β-lactoglobulin as a potential modulator of intestinal activity and morphology in neonatal piglets

Authors


Abstract

Studies were conducted to determine the effects of the whey protein β-lactoglobulin (blg) on the intestinal development and intestinal morphology in neonatal piglets. Two trials (5 and 28 days) were conducted on piglets in three separate groups. One group served as a control group while the remaining two were experimental groups based on diets. The two experimental diets were a bovine colostrum powder, one with supplementation with blg and the other without. The control group remained suckling on a sow. All groups were weaned on day 20 and placed onto a solid commercial piglet diet. Enzymatic activity, total DNA synthesis, crypt depth, and villus height were all parameters used to evaluate the effect of blg. Piglets fed a diet supplement with blg had the greatest total DNA after 5 days. Sow-reared piglets had the greatest intestinal morphology development with regards to villus height. No significant differences were found in enzymatic activity between treatments. Anat Rec Part A 288A:601–608, 2006. © 2006 Wiley-Liss, Inc.

Newborn animals receive their first nutritive meal as milk. This first meal not only provides nutrition in the form of protein, fat, and carbohydrates, but also has growth factors and other substances that provide for the development of the intestinal tissue. Milk is normally suckled from the mother and provides these first nutrients to the offspring. The initial milk received by the animal from the mother, colostrum, has many important nutrients critical to the survival of the animal, such as swine.

The first milk, colostrum, provides many different nutrients that, without them, the piglet would die. Several growth factors have been identified in porcine milk. These include insulin, insulin-like growth factor (IGF), and epidermal growth factor (EGF), but other trophic substances are yet to be identified (Read et al.,1984). Porcine colostrum has concentrations of approximately 10 nM of insulin and 10–20 nM of EGF (Read et al.,1984). These two growth factors in porcine milk have been the subject of great investigation into not only the development of the entire animal but also its gastrointestinal tract.

EGF has been shown to induce changes in the gastrointestinal (GI) tract with respect to development of brush border enzymes, increased DNA concentrations, and increased intestinal weight (Malo and Menard,1982). Several brush border enzymes, including sucrase, maltase, and alkaline phosphatase, indicate levels of gut maturity (Yamashiro et al.,1989). In a review by Donovan and Odle (1994), milk and more specifically colostrum were found to be potent stimulators of intestinal growth. Increases were found in gastrointestinal weight, length, and DNA concentration in colostrum-fed neonatal piglets (Widdowson et al.,1976). Simmen et al. (1990), using neonatal piglets, demonstrated that mature milk and especially colostrum led to increased stimulation of gastrointestinal DNA. Other data, with focus on EGF, insulin, and IGF, suggest that these milk-borne growth factors may provide important regulatory signals to the neonatal piglet intestine (Odle et al.,1996).

The importance of IGF has been investigated on intestinal growth and most notably on mucosal growth. Burrin et al. (1996), using orally administered IGF-1, investigated intestinal mucosal growth. Mucosal growth (jejunal and ileal) was found to be greatest in those pigs receiving the IGF-1. It is apparent that milk and colostrum of swine have certain components that determine beneficial growth of the intestinal tract.

Artificial liquid-fed diets have been employed to enhance the survival rate of piglets. The composition of these diets is similar to maternal colostrum but the piglets have ad libitum access to the milk. One such study has revealed that a milk replacer, liquid-fed gravity diet to early-weaned piglets increased overall weight gains (Zijlstra et al.,1996).

Another component that is interesting to note in porcine milk is the presence of the whey protein β-lactoglobulin (BLG). BLG is present in milks from animals that utilize colostrum as a means of establishing immunity in the newborn. BLG has been ascribed several possible functions but one suggestion was its possible role in developing passive immunity with IgG (Jenness,1974). Since its presence appears to be coincidental in those animals that utilize colostrum as a means of establishing passive immunity, it could also assist in establishing initial mucosal growth and DNA concentrations.

