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J. Dairy Sci. 87:1786-1795
© American Dairy Science Association, 2004.

Preterm as Compared with Full-Term Neonatal Calves Are Characterized by Morphological and Functional Immaturity of the Small Intestine*

S. Bittrich1,{dagger}, C. Philipona1, H. M. Hammon1, V. Romé2, P. Guilloteau2 and J. W. Blum1

1 Division of Animal Nutrition and Physiology, Institute of Animal Genetics, Nutrition and Housing, Vetsuisse Faculty, University of Berne, CH-3012 Berne, Switzerland
2 Unité Mixte de Recherches sur le Veau et le Porc, Institut National de la Recherche Agronomique, F-35042 Rennes, France

Corresponding author: J. W. Blum; e-mail: juerg.blum{at}itz.unibe.ch.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Intestinal diseases in neonatal calves may be due to morphological and functional immaturity. We have studied histomorphology, crypt cell proliferation rates (based on incorporation of 5-bromo-2'-deoxyuridine into DNA), presence of apoptotic cells (based on terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling), and brush border enzyme activities in preterm calves (277 d of gestation), euthanized on d 1 (P0) or 8 (P8), and in full-term calves (290 d of gestation), euthanized on d 1 (F0) or 8 (F8). Vacuolated epithelial cells were present in ileum of P0 and F0 but not in P8 and F8. During the first 8 d, villus sizes, crypt depths, and proliferation rates of crypt cells in the small intestine of preterm calves did not significantly change. In contrast, in full-term calves during the first 8 d, villus sizes in jejunum decreased, crypt depths increased in small intestine and colon, and crypt cell proliferation increased in duodenum and jejunum. Submucosal thickness in jejunum was highest in P0, but in ileum it increased with gestational age and feeding. Gestational age x feeding interactions indicated increased activities of aminopeptidase N and reduced lactase activities only in F8 and reduced dipeptidylpeptidase IV activities only in P8. In conclusion, in preterm calves the small intestinal epithelium was immature and brush border enzyme activities differed in part from those in full-term calves.

Key Words: intestine • nutrition • colostrum • preterm calf

Abbreviation key: BrdU = 5-bromo-2'-deoxyuridine, GIT = gastrointestinal tract, F0 = group born full term and euthanized immediately after birth, F8 = group born full term, fed for 7 d, and euthanized on d 8 of life, P0 = group born preterm and euthanized immediately after birth, P8 = group born preterm, fed for 7 d, and euthanized on d 8 of life, MR = milk replacer


