provenance: AgriculturalAndFoodSciences,1970-2015-Agricultural And Food Sciences-2012.jsonl.gz.gz

text:
PHYSICAL PROPERTIES OF CORN RESIDUES

Corn residues (cobs, leaves and stalks) are abundan tly available renewable materials that can be used as an energy source in gasification and combustion system . Proper understanding of the physical properties of these materials is necessary for their use in therm ochemical conversion processes. The physical proper ties (moisture content, particle size, bulk density and porosity) of corn cobs, leaves and stalks were dete rmin d in this study. The moisture contents were 6.38, 7.9 2 and 6.40% of the cobs, leaves and stalks, respect ively. The cobs had the highest weight percentage (18.23%) of the small particles (<0.212 mm) and the leaves had the highest weight percentage (40.10%) of large par ticles (>0.850 mm). Most of the stalk particles (84 .82%) were in the range of 0.212-0.850 mm. The cob partic le size had a normal concave (inward) distribution between particle sizes 0.106 mm (18.23 weight %) an d 0.925 mm (25.26 weight %) with the lowest weight percentage (5.30 weight %) at 0.390 mm particle siz while the stalk particle size had a normal convex (outward) distribution between particle sizes 0.106 mm (8.49 weight %) and 0.925 mm (6.69 weight %) with the highest weight percentage (23.47 weight %) at the 0.605 mm particle size. The leaves had an increasing trend of particle size distribution betw en the particle sizes 0.106 and 0.925 mm. The aver age particle sizes for the cobs, leaves and stalks were 0.56, 0.70 and 0.49 mm, respectively. The average bulk density was 282.38, 81.61 and 127.32 kg m −3 for the corn cobs, leaves and stalks, respectively . The average porosity was 67.93, 86.06 and 58.51% for the corn c bs, leaves and stalks, respectively. A positive re lationship between the average particle size and the porosity was observed for the corn residues. The differences in the physical properties among the corn residues (cobs, leaves and stalks) observed in this study are due t o variations in the compositions and structures of th ese materials.

INTRODUCTION
is an important food for many people in Africa, Asia and Latin America and is used in animal feeding in North America and some parts of the world. In sub-Saharan Africa, corn is a staple food for an estimated 50% of the population IITA, 2009 and it remains the most important agricultural crop for over 70 million farm families worldwide. Corn is used as human food in the form of tortillas, porridge, popcorn and barbecues and as forage and silage for animals. It is also a good source of industrial products such as starch (Zhang et al., 2012), vitamin (Warman and Havard, 1998), fiber (Pandya and Srinivasan, 2012), oil (Comin et al., 2012) and ethanol (Lamsal et al., 2011).
The global corn production increased from 599.35-867.52 million tonnes (44.74% increase) during the period of 2001-2011 as shown in Fig. 1 (USDA, 2011), which is much higher than the increase in the world population of 12.34% (6.16-6.92 billion) during the same period. The estimated value of global corn production in 2011 was US$ 199.53 billion. Table 1 shows the corn production by important producing countries. The per capita corn consumption, corn imports and exports of the top 10 counties in each category are presented in Fig. 2-4, respectively.
Cobs, leaves and stalks are important residues of corn processing and consumption. For every 1 kg of dry corn grains produced, about 0.15 kg of cobs, 0.22 kg of leaves and 0.50 kg of stalks are produced (Sokhansanj et al., 2002;(USDA, 2011. This results the production of about 130.13, 190.85 and 433.76   Currently, these residues have a number of limited applications including: (a) use of corn cobs as building material and activated carbon (Pinto et al., 2012;Cao et al., 2006), (b) use of corn leaves as a feedstock for fermentable sugars and supplemental fiber source for paper pulp Su et al., 2006) and (c) use of corn stalks as livestock feed and biofertilizer (Chen et al., 2010a;Li et al., 2007).   However, these materials can be (USDA, 2011; used as an energy source in thermochemical conversion processes such as gasification and pyrolysis (Kumar et al., 2008;Ioannidou et al., 2009).
The physical properties (moisture content, particle size, bulk density and porosity) of a given biomass material such as corn cobs, leaves and stalks greatly influence the design and operation of thermochemical conversion systems. High moisture content decreases the heating value of fuel, which in turn reduces the conversion efficiency as a large amount of energy would be used for the initial drying step during the conversion processes (Mansaray and Ghaly, 1997). The particle size distribution affects the flowability, heating, diffusion and rate of reaction (Guo et al., 2012;Hernandez et al., 2010). The bulk density affects the economics of collection, transportation and storage as well as feeding the material into the thermochemical conversion system (Natarajan et al., 1998). Porosity affects the interstitial airflow velocity and the heat and mass transfer conditions and ultimately influences reaction parameters such as heat conductivity, burning rate, conversion efficiency and emissions Hamel and Krumm, 2008). Therefore, a full understanding of the physical properties of cobs, leaves and stalks is essential for the design and operation of efficient thermochemical conversion systems such as gasifiers and combustors.
The main objectives of this study were: (a) to investigate the physical properties (moisture content, particle size distribution, bulk density and porosity) of corn plant residues (cobs, leaves and stalks) as related to pre-processing and design of thermochemical conversion systems and (b) to determine if there are differences in the physical properties of these three parts of corn plant.

Sample Preparation
Corn cobs, leaves and stalks were collected from China. The samples were ground through a coarse sieve (12.7 mm) and a 20-mesh sieve (0.85 mm) on a medium size Wiley Mill (Model X876249, Brook Crompton Parkinson Limited, Toronto, Ontario). The coarse ground samples were then ground through a 40-mesh sieve (0.425 mm) on the Wiley Mill in order to narrow the range of particle size and thus obtain homogeneous samples.

Moisture Content
The moisture content was determined using the oven-drying method (ASTM 2010). A large aluminum dish was weighed using a digital balance (Model PM 4600, Mettler Instrument AG, Greifensee, Zurich). The ground sample was placed in the dish and the dish and sample were weighed. The dish and sample were then placed in an air-forced drying oven (Heratherm, Thermo Fisher Scientific Inc., Waltham, USA) and kept at 105°C until a constant weight was achieved. The dish containing the dried sample was cooled to the room temperature in a desiccator and then weighed. The moisture content was calculated on a wet basis as follows Eq. 1: Where: MC = The moisture content (%) WW = The wet weight of the sample and dish (g) DW = The dry weight of the sample and dish (g)

Particle Size Distribution
The particle size distribution was determined using seven standard sieves (Canadian Standard Sieve Series, W.S. Tyler Company of Canada Limited, St. Catharines, Ontario) and a bottom pan that collects everything that passed though the seventh sieve. The sieves were mounted on an electrical sieve shaker driven by a 0.25-hp electric motor running at 1725 rpm (Model Rx-86, Hoskin Scientific Limited, Gastonia, North Carolina). The sample was placed in sieve 1, which was then covered with the sieve lid. The shaker was operated at the speed of 350 rpm for 30 min. The particles collected in each sieve were weighed. The sieve number, mesh number and mesh size of the seven sieves are shown in Table 2.

Bulk Density
An empty container (150 mL) was weighed using a digital balance (Model PM 4600, Mettler Instrument AG, Greifensee, Zurich) to the nearest 0.0001g. The container was filled with the sample and the material was slightly compacted to ensure absence of large void spaces. The container and the sample were then weighed. Three replicates were carried out. The wet bulk density of the sample was calculated from the following Eq. 2: Where: ρ b = The bulk density of the sample (g cm -3 ) W 2 = The weight of the container and sample (g) W 1 = The weight of the container (g) V = The volume of the container (cm 3 )

Porosity
The porosity of biomass was determined using the water pycnometer method. A sample of approximately 33 mL was placed in a 100 mL graduated cylinder. A wire mesh screen was placed on the top of the sample to prevent material from floating once submerged in water. Distilled water was slowly poured over the sample until the water level was above the top of the sample. The cylinder was gently rocked from side to side ten times to free trapped air bubbles before recording the final water level. The amount of added water and the water level were recorded to the nearest 1 mL. The cylinder was emptied and cleaned thoroughly after each test. Three replicates were carried out. The porosity of biomass was calculated from the following Eq. 3:

AJBB
Where: P = The porosity of the sample (%) V i = The combined volume of the sample plus added water (ml) V f = The final total volume of the sample and added water (ml) V s = The volume of the sample (ml)

