Abstract  A rapid and sensitive method is described for the determination of scopolamine and its metabolites in rat urine by combining liquid chromatography and tandem mass spectrometry (LC-MS/MS). Various extraction techniques (free fraction, acid hydrolyses and enzyme hydrolyses) and their comparison were carried out for investigation of the metabolism of scopolamine. After extraction procedure, the pretreated samples were injected into a reversed-phase C18 column with mobile phase of methanol/ ammonium acetate (2mM, adjusted to pH 3.5 with formic acid) (70:30, v/v) and detected by an on-line MS/MS system. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular masses (DeltaM), retention-times and full scan MS(n) spectra with those of the parent drug. The results revealed that at least 18 metabolites (norscopine, scopine, tropic acid, aponorscopolamine, aposcopolamine, norscopolamine, hydroxyscopolamine, hydroxyscopolamine N-oxide, p-hydroxy-m-methoxyscopolamine, trihydroxyscopolamine, dihydroxy-methoxyscopolamine, hydroxyl-dimethoxyscopolamine, glucuronide conjugates and sulfate conjugates of norscopolamine, hydroxyscopolamine and the parent drug) and the parent drug existed in urine after ingesting 55mg/kg scopolamine to healthy rats. Hydroxyscopolamine, p-hydroxy-m-methoxyscopolamine and the parent drug were detected in rat urine for up 106h after ingestion of scopolamine.

Abstract  Perchlorate, a known thyroid disruptor, is deposited in eggs of exposed female birds, raising concerns that the embryos from these eggs may become hypothyroid, which may in turn affect the development and function of thyroid-dependent organs. We hypothesized that exposure to ammonium perchlorate (AP) would decrease hen and embryonic thyroid function and affect the expression of thyroid-responsive genes in embryonic brain and liver. Laying Japanese quail hens were treated with 2000 mg/l or 4000 mg/l AP in drinking water. Thyroid status and expression of thyroid-responsive genes were examined in the embryos from eggs of exposed hens. Perchlorate exposure led to hypothyroidism in hens from both treatment groups; egg production was decreased in the high dosage group only. Embryos from eggs of perchlorate-exposed hens had hypertrophied thyroid glands and significantly lower thyroidal hormone storage, indicating hypothyroidism in these embryos. The embryonic hypothyroidism was associated with decreased embryonic growth, delayed hatching and greater mortality during hatching. The mRNA level of type 2 deiodinase (D2) in the liver of embryos from eggs of perchlorate-exposed hens was increased compared to the control embryos, a compensatory response that increases the production of metabolically active T(3). However, the mRNA levels of D2 and RC3 in the brain were not affected. These results suggest that the embryonic brain is protected from hypothyroidism by other mechanisms known to influence hormone entry into and exit from the brain. Our study shows that maternal perchlorate exposure led to embryonic hypothyroidism and may have interfered with embryonic development.

Abstract  The cation and the anion in the title salt, (C(6)H(16)N)[SnCl(4)(C(6)H(4)NO(2))], are linked by an N-H⋯O hydrogen bond. The Sn(IV) atom in the stannate anion is chelated by the pyridine-2-carboxyl-ate group and exists in a cis-SnCl(4)NO octa-hedral geometry. The cation is disordered over two positions in a 0.564 (1):0.436 (1) ratio.

Abstract  On-site disposal of sewage in septic systems can lead to groundwater plumes with NO(3) (-) -N concentrations exceeding the common drinking water limit of 10 mg/L. Currently, denitrification is considered as the principal natural attenuation process. However, at a large seasonal-use septic system in Ontario (256 campsites), a suboxic zone exists where nitrogen removal of up to 80% occurs including removal of NH(4) (+) -N. This zone has both NO(3) (-) -N and NH(4) (+) -N at >5 mg/L each. In the distal NH(4) (+) -rich zone, NH(4) (+) -N concentrations (8.1 ± 8.0 mg/L) are lower than in the proximal zone (48 ± 36 mg/L) and NH(4) (+) -N is isotopically enriched (concentration-weighted mean δ(15) N of +15.7‰) compared to the proximal zone (+7.8‰). Furthermore, δ(15) N-NH(4) (+) isotopic enrichment increases with depth in the distal zone, which is opposite to what would result if nitrification along the water table zone was the mechanism causing NH(4) (+) depletion. Bacterial community composition was assessed with molecular (DNA-based) analysis and demonstrated that groundwater bacterial populations were predominantly composed of bacteria from two Candidatus genera of the Planctomycetales (Brocadia and Jettenia). Together, these data provide strong evidence that anaerobic ammonium oxidation (anammox) plays an important role in nitrogen attenuation at this site.

