Die neue Bären-Anlage im NaturZoo Rheine – Planung,Konzept und Erfahrungen

In May 2009 NaturZoo Rheine, Germany, opened a new enclosure for Sloth bears (Melursus ursinus) combined with Golden jackals (Canis aureus). Introducing the article tells the 70 years long history of bear-maintenance at NaturZoo Rheine. The process of institutional collection planning resulting in Sloth bears as the taxon of choice is described as are the special ideas of modern animal-husbandry and –presentation for the concept of the facility. The enclosure and husbandry practice are detailed. A list of experiences made so far places special emphasise on behavioural enrichment and the mixed-species exhibit. Finally the visitors‘ response to the new enclosure gets mentioned.     

Die neue Flamingo-Anlage im NaturZoo Rheine

In spring 2006 NaturZoo Rheine opened a new enclosure for a colony of currently some 88 Chilean flamingos (Phoenicopterus chilensis). The zoo keeps flamingos for 40 years. First successful breeding happened in 1981 and from this time on regularly. So in this way the old enclosure proved to be succesful. However it has lost of appeal and there was also need for technical improvement.The new enclosure incorporates features which give the flamingos a wide variety of behavioural stimulations with such of design which tear the visitors` attention much more than in the past.For the first time a kind of fencing made of 6 m high street-lamp poles with a soft netting tightened in between was used in order to keep also full-flighted flamingos in an open-topped exhibit.The article describes in detail the new enclosure. Comprehensive experiences made over the first year in function - some parts of the enclosure are working even longer - are added.     

Neue Giraffenhaltung im Opel-Zoo Kronberg.

The „Africa Savanna“– exhibit is described. The biggest investment of the Opel-Zoo in recent years was opened for the public on the 50th anniversary of the so-called Georg von Opel – Freigehege für Tierforschung on September 16th, 2006. The developed area covers about 22500 m² and includes in addition to the animal related buildings, a new restaurant, a new main entrance and a new administration building. The mostly moated outside exhibit of about 10000 m² is dominated by artificial baobab tree. A waterhole of 200 m² separates the exhibit from the restaurant. Due to fairly long winters the inside area for the giraffes is rather large, so they can be kept as a herd, even when they must stay inside. Visitors can enter a small area of the giraffe house and may watch the animals through glass. The off-exhibit area for the zebras, gnus and impalas has 18 equal stables of 10 m² each. Five introduction pens connect the new animal house with the outside exhibit.This article also informs about the giraffe keeping history at Kronberg since 1961 and tells about the experiences made with the new development during the first year of use.     

Vom Ruhr Zoo zur ZOOM Erlebniswelt III: Der Bauabschnitt Asien

In the Zoom Erlebniswelt Gelsenkirchen the third and last biogeographical area dedicated to the Asian fauna was opened in March 2010 covering a total area of five hectares. In May 2013 a new enclosure for tigers was added. The emphasis of the Asia area lies on a 4700 m² tropical hall with mammals and birds, and Asian gastronomic facilities as well. Included is a mixed species exhibit for orangutans (Pongo abelii), northern plains grey langurs (Semnopithecus entellus) and Asian small clawed otters (Aonyx cinerea). Along a 1.3 km outside path enclosures for some species of colder climates were built, too. The transformation of the former Ruhr Zoo Gelsenkirchen to the modern ZOOM Erlebniswelt herewith is brought to a close.     

Immobilisation and characterisation of biocatalytic co-factor recycling enzymes, glucose dehydrogenase and NADH oxidase, on aldehyde functional ReSyn™ polymer microspheres

The use of enzymes in industrial applications is limited by their instability, cost and difficulty in their recovery and re-use. Immobilisation is a technique which has been shown to alleviate these limitations in biocatalysis. Here we describe the immobilisation of two biocatalytically relevant co-factor recycling enzymes, glucose dehydrogenase (GDH) and NADH oxidase (NOD) on aldehyde functional ReSyn? polymer microspheres with varying functional group densities. The successful immobilisation of the enzymes on this new high capacity microsphere technology resulted in the maintenance of activity of ～40% for GDH and a maximum of 15.4% for NOD. The microsphere variant with highest functional group density of ～3500 μmol g^?1 displayed the highest specific activity for the immobilisation of both enzymes at 33.22 U mg^?1 and 6.75 U mg^?1 for GDH and NOD with respective loading capacities of 51% (0.51 mg mg^?1) and 129% (1.29 mg mg^?1). The immobilised GDH further displayed improved activity in the acidic pH range. Both enzymes displayed improved pH and thermal stability with the most pronounced thermal stability for GDH displayed on ReSyn? A during temperature incubation at 65 °C with a 13.59 fold increase, and NOD with a 2.25-fold improvement at 45 °C on the same microsphere variant. An important finding is the suitability of the microspheres for stabilisation of the multimeric protein GDH.     

