Twenty weaned rabbits were fed ad libitum two granulated feeds containing lucerne meal, barley, oats, wheat bran, oilseed meals and sugarbeet pulp in different proportions. Phytate P in these feeds represented 28·6 and 29·3 % of the total P. Digestibility trials were carried out in rabbits 7 and 10 weeks old. Digestibility of phytate P was 82·1 %, on average. Apparent digestibility of total P was 48·1 and 35·5 % in rabbits aged 7 and 10 weeks, respectively. Concentration of P in the faecal DM of these rabbits averaged 11·9 and 14·7 m/. Most of the faecal P was phosphates P (68·1 %). Proportion of phytate P in total faecal P was 9·0 %. Age effect on total P digestibility and faecal P concentration was significant (P<0·05). In five in vitro experiments twenty-four rabbits were killed at the age of 11 weeks, digesta samples diluted with physiological saline containing phytic acid and incubated anaerobically. Calculations of phytase activity in segments of the digestive tract were based on the estimation of phytic acid hydrolysed during the first 2 h of incubation. The caecum contained 58·6 % of the phytase activity of the digestive tract. Corresponding relative values for the phytase activity in the stomach, small intestine and colon were 22·3, 7·7 and 11·4 %, respectively. In incubations of the caecal contents, phytic acid was hydrolysed more rapidly at pH 5–6 than in the neutral pH region. The hydrolysis was inhibited by Ca cations, and to a small extent also by phosphate anions. Commercial fungal phytase (Natuphos®) was highly active in incubations of the contents of the stomach at pH 1·9. It can be concluded that phytic acid is hydrolysed quite efficiently in the digestive tract of rabbits. This hydrolysis occurred mainly in the caecum. Absorption of soluble inorganic phosphates in the gut is incomplete.

The polymerase chain reaction (PCR) technique was used to investigate the fate of a transgene in the rumen of sheep fed silage and maize grains from an insect-resistant maize line. A 1914-bp DNA fragment containing the entire coding region of the synthetic cryIA(b) gene was still amplifiable from rumen fluid sampled 5 h after feeding maize grains. The same target sequence, however, could not be amplified from rumen fluid sampled from sheep fed silage prepared from the genetically modified maize line. PCR amplification of a shorter (211-bp), yet still highly specific, target sequence was possible with rumen fluid sampled up to 3 and 24 h after feeding silage and maize grains, respectively. These findings indicate that intact transgenes from silage are unlikely to survive significantly in the rumen since a DNA sequence 211-bp long is very unlikely to transmit genetic information. By contrast, DNA in maize grains persists for a significant time and may, therefore, provide a source of transforming DNA in the rumen. In addition, we have examined the biological activity of plasmid DNA that had previously been exposed to the ovine oral cavity. Plasmid extracted from saliva sampled after incubation for 8 min was still capable of transforming competent Escherichia coli to kanamycin resistance, implying that DNA released from the diet within the mouth may retain sufficient biological activity for the transformation of competent oral bacteria.

Vitamin D is metabolised by a hepatic 25-hydroxylase into 25-hydroxyvitamin D (25(OH)D) and by a renal 1α-hydroxylase into the vitamin D hormone calcitriol. Calcitriol receptors are present in more than thirty different tissues. Apart from the kidney, several tissues also possess the enzyme 1α-hydroxylase, which is able to use circulating 25(OH)D as a substrate. Serum levels of 25(OH)D are the best indicator to assess vitamin D deficiency, insufficiency, hypovitaminosis, adequacy, and toxicity. European children and young adults often have circulating 25(OH)D levels in the insufficiency range during wintertime. Elderly subjects have mean 25(OH)D levels in the insufficiency range throughout the year. In institutionalized subjects 25(OH)D levels are often in the deficiency range. There is now general agreement that a low vitamin D status is involved in the pathogenesis of osteoporosis. Moreover, vitamin D insufficiency can lead to a disturbed muscle function. Epidemiological data also indicate a low vitamin D status in tuberculosis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel diseases, hypertension, and specific types of cancer. Some intervention trials have demonstrated that supplementation with vitamin D or its metabolites is able: (i) to reduce blood pressure in hypertensive patients; (ii) to improve blood glucose levels in diabetics; (iii) to improve symptoms of rheumatoid arthritis and multiple sclerosis. The oral dose necessary to achieve adequate serum 25(OH)D levels is probably much higher than the current recommendations of 5–15 μg/d.