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Perinatal mortality and morbidity rates of neonates are generally high. This also holds for calves (Flemming et al., 2001). The disease problem is even greater if animals are born preterm (Sangild, 2001). Perinatal losses in preterm neonates are partly due to insufficient thermoregulatory, acid-base, and metabolic control due to immature organ function especially of some endocrine systems (Cabello and Levieux, 1980; Richet et al., 1985; Challis et al., 2000; Sangild, 2001), the lung (Pickel et al., 1989; Zaremba et al., 1997), and the gastrointestinal tract (GIT) (Sangild, 2001). Adequate adaptation responses of the GIT to feeding are essential for survival. As concerns feeding, colostrum provides essential and nonessential nutrients, minerals, and vitamins, but it also provides high amounts of nonnutrient substances, especially immunoglobulins, hormones, growth factors, and cytokines (Campana and Baumrucker, 1995; Blum and Hammon, 2000; Blum, 2002). Intake of colostrum is important for passive immunity and changes the nutritional, metabolic, and endocrine status of calves (Blum and Hammon, 2000; Blum and Baumrucker, 2002). Colostrum intake enhances postnatal GIT growth (Odle et al., 1996; Xu, 1996; Zhang et al., 1996), as also shown in calves (Guilloteau et al., 1997; Bühler et al., 1998; Blättler et al., 2001). Colostrum also increases digestive and absorptive capacity of the GIT (Zhang et al., 1996), as also shown in the calf (Guilloteau et al., 1997; Hammon and Blum, 1997). Furthermore, colostrum modifies glucocorticoid effects on the GIT (Sauter et al., 2004). Both nutrient and nonnutrient colostral factors exert GIT effects (Blum and Baumrucker, 2002). The IGF and insulin are present in high amounts in bovine colostrum (Campana and Baumrucker, 1995; Blum and Hammon, 2000), their receptors are found in the small intestine and colon of neonatal calves (Baumrucker et al., 1994; Hammon and Blum, 2002; Georgiev et al., 2003; Georgieva et al., 2003), and IGF-I can enhance GIT growth and function, as reviewed by Howarth (2003). Furthermore, GIT growth is modified by postprandially released intestinal and pancreatic humoral factors (Lipkin, 1981; Guilloteau et al., 1997; Sangild, 2001). In addition, effects on the GIT are expectedly mediated by blood hormones and growth factors, whose status in neonatal calves up to at least d 7 of life is markedly modified by feeding (Blum and Hammon, 2000; Blum and Baumrucker, 2002; Howarth, 2003). The IGF are produced in the GIT of neonatal calves (Pfaffl et al., 2002; Georgieva et al., 2003) and can basically influence GIT development by auto- and paracrine mechanisms through specific receptors (Hammon and Blum, 2002; Georgiev et al., 2003; Howarth, 2003). Therefore we have investigated GIT morphology, mucosal epithelial proliferation, apoptosis, and intestinal enzyme activities in preterm calves compared with full-term calves immediately after birth as well as in response to feeding. The hypothesis was tested that the GIT of preterm calves is immature and reacts insufficiently to feeding compared with full-term calves. To our knowledge, no such data are available for preterm calves.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Animals, Husbandry, Feeding, and Experimental Procedures
The experimental procedures followed the present Swiss Law on Animal Protection. They were approved by the committee for the permission of animal experiments of the Canton of Freiburg (Granges-Paccot, Switzerland) and were supervised by the Federal Veterinary Office, Berne.

We have studied 28 calves (19 Simmental x Red Holstein, 6 Holstein Friesian, and 3 Brown Swiss) that were randomly assigned to 4 groups. All calves were single-born between November and February and were separated from their dams immediately after birth. Calves of group F0 (1 female and 6 males; 4 Simmental x Red Holstein, 2 Holstein Friesian, and 1 Brown Swiss) were spontaneously born after normal length of pregnancy (290 ± 2 d) and were euthanized immediately after birth. Calves of F8 (7 males; 5 Simmental x Red Holstein, and 2 Holstein Friesian) were spontaneously born after normal length of pregnancy and were euthanized on d 8 of life. Calves of group P0 (4 females and 3 males; 4 Simmental x Red Holstein, 1 Holstein Friesian, and 2 Brown Swiss) and of P8 (4 females and 3 males; 6 Simmental x Red Holstein, and 1 Holstein Friesian) were born on d 277 of gestation within 42 ± 3 h after cows were injected with 500 µg of prostaglandin F2{alpha} (Estrumate; Essex Pharma GmbH, Friesoythe, Germany) and 5 mg of Flumethason (Flumilar; Veterinaria AG, Zürich, Switzerland). Calves of P0 were euthanized immediately after birth, and those of P8 were euthanized on d 8 of life.

Calves of F8 and P8 received colostrum of first milking on the first 3 d. On d 4, 5, 6, and 7, they were fed colostrum of first milking diluted with 25, 50, 75, and 75 parts of milk replacer (MR), respectively. The amounts of colostrum fed were 60 g/kg of BW on d 1, 80g/kg of BW on d 2, and 100g/kg of BW of colostrum or colostrum and MR from d 3 to 7. Calves received their first meal on average within 2 h after birth. The following feedings were at 8, 24, and 32 h after the first feeding and from d 3 on, they were fed at 0800 and 1600 h. Calves were always suckling from a bottle.

The colostrum was from a pool obtained from cows of the Federal Research Station for Animal Production (Posieux, Switzerland) and from farms in the neighborhood of the research station. Colostrum of first milking was deep-frozen in plastic bottles at –20°C. Before feeding, colostrum was warmed up to 40°C and then immediately fed. The MR (UFA-200-Natura; without antibiotics, Union de Fédération Agricole, Sursee, Switzerland) was prepared as 100 g/L of solution. Composition of colostrum and MR are shown in Table 1Go.


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  		Table 1. Composition of first colostrum and milk replacer fed to neonatal calves.
 
