| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
ANIMAL NUTRITION |
* Department of Animal Science, University of Manitoba, Winnipeg, MB, Canada R3T 2N2;
and
Degussa AG, Rodenbacher Chaussee 4, 63457 Hanau-Wolfgang, Germany; and
and
Department of Food Science, University of Manitoba, Winnipeg, MB, Canada R3T 2N2
 |
Abstract |
|---|
 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
|---|
Key Words: amino acid • protein • early-weaned pig • growth • intestinal health
|
INTRODUCTION |
|---|
|
TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
|---|
). A current interest in swine nutrition is to eliminate the use of in-feed antibiotics. Diets for early-weaned pigs usually contain high levels of protein, which may encourage proliferation of pathogenic bacteria in the gastrointestinal tract (Ball and Aherne, 1987). The elimination of in-feed antibiotics coupled with the high dietary protein content of pig starter diets may increase the incidence of postweaning diarrhea in weaned pigs, leading to poor performance. It is prudent to speculate that reducing dietary protein supply with appropriate AA supplementation could reduce the amount of substrate for bacterial proliferation in the gastrointestinal tract and that this would be a great benefit to piglets fed antibiotic-free diets. Therefore, the objective of the current study was to determine the effect of feeding early-weaned pigs low-protein, AA-supplemented diets on growth performance and on indicators of intestinal health.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
|---|
). Specifically, all diets contained equal amounts of standardized ileal digestible Lys, Met plus Cys, Thr, and Trp with the latter 3 AA supplied to maintain the ideal pattern suggested by Rademacher et al. (2000). The Ile content in the 19% and 17% CP diets was formulated to equal the amount in the 21% CP diet because commercial diets for pigs at this age are unlikely to contain less than 21% CP. Ingredients contributing AA were analyzed for AA composition before diet formulation and analyzed values were used in diet formulation. Amino acid content in the diets was balanced by supplementation with crystalline AA provided by Degussa-Hüls AG (Hanau-Wolfang, Germany). All other nutrients were supplied in amounts meeting or exceeding NRC (1998) recommendations for a 6- to 10-kg pig. All diets were fed in mash form.
|
). A total of 96 Cotswold pigs weaned at 18 ± 1 d of age were obtained from the University of Manitoba’s Glenlea Swine Research Farm for use in the current study. Pigs were weighed and blocked on the basis of BW and sex and from within block divided into groups of 4 pigs (2 barrows and 2 gilts) per pen. Each pen had a plastic-covered expanded metal floor. Before the experiment began, the piglets were fed a commercial diet from a local feed mill (FeedRite, A Division of Ridley, Inc., Winnipeg, Manitoba, Canada) for a 7-d adaptation period to a BW of 6.2 ± 0.1 (mean ± SD) kg. Six replicate pens were assigned to each diet in a completely randomized design. Pigs had unlimited access to feed and water throughout the 3-wk study. Each pen had a water meter for measuring consumption. Body weight and feed disappearance were monitored weekly, and the results were used to calculate ADG, ADFI, and G:F. Room temperature was initially set at 31°C and gradually reduced by 1.5°C per week.
Blood Sampling and Assessment of Severity of Diarrhea
On d 0, 7, and 14, blood samples (10 mL) were collected from all pigs via jugular vein puncture into vacutainer tubes coated with lithium heparin (Becton Dickinson, Rutherford, NJ) and immediately centrifuged at 2,000 x g for 10 min at 5°C to recover plasma. Plasma samples were immediately stored at –20°C until used for plasma urea N (PUN) analysis.
Severity of diarrhea was characterized by using the fecal consistency score described by Marquardt et al. (1999). To ascertain the health status of the pigs, fecal consistency scoring (0 = normal, 1 = soft feces, 2 = mild diarrhea, and 3 = severe diarrhea) and Visual Assessment Scoring System (1 = poor, 2 = good, and 3 = better) were performed independently by 4 trained individuals with no prior knowledge of the treatment allocation.