Responses in variables of amino acid (AA) metabolism across peripheral tissues to feed intake were studied in six sheep (mean live weight 32 kg) prepared with arterio–venous catheters across the hindquarters. Four intakes (0·5, 1·0, 1·5 and 2·5 × maintenance energy) were offered over 2-week periods to each sheep in a Latin square design with two animals replicated. Animals were infused intravenously with a mixture of U-13C-labelled AA for 10 h and integrated blood samples withdrawn from the aorta and vena cava hourly between 5 and 9 h of infusion. Biopsy samples were also taken from skin and m. vastus lateralis. Data from both essential (histidine, isoleucine, leucine, lysine, phenylalanine, threonine) and nonessential (glycine, proline, serine, tyrosine) AA were modelled to give rates of inward and outward transport, protein synthesis and degradation, plus the fraction of total vascular inflow that exchanged with the hindquarter tissues. Rates of inward transport varied more than 10-fold between AA. For all essential AA (plus serine), inward transport increased with food intake (P<0·04). There were corresponding increases in AA efflux (P<0·05) from the tissues for threonine and the branched-chain AA. Protein synthesis rates estimated from the kinetics of these AA also increased with intake (P<0·02). Rates of inward transport greatly exceeded the amount of AA necessary to support protein retention, but were more similar to rates of protein synthesis. Nutritional or other strategies to enhance AA transport into peripheral tissues are unlikely to increase anabolic responses.

The number of known glucose transporters has expanded considerably over the past 2 years. At least three, and up to six, Na+-dependent glucose transporters (SGLT1–SGLT6; gene name SLC5A) have been identified. Similarly, thirteen members of the family of facilitative sugar transporters (GLUT1–GLUT12 and HMIT; gene name SLC2A) are now recognised. These various transporters exhibit different substrate specificities, kinetic properties and tissue expression profiles. The number of distinct gene products, together with the presence of several different transporters in certain tissues and cells (for example, GLUT1, GLUT4, GLUT5, GLUT8, GLUT12 and HMIT in white adipose tissue), indicates that glucose delivery into cells is a process of considerable complexity.

Ingestion of carbohydrates from the small intestine is the major route of energy supply in animals. In mammals these functions develop both pre- and postnatally and are coordinated for the sucking period. In birds, the physiological requirements are different and hatchlings ingest diets rich in complex carbohydrates soon after hatching. The present study examined the ontogeny of intestinal carbohydrate uptake in the chicken. The expression of mRNA for a brush border enzyme, sucrase–isomaltase (SI), which is critical in disaccharide digestion, was determined, together with that of the Na–glucose transporter (SGLT)-1, which is the major apical glucose transporter, In addition, the homeobox gene cdx, which is involved in inducing SI expression in mammals was examined. It was found that the expression of cdxA mRNA and cdxA protein increased from day 15 of incubation until hatch, after which further changes were small. CdxA protein was shown to bind to the promoter region of SI in the chick indicating that cdxA is similar to the mammalian cdx2. The mRNA of SI was observed at 15 d incubation, increased from 17 d of incubation to a peak on day 19, decreased at hatch and had a further peak of expression 2 d post-hatch. In contrast, the mRNA of SGLT-1 was not detected until 19 d of incubation when a major peak of expression was observed followed by a decrease to low levels at hatch and small increases post-hatch. It appears that both SI and SGLT-1 mRNA are expressed before hatch in the chick, but the ontogeny of expression is controlled by different mechanisms.