Abstract  Due to eutrophication, high environmental ammonia (HEA) has become a frequent problem in aquatic environments, especially in agricultural or densely populated areas. During certain periods, e.g. winter, feed deprivation may occur simultaneously in natural waters. Additionally, under such stressful circumstances, fish may be enforced to swim at a high speed in order to catch prey, avoid predators and so on. Consequently, fish need to cope with all these stressors by altering physiological processes which in turn are controlled by genes expression. Therefore, in the present study, ammonia toxicity was tested in function of nutrient status (fed versus starved) and swimming performance activity (routine versus exhaustive). Goldfish, a relatively tolerant cyprinid, were exposed to HEA (1 mg/L; Flemish water quality guideline for surface water) for a period of 3 h, 12 h, 1 day, 4 days, 10 days, 21 days and 28 days and were either fed (2% body weight) or starved (kept unfed for 7 days prior to sampling). Results showed that the activity of Na⁺/K⁺-ATPase in the gills was stimulated by HEA and disturbance in ion balance was obvious with increases in plasma [Na⁺], [Cl⁻] and [Ca²⁺] after prolonged exposure. Additionally, osmoregulation and metabolism controlling hormones like cortisol and thyroid hormones (T3 and T4) were investigated to understand adaptive responses. The expression kinetics of growth, stress and osmo-regulatory representative genes such as Insulin-like growth factor 1 (IGF-I), growth hormone receptor (GHR), thyroid hormone receptor β (THRβ), prolactin receptor (PRLR), cortisol receptor (CR) and Na⁺/K⁺-ATPase α(3) were examined. Overall effect of HEA was evident since Na⁺/K⁺-ATPase activity, plasma cortisol, Na⁺ and Ca²⁺ concentration, expression level of CR and Na⁺/K⁺-ATPase α₃ mRNA in fed and starved fish were increased. On the contrary, transcript level of PRLR was reduced after 4 days of HEA; additionally T3 level and expression of GHR, IGF-I and THRβ genes were decreased following 10-21 days of HEA. Starvation, the additional challenge in the present study, significantly increased plasma cortisol level and CR transcript level under HEA compared to the fed exposed and control fish. Furthermore, a remarkable reduction in T3 and mRNA levels of THRβ, IGF-I and GHR genes was observed under starvation. The toxic effects in both feeding treatments were exacerbated when imposed to exhaustive swimming with more pronounced effects in starved fish. This confirms that starvation makes fish more vulnerable to external ammonia, especially during exercise.

Abstract  BACKGROUND: Natural honey is widely used all over the world as a complementary and alternative medicine in various disorders including gastrointestinal lesions.

AIM: To evaluate the effects of combination of low dosage of honey (0.312 g/kg) and sucralfate (0.125 or 0.250 g /kg) on gastric protection and to determine any potentiating interactions between them against ammonia-induced gastric lesions in rats.

MATERIAL AND METHODS: Twenty-four hours fasted rats were given I ml of ammonium hydroxide 1 % intragastrically and they were killed one hour later under deep ether anesthesia. The gastric lesion index was calculated according to the method of Takaishi et al 1998. Non protein sulthydryls level was determined spectrophotometrically as described by Sedlak and Lindsay 1968.

RESULTS: Administration of ammonium hydroxide produced red and black linear lesions and significant depletion of gastric nonprotein sulthydryls level. Oral administration of honey (0.312g/kg) or sucralfate (0.125 and 0.250 g/kg) 30 min before ammonium hydroxide reduced the severity of gastric mucosal lesions by 1 I or 18 and 42 % respectively, and has shown the changes in nonprotein sulfhydryls level induced by ammonium hydroxide. Furthermore, pretreatment with a combination of a low dose of honey (0.312 g /kg) and sucralfate (0.125 g or 0.250 g/kg) afforded significantly greater protection (58 and 77 %) than that obtained with either of them administered alone.

CONCLUSION: The present results suggest potentiation of gastric protection effect of sucralfate by honey and this may have a clinical value in the treatment of peptic ulcer diseases in Helicobacter pylori positive patients.

Abstract  Diuron, a substituted urea herbicide, is carcinogenic to the urinary bladder of rats at high dietary levels. Its proposed carcinogenic mode of action (MOA) includes urothelial cytotoxicity and necrosis followed by regenerative cell proliferation and sustained urothelial hyperplasia. Cytotoxicity could be induced either by urinary solids or by chemical toxicity by diuron and/or metabolites excreted in the urine. Diuron was not genotoxic in a previous single-cell gel (comet) assay, but possible cross-linking activity remained to be evaluated. The present study explored the MOA of diuron and the effect of urinary acidification on the development of urothelial lesions. Male Wistar rats were fed diuron (2500 ppm, about 130 mg/kg of body weight) either with or without NH(4)Cl 10,000 ppm to acidify the urine. Reversibility of urothelial changes was also examined. The animals were euthanized after 15, 25, or 30 weeks. Diuron-fed rats had urinary amorphous precipitate and magnesium ammonium phosphate crystals similar to control animals. Groups treated with diuron + NH(4)Cl showed decreased urinary pH and reduced amounts of urinary crystals and precipitate. Urothelial necrosis and simple hyperplasia were observed by light microscopy and scanning electron microscopy both in diuron- and in diuron + NH(4)Cl-treated groups. Cytotoxicity and proliferative changes were mostly reversible. A modified comet assay developed in vitro with Chinese hamster ovary cells showed that diuron did not induce DNA cross-links. These data suggest that cytotoxicity with consequent regenerative cell proliferation is the predominant MOA for diuron rat urothelial carcinogenesis, the cytotoxicity being chemically induced and not due to urinary solids.

