Specific regulation of the gene expression of some pancreatic enzymes during postnatal development and weaning in the calf.

The construction of cDNA library from calf pancreas allowed us to examine the mRNA levels of four pancreatic hydrolases (chymotrypsin, lipase, trypsin and amylase) during postnatal development in preruminant and ruminant animals. The lack of parallel variations in the levels of the enzyme specific activities suggested that protein synthesis was not coordinately regulated. In preruminant calves, the change in chymotrypsin and lipase mRNA concentrations (0-28 day period) and in trypsin mRNA concentrations (0-119 day period) was opposite to that in the corresponding specific activities. In contrast, both the activity and mRNA profiles of amylase during the latter period, on the one hand, and those of chymotrypsin and lipase during the 28-119 day period, on the other hand, were comparable. However, the extent to which the specific activity and mRNA concentration of each enzyme were increased did not necessarily coincide. The observed changes in mRNA levels probably resulted from some transcriptional control of the gene expression and/or variation in mRNA stability. Moreover, a translational regulation of the messengers could explain the existence of non-parallel mRNA and specific activity profiles. In sharp contrast with the multiple control of protein synthesis during postnatal development in preruminant calves, weaning was found to induce the same increase in enzyme activity and corresponding mRNA for each of the four pancreatic enzymes, suggesting that pretranslational modulation of gene expression was mainly, if not exclusively, concerned.

IgM, IgA, IgG1 and IgG2 specific responses in blood and gut secretion of calves fed soyabean products.

Calves fed soya proteins may develop severe gastrointestinal disorders. Whether these are predominantly associated with particular Ig subclasses and (or) dietary proteins remains unclear. Therefore, antibody responses to soyabean protein were analysed by dot- and blot-immunobinding in plasma and intestinal mucous secretions. One-month-old calves were fed for 2.5 months liquid diets based on skim milk powder (SMP) or a mixture (2:3, protein basis) of whey and soyabean products including a low antigenic hydrolysed soya protein isolate (HSPI) and a highly antigenic heated soya flour (HSF). Specific antibodies (Abs) of the main isotypes (IgM, IgA, IgG1, IgG2) were characterised by immunostaining of samples which had been previously incubated with nitrocellulose sheets coated with SMP, HSPI or HSF extracts. Plasma collected before feeding experimental diets showed very little specific Abs. By contrast, 2.5 months later, a three-fold increase (P < 0.05) in IgG1 and IgA titres against HSF antigens was observed in calves fed HSF compared with those fed the control or HSPI diet. IgG1 immunoblotting revealed many protein bands from soya in the molecular range of 22-32 and 38-42 kDa. Immunorecognition of specific proteins from SMP and HSPI remained low and similar among animal groups. Specific IgM, IgA and IgG1 titres against HSF, and to a lesser extent HSPI, were significantly higher (P < 0.05) in jejunal mucous secretion of calves fed HSF compared with other groups. Secretions from calves fed HSF bound to many soyabean proteins in the range of 17-23 and 26-38 kDa, with similar patterns for IgA and IgG1. By contrast, only weak bands were found for IgM and IgG2 in all groups of calves. Thus, calves fed antigenic HSF do present specific Abs including IgG1 and IgA isotypes, both systemically and locally. Therefore, IgG1 and (or) IgA rather than IgM and IgG2 Abs may be preferred for assessing the immunogenicity of soyabean products in calves. Interestingly, soyabean immunogenicity was drastically reduced by adequate proteolysis.

B and T lymphocytes are enhanced in the gut of piglets fed heat-treated soyabean proteins.

Gut immune responses have been suspected in food hypersensitivity reactions such as those to soyabean proteins in early-weaned piglets. The present study examines the lymphoid cell subset distribution in piglets fed heat-treated (HTSP) or ethanol-treated soyabean proteins (ETSP). Duodenal cryosections of 4-week-old HTSP piglets (n = 10) and ETSP piglets (n = 8) were analysed for IgA, IgM, IgG1 and IgG2 positive cells, CD2, CD4, CD8, WC1 T cell positive antigens using immunohistochemical peroxidase reactions. Densities of IgM+ and IgA+ cells were three times and, IgG1+ and IgG2+ six times higher in the lamina propria of HTSP piglets compared with ETSP (P < 0.05). Increased CD2+ T cells were accounted for by a rise in both CD4+ and CD8+ T cell subsets in the lamina propria (P < 0.01) as well as in the epithelium of the duodenal mucosa of piglets fed HTSP. The density of the WC1+ T cell subset in the epithelium was significantly higher in HTSP than in ETSP piglets (P < 0.01). Immune reactions in the duodenal mucosa, involving both B and T lymphocytes may be related to atrophy of the duodenal villi in HTSP piglets.