Gold nanoparticles functionalized by gadolinium–DTPA conjugate of cysteine as a multimodal bioimaging agent

The synthesis and characterization of gold nanoparticles coated with Gd-chelate (Au@GdL), where L is a conjugate of DTPA and cysteine, is described. These particles are obtained by the replacement of citrate from the gold nanoparticle surfaces with gadolinium chelate (GdL). The average size of Au@GdL is 14 nm with a loading of GdL reaching up to 2.9 × 10³ per particles, and they demonstrate very high R1 relaxivity (～10⁵ mM^?1 s^?1) as well as X-ray attenuation. The R1 relaxivity per [Gd] is 17.9 mM^?1 s^?1. The present system also exhibits macrophage-specific property, as demonstrated by histological and TEM images as well as CT and MR, rendering itself as a new class of T1 multimodal CT/MR contrast agent.     

Involvement of palmitate/Ca2 +(Sr2 +)-induced pore in the cycling of ions across the mitochondrial membrane

The palmitate/Ca^2 +-induced (Pal/Ca^2 +) pore, which is formed due to the unique feature of long-chain saturated fatty acids to bind Ca^2 + with high affinity, has been shown to play an important role in the physiology of mitochondria. The present study demonstrates that the efflux of Ca^2 + from rat liver mitochondria induced by ruthenium red, an inhibitor of the energy-dependent Ca^2 + influx, seems to be partly due to the opening of Pal/Ca^2 + pores. Exogenous Pal stimulates the efflux. Measurements of pH showed that the Ca^2 +-induced alkalization of the mitochondrial matrix increased in the presence of Pal. The influx of Ca^2 + (Sr^2 +) also induced an outflow of K⁺ followed by the reuptake of the ion by mitochondria. The outflow was not affected by a K⁺/H⁺ exchange blocker, and the reuptake was prevented by an ATP-dependent K⁺ channel inhibitor. It was also shown that the addition of Sr^2 + to mitochondria under hypotonic conditions was accompanied by reversible cyclic changes in the membrane potential, the concentrations of Sr^2 + and K⁺ and the respiratory rate. The cyclic changes were effectively suppressed by the inhibitors of Ca^2 +-dependent phospholipase A₂, and a new Sr^2 + cycle could only be initiated after the previous cycle was finished, indicating a refractory period in the mitochondrial sensitivity to Sr^2 +. All of the Ca^2 +- and Sr^2 +-induced effects were observed in the presence of cyclosporin A. This paper discusses a possible role of Pal/Ca^2 + pores in the maintenance of cell ion homeostasis.     

Interactions between nutritional and opioidergic pathways in the control of LH secretion in male sheep

Our aim was to determine the role of opioidergic processes in the effects of nutrition on the secretion of LH pulses in the mature male sheep. In the first of three experiments, adult Merino rams were acclimatised to a maintenance diet and then allocated to one of three dietary groups (n = 5): continuation of the maintenance diet (Group M); reduction to half of the maintenance allocation (Group HM); or supplementation of the maintenance diet with lupin grain (Group HD). An initial administration of naloxone (2 mg/kg body weight, i.v.) was followed at 40-min intervals by three further administrations (1 mg/kg). Blood was sampled every 20 min for 12 h before the initial naloxone administration and then for a further 6 h. LH pulse frequency after naloxone treatment was significantly higher in Group HD than in Group HM (P < 0.05). The second study tested whether the response to naloxone depended on calcium status. We used 22 adult Merino rams in two consecutive experiments, one in which the rams were fed a maintenance diet, and one in which the rams were fed with the maintenance diet plus 1 kg lupin grain for 5 weeks. In both experiments, rams were allocated to groups that received one of the following treatments: (a) 0.02 g/kg calcium borogluconate + 0.2 mg/kg naloxone hydrochloride (Nal + Ca²⁺; n = 6); (b) 0.2 mg/kg naloxone hydrochloride (Nal; n = 6); (c) 0.02 g/kg calcium borogluconate (Ca²⁺; n = 5); (d) 0.1 ml/kg NaCl 0.9% (Saline; n = 5). All treatments were given as a single i.v. administration daily for 5 days. Blood was sampled every 20 min for 24 h during the acclimatization period (Day 0) and on the last day (Day 5) of treatment. In the first study (under maintenance), none of the treatments affected LH pulse frequency. In the second study (the lupin-supplemented rams), LH pulse frequency was significantly increased (P < 0.05) by the administration of naloxone + Ca²⁺, naloxone alone and Ca²⁺ alone. Overall, rams on a low plane of nutrition showed the smallest response to naloxone, suggesting that an opioidergic mechanism is not involved in the suppressive effect of restricted nutrition on the gonadotrophic axis. Rather, because testosterone secretion was increased on the high plane of nutrition, the LH responses to naloxone are better explained by the effects of testosterone on opioidergic mechanisms. Finally, we failed to observe any interaction between opioids and calcium in the control of LH secretion.     

