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Articles

Feeding Colostrum, Its Composition and Feeding Duration Variably Modify Proliferation and Morphology of the Intestine and Digestive Enzyme Activities of Neonatal Calves^{1 ,2}

Urs Blättler^*,3, Harald M. Hammon^*, Claudine Morel^*, Chantal Philipona^*, Andrea Rauprich^*, Véronique Romé, Isabelle Le Huërou-Luron, Paul Guilloteau and Jürg W. Blum^*4

^* Division of Nutritional Pathology, Faculty of Veterinary Medicine, CH-3012 Berne, Switzerland and Unité Mixte de Recherches sur le Veau et le Porc, Institut National de la Recherches Agronomique, F-35042 Rennes, France

⁴To whom correspondence should be addressed at Division of Nutritional Pathology, Faculty of Veterinary Medicine, University of Berne, Bremgartenstr. 109a, CH-3012 Berne, Switzerland. E-mail: blum{at}itz.unibe.ch

	ABSTRACT

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We studied the effects of amounts of colostrum consumed on intestinal morphology and proliferation and digestive enzyme activities in neonatal calves. Group GrC_max calves were fed colostrum from the first milking undiluted on d 1–3 and diluted with 25, 50, 75 and 75 parts of a milk replacer on d 4–7. Group GrC_1–3 calves were fed colostrum from milkings 1–6 up to d 3 and then a milk replacer up to d 7. Group GrF_1–3 calves were fed a milk-based formula (containing only traces of growth factors and hormones) up to d 3 and then a milk replacer up to d 7. Calves were killed on d 8. Differences in feeding affected villus sizes and villus height/crypt depth ratios in the duodenum (GrC_max > GrC_1–3), villus areas and villus height/crypt depth ratios in the jejunum (GrC_1–3 > GrF_1–3) and crypt depths in the colon (GrF_1–3 > GrC_1–3). Furthermore, different feeding protocols affected the proliferation rates of epithelial cells in the duodenum (GrC_1–3 > GrC_max; GrC_1–3 > GrF_1–3) and the jejunum (GrF_1–3 > GrC_1–3; based on Ki-67 labeling). Lipase activities in the pancreas were influenced by colostrum feeding (GrC_max > GrC_1–3). Colostrum intake differentially affected intestinal epithelial surface and proliferation and enzyme activities. Feeding high amounts of first colostrum seemed to enhance the survival of mature mucosal epithelial cells in selected parts of the small intestine, whereas the lack of colostrum seemed to decrease epithelial growth.

KEY WORDS: • neonates • colostrum • intestine • pancreas • calves

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Bovine colostrum contains higher amounts of fats, proteins and peptides; fat-soluble vitamins; and various enzymes, hormones, growth factors, cytokines, minerals and nucleotides than mature milk, and except for lactose, the levels of these compounds rapidly decrease during the first 3 d of lactation to those typical for mature milk (Blum and Hammon 2000a and 2000b , Campana and Baumrucker 1995 ). Colostrum intake in neonatal calves is essential for passive immunity and influences metabolism, endocrine systems and the nutritional state, as reviewed by Guilloteau et al. (1997 ) and Blum and Hammon (2000a and 2000b ). Furthermore, ingested colostrum stimulates the development and function of the gastrointestinal tract (GIT)⁵ in neonatal calves (Blum and Hammon 2000a and 2000b , Bühler et al. 1998 , Guilloteau et al. 1997 , Hadorn at al. 1997 ) and neonatal pigs (Odle et al. 1996 , Reinhart et al. 1992 , Simmen et al. 1990 , Xu 1996 ). Ingested immunoglobulins, some proteins and enzymes are absorbed in particular during the first hours after birth (Baumrucker et al. 1994b , Hadorn and Blum 1997 , Hammon and Blum 1998 , Michanek et al. 1989 ). On the other hand, ingested growth factors [e.g., insulin-like growth factor (IGF)-I] and hormones (e.g., insulin and prolactin) are barely absorbed in newborn calves (Baumrucker et al. 1994a , Grütter and Blum 1991a and 1991b , Hadorn et al. 1997 , Hammon and Blum 1997b , Lee et al. 1995 , Vacher et al. 1995 ) and pigs (Burrin et al. 1996 , Odle et al. 1996 ). Therefore, colostral growth factors and hormones in neonatal calves seem to exert their effects primarily in the GIT and appear to modify GIT growth, differentiation and function (Baumrucker et al. 1994a , Guilloteau et al. 1997 ), as also indicated by studies in neonatal pigs (Burrin et al. 1995 and 1996 , Odle et al. 1996 , Xu 1996 ). Thus, intestinal absorptive capacity in neonatal calves was lower in calves fed milk replacer (MR) or a formula (which contained only trace amounts of IGF-I and insulin) than in those fed colostrum (Hammon and Blum 1997a , Kühne et al. 2000 , Rauprich et al. 2000a ).