Calves were subcutaneously injected with 20 mL of an immunoglobulin preparation (Gammaserin, 100 g of immunoglobulin G/L; Gräub AG, Bern, Switzerland) after the first blood sample, and between d 2 and d 5 all calves were subcutaneously injected with antibiotics 1 x d (Baytril 5%, 2.5 mg/kg of BW, Bayer AG, Leverkusen, Germany, and Betamox LA, 15 mg/kg of BW subcutaneous, Norbrook Laboratories, Neddry, UK) for protection against infections.

The BW of all calves was measured immediately after birth and, in F8 and P8, on d 8 of life.

Analyses in Colostrum and Milk Replacer
Aliquots of 50 mL of the colostrum pool and of the MR were lyophilized and DM, CP (by the Kjeldahl method), crude fat (by Soxleth extraction), and crude ash (after combustion at 550°C) were determined using standard procedures at the Swiss Federal Research Station for Animal Production (Posieux, Switzerland). Contents of water, nitrogen-free extracts (i.e., sugars and thus mainly lactose), and gross energy (based on energy equivalents of 36.6, 17.0, and 24.2 MJ/kg of fat, nitrogen free extract, and CP, respectively; Kamphues et al., 1999) were calculated.

Histomorphometry, Cell Proliferation, and Apoptosis of Intestinal Epithelium
After calves were slaughtered on d 1 or 8 of life, the abdominal cavity was opened, and the GIT was removed. The small intestine was dissected free and was divided into duodenum, jejunum, ileum, and colon (Bühler et al., 1998). A 4- x 4-cm tissue sample of each part was fixed on a piece of cork and transferred into paraformaldehyde (40 g/L) phosphate buffered solution. After 24 h, three 10-mm-long and 1-mm-thick cross sections were cut from each sample and embedded in a paraffin block. Then, ten 3- to 4-µm thick cuts were made of each block, thus resulting in 30 different samples per intestinal site and calf.

For the histomorphometrical analyses, tissues were put on Super Frost Plus slides (Medite, St. Gallen, Switzerland) and stained with hematoxilin and eosin. Morphometrical analyses were conducted with a Zeiss light microscope (Zeiss, Jena, Germany) connected with a video-based, computer-linked system, as described by Bühler et al. (1998) and Blättler et al. (2001). The quantitative measurements were made in at least 30 lengthwise cut and well-oriented crypt-villus systems for each intestinal sample. Villus circumferences, heights, and crypt depths, as well as the submucosa thickness and the diameter of Peyer Patches (in the ileum), were evaluated in the small intestine and crypt depths were measured in the colon. The coefficient of variation for villus circumferences, 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 (Blättler et al., 2001; Bittrich, unpublished observations).

Cell proliferation was based on counting cells that incorporate 5-bromo-2'-deoxyuridine (BrdU; Boehringer GmbH, Mannheim, Germany) into DNA, as recently described (Blättler et al., 2001). All calves were intravenously injected with 500 mg of BrdU, dissolved in 20 mL of PBS at 1 h before euthanasia. Slides were stained using a mouse monoclonal anti-BrdU antibody (# 1 170 376; Boehringer GmbH) for the detection of BrdU incorporation into DNA. BrdU incorporation was visualized using biotinylated goat anti-mouse immunoglobulins (Dako A/S; Zug, Switzerland), streptAB complex/AP-Kit (Dako), and Fast Red TR/Naphtol AS/MX (Sigma; St. Louis, MO). Next, 2000 intestinal epithelial cells were counted at the different intestinal sites for every calf. The BrdU-labeled intestinal epithelial cells were calculated relative to unlabeled epithelial cells as well as relative to length (µm) of the mucosal epithelial layer in a well-oriented crypt-villus-crypt system. This resulted in ratios of mitotic epithelial cells per total epithelial cells and the number of mitotic epithelial cells per micrometer in this system, respectively, and it served as a mirror of the cell proliferation rate. Proliferation rates could not be determined in colon in F8 because of technical reasons (destroyed material).