Abstract  N-Isobutyl-phthalimic acid hydrolyzes to the title salt, 2C(4)H(12)N(+)·C(8)H(4)O(4) (-)·H(2)O, which adopts a hydrogen-bonded layer structure. In the anion, the carboxyl-ate groups are twisted with respect to the benzene ring [dihedral angles = 43.8 (1) and 50.9 (1)°].

Abstract  INTRODUCTION: Metabolic acidosis is present in end stage renal disease. There is a link between enhanced endothelial permeability and accelerated atherosclerosis. In this study, we investigated the effect of experimentally induced metabolic acidosis on aortic endothelial permeability and serum nitric oxide (NO) concentration in normal and high-cholesterol fed rabbits.

MATERIAL AND METHODS: Twenty-four male rabbits were divided into four groups: normal, hypercholesterolemic, acidemic, and hypercholesterolemic plus acidemic. Acidosis and hypercholesterolemia were induced by drinking water containing ammonium chloride (NH(4)Cl), and cholesterol-rich animal chow (1%), respectively. After 6 weeks, blood samples were taken and endothelial permeability was measured using the Evans blue dye injection method.

RESULTS: Hypercholesterolemic animals had higher aortic endothelial permeability compared with normal groups (16.18 ±0.91 µg EB/g tissue vs. 12.89 ±0.66 µg EB/g tissue, p < 0.05). Acidosis significantly increased endothelial permeability in the normal group (17.10 ±0.56 µg/g tissue vs. 12.89 ± 0.66 µg/g tissue; p < 0.05) but did not further increase endothelial permeability in hypercholesterolemic animals (16.18 ±0.91 µg EB/g tissue vs. 17.29 ±0.46 µg EB/g tissue; p > 0.05). Serum total cholesterol, low density lipoprotein (LDL) and NO concentrations in hypercholesterolemic animals were significantly higher than the normal group and acidosis could not change them either in the normal or in the high-cholesterol diet group.

CONCLUSIONS: Alterations of serum lipids and NO are not the main mechanism for accelerated atherosclerosis during metabolic acidosis. Acidosis increases aortic endothelial permeability at least in a normal diet which may be a possible mechanism for progression of atherosclerosis processes in end-stage renal disease.

Abstract  In the crystal structure of the title compound, C(5)H(8)N(2)O(3), the mol-ecules exist in the zwitterionic form. The pyrrolidine ring adopts an envelope conformation with the unsubstituted endocyclic C atom situated at the flap. The other four endocyclic atoms are coplanar with the exocyclic carbonyl O atom, with an r.m.s. deviation from the mean plane of 0.06 Å. The carboxyl-ate substituent is located axially, while the ammonium group occupies an equatorial position. In the crystal structure, the mol-ecules are linked through N-H⋯O hydrogen bonds, forming a three-dimensional network.

Abstract  The effect of different phytogenic feed additives on reducing odorous compounds in swine was investigated using in vitro fermentation and analyzed their microbial communities. Soybean meal (1%) added with 0.1% different phytogenic feed additives (FA) were in vitro fermented using swine fecal slurries and anaerobically incubated for 12 and 24 h. The phytogenic FAs used were red ginseng barn powder (Panax ginseng C. A. Meyer, FA1), persimmon leaf powder (Diospyros virginiana L., FA2), ginkgo leaf powder (Ginkgo biloba L., FA3), and oregano lippia seed oil extract (Lippia graveolens Kunth, OL, FA4). Total gas production, pH, ammonia-nitrogen (NH3-N), hydrogen sulfide (H2S), nitrite-nitrogen (NO2 (-)-N), nitrate-nitrogen (NO3 (-)-N), sulfate (SO4 (--)), volatile fatty acids (VFA) and other metabolites concentration were determined. Microbial communities were also analyzed using 16S rRNA DGGE. Results showed that the pH values on all treatments increased as incubation time became longer except for FA4 where it decreased. Moreover, FA4 incubated for 12 and 24 h was not detected in NH3-N and H2S. Addition of FAs decreased (p<0.05) propionate production but increased (p<0.05) the total VFA production. Ten 16S rRNA DGGE bands were identified which ranged from 96 to 100% identity which were mostly isolated from the intestine. Similarity index showed three clearly different clusters: I (FA2 and FA3), II (Con and FA1), and III (FA4). Dominant bands which were identified closest to Eubacterium limosum (ATCC 8486T), Uncultured bacterium clone PF6641 and Streptococcus lutetiensis (CIP 106849T) were present only in the FA4 treatment group and were not found in other groups. FA4 had a different bacterial diversity compared to control and other treatments and thus explains having lowest odorous compounds. Addition of FA4 to an enriched protein feed source for growing swine may effectively reduce odorous compounds which are typically associated with swine production.

