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Carbohydrates, which were not digested in the jejunum, will be fermented by micro-organisms to short chain fatty acids. These are transported by the monocarboxylate transporter 1 (MCT1) through the gut wall and serve as fuels for colonic cells. To deliver butyrate to the distal part of the intestine, inulin with a low precaecal digestibility was chosen as a coating material. Approximately 150 g of inulin-coated butyrate (containing 81 g butyrate) per day was fed to pigs (mean weight: 97 kg) over a period of 6 days after an adaptation period of 6 days with linear increasing amounts of butyrate. The following observations compared to controls were observed: (1) coating was digested microbially in the ileum; (2) MCT1-mRNA showed a higher expression in the ileum; (3) apoptosis was reduced in the ileum but mitosis was not changed; and (4) length of villi increased by approximately 25% in the ileum. Feeding inulin-coated butyrate resulted in an increased ileal surface. Delivery of butyrate to the colon requires a more resistant inulin-coating.

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Sexual differentiation in Placentalia consists of several consecutive steps during fetal, postnatal, and premature development. It is known from male rats that an elevation in testosterone synthesis is observable within 2 d of birth, which leads to a male pattern of growth hormone (GH) secretion with low base levels and high amplitudes compared with that in females. In the male pig, a transient rise in testosterone concentration occurs about 4 wk after birth, but it is unknown whether it results in a later male pattern of GH secretion. In this study, male pigs (sus scrofa) were castrated either at 1 wk of age (Group 1, n=8) or at 6 wk of age (Group 2, n=8). Blood was sampled daily via cephalic vein catheters between 17 and 29 wk of age and analyzed for testosterone, GH, insulin-like growth factor-1 (IGF-1), and urea. High-frequency blood sampling (every 20 min over 24h) for determination of GH pulsatility was performed at ages 19 and 24 wk. Total fat content and protein synthesis were determined at age 25 wk and at slaughter, respectively. Comparing Groups 1 and 2, there were no differences in daily GH concentrations or pulsatile secretion patterns, but in both groups, mean GH levels and pulsatility decreased from Week 19 to Week 24. Consequently, IGF-1, protein synthesis, urea, and body fat showed no differences when comparing both groups. It is concluded that the postnatal rise of testicular steroidogenesis in male pigs is not responsible for the later male pattern of GH secretion.

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Immunocastration of boars leads to a maintenance of growth harmone (GH) and a loss of anabolic hormones [androgens, oestrogens, insulin-like growth factor (IGF-I)] but an increase of voluntary feed intake. The aim of the experiment was to clarify whether IGF-I is increased by increasing feed supply in immunocastrated boars leading to improved anabolism. Two groups of six boars were given 2 or 3 kg of feed (13.5 MJ ME/kg) daily from 18­28 weeks of age. Because in boars feed intake is limited by gonadal hormones, a group with further increased feed supply could not be included. Until week 22 (second vaccination) gonadal steroids in blood were normal but dropped rapidly thereafter. Growth harmone levels did not change following vaccination. Pigs allocated 3 kg feed had 28% higher circulating IGF-I after the second immunization compared with pigs fed 2 kg feed daily. Higher IGF-I was associated with increased weight gain (682.4 g/day vs. 466.7 g/day; p < 0.01) and protein synthesis ((13)C-leucine infusion; 405 g/day vs. 247 g/day, p < 0.01). Protein breakdown (urea) was not different. Body fat (D(2)O) decreased in the low feed group from 15.2% (week 19) to 6.1% (week 25). In the high feed group it remained at the level found before second vaccination (13.7% vs. 15.0%). It is concluded that in the phase of reduced testicular steroids which inhibit appetite it is possible to increase feed intake which in turn increases IGF-I and protein deposition without accumulating excessive fat.

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Surgically castrated male piglets (barrows) reveal an increase in LH and a decrease in GH compared to untreated boars. Boars that were castrated by immunization against gonadotropin releasing hormone (GnRH) have decreased LH but maintain GH. The difference in GH levels between barrows and immunological castrated boars cannot be explained by testicular steroids because they are low in surgical and immunocastrated boars as well. Therefore, differences in GH concentrations might be due to an interaction between GnRH and growth hormone releasing hormone (GRH) in the hypothalamus or the pituitary. This hypothesis was tested with twelve male piglets that had been castrated within 1 week postnatally and fitted with indwelling cephalic vein catheters at 17 weeks of age. They were split into a control group and an immunized group (each n = 6). Vaccination with Improvac® was performed at 18 and 22 weeks of age. Specific radioimmunoassays were used for hormone determinations (GH, LH, FSH, testosterone and IGF-I). Additionally, metabolic responses were evaluated by measuring analytical parameters that characterize protein synthesis and breakdown, and body fat content. The second vaccination led to a rapid decrease of LH below the limit of detection whereas FSH decreased more slowly, over a period of 5 weeks, from 2.2 to 0.5 ng/ml. This level of FSH, which corresponds to boar-specific concentrations, was maintained thereafter. GH decreased with increasing age but was not influenced by vaccination and remained at a low concentration typical for barrows. Similarly, IGF-I was not altered by vaccination. Consequently, metabolic status was not changed by immunization. It is concluded that the difference in GH levels between surgical and immunocastrated boars is not explained by an interaction between GnRH and GRH.

