Stimulation of Stevioside Accumulation on Stevia rebaudiana (Bertoni) Shoot Culture Induced with Red LED Light in TIS RITA® Bioreactor System

Leaves of Stevia rebaudiana contain steviol glycoside which mainly comprise of stevioside, a natural sweetener compound that is 100-300 times sweeter than sucrose. Current cultivation method of Stevia rebaudiana in Indonesia has yet to reach its optimum efficiency and productivity to produce stevioside as a safe sugar substitute sweetener for people with diabetes. An alternative method that is not limited by environmental factor is in vitro temporary immersion system (TIS) culture method using recipient for automated immersion (RITA®) bioreactor. The aim of this research was to evaluate the effect of red LED light induction towards shoot growth and stevioside accumulation in TIS RITA® bioreactor system, as an endeavour to increase the secondary metabolite synthesis. The result showed that the stevioside accumulation in TIS RITA® bioreactor system induced with red LED light for one hour during night was higher than that in TIS RITA® bioreactor system without red LED light induction, i.e. 71.04 ± 5.36 μg/g and 42.92 ± 5.40 μg/g respectively. Biomass growth rate reached as high as 0.072 ± 0.015/day for red LED light induced TIS RITA® bioreactor system, whereas TIS RITA® bioreactor system without induction was only 0.046 ± 0.003/day. Productivity of Stevia rebaudiana shoots induced with red LED light was 0.065 g/L medium/day, whilst shoots without any induction was 0.041 g/L medium/day. Sucrose, salt, and inorganic consumption in both bioreactor media increased as biomass increased. It can be concluded that Stevia rebaudiana shoot in TIS RITA® bioreactor induced with red LED light produces biomass and accumulates higher stevioside concentration, in comparison to bioreactor without any light induction.

In vitro Effects of Amygdalin on the Functional Competence of Rabbit Spermatozoa

The present in vitro study was designed to reveal whether amygdalin (AMG) is able to cause changes to the motility, viability and mitochondrial activity of rabbit spermatozoa. New Zealand White rabbits (n = 10) aged four months were used in the study. Semen samples were collected from each animal and used for the in vitro incubation. The samples were divided into five equal parts and diluted with saline supplemented with 0, 0.5, 1, 2.5 and 5 mg/mL AMG. At times 0h, 3h and 5h spermatozoa motion parameters were assessed using the SpermVision™ computer-aided sperm analysis (CASA) system, cell viability was examined with the metabolic activity (MTT) assay, and the eosin-nigrosin staining technique was used to evaluate the viability of rabbit spermatozoa. All AMG concentrations exhibited stimulating effects on the spermatozoa activity, as shown by a significant preservation of the motility (P

Biocontrol Effectiveness of Indigenous Trichoderma Species against Meloidogyne javanica and Fusarium oxysporum f. sp. radicis lycopersici on Tomato

In this study, three local isolates of Trichoderma (Tr1: T. viride, Tr2: T. harzianum and Tr3: T. asperellum) were isolated and evaluated for their biocontrol effectiveness under in vitro conditions and in greenhouse. In vitro bioassay revealed a biopotential control against Fusarium oxysporum f. sp. radicis lycopersici and Meloidogyne javanica (RKN) separately. All species of Trichoderma exhibited biocontrol performance and (Tr1) Trichoderma viride was the most efficient. In fact, growth rate inhibition of Fusarium oxysporum f. sp. radicis lycopersici (FORL) was reached 75.5% with Tr1. Parasitism rate of root-knot nematode was 60% for juveniles and 75% for eggs with the same one. Pots experiment results showed that Tr1 and Tr2, compared to chemical treatment, enhanced the plant growth and exhibited better antagonism against root-knot nematode and root-rot fungi separated or combined. All Trichoderma isolates revealed a bioprotection potential against Fusarium oxysporum f. sp. radicis lycopersici. When pathogen fungi inoculated alone, Fusarium wilt index and browning vascular rate were reduced significantly with Tr1 (0.91, 2.38%) and Tr2 (1.5, 5.5%), respectively. In the case of combined infection with Fusarium and nematode, the same isolate of Trichoderma Tr1 and Tr2 decreased Fusarium wilt index at 1.1 and 0.83 and reduced the browning vascular rate at 6.5% and 6%, respectively. Similarly, the isolate Tr1 and Tr2 caused maximum inhibition of nematode multiplication. Multiplication rate was declined at 4% with both isolates either tomato infected by nematode separately or concomitantly with Fusarium. The chemical treatment was moderate in activity against Meloidogyne javanica and Fusarium oxysporum f. sp. radicis lycopersici alone and combined.