Digesta Collection and Histological Measurements
At the end of the 3-wk study period, 2 pigs (1 of each gender) selected at random from each pen were held under halothane general anesthesia and killed by an intra-cardiac injection of sodium pentobarbital. Stomach, spleen, small intestine, and liver were removed, flushed with ice-cold physiological saline solution containing phenylmethyl sulfonyl fluoride (2 L of 0.9% saline, pH 7.4 + 2 mL of 100 mM phenylmethyl sulfonyl fluoride) to remove any excess blood, and 20 mL each of digesta from the stomach and the small intestine were obtained for pH measurement. After blotting the organs with an absorbent paper, weight and length (small intestine) were determined and 10-cm segments of the duodenum, jejunum, and ileum were taken and stored in 10% formalin to fix the villi and the crypts for subsequent histological measurement according to the procedures described by Owusu-Asiedu et al. (2003). Briefly, 6 cross-sections were obtained from each formalin-fixed segment and processed for histological examination using the standard hematoxylin and eosin method. Villous height was measured from the tip to the cryptvillus junction, and crypt depth was measured from the cryptvillus junction to the base on 10 well-oriented villi per specimen using a Zeiss photomicroscope equipped with a Sony 3 chip CCD color camera (Carl Zeiss Canada Ltd., Toronto, Ontario, Canada). Duodenum sample was taken at 30 cm away from the stomach. Jejunum sample was taken at 2 m before the ilealcecal junction. Ileum was sampled at 30 cm before the ilealcecal junction. The images were captured using Northern Eclipse Image Processing Software (Empix Imaging, Inc., Mississauga, Ontario, Canada).
Chemical Analysis and Microbial Population
The AA composition of the ingredients and diets was determined by Degussa AG. Dietary AA contents were determined by using ion-exchange chromatography with postcolumn derivatization with ninhydrin. Amino acids were oxidized with performic acid, which was neutralized with sodium metabisulfite (Commission Directive, 1998). Samples were hydrolyzed in 6 N HCl for 24 h at 110°C, and AA were quantified with the internal standard method by measuring the absorption of reaction products with ninhydrin at 570 nm. Tryptophan was determined by HPLC with fluorescence detection (extinction 280 nm, emission 356 nm) after alkaline hydrolysis with barium hydroxide octahydrate for 20 h at 110°C (Commission Directive, 2000).
Plasma samples were analyzed for urea N using a Nova Stat profile M blood gas and electrolyte analyzer (Nova Biomedical Corporation, Waltham, MA). Ammonia N concentration in digesta samples was determined using the method described by Novozamsky et al. (1974). Briefly, 1.5 mL of a reagent containing 200 mL 0.05% sodium nitroprusside and 10 mL of 4% EDTA was added to 50 µL of digesta fluid in a 10-mL test tube. A solution containing 10% NaOCl (2.5 mL) was then added to the mixture. Test tubes containing the resulting mixture were placed in a test tube rack wrapped with green plastic sheets and placed in complete darkness for 30 min followed by the reading of absorbance of the mixture at 630 nm. Ammonia N concentrations were determined by calculating the concentrations from a regression equation of the standard curve (range: 2.5 to 20 mg/L).
Volatile fatty acid determinations were conducted using gas chromatographic methods described by Erwin et al. (1961). Briefly, 1 mL of 25% meta-phosphoric acid was mixed with 5 mL of digesta fluid in a centrifuge tube. The mixture was frozen overnight and then incubated at room temperature for 30 min. The samples were centrifuged for 10 min at 2,400 x g in a Centra GP8 centrifuge (International Equipment Co., Need-ham Heights, MA) and the supernatants further centrifuged at 12,000 x g. The clean sample was analyzed for VFA (i.e., acetate, butyrate, valerate, isobutyrate, and isovalerate) concentrations using a Varian model 3400 gas chromatograph (Varian, Walnut Creek, CA).