Water use and feeding patterns of the marsupial western grey kangaroo (Macropus fuliginosus melanops) grazing at the edge of its range in arid Australia,as compared with the dominant local livestock,the Merino sheep (Ovis aries)

Western grey kangaroos (Macropus fuliginosus melanops) are large, foregut fermenting herbivores common to Australia's southern woodlands and shrublands, but they extend well into semiarid habitats at the north-eastern edge of their range. At this range boundary, western grey kangaroos occupy open chenopod (saltbush) shrubland, along with Australia's other large native kangaroos, as well as with extensive pastoral stock, primarily the wool-breed merino sheep. In this habitat, within a large naturally vegetated enclosure (16 ha), western grey kangaroos grazing sympatrically with merino sheep spent much of the day resting under shade trees, and fed mainly during the evening and early morning, mainly on grasses and flat-leaved chenopods. On this diet, western grey kangaroos had water turnovers similar to those of red kangaroos, at 1.1 L d⁻¹. Sheep, however, used 7.7 L of water each day. Thus, although the sheep were twice the average body mass of kangaroos, the sheep used more than seven times as much water. This level of water use by sheep was almost half that previously reported for sheep at the same site feeding mainly on salt-laden chenopods (ca. 12 L d⁻¹), but was consistent with other studies showing lower water usage by sheep feeding on trees and low-salt shrubs; foregut (rumen) contents of our sheep comprised 35% tree browse. Overall, our data do not support suggestions that western grey kangaroos are limited mainly by water at this arid range-boundary. Notably, the western grey kangaroos’ feeding behaviours were consistent with those of other arid-zone kangaroos, highlighting distinctive differences in the ecological physiology of the foregut fermenting kangaroos and the ruminant sheep.     

Quantification of the water,energy and carbon footprints of wastewater treatment plants in China considering a water–energy nexus perspective

Water and energy are closely connected and both are very important for human development. Wastewater treatment plants (WWTPs) are central to water–energy interactions as they consume energy to remove pollutants and thus reduce the human gray water footprint on the natural water environment. In this work, we quantified energy consumption in 9 different WWTPs in south China, with different treatment processes, objects, and capacities. The energy intensity in most of these WWTPs is in the range of 0.4–0.5 kWh/m³ in 2014. Footprint methodologies were used in this paper to provide insight into the environmental changes that result from WWTPs. A new indicator “gray water footprint reduction” is proposed based on the notion of gray water footprint to better assess the role of WWTPs in reducing human impacts on water resources. We find that higher capacity and appropriate technology of the WWTPs will result in higher gray water footprint reduction. On average, 6.78 m³ gray water footprint is reduced when 1 m³ domestic sewage is treated in WWTPs in China. 13.38 L freshwater are required to produce the 0.4 kWh electrical input needed for treating 1 m³ domestic wastewater, and 0.23 kg CO₂ is emitted during this process. The wastewater characteristics, treatment technologies as well as management systems have a major impact on the efficiency of energy utilization in reducing gray water footprint via these WWTPs. The additional climate impact associated with wastewater treatment should be considered in China due to the enormous annual wastewater discharge. Policy suggestions are provided based on results in this work and the features of China's energy and water distribution.     