Comparative Growth Rates of Treculia africana Decne: Embryo in Varied Strengths of Murashige and Skoog Basal Medium

This study provides a regeneration protocol for Treculia africana Decne (an endangered plant) through embryo culture. Mature zygotic embryos of T. africana were excised from the seeds aseptically and cultured on varied strengths (full, half and quarter) of Murashige and Skoog (MS) basal medium supplemented. All treatments experienced 100±0.00 percent sprouting except for half and quarter strengths. Plantlets in MS full strength had the highest fresh weight, leaf area, and longest shoot length when compared to other treatments. All explants in full, half, quarter strengths and control had the same number of leaves and sprout rate. Between the treatments, there was a significant difference (P>0.05) in their effect on the length of shoot and root, number of adventitious root, leaf area, and fresh weight. Full strength had the highest mean value in all the above-mentioned parameters and differed significantly (P>0.05) from others except in shoot length, number of adventitious roots, and root length where it did not differ (P

Evaluation of Hazelnut Hulls as an Alternative Forage Resource for Ruminant Animals

The aim of this study was to estimate the digestibility of the fruit internal skin of different varieties of hazelnuts to propose hazelnut fruit skin as an alternative feed source as roughage in ruminant nutrition. In 2015, the fruit internal skins of three different varieties of round hazelnuts (RH), pointed hazelnuts (PH) and almond hazelnuts (AH) were obtained from hazelnut processing factory then their crude nutrients analysis were carried out. Organic matter digestibility (OMD) and metabolisable energy (ME) values of hazelnut fruit skins were estimated from gas measured by in vitro gas production method. Their antioxidant activities were determined by spectrophotometric method. Crude nutrient values of three different varieties were; organic matter (OM): 87.83, 87.81 and 87.78%), crude protein (CP): 5.97, 5.93 and 5.89%, neutral detergent fiber (NDF): 30.30, 30.29 and 30.29%, acid detergent fiber (ADF): 48.68, 48.67 and 48.66% and acid detergent lignin (ADL): 25.43, 25.43 and 25.39% respectively. OMD from 24 h incubation time of RH, PH and AH were 22.04, 22.46 and 22.74%; MEGP values were 3.69, 3.75 and 3.79 MJ/kg DM; and antioxidant activity values were 94.60, 94.54 and 94.52 IC 50 mg/mL respectively. The fruit internal skin of different varieties of hazelnuts may be considered as an alternative roughage for ruminant nutrition regarding to their crude and digestible nutritive values. Moreover, hazelnut fruit skin has a rich antioxidant content so it may be used as a feed additive for both ruminant and non-ruminant animals.