Digesta (APHA, 1992) samples (1 g) for microbial counts were initially diluted in sterile peptone (0.1%; 9 mL) and serially diluted before analysis. Samples were analyzed for aerobic sporeformers, anaerobic sporeformers, Enterobactericeae, Enterococci, Escherichia coli, and total coliforms. All samples were analyzed in duplicate except for total coliforms and E. coli, which were analyzed only once. For aerobic spore counts, 5 mL of diluted (1:10) samples contained in screw-capped test tubes were heated in a thermostatically controlled water bath at 80°C for 20 min and then cooled in an ice bath. Decimal dilutions were plated in duplicate using Standards Methods Agar (Difco, Detroit, MI). Spore counts were evaluated after incubation at 35°C for 48 h. Samples for anaerobic spore counts were processed similarly except that incubation was performed using anaerobic jars containing a gas generating kit (Oxoid, UK). Diluted samples for enterococci counts were plated in duplicate using KF Streptococcus Agar (Difco, Detroit, MI); presumptive enterococci were enumerated after incubation at 37°C for 48 h. Total coliforms and E. coli were enumerated using Petrifilm coliform and E. coli count plates (3M, St. Paul, MN). Plates were evaluated after incubation at 35°C for 24 h.
Statistical Analyses
Data were analyzed as a completely randomized block design using the GLM procedures of SAS (SAS Inst., Inc., Cary, NC). Linear, quadratic, and cubic contrasts were performed to assess the effect of declining levels of dietary protein. For performance data, the pen was used as the experimental unit, but for other response criteria, pig within diet was used as the error term. Fisher’s protected least significant difference was used to compare means when a significant quadratic or cubic response was observed. Chi-square analysis was used to test scour and BCS (Cody and Smith, 1991).
|
RESULTS |
|---|
|
TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
|---|
and 3, respectively. For most AA and CP, with the exception of Ile (which had levels in the 19 and 17% diets that were lower than expected), the analyzed contents in experimental diets were similar to values calculated from analyzed amounts in the individual ingredients and their respective inclusion levels in the diets.
|
|
. The initial BW was similar among diets at 6.22 ± 0.1 kg (mean ± SD). Final BW decreased (linear, P < 0.001; quadratic, P < 0.05) as dietary CP decreased; the effect was more pronounced in pigs fed 19 and 17% CP diets.
|
). With a reduction in dietary CP content, G:F declined linearly (P < 0.05) during wk 1, 2, and overall, and quadratically (P < 0.05) during wk 3 (Table 4). Overall, pigs fed the 17% CP diet had the lowest G:F compared to all other treatments.
Although water usage was not different among treatments, there was a numerical decline in the amount of water consumed by piglets fed the low-CP AA-supplemented diets compared with the control diet (Table 4). Body condition scores and fecal consistency scores were not influenced by dietary treatments (P > 0.10; Table 4).
Plasma Urea N
Plasma urea N levels were similar (P = 0.16) in piglets at the start of the trial. However, after a week of consuming experimental diets, piglets fed diets 19% and 17% CP had lower (linear, P < 0.001) PUN levels compared with those fed the 23% CP control diet (Figure 1). On d 14 and 21, PUN was reduced dramatically (linear, P < 0.01; quadratic, P < 0.05) as dietary CP decreased, but the largest decline occurred when CP was reduced from 23 to 21% (Figure 1). The PUN levels in piglets fed the 19 and 17% CP diets were not different throughout the study (P > 0.10).
|
. Dietary treatment had no effect (P > 0.10) on spleen weight and length of the small intestine. However, as dietary CP was reduced from 23 to 17%, there was a linear (P < 0.05) and cubic (P < 0.05) effect on stomach and liver weights, respectively (Table 5).
|
Digesta pH, Ammonia N, and VFA Levels and Intestinal Microbial Populations
Decreasing dietary CP while maintaining the balance of essential AA had no effect (P > 0.10) on the pH of duodenal and jejunal digesta. However, as dietary CP declined, ileal digesta pH was quadratically (P < 0.01) reduced, with the 21% CP diet having the lowest value compared with the control diet (Table 6).