Moisture Content
The moisture content results of the corn cobs, leaves and stalks are shown in Table 3. The moisture content was 6.38, 7.92 and 6.40% for the corn cobs, leaves and stalks, respectively. The moisture contents of corn cobs and stalks are in the range of 5.3-7.2% reported by Ileleji and Zhou (2008) for the corn stover (whole corn plant tissue except the ear) from USA. The moisture content of corn leaves is similar to the values of 8-9% reported by Kumar et al. (2011) from the leaves of corn stover from USA. However, the moisture contents reported in this study (6.38-7.92%) are lower than the value of 9% reported by Bitra et al. (2009) for the corn stover from USA, the value of 11.3% reported by Igathinathane et al. (2009) for the corn stalk from the USA and the value of 11.4% reported by Luo et al. (2011) for the corn stalk from China. These variations could be due to differences in the collection and storage procedures and the use of different techniques to determine the moisture content.    Liang et al. (2003) and Pommier et al. (2008) stated that the moisture content provides a medium for the transport of dissolved nutrients required for the metabolic and physiological activities of microorganisms in the solid fuels and an increase in moisture content will increase the biodegradation rate of organic material, resulting in the loss of solid fuels. The high moisture content of wheat straw will substantially affect its quality as a fuel source and decreases its heating value, which in turn reduces the conversion efficiency and performance of the system, because a large amount of energy would be used for vaporization of the fuel moisture during the conversion processes (Chen et al., 2009;Ghaly and Al-Taweel, 1990). A dry material is thus preferred for storage, size reduction, gasification and combustion. On the other hand, a certain amount of moisture in the fuel may be beneficial for densification and gasification (Mani et al., 2006;Swierczynski et al., 2007). Table 4 shows the particle size distribution results of the cobs, leaves and stalks. There were significant differences among the corn cobs, leaves and stalks. Cobs had more (18.23%) small particles (<0.212 mm) than leaves (4.03%) and stalks (8.49%). Leaves had more (40.10%) larger particles (>0.850 mm) than cobs Science Publications AJBB (25.26%) and stalks (6.69%). However, the medium particles (0.212-0.850 mm) for the cobs, leaves and stalks were 56.51, 55.87 and 84.82%, respectively. Figure 5 shows the particle size distribution for the three corn residues. The particle size distribution of the cobs had a normal concave (inward) distribution between particle sizes 0.106 mm (18.23 weight %) and 0.925 mm (25.26 weight %) with the lowest weight percentage (5.30 weight %) at 0.390 mm particle size. The particle size distribution of the stalks had a normal convex (outward) distribution between particle sizes 0.106 mm (8.49 weight %) and 0.925 mm (6.69 weight %) with the highest weight percentage (23.47 weight %) at the 0.605 mm particle size. However, the particle size of the leaves had an increasing trend distribution between particle sizes 0.106 mm and 0.925 mm, the smaller the particle size the lower the weight percentage and the larger the particle size the greater the weight percentage.  The variations in particle size distribution among the three corn residues observed in this study could be due to variations in their compositions. Duguid et al. (2009) reported that the glucan, xylan, acid insoluble lignin, acid soluble lignin and ash contents were 36.6, 29.2, 17.8, 4.9 and 0.3% for corn cobs, 35.8, 20.0, 17.0, 5.4 and 2.0% of corn leaves and 35.4-37.3, 18.5-20.5, 17.8-20.2, 4.4-4.6 and 0.5-1.2% of corn stalks, respectively. Montross and Crofcheck (2004) reported that the glucose concentrations for corn cobs, leaves and stalks were 55.9, 35.7 and 29.6%, respectively. There are also reported variations in the cellulose, hemicellulose and lignin contents among the corn cobs, leaves and stalks. The reported cellulose, hemicellulose and lignin contents were 30, 38 and 3% for corn cobs (Mullen et al., 2010), 38.77, 23.74 and 7.15% for corn leaves (Wang et al., 2012) and 40.4, 71.3 and 18.3% of corn stalks (Luo et al., 2011), respectively.

Particle Size Distribution
The average particle sizes for corn cobs, leaves and stalks were 0.56, 0.71 and 0.49 mm, respectively ( Table  3 and Fig. 6). These values are within the range of 0.25-4.42 mm reported by Ileleji and Zhou (2008) for the corn stover from USA and similar to the value of 0.77 mm reported by Li et al. (2008) for the corn leaves, stalks and bracts from China. They are, however, lower than the values of 1-2 mm reported by Chen et al. (2010b) for the corn cobs from China and the value of 5.6 mm reported by Mani et al. (2006) for the corn stover from Canada. These differences could be the result of using different equipment and different grinding procedures. The sample presented by Chen et al. (2010b) was chopped in a knife mill, the sample reported by Mani et al. (2006) was ground in a tub grinder, whereas the samples in this study were ground through a Wiley mill with three sieves: a coarse sieve (12.7 mm), a 20-mesh sieve (0.85 mm) and a 40-mesh sieve (0.425 mm). Ryu et al. (2006) stated that large particles are thermally thick thereby having a slow devolatilization rate and more distributed heat transfer to nearby particles. On the other hand, small particles of fuel may enhance the reaction area and result in high burning rates and ignition front speeds (Kwong et al., 2007). Small particle size can also significantly increase the bulk density of biofuels and eventually increase the energy density and reduce the cost of transport and storage (Sangnark and Noomhorm, 2004;Chiueh et al., 2012;Deng et al., 2009). Size reduction therefore appears to be beneficial and important for pretreatment of biofuels before the utilization (Zhang and Zhang, 1999).

Bulk Density
The bulk density results of the corn cobs, leaves and stalks are shown in Table 3. The average bulk density Science Publications AJBB was 282.38, 81.61 and 127.32 kg m −3 for the corn cobs, leaves and stalks, respectively. The average bulk density of corn stalks is similar to the value of 127.5 kg m −3 reported by Sciban et al. (2008) for the corn stalks from Serbia and the value of 129 kg m −3 reported by Raveendran et al. (1995) for the corn stalks from India. The bulk density values of corn leaves and stalks are within the values of 37-150 kg m −3 reported by  for the corn stover from USA. The average bulk density of corn cobs is within the range of 170.00-297.78 kg m −3 reported by Pinto et al. (2012) for the corn cobs from Portugal. However, these values (81.61-282.38 kg m −3 ) are lower than the values of 695.5-1169.9 kg m -3 reported by Kaliyan and Morey (2009) and higher than the values of 34.44-80.24 kg m −3 reported by Chevanan et al. (2010) for the corn stovers from USA. These differences could also be the result of using different equipment and different grinding procedures. The samples reported by Kaliyan and Morey (2009) were densified by a cylinder, the samples reported by Chevanan et al. (2010) were chopped in a knife mill, whereas the samples used in this study were ground in a Wiley mill (12.7, 0.85 and 0.425 mm).
The bulk density of the cobs was 121.79% higher than that of the stalks and 246.01% higher than that of the leaves. The bulk density of the stalks was only 56.01% higher than that of the leaves. These differences may be due to variations in the particle size distribution, the chemical composition and the structures of these materials. Figure 7 shows SEM photographs of the corn residues which indicate structural differences among these residues. The structure of cobs is more compact than the leaves and stalks which results in a higher bulk density. Rozainee et al. (2008) stated that the bulk density of fuel affects its residence time in the reactor. Lower bulk density may result in lower conversion efficiency, as it gives rise to poor mixing characteristics and a nonuniform temperature distribution, both of which create unfavorable operating conditions of the thermochemical conversion systems. Densification of corn stover can increase its density to more than 600 kg m -3 (Petrolia, 2008) through a squeezer and to more than 1100 kg m -3 (Kaliyan and Morey, 2009) through a compression cylinder. The major advantages of this technique include high volumetric density and energy content, lower transportation and storage costs and lower emissions during combustion Ryu et al., 2006;Mani et al., 2006;Khan et al., 2009). The high investment on equipment and energy input required for pelletization and compression are the major constraints of the densification process (Adapa et al., 2009). However, the high cost of oil, current demand for biomass utilization and technology improvement will make the processes of densification and compression more attractive.

Porosity
The porosity results of the corn cobs, leaves and stalks are shown in Table 3. The average porosity was 67.93, 86.06 and 58.71% for the corn cobs, leaves and stalks, respectively. Corn leaves have the highest porosity, because they have larger particles and the greatest average particle size than those of the cobs and stalks.
The porosity values (58.51-86.06%) obtained in this study are lower than the values of 88.21-88.80% (calculated from the bulk density and particle density) presented by Mani et al. (2004) for the corn stover from Canada. They are, however, higher than the values of 11.36-29.06% presented by Theerarattananoon et al. (2011) for the corn stover from USA. These may be due to the fact that the samples presented by Mani et al. (2004) were milled though 3.2 mm, 1.6 mm and 0.8 mm sieves, the samples presented by Theerarattananoon et al. (2011) were made into pellets, while the samples in this study were milled though 12.7, 0.85 0.425 mm sieves and not made into pellets. A positive relationship between average particle size and porosity was observed for the corn residues (Fig. 8), the smaller the particle size the lower the porosity. The relationship can be described by the following Eq. 4: 2 P .5.55 130.95PS (R 1.00) = + = Where: P = The porosity (%) PS = The average particle size (mm) The porosity of biomass samples depends on a number of factors including particle size distribution, particle shape, shaking and pressing . A decrease in the porosity will increase the interstitial airflow velocity and brings changes in heat and mass transfer conditions and ultimately influences the combustion parameters such as heat conductivity, burning rate, conversion efficiency and emissions Hamel and Krumm, 2008). Pelletizing or compacting can decrease the porosity of biomass samples. However, these dense materials may deteriorate the flow characteristics in the gasifier or combustor, thereby causing post-processing problems (Chen et al., 2009).

CONCLUSION
The results showed that the cobs, leaves and stalks of corn plant had different physical properties (moisture content, particle size distribution, bulk density and porosity). The moisture contents were 6.38, 7.92 and 6.40% for the cobs, leaves and stalks, respectively. The cobs had the highest weight percentage (18.23%) of most of the small particles (<0.212 mm) while the leaves had the highest weight percentage (40.10%) of large particles (>0.850 mm). Most of the stalk particles (84.82%) were in the range of 0.212-0.850 mm. The cob particle size had a normal concave (inward) distribution between particle sizes 0.106 mm (18.23 weight %) and 0.925 mm (25.26 weight %) with the lowest weight percentage (5.30 weight %) at 0.390 mm particle size while the stalk particle size had a normal convex (outward) distribution between particle sizes 0.106 mm (8.49 weight %) and 0.925 mm (6.69 weight %) with the highest weight percentage (23.47 weight %) at the 0.605 mm particle size. The leaves had an increasing trend of particle size distribution between the particle sizes 0.106 and 0.925 mm. The average particle sizes for the cobs, leaves and stalks were 0.56, 0.70 and 0.49 mm, respectively. The average bulk density was 282.38, 81.61 and 127.32 kg m −3 for the corn cobs, leaves and stalks, respectively. The average porosity was 67.93, 86.06 and 58.51% for the corn cobs, leaves and stalks, respectively. A positive relationship between the average particle size and the porosity was observed for the corn residues. The differences in the physical properties among the corn residues (cobs, leaves and stalks) observed in this study are due to variations in the compositions and structures of these materials.