Based on these premises, we tested the hypothesis that feeding the colostrum of the first milking in very high amounts for 7 d stimulates GIT growth and digestive enzyme activities of the GIT and pancreas compared with feeding the first colostrum for 3 d in amounts mimicking usual husbandry conditions. In addition, we tested the hypothesis that feeding colostrum has specific and greater effects on indicators of GIT growth and on digestive enzyme activities of the intestine and pancreas than does feeding a formula (which contained very similar amounts of nutrients but only traces of bioactive substances, including growth factors and hormones like IGF-I and insulin).

	MATERIALS AND METHODS

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Animals, husbandry, feeding and experimental procedures.

The experimental procedures followed the Swiss Law on Animal Protection and were approved by the Committee for the Permission of Animal Experiments of the Canton of Freiburg, Granges-Paccot, Switzerland.

Twenty-one male calves of dairy breeds (Simmental x Red Holstein, Holstein Friesian, and Brown Swiss) were available. The number of animals tested per group (n = 7) was chosen based on previous experiments (Bühler et al. 1998 , Le Huërou-Luron et al. 1992 ). Because calves were killed and the studies were very expensive and time consuming, the number of experimental animals was kept as low as possible. All calves were single born after a normal-length pregnancy (290 d) and normal parturition. They were obtained immediately after birth, weighed and kept on straw litter in single boxes for 7 d at the Research Station of the Division of Nutritional Pathology (Posieux, Switzerland).

The body weight (BW) was measured immediately after birth and on d 4 and 7 of life, i.e., shortly before being killed (on d 8 of life). The health status was evaluated daily using a scoring system as described by Kühne et al. (2000 ).

Calves were divided into three groups (GrC_max, GrC_1–3 and GrF_1–3). To protect against infections, all calves received 20 mL immunoglobulins subcutaneously (Gammaserin, containing 100 g immunoglobulin G/L; Dr. E. Gräub AG, Berne, Switzerland) on average within the first 4.5 h after birth. From d 2 on, calves were fed twice daily chicken egg–derived immunoglobulins (Globigen 88; Lohmann Animal Health, Cuxhaven, Germany) in the amounts of 5 g (d 2), 4 g (d 3), 3 g (d 4) and 1 g (d 6 and 7). The product contained high amounts of specific antibodies against rotavirus and pathogenic Escherichia coli type K 99 but no bovine IGF-I or insulin. In addition, all navels were disinfected at birth.

Details of the feeding plans are shown in Table 1 . The GrC_max calves were fed colostrum from the first milking that was undiluted on d 1–3 and was progressively diluted with 25, 50, 75 and 75% of an MR (100 g/L water), respectively, on d 4–7. The GrC_1–3 calves were fed the colostrum from milkings 1–6 during the first 3 d and then an MR (100 g/L water), up to d 7. The GrF_1–3 calves were fed a milk-based formula during the first 3 d and then an MR (100 g/L water) up to d 7.

The compositions of colostrum milkings, formulas and MR are shown in Table 2 . The colostrum was obtained from cows of the Swiss Federal Research Station for Animal Production (Posieux, Switzerland). Colostrum for calves of GrC_max was from a pool of first milking from 20 cows. Colostrum for calves of GrC_1–3 was from cows milked twice daily and was separately stored at -20°C to make pools of milkings 1–6. Before feeding, the colostrum was warmed to 40°C and then immediately fed. For GrF_1–3, three formulas (for meals on d 1, 2 and 3) were produced in cooperation with UFA AG (Sursee, Switzerland) and contained comparable amounts of nutrients as colostrum obtained on d 1 (milking 1), d 2 (milking 3) and d 3 (milking 5), respectively. The formulas consisted of calcium caseinate (a gift from Emmi Milch AG, Lucerne, Switzerland), lactalbumin (a gift from Emmi Milch AG), lactose (UFA AG), milk fat in the form of commercially available dairy double cream (Agricultural Institute of the Canton of Fribourg, Grangeneuve/Posieux, Switzerland) and a vitamin and mineral premix routinely used in veal calves (Provimi S.A., Cossonay-Gare, Switzerland). The MR (UFA-200 Natura; without antibiotics) was purchased from UFA AG and was prepared as a 100 g/L solution.

Food and drug analyses.

Aliquots of 50 mL colostrum from milkings 1–6, of MR and of formulas (fed on d 1–3) were lyophilized, and dry matter, crude protein (by the Kjeldahl method), crude lipid (by Soxleth extraction) and crude ashes (after combustion at 550°C) were determined using standard procedures at the Swiss Federal Research Station for Animal Production (Posieux, Switzerland). Contents of nitrogen-free extracts and gross energy (based on energy equivalents of 36.6, 17.0 and 24.2 MJ/kg crude fat, nitrogen-free extracts and crude protein, respectively) were calculated (Table 2) . Contents of MR were provided by the producer (UFA AG Sursee, Switzerland). Food IGF-I and insulin concentrations were determined as previously described (Hammon and Blum 1997b ). Moreover, the concentration of IGF-I was measured in Globigen 88 (where it was below the detection limit) and in Gammaserin, the subcutaneously injected immunoglobulin preparation (which contained 288 µg IGF-I/L).