Abstract  The experiment was conducted by in vitro fermentation and bacterial community analysis to investigate the reduction of odorous compounds in response to the use of feed additives (FA) during carbohydrate overload in growing pigs. Soluble starch at 1% (control) and various FA at 0.1% Ginseng meal (FA1); Persimmon leaf (FA2); Gingko nut (FA3) and Oregano lippia (FA4) were added to fecal slurry and incubated anaerobically for 12 and 24 h. In vitro parameters and microbial diversity of the dominant bacteria following fermentation were analyzed using Denaturing Gradient Gel Electrophoresis (DGGE), band cloning and sequencing of the V3 region. Results showed that total gas production increased with the advancement of incubation (p<0.05). pH values of FAs and control groups were decreased except the FA4 group which increased somewhat from 12 to 24 h (p<0.05). Ammonia nitrogen (NH3-N) and H2S gas concentrations were comparatively lower in both stages in FA4 treatment than in the other groups (p<0.05). Hence, NH3-N concentrations in liquid phases were increased (p<0.05) from 12 to 24 h, but the trend was lowest in FA4 than in the other groups at both stages. The total VFA production was comparatively lower and butyrate levels were moderate in FA4 group than in the the other groups during both stages (p<0.05). Indirect odor-reducing compounds such as NO2, NO3 and SO4 concentrations were higher in the FA4 and FA3 than in the other groups at 24 h (p<0.05). After fermentation, ten dominant bands appeared, six of which appeared in all samples and four in only the FA4 treated group. The total number of DGGE bands and diversity was higher in the FA4-group compared to other groups. Additionally, similarity indices were lowest (71%) in the FA4, which represented a different bacterial community compared with the other groups. These findings indicate that NH3-N, H2S and VFA production was minimal, and pH was also better in the FA4 group than in the other groups. Furthermore, the conversion of odor-reducing indirect compounds or their intermediates was higher in the FA4 group in compared to the other groups. FA4 group generated less odorous products and more indirect products by in vitro fermentation at 24 h, and their microbial pattern appeared to differ from that of the other groups. These findings suggest that this particular FA could change the microbial population, which may have a beneficial effect on odor reduction. It is recommended that the oregano lippia may be supplied to growing pigs as FA along with excess carbohydrate sources to reduce the production of odorous compounds.

Abstract  AIMS: The objective of this study was to comprehensively evaluate quillaja (QSP) and yucca saponin (YSP) products with respect to their effects on diversity of rumen bacteria and archaea, abundance of selected microbes, and feed degradability and fermentation.

METHODS AND RESULTS: Both QSP and YSP at doses 0-0.6 g l(-1) tended to increase degradability of feed substrate in in vitro rumen cultures, but to different extents. Neither one of the saponins affected the concentrations of ammonia, total volatile fatty acids, or molar proportion of acetate. However, QSP increased molar proportion of propionate and decreased that of butyrate, whereas YSP tended to decrease that of butyrate. As determined by qPCR, QSP and YSP did not affect the abundance of total bacteria or Ruminococcus albus. The QSP did not affect the abundances of Fibrobacter succinogenes or genus Prevotella, but tended to decrease that of Ruminococcus flavefaciens, whereas YSP significantly increased the abundance of R. flavefaciens and Prevotella, and numerically increased that of F. succinogenes. Both saponins increased archaeal abundance, although to small magnitudes (0.3-0.4 log). The protozoal populations were decreased significantly by QSP, but not by YSP. Based on DGGE and T-RFLP analysis, both saponins altered the bacterial community and species organization, but less so the archaeal community.

CONCLUSIONS: This study demonstrated that saponins, although not effective in mitigating methane emission, may improve feed utilization at low doses, and modulate ruminal microbial communities in a dose-dependent manner.

SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study suggest that saponins at low doses may directly stimulate the growth of some rumen bacteria including cellulolytic bacteria, thus improving digestibility of feeds, independent of their defaunation activity. In contrast, saponins at high doses modulate rumen fermentation characteristically similar to defaunation.