ACKNOWLEDGEMENT
The project was funded by National Science and Engineering Council (NSERC) of Canada. The authors would like to thank Mr. Rye Dube and Mr. Weida Shi for their technical support during the grinding experiments.


extfieldsofstudy: Biology Medicine

text:
Effects of Alfalfa Meal on Growth Performance and Gastrointestinal Tract Development of Growing Ducks

A study was conducted to evaluate effects of alfalfa meal on growth performance and gastrointestinal tract development of growing layer ducks to provide evidence for application of alfalfa meal in the duck industry. Two hundred and fifty-six healthy Shaoxing 7-wk old growing layer ducks were selected and randomly allocated to 1 of 4 dietary treatments based on corn and soybean meal and containing 0, 3, 6, and 9% of alfalfa meal for 8 wks. Each treatment consisted of 4 replicates of 16 ducks each. Briefly, birds were raised in separate compartments, and each compartment consisted of three parts: indoor floor house, adjacent open area and a connecting water area. The results showed: i) Growing ducks fed alfalfa meal diet were not significantly different in average daily gain, feed intake and gain-to-feed ratio from those fed no alfalfa diet (p>0.05). ii) Alfalfa meal increased the ratio crop, gizzard to live weight, caecum to live weight, the caecum index of growing ducks (p<0.05). iii) Villus height in duodenum and jejunum of growing ducks increased significantly with the increase of alfalfa meal levels (p<0.05). Crypt depth in duodenum and jejunum of growing ducks decreased significantly with the increase of alfalfa meal levels (p<0.05). This experiment showed that feeding of alfalfa meal to growing layer ducks could improve gastrointestinal tract growth and small intestinal morphology without effect on performance. This experiment provides evidence that alfalfa meal is a very valuable feedstuff for growing layer ducks.

INTRODUCTION
Alfalfa meal is one of the best sources for protein, and it is a rich source of minerals as well as vitamins (Ensminger, 1992). Specifically, alfalfa contains vitamins A, D, E, K, C, B1, B2, B6, B12, Niacin, Panthothanic acid, Inocitole, Biotin, and Folic acid. Alfalfa also contains the following minerals: Phosphorus, Calcium, Potassium, Sodium, Chlorine, Sulfur, Magnesium, Copper, Manganese, Iron, Cobalt, Boron, and Molybdenum. Alfalfa is well balanced in amino acids and rich in carotenoids, and xanthophylls that give poultry carcasses their desirable yellow color (Sen et al., 1998;Ponte et al., 2004). Alfalfa also contains high levels (2 to 3% of DM) of saponins, which have been shown to have hypocholesterolemic, anticarcinogenic, antiinflammatory, and antioxidant properties (Klita et al., 1996;Ponte et al., 2004). Alfalfa is a commercially available feedstuff that is high in protein (17.5%) and crude fiber (24.1%) while exhibiting a relatively low metabolizable energy (1,200 kcal/kg) (National Research Council, 1994). Alfalfa, with high fiber content, has been shown to slow the passage rates in the avian gastrointestinal tract (Sibbald, 1979). This is very advantageous, since indigenous microorganisms in the gastrointestinal tracts of poultry can ferment dietary fiber sources to some extent (Ricke et al., 1982) and a slow passage rate would further breakdown of feed and extend microbial fermentation.
There are many researches on application of alfalfa meal as a feed supplement in laying hens and broilers, previous studies have shown that effects of alfalfa meal on performance in broilers, geese, turkeys and quails is variable. Lepkovsky et al. (1950) suggested that an addition of more than 5% alfalfa meal adversely affected performance in broilers. Heywang (1950) reported adding 5, 10, 15, 20% alfalfa meal to diets had no effect on feed consumption, but adding more than 5% alfalfa meal decreased egg production. Potter and Shelton (1973) reported diet with 5% alfalfa meal did not affect the average body weight gain but increased food consumption and decreased the efficiency of food utilization in turkeys. Diets containing 10% or more alfalfa meal affected feed consumption in geese, turkeys, quails and broilers (Cheeke et al., 1983) and daily weight gain and feed efficiency in geese (Yu et al., 1998). Adding 3, 6 and 9% alfalfa meal to diets of laying quails had no significant effect on live weight, egg production, feed consumption or feed efficiency (Güçlu et al., 2004).
The influence of alfalfa meal in ducks is not clear. Duck is a waterfowl and has a different physiology than other poultry (Ali et al., 2008). Ducks intake much fodder succulence in practice, which showing ducks should be able to utilize alfalfa. To the authors' knowledge, so far, insufficient or no investigations at all have been carried out into the effects of the alfalfa meal upon duck. Therefore in the present experiment, effects of alfalfa meal added to the diet of ducks on performance and gastrointestinal tract development were investigated.

Experimental animals and diets
Shaoxing duck is a local breed in China, characterized by small body size, early maturity and high egg production. In the present experiment, 256 Shaoxing 7-w-old growing ducks for egg production from National Shaoxing Duck Breeding Farm (Zhejiang, China) were selected and randomly allocated to the control and alfalfa-treated groups, with alfalfa supplemented to the basal diet at the level of 3, 6 and 9%, respectively, for a period of 8 wks. Each treatment consisted of 4 replicates of 16 ducks each. The place for raising ducks was comparted into 16 compartments. Each replicate was individually arranged in one compartment and each compartment has three parts: indoor floor pens, adjacent open sports ground and a connecting water area for ducks to paddle in. The dimensions of each indoor floor pens were 9 m2 m1.5 m, the dimensions of each adjacent open field were 10 m2 m 1.5 m, and the dimensions of water area were 10 m2 m 1.5 m. The whole indoor house including all indoor floor pens has enclosing wall at 2.5 m and sloping roof at height 2.5 m. Alfalfa was harvested at the start of flowering, dried in the sun by 5 h, passed through a hot air drier at 500C by 3 min and graded by mesh size (1 mm to 2 mm). Alfalfa meal composition: CP 14.2, EE 2.6, CF 21, ASH 8.3, Ca 1.52, P 0.22. CP, EE, CF, ASH, Ca and P were respectively analyzed by kjeldahl nitrogen determination method, soxhlet extraction method, acid-alkali method, ashing method, EDTA complexometric titration method, molybdate yellow spectrophotometric method.
Ducks were maintained on the growing diet ad libitum. The main characteristics and composition of diets are shown in Table 1 following the feeding and housing standards of the breed. The breeders' diet for 5-to 17-wkold formulate containing 10.8 MJ/kg energy, 16.0% crude protein, 0.9% calcium, 0.5% phosphorus, 0.3% methionine and 0.7% lysine. The place for raising ducks consists of three parts: indoor floor house, adjacent open sports ground and a connecting water area. Birds were raised in separate compartments with 8 male and 8 female ducks each. Ducks were given free access to water and food with plastic drinkers and plastic feeders under natural 13 h lighting and low lighting throughout the night (the illumination intensity was 5 Lx in the house). The trial was conducted in 2010, and environmental temperature during trial was 19 to 29C. All procedures of this experiment were approved by the Animal Care and Use Committee of Zhejiang Province, China.

Parameters measured
Growth performances were determined from 7 to 15 wks old, feed consumption was recorded daily. Weight gain, feed intake and feed/gain of ducks were measured.
At 15-wk old, ducks were weighed after a period of fasting (12 h) with free access to water and then 2 male and 2 female ducks selected randomly from each group were slaughtered by neck cut for tissue sampling. The gastrointestinal tract was quickly removed from the body.

Measurement of gastrointestinal tract indices
The crop and gizzard tissues were cleaned and weighed. Cecal contents were immediately removed and the cecal wall was flushed clean with 0.9% saline, blotted dry on filter paper, and weighed, the same procedure was applied to the small intestine. The length of the small intestine and caecum was measured to the nearest 1 mm under the same conditions.
The ratio crop, gizzard to live weight was expressed as percentages of weights of the tissues to live body weight. Small intestine index and caecum index were expressed as the value of small intestine and caecum lengths and to live body weight (mm/g of BW).

Measurement of small intestinal mucosa morphology
Samples of duodenum, jejunum and ileum were fixed immediately in formalin for the measurement of villus height and crypt depth after ducks were slaughtered. Samples were fixed in 10% formalin, dehydrated by immersion in a graded series of alcohols of increasing concentration (from 70% to absolute), infiltrated with xylene, and embedded in paraffin. A microtome was used to make 5-m cuts that were mounted in glass slides and stained with hematoxylin and eosin. Villus height (m) and crypt depth (m) from each slice were measured in approximately 50 microscopic fields by using an image analysis system. (Image-Pro, Media Cybernetics, Inc., Silver Springs, MD). The villus height was measured from the tip of the villi to the villus crypt junction, and the crypt depth was measured from the villus crypt junction to the base of the crypt.

Statistical analysis
All data were arranged to evaluate by analysis of variance following the mixed procedure in a completely randomized design using the SAS software program (SAS, 1998), the statistical model included the main and interactive effects of alfalfa meal and sex. Treatment differences were considered significant at p0.05.

