00) has the highest  than positive control group (K+=3,83) and treatment (P=3,25). <strong>Conclusion: </strong>Therapy HBO 2,4 ATA 7 days starting on day 8 to day 14 is could increase osteocyte in the tissue to stimulate process of bone remodelling.</em></p><pre><strong> </strong></pre><p><strong><em>Keywords:</em></strong><em> Hyperbaric Oxygen, Tooth movement, Bone remodeling, </em><em>Osteocyte</em><em></em></p><p><em> </em></p><p><strong><em>Correspondence:</em></strong><em> </em><em>Arya Brahmanta</em><em>, Department of Orthodonty, Faculty of Dentistry, Hang Tuah University, Arif Rahman Hakim 150, Surabaya, Phone 031-5945864, Email:</em><em> </em><a href="mailto:[email protected]"><em>arya.brahmanta</em><em>@</em><em>hangtuah.ac.id</em></a></p>

UJI AKTIVITAS HEPATOPROTEKTOR KOMBINASI EKSTRAK ETANOL DAUN PANDAN WANGI (Pandanus amaryllifolius roxb) DAN KAYU MANIS (Cinnamomum burmanii) TERHADAP KADAR MDA YANG DIINDUKSI PARASETAMOL

2020 ◽  
pp. 68-73
Author(s):  
Yuni Asri Mulatsih Agami ◽  
Eka Wisnu Kusuma
Keyword(s):  
Oxidative Stress ◽  
Ethanol Extract ◽  
Optimal Dose ◽  
Experimental Methods ◽  
Positive Control ◽  
Negative Control ◽  
Male Rats ◽  
Negative Group ◽  
Ic50 Value ◽  
Dose Combination

Kasus penyakit hati semakin meningkat seiring penggunaan senyawa hepatotoksin salah satunya karena penggunaan parasetamol dengan dosis berlebih. Hal tersebut dapat meningkatkan produksi radikal bebas sehingga memicu terjadinya stress oksidatif yang dapat menimbulkan kerusakan jaringan yang ditandai dengan peningkatan kadar Malondialdehyde (MDA). Stress oksidatif dapat diatasi dengan antioksidan dari berbagai tanaman. Kulit kayu manis memiliki aktivitas antioksidan dengan nilai IC50 53ppm dan daun pandan wangi 39,7%  Penelitian ini bertujuan untuk mengetahui aktivitas kombinasi ekstrak etanol daun pandan wangi dan kayu manis dalam menurunkan kadar MDA. tikus yang diinduksi parasetamol. Penelitian menggunakan metode eksperimental, dilakukan selama 9 hari dengan 30 ekor tikus jantan dibagi menjadi 6 Kelompok, yaitu: Normal diberi aquadest, Kontrol Positif diberi silimarin 100 mg/kgBB, Kontrol Negatif diberi CMC-Na 0,05%, serta 3 kelompok lainnya diberi kombinasi ekstrak daun pandan wangi:kayu manis berturut-turut dosis I (25:75), dosis II (50:50), dosis III (75:25). Semua kelompok diinduksi parasetamol 2,5 g/kgBB pada hari ke-7  setelah 30 menit perlakuan, kecuali kelompok normal. Pada hari ke 9 dilakukan pengukuran kadar MDA dengan metode TBARs menggunakan spektrofotometri. Pemberian kombinasi ekstrak etanol daun pandan wangi dan kayu manis dapat menurunkan kadar MDA dengan kombinasi dosis yang paling optimal adalah 75:25 berdasarkan statistik dengan nilai signifikan 0,000<0,05 dibandingkan dengan kelompok negatif.    Cases of liver disease have increased with the use of hepatotoxin compounds, one of which is due to the use of paracetamol with excessive doses. This can increase the production of free radicals so that it triggers oxidative stress which can cause tissue damage which is characterized by increased levels of Malondialdehyde (MDA). Oxidative stress can be overcome with antioxidants from various plants. Cinnamomum burmanii has antioxidant activity with IC50 value of 53ppm and Pandanus amarrylifolius 39.7%. This study aims to determine the combined activity of ethanol extract of Pandanus amarrylifolius and Cinnamomum burmanii  in reducing MDA levels. Paracetamol-induced rats. Research using experimental methods, conducted for 9 days with 30 male rats divided into 6 groups, namely: Normal given aquadest, Positive Control were given silimarin 100 mg / kgBB, Negative Control was given CMC-Na 0.05%, and 3 other groups were given a combination of Pandanus amarrylifolius extract: Cinnamomum burmanii dose I (25:75), dose II (50:50), dose III (75:25). All groups induced paracetamol 2.5 g / kgBB on the 7th day after 30 minutes of treatment, except the normal group. On the 9th day MDA levels were measured using the TBARs method using spectrophotometry. Giving a combination of Pandanus amarrylifolius and Cinnamomum burmanii ethanol extract can reduce MDA levels with the most optimal dose combination is 75:25 based on statistics with a significant value of 0,000<0.05 compared with the negative group.