Histomorphometry of intestinal mucosa.

After killing, the intestine was divided into duodenum, mid-jejunum, ileum and colon, as described by Bühler et al. (1998 ). Then, a tissue sample of each section of 4 x 4 cm was removed, stuck onto a piece of cork (to inhibit shrinking) and transferred into a phosphate-buffered paraformaldehyde (40 g/L) solution. After 24 h, three 1-mm-thick cross-sectional pieces were cut from each sample and embedded in a paraffin block. Ten cuts, 3–4 µm thick, were made of each block, thus resulting in 30 different samples per intestinal site and calf.

For the histomorphometric analyses, from each paraffin block ten 3- 4-µm-thick sections of different regions were taken, put onto Super Frost slides and stained with hematoxylin and eosin. Morphometric analyses were conducted with a Zeiss light microscope connected to a video-based, computer-linked system, as described by Bühler et al. (1998 ). Quantitative measurements were made in at least 30 lengthwise-cut and well-oriented crypt-villus preparations for each intestinal sample. Villus circumferences, areas, heights and crypt depths were evaluated. The coefficient of variation for villus circumferences, areas, heights and crypt depths could be reduced <20%, if at least 30 villi and crypts in the small intestine or crypts in the colon were evaluated. Mean repeatability (variation within calves) of the 30 villi and crypts determined in duodenum, jejunum and ileum was 0.51, and in the colon, the repeatability for the determination of the 30 crypt depths was 0.71. To evaluate the day-to-day repeatabilities of determinations in the small intestine and colon, we measured the same five villi and/or five crypts on 5 consecutive d. In the small intestine, the repeatabilities for villi parameters were 0.98, and in the colon, the repeatability for crypt depths was 0.99. Furthermore, we measured circumferences, areas and the vertical and horizontal diameters of Peyer’s patches in the ileum (data not shown), and mean repeatability was 0.91. We also counted goblet cells per length unit in at least 30 villus-crypt systems in the ileum (data not shown), and in this case, the repeatability was 0.96.

Cell proliferation of intestinal mucosa.

Cell proliferation was based on counting cells that incorporate 5-bromo-2'-deoxyuridine (BrdU; Boehringer-Mannheim, Mannheim, Germany) into DNA (Matsuura and Suzuki 1997 ). Calves were administered 500 mg BrdU intravenously, dissolved in 20 mL of PBS, at 60 min before killing. Slides of intestinal tissue sections were stained with a mouse monoclonal anti-BrdU antibody (No. 1 170 376; Boehringer-Mannheim). BrdU incorporation was visualized with biotinylated goat anti-mouse immunoglobulins (Dako A/S, Zug, Switzerland), streptABComplex/AP-kit (Dako A/S) and Fast Red TR/Naphtol AS-MX (Sigma Chemical Co., St. Louis, MO).

Furthermore, the proliferation of epithelial intestinal cells was evaluated based on counting cells that are positive for protein Ki-67. This protein is expressed in G₁, S and G₂ phases of all proliferating cells but not in resting cells (Scholzen and Gerdes 2000 ). To detect the Ki-67 protein, a monoclonal antibody against Ki-67 (MIB 1; Dianova GmbH, Hamburg, Germany) was used. Ki-67 was visualized using biotinylated anti-MIB immunoglobulins and, finally, the peroxidase visualization system.

Two thousand intestinal epithelial cells were counted at the different intestinal segments for every calf. BrdU- or Ki-67–labeled intestinal epithelial cells were calculated relative to unlabeled epithelial cells as well as relative to length (µm) of the mucosal epithelial layer, thus resulting in ratios of mitotic cells per total cells and the number of mitotic epithelial cells per µm, respectively, and served as a mirror of the cell proliferation rate.

Total amounts of DNA, RNA and protein in intestine and pancreas.

The DNA and RNA were quantitatively isolated with DNAzol reagent (GIBCO BRL, Basle, Switzerland) and TRIzol reagent (GIBCO BRL), respectively. Homogenization of gut segment samples and pancreas was performed with a Mini-Beadbeater-8 cell disrupter (Biospec Products, Bartlesville, OK) for 3 min. DNA and RNA were extracted according to the instructions of the suppliers, and the pellets were resuspended in water or diethylpyrocarbonate water, respectively. DNA (which served as a measure of cell number or cell density) and RNA were quantified by fluorometry with PicoGreen and RiboGreen RNA Quantitation kits, respectively (Molecular Probes, Leiden, the Netherlands). The RNA/DNA ratio was calculated and served as a measure of protein synthetic capacity. The protein concentration was determined using the Pierce (Rockford, IL) BCA protein assay as previously described (Bühler et al. 1998 ). The protein/RNA ratio served as a measure of translation activity.

Activities of digestive enzymes in intestinal mucosa and pancreas.