Comparative Analysis of Chemical Composition and Biological Activities of Ajuga genevensis L. in in vitro Culture and Intact Plants

One of the tasks in contemporary biotechnology, pharmacology and other fields of human activities is to obtain biologically active substances from plants. They are very essential in the treatment of many diseases due to their actually high therapeutic value without visible side effects. However, sometimes the possibility of obtaining the metabolites is limited due to the reduction of wild-growing plants. That is why the plant cell cultures are of great interest as alternative sources of biologically active substances. Besides, during the monitored cultivation, it is possible to obtain substances that are not synthesized by plants in nature. Isolated culture of Ajuga genevensis with high growth activity and ability of regeneration was obtained using MS nutrient medium. The agar-diffusion method showed that aqueous extracts of callus culture revealed high antimicrobial activity towards various gram-positive (Bacillus subtilis A1WT; B. mesentericus WDCM 1873; Staphylococcus aureus WDCM 5233; Staph. citreus WT) and gram-negative (Escherichia coli WKPM M-17; Salmonella typhimurium TA 100) microorganisms. The broth dilution method revealed that the minimal and half maximal inhibitory concentration values against E. coli corresponded to the 70 μg/mL and 140 μg/mL concentration of the extract respectively. According to the photochemiluminescent analysis, callus tissue extracts of leaf and root origin showed higher antioxidant activity than the same quantity of A. genevensis intact plant extract. A. genevensis intact plant and callus culture extracts showed no cytotoxic effect on K-562 suspension cell line of human chronic myeloid leukemia. The GC-MS analysis showed deep differences between the qualitative and quantitative composition of callus culture and intact plant extracts. Hexacosane (11.17%); n-hexadecanoic acid (9.33%); and 2-methoxy-4-vinylphenol (4.28%) were the main components of intact plant extracts. 10-Methylnonadecane (57.0%); methoxyacetic acid, 2-tetradecyl ester (17.75%) and 1-Bromopentadecane (14.55%) were the main components of A. genevensis callus culture extracts. Obtained data indicate that callus culture of A. genevensis can be used as an alternative source of biologically active substances.

Efficient Callus Induction and Plant Regeneration from Mature Embryo Culture of Barley (Hordeum vulgare L.) Genotypes

Crop improvement through genetic engineering depends on effective and reproducible plant regeneration systems. Immature embryos are the most widely used explant source for in vitro regeneration in barley (Hordeum vulgare L.). However, immature embryos require the continuous growth of donor plants and the suitable stage for their culture is also certainly limited. On the other hand, mature embryos can be procured and stored easily; they can be studied throughout the year. In this study, an effective callus induction and plant regeneration were aimed to develop from mature embryos of different barley genotypes. The effect of medium (MS1 and MS2), auxin type (2,4-D, dicamba, picloram and 2,4,5-T) and concentrations (2, 4, 6 mg/l) on callus formation and effect of cytokinin type (TDZ, BAP) and concentrations (0.2, 0.5, 1.0 mg/l) on green plant regeneration were evaluated in mature embryo culture of barley. Callus and shoot formation was successful for all genotypes. By depending on genotype, MS1 is the best medium, 4 mg/l dicamba is the best growth regulator in the callus induction and MS1 is the best medium, 1 mg/l BAP is the best growth regulator in the shoot formation were determined.

Accuracy of Small Field of View CBCT in Determining Endodontic Working Length

An in vitro study was carried out to evaluate the feasibility of small field of view (FOV) cone beam computed tomography (CBCT) in determining endodontic working length. The objectives were to determine the accuracy of CBCT in measuring the estimated preoperative working lengths (EPWL), endodontic working lengths (EWL) and file lengths. Access cavities were prepared in 27 molars. For each root canal, the baseline electronic working length was determined using an EAL (Raypex 5). The teeth were then divided into overextended, non-modified and underextended groups and the lengths were adjusted accordingly. Imaging and measurements were made using the respective software of the RVG (Kodak RVG 6100) and CBCT units (Kodak 9000 3D). Root apices were then shaved and the apical constrictions viewed under magnification to measure the control working lengths. The paired t-test showed a statistically significant difference between CBCT EPWL and control length but the difference was too small to be clinically significant. From the Bland Altman analysis, the CBCT method had the widest range of 95% limits of agreement, reflecting its greater potential of error. In measuring file lengths, RVG had a bigger window of 95% limits of agreement compared to CBCT. Conclusions: (1) The clinically insignificant underestimation of the preoperative working length using small FOV CBCT showed that it is acceptable for use in the estimation of preoperative working length. (2) Small FOV CBCT may be used in working length determination but it is not as accurate as the currently practiced method of using the EAL. (3) It is also more accurate than RVG in measuring file lengths.