|
). In general, diet had no effect on the concentration of VFA in digesta obtained from the duodenum, except for propionic acid and valeric acid, which had concentrations linearly reduced (P < 0.05) with declining dietary CP (Table 7). The concentration of VFA in jejunal digesta was not influenced by diet (P > 0.10). In the ileal digesta, the concentration of all VFA measured was linearly reduced (P < 0.05) with decreasing dietary CP levels (Table 7). Bacterial counts in the ileal digesta are presented in Table 8. None of the bacterial populations determined was influenced by dietary treatment (P > 0.10).
|
|
|
DISCUSSION |
|---|
|
TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
|---|
) was close to the calculated values (Table 1). The CP content of the ingredients used in the current study was within the broad range of reported values (NRC, 1998; Degussa, 2001; Owusu-Asiedu et al., 2003).
It was anticipated that reducing dietary CP while balancing the diets for essential AA would support similar piglet performance. However, in the current study, final BW and the overall ADG and ADFI were reduced by feeding diets containing 19% or less CP, which is contrary to the results of previous studies (Hansen et al., 1993; Le Bellego and Noblet, 2002). Although ADG and ADFI were reduced when CP content was lowered to 19%, feed efficiency was maintained, which is in agreement with results of Southern and Baker (1982) and Le Bellego and Noblet (2002). As low-protein AA-supplemented diets are expected to supply a better balance of AA while minimizing excess supply, it has been argued that limiting dietary protein content while balancing for essential AA may boost feed intake in piglets (Le Bellego and Noblet, 2002). The discrepancies between our study and those of others may be explained by the differences in the age of piglets used. In the current study, piglets were weaned at 18 ± 1 d of age, but Le Bellego and Noblet (2002) used piglets that were weaned at 28 d and Jin et al. (1998) used growing pigs with an initial BW of 14.2 kg. Clearly, the apparent inconsistency in effect of dietary CP content and AA supplementation on the performance of early-weaned piglets should be investigated further.
As noted by Lewis (2001), conflicting results have been obtained when CP is reduced by more than 2 percentage units. Whereas the cause(s) of poor performance when low-protein AA-supplemented diets are fed have not been clearly elucidated, AA imbalance and/or deficiencies of other nutrients, including AA, may become limiting factors. As stated previously, diets used in the current study were formulated to contain equal amounts of standardized ileal digestible contents of Lys, Met plus Cys, Thr, and Trp according to the ideal pattern suggested by Rademacher et al. (2000). Because CP levels in commercial diets are still in the range of 23% and certainly not below 21% for pigs this age, the 19 and 17% CP diets were formulated to contain standardized digestible Ile levels equal to the 21% CP diet. All diets met or exceeded the specified total Ile requirements (NRC, 1998) for pigs within the weight range used in the present study (Table 1).
However, a careful examination of the analyzed AA composition of the experimental diets used in the current study reveal a potential Ile and Val deficiency in the 19 and 17% CP diets. Indeed, the calculated ratios of Ile and Val relative to Lys (standardized digestible basis) in the 19 and 17% CP were substantially lower than the ratios suggested by Rademacher et al. (2000). The ratio of Ile to Lys in the 19% and 17% CP diets based on the calculated standardized digestible ileal basis was 52%. Respective values for Val were calculated to be 55 and 48%. The ideal patterns for Ile and Val for 5- to 20-kg pigs have been proposed to be 60 and 68%, respectively (Baker et al., 1993). The calculated ratios of these two AA (based on total analyzed AA contents) were 49 and 46% for Ile and 53 and 46% for Val in the 19 and 17% CP diets, respectively. The ratios of Ile to Lys in the 21% CP diet were 52 and 50% on a standardized digestible and analyzed total basis, respectively. Corresponding values in the 23% CP diet were 58 and 55%, respectively. Because pig performance was not different between the 23 and 21% CP diets, it can be argued that Ile might not have limited performance in the 19 and 17% CP diets whose Ile to Lys ratios were similar to the 21% diet. Thus, it seems that only Val and or some other essential AA might have limited piglet performance in these treatments. Nonetheless, it has been suggested that other AA such as Val and Ile may limit growth performance when dietary CP is reduced by more than 4 percentage units (Figueroa et al., 2002). Also, Kerr et al. (2004), in a study with weaned pigs, demonstrated a positive response to Ile added to low-protein diets and concluded that low-protein AA-supplemented diets may require Ile supplementation in order to maintain optimal performance.