Abstract  At weaning (33 days of age), 246 hybrid rabbits (782 ± 53 g live weight) were divided into six experimental groups and fed ad libitum six iso-ADF diets formulated according to a bifactorial arrangement with two protein levels (152 and 162 g/kg) and three soluble fibre-to-starch ratios (0.2, 0.6 and 1.5), the latter obtained by replacing starch (from 209 to 91 g/kg) with soluble fibre (from 48 to 136 g/kg). The trial lasted for 42 days until slaughter. The rabbits that were fed the diet with the highest protein level and the lowest soluble fibre-to-starch ratio showed the highest mortality rate (17.1% v. 1.7% on average; P < 0.001) and sanitary risk (mortality + morbidity: 20.0% v. 8.1%; P = 0.04) compared with the rabbits fed the other diets. With increasing dietary crude protein level, the digestibility of dry matter (DM; 0.615 to 0.626) and gross energy (0.620 to 0.630) as well as aNDF (without sodium sulphite; 0.298 to 0.323) and hemicelluloses (0.417 to 0.461) significantly (0.001 < P < 0.10) improved. Moreover, total volatile fatty acids (VFAs) in the caecal content increased (59.0 to 68.4 mmol/l; P = 0.01) and ileum crypt depth tended to reduce (P = 0.07). Neither growth performance nor slaughter results were affected by the protein level. When increasing soluble fibre-to-starch ratio, the digestibility of DM and gross energy did not change, whereas the digestibility of aNDF (0.264 to 0.352), ADF (0.167 to 0.267) and hemicelluloses (0.400 to 0.470) linearly increased (P < 0.001). At caecum, N-ammonia tended to decrease linearly (P = 0.08), total VFA concentration (56.0 to 67.3 mmol/l) and acetate proportion (80.4 to 83.3 mmol/100 mmol VFA) linearly increased (P < 0.01), whereas butyrate and valerate proportions decreased (0.01 < P < 0.05). Growth performance was similar among groups, whereas at slaughter the proportion of the gastrointestinal tract linearly increased (177 to 184 g/kg; P < 0.01) without effect on dressing percentage, however. As soluble fibre-to-starch ratio increased, meat pH linearly decreased and lightness (L*), redness (a*) and yellowness (b*) colour indexes increased (0.01 < P < 0.05).

Abstract  The ammonia transporter family member, Rh B Glycoprotein (Rhbg), is an ammonia-specific transporter heavily expressed in the kidney and is necessary for the normal increase in ammonia excretion in response to metabolic acidosis. Hypokalemia is a common clinical condition in which there is increased renal ammonia excretion despite the absence of metabolic acidosis. The purpose of this study was to examine Rhbg's role in this response through the use of mice with intercalated cell-specific Rhbg deletion (IC-Rhbg-KO). Hypokalemia induced by feeding a K(+)-free diet increased urinary ammonia excretion significantly. In mice with intact Rhbg expression, hypokalemia increased Rhbg protein expression in intercalated cells in the cortical collecting duct (CCD) and in the outer medullary collecting duct (OMCD). Deletion of Rhbg from intercalated cells inhibited hypokalemia-induced changes in urinary total ammonia excretion significantly and completely prevented hypokalemia-induced increases in urinary ammonia concentration, but did not alter urinary pH. We conclude that hypokalemia increases Rhbg expression in intercalated cells in the cortex and outer medulla and that intercalated cell Rhbg expression is necessary for the normal increase in renal ammonia excretion in response to hypokalemia.

Abstract  The Rhesus factor protein, Rh C glycoprotein (Rhcg), is an ammonia transporter whose expression in the collecting duct is necessary for normal ammonia excretion both in basal conditions and in response to metabolic acidosis. Hypokalemia is a common clinical condition associated with increased renal ammonia excretion. In contrast to basal conditions and metabolic acidosis, increased ammonia excretion during hypokalemia can lead to an acid-base disorder, metabolic alkalosis, rather than maintenance of acid-base homeostasis. The purpose of the current studies was to determine Rhcg's role in hypokalemia-stimulated renal ammonia excretion through the use of mice with collecting duct-specific Rhcg deletion (CD-Rhcg-KO). In mice with intact Rhcg expression, a K(+)-free diet increased urinary ammonia excretion and urine alkalinization and concurrently increased Rhcg expression in the collecting duct in the outer medulla. Immunohistochemistry and immunogold electron microscopy showed hypokalemia increased both apical and basolateral Rhcg expression. In CD-Rhcg-KO, a K(+)-free diet increased urinary ammonia excretion and caused urine alkalinization, and the magnitude of these changes did not differ from mice with intact Rhcg expression. In mice on a K(+)-free diet, CD-Rhcg-KO increased phosphate-dependent glutaminase (PDG) expression in the outer medulla. We conclude that hypokalemia increases collecting duct Rhcg expression, that this likely contributes to the hypokalemia-stimulated increase in urinary ammonia excretion, and that adaptive increases in PDG expression can compensate for the absence of collecting duct Rhcg.