BLG has been shown to pass through the acidic gastric contents without degradation (McAlpine and Sawyer,1990). Additionally, BLG can pass through the stomach intact and once presented in the gut, it can pass through into the intestinal epithelium (Lovegrove et al.,1993). These same researchers found that once BLG is presented to the intestinal epithelium, it will be transported through the intestinal cell and be presented on the basolateral side and eventually to the serum.

Other studies have examined the transport of BLG in the small intestine. Using the rabbit ileum, Caillard and Tome (1994) found that BLG is absorbed in an intact form by a transcellular pathway. Another study by Marcon-Genty et al. (1989) confirms the observation that BLG is transported intact across the gut epithelium. The purposes of the present study were to determine if supplemented BLG could modulate intestinal growth at the level of the villus and crypt and whether or not DNA concentration could be affected by BLG. Intestinal maturation levels were also analyzed, thereby indicating gut maturation levels.

MATERIALS AND METHODS

Animals

Two separate trials were performed, the first lasting 5 days (trial 1) and the second 28 days (trial 2). For both trials, 3 days prior to farrowing, three sows were brought from the Swine Field Lab at North Carolina State University to Grinnell's Laboratory. Sows were given a 3 cc injection of lutylase 36 hr prior to their estimated farrowing time. As piglets were delivered, 18 were removed from the sow for initial weight determination and then assigned to one of three groups: one control (treatment 3, n = 6) and two experimental groups. These two experimental groups were divided into two different dietary treatments (treatment 1, n = 6; treatment 2, n = 6). Both treatments 1 and 2 received experimental diets for 36 hr followed by a commercial piglet milk replacer. Two additional piglets were placed on the sow to simulate natural milk production. Control piglets remained on the sow for the 5 days in trial 1 while experimental piglets were individually housed and caged in a separate sterile room in a university swine facility (Grinnell's, Raleigh, North Carolina). Heat lamps were used on control piglets and a heating system kept the separate room for experimental piglets at 86°F. In trial 2 (n values for all treatments identical to that of trial 1, n = 6), all piglets, regardless of treatment, were weaned at day 20 and transferred to a heated rearing pen. Piglets surviving until the end of both trials (all survived trial 1; n = 6 for treatments 1 and 2 and n = 5 for treatment 3 at 28 days) were euthanized using a xylazine:rompun cocktail (1:4) followed by a fatal injection of sodium pentobarbital in compliance with the Institutional Animal Care and Use Committee.

Intestinal sections were removed from the animal and immediately placed on ice. Three sections per region of the small intestine (duodenal, jejunal, ileal) were cut and cleaned with a saline solution (1%). Sections for DNA determination were placed into conical vials and frozen at −20°C. Sections for intestinal morphology were placed into 70% ethanol for storage at 4°C to be embedded in paraffin later. Sections for enzymatic activity [maltase, lactase (β-galactosidase), alkaline phosphatase] were rinsed in saline solution (1%) and frozen at −20°C until analysis.

Diets

In trial 1, the first group was given Colostrum Plus (LaBelle, Bellingham, WA) with whey protein concentrate (WPC; Milk Specialties, Dundee, IL) enriched with 10% BLG (treatment 1). The second group was given Colostrum Plus (LaBelle) without BLG supplementation (treatment 2). The rest of the piglets remained on the sow for the duration of the study (treatment 3) while animals in treatments 1 and 2 were placed onto a commercial piglet milk replacer diet after 36 hr. Dietary treatments for groups 1 and 2 were derived from a prepackaged colostral powder (LaBelle) containing dehydrated bovine colostrum (350 g) with approximately 57 g as IgG, essential vitamins A, D, E, and viable lactic acid bacteria. The 350 g packets were dissolved into 3 L of tap water (dry matter content of 10%) and then served into 0.5 L cooled containers to prevent excessive spoiling.

As described previously, after 36 hr, piglets from treatments 1 and 2 were removed from the bovine colostrum powder and placed onto a liquid milk replacer diet.