Wire tension versus wire frequency: An experimental Ilizarov frame study

Stability of an Ilizarov frame highly depends on maintenance of adequate tension in the wires. Wire tension should be measured accurately in experimental laboratory studies when new types of wire fixators are tested. In this study, 20 wires were tested using two different wire fixators. The wires were sequentially tensioned from 0 to 1275 N in 50 N intervals. For each tension value, corresponding vibration frequency was recorded. We then described the relationship between wire tension and wire vibration frequency in an empirical equation (R²=99.8). Wire vibration frequency can also be described theoretically by the Euler–Bernoulli equation for a thin beam. Theoretical frequencies were calculated and compared with corresponding experimental frequencies. A close agreement was found (95% limits of agreement, ±3.2 Hz). This empirical equation represents a simple tool, applicable when investigating the effect of new wire fixators, pre-tensioning and frame constructions on wire tension.     

Advantage of menadione-catalyzed chemiluminescent assay for the determination of viable mammalian cell number

A chemiluminescent assay composed of TCPO [bis(2,4,6-trichlorophenyl)oxalate] and harmless rhodamine B is proposed to be superior in the determination of menadione-catalyzed hydrogen peroxide (H₂O₂) production by viable mammalian cells to that composed of TCPO and harmful pyrene [Anal. Biochem. 207 (1992) 255–260]. In tests, the proposed assay showed that the measurable concentration of H₂O₂ and the viable cell number ranged from 10^?9 to 10^?3 M and from 2 × 10² to 2 × 10⁶ cells/100 μl/well in the presence of 10% bovine serum, respectively. The measuring time was approximately 10 min. On the other hand, the measurable cell numbers by the colorimetric WST-1 and MTT assays requiring several hours ranged only from 10³ to 10⁴ cells/100 μl/well and from 10⁴ to 10⁵ cells/100 μl/well, respectively. The cytotoxicity of sodium dodecyl sulfate was also observed at intervals of 1 min by the proposed assay, but not by the above colorimetric assays.     

Fungal network responses to grazing

Mycelial networks operate on scales from microscopic to many m² and naturally persist for extended periods. As fungi exhibit highly adaptive development, it is important to test behavioural responses on natural substrata with realistic nutrient levels across a range of spatial scales and extended time periods. Here we quantified network responses over 7.5 months in large (57 × 57 cm) microcosms to test whether grazing shifts the network to a more resilient architecture. Resource limitation constrained any ability to respond at all, with both grazed and ungrazed networks gradually thinning out over time. Added resources sustained further exploratory growth, but only transiently increased cross-connectivity and network resilience, when tested by simulated damage in silico. Grazed networks were initially weaker and emergence of new exploratory growth was curtailed. However, increased interstitial proliferation led to new cross-links, consolidating the existing mycelial network and increasing the resilience of the network to further attack.     

Control of ichthyotoxic Cochlodinium polykrikoides using the mixotrophic dinoflagellate Alexandrium pohangense: A potential effective sustainable method

Red tides dominated by Cochlodinium polykrikoides often lead to great economic losses and some methods of controlling these red tides have been developed. However, due to possible adverse effects and the short persistence of their control actions, safer and more effective sustainable methods should be developed. The non-toxic dinoflagellate Alexandrium pohangense is known to grow well mixotrophically feeding on C. polykrikoides, and populations are also maintained by photosynthesis. Thus, compared with other methods, the use of mass-cultured A. pohangense is safer and the effects can be maintained in the long term. To develop an effective method, the concentrations of A. pohangense cells and culture filtrate resulting in the death of C. polykrikoides cells were determined by adding the cells or filtrates to cultured and natural populations of C. polykrikoides. Cultures containing 800 A. pohangense cells ml⁻¹ eliminated almost all cultured C. polykrikoides cells at a concentration of 1000 cells ml⁻¹ within 24 h. Furthermore, the addition of A. pohangense cultures at a concentration of 800 cells ml⁻¹ to C. polykrikoides populations from a red-tide patch resulted in the death of most C. polykrikoides cells (99.8%) within 24 h. This addition of A. pohangense cells also lowered the abundances of total phototrophic dinoflagellates excluding C. polykrikoides, but did not lower the abundance of total diatoms. Filtrate from 800 cells ml⁻¹ A. pohangense cultures reduced the population of cultured C. polykrikoides by 80% within 48 h. This suggests that A. pohangense cells eliminate C. polykrikoides by feeding and releasing extracellular compounds. Over time, A. pohangense concentrations gradually increased when incubated with C. polykrikoides. Thus, an increase in the concentration of A. pohangense by feeding may lead to A. pohangense cells eliminating more C. polykrikoides cells in larger volumes. Based on the results of this study, a 1 m³ stock culture of A. pohangense at 4000 cells ml⁻¹ is calculated to remove all C. polykrikoides cells in ca. 200 m³ within 6 days. Furthermore, maintenance of A. pohangense populations through photosynthesis prepared A. pohangense to eliminate C. polykrikoides cells in future red-tide patches. Moreover, incubation of A. pohangense at 2000 cells ml⁻¹ with juvenile olive flounder Paralichthys olivaceus for 3 days did not result in the death of fish. Therefore, the method developed in this study is a safe and effective way of controlling C. polykrikoides populations and can be easily applied to aqua-tanks on land.     