High plasma total homocysteine (tHcy) concentration is reported to be a risk factor for vascular diseases. We investigated the extent to which serum folate and plasma tHcy respond to a high intake of natural folate from food. Thirty-seven healthy females volunteered to participate in a crossover dietary intervention. The study included a baseline period and two 5-week diet periods (low- and high-folate diets) with a 3-week washout in between. The low-folate diet contained one serving of both vegetables and frui/, while during the high-folate diet the subjects ate at least seven servings of vegetables, berries, and citrus frui/. Serum and erythrocyte (RBC) folate, serum vitamin B12, and plasma tHcy concentrations were measured at the baseline and at the end of each diet period. The mean concentrations of serum and RBC folate were 11·0 (sd 3·0) nmo/ and 412 (sd 120) nmo/ at the end of the low-folate diet and 78 (95 % CI 62, 94) % and 14 (95 % CI 8, 20) % higher in response to the high-folate diet (P<0·001). The serum concentration of vitamin B12 remained unchanged during the intervention. The mean plasma tHcy concentration was 8·0 μmo/ at the end of the low-folate diet and decreased by 13 (95 % CI 9, 18) % in response to the high-folate diet (P<0·001). In conclusion, a diet high in fresh berries, citrus fruit, and vegetables effectively increases serum and RBC folate and decreases plasma homocysteine.

In rats, in contrast with human subjects who develop megaloblastic anaemia due to vitamin B12 deficiency, haematological abnormalities with anaemia were not observed under normoxic conditions even though plasma vitamin B12 concentration was reduced to <15 % of a normal concentration by depleting dietary vitamin B12. To elucidate whether erythropoiesis was affected by vitamin B12 deficiency in rats, these vitamin B12-deficient rats were exposed to hypoxia (10·5 % O2) to stimulate erythropoiesis. In the vitamin B12-sufficient control rats, erythrocyte count was significantly (P<0·05) increased 1 week after starting the hypoxic exposure. However, the hypoxia-induced erythropoiesis was affected by vitamin B12 deficiency, and no significant increase in the erythrocyte count was observed even after 6-week exposure to hypoxia in the vitamin B12-deficient rats. In the vitamin B12-deficient rats in hypoxia, erythrocytes became abnormally enlarged, and haemoglobin concentration in peripheral blood was increased in proportion to the increase of mean corpuscular volume. However, the level of the increase in the haemoglobin concentration was significantly (P<0·05) lower in the vitamin B12-deficient rats compared with that in the -sufficient controls. In addition, in the vitamin B12-deficient rats, in contrast to the -sufficient rats, serum erythropoietin concentration was not normalized even after 6-week exposure to hypoxia. These results indicate that a megaloblastic anaemia-like symptom is induced when the vitamin B12-deficient rats are exposed to hypoxia.

The objective of the present study was to examine the effects of fish oil (FO)- and olive oil (OO)-rich diets on Fe metabolism and oxidative stress. Rats were fed for 16 weeks with diets containing 50 g lipid/g; either OO, maize oil (MO) or FO. OO or MO diets contained a standard amount (100 m/g) of all-rac-α-tocopheryl acetate. FO diets were supplemented with 0, 100 or 200 mg all-rac-α-tocopheryl acetat/g (FO-0, FO-1 or FO-2 diets, respectively). At the end of the feeding period, we measured non-haem Fe stores in liver and spleen, and erythrocyte and reticulocyte count. We also determined antioxidants and products derived from lipid peroxidation in plasma and erythrocytes. Our results showed reduced non-haem Fe stores in rats fed any of the FO diets. Reticulocyte percentage was higher in the rats fed FO-0 and FO-1. Plasma α-tocopherol was very low in rats fed the FO-0 diet. Rats fed the FO-1 and FO-2 diets showed higher α-tocopherol in plasma than the FO-0 group but lower than the MO or OO groups. We did not observe such differences in the α-tocopherol content in erythrocyte membranes. Superoxide dismutase and glutathione peroxidase activities were lower in the erythrocytes of rats fed the FO-0 diet. The products derived from lipid peroxidation were also higher in the FO groups. The administration of FO-rich diets increased lipid peroxidation and affected Fe metabolism. On the other hand, the OO-rich diet did not increase oxidative stress and did not alter Fe metabolism. Based on these results, we conclude that FO supplementation should be advised carefully.