The composition of this diet was the following: nonfat dry milk (Milk Specialties), 90.0 g/L; Fat Pack 60 (7/60, a product supplying 7% crude protein and 60% ether extract; Milk Specialties), 26.0 g/L; Fat Pack 80 (4/80, a product supplying 4% crude protein and 80% ether extract; Milk Specialties), 10 g/L; lecithin, 1 g/L; vitamin and mineral premix (Consolidated Nutrition, Fort Wayne, IN), 1.6 and 5.0 g/L, respectively. The dry matter (DM) content of this replacer diet was 12.2%, and the calculated analysis (DM basis) was as follows: crude protein, 23%; lactose, 37%; fat, 39%. This diet was homogenized in 890.0 ml (to make 1 L) in a Gaulin homogenizer (Manton-Gaulin Manufacturing, Everett, MA) at a final pressure of 140 kg/cm2.

In the second trial, lasting 28 days, the same diet formulations for all three treatments were prepared as previously described. Piglets from treatments 1 and 2 remained on their respective diets for 36 hr and were then changed to the commercial piglet milk replacer diet, as described above, until day 20. Control piglets (treatment 3) remained on the sow until day 20. All piglets, regardless of group, were weaned at day 20 and placed onto the same commercial solid starter piglet weaning diet (McNess Launcher-Plus, Furst-McNess Nutrition, Freeport, IL).

DNA Analysis of Intestinal Tissue

Tissues sections were cut (2.5 cm) from the three regions of the small intestine, duodenum, jejunum, and ileum. When the assay was ready to be performed, a TNE stock buffer was made from 100 mM Tris Base, 10 mM EDTA, and 1.0 M NaCl at a pH of 7.4.

The stock buffer was diluted 1:10 for homogenizing tissue and for dilution of samples. The dilution for homogenizing tissue was created with 2.0 M NaCl and for dilution of samples, a 1:10 dilution with distilled water was used. The source of DNA standard was commercially prepared calf thymus (Sigma, D4522) and was diluted with distilled water to give 100 μg/ml of DNA using the 1:10 TNE buffer. A Hoechst working dye (Sigma, B2883) was prepared the day of the assay to minimize alterations of the dye. To a 100 ml volumetric flask was added 10 μl of Hoechst stock solution (1 mg/ml), 10 ml TNE, and brought up to 100 ml with distilled water.

Intestinal sections were weighed and placed in polystyrene scintillation vials with approximately 100 mg of tissue per vial. Sample tissue was then homogenized in 2 M NaCl TNE for 2 min. Tissue was kept on ice until the assay was performed. When samples and standards were ready for analysis, the fluorometer was turned on and allowed to warm up (20 min). A clean cuvette was placed into the machine with 2.0 ml of blank working dye solution for calibration. DNA standard was added (100 μg/ml) and scale was set to 100. The working Hoechst reagent was added and then diluted and intestinal samples were added (10 μl aliquots) accordingly and mixed with a Pasteur pipette to ensure proper homogeneity. Hoechst dye and samples were allowed 3 min to interact before fluorescence was determined. For every 10 unknown samples processed, the fluorometer was recalibrated. To ensure accuracy, the mixing pipette was thoroughly rinsed to remove adherent DNA solution and the cuvette was thoroughly rinsed between samples. From derived DNA standard values, sample concentrations were calculated.

Intestinal Morphology

Villus height and crypt depth of intestinal tissue were examined by light microscopy. Samples were taken (2.5 cm) from all three sections of the small intestine and placed in Carnoy's solution overnight (4°C). Tissue was then stored in 70% alcohol for later use. For processing, tissues were dehydrated through a series of alcohols (70%, 95%, 100%) followed by clearing in methyl salicylate and embedded in paraffin. Embedded tissues were then molded onto blocks for sectioning.

Tissues were sectioned at 10 μm and four cut sections were placed onto each slide. Slides were organized not only by treatment but also by small intestinal section. After sections were deparaffinized, they were stained with hemotoxylin and eosin and dried at room temperature. Sections were viewed under a binocular Olympus Image Analysis System microscope for villus height and crypt depth determination. Villus height was measured from the apical tip of the villus to the base of the villus on the intestinal fold. Crypt depth was measured from the intestinal fold to the proximal portion of the basement membrane.