About 3 g of mucosa was scraped off with a microscope slide in the middle of duodenum, jejunum, ileum and colon at sites where samples were taken for histomorphometric measurements and were deep-frozen in liquid nitrogen. Frozen intestinal mucosa of each gut segment was homogenized in ice-cold water (200 g/L) and, for peptidase activity analysis, centrifuged for 5 min at 1000 x g at 4°C. The activities of aminopeptidases A and N (EC 3.4.11.7 and EC 3.4.11.2, respectively) were assayed with L-glutamyl-p-nitroanilide and L-leucyl-p-nitroanilide as substrates (Maroux et al. 1972), and that of dipeptidyl peptidase IV (EC 3.4.14.5) was assayed with glycyl-L-prolyl-p-nitroanilide (Nagatsu et al. 1976 ). The resulting enzymatic units (IU) were expressed as µmol of p-nitroanilide released/min at 37°C. Lactase (EC 3.2.1.23) and maltase (EC 3.2.1.20) activities were determined using lactose in the absence of p-chloromercuribenzoate and maltose as substrates (Dahlquist 1964 ). One disaccharidase unit (IU) corresponded to the release of 1 µmol of glucose/min at 37°C.

Samples of the pancreas (corpus pancreatis) were frozen in liquid nitrogen and stored at -80°C until assayed. Before enzymatic analysis, zymogens in tissue homogenates were activated by trypsin (EC 3.4.21.4) as previously described (Le Huërou-Luron et al. 1992 ). Trypsin (EC 3.4.21.1) and chymotrypsin (EC 3.4.21.2) activities were assayed with N--benzoyl-L-arginine-p-nitroanilide (L-BA-pNA, B3133; Sigma Chemical Co.) and succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide (Suc-Ala₂-Pro-pNA, S7388; Sigma Chemical Co.) as substrates, respectively (Lain et al. 1993 ). Lipase (EC 3.1.1.3) activity was determined by titrimetry using tributyrate as a substrate as reported by Le Huërou-Luron et al. (1992 ). Elastase I (EC 3.4.21.36) and II (EC 3.4.21.71) activities were assayed with succinyl-L-alanyl-L-alanyl-L-alanine-p-nitroanilide (Suc-Ala₃-pNa, L1385; Bachem AG, Bubendorf, Switzerland) and succinyl-L-alanyl-L-alanyl-L-prolyl-L-leucine-p-nitroanilide (Suc-Ala₂-Pro-Leu-pNa, L1390; Bachem AG) as substrates, respectively. Elastase II substrate is also hydrolyzed by both chymotrypsin and, in particular, elastase I. Therefore, the proportion of elastase II substrate hydrolyzed by these two enzymes was quantified and subtracted to express the true elastase II activity in pancreatic homogenates (Gestin et al. 1997 ). The resulting enzymatic activities were expressed as µmol of substrates released/min (IU).

Statistical procedures.

BW, total 7-d intakes, histomorphometric and histoproliferation values, as well as enzyme activities and DNA, RNA and protein concentrations in tissues, were expressed as means ± SEM. Intestinal data are shown for individual segments of the small intestine and for the colon.

Variations in individual measurements of histomorphometric analyses of the intestine were calculated by the SAS program with the VARCOMP procedure. Repeatability of individual measurements and (technical) repeatabilities were calculated as the ratio of variation from individual calves or the variation from several measurements of the same sample and the sum of variation from individual calves and errors (individual repeatabilities) or the sum of variation from several measurement and errors (technical repeatabilities).

To test differences in feeding (GrC_max versus GrC_1–3 and GrC_1–3 versus GrF_1–3), histomorphometric and histoproliferation values and enzymatic activities were evaluated using the RANDOM and REPEATED methods of the MIXED procedure with an interanimal random effect of differences between animals and a correlation structure within animals (SAS 1994 ). Feeding and gut segments were used as fixed effects within animals. P-values were derived from t tests using estimates after the MIXED procedure and were considered significant if P < 0.05. The significance of differences in intake was evaluated by Student’s t test.

Group differences of DNA, RNA and protein concentrations in tissues were evaluated by means of Wilcoxon’s two-sample test (SAS 1994 ) because these variables were not normally distributed.

	RESULTS

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Feed intake, BW and health status.

BW was 47.1 ± 4.5, 45.7 ± 6.7 and 47.7 ± 5.7 kg immediately after birth and 48.1 ± 1.7, 44.6 ± 2.4 and 43.6 ± 2.5 kg at age 7 d in GrC_max, GrC_1–3 and GrF_1–3, respectively. There were no significant group differences, but GrC_max maintained its BW, in contrast to the other groups.

Feed refusals were seldom, and calves that did not completely drink their feed were fed with an esophageal tube. The total 7-d intakes of groups GrC_max, GrC_1–3 and GrF_1–3 of dry matter (110 ± 6, 79 ± 2 and 68 ± 2 g · kg body^-1 · wk^-1, respectively), gross energy (2.6 ± 0.2, 1.7 ± 0.1 and 1.4 ± 0.1 MJ · kg body^-1 · wk^-1, respectively), crude protein (54 ± 3, 24 ± 0.7 and 22 ± 0.7 g · kg body^-1 · wk^-1, respectively) and crude fat (28 ± 2, 20 ± 0.1 and 17 ± 0.6 g · kg body^-1 · wk^-1, respectively) were always much higher (P < 0.05) in the GrC_max group than in the GrC_1–3 group. The total 7-d intakes of nitrogen-free extracts (15 ± 0.7, 22 ± 1.7 and 18 ± 0.5 g · kg body^-1 · wk^-1for GrC_max, GrC_1–3 and GrF_1–3, respectively) were lower (P < 0.05) in the GrC_max group than in the GrC_1–3 group. The 7-d intakes in GrC_1–3 and GrF_1–3 were similar, as planned.