Effect of alfalfa meal on growth performance of growing ducks
All ducks fed alfalfa meal diet were not significantly different in body weight, average daily gain, mortality, feed intake and gain-to-feed ratio from those on the control (p>0.05) ( Table 2). The results showed 3 to 9% alfalfa meal in diet didn't affect the growth of growing ducks.
Previous studies have shown that effects of alfalfa meal on performance in animals may vary. Heywang (1950) reported adding 5, 10, 15, 20% alfalfa meal to diets had no effect on feed consumption, but adding more than 5% alfalfa meal decreased egg production. Lepkovsky et al. (1950) suggested that an addition of more than 5% alfalfa meal adversely affected performance in broilers. Potter and Shelton (1973) reported diet with 5% alfalfa meal did not affect the average body weight gain but increased food consumption and decreased the efficiency of food utilization in turkeys aged between 4 and 8 wks. Diets containing 10% or more alfalfa meal affected feed consumption in geese, turkeys, quails and broilers (Cheeke et al., 1983) and daily weight gain and feed efficiency in  (Yu et al., 1998). Adding 3, 6 and 9% alfalfa meal to diets of laying quails had no significant effect on live weight, egg production, feed consumption or feed efficiency (Güçlu et al., 2004). The variability in performance may be due to there was difference in the ability in utilization the alfalfa meal among poultry species, because there was difference in physiology among poultry species.

Effect of alfalfa meal on gastrointestinal tract growth of growing ducks
The effects of alfalfa meal on gastrointestinal tract development of growing ducks are shown in Table 3. Alfalfa meal increased significantly the ratio crop, gizzard to live weight, caecum to live weight, and the caecum index of ducks (p<0.05). The effect of alfalfa meal on the ratio small intestine to live weight and small intestine index of growing ducks were not significant (p>0.05). Sex had significant effect on the ratio small intestine to live weight and small intestine index of growing ducks (p<0.05), and had no significant effects on the ratio crop, gizzard to live weight, caecum to live weight, and the caecum index of growing ducks (p>0.05) ( Table 3).
Alfalfa meal increased significantly the ratio crop, gizzard to live weight, caecum to live weight, and the caecum index of ducks, which showed alfalfa meal enhanced gastrointestinal tract development of ducks. Alfalfa is a commercially available feedstuff that is high in crude fiber (24.1%) (National Research Council, 1994). The increase of the fiber content in a diet benefits the digestive system by normalizing colonic function and by increasing fecal weights and evacuation frequency (Salvin et al., 1985). Birds can digest fibre through fermentation, mainly in the caecum (Clench and Mathis, 1995). Their capabilities of fibre digestion depend on individual differences, age of birds and feed composition. It has been shown that alfalfa, with high crude fiber, has a very long transit time in the gastrointestinal tract of chickens. The increase in transit time favors bacteria degrading dietary fiber into fermentable substrates. These actions increased mucosal structure and function as well as increased the populations of commensal bacteria in the gastrointestinal tract, which would help maintain the small and large intestine (Buddington et al., 1999;McReynolds et al., 2009).

Effect of alfalfa meal on small intestinal morphology of growing ducks
The effects of alfalfa meal on the small intestinal morphology in growing ducks are shown in Table 4. The effects of alfalfa meal on villus height in duodenum and jejunum were significant, villus height in duodenum and jejunum increased with the increase of alfalfa meal levels (p<0.05). Villus height were similar in ileum among all groups (p>0.05). The effects of alfalfa meal on crypt depth in duodenum and jejunum were significant, crypt depth in duodenum and jejunum decreased with the increase of alfalfa meal levels (p<0.05). Crypt depth were similar in ileum among all groups (p>0.05). Sex had no significant effects on villus height and crypt depth in duodenum, jejunum and ileum (p>0.05) ( Table 4).  The morphology of the intestinal epithelium is particularly affected by the diet. Chickens fed wet diets had increased villus height and less depth of crypts in duodenum, jejunum, ileum, ceca and colon compared to broiler chickens given dry diets (Yasar et al., 1999). Chicks fed with B. subtilis had increased villus height of duodenum and ileum in 28 d old (Samanya et al., 2002). Villus height was greater in birds fed yeast cell wall than in the control birds, no significant differences were found in crypt depth in small intestinal (Zhang et al., 2005). The addition of synbiotic increased the villus height and decreased the crypt depth in ileum in broiler chickens (Awad et al., 2008). Dietary supplementation of mannan-oligosaccharides and direct feed microbials led to an increase in villus height and width, as well as a decrease in crypt depth in all parts of the intestine in broiler (Markovicva et al., 2009). The addition of prebiotic or antibiotic increased the villus height in duodenum, and prebiotic increased villus width of duodenum and ileum compared with antibiotic and control group. However antibiotic increased duodenal crypt depth compared with the prebiotic and control group in broiler chickens (Sayrafi et al., 2011). The supplementation with organic acids significantly increased the villus height in the duodenum, jejunum, the crypt depth in the duodenum, jejunum, and ileum was not affected in broiler chickens (Adil et al., 2010).
The intestinal villus and crypt morphology has been associated with intestine function and growth in animals. According to Tivey and Smith (1989), changes in enterocytes development and villi structure determine the digestive and absorptive capacity of the small intestine. Histological examination reveals the crypts to be the place of epithelial regeneration (Paulus et al., 1992). A lengthening of the villi increases the surface area for nutrient absorption. The crypt can be considered as the villus factory, and a large crypt indicates fast tissue turnover and high demand for new tissue (Yason et al., 1987).
It can be concluded that alfalfa meal in diet has the potential to improve gastrointestinal tract growth and the intestinal morphology of growing ducks for egg production. The search indicates alfalfa meal is very available feedstuff on growing egg-type duck industry. Application of alfalfa meal as a feed supplement in growing egg-type ducks not only will benefit gastrointestinal development of growing ducks, but also will economize traditional ingredients which are becoming costly or less available.


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Does dietary inulin affect biological activity of a grapefruit flavonoid-rich extract?

Background The aim of the study was to verify that the concomitant presence of grapefruit flavonoid extract with inulin in a Western-type diet may provide synergistic effects to the hindgut metabolism, as well as blood lipid and mineral profiles. Methods Forty male Wistar rats were distributed into 4 groups and fed for 28 days with diets rich in fat, cholesterol and protein. A two-way repeated measures ANOVA was applied to assess the effects of inulin (v. sucrose, 5% of the diet), the addition of dietary grapefruit flavonoid extract (diets without or with 0.3% of an extract from hard parts of grapefruit) and the interaction between these two dietary factors. Results When compared to the control sucrose-containing diet, the diet enriched with inulin led to typical changes within the caecum, the main part of hindgut fermentation in rats, such as acidification of the digesta, support of bifidobacteria growth and increase of propionate and butyrate production. The dietary grapefruit flavonoid extract without inulin increased the bulk and pH value of caecal digesta, whereas short-chain fatty acid concentration and the bifidobacteria population were lowered compared to the extract-free diets. Simultaneous dietary addition of both tested components decreased slightly the pH value and increased somewhat the bifidobacteria number and the propionate concentration, however to the level observed with the control sucrose-containing diet. With regard to blood lipids, dietary grapefruit flavonoid extract decreased the triglyceride concentration regardless of the dietary carbohydrate type. Conclusion Inulin does not provide any additional benefit to the blood lipid profile caused by the dietary application of grapefruit flavonoid extract and it does not counteract clearly detrimental effects of the extract in the hindgut. Adding grapefruit extract to the diet must be performed with caution due to possible adverse hindgut responses with overdoses.

Background
In economically developed countries, a high intake of processed, energy-dense foods, such as French fries, processed red meats, high-fat dairy products and refined grains and sweets, that contain high amounts of saturated fat, cholesterol and protein or easily absorbable carbohydrates is often called a Western diet. This dietary pattern is thought to be an important factor for the development of many diet-related diseases, such as type 2 diabetes, cardiovascular disease (CVD) or cancers [1,2]. Saturated fat and cholesterol induce dyslipidaemia and thus contribute to CVD development, whereas chronic, excessive ingestion of easily absorbable sugars that affect postprandial glycaemia and insulinaemia seem to underlay type 2 diabetes [3]. There is also a growing body of evidence that the Western diet may be a risk factor for osteoporosis through the induction of chronic, low-grade metabolic acidosis leading to the dissolution of bone minerals [4]. However, a diet abundant in whole-grain cereals, vegetables and fruits is associated with a decreased risk of diet-related diseases [5]. Notably, these health-promoting effects are due not to a single dietary constituent but rather to many factors working together, such as phytochemicals or fibrepolyphenolic complexes.
Inulin is a group of non-digestible polysaccharides occurring in many plants that are not hydrolysed by pancreatic enzymes but are broken down by specific bacteria in the hindgut, to help the large bowel function and improve the health of the organism. Among others, inulin consumption leads to the acidification of digesta and an enhancement of short-chain fatty acid (SCFA) production in the hindgut, especially propionate and butyrate. After absorption, propionate is thought to inhibit hepatic lipogenesis and positively influence the blood lipid profile [6], whereas the decreased digesta pH is probably responsible for the stimulation of mineral absorption and bone health in laboratory animals [7].
Grapefruit flavonoids are a part of the flavanone subclass, to which a number of positive and negative biological activities have been attributed. For example, flavanones are partly responsible for the interaction of grapefruit with some drugs [8]. Interestingly, some similar systemic effects following the ingestion of flavanones and inulin can occur. It has been reported that naringenin and hesperetin diglycosides (naringin and hesperidin, respectively), the main grapefruit flavonoids, significantly hindered an increase in the plasma lipids of rats fed a cholesterol-containing diet [9]. Furthermore, it has been shown that both naringin and hesperidin can improve mineral metabolism and bone health, but the exact mechanism of action remains unknown [10][11][12][13]. Interestingly, the bioavailability of flavonoids, including some grapefruit flavanones, is rather low, and the majority of these flavonoids reach the hindgut where they interact with local microbiota in a similar manner as inulin. As a result, low-molecular weight metabolites of flavanones are formed, which may contribute to their bioactivity [14].
Recent studies performed in our laboratory suggest that dietary inulin-type fructans and polyphenols can benefit the health status of the host when applied together. For example, our preliminary study showed that grapefruit flavonoids in a standard rodent diet had a negative influence on the caecum, the main part of the hindgut fermentation in rats, whereas inulin can decrease that detrimental effect [15]. Additionally, we have shown that the concomitant dietary presence of apple polyphenols and fructo-oligosaccharides increased the susceptibility of quercetin glycosides to microbial metabolism in the caecum [16]. In the present study, we verified that the concomitant presence of a grapefruit flavonoid-rich extract and inulin in a rat's Western-type diet may provide synergistic effects to caecal metabolism, as well as blood lipid and mineral profiles. A modified diet with a relatively high protein, fat and cholesterol content was used to obtain an animal model of metabolic changes related to the Westernisation of human eating habits.