Enzymatic Activity

For lactase and maltase activity, the mucosa was scraped from small intestinal samples using a glass microscope slide. Substrates for the two enzymes were lactose and maltose, respectively, and were prepared in 0.1 M maleate buffer of optimum pH, which depended on the substrate. The maleate buffer was prepared using 1.16 g of maleic acid in either 15.3 or 17.8 ml of 1 N NaOH (depending on optimum pH of substrate) and diluted to 100 ml with distilled water. The substrate solution for lactose was prepared by dissolving 2.02 g of lactose monohydrate in maleate buffer of pH 6.0 and for maltose by dissolving 2.02 g of maltose monohydrate in maleate buffer of pH 6.5. Intestinal tissue was first sectioned into the three respective portions of the small bowel: duodenum, jejunum and ileum; 3 cm sections were taken from each portion of the small intestine and rinsed with 0.9% NaCl and the mucosa was scraped into a conical vial using a flat microscope slide. Scrapings were homogenized in 0.9% NaCl and dilutions were made in distilled water for each sample (maltase and lactase, 0.1 ml of homogenate in 5 ml distilled water). Forty μl of 0.9% NaCl was added in triplicate to wells to serve as blanks. Glucose was added in triplicate and in nonadjacent wells to serve as standards. Standards were made by using increasing amounts of glucose and bringing dilution up to 200 μl for final concentrations of 1, 2, 4, 6, and 10 μg glucose/40 μl. Homogenized tissue of proper dilution was added to wells in triplicate, nonadjacent to standards. Respective substrates were added to two of the three wells in homogenate triplicate in the maleate buffer. The third well served as a blank. Two separate plates were used to measure the activity of lactase and maltase. The plate was incubated for 60 min at 37°C followed by an ice bath. Tris-glucose oxidase (TGO), 250 μl, was then added to all wells including blanks and standards. TGO was prepared by using 50 ml Tris buffer (pH 7.0; dissolve 61.0 g Tris base in 900 ml distilled water, check pH, and bring up to 1.0 L), 125 mg glucose oxidase (Sigma, G 7141), 0.5 ml peroxidase solution (dissolve 50.0 mg peroxidase in exactly 50.0 ml of distilled water), 0.5 ml of o-dianisidine (5.0 ml of distilled water to commercially purchased 50 mg vial of o-dianisidine; Sigma, D 3252), 1.0 ml Triton-X 100 solution (20.0 ml Triton-X 100 in 80 ml 95% EtOH). TGO was brought up to 100 ml with Tris buffer. Finally, the appropriate substrate for either maltase or lactase was added to the third well of the homogenates and mixed. Plates were incubated again at 37°C for 60 min, removed, and absorbance was read at 415 nm on the microplate reader (BioTek EL310, Burlington, VT).

For alkaline phosphatase, intestinal mucosa was scraped and washed in a saline solution (1%). After tissue was homogenized in 0.9% NaCl and proper dilutions were made (0.1 ml of homogenate in 10 ml distilled water), tubes were set up in triplicate before homogenate sample was added. Into each tube, 1.0 ml of alkaline phosphatase substrate, disodium phenylphosphate, was added. The substrate was prepared by using 40 ml of 240 mM phenylphosphate, 50 ml of carbonate buffer (pH 10.2), and 10 ml 0.1 M MgCl2. Tubes were placed into 37°C water bath and warmed for 5 min. The sample homogenate (0.2 ml) was added to two of the three tubes while the third tube served as an assay blank. Tubes were incubated for 20 min in a water bath and removed. The reaction was stopped by adding 0.4 ml of Folin-Ciocalteu reagent (100 ml of 2 N Folin-Ciocalteu phenol reagent, 285 ml distilled water, 15 ml concentrated HCl) to all three tubes in each set. To the blank, 0.2 ml of diluted homogenate was added and all tubes received 0.64 ml 20% Na2CO3 (10.6 g Na2CO3/500 ml distilled water). Color was fully developed in 8–10 min and absorbances were recorded at 520 nm. Absorbances of the samples were then compared to the standards based on known concentrations of tyrosine.

