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ANIMAL NUTRITION |
Department of Animal Sciences, Purdue University, West Lafayette, IN 47907-2054
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: digestive capacity • hydrolase • intestine • pig • postnatal development
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Hydrolases and nutrient transporters anchored on the brush border of enterocytes play significant roles in the final stages of digestion and assimilation of nutrients, and the capacity of an animal in this regard dictates its ability to attain conditions of optimal growth when nutrients are provided in adequate amounts. Studies on the development of digestive functions including hydrolases (Matasushita, 1985; Ferraris et al., 1992; Biviano et al., 1993) and transport systems (Buddington and Diamond, 1990; Tolza and Diamond, 1992; Soriano and Planas, 1998) have reported age-related differences in small intestine hydrolase activities and AA transport rates. Investigations of postnatal development of small intestinal hydrolase are largely limited to the use of whole mucosal homogenates. We have used purified enterocyte brush-border preparations to investigate the ontogeny of intestinal digestive functions in ducks (King et al., 2000) and pigs (Fan et al., 2002) and have observed age-related distinctions. In the current study, the observations are extended for both morphometry and hydrolases to pigs between the ages of 1 and 9 wk, a period that includes the transition from nursing to weaning. Therefore, the objective of this study was to investigate developmental patterns of digestive capacity in growing pigs using weights, linear and area dimensions of each section of the small intestine, and jejunal brush-border sucrase and alkaline phosphatase (ALP) activities as response criteria. |
MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The intestinal morphometric measurements and mucosa samples in these studies were collected from 20 healthy pigs (2 barrows and 2 gilts from each of 5 litters) obtained from Purdue University Swine Research Center. The experimental protocol was approved by the Purdue University Animal Care and Use Committee. Littermate pigs were assigned for collection of intestinal morphometric measurements and mucosa at ages 1, 3, 5, or 9 wk. Pigs stayed with dams for 3 wk after parturition, after which they were provided free access to feed and water. Diets were based on corn and soybean meal and were formulated with CP concentrations, trace minerals, and vitamins to meet or exceed NRC (1998) recommendations for the different growth phases. During the period from 3 to 5 wk, the corn-soybean meal diet offered to pigs contained 10% whey, 4% Menhaden fish meal, and 0.375% ZnO and was formulated to contain 21% CP, 1.35% lysine, 0.75% Ca, 0.65% total P, and 0.48% nonphytate P (as-fed basis). From 5 to 9 wk old, the corn-soybean meal diet was formulated to contain 19% CP, 1.1% lysine, 0.7% Ca, 0.6% total P, and 0.41% nonphytate P (as-fed basis).
Chemicals
Dye reagent for protein determination was purchased from BIO-RAD Laboratories (Richmond, CA). Bovine serum albumin (Fraction V), p-nitrophenyl phosphate, D-mannitol, Trizma-HCI, HEPES, phenylmethylsulfonyl fluoride (PMSF), and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).
Small Intestinal Morphometry and Isolation of Mucosal Scrapings
At 1, 3, 5, and 9 wk of age, 6, 6, 4, and 4 pigs, respectively, were i.m. injected with ketamine (Fort Dodge Laboratories Inc., Fort Dodge, IA), Telazol (Fort Dodge Laboratories Inc.), and xylazine (Bayer Corp., Shawnee Mission, KS) at doses of 4, 2.2, and 2.2 mg/kg of BW, respectively. After the pre-anesthetic procedure, halothane was administered to the pigs at a 5% concentration via a facemask to achieve a surgical plane of anesthesia. While the pigs were under anesthesia, their abdomens were opened, and the entire small intestine was transected 5 cm from both the pylorus and ileocecal sphincter. The intestinal lumen was flushed with ice-cold saline solution (154 mM NaCl, 100 nM PMSF, Trizma-HCI; pH 7.4). For 1-wk-old pigs, the entire length of the small intestine was divided into 3 equal-length segments. For 3-, 5-, and 9-wk-old pigs, gut morphology was used to identify intestinal segments, and the entire duodenum (from 5 cm posterior to the pylorus to the junction with jejunum), jejunum (from the junction with duodenum to the junction with ileum), and ileum (from the junction with jejunum to the junction with cecum) were transected. The total weight, mucosa weight, length, and surface area (minus amplification by villi and microvilli using the width x length approach) of the duodenum, jejunum, and ileum were determined for the different age groups. These isolated intestinal segments were divided into sections. The sections were opened longitudinally, and mucus was removed by patting with paper towels. The mucosa was collected by scraping the luminal surfaces firmly with glass slides that were placed over ice. The mucosal scrapings were pooled within the defined duodenal, jejunal, and ileal segments for the same pig, divided, and placed in screw-capped plastic tubes. The tubes were capped, frozen in liquid N2, and stored at –70°C.