Antioxidant Capacity and Total Phenolic Content of Aqueous Acetone and Ethanol Extract of Edible Parts of Moringa oleifera and Sesbania grandiflora

Aqueous ethanol and aqueous acetone extracts of Moringa oleifera (outer pericarp of immature fruit and flower) and Sesbania grandiflora white variety (flower and leaf) were examined for radical scavenging capacities and antioxidant activities. Ethanol extract of S. grandiflora (flower and leaf) and acetone extract of M. oleifera (outer pericarp of immature fruit and flower) contained relatively higher levels of total dietary phenolics than the other extracts. The antioxidant potential of the extracts were assessed by employing different in vitro assays such as reducing power assay, DPPH˙, ABTS˙+ and ˙OH radical scavenging capacities, antihemolytic assay by hydrogen peroxide induced method and metal chelating ability. Though all the extracts exhibited dose dependent reducing power activity, acetone extract of all the samples were found to have more hydrogen donating ability in DPPH˙ (2.3% - 65.03%) and hydroxyl radical scavenging systems (21.6% - 77.4%) than the ethanol extracts. The potential of multiple antioxidant activity was evident as it possessed antihemolytic activity (43.2 % to 68.0 %) and metal ion chelating potency (45.16 - 104.26 mg EDTA/g sample). The result indicate that acetone extract of M. oleifera (OPIF and flower) and S. grandiflora (flower and leaf) endowed with polyphenols, could be utilized as natural antioxidants/nutraceuticals.

Preparation, Characterisation, and Measurement of the in vitro Cytotoxicity of Mesoporous Silica Nanoparticles Loaded with Cytotoxic Pt(II) Oxadiazoline Complexes

Cytotoxic platinum compounds play a major role in the chemotherapy of a large number of human cancers. However, due to the severe side effects for the patient and other problems associated with their use, there is a need for the development of more efficient drugs and new methods for their selective delivery to the tumours. One way to achieve the latter could be in the use of nanoparticular substrates that can adsorb or chemically bind the drug. In the cell, the drug is supposed to be slowly released, either by physical desorption or by dissolution of the particle framework. Ideally, the cytotoxic properties of the platinum drug unfold only then, in the cancer cell and over a longer period of time due to the gradual release. In this paper, we report on our first steps in this direction. The binding properties of a series of cytotoxic Pt(II) oxadiazoline compounds to mesoporous silica particles has been studied by NMR and UV/vis spectroscopy. High loadings were achieved when the Pt(II) compound was relatively polar, and has been dissolved in a relatively nonpolar solvent before the silica was added. Typically, 6-10 hours were required for complete equilibration, suggesting the adsorption did not only occur to the outer surface but also to the interior of the pores. The untreated and Pt(II) loaded particles were characterised by C, H, N combustion analysis, BET/BJH nitrogen sorption, electron microscopy (REM and TEM) and EDX. With the latter methods we were able to demonstrate the homogenous distribution of the Pt(II) compound on and in the silica particles, and no Pt(II) bulk precipitate had formed. The in vitro cytotoxicity in a human cancer cell line (HeLa) has been determined for one of the new platinum compounds adsorbed to mesoporous silica particles of different size, and compared with the corresponding compound in solution. The IC50 data are similar in all cases, suggesting that the release of the Pt(II) compound was relatively fast and possibly occurred before the particles reached the cells. Overall, the platinum drug is chemically stable on silica and retained its activity upon prolonged storage.