Feeding heated soyabean flour increases the density of B and T lymphocytes in the small intestine of calves.

The effect of dietary antigens on the gut morphology and density of immune cells was studied in preruminant calves fed milk substitutes containing skim milk powder (SMP), non-antigenic hydrolysed soya protein isolate (HSPI) or antigenic heated soyabean flour (HSF) as their main protein source for 3 months. The height and perimeter of proximal jejunum villi were highest in the calves fed SMP and lowest in those fed HSF (P < 0.05). In contrast, the crypt depth and perimeter were apparently not influenced by the dietary treatments studied. This morphological alteration was associated with a dramatic infiltration of the lamina propria by B and T lymphocytes in the calves fed HSF (P < 0.01). Increased B cell density was essentially accounted for by IgA-, IgG1- and IgG2-bearing cells. The density of CD2-positive T lymphocytes increased (P < 0.01) in the jejunal lamina propria of HSF calves, involving helper (CD4+) and suppressor-cytotoxic (CD8+) T cell subsets. The density of gamma-delta (WC1+) T cells also increased (P < 0.01). The major change concomitantly observed in the villus epithelium was an increased density of CD8+ cells (P < 0.05) and WC1 + cells (P < 0.01).

Immunochemical studies on gastric and intestinal digestion of soybean glycinin and beta-conglycinin in vivo.

Two experiments were conducted to study gastric and small intestinal digestion of soybean glycinin and beta-conglycinin in preruminant calves fed milk replacers containing a mixture of skim milk powder and antigenic heated soybean flour. In experiment 1, duodenal passage of immunoreactive beta-conglycinin lasted for a much longer time after the morning meal than that of glycinin. Western blotting revealed the early abomasal outflow of glycinin subunits that associated nearly intact basic polypeptides to partially degraded acidic polypeptides. Intact beta-conglycinin was evidenced at most sampling times. In experiment 2, intact basic glycinin (M(r) = 21000) associated with partially digested acidic glycinin (7000 < M(r) < 25000) was demonstrated in ileal digesta up to 8-10 h after the meal. beta-Conglycinin immunoreactivity could not be evidenced by Western blotting in ileal digesta.

Systemic and local gut-specific antibody responses in preruminant calves sensitive to soya.

The systemic and local (gut) patterns of antibodies against various proteins from soyabean were analysed in preruminant calves fed milk substitutes based on skim milk powder (SMP) or heated soyabean flour (HSF) as the main protein sources. The titres of IgM, IgA, IgG1 and IgG2 antibodies were determined against feed extracts and purified soyabean proteins by dot-blotting in plasma after three months and jejunal mucous secretions after six months of feeding the experimental diets. The calves fed HSF had higher levels of circulating IgA, IgG1 and IgG2 antibodies against raw or heated soya extracts and purified proteins including alpha-conglycinin, beta-conglycinin, Bowman-Birk protease inhibitors and lectins than the calves fed SMP. In contrast, the differences between the IgM titres of the groups were most often not significant. The patterns of specific antibodies present in the jejunum were broadly similar to those observed in the blood, although the differences between the groups of calves more often reached significance for IgG2 and IgM than for IgA and IgG1, when the purified soyabean proteins were considered.

Identification of soyabean allergens and immune mechanisms of dietary sensitivities in preruminant calves.