Batch cultures of mixed rumen micro-organisms were used to study the effects of different concentrations of malate (Rumalato®; Norel & Nature S.A., Barcelona, Spain; composed of disodium malate–calcium malate (0·16:0·84, w/w)) on the fermentation of four cereal grains (maize, barley, wheat and sorghum). Rumen contents were collected from four Merino sheep fed lucerne hay ad libitum and supplemented with 300 g concentrate/d. Rumalato® was added to the incubation bottles to achieve final concentrations of 0, 4, 7 and 10 mM-MALATE. Gas production was measured at regular intervals up to 120 h. Malate increased (P<0·01) the average fermentation rate of all substrates, and the lag time decreased (P<0·05) linearly with increasing concentrations of malate for all substrates, with the exception of sorghum. in 17 h incubations, the final pH and total volatile fatty acid production increased (P<0·001) linearly for all substrates as malate concentration increased from 0 TO 10 mM. Propionate and butyrate production increased (P<0·05), while the value of the acetate: propionate ratio and L-lactate concentrations decreased (P<0·05) linearly with increasing doses of malate. Malate treatment increased (P<0·05) the CO2 production and decreased the production of CH4, although this effect was not significant (P>0·05) for maize. Malate at 4 and 7 mm increased (P<0·05) optical density of the cultures measured at 600 nm for maize, with no differences for the other substrates. The results indicate that malate may be used as a feed additive for ruminant animals fed high proportions of cereal grains, because it increased pH and propionate production and decreased CH4 production and L-lactate concentrations; however, in general, no beneficial effects of 10 compared with 7 mM-malate were observed.

The purpose of the present study was to determine the effect of abomasal casein infusion on glucose uptake and abundance of the Na+/glucose co-transporter (SGLT1) 1 in the ovine small intestine. Lambs (body weight 35 (SEM 1·0) kg) were surgically fitted with abomasal infusion catheters and were fed diets containing equal portions of wheat hay and cracked maize. Lambs were infused with either 500 g water/d or with 500 g water containing 35 g casein/d. The infusion period lasted 10 d, after which lambs were killed, exsanguinated and eviscerated. Brush border membrane vesicles (BBMV) were prepared using mucosa from different small intestinal regions. Intake and total tract digestibility of nutrients were similar between treatments and averaged 1134, 1142 and 486 g/d and 67, 70, and 94 % for DM, organic matter and non-structural carbohydrates respectively. Crude protein (N×6·25) digestibility was 15 % greater in the casein-infused than control lambs. Glucose uptake to BBMV ranged from 101 to 337 pmol/mg protein per s along the small intestine and was greatest in the mid-section of the small intestine. In the mid-jejunum, glucose uptake was greater (P<0·07) in lambs infused with casein and averaged 120 pmol/mg protein per s compared with 68 pmol/mg protein per s in the control group. SGLT1 affinity was similar between treatments and averaged 104 μM in the different segments of the small intestine of lambs. However, lambs infused with casein exhibited similar values along the small intestine and affinity averaged 106 μm, while in the control group a greater affinity (85 μm) was measured in the mid-jejunum. SGLT1 protein abundance was correlated with glucose uptake in the BBMV in the casein-treated lambs, but not in the control group. These results suggest that glucose uptake along the small intestine of lambs is influenced by casein or its derivatives in the small intestine via SGLT1 affinity and activity at the brush border membrane, and that SGLT1 activity may be regulated by post-translational events affected by amino acids and peptides.

Recently, dietary phyto-oestrogens (PO) have been suggested as possible alternatives to oestrogen therapy (hormone replacement therapy) as a means of preventing bone loss associated with ovarian hormone deficiency. While PO, which exhibit oestrogen-like activity, act directly on bone cells, their protective effect on bone may be partly due to their ability to enhance Ca absorption. Therefore, the aim of the present study was to investigate the effect of 17β-oestradiol and two commonly consumed soyabean PO (genistein and daidzein) on Ca absorption in the human Caco-2 intestinal-like cell model. Caco-2 cells were seeded onto permeable filter supports and allowed to differentiate into monolayers. On day 21, the Caco-2 monolayers (n 8–18 per treatment), grown in oestrogen-replete or -deplete media, were then exposed to 10 nM-17β-oestradiol, 1 nM-1,25-dihydroxycholecalciferol, or 50 μM-genistein or -daidzein for 24 h. After exposure, transepithelial and transcellular transport of 45Ca and fluorescein transport (a marker of paracellular diffusion) were measured. As expected, 1,25-dihydroxycholecalciferol stimulated Ca absorption in Caco-2 cells, by up-regulating transcellular transport, whereas 17β-oestradiol had no effect on Ca absorption. Unexpectedly, both PO decreased Ca absorption (by about 17–19 % compared with control, P<0·05), by reducing transcellular Ca transport in Caco-2 cells grown in oestrogen-replete media. This inhibitory effect disappeared when monolayers were grown in oestrogen-deplete media. In conclusion, PO at high luminal concentrations either had no effect or reduced Ca absorption in Caco-2 cells, dependent on oestrogen status. The effect of lower concentrations of these compounds needs to be investigated.