Ultrasound Therapy: Amplitude Modulation Technique for Tissue Ablation by Acoustic Cavitation

In recent years, non-invasive Focused Ultrasound (FU) has been utilized for generating bubbles (cavities) to ablate target tissue by mechanical fractionation. Intensities >10 kW/cm2 are required to generate the inertial cavities. The generation, rapid growth, and collapse of these inertial cavities cause tissue fractionation and the process is called Histotripsy. The ability to fractionate tissue from outside the body has many clinical applications including the destruction of the tumor mass. The process of tissue fractionation leaves a void at the treated site, where all the affected tissue is liquefied to particles at sub-micron size. The liquefied tissue will eventually be absorbed by the body. Histotripsy is a promising non-invasive treatment modality. This paper presents a technique for generating inertial cavities at lower intensities (< 1 kW/cm2). The technique (patent pending) is based on amplitude modulation (AM), whereby a low frequency signal modulates the amplitude of a higher frequency FU wave. Cavitation threshold is lower at low frequencies; the intensity required to generate cavitation in water at 10 kHz is two orders of magnitude lower than the intensity at 1 MHz. The Amplitude Modulation technique can operate in both continuous wave (CW) and pulse wave (PW) modes, and the percentage modulation (modulation index) can be varied from 0 % (thermal effect) to 100 % (cavitation effect), thus allowing a range of ablating effects from Hyperthermia to Histotripsy. Furthermore, changing the frequency of the modulating signal allows controlling the size of the generated cavities. Results from in vitro work demonstrate the efficacy of the new technique in fractionating soft tissue and solid calcium carbonate (Chalk) material. The technique, when combined with MR or Ultrasound imaging, will present a precise treatment modality for ablating diseased tissue without affecting the surrounding healthy tissue.

Properties of Adipose Tissue Derived Mesenchymal Stem Cells with Long-Term Cryopreservation

Adult mesenchymal stem cells (MSCs) have been investigated using preclinical approaches for tissue regeneration. Porcine MSCs (pMSCs) are capable of growing and attaching to plastic with a fibroblast-like morphology and then differentiating into bone, adipose, and cartilage tissues in vitro. This study was conducted to investigate the proliferating abilities, differentiation potentials, and multipotency of miniature pig adipose tissue-derived MSCs (mpAD-MSCs) with or without long-term cryopreservation, considering that cryostorage has the potential for use in clinical applications. After confirming the characteristics of the mpAD-MSCs, we examined the effect of long-term cryopreservation (> 2 years) on expression of cell surface markers (CD34, CD90 and CD105), proliferating abilities (cumulative population doubling level, doubling time, colony-forming unit, and MTT assay) and differentiation potentials into mesodermal cell lineages. As a result, the expression of cell surface markers is similar between thawed and fresh mpAD-MSCs. However, long-term cryopreservation significantly lowered the differentiation potentials (adipogenic, chondrogenic, and osteogenic) of mpAD-MSCs. When compared with fresh mpAD-MSCs, thawed mpAD-MSCs exhibited lower expression of mesodermal cell lineage-related genes such as peroxisome proliferator-activated receptor-g2, lipoprotein lipase, collagen Type II alpha 1, osteonectin, and osteocalcin. Interestingly, long-term cryostoraged mpAD-MSCs exhibited significantly higher cell viability than the fresh mpAD-MSCs. Long-term cryopreservation induced a 30% increase in the cell viability of mpAD-MSCs when compared with the fresh mpAD-MSCs at 5 days after thawing. However, long-term cryopreservation significantly lowered expression of stemness markers such as Oct3/4, Sox2, and Nanog. Furthermore, long-term cryopreservation negatively affected expression of senescence-associated genes such as telomerase reverse transcriptase and heat shock protein 90 of mpAD-MSCs when compared with the fresh mpAD-MSCs. The results from this study might be important for the successful application of MSCs in clinical trials after long-term cryopreservation.