Protein requirements of Texel crossbred lambs

A comparative slaughter trial was conducted to determine the protein requirements for maintenance and growth of feedlot Texel crossbred lambs. Thirty 11/16 Texel × 5/16 Ile de France crossbred noncastrated male lambs weaned at 42 days of age (16.2 ± 2.1 kg of shrunk body weight; SBW) were used. Five lambs were chosen randomly and slaughtered after 10 days of experimental management and diet adaptation (baseline group). Fifteen lambs were fed ad libitum and slaughtered at 25, 30 or 35 kg of SBW. The remaining 10 lambs were then assigned randomly to two levels of dry matter intake, either 70 or 55% of the ad libitum intake, and were slaughtered concomitantly with lambs slaughtered at 35 kg of SBW but given free choice access to feed. Total body N content and retention were determined. Additionally, six Texel × Ile de France crossbred lambs (30.4 ± 2.6 kg of SBW) housed in individual metabolic cages were used in a replicated 3 × 3 Latin square digestibility trial to evaluate diet digestibility and microbial protein synthesis at the different levels of feed intake. The endogenous N loss was 243 ± 29 mg/kg^0.75 of SBW, corresponding to a net protein requirement for maintenance of 1.52 ± 0.18 g/kg^0.75 of SBW. The metabolizable protein (MP) requirement for maintenance was 2.31 g/kg^0.75 of SBW, and the efficiency of MP use for maintenance was 0.66. Fleece-free body protein content decreased from 163 to 155 g/kg of empty body weight (EBW) as EBW increased from 13 to 28 kg. However, when protein in fleece was considered the whole-body protein content remained nearly constant. Net protein requirements for body weight gain and wool growth of lambs at 15 and 35 kg of SBW, and an average daily gain of 250 g, were 28.7 and 27.3 g/day and 3.8 and 5.8 g/day, respectively. Estimated efficiencies of MP use for body weight gain (k_pg) and wool growth (k_pw) were, respectively, 0.71 and 0.46. Growth pattern of the wool has a high influence on protein requirements of lambs. Texel crossbred growing lambs used in our study showed protein requirements for growth lower than those reported by the most nutritional systems.     

Multiple effects of anthracene-9-carboxylic acid on the TMEM16B/anoctamin2 calcium-activated chloride channel

Ca^2 +-activated Cl⁻ currents (CaCCs) play important roles in many physiological processes. Recent studies have shown that TMEM16A/anoctamin1 and TMEM16B/anoctamin2 constitute CaCCs in several cell types. Here we have investigated for the first time the extracellular effects of the Cl⁻ channel blocker anthracene-9-carboxylic acid (A9C) and of its non-charged analogue anthracene-9-methanol (A9M) on TMEM16B expressed in HEK 293T cells, using the whole-cell patch-clamp technique. A9C caused a voltage-dependent block of outward currents and inhibited a larger fraction of the current as depolarization increased, whereas the non-charged A9M produced a small, not voltage dependent block of outward currents. A similar voltage-dependent block by A9C was measured both when TMEM16B was activated by 1.5 and 13 μM Ca^2 +. However, in the presence of 1.5 μM Ca^2 + (but not in 13 μM Ca^2 +), A9C also induced a strong potentiation of tail currents measured at − 100 mV after depolarizing voltages, as well as a prolongation of the deactivation kinetics. On the contrary, A9M did not produce potentiation of tail currents, showing that the negative charge is required for potentiation. Our results provide the first evidence that A9C has multiple effects on TMEM16B and that the negative charge of A9C is necessary both for voltage-dependent block and for potentiation. Future studies are required to identify the molecular mechanisms underlying these complex effects of A9C on TMEM16B. Understanding these mechanisms will contribute to the elucidation of the structure and functional properties of TMEM16B channels.     