Detection of apoptotic epithelial cells was based on terminal transferase 3'-end labeling of DNA in paraffin sections (David et al., 2003). Slides were twice dewaxed with xylem (for 2 and 5 min, respectively), defatted with alcohol (10, 100, 90, 70, and 50% for 2, 5, 2, 5, and 5 min, respectively), washed with distilled water (for 5 min), deproteinized with proteinase K (Roche, Basle, Switzerland; 15.6 mg or >30 units/mg for 10 min at 37°C), and washed with PBS and distilled water (for 2 and 5 min, respectively). This was followed by terminal transferase reaction (for 60 min at 37°C in a moist chamber) with a mixture (40 to 100 µL/parafilm-sealed slide) containing (in 250 µL) the DNA deoxynucleotidylexotransferase (EC 2.7.7.31; extracted from calf thymus; 20 units/µL; Roche), CoCl2 (25 mM; 25 µL; Roche), fluorescein-12-2'-deoxyuridine-5'-triphosphate (6 µL; Roche), distilled water (170 µL), and reaction buffer 5 x (50 µL). Slides were then washed 2x with PBS (for 5 and 10 min, respectively), fixed, and mounted for microscopy.

Activities of Digestive Enzymes in the Mucosa of the Small Intestine
About 3 g of mucosa was scraped off with a microscope slide in the middle of duodenum, jejunum, and ileum at sites where samples were taken for histomorphometrical measurements and were deep-frozen in liquid nitrogen. Frozen intestinal mucosa of each gut segment was homogenized in ice-cold water (200 mg/mL). For peptidase activity analysis, the material was centrifuged for 5 min at 1000 x g at 4°C. Assays were performed as recently described (Blättler et al., 2000). 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 and that of dipeptidyl peptidase IV (EC 3.4.14.5) with glycyl-L-prolyl-p-nitroanilide. The resulting enzymatic units (IU) were expressed as µmol of p-nitroanilide released per minute at 37°C. Lactase (EC 3.2.1.23) activities were determined using lactose in the absence of p-chloromercuribenzoate as substrate. One µmol corresponded to the release of disaccharidase unit (IU) glucose per minute at 37°C. For technical reasons, enzyme activities in duodenum and ileum were only measured in P0, P8, and F0 but not in F8.

Statistical Analyses
In this study there were 2 main effects: conceptional or gestational age ({delta}) and feeding or postnatal age ({phi}). Data were therefore analyzed by ANOVA based on a 2 x 2 completely randomized design that reflected main effects and interactions between the 2 factors ({delta} x {phi}) using the general linear model procedure (SAS, 1994). When the F test was significant (P < 0.05), differences were localized by Bonferroni t test (P < 0.05). Furthermore, differences between gut segments were evaluated by general linear model (SAS, 1994) and localized by Bonferroni t test (P < 0.05). Effects of sex and breed in this study did not affect the data, as tested in preliminary statistical models and therefore were not included in the final statistical model. Values are expressed as means ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Body Weight, Growth Performance, and Health Status
Mean BW at birth of P0 and P8 (42 ± 2 and 39 ± 2 kg, respectively) were lower (P < 0.05) than those of F0 and F8 (50 ± 2 and 47 ± 2 kg, respectively). The BW gain from birth to d 8 of life of P8 and F8 was similar (2.2 ± 0.3 kg and 1.4 ± 0.7 kg, respectively). There were no group differences and feeding effects on BW gain.

Intestinal Traits
General aspects.
There were significant differences between intestinal sites of villus circumferences and heights (jejunum > duodenum and ileum, P < 0.01), of crypt depths (colon > duodenum > ileum > jejunum, P < 0.05), of villus height:crypt depth ratios (jejunum > ileum > duodenum, P < 0.01), and of submucosa thickness (ileum > duodenum, jejunum, and colon, P < 0.001). Interestingly, villus-like structures were not only seen in the small intestine, but also present in the colon in unfed preterm and full-term calves. Vacuolated intestinal epithelial cells were present in the ileum of all calves at birth (277 and 290 d) but were absent in all calves receiving food during 7 d regardless of gestational age. Vacuolated cells were not detectable in duodenum, jejunum, and colon in any calf of the 4 groups.

The BrdU was incorporated in cells of the mucosa and submucosa (such as in Peyer patches) of the small intestine and colon, but BrdU-labeled epithelial cells were exclusively seen in the crypt cell area. Apoptotic cells were readily detectable in the stroma of villi of the small intestine and in the submucosa (especially in Peyer patches) of the ileum and colon, but there were no apoptotic epithelial cells detectable in the crypts and in the intestinal epithelia up to the tips of villi.