Preparation of Intestinal Brush-Border Membrane Vesicles
Brush-border membrane vesicles were prepared from 5-wk-old pig jejunal mucosa according to the modified Mg2+ precipitation procedure adapted from Maenz and Patience (1993). Approximately 50 g of mucosa scrapings was thawed in ice-cold homogenate buffer (50 mM D-mannitol, 10 mM Trizma-HCl, 100 uM PMSF; pH 7.4 adjusted with Trizma-base). The thawed tissue and homogenate buffer were divided into eight 45-mL plastic tubes and homogenized with an Omni 2000 polytron homogenizer (Omni International Inc., Waterbury, CT) with a 10-mm diameter generator at a speed of 20,000 rpm for 1 min. The resulting homogenates were pooled and centrifuged at 2,000 x g for 15 min with a preparative ultracentrifuge (Beckman Model L5-65; Beckman Instruments, Inc., Palo Alto, CA). After removing the top foam layer and discarding the pellets, the supernatant fraction was mixed with 1 M MgCl2 solution to achieve 10 mM MgCl2, stirred for 15 min, and then centrifuged at 2,400 x g for 15 min. The top foam layer and pellets were removed, and the resulting supernatant fraction was centrifuged at 19,000 x g for 30 min to generate crude brush-border membrane pellets. The supernatant fraction was poured off, and 1 mL of resuspension buffer (300 mM D-Mannitol, 50 mM HEPES; pH 7.4 adjusted with Trizma-base) was added to the pellets. The pellets were resuspended by repeated passage through a 26-gauge needle and pooled among the centrifuge tubes. The resultant crude brush-border membrane vesicle suspension was transferred into cryogenic vials in 2-mL aliquots (Nalgene Company, Rochester, NY) and frozen in liquid N2 until use. For a given hydrolase assay, a suitable number of aliquots of brush-border membrane vesicle suspensions was thawed in the vesicle resuspension buffer (150 mM D-mannitol, 200 mM KSCN, 50 mM HEPES; pH 7.4 adjusted with Trizma-base). The resuspended crude vesicles were then homogenized in a prechilled glass Wheaton tissue grinder (Wheaton, Millville, NJ) with 8 strokes before centrifugation at 39,000 x g for 30 min to generate the brush-border membrane pellets. The pellets were resuspended with a 26-gauge needle in a suitable volume of vesicle resuspension buffer (150 mM D-mannitol, 200 mM KSCN, 50 mM HEPES; pH 7.4 adjusted with Trizma-base) to give the final brush-border membrane vesicle suspension, which was analyzed for protein and hydrolase.
Protein and Hydrolase Assays
Protein concentrations of the homogenates and brush-border suspensions were determined colorimetrically with a DU-640 spectrophotometer at 595 nm (Beckman Instruments, Inc., Fullerton, CA) according to the method of Bradford (1976).
Sucrase (EC 3.2.1.48) activity was determined according to the procedure of Dahlqvist (1964). The intestinal mucosal homogenate or brush-border membrane vesicles were incubated with sucrose at 38°C, and the liberated glucose was measured by a glucose-specific hexokinase reaction. Alkaline phosphatase (EC 3.1.3.1) was assayed according to the method of Engstrom (1964). The intestinal mucosal homogenate or brush-border membrane vesicles were incubated with p-nitrophenyl phosphate at 38°C. Enzyme activities were normalized to protein content. Total jejunal hydrolase activities (µmol hydrolyzed/min) over the entire jejunal segment were estimated as enzyme activity per unit of mucosal homogenate protein x homogenate protein content per gram mucosa x total jejunum mucosa weight (Zhang et al., 1997).
Statistical Analyses
Statistical analyses were preformed using the GLM procedures of SAS (SAS Inst., Inc., Cary, NC) as a completely randomized design; individual pig was the experimental unit. Gender effect was tested in the model; however, no gender effects were detected. Means were separated using the LSD procedure at a significance level of P < 0.05.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Body weights and small intestinal weights, length, and area presented in Table 1 show the expected increase (P < 0.05) from 1 to 9 wk postnatal. There was a 7-fold increase in BW from 2.7 kg at 1 wk to 23.32 kg at 9 wk. Weight gains averaged 111, 388, and 486 g/d from 1 to 3 wk, 3 to 5 wk, and 5 to 9 wk, respectively. Body weight gains were greater (P < 0.05) from wk 3 to 5 than from wk 1 to 3. Total intestinal weights and intestinal mucosa weights increased more rapidly from 3 to 5 wk than from 1 to 3 wk; the slowest rate of increase was from 5 to 9 wk. Intestinal length and surface area decreased relative to gain as pigs grew during the 3- to 9-wk postnatal period. Intestinal weight as a proportion of BW was greatest (P < 0.05) at 5 wk for the age groups studied.