Calves were healthy, and there were no obvious differences on the various health scores. However, loose feces were apparent in six GrC_max calves (lasting for 1 d in one calf, for 2 d in three calves and for 3 d in two calves), two GrC_1–3 calves (lasting in one calf for 1 d and in one calf for 2 d) and in GrF_1–3 calves (lasting for 2 d in all 7 calves), but fecal scores were very similar among the groups.

Villus circumferences, areas and heights and crypt depths in the small intestine and crypt depths in the colon.

Villus circumferences, areas and heights in the duodenum were greater (P < 0.01) in GrC_max than in GrC_1–3 calves (Table 3 ). Duodenal villus height/crypt depth ratios were also higher (P < 0.01) in GrC_max than in GrC_1–3 calves.

Proliferation of small intestinal epithelial cells.

Intestinal cells labeled with BrdU and Ki-67 were almost exclusively found in the crypt regions of the intestinal mucosa. The numbers of BrdU- and Ki-67–labeled cells per µm and per total cells in the small intestine were positively correlated (r = 0.5 and r = 0.56, respectively; P < 0.001).

The number of BrdU-labeled cells in the duodenum was lower (P < 0.05) in GrC_max than in GrC_1–3 calves (Table 4 ). Similar tendencies (P < 0.1) were observed in the ileum. In addition, the number of duodenal Ki-67–labeled cells was lower (P < 0.05) in GrC_max than in GrC_1–3 calves.

Concentrations of DNA, RNA and protein in the intestine and pancreas.

Concentrations of DNA and RNA of the three groups in the small intestine (4.9 ± 0.6 and 16 ± 0.8 mg/g wet tissue, respectively) were not affected by differences in feeding. In the colon, the DNA concentration of the three groups was 0.6 ± 0.1 mg/g wet tissue and tended to be lower (P < 0.1) in GrC_max than in GrC_1–3 calves (0.4 ± 0.08 and 0.9 ± 0.3 mg/g wet tissue, respectively). In the colon, the RNA concentration of the three groups was 11 ± 0.8 mg/g wet tissue and did not differ among groups. The protein concentration of the three groups in the small intestine and the colon was 94 ± 1.2 and 80 ± 3.7 mg/g wet tissue, respectively. The protein concentration was higher in GrC_max than in GrC_1–3 calves in the jejunum (94.4 ± 1.9 and 86.9 ± 2.7 mg/g wet tissue, respectively; P < 0.05). In addition, the protein concentration in ileum tended to be lower (P < 0.1) in GrC_1–3 than in GrC_max and GrF_1–3 calves (91.7 ± 2.5, 98.0 ± 2.3, and 97.5 ± 2.4 mg/g wet tissue, respectively).

In the pancreas, RNA concentrations of the three groups (21 ± 1.1 mg/g wet tissue) did not differ among groups. DNA concentration of the three groups was 0.9 ± 0.2 mg/g wet tissue and tended to be lower (P < 0.1) in GrC_max than in GrC_1–3 calves (0.5 ± 0.07 and 0.8 ± 0.2 mg/g wet tissue, respectively). Protein concentration of the three groups was 137 ± 3.4 mg/g wet tissue and tended to be higher (P < 0.1) in GrC_max than in GrC_1–3 calves (144 ± 3.8 and 134 ± 5.7 mg/g wet tissue, respectively). There were no significant differences of RNA, DNA and protein concentrations between the GrF_1–3 and GrC_1–3 groups.

Jejunal and pancreatic enzyme activities.

In the jejunum, activities among the three groups of dipeptidyl peptidase, amino peptidase N, amino peptidase A, lactase and maltase were 223 ± 33, 383 ± 63, 80 ± 25, 7.9 ± 1.4 and 0.38 ± 0.04 IU/g mucosa, respectively. Dipeptidyl peptidase IV and maltase activities tended to be lower and higher (P < 0.1), respectively, in GrF_1–3 than in GrC_1–3 calves (dipeptidyl peptidase IV: 150 ± 18 and 232 ± 50 IU/g mucosa; maltase: 0.49 ± 0.1 and 0.33 ± 0.04 IU/g mucosa, respectively). There were no significant differences of intestinal enzyme activities between GrC_max and GrC_1–3 calves.