Materials and methods
Preparations A long-chain, sugar-free inulin (Frutafit ® TEX) was purchased from Sensus (Roosendaal, The Netherlands). Approximately 98% of the inulin preparation contains fructan molecules with a degree of polymerisation above 10. A commercial grapefruit extract (Cintamani, Poland) made from the stone, peel, and pith was also used in the study. The preparation consisted of flavonoids (52.8%), silicon dioxide (25%), glycerol (17.8%) and vitamin C (4.4%). The main flavonoids were diglycosides and glycosides of naringenin and hesperetin. The flavonoid composition of the preparation was characterised using a reversed-phase HPLC method (Waters system equipped with photodiodearray detector, Waters Co., Milford, MA, USA, equipped with Nova Pak C18 column, 30 cm × 3.9 mm with 4-μm packing). Two mobile phases were employed for elution: water acetic acid (98:2, v/v; A) and water acetonitrile acetic acid (78:20:2, v/v; B). The gradient profiles were 0-55 min and 100-20% for phase A and 55-70 min and 20-10% for phase B. More details regarding the flavonoid composition and method applied were given previously [15].

Animals and diets
The local Institutional Animal Care and Use Committee approved the animal protocol used in the present study. The experiment was conducted on 40 young (4 weeks old) male Wistar rats with an average initial body weight of 81.4 ± 0.5 g that were distributed into 4 groups of ten rats each. The animals were fed for 4 weeks with an experimental diet that was a modified, standard, semipurified casein diet for laboratory rodents recommended by the American Institute of Nutrition (Table 1). To mimic the human eating habits of economically developed countries, each diet had a high and equal content of fat, cholesterol and protein (14%, 1% and 23% of the diet, respectively). Half of the dietary fat and protein was derived from soybean oil and casein, respectively, and the rest was supplemented with pork lard and soy protein isolate, respectively. Two groups of rats were fed control (C) diets that, apart from the aforementioned, contained 5% of sucrose, and the other two groups were fed inulin (IN) diets in which 5% of the IN preparation was added instead of sucrose. Additionally, one C and IN diet was supplemented with 0.3% of the grapefruit flavonoid extract (+GFE) added to maize starch. The animals were housed in individual cages under the following standard conditions: temperature 21-22°C, relative humidity 50-70%, intensive room ventilation (15×/h), 12 h light/dark cycle, permanent access to the diets and water.

Sample collection and analysis
Upon termination of the experiment, the rats were weighed and anaesthetised with sodium pentobarbital.
Blood samples were taken from the caudal vein, and subsequently the liver, kidneys and caecum were removed and weighed. The blood was allowed to clot over 1.5 h at 37°C, and the serum was collected after centrifugation. Fresh caecal digesta was used to determine the pH, dry matter, ammonia concentration and microbiota, whereas the determination of enzyme activity and SCFA concentration were performed after storing the samples at -70°C.
The pH of the caecal digesta was measured using a microelectrode and a pH/ION meter (model 301, Hanna Instruments), and dry matter was determined at 105°C. Ammonia was extracted and trapped in a solution of boric acid then determined by a direct titration with sulphuric acid [17]. The caecal microbiota was assessed by measuring the number of bacteria in chosen populations of probiotic and potentially pathogenic microorganisms. All bacteria determinations were done immediately after sampling. Fresh caecal digesta was collected directly from each rat, weighed and immediately homogenised with 1% peptone water as a diluter. The populations of bacteria were enumerated after their cultivation on selective culture media and/or in specific incubation conditions. Bifidobacteria count was determined on Garche's agar after undergoing incubation at 37°C for 48 h in anaerobic conditions (anaerostat + AnaeroGen, Oxoid) [18]. Their identification was based on appearance of colonies, specific morphology of cells checked under phase contrast with microscope Microphot FXA (Nikon, Japan), as well as on the presence of an enzyme specific for Bifidobacteria (fructose-6-phosphate phosphoketolase, EC 4.1.2.22). The lactic acid bacteria were enumerated on MRS medium (BTL, Poland) after undergoing cultivation at 37°C for 72 h in microaerophilic conditions (the double agar layer technique). Their identification was based on appearance of colonies and specific morphology of cells checked under the phase contrast microscope. The quantity of Escherichia coli was evaluated after a 24 h incubation at 37°C on McConkey agar (Merck) in an oxidative atmosphere. Spore-forming bacteria were determined after heating of adequate dilutions at 80°C/20 min and cooling. The saccharolytic spore formers were counted after the growth on an SPC medium [19] in anaerobic conditions for 7 days at 37°C, whereas proteolytic spore formers were enumerated in a broth-gelatine medium after 5 days of incubation at 22-25°C in an anaerobic atmosphere [20]. The results were expressed as log cfu number/g digesta. Enzyme activity (α-and β-glucosidase, αand β-galactosidase, and β-glucuronidase) was measured by the rate of p-or o-nitrophenol release from nitrophenyl glycosides for 10 min [15] and expressed as the μmol of product formed per one min per g of caecal digesta. The SCFA concentration was measured using gas chromatography under the conditions described previously [15]. Briefly, a known amount of fresh caecal digesta was mixed with 0.2 mL of formic acid and stored at -80°C. Afterwards, the sample was diluted with deionized water, centrifuged at 10 000 g for 5 min, filtered through a 0.45 μm membrane, and then the supernatant was decanted for injection into a gas chromatograph (Shimadzu GC-14A, Shimadzu Co., Kyoto, Japan, equipped with a glass column, 2.5 m × 2.6 mm, containing 10% SP-1200/1% H 3 PO 4 on 80/100 Chromosorb W AW; column temperature 110°C; flame ionization detector temperature 180°C; injector temperature 195°C).
The blood serum concentrations of minerals (magnesium, calcium, phosphorous), glucose, triglycerides, and total cholesterol and its HDL fraction, as well as the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were estimated with reagents from Alpha Diagnostics Ltd. (Warsaw, Poland).

Statistical analysis
The data were analysed using the STATISTICA software package version 6.0 (Statsoft Corporation, Krakow, Poland). A two-way repeated measures ANOVA was applied to assess the effects of the IN preparation, GFE and the interaction between these dietary factors (IN × GFE). If the analysis revealed a significant interaction or that both dietary factors had a significant influence (P ≤ 0.05), the differences among the individual groups were then analysed with Duncan's multiple range post hoc test (P ≤ 0.05). The pooled standard error of the mean (SEM) was calculated as the standard deviation from all measurements divided by their square root.