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). Weights of the mucosa in each section of the small intestine were greater (P < 0.05) at 9 wk than at 1, 3, or 5 wk of age. The length of jejunum was greater (P < 0.05) in pigs at 9 wk than at 5 wk, which was greater than in those at 3 wk of age. Surface area of each section of the small intestine increased (P < 0.05) from 3 to 9 wk (Table 2). Between the ages of 3 to 9 wk, the increase in mucosa weight was greatest for the jejunum followed by the duodenum and the ileum. Similarly, the increase was greatest for the duodenum followed by the jejunum and then the ileum when mucosal weight was expressed per unit of the appropriate intestinal segment weight (Table 2).
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Table 3 shows the jejunal mucosa protein content and hydrolase activities of the pigs. Mucosal homogenate protein concentrations of the harvested jejunums did not differ among the age groups studied. There were large variations in enzyme activities among pigs. Sucrase activity was detectable, but very low, at 1 wk of age. There was a 55-fold increase in jejunal sucrase activity from 1 to 9 wk; the greatest rate of increase occurred between 5 and 9 wk. Total jejunal sucrase activity, total jejunal sucrase activity expressed per unit of jejunal mucosa weight, and total sucrase activity expressed per unit of jejunal surface area did not differ among pigs of ages 1, 3, and 5 wk but was greater (P < 0.05) at 9 wk of age. Alkaline phosphatase activity was numerically greater at 3 wk, but it was not different from that at 1, 5, or 9 wk of age. Total jejunal ALP in pigs at 9 wk was greater (P < 0.05) than activity at 5 wk, which was greater than that at 1 wk of age. Total ALP activity normalized to mucosa weight of the jejunum was greater (P < 0.05) at 3 wk than at other ages (Table 3). The quality and purity of the brush-border membrane vesicles assessed by characterization of mucosal hydrolase showed that relative to the intestinal homogenate, the final brush-border membrane preparations from the 1-, 3-, 5-, and 9-wk-old pigs had respective enrichments of 18-, 26-, 38-, and 5-fold in sucrase activity. Corresponding numbers for ALP were 4-, 5-, 8-, and 10-fold in enrichments in ALP activities, respectively (data not shown).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Rapid growth and development during the early postnatal period significantly affects subsequent growth and thereby pig productivity. In the current study, developmental patterns of small intestine digestive capacity in pigs between 1 and 9 wk of age were investigated. The pig’s intestinal weight increased in direct proportion to its BW. In contrast, rabbit intestinal weight increased disproportionately slowly (Buddington and Diamond, 1990), and rat intestinal weight increased disproportionately rapidly (Tolza and Diamond, 1992) relative to BW. However, both studies reported disproportionate changes in small intestinal lengths and nominal surface areas to BW as in the pig. Moreover, in the cat, a strict carnivore, intestinal weight increased disproportionately slower to BW, whereas intestinal length and nominal surface area increased in proportion to BW (Buddington and Diamond, 1992). During the early postnatal period in life of the cat, its small intestine undergoes no significant increase in weight, length, or nominal surface area (Buddington and Diamond, 1992), whereas in the pig, an omnivore, the intestine undergoes intense hyperplasia within the first 24 h after birth (Widdowson et al., 1976). Indeed, a 126% increase in pig mucosal homogenate protein has been reported during the first 6 h after parturition (Zhang et al., 1997). In another study, Tarvid et al. (1994) observed that intestinal weight and length increased in direct proportion to BW in pigs from 1 to 3 wk of age. During growth between 1 to 9 wk in the current study, BW, small intestine weight, and small intestine length increased 8.5-, 8.1-, and 3.2-fold, respectively. Furthermore, associated with the increase in tissue weight was a significant increase in weight of the mucosa from 1 to 9 wk of age. This finding is an indication that, during the first 9 wk of age, the small intestine of pigs undergoes substantial increases in physical size to accommodate the processing of ingested nutrients. Intestinal weight and length expressed per unit of BW reported for 1- and 3-wk-old pigs by Tarvid et al. (1994) were similar to observations in the current study.
Anatomical distinction of duodenum, jejunum, and ileum at 1 wk of age is fraught with technical problems; thus, the small intestine was divided into three equal-length segments for 1-wk-old pigs. As a result, wk 1 morphometric data for duodenum (segment 1), jejunum (segment 2), and ileum (segment 3) may not truly represent each of these 3 segments. This uncertainty probably explains some of the observations of heavier and longer segment 1, 2, or 3 at wk 1 than of the duodenum, jejunum, and ileum at wk 3. The differential changes in weights among the duodenum, jejunum, and ileum were evident in mucosa weight. It has been reported the duodenum, jejunum, and ileum represent 4 to 5%, 88 to 91%, and 4 to 5%, respectively, of length of the small intestine in the fully grown pig (Yen, 2001). Observations from the current study indicate that the duodenum accounts for approximately 10, 9, and 7% of the length of the small intestine in 3-, 5-, and 9-wk-old pigs, respectively. Corresponding numbers for the jejunum are approximately 78, 81, and 84% with values of 12, 10, and 9% for the ileum. The jejunum, therefore, represents more of the length of the small intestine as the pig grows.