Activities of aminopeptidase A, aminopeptidase N, and dipeptidyl peptidase IV for P0, P8, and F0 were lower (P < 0.05) in duodenum than in jejunum and ileum (means of aminopeptidase A in duodenum, jejunum, and ileum: 0.7 ± 0.1, 3.2 ± 0.4, and 3.6 ± 0.4 µmol/g of mucosa, respectively; means of aminopeptidase N in duodenum, jejunum, and ileum: 0.7 ± 0,1, 2.3 ± 0.3, and 2.0 ± 0.2 µmol/g mucosa, respectively; means of dipeptidyl peptidase IV in duodenum, jejunum, and ileum: 0.6 ± 0.1, 2.9 ± 0.4, and 3.1 ± 0.4 µmol/g of mucosa, respectively). Activities of lactase were highest (P < 0.05) in jejunum and were higher (P < 0.05) in duodenum than in ileum (means for duodenum, jejunum, and ileum: 9.0 ± 1.1, 15.4 ± 1.3, and 2.1 ± 0.4 µmol/g of mucosa, respectively).

Effects of gestational age and postnatal feeding in the duodenum.
Table 2Go shows that crypt depths were affected by gestational age and by feeding (P < 0.05 and P < 0.001, respectively), and there were significant (P < 0.01) gestational age x feeding interactions because crypt depths increased (P < 0.01) during the first 8 d of life only in full-term calves and on d 8 of life were greater (P < 0.01) in full-term than in preterm calves. For villus height:crypt depth ratios, there was a significant gestational age x feeding interaction (P < 0.05), and ratios of villus height:crypt depth were higher (P < 0.05) in F0 than in F8. The number of BrdU-labeled crypt cells showed a significant (P < 0.01) gestational age x feeding interaction because the number of BrdU-labeled crypt cells increased during the first 8 d of life only in full-term calves and on d 8 of life were greater (P < 0.05) in full-term than in preterm calves. Villus circumferences and heights and the submucosa thickness were not affected by gestational age or feeding.


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  		Table 2. Villus circumferences and heights, crypt depths, thickness of submucosa, and mucosal epithelial cell proliferation in duodenum of preterm and full-term calves.1
 
Effects of gestational age and postnatal feeding in the jejunum.
Table 3Go shows villus circumferences and heights tended to be affected by gestational age (P < 0.1) and were significantly affected by feeding (P < 0.01). For villus circumferences and heights there were significant gestational age x feeding interactions (P < 0.01), and both parameters showed higher levels (P < 0.01) in F0 than in F8 and P0. Crypt depths were affected by gestational age (P < 0.001) and by feeding (P < 0.001), and there were significant (P < 0.001) gestational age x feeding interactions because crypt depths increased (P < 0.05) only in full-term calves and were greater (P < 0.05) at the age of 8 d in full-term than in preterm calves. Villus height:crypt depth ratios were affected by feeding (P < 0.001), and there were significant (P < 0.001) gestational age x feeding interactions because these ratios decreased (P < 0.05) only in full-term calves. On d 8 of life villus height:crypt depth ratios were smaller (P < 0.05) in full-term than in preterm calves. The number of BrdU-labeled crypt cells was affected by feeding (P < 0.05), and there were significant gestational age x feeding interactions (P < 0.05) because feeding enhanced (P < 0.05) the number of BrdU-labeled cells only in full-term but not in preterm calves. There was also a gestational age x feeding interaction (P < 0.001) because the submucosa was thicker (P < 0.05) in P0 than in P8 and F0.


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  		Table 3. Villus circumferences and heights, crypt depths, thickness of submucosa, mucosal epithelial cell proliferation, and brush border enzyme activities in jejunum of preterm and full-term calves.1
 
Aminopeptidase A activities were reduced (P < 0.001) during feeding in both pre- and full-term calves. For aminopeptidase N activity there were significant (P < 0.05) gestational age x feeding interactions because activities increased (P < 0.05) only in full-term calves. On d 8 of life aminopeptidase N activities were higher (P < 0.05) in full-term calves than in preterm calves. Dipeptidylpeptidase IV activities were affected by feeding (P < 0.05), and there were significant gestational age x feeding interactions (P < 0.05) because activities decreased only in preterm calves but not in full-term calves. Lactase activities tended to be affected (P < 0.1) by gestational age, and there tended to be gestational age x feeding interactions (P < 0.1) because activities tended to decrease (P < 0.1) only in full-term calves. On d 8 of life, lactase activities were lower (P < 0.05) in full-term than in preterm calves.