Water usage in the current study was only numerically reduced with reduction in dietary CP content, which is consistent with the results of Le Bellego and Noblet (2002), which showed numerical reductions in water consumption by piglets fed low-protein diets from 12 to 27 kg of BW. Although we did not determine urine output in the present study, reduced water consumption might result in reduced urine output as reported by Le Bellego and Noblet (2002). Similar to findings in the study by Le Bellego et al. (2002), fecal consistency score in the current study revealed no appreciable incidences of diarrhea in any group. Feeding high-protein diets has been reported to increase the incidences of diarrhea in swine (Ball and Aherne, 1987). However, this was not the case in the current study, perhaps due to the fact that piglets were housed in a relatively clean research facility.
The pig’s gastrointestinal tract is inhabited by both commensal and pathogenic bacteria that may directly affect intestinal nutrient requirements and limit the availability of dietary nutrients for growth (Burrin and Stoll, 2003). The population and activity of microflora in the upper gut depend on diet type and age (Stein and Nyachoti, 2003). Undigested nutrients such as protein and carbohydrates in the gut lumen provide a good source of substrates for microbial fermentation and proliferation (Cranwell and Moughan, 1989; Van Kol, 2000). Therefore, in the current study, it was hypothesized that feeding early-weaned pigs low-protein AA-supplemented diets would minimize microbial proliferation in the intestinal tract and also limit their byproducts, thus giving pigs an opportunity to better utilize dietary nutrients for growth. It is important to note that the levels of dietary fiber in the experimental diets changed with differences in CP content (Table 1). Because dietary fiber affects microbial activities in the gut, especially in the cecum and colon, this should be considered when interpreting the current results.
Intestinal pH, microbial populations, and by-products of microbial fermentation were measured to provide an indication of the effect of feeding low-protein AA-supplemented diets on indicators of intestinal health. The pH values in the current study are in close agreement with those reported previously (Owusu-Asiedu et al., 2003). The greater ileal digesta pH observed for piglets fed the 23% CP diet could be explained in part by the high buffering effect of protein (Ewing and Cole, 1994). Higher intestinal pH is thought to provide an optimal environment for enterotoxigenic E. coli to colonize the villi, leading to diarrhea (Smith and Jones, 1963; Ewing and Cole, 1994). Low pH, on the other hand, may favor development of beneficial bacteria and/or inhibit development of harmful bacteria (Fuller, 1977).
Digesta ammonia concentration was reduced linearly with the reduction in dietary CP content, which suggests a reduction in bacterial hydrolysis of nitrogenous compounds in pigs fed these diets (Blank et al., 2001). This observation is important because it implies that feeding low-protein AA-supplemented diets to weaned pigs may reduce the metabolic demand associated with ammonia detoxification by the liver and may improve the efficiency of energy and AA utilization (Jensen, 1998).
The VFA data indicate, as expected, an increase in microbial activity along the small intestine from the duodenum to the ileum. The concentration of VFA in ileal digesta was considerably higher than in the jejunum and duodenum. Franklin et al. (2002) made similar observations and reported that increased VFA concentration in the cecum coincided with increased microbial population in this segment of the gastrointestinal tract. The authors interpreted this to indicate increased microbial activity.
Reducing dietary CP with AA supplementation resulted in significant linear reductions in VFA in ileal digesta. Like the ammonia data, this observation suggests that feeding low-protein AA-supplemented diets dramatically reduced or altered microbial fermentation in the small intestine. Thus, it would seem that low-protein AA-supplemented diets could be used to manipulate the by-products of microbial activity, which in turn may provide opportunities to increase productivity and reduce the impact of livestock on the environment (Franklin et al., 2002).