UJI IMUNOMODULATOR DARI EKSTRAK ETANOL DAUN WARU (HIBISCUS TILIACEUS) DENGAN METODE HIPERSENSITIVITAS TIPE LAMBAT

2019 ◽  
Vol 1 (2) ◽  
pp. 11-16
Author(s):  
Yanna Rotua Sihombing ◽  
Debi Dinha Sitepu
Keyword(s):  
Treatment Group ◽  
Leaf Extract ◽  
Positive Control ◽  
Negative Control ◽  
Delayed Type Hypersensitivity ◽  
Hypersensitivity Response ◽  
Delayed Type Hypersensitivity Response

Immunomodulator is a compound that can increaase the imuno system. One of the plants that have immunomodulator’s activity is Waru Leaf (Hibiscus tiliaceus). the purpose of this research was to test the effect of immunomodulator by extract of Waru Leaf ethanol on rat male. The activity of immunomodulator was determined by using digital pletysmometer by measuring the differences between the last leg swelling’s volume and the first leg swelling’s volume. The treatment group were divided into 5 groups. Each group consistof 5 rats CMC-Na 0,5% (negative control), Stimuno®  32,5 mg/kgBW (positive control), dose of EEDW 50, 100 and 200 mg/kgBW, and bacteria E.coli as antigen. The results slowed that distribution of EEDW dose 200 mg/kgBW can give the effect of immunostimulant by swelling enthancement compared by CMC-Na 0,5 %. EEDW 200 mg/kgBW that have activity comparable with Stimuno®  32,5 mg/kgBW. Thus, it is concluded that of Waru Leaf extract has immunomodulator effects on delayed-type hypersensitivity response of rat male.

In vitro and in vivo Assessment of Silver Nanoparticles Against Clostridium botulinum Type A Botulinum

2019 ◽  
Vol 16 (1) ◽  
pp. 113-119 ◽  
Author(s):  
Mohammad Aminianfar ◽  
Siavash Parvardeh ◽  
Mohsen Soleimani
Keyword(s):  
Silver Nanoparticles ◽  
Botulinum Toxin ◽  
Clostridium Botulinum ◽  
Lethal Dose ◽  
Type A ◽  
Positive Control ◽  
Negative Control ◽  
Laboratory Animals ◽  
Control Group ◽  
Nano Silver

Background: Clostridium botulinum causes botulism, a serious paralytic illness that results from the ingestion of a botulinum toxin. Because silver nanoparticle products exhibit strong antimicrobial activity, applications for silver nanoparticles in healthcare have expanded. Therefore, the objective of the current study was to assess a therapeutic strategy for the treatment of botulism toxicity using silver nanoparticles. Methods: A preliminary test was conducted using doses that produce illness in laboratory animals to determine the absolute lethal dose (LD100) of botulinum toxin type A (BoNT/A) in mice. Next, the test animals were divided into six groups containing six mice each. Groups I, II and III were the negative control (botulinum toxin only), positive control-1 (nano-silver only) and positive control-2 (no treatment), respectively. The remaining groups were allocated to the toxin that was supplemented with three nano-silver treatments. Results: The mortality rates of mice caused by BoNT/A significantly reduced in the treatment groups with different doses and injection intervals of nano-silver when compared to the negative control group. BoNT/A toxicity induced by intraperitoneal injection of the toxin of Clostridium botulinum causes rapid death while when coupled with nano-osilver results in delayed death in mice. Conclusion: These results, while open to future improvement, represent a preliminary step towards the satisfactory control of BoNT/A with the use of silver nanoparticles for human protection against this bioterrorism threat. Further study in this area can elucidate the underlying mechanism for detoxifying BoNT/A by silver nanoparticles.

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