Digestive actions of mucosal enzymes occur on the brush-border surface of enterocytes compared with pancreatic enzymes, whose digestive actions take place in the intestinal lumen. Postnatal increases in pancreatic enzymes during the first 6 to 8 wk of life in pigs have been reported (Lindemann et al., 1986; Owsley et al., 1986). The current study examined postnatal changes in jejunal brush-border membrane-bound enzymes sucrase and ALP. Jejunum was selected because it represents >75% of the entire small intestine. Brush-border maltase and sucrase that serve as the final step in small intestinal digestion of starch (linear regions of its structure) to glucose were recently shown to have a common ancestral gene with shared exon structures and peptide domains (Nichols et al., 2003). The current study confirms the presence of sucrase at 1 wk of age and, thus, the potential for sucrose hydrolysis in pigs. Previously, sucrase activities generally have been found to be absent from fetal pigs (Buddington and Malo, 1996) and present at very low levels at birth (Manners and Stevens, 1972; Zhang et al., 1997). Furthermore, sucrase activity was found to be virtually nonexistent in the pig jejunum until 6 d after birth; a marked increase occurred at the second week of postnatal life (James et al., 1987). Manners and Stevens (1972) reported that maximum sucrase activity was found at the 20 and 40% sites from the proximal duodenum in younger pigs (1 to 8 wk) and at a wider range of sites 40, 60, and 80% from the proximal duodenum in older pigs (17 to 19 wk). Those researchers concluded that a trend toward extension of sucrase in a proximal to distal direction along the small intestine existed as the pigs aged. The 10-fold increase in jejunal mucosa brush-border sucrase activity between 5 and 9 wk of age is more than twice the increase from 1 to 3 wk of age, which perhaps reflects the switch in dietary carbohydrate from predominantly lactose to starch. This hypothesis is based on the known substrate effects on enzyme production in the digestive tract, which in the current situation would be expected to increase the production of sucrase for the hydrolysis of digestion products of starch by amylase. Age-related developmental changes in brush-border sucrase have been observed in ducks (King et al., 2000), pigs (Fan et al., 2002), and rats (Pacha et al., 2003; Sabat and Veloso, 2003). There was no significant change in intestinal brush-border ALP activity of pigs among the age groups examined in the current study. This observation is similar to those reported for ducks and rats (King et al., 2000; Pacha et al., 2003) but different from the findings of Fan et al. (2002), who observed that ALP was greatest in 28-d-old pigs. Indeed, ALP was numerically greatest in 3-wk-old pigs in the current study, but high variation in ALP data masked statistical detection of the difference.
The magnesium-precipitation method used in the current study produced stable preparations of purified porcine jejunal brush-border membrane vesicles. The purity of intestinal membrane preparations is usually assessed in terms of enrichments of marker enzymes. The enzymes used are anchored in the brush-border membranes of the small intestine. Traditionally, disaccharidases (sucrase and maltase), peptidases (amino-peptidase), and ALP (Salloum et al., 1993) have been used as marker enzymes for enzyme enrichment. The sucrase enrichment in the current study was greater than the 20-fold enrichment reported in rat and rabbit jejunum (Hopfer, 1987). The 8-fold ALP enrichment in the present study for 5-wk-old, 10-kg pigs was less than the 21-fold enrichment reported for 15-kg pigs by Maenz and Patience (1993) and the 13-fold enrichment reported by Fan et al. (1998) for 110-kg pigs. The different enrichment values reported in the literature are suggestive of species- and age-related enzyme-specific ontogenetic patterns of biosynthesis and insertion into brush-border membranes of the small intestine (Buddington and Malo, 1996).
In conclusion, relative BW, intestinal weight, and mucosa weight normalized to BW in pigs were significantly greater from 3 to 5 wk than from 1 to 3 wk, but specific jejunal sucrase and ALP activities were unchanged from 1 to 5 wk. In avian species, it was proposed that whole body growth rates are determined in part by the allocation of tissue to the gastrointestinal tract (Obst and Diamond, 1992). This assertion also might be true in mammals because in the present study, the most rapid intestinal growth rates correlated with the greatest whole body growth rates. These results support the premise that age-related increases in intestinal brush-border enzymes support developmental changes in hydrolytic capacity and that amplified intestinal growth is observed during postnatal growth in the pig.
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Footnotes |
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2 Corresponding author: ladeola{at}purdue.edu
Received for publication February 25, 2005. Accepted for publication August 16, 2005.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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