The allergenicity of soya proteins was assessed by direct skin testing and by in vitro lymphoproliferation tests in calves fed milk substitutes containing skim milk powder (SMP) or an antigenic heated soya flour (HSF). During the last three weeks of treatment, the calves were injected intradermally with raw soya flour (RSF), HSF, hydrolysed soya protein isolate (HSPI), SMP or purified soya proteins, after being premedicated with anti-histamine or not. Peripheral blood leucocytes (PBL) were grown over five days with various mitogens or dietary antigens, and the incorporation of tritiated thymidine was measured. Strong skin oedema reactions to RSF, HSF and all the purified proteins were observed in the calves fed HSF at various times up to 24 hours after injection. The skin oedema was largely prevented by premedication with anti-histamine. A strong delayed skin thickening was observed in the calves fed HSF for up to five days with beta-conglycinin. PBL from the calves fed HSF proliferated in vitro with HSF, HSPI and beta-conglycinin, but not with glycinin. Thus, most proteins from soyabean were implicated in the immediate and semi-delayed immune reactions, whereas beta-conglycinin was strongly involved in a delayed type hypersensitivity in calves.

Intestinal digestion of dietary and endogenous proteins along the small intestine of calves fed soybean or potato.

This study was conducted to investigate the quantitative and qualitative changes in dietary and endogenous protein components along the small intestine of calves. They were fed milk replacers in which protein was provided either by skim milk powder alone, as control diet, or by skim milk powder and a soy concentrate, a partially hydrolyzed soy protein isolate, or a potato protein concentrate (1:1 on a CP basis). The calves were continuously infused with the liquid milk substitute diets into the abomasum. Duodenal, jejunal, and ileal digesta were collected through T-piece cannulas. Digestibility (corrected for total endogenous protein) of dietary protein and the concentration of dietary, host endogenous, and bacterial proteins were estimated from the AA composition of digesta using multiple linear regression. The apparent digestibility of feed components increased linearly between the duodenum and the ileum. It was lower with the diets containing plant protein than with the control, especially at the ileum (85% for CP with soy concentrate, 73% with soy isolate, and 81% with potato concentrate vs 91% with skim milk powder). The real digestibility of dietary protein at the ileum was estimated to be 96, 95, 94, and 99%, respectively, for each of the dietary components. The partition of protein components in digesta between dietary, nonspecific endogenous, specific host endogenous, and(or) specific bacterial proteins varied along the small intestine and between diets at a given site. Intestinal apparent reabsorption of host endogenous protein was estimated to be equivalent to at least 86% of the jejunum flow. To conclude, the lower apparent digestibility of the plant protein sources studied resulted more from an enhanced loss of host and bacterial endogenous proteins than from decreased hydrolysis of dietary protein and absorption of their AA.

Local and systemic immune responses to soybean protein ingestion in early-weaned pigs.

The development of local and systemic immune responses to soybean proteins was investigated in early-weaned pigs. Pigs were given either antigenic (ASP, n = 10 pigs) or non-antigenic (NASP, n = 8 pigs) soybean products (6 g of protein/d) from d 5 to 9 of age by stomach tube. After weaning at d 21, pigs were fed diets containing the corresponding soybean products and slaughtered between d 28 to 30. Diarrhea was 2.4-fold more frequent, the size of duodenal villi was reduced by 24 to 36%, and the eosinophil density in the duodenal mucosa was 13 times greater (P < .02) in the ASP pigs compared with the NASP pigs. A larger erythema area (P = .006) was observed in the ASP group than in the NASP group 30 min after an intradermal injection of glycinin, but no significant difference could be detected with alpha- or beta-conglycinin or whole soybean extracts. No difference in skin fold thickness was apparent between groups 24 h later. Intestinal, mesenteric lymph node, and blood lymphocytes did not proliferate when cultured with soybean proteins, regardless of dietary treatment. By ELISA, no difference between groups was observed in the circulating levels of total immunoglobulins (Ig) and IgM. Immunoblotting patterns of raw soybean with sera from 28-d-old pigs showed two bands (22 and 36 kDa) recognized by IgA and IgM, respectively, in the ASP group only. Finally, the adverse effects observed with antigenic soybean flour can be overcome by the use of alcohol-treated products.(ABSTRACT TRUNCATED AT 250 WORDS)

[Distribution of enzyme concentrations in the abomasal mucosa of the preruminant calf. Age changes]. / Répartition des concentrations enzymatiques dans la muqueuse abomasale du veau préruminant. Evolution avec l'âge.