Electro-Thermal Imaging of Breast Phantom: An Experimental Study

To increase the temperature contrast in thermal images, the characteristics of the electrical conductivity and thermal imaging modalities can be combined. In this experimental study, it is objected to observe whether the temperature contrast created by the tumor tissue can be improved just due to the current application within medical safety limits. Various thermal breast phantoms are developed to simulate the female breast tissue. In vitro experiments are implemented using a thermal infrared camera in a controlled manner. Since experiments are implemented in vitro, there is no metabolic heat generation and blood perfusion. Only the effects and results of the electrical stimulation are investigated. Experimental study is implemented with two-dimensional models. Temperature contrasts due to the tumor tissues are obtained. Cancerous tissue is determined using the difference and ratio of healthy and tumor images. 1 cm diameter single tumor tissue causes almost 40 °mC temperature contrast on the thermal-breast phantom. Electrode artifacts are reduced by taking the difference and ratio of background (healthy) and tumor images. Ratio of healthy and tumor images show that temperature contrast is increased by the current application.

In vitro Susceptibility of Madurella mycetomatis to the Extracts of Anogeissus leiocarpus Leaves

Anogeissus leiocarpus (Combretaceae) is well known for its medicinal uses in African traditional medicine, for treating many human diseases mainly skin diseases and infections. Mycetoma disease is a fungal and/ or bacterial skininfection, mainly cause by Madurella mycetomatis fungus. This study was carried out in vitro to investigate the antifungal activity of Anogeissus leiocarpus leaf extracts against the isolated pathogenic Madurella mycetomatis, by using the NCCLS modified method compared to Ketoconazole standard drug, and MTT assay. The bioactive fraction was subjected to chemical analysis implementing different chromatographic analytical methods (TLC, HPLC, and LC-MS/MS). The results showed significance antifungal activity of A. leiocarpus leaf extracts against the isolated pathogenic M. mycetomatis, compared to negative and positive controls. The chloroform fraction showed the highest antifungal activity. The chromatographic analysis of the chloroform fraction with the highest activity showed the presence of important bioactive compounds such as ellagic and flavellagic acids derivatives, flavonoids and stilbenoid, which are well known for their antifungal activity.

Phytochemical Screening, Antioxidant Activity and Lipid Profile Effects of Citrus reticulata Fruit Peel, Zingiber officinale Rhizome and Sesamum indicum Seed Extracts

Many herbal medicinal products are considered potential anti-hypercholesterolemic agents with encouraging safety profiles, however only a limited amount of clinical research exists to support their efficacy. The present study was designed to compare the antihypercholesterolemic and antioxidant activities of the crude ethanolic extracts of Citrus reticulata fruit peel, Zingiber officinale rhizome and Sesamum indicum seeds. Forty-five rats were used throughout the experiment which are extended for four weeks. These were divided into nine groups, five rats per each group as follows; group 1 was the normal control group (rats only fed standard normal rat diet), group 2 was the hypercholesterolemic control group (rats fed only hypercholesterolemic diet which contained 1% cholesterol plus 10% saturated animal fat added to the normal rat diet), groups 3 and 4 were fed hypercholesterolemic diet in addition to Citrus reticulata ethanolic extract at doses of (250mg/kg (group 3) and 500mg/kg (group 4)) administered daily via oral route, groups 5 and 6 were given hypercholesterolemic diet in addition to Zingiber officinale ethanolic extract at doses of (250mg/kg (group 5) and 500mg/kg (group 6)) daily through oral route, groups 7 and 8 fed on hypercholesterolemic diet in addition to Sesamum indicum ethanolic extract at doses of (250mg/kg (group 7) and 500mg/kg (group 8)) daily orally; and group 9 rats were given hypercholesterolemic diet in addition to atorvastatin (0.18mg/kg) daily via oral route as a standard reference antihypercholesterolemic drug. Blood samples from all groups were drawn from the retro-orbital venous plexus four weeks following treatment after overnight fasting and the lipid profile (total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C) and triglyceride levels) were measured and the risk ratio (TC/HDL-C) was assessed. The antioxidant activity of the three plants extracts was determined using DPPH free-radical antioxidant assay. Results of in vivo and in vitro antihypercholesterolemic and antioxidant assay respectively, revealed that the three extracts possess comparable antioxidant and antihypercholesterolemic activities.