Effects of gestational age and postnatal feeding in the ileum.
Table 4Go shows villus circumferences and heights were affected by feeding (P < 0.01 and P < 0.05, respectively). Crypt depths tended to be influenced by gestational age (P < 0.1) and by feeding (P < 0.001), and there were significant (P < 0.001) gestational age x feeding interactions because there was only an increase (P < 0.001) of crypt depths in full-term calves. On d 8 of life, the crypt depth was greater (P < 0.05) in full-term than in preterm calves. The villus height:crypt depth ratio was affected by feeding (P < 0.05), and there were significant (P < 0.01) gestational age x feeding interactions because this ratio only decreased (P < 0.05) in full-term calves. The number of BrdU-labeled crypt cells was affected by gestational age (P < 0.05). The submucosa thickness was greater (P < 0.01) in full-term than preterm calves and tended to increase (P < 0.1) with feeding.


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  		Table 4. Villus circumferences and heights, crypt depths, thickness of submucosa, and mucosal epithelial cell proliferation in ileum of preterm and full-term calves.1
 
Effects of gestational age and postnatal feeding in the colon.
Table 5Go shows crypt depths were affected (P < 0.001) by gestational age and by feeding, and there were significant (P < 0.001) gestational age x feeding interactions because the crypt depths increased more (P < 0.05) in full-term than in preterm calves. On d 8 of life, crypt depths were greater (P < 0.001) in full-term than in preterm calves. The number of BrdU labeled crypt cells was lower (P < 0.01) in P8 than in P0.


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  		Table 5. Crypt depths and mucosal epithelial cell proliferation in colon of preterm and full-term calves.1
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 REFERENCES
 
Although in the present study the heart and respiratory rates were relatively high in preterm calves and 1 calf of P8 once exhibited postprandial colic, diarrhea was not observed and the metabolic and endocrine status was very similar to full-term neonatal calves (Bittrich et al., 2002). The BW was lower in preterm than in full-term calves, as expected. However, the preterm calves gained weight in a manner similar to that of full-term calves, demonstrating that they were able to utilize the ingested feed efficiently. Thus, preterm calves evaluated in the present study postnatally adapted clinically well.

Differences in feeding of neonatal calves variably influence small intestinal growth, epithelial and subepithelial morphology (Bühler et al., 1998; Blättler et al., 2001; David et al., 2003), and GIT function (Guilloteau et al., 1997). In the present study, significant feeding effects were not seen in all parts of the small intestine. Thus, the duodenum was less affected by feeding than the jejunum and ileum. This was surprising because nutritional and nonnutritional factors of the ingested colostrum and milk replacer are expected to exert effects primarily in the duodenum (Bühler et al., 1998). It can be speculated that additional digested feed components, such as those derived from casein, to which the duodenum may not be exposed, are necessary to exert effects. There might also be local association differences in the release of trophic factors, such as glucagon-like peptide-2 and gastrin (Lipkin, 1981; Burrin et al., 2003) and of IGF and their receptors (Georgiev et al., 2003; Georgieva et al., 2003).

Vacuolated cells were present in this study in the ileum of preterm and full-term calves immediately after birth, but they were absent on d 8 in both groups. Prior to gut closure, vacuolated epithelial cells are involved in the transport of macromolecules, such as immunoglobulin G (Bainter, 2002). Therefore, the absence of vacuolated cells on d 8 of life suggests that the transport of macromolecules had likely ceased in both groups by this time. Thus, in this respect the small intestine of preterm calves behaved as those in full-term calves.