Results
After four weeks of experimental feeding, the IN and GFE dietary additions did not affect the body weight of rats, their relative liver and kidney mass, serum AST and ALT activity and urea concentration of the rats ( Table 2). Dietary GFE treatment significantly increased the tissue and digesta mass and the digesta pH, whereas the digesta dry matter, ammonia and protein concentration were substantially decreased (P < 0.001). IN tended to increase the relative caecal tissue mass (P = 0.09) but significantly reduced the caecal pH value (P < 0.001). Moreover, an interaction between IN and GFE was noted with regard to the caecal digesta bulk and its pH value (P < 0.05 and P < 0.001, respectively). The lowest digesta mass followed the consumption of control (C) and IN diet, whereas the highest digesta mass was in rats on the C + GFE diet (P ≤ 0.05). The lowest digesta pH value was after the consumption of the IN diet. The digesta pH was significantly higher with the C diet than with the IN diet, whereas the highest digesta pH was in the caecum of rats fed the C + GFE diet (P ≤ 0.05). The IN + GFE diet had a slightly decreased pH value that was comparable to that observed in rats on the C diet. Microbiota and microbial enzyme activity in the caecal digesta of rats are shown in Table 3. Both dietary factors substantially contributed to changes in the bifidobacteria population (P < 0.05) but did not affect lactic acid bacteria. The bifidobacteria count was the lowest with the C + GFE diet, slightly and significantly higher with the IN + GFE and C diets, respectively, and highest with the IN diet (P ≤ 0.05). In addition, the E. coli population was considerably decreased after IN supplementation (P < 0.001). Dietary GFE reduced significantly the number of saccharolytic spore-forming bacteria (P < 0.01), and the number of proteolytic spore-forming bacteria was significantly influenced both by GFE and IN (P < 0.001 and P < 0.01, respectively). The highest number of anaerobic proteolytic spore formers was observed with the GFE diets. The number of anaerobic proteolytic spore formers was significantly lower with the C diet, and the lowest number of them was observed with the IN diet (P ≤ 0.05). The activity of all microbial enzymes in the caecal digesta (α-and βglucosidase, αand β-galactosidase, and β-glucuronidase, Table 3) was significantly affected by GFE, whereas IN had a substantial influence on αand β-galactosidase and βglucuronidase activity; however, a trend of IN towards decreased α-glucosidase activity was also noted (P = 0.08). Additionally, co-ingestion of IN and GFE led to an interactive effect on α-glucosidase and αand β-galactosidase activity (P < 0.05, P < 0.001 and P < 0.001, respectively). Specifically, the activity was similar among rats on the C, IN and C + GFE diets and significantly greater in rats on the IN + GFE diet (P ≤ 0.05). Interestingly, among the aforementioned enzyme activity, αand β-galactosidase activity in rats on the IN + GFE diet was more than two times and more than five times greater, respectively. The β-glucosidase activity was significantly less with GFE (P < 0.001) and was significantly decreased in the caecal digesta of rats consuming IN, C + GFE and IN + GFE diets than in those consuming the C diet (P ≤ 0.05).
The total and all particular SCFA concentrations (acetic, propionic, iso-butyric, butyric, iso-valeric and valeric acids) in the caecal digesta of rats were significantly influenced by GFE (P < 0.01), whereas IN had a substantial influence on the concentration of propionic, butyric and iso-valeric acids (P < 0.001, P < 0.001 and P < 0.05, respectively, Table 4). However, valeric acid concentration only trended towards being influenced by the addition of IN (P = 0.07). Moreover, an interaction and a trend towards interaction were noted when considering the butyric acid and iso-butyric acid concentration (IN × GFE, P < 0.001 and P = 0.08, respectively). The highest butyric acid concentration was observed with the IN diet, significantly lower with the C diet and the lowest with GFE supplemented diets (P ≤ 0.05). The total SCFA concentration was almost two times lower in rats on GFE-supplemented diets (P < 0.001); the concentration of acetic, iso-butyric and valeric acids was also lower in rats consuming these diets (P < 0.01). The highest propionic acid concentration was observed with the IN diet, significantly lower with the C and IN + GFE diets and the lowest with the C + GFE diet (P ≤ 0.05). The iso-valeric acid concentration was greater in the caecal digesta of rats consuming the C diet than in rats fed other diets (P ≤ 0.05). SCFA profile in the caecal digesta of rats were significantly influenced both by GFE and IN (P < 0.01). In addition, an interaction was noted when considering the acetic and butyric acid proportion (IN × GFE, P < 0.01 and P < 0.001, respectively). The highest acetic acid proportion followed the consumption of GFE supplemented diets, significantly lower proportion was observed with the IN diet and the lowest with the C diet. The propionic acid proportion was significantly higher with the IN, C + GFE and IN + GFE compared to the C diet. As in the case of the concentration of butyric acid, its proportion was the lowest in rats on GFE supplemented diets, significantly higher on the C diet and the highest on the IN diet. The concentration of glucose, magnesium, calcium and phosphorous did not differ among all diet groups (data not shown). Lipid concentrations in the blood serum of rats are shown in Table 5. Total cholesterol and HDL cholesterol concentrations were comparable among all diet groups, whereas triglyceride concentration was significantly lower in those rats that were fed diets with GFE added.

Discussion
The absorption of flavonoids and their glycosides occurs along the entire gut. Often metabolic processes in the large intestine are predominant and are not fully understood thus far. Among the flavonoids, the bioavailability of the grapefruit flavanones is considered to be relatively high, which seems to be related to their glycosidic moiety. For example, the bioavailability of hesperidin (hesperetin-7-rutinoside) in humans was increased after its enzymatic conversion to hesperetin-7-glucoside, which changed the absorption site from the colon to the small intestine [21]. In another study, naringenin and its glucoside were absorbed across the small intestine of rats, whereas the bioavailability of naringin (naringenin-7-rhamnoglucoside) was significantly lower with the simultaneous compound movement into the caecum [22]. Although there is also a suggestion that naringin may be efficiently absorbed into the bloodstream in its unchanged form, as reported in study on beagles [23], most of the reports suggest that this flavanone and other flavanone glycosides undergo microbial deglycosylation and formation of metabolites, such as phloroglucinol and phenylpropionic acids, in the hindgut [12]. Afterwards, these low-molecular metabolites can be absorbed and might contribute to flavanone bioactivity. In the present study, a 4-week ingestion of a diet containing 0.3% GFE caused a considerable enlargement of the caecal tissue and digesta ( Table 2). The amount of GFE used in this study was comparable to the amount of polyphenol extracts usually used in such nutritional experiments. Interestingly, Felgines et al. have implied a good adaptation of caecal microbiota to naringin in rats [22]. Our results suggest that the caecal microbiota were not able to sufficiently metabolise the flavonoid glycosides of GFE, and we speculate that it induced additional osmotic pressure that kept fluids within the caecum and led to its Table 4 Short-chain fatty acids (SCFA) in the caecal digesta of rats consuming control (C) and inulin (IN) diets without or with the addition of grapefruit flavonoid extract (GFE)  enlargement. Nevertheless, because an extract has been used it is difficult to identify specific role of grapefruit phenolics responsible for such observed effects and the influence of other components of GFE cannot be excluded. Interestingly, in our laboratory, high doses of polyphenol extracts (up to 1.2% of the diet) from different sources, including green tea, chokeberry and honeysuckle, have been used so far without a sizeable increase of the caecal tissue and digesta mass [24]. It should be emphasized also that an increase of the caecal digesta mass may cause discomfort to the host. Moreover, such an enlargement of the caecum as in the present study was also observed in a previous experiment performed in our laboratory on rats with lactulose-induced diarrhea [25]. Indeed, in the present experiment, an increased and looser stool output was observed with GFE feeding (data not shown). Notably, the dietary IN beneficially suppressed the increased digesta mass observed after GFE feeding. This finding is especially of interest because IN is well known for increasing faecal biomass and the water content [13] and thus indicates a more complex modulatory effect of IN on the physiology of the large intestine.
Bifidobacteria are known to improve gut health by maintaining a balance between potentially harmful and health-promoting species. Selective stimulation of their growth is one of the most important attributes of IN and other inulin-type fructans [13]. The bifidogenic effect of IN was also observed in our study with an additional decrease in E. coli numbers, which some strains can be pathogenic ( Table 3). The addition of GFE adversely reduced the number of Bifidobacterium spp., but co-ingestion of IN increased the number to a level not significantly different from that observed in rats on the control diet without GFE supplementation. Antibacterial activity of flavonoids has been frequently suggested, but the research is widely conflicting [26]. Although flavonoids are less potent than antibiotics, they are thought to be able to inhibit the nucleic acid synthesis, cytoplasmic membrane function and energy metabolism of bacteria. In our study, dietary GFE affected the growth of only one genus. However, the bacteria investigated are only a small part of the indigenous microbiota, and other caecal indices show more extended microbiota-GFE interactions. Indeed, due to GFE supplementation significantly fewer anaerobic saccharolytic bacteria and more anaerobic proteolytic bacteria were incubated (spore-forming bacteria, Table 3) and it reflects the number of spores present in the caecum. Because spores are formed as response to unfavourable conditions of the environment, it can be concluded that the growth of saccharolytic bacteria was promoted, and the growth of proteolytic bacteria were inhibited, in the caecum of rats fed GFE. Such supposition seems to, at least partly, agree with other indices of caecal function in our study. Evidence of less proteolytic fermentation or less putrefactive processes may be supported by a decreased concentration of their metabolites in the digesta of rats on GFE diets (Tables 2 and 4), such as ammonia, iso-butyric, valeric and iso-valeric acids [27,28]. The decreased ammonia concentration is considered a positive change because this compound can destroy cells, alter nucleic acid synthesis, induce cancerous cell growth and increase viral infections at concentrations in the lower bowel of usual Western diets [27]. More intensive saccharolytic fermentation indicates microbial glycolytic activity, the activity of αglucosidase and both galactosidases, which strongly increased after the consumption of the IN + GFE diet (Table 4). However, a several fold increase in β-galactosidase activity is surprisingly high and difficult to explain. Nevertheless, this finding supports the earlier study performed in our laboratory [15]. Interestingly, the β-glucuronidase activity in the caecal digesta of rats fed IN, GFE or both decreased. An increased β-glucuronidase activity has been suggested to be unfavourable due to its ability to liberate toxic or carcinogenic compounds from glucuronide conjugates that are coupled earlier in the liver and excreted into the gut with bile [29].
In the reported experiment, an increased caecal digesta pH was another important disadvantage of GFE consumption (Table 2). Lower pH values are believed to prevent the overgrowth of pH-sensitive pathogenic bacteria and favour the growth of health-promoting species, such as bifidobacteria [30,31]. In our study, the pH values were negatively correlated with the number of bifidobacteria and positively correlated with the SCFA concentration in the caecal digesta. Interestingly, the co-ingestion of IN slightly decreased the pH to a level not significantly different from that observed with the control diet without GFE supplementation; yet, the digesta was acidified only in rats consuming IN as a single dietary supplement. The total SCFA concentration was almost two times lower with diets with GFE supplementation (Table 4). There is mounting evidence that SCFA play a key role in colonic health as an energy source for colonocytes, and some may be an important factor for the prevention and management of certain diseases, such as gastrointestinal disorders, cancer or CVD [31]. In our study, the acetic acid concentration in the caecal digesta decreased significantly after GFE supplementation. This finding could be considered a positive change because it is believed that after absorption, acetate stimulates cholesterol synthesis in the liver; however, GFE consumption simultaneously decreased the propionate concentration, which is suggested to be an inhibitor of hepatic cholesterol synthesis [31]. A lack of a cholesterol-lowering effect with GFE in our study might be a consequence of that interaction. Nevertheless, GFE and IN in the diet together significantly increased the propionic acid concentration, which was comparable to that observed in rats on the control diet without additives. The highest concentration of propionic acid and butyric acid was after ingesting IN as a single supplement. This finding is consistent with the well-known ability of IN to stimulate bacterial propionate and butyrate production in the large intestine [13]. In the present experiment, a several fold decrease in the butyric acid concentration was also observed in rats on GFE diets. Butyrate is thought to be the preferred energetic substrate for colonocytes, and it appears to promote a normal phenotype of these cells [30,31].
The serum cholesterol concentration was similar among all dietary groups in our study; however, the serum triglyceride concentration decreased significantly in rats consuming GFE (Table 5). This finding supports that of the study by Cho et al. performed on rats consuming a high-sucrose diet and dietary levels of naringenin at concentrations equivalent to 1-4 cups of grapefruit juice, which significantly lowered blood triglyceride concentrations but had an ambiguous effect on blood cholesterol [32]. The authors suggest that the lipid-lowering effects may result from the upregulation of hepatic peroxisome proliferator-activated receptor alpha and its target genes by naringenin. Surprisingly, in another study of rats fed a cholesterol-containing diet, a solution of hesperidin or naringin given once daily by gavage significantly hindered an increase in the plasma total cholesterol and LDL but had no influence on plasma triglycerides after 30 days [7]. Additionally, human trials are inconsistent in terms of the lipid-lowering effects of grapefruits and their flavonoids. For example, fresh red grapefruit positively influenced all serum lipid levels, especially serum triglycerides, in hyperlipidaemic patients after coronary bypass surgery [33]. However, in a randomised, placebo-controlled, parallel trial, pure hesperidin and naringin did not affect serum lipids in moderately hypercholesterolaemic men and women [34]. Other grapefruit components may also play a role in the lipid-lowering effects. In the present study, contrary to the expectations, dietary IN did not affect the serum lipids despite an increased concentration of propionic acid in the caecal digesta. A meta-analysis of randomised controlled trials shows that the triglyceride-lowering effect of inulin-type fructans is well proven [35]. In the reported study, the serum mineral concentrations were not affected by IN or GFE. This finding does not support the literature that reports a potent improved mineral metabolism with IN and grapefruit flavanones [8,11,14]. Perhaps the period of low-grade metabolic acidosis from the Western-type diet was too short in our study to disturb the mineral homeostasis of rats.