Experimental Study on Capturing of Magnetic Nanoparticles Transported in an Implant Assisted Cylindrical Tube under Magnetic Field

Targeted drug delivery is a method of delivering medication to a patient in a manner that increases the concentration of the medication in some parts of the body relative to others. Targeted drug delivery seeks to concentrate the medication in the tissues of interest while reducing the relative concentration of the medication in the remaining tissues. This improves efficacy of the while reducing side effects. In the present work, we investigate the effect of magnetic field, flow rate and particle concentration on the capturing of magnetic particles transported in a stent implanted fluidic channel. Iron oxide magnetic nanoparticles (Fe3O4) nanoparticles were synthesized via co-precipitation method. The synthesized Fe3O4 nanoparticles were added in the de-ionized (DI) water to prepare the Fe3O4 magnetic particle suspended fluid. This fluid is transported in a cylindrical tube of diameter 8 mm with help of a peristaltic pump at different flow rate (25-40 ml/min). A ferromagnetic coil of SS 430 has been implanted inside the cylindrical tube to enhance the capturing of magnetic nanoparticles under magnetic field. The capturing of magnetic nanoparticles was observed at different magnetic magnetic field, flow rate and particle concentration. It is observed that capture efficiency increases from 47-67% at magnetic field 2-5kG, respectively at particle concentration 0.6mg/ml and at flow rate 30 ml/min. However, the capture efficiency decreases from 65 to 44% by increasing the flow rate from 25 to 40 ml/min, respectively. Furthermore, it is observed that capture efficiency increases from 51 to 67% by increasing the particle concentration from 0.3 to 0.6 mg/ml, respectively.

In vitro Environmental Factors Controlling Root Morphological Traits of Pineapple (Ananas comosus L. Merr)

Developing our knowledge of when pineapple roots grow can lead to improved water, fertilizer applications, and more precise culture management. This paper presents current understanding of morphological traits in pineapple roots, highlighting studies using incubation periods and various solid MS media treated with different sucrose concentrations and pH, which directly assess in vitro environmental factors. Rooting parameters had different optimal sucrose concentrations and incubation periods. All shoots failed to root in medium supplemented with sucrose at 5 g/L and no roots formed within the first 45 days in medium enriched with sucrose at 10 g/L. After 75 days, all shoots rooted in medium enriched with 10 and 20 g/L sucrose. Moreover, MS medium supplied with 20 g/L sucrose resulted in the longest and the highest number of roots with 27.3 mm and 4.7, respectively. Root function, such as capacity for P and N uptake, declined rapidly with root length. As a result, the longer the incubation period, the better the rooting responses would be.

Crude Glycerol Affects Canine Sperm Motility: Computer Assisted Semen Analysis in vitro

Target of this study was the analysis of the impact of crude glycerol on canine spermatozoa motility, morphology, viability, and membrane integrity. Experiments were realized in vitro. In the study, semen from 5 large dog breeds was used. They were typical representatives of large breeds, coming from healthy rearing, regularly vaccinated and integrated to the further breeding. Semen collections were realized at the owners of animals and in the veterinary clinic. Subsequently the experiments were realized at the Department of Animal Physiology of the SUA in Nitra. The spermatozoa motility was evaluated using CASA analyzer (SpermVisionTM, Minitub, Germany) at the temperature 5 and 37°C for 5 hours. In the study, 13 motility parameters were evaluated. Generally, crude glycerol has generally negative effect on spermatozoa motility. Morphological analysis was realized using Hancock staining and the preparations were evaluated at magnification 1000x using classification tables of morphologically changed spermatozoa. Data clearly detected the highest number of morphologically changed spermatozoa in the experimental groups (know twisted tails, tail torso and tail coiling). For acrosome alterations swelled acrosomes, removed acrosomes and acrosomes with undulated membrane were detected. In this study also the effect of crude glycerol on spermatozoa membrane integrity were analyzed. The highest crude glycerol concentration significantly affects spermatozoa integrity. Results of this study show that crude glycerol has effect of spermatozoa motility, viability, and membrane integrity. Detected changes are related to crude glycerol concentration, temperature, as well as time of incubation.