With the observed reductions in VFA concentration in ileal digesta as dietary CP was decreased, it would seem contradictory that digesta pH in this intestinal segment was also reduced. However, digesta pH in pigs is not always well correlated with VFA concentrations. For instance, Pluske et al. (2003) observed that digesta pH and VFA concentrations were not well correlated in piglets fed diets containing different sources and levels of dietary fiber. It has been suggested that digesta pH is dependent on the pKa and proportion of specific VFA present in the intestinal digesta and the buffering capacity of dietary nutrients such as protein (Ewing and Cole, 1994; Pluske et al., 1998).
The current observation that PUN was reduced linearly with reduced dietary CP content is in agreement with results of other investigators (Figueroa et al., 2002). Decreasing dietary protein from 23% to 21% resulted in the largest drop in PUN levels, but a reduction from 21 to 19% or lower had no significant effect except on d 21. However, decreasing dietary protein from 19 to 17% had no effect on PUN levels throughout the study, perhaps indicating a limit as to how much this response criterion can be influenced by dietary protein content.
Results of the current study indicate that changes in PUN levels in pigs fed diets differing in CP are not dependent on feed intake, which is consistent with the finding of Figueroa et al. (2002) that indicated no change in PUN despite a significant reduction in feed intake when dietary CP was reduced from 12 to 11%. Furthermore, Figueroa et al. (2002) observed a significant reduction in PUN when dietary CP was reduced from 16 to 14% despite the fact that pigs fed the 14% CP diet had higher feed intake. In general, a reduction in PUN level is indicative of a more efficient use of dietary N (Figueroa et al., 2002; Owusu-Asiedu et al., 2003) and is probably dependent more on the amounts and balance of AA available systemically rather than simply feed intake per se.
Diet composition is one of the major factors that can influence microbial population in the gastrointestinal tract, mainly through its effects on substrate availability. Thus, it was anticipated that reducing dietary CP content coupled with AA supplementation would influence the microbial population in the gut of young pigs. However, the results of the current study suggest that low-protein AA-supplemented diets had no effect on gut microbial populations, which is consistent with the observed scores of fecal consistency. Because the current study was conducted in a clean research facility, caution should be exercised in translating these results to a commercial situation. Bacterial load in the gastrointestinal tract can be influenced by sanitary conditions in the environment where pigs are kept. Indeed, it has been suggested that a greater effect of dietary protein content on piglet diarrhea and performance is likely under commercial conditions (Göransson et al., 1995).
|
IMPLICATIONS |
|---|
|
TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
|---|
|
Footnotes |
|---|
2 Corresponding author: martin_nyachoti{at}umanitoba.ca
Received for publication November 30, 2004. Accepted for publication September 14, 2005.
|
LITERATURE CITED |
|---|
|
TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
|---|
This article has been cited by other articles:
|
|
 |
|
  J. K. Htoo, B. A. Araiza, W. C. Sauer, M. Rademacher, Y. Zhang, M. Cervantes, and R. T. Zijlstra Effect of dietary protein content on ileal amino acid digestibility, growth performance, and formation of microbial metabolites in ileal and cecal digesta of early-weaned pigs J Anim Sci, December 1, 2007; 85(12): 3303 - 3312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Kiarie, C. M. Nyachoti, B. A. Slominski, and G. Blank Growth performance, gastrointestinal microbial activity, and nutrient digestibility in early-weaned pigs fed diets containing flaxseed and carbohydrase enzyme J Anim Sci, November 1, 2007; 85(11): 2982 - 2993. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Bikker, A. Dirkzwager, J. Fledderus, P. Trevisi, I. le Huerou-Luron, J. P. Lalles, and A. Awati The effect of dietary protein and fermentable carbohydrates levels on growth performance and intestinal characteristics in newly weaned piglets J Anim Sci, December 1, 2006; 84(12): 3337 - 3345. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Hedemann, M. Eskildsen, H. N. Laerke, C. Pedersen, J. E. Lindberg, P. Laurinen, and K. E. B. Knudsen Intestinal morphology and enzymatic activity in newly weaned pigs fed contrasting fiber concentrations and fiber properties J Anim Sci, June 1, 2006; 84(6): 1375 - 1386. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