Conclusion
Inulin does not provide any additional benefit to the blood lipid profile caused by dietary application of the grapefruit flavonoid extract and it does not counteract clearly detrimental effects of the extract in the hindgut. Adding grapefruit extract to the diet must be performed with caution due to possible adverse hindgut responses with overdoses.


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OPTIMIZATION OF THE PROCESSES OF STERILIZATION AND MICROPROPAGATION OF CUP PLANT (Silphium perfoliatum L.) FROM APICAL EXPLANTS OF SEEDLINGS IN in vitro CULTURES

Cup plant (Silphium perfoliatum L.) is a tall, reaching up to 2.5 m, perennial plant that represents the Asteraceae family. Silphium perfoliatum L can be applied in medicine, animal feeding, and as a decorative, honey-producing and energy production plant which proves its multi-functional features. Sowing material currently available in Poland is insufficient, which justifies the present attempts at propagating these plants in in vitro cultures. Therefore, Silphium perfoliatum L. seed sterilization and micropropagation processes were studied under controlled conditions in in vitro cultures. Among the tested methods, ACE proved to be the most effective for sterilization. The apical parts of seedlings were used as explants; they were placed onto MS media with growth regulators added. 4 growth regulator concentration variants were applied. The highest percentage of explants with lateral shoots (41.7%) and callus (90.3%) was obtained on MS medium with 5 mg x dm BAP + 1 mg × dm NAA added. Shoots were isolated and transferred onto MS rooting medium without growth regulators. The rooted plants were transferred to the greenhouse and acclimatised to ex vitro conditions.

INTRODUCTION
The genus Silphium of the family Asteraceae comprises about 23 species (R u t k o w s k i , 1998). The species occurs in the natural environment in the central and eastern parts of the United States of America and in the southern part of Canada (P o d b i e lk o w s k i , 1995). Due to its numerous functional traits, there has been a growing interest in cup plant over the recent years. Its herbage offers a potentially valuable material for pharmaceutical and foodstuffs industries since Silphium perfoliatum L. roots and rhizomes contain inulins (K o w a l s k i and W i e r c i ńs k i , 2004), while its leaves, flowers, and rhizomesbiologically active substances of the isoprenoid group, phenol, and polyphenol compounds. Extracts from tissues of this species demonstrate analgesic, anti-inflammatory, sudorific, restorative, antibacterial, antifungal, and expectorant properties as well as they reduce the cholesterol level (E l -S a y e d et al. 2002 2007). Similarly, this species is used as a fodder plant thanks to a high content of exogenous amino acids (P i ł a t et al. 2007).
Due to its long flowering period and production of high amounts of pollen grains as well as uneven development of flowers, the cup plant is grown to provide pollen and nectar for bees (W r ó b l e w s k a , 1997). Since the species is a perennial on nutrient-poor soil and because it can adapt to different environments, it can also be used as a pioneer plant for land rehabilitation in areas destroyed by industry and municipal waste disposal (W o ź n i a k and G ó r a l , 1998; K l i m o n t , 2007). Silphium perfoliatum plantations also provide considerable amounts of biomass used as energy-producing material (M a j t k o w s k i , 2007; S t o l a r s k i et al. 2008).
The common technology offering vegetative material for propagation purposes involves micropropagation of plants in in vitro cultures. Currently, the available literature seems to provide few reports on how to produce cup plants under controlled conditions (F i g a s et al. 2010).
The technology that allows regenerant plants to be obtained, preserving the traits of the genotype and virus-free plants, is the method using the apical parts of seedlings which can be applied once the factors favourable to plant organogenesis are determined; e.g. culture conditions, method and stages of isolation of the explant, or the medium composition. The aim of the present research was to optimise the process of sterilization and micropropagation of cup plants (Silphium perfoliatum L.) from apical explants of seedlings in in vitro cultures.
In the present experiment, the following were analyzed: methods of seed sterilization, number of seedlings obtained, number of shoots, and rooting efficiency.

MATERIALS AND METHODS
The initial research material comprised seeds of cup plant (Silphium perfoliatum L). Selected, surface-treated seeds of cup plants were placed in Petri dishes with filter paper soaked with bidistilled water. The plates were kept for 48 hours in the dark at 24 o C.
The seeds were sterilized according to different variants (Table 1 and 2). Swollen seeds were sterilized and defatted in 70% ethanol for 30 seconds, then sterilized in 5% calcium hypochlorite with 2-3 drops of Tween 20 for 20 minutes, 3% hydrogen peroxide for 5 min. or 30% of ACE (commercial bleach, which is a component of sodium hypochlorite (4.85%) and as well as other compounds containing chlorine, <5%) for 7 min., then the seeds were rinsed with sterile bidistilled water three times. In addition, another part of seeds was subjected to high temperature sterilization, by placing the seeds in 96% alcohol, and then in a flame for a few seconds (Table 1).
In another variant of the experiment, the seeds surface sterilized with 70% ethanol for 30 seconds were subjected to swelling. Therefore, they were placed in Petri dishes with filter paper soaked with bidistilled water and kept in the dark at 24 o C for 48 hours. The embryos isolated with a preparation needle were sterilized in: 5% calcium hypochlorite with 2-3 drops of Tween 20 for 5 minutes, 3% hydrogen peroxide for 5 minutes, or 30% solution ACE for 7 and 8 minutes, and then rinsed with bidistilled water three times (Table 2). To induce germination, the embryos were placed onto Murashige and Skoog medium (MS) (1962), diluted at a ratio of 1:1 and solidified with 0.7% Vitro Lab -Agar (Biocorp). The medium was brought up to pH 5.6-5.8, poured into tubes and autoclaved at a temperature of 121 o C for 20 minutes. The embryos were placed on the medium so prepared by gentle pressing. Over the next weeks of culture, the sprouting process took place producing seedlings. Seedlings obtained from the seeds sterilized by ACE for 7 minutes were used for isolation of apical explants. The germination process occurred under controlled conditions: 16-hour photoperiod, light intensity of 2500 μmol×m--2 ×s -1 , temperature of 25 +/-2 o C, and air humidity of 70%. The apical parts of shoots, having been deprived of cotyledons and leaves, were isolated from 6-week sterile seedlings. The explants were put into test tubes on MS regeneration medium which, in addition to mineral compounds, contained organic substances and growth regulators: NAA (1-naphthaleneacetic acid), IAA (3-indoleacetic acid), BAP (6-benzylaminopurine), KIN (kinetin), and ABA (abscisic acid).
The experiment involved the use of four MS regeneration medium variants: Variant 1: MS containing 2.5 mg × dm -3 BAP + 0.45 mg × dm -3 ABA; Variant 2: MS containing 5 mg × dm -3 BAP + 1 mg × dm -3 NAA; Variant 3: MS containing 2 mg × dm -3 BAP + 1 mg × dm -3 IAA; Variant 4: MS containing 0.5 mg × dm -3 IAA + 4 mg × dm -3 KIN. The medium was solidified with 0.7% Vitro Lab -Agar. The stands with the tubes were placed in the breeding room with the same conditions as during sprouting. The experiment was carried out in three replications. In each replication, 24 explants were placed on the medium. After 8 weeks of growth the regenerated shoots were excised and transferred from the regeneration medium to MS rooting medium without growth regulators. The culture was exposed to 16-hour photoperiod, light intensity of 2500 μmol×m -2 ×s -1 , temperature of 25 +/-2 o C, and air humidity of 70%. The rooted plants were transferred into the mixture of soil and perlite (1:1) and acclimatized under greenhouse conditions.