Chemical Composition of Essential Oil and in vitro Antibacterial and Anticancer Activity of the Hydroalcolic Extract from Coronilla varia

The aims of study were investigation on chemical composition essential oil and the effect of extract of Coronilla varia on antimicrobial and cytotoxicity activity. The essential oils of Coronilla varia is obtained by hydrodistillation and analyzed by (GC/MS) for determining their chemical composition and identification of their components. Antibacterial activity of plant extract was determined by disc diffusion method and anticancer activity measured by MTT assay. The major components in essential oil were Caryophyllene Oxide (60.19%), Alphacadinol (4.13%) and Homoadantaneca Robexylic Acid (3.31%). The extracts from Coronilla varia had interesting activity against Proteus mirabilis in the concentration of 700 μg/disc and did not show any activity against Staphylococus aureus, Bacillus subtillis, Klebsiella pneumonia and Entrobacter cloacae. The positive control, Ampicillin, Chloramphenicol and Cenphalothin had shown zone of inhibition resistant all bacteria. The ethanol extract of Corohilla varia inhibited on MCF7 cell lines. IC50 0.6(mg/ml) was the optimum concentration of extract from Coronilla varia inhibition of cell line growth. The MCF7 cancer cell line and Proteus mirabilis were more sensitive to Coronilla varia ethanol extract.

A Piscan Ulcerative Aeromonas Infection

In the immunologic sense, clinical infection is a state of failure of the immune system to combat the pathogenic weapon of the bacteria invading the host. A motile gram negative vibroid organism associated with marked mono and poly nuclear cell responses was traced during the examination of a clinical material from an infected common carp Cyprinus carpio. On primary plate culture, growth was shown to be pure, dense population of an Aeromonas-like colony morphotype. The pure isolate was found to be; Aerobic, facultatively anaerobic, non-halophilic, grew at 0C, and 37C, oxidase positive utilizes glucose through fermentative pathway, resist 0/129 and novobiocin, produces alanine and lysine decarboxylases but non-producing ornithine dehydrolases. Tests for the in vitro determinants of pathogenicity has shown to be; Betahaemolytic onto blood agar, gelatinase, casienase and amylase producer. Three in vivo determinants of pathogenicity were tested as, the lethal dose fifty, the pathogenesis and pathogenicity. It was evident that 0.1 milliliter of the causal bacterial cell suspension of a density 1 x 107 CFU/ml injected intramuscularly into an average of 100gms fish toke five days incubation period, then at the day six morbidity and mortality were initiated. LD50 was recorded at the day 12 post-infection. Use of an LD50 doses to study the pathogenicity, reveals mononuclear and polynuclear cell responses, on examining the stained direct films of the clinical materials from the experimentally infected fish. Re-isolation tests confirm that the reisolant is same. The course of the infection in natural case was shown manifestation of; skin ulceration, haemorrhage and descaling. On evisceration, the internal organs were shown; congestion in the intestines, spleen and, air sacs. The induced infection showed a milder form of these manifestations. The grading of the virulence of this organism was virulent causing chronic course of infections as indicated from the pathogenesis and pathogenicity studies. Thus the infectious bacteria were consistent with Aeromonas hydrophila, and the infection was chronic.