The aim of this trial was to draw an "enzyme map" of the abomasum of the preruminant calf in order to determine the secretory capacities of various areas and to study their age changes. Three pairs of calves were slaughtered at 1.5 days and at 10 and 19 weeks. The abomasum was divided into 12 parts (fig. 1). The chymosin and pepsin extracted from each part were separated and assayed. We found large individual variations in the amount and distribution of the enzymes (fig. 2, table 1) but, in all cases, more than 78 p. 100 were present in the fundus, while the antrum usually contained very little. The cardiac part contained as much as the fundic parts of similar size. The distribution of enzymes in the fundus between the parietal and visceral surfaces or between the proximal, median and distal plicae did not seem to change with age. On the contrary, that part of the enzymes provided by the ventral surface (along the greater curvature) increased with age, while the part provided by the dorsal surface (along the lesser curvature) decreased (fig. 2; table 2). However, the supply of the distal region (parts 3 + 7) of the latter surface varied within much smaller limits, and the pepsin/chymosin ratio remained close to that observed in the whole abomasum. Therefore, the distal region would be the most suitable for making an abomasal pouch which would retain the same representativeness in relation to the whole of the abomasum. It could eventually be extended on the side of the distal plica.

Refractive index of GaSe between 0.45 microm and 330 microm.

Using the natural properties of the lamellar compounds, the ordinary refractive index of GaSe has been induced from interferential patterns of transmission and reflection spectra in the low absorption range and from reflectivity spectra in the polar modes range. An analytic development of the refractive index vs energy can reproduce the measured values of this index and provides the energy gap in the visible range, and values for omega(LO) and omega(TO) in the ir range infinity have been used as a parameter to get this fitting.

Calf intestinal mucin: isolation, partial characterization, and measurement in ileal digesta with an enzyme-linked immunosorbent assay.

The aim of this study was to develop a specific ELISA for calf intestinal mucin to quantify its contribution to ileal endogenous losses. Mucin was isolated from intestinal scrapings by cesium chloride density gradient ultracentrifugation. The isolated mucin had a high concentration of glutamic and aspartic acids, threonine, and serine (13.2, 11.2, 9.6, and 9.2 mol % of total amino acids assayed, respectively). The carbohydrates present were (mol % of total hexoses): galactose 42.1, N-acetylglucosamine 24.1, N-acetylgalactosamine 23.6, fucose 4.7, mannose 3.1, and sialic acids 2.4. Amino acids and carbohydrates represented 52.6 and 47.4% of the mucin by weight, respectively. A rabbit hyperimmune plasma was raised against purified mucin and used to set up an ELISA. The linear range of this assay was 20 to 640 ng/ml. The plasma cross-reacted with calf abomasal and colonic mucin. It showed no cross-reactivity with nonmucin components and no reactivity with intestinal mucin from other animal species except for the rat. Mucin accounted for approximately 3.5% of the dry matter output at the ileum of calves fed a substitute milk based on skim milk powder. This represented a flow of 3.4 g of mucin/kg of dry matter intake. Mucin flow increased when dietary protein was provided by cow's colostrum. Finally, the developed assay is a suitable tool to investigate the impact of dietary factors on the flow of mucin along the gut of preruminant calves.

Influence of soya antigen levels in milk replacers on the disruption of intestinal motility patterns in calves sensitive to soya.

An experiment was designed to determine the soya antigen levels in milk replacers above which gastrointestinal disorders appeared in preruminant calves previously sensitized to antigenic soya by feeding a soya-based milk replacer for 3 months. These calves were then equipped with wire electrodes on the duodenum and the mid-jejunum. The sensitization was visualized using direct skin testing, plasma anti-soya antibody determination and intestinal myoelectric activity recording. After sensitization, the calves were occasionally fed liquid test meals containing various proportions of antigenic soya and whey. The soya-fed calves displayed larger 24 h skin reactions to beta-conglycinin and higher plasma anti-soya antibody titres than the controls maintained on a skim-milk based milk replacer. Disturbances of the myoelectric activity patterns were recorded on the duodenum and mid-jejunum after feeding antigenic soya, but not non-antigenic soya or milk protein, in the soya-sensitized calves. When the level of antigens was varied, disorders in the jejunal motility patterns appeared when antigenic soya provided one-third or more of the dietary protein in the test meals, although some abnormalities were evident at lower incorporation rates. The major change was a reduction in the mean duration of the jejunal migrating motor complexes which was essentially accounted for by a decrease in the mean duration of the phase I (quiescence). This level of antigens corresponded approximately to 14 and 12 mg of immunoreactive glycinin and beta-conglycinin respectively, per gram of protein intake, i.e. 80 and 70 mg/kg0.75 per test meal.