Abstract  Two experiments were conducted with the objective of investigating the effects of rumen-protected methionine (RPMet) supplementation of metabolizable protein (MP)-deficient or MP-adequate but Met-deficient diets on dairy cow performance. Experiment (Exp.) 1 utilized 36 Holstein dairy cows blocked in 12 blocks of 3 cows each. Cows within block were assigned to one of the following dietary treatments: (1) MP-adequate diet [AMP; positive MP balance according to the National Research Council (2001) dairy model]; (2) an MP-deficient diet supplemented with 100g of rumen-protected Lys (RPLys)/cow per day (DMPL); and (3) DMPL supplemented with 24 g of RPMet/cow per day (DMPLM). Experiment 2 utilized 120 Holstein cows assigned to 6 pens of 20 cows each. Pens (3 per treatment) were assigned to one of the following dietary treatments: (1) AMP diet supplemented with 76 g of RPLys/cow per day (AMPL); and (2) AMPL (74 g of RPLys/cow per day) supplemented with 24 g of RPMet/cow per day (AMPLM). Each experiment lasted for 10 wk (2-wk adaptation and 8-wk experimental periods) following a 2-wk covariate period (i.e., a total of 12 wk). In Exp. 1, the MP-deficient diets decreased apparent total-tract nutrient digestibility but had no statistical effect on dry matter intake (DMI), milk yield, or milk fat percentage and yield. Compared with AMP, DMPL decreased milk protein content; both DMPL and DMPLM diets decreased milk protein yield. Urinary N losses and milk urea-N concentration were decreased by the MP-deficient diets compared with AMP. The ammonia emitting potential of manure from the MP-deficient diets was decreased by about 37% compared with that of AMP manure. Plasma Lys and Met concentrations were not affected by treatment, but concentrations of His, Thr, and Val were lower for the MP-deficient diets compared with AMP. In Exp. 2, the AMPLM diet had lower milk yield than AMPL due to numerically lower DMI; no other effects were observed in Exp. 2. In conclusion, feeding MP-deficient diets supplemented with RPLys and RPMet did not statistically decrease milk yield in dairy cows in Exp. 1. However, without RPMet supplementation, milk protein content was decreased compared with the MP-adequate diet. Other amino acids, possibly His, may limit milk production in MP-deficient, corn or corn silage-based diets. A summary of 97 individual cow data from trials in which MP-deficient diets were fed suggested the National Research Council (2001) model under-predicts milk yield in cows fed MP-deficient diets (MP balance of -20 to -666 g/d) in a linear manner: milk yield under-prediction [National Research Council (2001) MP-allowable milk yield, kg/d - actual milk yield, kg/d] = 0.0991 (±0.0905) + 0.0230 (±0.0003) × MP balance, g/d (R(2)=0.99).

Abstract  The objective of this study was to determine the underlying physiological and molecular responses to long-term sublethal ammonia exposure in Atlantic salmon (Salmo salar) parr. Previous studies have predominately focused on mechanisms during acute, short-term exposure. For that purpose Atlantic salmon parr were exposed to four ammonia concentrations between 4 and 1800 μmol l(-1) total ammonia nitrogen (TAN), and subjected to two feeding regimes for 15 weeks. Elevated environmental ammonia and full feeding strength caused an initial increase in plasma ammonia levels ([T(amm)]) after 22 days of exposure, which thereafter declined and remained similar to the control animals towards the end of the study. On the other hand, a progressive decrease in plasma urea levels was evident throughout the entire exposure period and depended on the concentration of environmental ammonia, with the largest decrease in urea levels observed at the highest ammonia concentrations (1700 and 1800 μmol l(-1) TAN). We hypothesized that the successful adaptation to long-term elevated ammonia levels would involve an increased capacity for carrier-facilitated branchial excretion. This hypothesis was strengthened by the first evidence of an up-regulation of branchial transcription of the genes encoding the Rhesus (Rh) glycoproteins, Rhcg1 and Rhcg2, urea transporter (UT) and aquaporin 3a (Aqp3a), during long-term exposure. Of the Rhesus glycoprotein (Rh) mRNAs, Rhcg1 was up-regulated at all tested ammonia levels, while Rhcg2 showed a concentration-sensitive increase. Increased transcription levels of V-type H(+)-ATPase (H(+)-ATPase) were observed at the highest ammonia concentrations (1700 and 1800 μmol l(-1) TAN) and coincided with an up-regulation of Rhcg2 at these concentrations. Transcription of UT and Aqp3a was increased after 15 weeks of exposure to low ammonia levels (470 and 480 μmol l(-1) TAN). A significant increase in brain glutamine (Gln) concentration was observed for full fed Atlantic salmon after 22 days and in fish with restricted feeding after 105 days of exposure to 1800 and 1700 μmol l(-1) TAN, respectively, without any concomitant decrease in brain glutamate (Glu) concentrations. These results suggest that Gln synthesis is an ammonia detoxifying strategy employed in the brain of Atlantic salmon parr during long-term sublethal ammonia exposure. Full feed strength had an additive effect on plasma [T(amm)], while the restricted feeding regime postponed the majority of the observed physiological and molecular responses. In conclusion, Atlantic salmon parr adapts to the long-term sublethal ammonia concentrations with increased branchial transcription levels of ammonia and urea transporting proteins and ammonia detoxification in the brain.