Villus-like structures were present in the colon only in preterm calves immediately after birth but not in full-term calves, and they were absent in preterm calves on d 8 of life. During fetal life the villus-like structures of the colon are thought to be involved in the absorption of components of ingested amniotic fluid (Trahair et al, 1986; Avila and Harding, 1991; Xu, 1996). Their presence in preterm calves at birth expressed a premature state of colon morphology and function. However, their absence in preterm calves on d 8 of life indicated that colonic absorption through villus structures had likely ceased and that the colon morphology and function had matured during the first week of life.

Villus circumferences and heights in the present study were greatest in jejunum, as shown previously (Blättler et al., 2001). Villus circumferences and heights at birth were greater in preterm than in full-term calves, but a significant effect of gestational age was only seen in the jejunum. Growth of the epithelium of this part of the gut during the last days of the fetal period was particularly marked. Villus sizes are negatively associated with epithelial cell proliferation rates (Blättler et al., 2001; Sauter et al., 2003), expressing negative feedback control of intestinal epithelial growth (Creamer et al., 1961; Galjaard et al., 1972; Rijke et al., 1976). Feeding and gestational age modified villus sizes in the jejunum, and villus sizes decreased in the jejunum of full-term calves and tended to increase in preterm calves. In the ileum, villus size was modified only by feeding and was similar in preterm and full-term calves during the first 8 d of life. The decrease of villus sizes in response to feeding was unexpected because feeding intensity usually enhances villus growth in calves (Bühler et al., 1998; Blättler et al., 2000) and other species, such as pigs (Zhang et al., 1996).

In the colon, crypt depths were greatest, as shown earlier in neonatal calves (Bühler et al., 1998). Crypt depths can be taken as a measure of the epithelial cell proliferation potential. Because crypt depths were affected by gestational age and by feeding and because there were significant gestational age x feeding interactions at all intestinal sites, effects on crypt sizes were much more consistent than effects on villus sizes. This also indicates that factors regulating crypt depths and villus sizes were different. Crypt depths were considerably greater in F8 than in F0 at all sites of the gut, indicating that the potential for intestinal epithelial proliferation increased with age and (or) in response to feeding in full-term calves. The inability of preterm calves to enhance crypt sizes in response to feeding during the first days of life is expected to reduce the cell proliferation potential.

Villus height:crypt depth ratios in the jejunum and ileum were influenced by feeding, and there were significant gestational age x feeding interactions for all 3 small intestinal sites. The reduction of the villus height:crypt depth ratios in full-term calves during the first 8 d of life expresses marked alterations in the intestinal structure that were absent in preterm calves.

The thickness of the submucosa behaved differently in jejunum and ileum. Thus, in the jejunum, the submucosa thickness decreased in preterm calves, but it increased in full-term calves with feeding and was greater in P0 than in F0, whereas the thickness of the total submucosa in the ileum was affected by gestational age (i.e., differed between preterm and full-term calves and increased with feeding at both ages). Peyer patches in calves are primarily localized in the ileum and are mainly responsible for the obviously much greater thickness of the GT wall in the ileum than in the jejunum and duodenum (David et al., 2003). However, additional analyses (Bittrich and Blum, unpublished observations) showed that in the ileum the greater thickness of the subepithelial layer in full-term rather than preterm calves was not due to differences in the size of Peyer patches. Thus, other components in the submucosa, such as connective tissues, must have contributed to the different submucosa thickness in preterm and full-term calves.

Based on BrdU incorporation, proliferating cells were primarily seen in the crypts of the epithelium and especially in Peyer patches, as expected and shown previously (Blättler et al., 2001; David et al., 2003; Roffler et al., 2003; Sauter et al., 2004). The number of proliferating crypt cells in the small intestine was variably affected by gestational age and by feeding. Whereas by d 8 of life the number of proliferating crypt cells in the small intestine had increased 1.4- to 2.8-fold in full-term calves, changes were numerically much smaller and nonsignificant in preterm calves. The data were consistent with those on crypt depths and indicated that crypt cells of preterm calves did not respond to feeding. As a consequence, the regeneration of the intestinal epithelium in preterm calves is expectedly reduced, thus possibly leading to functional deficits and intestinal diseases.