In the pancreas, activities of the three groups of trypsin, chymotrypsin, lipase, elastase I and elastase II were 22 ± 3, 494 ± 73, 1061 ± 124, 11 ± 1 and 747 ± 133 IU/g pancreas. Pancreatic activity of lipase was higher (P < 0.05) and activities of trypsin and elastase II tended to be higher (P < 0.1) in GrC_max than in GrC_1–3 calves (lipase: 1481 ± 110 and 945 ± 171 IU/g pancreas; trypsin: 28 ± 2 and 20 ± 4 IU/g pancreas; elastase II: 958 ± 118 and 626 ± 144 IU/g pancreas, respectively). There were no significant differences in pancreatic activities between GrC_1–3 and GrF_1–3 calves.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Feeds, feeding, growth performance and health status.

A decrease in dry matter, gross energy, crude protein, crude fat, crude ashes, IGF-I and insulin concentrations and an increase in nitrogen-free extracts (mainly of lactose) in the colostrum from milkings 1–6 in the diets fed to the calves in this study were typical (Campana and Baumrucker 1995 , Blum and Hammon 2000b ).

With feeding the formula, the experiment, which was designed to study effects on the GIT and pancreas, was conducted under conditions not yet performed in neonatal calves. Thus, the formula contained only trace amounts of hormones (IGF-I, insulin), enzymes (e.g., -glutamyltransferase, alkaline phosphatase and alanine transferase; J. W. Blum and H. M. Hammon, unpublished observations) and other bioactive substances (e.g., lactoferrin; not shown) that are otherwise present in high amounts in colostrum. However, the formula contained comparable amounts of dry matter, gross energy, crude protein, nitrogen-free extracts, crude fat and crude ashes as colostrum of first 3 d of lactation and fed to GrC_1–3 calves. Because the colostrum and formula were similarly composed with respect to nutrients and because the MR (d 4–7) was fed to groups GrC_1–3 and GrF_1–3 in the same amounts, intakes of dry matter, gross energy, crude protein and crude fat during the 1st wk of life in these two groups were comparable. However, because the GrC_1–3 and GrF_1–3 calves on d 4–7 were fed the same MR, prolonged effects of differences in the feeding of non-nutrient components during the first 3 d of life could be studied when they were killed (on d 8).

The GrC_max calves ingested much greater amounts of dry matter, gross energy, crude protein and crude fat but lower amounts of nitrogen-free extracts than the GrC_1–3 calves. Calves of GrC_max were probably fed maximally tolerable amounts of (first-milked) colostrum. This allowed us to study effects of an excessive feeding intensity and of non-nutrient components on the GIT and on the pancreas, thus extending our previous investigations (Kühne et al. 2000 ).

Effects on health status, growth performance and metabolic and endocrine traits in GrC_max, Gr_1–3 and GrF_1–3 calves have been previously described (Rauprich et al. 2000a and 2000b ). In short, calves were generally healthy, and there were no significant group differences in clinical traits. Loose feces indicated mild disturbances of GIT function, which were transient but could have caused reduced absorptive capacities in GrF_1–3 compared with GrC_1–3 calves (Rauprich et al. 2000a ).

Effects of maximizing compared with conventional colostrum intake.

Growth of the intestine in the first postnatal days follows an inherent pattern determined by endogenous factors, but exogenous factors, including nutrition, play roles as well. The postnatal development of the GIT is well known to be greatly influenced by colostrum intake (Bühler et al. 1998 , Odle et al. 1996 , Reinhart et al. 1992 , Widdowson et al. 1976 , Xu 1996 ). Based on histomorphometric analyses, our study demonstrates that excessive intake of first colostrum in GrC_max compared with GrC_1–3 calves especially stimulates the development of the duodenal villus size during the first 8 d of life. Villi of more distal segments of the small intestine were not significantly influenced by differences in colostrum feeding intensity. That the duodenum was more influenced by effects of colostrum intake than were more distal parts of the small intestine also occurred in a previous study (Bühler et al. 1998 ). Nutrient and non-nutrient components were likely to be present in greater amounts in proximal than in distal parts of the intestine. Hence, their effects were expectedly greater in the duodenum than in the jejunum and ileum, i.e., they may have particularly stimulated duodenal growth. However, the greater absorptive surface in GrC_max than in GrC_1–3 calves was not reflected by an enhanced xylose absorption on d 5 of life (Rauprich et al. 2000b ), indicating that absorptive capacity could not be further enhanced by maximizing and prolonging colostrum intake.

That intestinal cells labeled with BrdU and Ki-67 were almost exclusively found in the crypt regions of the intestinal mucosa was not surprising because new mucosal epithelial cells arise through mitosis of stem cells and migrate up along the epithelial surface to the tip of villi (Johnson 1988 ). Most surprisingly, both BrdU and Ki-67 labeling in the duodenum indicated smaller proliferation rates of the mucosa of GrC_max compared with GrC_1–3 calves. Thus, although maximizing colostrum feeding enhanced small intestinal villus size, it reduced mucosal cell proliferation on d 8. However, because there were no significant effects on DNA concentrations per gram of tissue in GIT (and pancreas), cell density was not influenced. These results strongly suggest that maximizing colostrum intake decreased apoptosis of the mucosal epithelial cells. Reduction in apoptosis via colostrum intake in high amounts may be a possibility (Playford et al. 2000 ) and possibly was mediated by growth factors such as IGF-I (Mylonas et al. 2000 ), which is present in high amounts in bovine colostrum (Blum and Hammon 2000b , Campana and Baumrucker 1995 ). Because expression of IGF-I in the duodenum, jejunum and ileum was not influenced by differences in feed intake, whereas expression of IGF-I in the liver was enhanced in GrC_max calves and was closely and positively correlated with plasma IGF-I concentrations (Cordano et al. 2000 and unpublished observations), circulating IGF-I may be of greater importance than IGF-I synthesized in the GIT.