Morphology and enzyme activities of the small intestine are modulated by dietary protein source in the preruminant calf.

A study was undertaken to assess the impact of the protein nature and soya antigenicity on the morphology and some enzyme activities of the jejunum in preruminant calves. Twenty Holstein calves fitted with a duodenal cannula were fed a liquid diet based on skimmed milk powder (SMP) for 2 weeks. They were then switched onto diets containing a mixture of SMP and either antigenic heated soybean flour (HSF; n = 12) or hypo-antigenic soya protein concentrate (SPC; n = 8) for 8 weeks, after which they were reverted back to the SMP diet for 2 weeks. The diets contained similar amounts of digestible nitrogen and energy, and were fed at a rate of 55 g DM/kg(0.75)/d. Proximal jejunal biopsies were collected just before (week 0), during (weeks 2 and 8) and after (week 10) feeding of the soya-based diets, and were used for morphology measurements and the determination of total alkaline phosphatase, lactase, amino-peptidases A and N, and dipeptidyl peptidase IV activities. Feed intake and growth were similar between the HSF and SPC groups during the experimental period. The effects of antigenicity and the antigenicity x time interaction were never significant (P > 0.05). Villus height decreased (P < 0.01) between weeks 0 and 2, and increased (P < 0.05) between weeks 8 and 10. Villus width increased between weeks 2 and 8 (P < 0.001). Crypt depth also increased between weeks 0 and 2 (P < 0.001). Specific activities of alkaline phosphatase (P < 0.01) and amino-peptidase N (P < 0.05) decreased between weeks 0 and 2. Conversely, those of alkaline phosphatase (P < 0.0001), lactase (P < 0.01) and dipeptidyl-peptidase IV (P < 0.0001) increased between weeks 8 and 10. Specific activities for lactase and amino-peptidase N decreased (P < 0.01) between weeks 2 and 8. The treatments had little effects on the amino-peptidase A activity. In conclusion, the present work demonstrated that soybean protein markedly depressed the morphology and most enzyme activities of the calf small intestine. On the contrary, the in vitro antigenicity of soybean protein had little influence on these parameters in this study.

[The effect of postruminal supplementation of energy or energy and proteins on feed intake in the ruminant calf]. / Effet d'un apport postruminal d'énergie ou d'énergie et de protéines sur la prise alimentaire chez le veau ruminant.

Forty-eight Friesian bull calves were weaned at 8 weeks of age. They were divided into four groups (T, L, LS1 and LS2) 4 weeks before weaning. All the animals were fed a concentrate diet ad libitum, containing 16 p. 100 of crude protein on a DM basis, and hay. The animals of groups L, LS1 and LS2 also received a limited amount (24 p. 100 of the ingested DM) of a liquid supplement of deproteinized whey fed alone (group L) or mixed with 25 (group LS1) or 50 p. 100 (group LS2) of soybean oil-meal. The voluntary intake of DM/kg live weight0.75 was increased before weaning by giving the three liquid supplements (table 1). That effect lasted until 10 weeks of age in groups LS1 and LS2. On the contrary, the DM intake/kg0.75 decreased from 10 to 14 weeks of age in group L. From 15 to 19 weeks, the DM intake/kg0.75 was the same in group L as in group T, but was lower in groups LS1 and LS2. The plasma glucose level was higher in groups LS1 and LS2 than in group T; it was noticeably higher in group L than in group T (table 1). The plasma urea level was always very low in group L; the urea level of groups LS1 and LS2 was lower at 10 weeks of age than that of group T, but was as high (group LS1) or higher (group LS2) at 16 weeks (table 1). It is concluded that the voluntary intake of young ruminant calves is limited by rumen volume until 3 months of age; the supply of lactose or lactose and protein through the rumen bypass allows an increase in intake. After that period, voluntary intake is rapidly regulated by metabolic mechanisms in relation to the degree of satisfaction of energy and protein requirements; glycemia does not play a role in this regulation. As in monogastric animals, the nitrogen/energy ratio has a determining effect on the control of voluntary intake.