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Many single effects of glucocorticoids are known but complex metabolic reactions in pigs in response to a glucocorticoid challenge were not reported. Seven pigs (mean weight 69 kg) with indwelling catheters were kept in metabolic crates. After a 7-day control period they were fed for 9 days with 0.4 mg dexamethasone (dex) per kg body weight daily, followed by another 9-day post-treatment period. Hormones and metabolic parameters were continuously determined in urine or blood plasma. Treatment significantly changed all parameters except non-esterified fatty acids. Cortisol decreased from 84.5 to 4.9 nmol/l, insulin-like growth factor I (IGF-I) from 17.5 to 10.8 nmol/l and aldosterone from 0.36 to 0.13 nmol/l. The N-retention decreased from 1.07 to 0.53 g/kg(0.75) and hydroxyproline from 2.97 to 1.05 mmol/day. An increase was found for urine volume (5.2 versus 13.6 l/day), urea-N (0.90 versus 1.43 g/kg(0.75)), allantoin (6.40 versus 8.75 mmol/day), glucose (3.9 versus 4.34 mmol/l) and insulin (6.21 versus 11.16 mU/l). In the post-treatment period IGF-I revealed a compensatory pattern (control period versus post-treatment period: 17.5 versus 22.9 nmol/l) whereas the other parameters were not significantly elevated. Data suggest that dex increased N-excretion both by inhibiting mitosis and resynthesis of proteins partly via a reduced collagen synthesis. Increased allantoin concentrations additionally pointed to increased apoptosis.

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Evidence exists that butyrate inhibits apoptosis of colon crypt cells in vivo so that less tryptophan from cell debris is available for skatole formation by microbes in the pig colon. In this study, potato starch containing a high proportion of resistant starch was fed to test the hypothesis that increased butyrate formation will occur in the colon and contribute to reduced epithelial cell apoptosis, thus leading to reduced skatole formation and absorption. Two groups of six barrows were provided with catheters in the jugular vein and fed either a ration with pregelatinized starch (high ileal digestibility; controls) or potato starch (low ileal digestibility; PS) as the main carbohydrate. All pigs were fed 31 MJ of metabolizable energy and 381 g of crude protein per day. The controls were fed for 19 d. The PS group received the same control ration for 10 d, and then changed to the PS ration. The total feeding period of PS consisted of a 5 d adaptation period followed by another 19 d. In the continously sampled feces, pH, short chain fatty acids, and skatole were determined. Skatole was additionally measured in blood plasma that was sampled daily. After killing barrows at the end of the feeding period, fat tissue for skatole measurement and colon tissue for histological quantification of mitosis and apoptosis were obtained. Feeding potato starch led to a rapid 2.2 fold increase of fecal butyrate when compared both with the control period of the PS group and the control group (P < 0.001). PS feeding resulted in a decrease in pH from 7.3 to 5.3 (P < 0.001) and apoptosis from 2.06 cells/crypt to 0.90 cells (P < 0.01), whereas there was no change in mitosis. Consequently, skatole decreased both in feces (controls vs PS group: 120.0 vs 1.9 microg/g; P < 0.001) and in blood plasma (1.6 vs 0.2 ng/mL; P < 0.001). The mean concentration of skatole in fat tissue was 167 ng/g tissue in controls, and below the detection limit (0.8 ng/g) in the PS group (P < 0.001). It is concluded that butyrate-dependent inhibition of apoptosis in the colon due to potato starch feeding efficiently inhibits skatole production in barrows. Because of the depressed skatole levels, improved sensory quality of pork is possible.

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11beta-Hydroxysteroid dehydrogenase 2 (11beta-HSD 2) converts active cortisol to inactive cortisone and thus modifies the availability of glucocorticoids for the target tissue. An additional function is the protection of the aldosterone receptor in mineralocorticoid-sensitive tissues such as the kidney and the gut. The occurrence of 11beta-HSD 2 activity was investigated in several species. Data for the pig, however, so far are missing. The activity was determined by a radio-enzyme-assay based on the conversion of tritiated cortisol to cortisone under standardized incubation conditions in supernatants of homogenates prepared from tissues of four castrates. Tissues comprised several locations along the intestinal tract and in addition kidney, lung, muscle, heart, spleen and pancreas. Highest values of the enzyme activity were found in kidney and very low activities in lung tissue but no activity in muscle, spleen, heart and pancreas. In the gut, there was a continuous increase in enzyme activity from the duodenum (0.60 pmol x min(- 1) x mg protein(- 1)) towards the colon with maximum values in the colon transversum (23.32 pmol x min(- 1) x mg protein(- 1)). In the colon the activity was 10-fold higher than in jejunum and 3-fold higher compared to ileum. The activities did not differ significantly between the colon transversum and colon descendens.