Abstract  Fish need to balance their energy use between digestion and other activities, and different metabolic compromises can be pursued. We examined the effects of fasting (7 days) on metabolic strategies in goldfish and common carp at different swimming levels. Fasting had no significant effect on swimming performance (U(crit)) of either species. Feeding and swimming profoundly elevated total ammonia (T(amm)) excretion in both species. In fed goldfish, this resulted in increased ammonia quotients (AQ), and additionally plasma and tissue ammonia levels increased with swimming reflecting the importance of protein contribution for aerobic metabolism. In carp, AQ did not change since oxygen consumption (MO(2)) and T(amm) excretion followed the same trend. Plasma ammonia did not increase with swimming suggesting a balance between production and excretion rate except for fasted carp at U(crit). While both species relied on anaerobic metabolism during exhaustive swimming, carp also showed increased lactate levels during routine swimming. Fasting almost completely depleted glycogen stores in carp, but not in goldfish. Both species used liver protein for basal metabolism during fasting and muscle lipid during swimming. In goldfish, feeding metabolism was sacrificed to support swimming metabolism with similar MO(2) and U(crit) between fasted and fed fish, whereas in common carp feeding increased MO(2) at U(crit) to sustain feeding and swimming independently.

Abstract  How to acquire sufficient quantity of nitrogen is a pivotal issue for herbivores, particularly for lepidopterans (butterflies and moths) of which diet quality greatly differs among their life stages. Male Lepidoptera often feed from mud puddles, dung, and carrion, a behavior known as puddling, which is thought to be supplementary feeding targeted chiefly at sodium. During copulation, males transfer a spermatophore to females that contains, besides sperm, nutrients (nuptial gifts) rich in sodium, proteins, and amino acids. However, it is still poorly understood how adults, mostly nectarivores, extract nitrogen from the environment. We examined the availability of two ubiquitous inorganic nitrogenous ions in nature, viz. ammonium (or ammonia) and nitrate ions, as nutrients in a butterfly, and show that exogenous ammonia ingested by adult males of the swallowtail, Papilio polytes, can serve as a resource for protein biosynthesis. Feeding experiments with (15)N-labeled ammonium chloride revealed that nitrogen was incorporated into eupyrene spermatozoa, seminal protein, and thoracic muscle. Ammonia uptake by males significantly increased the number of eupyrene sperms in the reproductive tract tissues. The females also had the capacity to assimilate ammonia into egg protein. Consequently, it is evident that acquired ammonia is utilized for the replenishment of proteins allocable for reproduction and somatic maintenance. The active exploitation of exogenous ammonia as a nutrient by a butterfly would foster better understanding of the foraging and reproductive strategies in insects.

Abstract  The nature of ammonia and urea excretion was examined in the Pacific hagfish (Eptatretus stoutii), which, under resting conditions, excreted similar quantities of nitrogen as either ammonia or urea. In the presence of high external ammonia (HEA) concentrations, ammonia was taken up at high rates and then excreted at similarly high rates upon return to normal water. However, although elevated by HEA, plasma ammonia levels were maintained at approximately 1-4 μmolNg⁻¹, reflecting time-dependent decreases in the rates of ammonia uptake, the possible conversion of ammonia to urea, and the potential active excretion of ammonia against a gradient. Internal injections of NH₄Cl caused marked increases in the rate of ammonia excretion and a delayed increase in urea excretion that may have resulted from increasing urea levels in the plasma. Conversely, when the rate of urea excretion was reduced in the presence of 0.1 mM phloretin, ammonia excretion was significantly elevated. Rates of urea excretion were initially increased by approximately 1000-fold following internal urea injections while the presence of high external urea levels (5-100 mM final concentration) resulted in associated linear increases in plasma urea levels. Using hagfish skin mounted in Ussing chambers, the rate of diffusion of ammonia across the skin exceeded that of urea by approximately four times when equivalent gradients were imposed. Based on western blotting and immunocytochemistry, hagfish gill appears to possess Rh proteins (Rhag, Rhbg and Rhcg1) and urea transporter proteins. Despite the tolerance of hagfish to high levels of ammonia and urea, it is suggested that the presence of ammonia and urea transporter proteins may be required during the period of time hagfish spend in burrows or while feeding, when conditions of high ammonia and/or urea might be encountered.

Abstract  Ammonia absorption by the medullary thick ascending limb of Henle's loop (MTALH) is thought to be a critical step in renal ammonia handling and excretion in urine, in which it is the main acid component. Basolateral Na+/H+ exchangers have been proposed to play a role in ammonia efflux out of MTALH cells, which express 2 exchanger isoforms: Na+/H+ exchanger 1 (NHE1) and NHE4. Here, we investigated the role of NHE4 in urinary acid excretion and found that NHE4-/- mice exhibited compensated hyperchloremic metabolic acidosis, together with inappropriate urinary net acid excretion. When challenged with a 7-day HCl load, NHE4-/- mice were unable to increase their urinary ammonium and net acid excretion and displayed reduced ammonium medulla content compared with wild-type littermates. Both pharmacologic inhibition and genetic disruption of NHE4 caused a marked decrease in ammonia absorption by the MTALH. Finally, dietary induction of metabolic acidosis increased NHE4 mRNA expression in mouse MTALH cells and enhanced renal NHE4 activity in rats, as measured by in vitro microperfusion of MTALH. We therefore conclude that ammonia absorption by the MTALH requires the presence of NHE4 and that lack of NHE4 reduces the ability of MTALH epithelial cells to create the cortico-papillary gradient of NH3/NH4+ needed to excrete an acid load, contributing to systemic metabolic acidosis.