Mild-to-moderate vitamin C depletion in weanling guinea-pigs affects pyridinoline:deoxypyridinoline (collagen cross-link) ratios in femur shaft and urine, attributed to impairment of hydroxylation of collagen lysine. We investigated: (1) whether the picture at two time points is compatible with progressive accumulation of abnormal collagen; (2) whether any changes are seen in skin, where little deoxypyridinoline occurs; (3) whether total food restriction has similar effects. Male weanling Dunkin–Hartley guinea-pigs were fed diets containing either 0·5 (vitamin C-restricted) or 160·0–320·0 (vitamin C-adequate) mg vitamin /. Two groups receiving the vitamin C-adequate diet received it ad libitum. Two other groups received the vitamin C-adequate diet in a restricted amount, limited to that which permitted nearly the same growth rate as in the vitamin C-restricted groups. Animals were fed for 4 or 8 weeks; urine was collected, and vitamin C and collagen indices were measured. In the femur shaft, the hydroxyproline content per unit weight was unaffected by vitamin C restriction or by total food restriction. Deoxypyridinoline was increased and the pyridinoline:deoxypyridinoline ratio was decreased in vitamin C-restricted groups, but not in food-restricted groups. Changes in the value of the ratio were greater after 8 than after 4 weeks. Urine indices mirrored bone indices. In skin, the main effect of vitamin C restriction was to reduce hydroxyproline. Here, the cross-link ratios changed less markedly than in bone, and there was less deoxypyridinoline. We conclude that the picture at two time points is compatible with a progressive accumulation of pyridinoline-enriched collagen in vitamin C-deprived animals, that the picture in skin differs from that of bone and urine, and that cross-link changes are not produced by total food restriction.

Tropical livestock is often maintained on roughage-based diets deficient in N, and therefore requires supplementation with protein-rich substrates to achieve reasonable production levels. The optimum inclusion rate of a potential supplement is usually determined by in vivo feeding trials or by in vitro incubation of the diet components to estimate the feed value of the complete diet. The present work simulates a supplementation experiment in vitro, by incubating a pure roughage (barley straw), a pure supplement (Sesbania pachycarpa leaves) and mixtures of the two, with increasing inclusion levels of the supplement, in a short-term batch incubation system. Fermentation kinetics were followed by the release of fermentation endproducts (gas and short-chain fatty acids). Microbial biomass was estimated using ribosomal (r) RNA as internal marker for bacteria and eukaryotes separately. Cell-wall-degrading subpopulations were quantified by hybridisation with taxon-specific oligonucleotide probes targeting Chytridiomycetes, Fibrobacter spp., Ruminococcus albus and R. flavefaciens. Carboxymethylcellulase (CMCase) was assayed as an indicator for cell-wall-degrading activity. The addition of S. pachycarpa leaves stimulated fermentation in all cases. Gas production, and especially rRNA concentration, showed clear maxima at 40 % S. pachycarpa inclusion, rates that significantly exceeded the values interpolated from the incubations of the pure substrates. Short-chain fatty acid yield changed only slightly, but in the same way. The analysis of the microbial population structure showed that the positive effects were mainly mediated through enhanced growth of Ruminococcus spp. Increasing proportions of S. pachycarpa leaves in the diet led to a drastic decline in the total eukaryotic population. This points to a defaunation, which may also have added to the positive effects. The eukaryotic subpopulation of the rumen fungi were affected to a lesser degree. Although the cell-wall-degrading organisms showed positive responses to the supplementation, the CMCase activity was not affected significantly by the supplementation. The present work shows that it is possible to predict optimum inclusion levels for a new feed supplement in vitro and thus reduce in vivo experiments. It was also demonstrated that true supplementation effects occur particularly for the microbial biomass production, which is the primary source of amino acids for the ruminant animal. The analysis of microbial population structure in context with conventional metabolic measurements adds valuable information to interpret the observed effects on production-related variables.