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A rapid, reproducible, and sensitive ion-exchange chromatography (IEC) method combined with graphite-furnace atomic absorption spectrometry (GF-AAS) was developed to separate and quantify basal amounts of both metallothionein (MT) isoforms in dab (Limanda limanda) liver samples. Dab liver homogenates were saturated with Cd, and obtained cytosols were purified by a two-step acetone precipitation prior to chromatographic analysis. Metallothionein isoforms were separated by IEC and were subsequently quantified indirectly by GF-AAS via their Cd contents. The amount of Cd needed for saturation was optimized. The efficiency of Cd saturation and acetone precipitation was proven by gel permeation chromatography (GPC) and metal distribution analysis. Based on the method of standard addition, a recovery for MT was 98% after acetone precipitation and 68% after IEC. The repeated determination of MT isoforms in a dab liver homogenate resulted in coefficients of variation of approximately 12% for both isoforms. Based on the detection limit for GF-AAS, the calculated detection limit for MT isoforms is 2 ng/mg protein. Therefore, this method is suitable for monitoring purposes. The implications of isoform-specific measurement for biomarker monitoring are discussed.

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The present study was undertaken to investigate the influence of natural and anthropogenic stressors on the induction of apoptosis, metallothionein (MT) isoforms, heat shock proteins and DNA strand breaks in the marine flatfish dab (Limbanda limanda) Seasonal changes and possible physiological influences were evaluated over a 1-year period at a fixed location northwest of Helgoland in the German Bight. These results were compared with data from sampling sites in the North Sea and the Baltic Sea. Annual cycles could be observed for all parameters except for Cd. The data revealed that changes in biomarker are not only linked to physiological processes related to reproduction but also to factors like water temperature changes, lipid content and zinc content. Cd and organochlorines had no influence on biomarkers whereas an influence of Cd on MT levels revealed in the regional observations was possibly masked by the major changes in Zn content during the annual cycle. Due to different abiotic factors we supposed that the annual cycles at each sampling site in the North and the Baltic Sea might be shifted temporally and therefore measurements at different locations during a small time window of a few weeks may lead to misinterpretation in biomarker research.

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Apoptosis, also known as programmed cell death, is a physiological and irreversible process in tissue homeostasis that leads to DNA fragmentation of multiples of 180-200 bp. Because apoptosis can be initiated not only by physiological stimuli but also by various chemical substances, the present paper investigates the suitability of apoptosis as a biomarker for biological effect monitoring in the marine environment. Aquarium experiments with dab (Limanda limanda) were carried out to examine the effects of exposure to cadmium, PCB 118, and PCB 77 (each 1 mg/kg fish wt) on apoptosis in dab liver. Determination of apoptosis was carried out by DNA gel electrophoresis and quantification of DNA fragments smaller than 1500 bp. In addition, accumulated amounts of cadmium, PCB 118, and PCB 77 in dab liver were analyzed. Quantification of the three xenobiotics resulted in an accumulation of about factor 10(2)-10(4). Exposure to PCB 118 and cadmium resulted in an increase in apoptotic DNA fragmentation. Exposure to PCB 77 led mainly to cell death by necrosis.

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The marine sponge Suberites domuncula was used as a bioindicator to study the effects of cadmium on the occurrence of DNA strand breakage and on the induction of the expression of the stress biomarkers, heat shock protein 70 (HSP70) and glucose-regulated protein 78 (GRP78) homolog. The cDNA encoding GRP78 homolog from S. domuncula was isolated and characterized. The GRP78 cDNA has a length of 2.1 kb and displays characteristic features of the HSP70 family; it encodes an aa sequence of Mr 72,000. Exposure of S. domuncula to 1 mg/L of cadmium chloride for 24 h caused a strong (16. 6-fold) increase in cadmium content to 7.7 microg/g wet weight of sponge tissue; after an incubation period of 6 days, the accumulation was 20.4-fold. The increase in cadmium content was paralleled by a transient decrease in zinc content at days 1 and 3. Exposure of S. domuncula to cadmium chloride also resulted in a marked increase in the number of DNA single strand breaks, as assessed by a recently developed fast and sensitive microplate assay. The maximum increase in DNA damage was observed after an incubation of 12 h in the presence of 1 mg/L of cadmium chloride; after longer incubation, the number of damaged sites decreased, most likely due to DNA repair. Quantitative analysis of the expression of HSP70 (Mr 73 kDa) revealed that onset of maximal levels of HSP70 depends on the concentration of cadmium chloride in the ambient seawater. Maximal induction (8.9-fold increase compared to control) of HSP70 following exposure to 1 mg/L of cadmium chloride was found after 12 h, while longer incubation periods (3-6 days) were needed to reach maximum levels of HSP70 in the presence of lower concentrations of cadmium chloride (0.1 mg/L and 0.01 mg/L). Northern blot analysis revealed that the level of the 2.0 kb sponge GRP78 homolog mRNA transiently increased under cadmium stress; the maximum increase in the presence of 0.1 mg/L of cadmium chloride was observed at day 3. Our results suggest that sponges are useful indicator organisms to assess the genotoxic risks of cadmium pollution in marine environments.

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