Abstract  This study aimed to examine the hypothesis that intestinal glutamate dehydrogenase (GDH) and glutamine synthetase (GS) could be involved in ammonia detoxification in the euryhaline Bostrychus sinensis exposed to ammonia in a hyperosmotic environment, whereby drinking was essential for osmoregulation. Our results indicate that there was a significant increase in ammonia content in the intestine of B. sinensis exposed to 15 mmol l(-1) NH(4)Cl in seawater (pH 7.0) for 6 days. There were also significant increases in the amination and deamination activities and protein abundance of intestinal GDH. The GDH amination/deamination ratio remained unchanged, indicating that there could be increases in the turnover of glutamate. However, the difference between the amination and deamination activities increased 2-fold, implying that there could be an increase in glutamate formation in the intestine. Since the intestinal glutamate content remained unchanged, excess glutamate formed might have been channeled into other amino acids and/or transported to other organs. Indeed, the intestinal glutamine content increased significantly by 2-fold, with a significant increase in the activity and protein abundance of intestinal GS. Since the magnitude of glutamine accumulation in the intestine was lower than those in liver and muscle, which lacked changes in GDH activities, intestinal glutamate could have been shuttled to liver and muscle to facilitate increased synthesis of glutamine therein. By contrast, when fish were exposed to a much higher concentration (30 mmol l(-1)) of NH(4)Cl in 5 per thousand water (pH. 7.0), the magnitude of increase in ammonia content in the intestine was less prominent, and there were no changes in activities and kinetic properties of intestinal GDH. Therefore, it can be concluded that the intestine of B. sinensis was involved in the defense against ammonia toxicity during exposure to ammonia in a hyperosmotic medium.

Abstract  An ion chromatographic method has been developed for the determination of alkali (Li(+), Na(+), K(+)), alkaline earths (Ca(2+), Mg(2+), Ba(2+), Sr(2+)) and ammonium ion in waters. The usual difficulties encountered during traditional cation-exchange separations (incomplete resolution for Na(+) and NH(4) (+) present in disproportionate concentration ratios) have been overcome tuning the selectivity of the separation by the introduction of 18-crown-6 ether in the mobile phase using an IonPac CS12A (150x3 mm id) column. After a detailed study of the effect of mobile phase components on separation, a gradient elution from 26 mM methanesulphonic acid (MSA) with a step change at 9 min to 60 mM MSA (0.5 mM 18-crown-6) provided the required baseline separation for the eight selected analytes. The method developed provides the advantage of the determination, in the same analytical run, also of strontium and barium, which is usually performed by spectroscopic techniques. Within-day and between-day repeatability have been assessed, observing between-day RSD included between 0.3 and 1.8% for retention times and 0.6 and 7.2% for peak areas. The method has been finally tested for the analysis of water samples of different provenience (well, tanks, water system) and results compared with those obtained by the laboratory in charge of the control of drinking water for the city of Torino (Italy).

Abstract  Effects of two fertilizers, NH(4)Cl and KCl, on the growth of the edible cyanobacterium Ge-Xian-Mi (Nostoc) and four other cyanobacterial strains were compared at pH 8.3+/-0.2 and 25 degrees C. Their growth was decreased by at least 65% at 10 mmol L(-1) NH(4)Cl but no inhibitory effect was observed at the same level of KCl. Meanwhile, the strains exhibited a great variation of sensitivity to NH(4)(+) toxicity in the order: Ge-Xian-Mi>Anabaena azotica FACHB 118>Microcystis aeruginosa FACHB 905>M. aeruginosa FACHB 315>Synechococcus FACHB 805. The 96-h EC(50) value for relative growth rate with regard to NH(4)(+) for Ge-Xian-Mi was 1.105 mmol L(-1), which was much less than the NH(4)(+) concentration in many agricultural soils (2-20 mmol L(-1)). This indicated that the use of ammonium as nitrogen fertilizer was responsible for the reduced resource of Ge-Xian-Mi in the paddy field. After 96 h exposure to 1 mmol L(-1) NH(4)Cl, the photosynthetic rate, F(v)/F(m) value, saturating irradiance for photosynthesis and PSII activity of Ge-Xian-Mi colonies were remarkably decreased. The chlorophyll synthesis of Ge-Xian-Mi was more sensitive to NH(4)(+) toxicity than phycobiliproteins. Thus, the functional absorption cross section of Ge-Xian-Mi PSII was increased markedly at NH(4)Cl levels >or=1 mmol L(-1) and the electron transport on the acceptor side of PSII was significantly accelerated by NH(4)Cl addition >or=3 mmol L(-1). Dark respiration of Ge-Xian-Mi was significantly increased by 246% and 384% at 5 and 10 mmol L(-1) NH(4)Cl, respectively. The rapid fluorescence rise kinetics indicated that the oxygen-evolving complex of PSII was the inhibitory site of NH(4)(+).