RESULTS AND DISCUSSION
Describing the proper method of sterilization of plant material is an essential step to achieve high efficiency in in vitro cultures of plant tissues. The effectiveness of sterilization may be dependent on the type, concentration and time of treatment with the sterilizing agent (R o x a s et al., 1996). In this experiment, sterilization of Silphium perfoliatum seeds started with 5% calcium hypochlorite, as an appropriate disinfectant. However, in this experiment only 45.83% of sterile seeds were obtained and they sprouted in 8.33% (Fig. 1), while in the case of isolated embryos 33.3% of seedlings were obtained from 83.3% sterile embryos (Fig. 2). The seedlings were deformed, with their cotyledons curled, often brownish at the time of in vitro culture and with a single embryonic root or completely devoid of it. The low effectiveness of sterilization of cup plant seeds or embryos in 5% calcium hypochlorite and the deformation of germinated seedlings are explained by too long an action of the disinfectant or the phytotoxic properties of this species (P a r k i ns o n et al. 1996).
The best method used to sterilize the buds of yellow everlasting Helichrysum aenarium (L.) was to disinfect them in 70% ethanol for 1-2 seconds, and then in 9% calcium hypochlorite solution for 10 minutes. 63.33% of sterile seeds, capable of regenerating explants, are obtained with this method (S a w i l s k a and F i g a s , 2006). The sterilization method in which the seeds were immersed in 96% ethanol and fired over a flame burner allowed 91.67% of sterile samples to be obtained, but further development took place in only 37.5% of all embryos (Fig. 1).
Seedlings in this experiment were characterized by deformed, fine and thick cotyledons. Due to the adverse effects of temperature directly on the tissue, this method is not used for sterilization of isolated embryos. Another known technique for disinfecting plant material is, for example, treatment of explants with solutions of mercuric chloride. During the sterilization of gerbera seeds, A l t a f et al. (2009) used a 0.1% solution of mercuric chloride for 2 minutes at high efficiency. During the experiment, it was found that mercuric chloride caused the death of explants of some varieties, because in the case of those varieties a method was introduced using 5% and 10% hydrogen peroxide solution acting, respectively, for 5 and 10 minutes, which allowed one to obtain the maximum number of sterile explants (R o x a s et al. 1996). In this experiment, the use of hydrogen peroxide for the disinfection of seeds also increased the effectiveness of sterilization, however, the percentage of germinated embryos was 31.25% in the variant 2/1 (Fig. 1) and 56% in the variant 2/2 (Fig. 2). The resulting seedlings were phenotypically different from the normal morphology of plants of this species. The seedlings were low and small and they had undeveloped cotyledons. An earlier study shows that hydrogen peroxide is typically used to disinfect seeds of gymnosperm plants (W e n n y and D u m r o e s e , 1987). The low efficiency of these methods induced us to seek alternative sterilization techniques. Therefore, seeds were sterilized and embryos isolated using ACE. As a result of the use of ACE, as an appropriate disinfectant, 25% of germinated embryos and cultured seedlings were obtained from whole seeds (Fig. 1), but in the case of sterilization of embryos 100% of sterile embryos were obtained as well as 100% sterile and well-developed seedlings (Fig. 2). The seedlings obtained from seeds sterilized with ACE for 7 minutes had a better appearance and vigour, because it was used for isolation of apical explants. In a study on organogenesis of Gerbera jamesonii from seeds, H a s b u l l a h et al. (2008) obtained the same result after using 40% sodium hypochlorite for 30 minutes, while B a i y e r i and M b a h (2006) achieved optimal results by sterilizing the seeds of African breadfruit (Treculia africana Decne) with 3.5% sodium hypochlorite solution.
Shoot meristems set in the leaf corners have the same histological and genetic structure like shoot apical meristems; hence a possibility of producing plants genetically identical as the parent plant (H u and W a n g , 1983; A s s i m et al., 2008). As for the Asteraceae family plants, including the cup plant, growing tips used to initiate in vitro cultures guarantee a high genetic stability and enhance the potential of producing sterile tissue cultures (K a n w a r and K u m a r , 2008). The isolated cup plant apical explants, incubated on MS medium with growth regulators, kept on developing in the first weeks of culture and callus tissue formed on the cut surface, on which no adventitious bud growth was observed. Lateral shoots developed on the explants of lateral buds, located in the corners of leaves and stipules, during the successive weeks of culture on MS media with 2.5 mg × dm -3 BAP and 0.45 mg × dm -3 ABA, 5 mg × dm -3 BAP and 1 mg × dm -3 NAA as well as 2 mg × dm -3 BAP and 1mg × dm -3 IAA (Table 3, Fig. 3). Analyzing the results reported over the few weeks of culture, it can be observed that morphogenetic potential of cup plant apical explants depends on the ratio of the amount of auxin to cytokinin; the highest share of explants with lateral shoots (41.7%) and callus (90.3%) was recorded on MS medium with 5 mg × dm -3 BAP and 1 mg × dm -3 NAA. Figas et al. (2010), investigating cup plant micropropagation and applying the same BAP and NAA concentrations, reported 84.2% of explants with lateral shoots and 92.8 % with callus tissue, which coincides with the observations that a high content of cytokinins and an inconsiderable content of auxins enhance plant proliferation in in vitro cultures. Cytokinins originating from adenine (BAP, KIN) can tolerate the dominance of the apical bud, thus stimulating lateral shoots growing from the corner buds. Media of a similar composition as for the cup plant were reported by H u s s e i n et al. (2008) for gerbera (Gerbera jamesonii). The highest regeneration efficiency (36.6%), however, was reported by the authors on the medium with BAP and ABA added. The application of ABA resulted in an effective induction of the processes of callus formation and, as a result of subsequent passages, also in successful shoot organogenesis. On MS medium enriched with 0.5 mg × dm -3 IAA and 4 mg × dm -3 KIN on the cup plant explants, no formation of lateral shoots was observed. However, the growth of roots was reported, which accounted for 73.6%. Isolating shoot apexes of dwarf everlast (Helichrysum arenarium (L.) Moench), representing the Asteraceae family, and incubating them on MS medium of the same composition with growth regulators, S a w i l s k a and F i g a s (2006), recorded 16.1 lateral shoots per explant. The results demonstrate that applying plant regulators of the same group in in vitro cultures can trigger a completely different response of the explants of the species taxonomically remote from each other and clones of the same species. It is suggested that genes conditioning the control of the process of synthesis of endogenous or using exogenous growth regulators can be responsible for the regeneration (R a k o c z y -T r o j a n o w s k a and M al e p s z y , 1993).
The induction of rhizogenesis in cup plant in vitro cultures was reported on MS media without growth regulators. In case of many plants representing the Asteraceae family, the process occurred on MS media with a reduced content of mineral salts (½ MS) and a lowered concentration of saccharose (1.5%) (W e b b and S t r e e t , 1977). Rooting of 90% of shoots in costus (Saussurea lappa) has been recorded on the media with 0.5-1.0 mg × dm -3 NAA (A r o r a and B h o j w an i , 1989). Rooting gerbera shoots, R e y n o i r d et al. (1993) applied MS medium containing 0.25 μM NAA, and thus producing 100% of rooted shoots. Many plants form roots on media without growth regulators, however, the addition of IAA, IBA or NAA intensifies the process considerably (F a r i a and I l l g , 1995). In the present experiment, the application of MS medium without growth regulators, proposed by Aswath and C h o u d h a r y (2002) for cup plant rooting, resulted in 100% of rooted plants. The characteristic feature of the roots was the formation of compact and long systems and their short formation time (Fig. 4).  A. B. C. Fig. 3. Axillary shoots formed from shoot tip explants cultured on MS medium supplemented with 0.45 mg × dm -3 ABA and 2.5 mg × dm -3 BAP (A), 1 mg × dm -3 NAA and 5 mg × dm -3 BAP (B), 1 mg × dm -3 IAA and 2 mg × dm -3 BAP (C) after 8 weeks of culture. Fig. 4. Rooted plant of (Silphium perfoliatum L.) in in vitro cultures (A); the cup plant after its transfer from in vitro to ex vitro conditions (B). Phot. by Katarzyna Zadworna.

CONCLUSIONS
1. Numerous trials of sterilization indicate that the best method of obtaining sterile in vitro cultures is sterilization of isolated embryos of Silphium perfoliatum L. using ACE, which contains sodium hypochlorite as the active substance.

As a result of cup plant micropropagation in in vi-
tro cultures from shoot apical explants, the highest number of shoots was produced, formed from lateral buds without adventitious organogenesis. 3. The applied method of micropropagation from shoot apical explants offers a potential of producing offspring plants which would be genetically stable and with the genotype identical as the parent plant. 4. The need of using growth regulators in the process of rhizogenesis was eliminated, since numerous and well-branched root systems were formed on MS medium without phytohormones. Using this method, 100% of shoots produced compact and long roots. 5. Considering many possible applications of cup plant (Silphium perfoliatum L.) in various industries, medicine and in agriculture, continued research to develop an optimal and efficient system of plant regeneration of that species is well justified.