The protein content of jejunum and ileum was higher in GrC_max than in GrC_1–3 calves. The ingestion of colostrum in neonatal rats and dogs was associated with significant increases in intestinal mucosal protein contents (Berseth et al. 1983 , Schwarz and Heird 1994 ). Higher protein concentrations in the intestine might have been the consequence of greater incorporation into epithelial cells of immunoglobulins after enhanced colostrum feeding, as demonstrated in neonatal calves (Patureau-Mirand et al. 1990 ) and pigs (Burrin et al. 1992 ). On the other hand, no change was observed in intestinal digestive enzyme activities.

The trend (although not significant) to higher protein concentrations in the pancreas of GrC_max than that of GrC_1–3 calves might, too, have been an effect of greater protein synthesis in response to excessive colostrum intake, in accordance with metabolic and endocrine data obtained from calves of this study (Cordano et al. 2000 , Rauprich et al. 2000b ). Trypsin and lipase activities were higher in GrC_max than in GrC_1–3 calves. This might have been mainly the consequence of higher protein, fat and energy intakes in GrC_max calves compared with GrC_1–3 calves. In neonatal pigs, dietary composition changes pancreatic enzyme activities, especially of lipase (Le Huërou-Luron and Guilloteau 1999 ).

Effects of milk-based formula intake compared with colostrum.

Replacement of colostrum by a milk-based formula (GrF_1–3) with a similar nutrient content as colostrum (GrC_1–3) caused reduced jejunal villus size and showed a trend toward greater jejunal crypt depths on d 8. This indicated that villus growth was reduced by formula feeding during the first 3 d but suggested that GrF_1–3 calves up to d 8 had started with the compensation of epithelial cell growth. We found more markedly reduced villus sizes in the small intestine when an MR with lower nutrient content than our formula was fed instead of colostrum (Bühler et al. 1998 ). Besides nutrients, biologically active factors in colostrum may be important for intestinal growth. Thus, growth factors like IGF-I are stable in the neonatal GIT (Playford et al. 2000 , Shen and Xu 2000 ) and may enhance growth of the GIT (Baumrucker et al. 1994a , Burrin et al. 1996 , Odle et al. 1996 ). As shown for IGF-I and insulin and as expected for other factors like lactoferrin, the near absence of these biologically active factors in GrF_1–3 calves may have been in part responsible for reduced jejunal villus sizes compared with GrC_1–3 calves. Because (in the ileum) there were no significant group differences in the size (and of the proliferation rate) of Peyer’s patches and the number of goblet cells (S. Bittrich, H. M. Hammon and J. W. Blum, unpublished observations), this indicates that nutritional effects were relatively specific and, in these tissues, small. In the colon, surprisingly the crypt size was the greatest in GrF_1–3, suggesting that the proliferation of epithelial cells in this part of the intestine was enhanced. The cause or causes for this effect are not obvious.

The absence of significant nutritional effects on total DNA content in the GIT and pancreas in GrF_1–3 compared with GrC_1–3 calves indicates that the cell density was not affected. In neonatal pigs, total DNA concentration also did not differ when lactose or mature milk was fed instead of colostrum (Simmen et al. 1990 ). Furthermore, oral IGF-I, IGF-II or insulin had no effect on mucosal DNA concentration in newborn pigs (Shulman 1990 , Xu et al. 1994 ). However, in studies with neonatal rats and dogs, the ingestion of colostrum was associated with significant increases in mucosal DNA concentrations (Berseth et al. 1983 , Schwarz and Heird 1994 ).

The proliferation rate of epithelial cells in the duodenum (based on BrdU labeling) was lower in GrF_1–3 than GrC_1–3 calves. These data support previous findings that colostrum intake stimulates intestinal epithelial cell proliferation more than mature milk or MR in neonatal calves (Baumrucker et al. 1994a ) and pigs (Odle et al. 1996 , Ulshen et al. 1991 ). Studies with neonates fed insulin or IGF-I analogues showed enhanced proliferation rates of the intestinal mucosa of calves, pigs, rats and humans (Baumrucker et al. 1994a , Ménard et al. 1999, Staley et al. 1998 , Xu 1996 ). Therefore, colostral factors (e.g., IGF-I and insulin) rather than nutrients may have been responsible for enhanced stimulation of cell proliferation in the duodenal mucosa of GrC_1–3 compared with GrF_1–3 calves. Furthermore, systemic IGF-I may have had an additional influence on gut growth, as seen in rats (Read et al. 1992 ). In addition to IGF-I, milk contains many other factors (hormones, growth factors, gut regulatory peptides, etc.) that could also be good candidates to stimulate small intestinal growth. Surprisingly, the proliferation rate in the jejunum (based on Ki-67 labeling) in GrF_1–3 was higher than that in GrC_1–3 calves. Causes for this deviating effect are not obvious.