Apoptotic cells in calves of the present study were numerous in the lamina propria of villi, in subepithelial cell layers (and especially in Peyer patches) (David et al., 2003), and were exceptionally present in crypts, but they could not be detected within the epithelial layer of villi (David and Blum, unpublished observations). This was surprising because apoptotic cells are expected on the tips of villi before they are released into the intestinal lumen. Because there were no significant differences in proliferation rates of crypt cells between F0 and P0 in the jejunum, the greater jejunal villus size in F0 than P0 was possibly in part due to reduced apoptotic rates or greater survival times.

The highest activities of peptidases and lactase in jejunum and lowest activities in duodenum in the present study were in accordance with results obtained by Le Huërou-Luron et al. (1992) and Guilloteau et al. (1997). In the jejunum, enzyme activities in the 4 experimental groups were variably affected by gestational age and by feeding, and there were differences with respect to gestational age x feeding interactions. In the jejunum, there were no significant differences between preterm and full-term calves at birth of aminopeptidases A and N and of lactase activities and only a tendency for a greater decrease of dipeptidylpeptidase IV activity in F0 than P0. This indicates that stored brush border enzyme activities in preterm calves were in the normal range of full-term calves. Decreased aminopeptidase A and lactase activities were associated with the decreased jejunal villus sizes, but the response pattern of aminopeptidase N and dipeptidylpeptidase differed from that seen for histomorphometrical traits. Whereas aminopeptidase N and lactase activities were not significantly affected by feeding, aminopeptidase A activity was reduced in both preterm and full-term calves. This was possibly a result of reduced synthesis or enhanced degradation or increased secretion rates. On the other hand, gestational age x feeding interactions indicated that the aminopeptidase N activity was only significantly reduced in preterm calves, lactase activity also tended to be reduced only in full-term calves, and dipeptidylpeptidase IV activity was only reduced in preterm calves. The digestion of proteins and peptides between preterm and full-term calves was therefore likely different.

In conclusion, this study demonstrates that there were marked differences with respect to various histomorphological and functional traits of the small intestine and colon between preterm and full-term calves. The data indicate that the small intestinal epithelium in preterm calves was immature compared with that of full-term calves, thus confirming our hypothesis. This statement is basically in agreement with conclusions drawn from studies in neonatal pigs (Sangild et al., 2002; Petersen et al., 2003). One or several causes may be responsible for these differences. Constitutional factors were likely responsible for these different responses, such as a reduced number or sensitivity of receptors or nonfunctioning or ineffective signal-transduction pathways after exposure to nutrients and endogenous or exogenous (colostral) hormones and growth factors. In fact, there were marked differences at the protein and mRNA level of receptors for IGF-I and -II and insulin in the small intestine and colon of calves of the present study (Georgiev et al., 2003; Georgieva et al., 2003). In addition, preterm calves may suffer from an insufficient cortisol surge during the perinatal time period because perinatal glucocorticoids promote the maturation of the GIT functioning (Sangild, 2001; Sauter et al., 2003). Although in preterm calves some of the jejunal protease activities were altered during the first days of life, changes were in part different from those in full-term calves, and differences between pre- and full-term calves in digestive capacities could therefore be expected. Morphological and functional differences between preterm and full-term calves especially of the small intestine are likely important causes for greater GIT problems seen in preterm as compared with full-term calves under practical conditions.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
REFERENCES
 
This study was supported by the Swiss National Science Foundation (Grant # 32-51012.97). S. Bittrich received a grant from the H. W. Schaumann-Stiftung, Hamburg, Germany. We thank G. Savary (Unité Mixte de Recherches sur le Veau et le Porc, Institut National de la Recherches Agronomique, Rennes, France), M. Biernat, and J. Wolinski (Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Jablonna, Poland) for their significant contributions in the analyses of enzymes and M. Iburg (Lohmann Animal Health, Cuxhaven, Germany) for providing the chicken-derived immunoglobulin.


   FOOTNOTES
 
* The data have been in part presented at the 11th International Conference on Production Diseases of Farm Animals, Copenhagen, Denmark, August 13–16, 2001, and at the 3rd Annual Meeting of the European Society of Veterinary Comparative Nutrition, Sursee, Switzerland, Sept. 13–14, 2001. Back

{dagger} Part of a thesis for DVM, accepted by the Faculty of Veterinary Medicine, University of Berne, Berne, Switzerland in 2001. Back

Received for publication September 12, 2003. Accepted for publication November 14, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 


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