Thiadiazole derivatives as New Class of β-glucuronidase inhibitors

Thiadiazole derivatives 1–24 were synthesized via a single step reaction and screened for in vitro β-glucuronidase inhibitory activity. All the synthetic compounds displayed good inhibitory activity in the range of IC₅₀ = 2.16 ± 0.01–58.06 ± 1.60 μM as compare to standard d-saccharic acid 1,4-lactone (IC₅₀ = 48.4 ± 1.25 μM). Molecular docking study was conducted in order to establish the structure–activity relationship (SAR) which demonstrated that thiadiazole as well as both aryl moieties (aryl and N-aryl) involved to exhibit the inhibitory potential. All the synthetic compounds were characterized by spectroscopic techniques ¹H, ¹³C NMR, and EIMS.     

Influence of hydrology process on wetland landscape pattern: A case study in the Yellow River Delta

A comprehensive study using ecological engineering analysis was conducted on the influence of the hydrology process in the Yellow River Delta. It was found that water and sediment played an important role in the formation and maintenance of the estuarine wetland. Based on hydrological data (1950–2005), meteorological data (1954–2005) and landscape data produced from integrated Landsat TM images of the Yellow River Delta, the relationship among all the above factors has been analyzed. The results indicated that runoff, sediment discharge and the area of the reed marsh wetland, meadow wetland and tidal wetland had evidently decreased from 1986 to 2001. The runoff and sediment discharge into the Yellow River Delta at a rate of 200–300 × 10⁸ m³ and at 5–8 × 10⁸ t, respectively, were probably the most optimal range for maintaining the stable wetland landscape pattern. Regression analysis and principal component analysis showed that there was a strong positive correlation between the area of wetland landscape and runoff, sediment discharge, whereas there was a negative correlation between the area and the temperature, and there was no significant trend with precipitation levels. The water and sediment discharge are the dominant variable factors of the wetland. Human activities also have an important influence on transformation of wetland types as well as wetland degradation. Therefore, with economic development, climate change and sustainable utilization of resources, great attention should be paid to the changes of natural landscape and their causes.     

New insights into the chemical and isotopic composition of human-body biominerals. I: Cholesterol gallstones from England and Greece

We have analyzed gallstones from four patients of Europe and particularly from England (including samples from a mother and a daughter) and Greece. According to the XRD, FTIR, NMR and laser micro-Raman results the studied materials correspond to typical cholesterol monohydrate (ChM). The micro-morphology of cholesterol microcrystals was investigated by means of SEM–EDS. The XRF results revealed that Ca is the dominant non-organic metal in all gallstones (up to ～1.95 wt.%) together with Fe, Cu, Pb and Ni (up to ～19 ppm for each metal). Gallstones from England contain additional Mn (up to ～87 ppm) and Zn (up to ～6 ppm) while the sample of the mother contains negligible Zn and Mn, compared to that of her daughter, but significant As (～4.5 ppm). All cholesterol gallstones examined are well enriched in potentially toxic metals (Pb, as well as Ni in one case) and metalloids (As also in one case) as compared to the global average. The position of Zn, which is a characteristic biometal, in the structure of cholesterol, was investigated by molecular simulation using the Accelrys Materials Studio^® software. On the basis of IRMS results, all gallstones examined exhibit a very light δ¹³C signature (average δ¹³C ～?24‰ PDB). Gamma-ray spectrometry measurements indicate the presence of ²¹⁴Pb and ²¹⁴Bi natural radionuclides due to the ²³⁸U series as well as an additional amount of ⁴⁰K.     

In vitro cultures of Silybum marianum and silymarin accumulation

In this study, a protocol for initiation of callus and shoot cultures from leaves and shoot tips explants of different silybium genotypes collected from different locations in Egypt was established. Callus cultures were initiated from leaves explants and exposed to different concentrations of the precursor (coniferyl alcohol). Shoot cultures were initiated from shoot tips explants. Moreover, the produced plants of the different Silybium shoots as well as intact plants were subjected to protein screening using SDS–PAGE analysis.Results obtained revealed that the optimum medium for growth and maintenance of friable callus was MS medium supplemented with 0.25 mg L⁻¹ 2,4-Dichlorophenoxy acetic acid (2,4-D) + 0.25 mg L⁻¹ Kinetin (Kin). The best medium for proliferation of high number of shoots was MS-medium with 0.25 mg L⁻¹ each of Benzyl Adinine (BA) and Naphthalene Acetic Acid (NAA). Coniferyl alcohol in concentration of 30 μM caused an increase in accumulation of silymarin contents in most callus cultures. SDS–PAGE of different Silybium shoots revealed that the protein profiles of 100% of in vitro produced plantlets similar to their control.