Our study failed to show reduced intestinal and pancreatic protein concentrations in GrF_1–3 compared with GrC_1–3 calves. This was in contrast to Burrin et al. (1995 ), who found enhanced stimulation of protein synthesis in jejunum, but not in pancreas, in colostrum-fed compared with formula-fed neonatal pigs, an effect that appeared to be mediated by non-nutritional factors rather than nutrients. Again, one explanation for the failure to show an effect in our calves might have been the relatively long time between formula feeding (up to d 3 of life) and tissue sampling (on d 8 of life). Differences between GrF_1–3 and GrC_1–3 calves in intestinal enzyme activities may have reflected specific regulation of enzyme synthesis, possibly caused by differences in the amount of ingested bioactive substances, but they were unimpressive.

Although differences in villus size between GrF_1–3 and GrC_1–3 calves on d 8 of life were small, they were expectedly more marked on d 5 and likely affected absorptive surface and explained differences in nutrient absorptive capacity at this stage. In accordance, we have found a smaller xylose absorption rate in GrF_1–3 than GrC_1–3 calves on d 5 of life (Rauprich et al. 2000a ). Furthermore, plasma concentrations of protein, triglycerides and insulin were lower and that of cortisol was higher in GrF_1–3 than in GrC_1–3 calves (Rauprich et al. 2000a ). This expressed a reduced nutritional status, possibly as a consequence of decreased nutrient digestibilities and absorption rates in GrF_1–3 than in GrC_1–3 calves, even though nutrient intakes did not differ.

In this study, maximization of colostrum intake enhanced duodenal villus size but reduced epithelial cell proliferation, suggesting that high and prolonged intake of colostrum may enhance the survival of intestinal epithelial cells and reduce mucosal epithelial cell turnover. The effects of high and prolonged colostrum intake on villus size and epithelial (crypt) cell proliferation were most marked in duodenum, i.e., at a site at which only partially digested components and likely still biologically active components of the chyme are present. Feeding only colostrum for 3 d seems to result in stimulation of small intestinal cell proliferation. Investigations into the mechanisms of regulation of apoptotic processes must be developed to clarify the role of colostrum intake on mucosal programmed cell death.

Feeding a formula containing only trace amounts of growth factors (e.g., IGF-I) and hormones (e.g., insulin) but similar amounts of nutrients as colostrum caused reduced villus size in jejunum and decreased crypt cell proliferation in the duodenum and ileum, indicating that colostral bioactive components (e.g., growth factors and hormones) had effects beyond those of nutrients and increased proliferation in selected parts of the small intestine.

	ACKNOWLEDGMENTS

 
We thank Hans Schnyder and Josef Sturny for supplying neonatal calves and Ernst Husmann (UFA AG, Sursee, Switzerland) for helping us to develop the milk-based formula. The help of Stephan Grimm (Institute of Veterinary Pathology, University of Berne) in the setup of histomorphometric analyses is greatly appreciated, and we thank Silvia Sahner, Claudia Thriller and Werner Wegmann (Cantonal Hospital of Basel-Land, Liestal, Switzerland) for Ki-67 labeling.

	FOOTNOTES

 
¹ Presented in part at the 14th Meeting of the Fachgruppe Physiologie und Biochemie der Deutschen Veterinrmedizinischen Gesellschaft, April 3–4, 2000, University of Munich, Munich, Germany [Blättler, U., Hammon, H. M., Le Huërou-Luron, I., Guilloteau, P. & Blum, J. W. (2000) Effects of different colostrum intake on morphology, cell proliferation and enzyme activities in the gut of neonatal calves].

² Submitted as the thesis of U. Blüttler to the Faculty of Veterinary Medicine, University of Berne, Berne, Switzerland, February 2001.

³ Supported by the Swiss National Science Foundation (Grant 35 3200-05102.97/1).

⁵ Abbreviations used: BrdU, 5-bromo-2'-deoxyuridine; BW, body weight; GIT, gastrointestinal tract; GrC_max, fed colostrum of first milking that was fed undiluted on d 1–3 and diluted with 25, 50, 75 and 75 parts of a milk replacer on d 4–7, respectively; GrC_1–3, fed colostrum of milkings 1–6 during the first 3 d and then a milk replacer up to d 7; GrF_1–3, fed a milk-based formula (containing only traces of growth factors and hormones such as insulin-like growth factor-I and insulin) during the first 3 d and then a milk replacer up to d 7; IGF, insulin-like growth factor; MR, milk replacer.

Manuscript received September 22, 2000. Initial review completed October 12, 2000. Revision accepted December 15, 2000.

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