The amino acid tyrosine is a phenol. The use of tyrosine as a standard allows for comparison of known alkaline phosphatase substrates, disodium phenylphosphate, as a measurement of phenol production. The greater the amounts of either tyrosine or phenol lead to greater absorbances and comparison of measured amounts of phenol versus standard amounts of tyrosine would allow for measurement of alkaline phosphatase activity. Final volumes of tyrosine were 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 ml, which correlated to 2, 4, 8, 12, 16, and 20 μg phenol released, respectively. The greater the activity of alkaline phosphatase, the greater absorbance as a result of greater production of phenol from the phenylphosphate substrate.

Statistics

Data were compared using SAS 6.12 (SAS Institute, Cary, NC) as well as Prism Graph Pad version 3. Treatments were compared using a one-way analysis of variance with Duncan's posthoc test (SAS) as well as Newman-Keuls Multiple Range Comparison posthoc test (Prism).

RESULTS AND DISCUSSION

The passage of nutrients in the gut generally follows a simple pattern of apical-to-basolateral passage through the small intestinal cell. For the first 24 hr of life, there are leaky portions in the gut that allow nutrients and immunoglobulins, specifically IgG, from milk to pass directly into the blood. Present in colostrum with IgG is BLG and the presence of the two together in high concentrations suggests a possible connection between the two. Other nutrients pass from colostrum into the gut such as insulin and epidermal growth factor, allowing for the proper growth and maintenance of intestinal tissue. The development of intestinal tissue is vital for the success of the animal receiving these nutrients. The importance of IgG in establishing immunity is also important. The presence of BLG in increased concentrations in colostrum leads to speculation that the protein may be a potential modulator of intestinal activity. The following tables and figures outline this possible connection between the presence of IgG and BLG and intestinal activity.

Piglets that were on either Colostrum Plus or Colostrum Plus/BLG supplementation had the most increases in weight (P < 0.001), whereas the piglets remaining on the sow for the duration had the least amount of weight gain (Fig. 1). It has been shown that liquid-fed gravity diets facilitate increased weight gain in swine (Zijlstra et al.,1996) because piglets who can access ad libitum would ingest more nutrients than piglets that could only suckle when milk was received from the sow (Zijlstra et al.,1996; Gomez and Phillips,1998). Other research has also illustrated restriction in piglet growth in piglets naturally reared rather than artificially (Harrell et al.,1993).

Figure 1.

Overall weight gains, trial 1. Weight gains of piglets on artificially reared diets (treatments 1 and 2) were not significantly different from each other but both were significantly greater (P < 0.001) than sow-reared piglets (treatment 3).

DNA Concentrations

Table 1 displays DNA concentrations for the three treatments in each region of the small intestine for the 5-day trial. Piglets from treatment 1, the Colostrum Plus supplemented group, had the highest concentration of DNA after 5 days compared to those of the sow-reared group in each region of the small bowel with an increase of almost 40% (P < 0.05). The piglets receiving the bovine colostrum without BLG supplementation, treatment 2, had higher concentrations than those of the controls but not to the extent of those with BLG supplementation. Initial exposure to colostrum in the piglets on the sow was low because of various factors such as competition among piglets for available teats, the sow turning away because of excessive heat from heating source and general lethargy of piglets due to age. Piglets individually housed had ad libitum access to bovine colostrum and therefore access to the growth-promoting substances that would lead to greater DNA synthesis.

Table 1. 
 Col +, BLG Treatment 1Col + Treatment 2Sow Group Treatment 3
Duodenum74.8* ± 8.164.7 ± 4.246.3 ± 6.1
Jejunum80.9* ± 8.373.6 ± 5.151.0 ± 6.4
Ileum75.3* ± 7.370.1 ± 7.149.2 ± 7.4

The increased weights of piglets in treatments 1 and 2 illustrate the concept of artificially rearing piglets instead of conventional suckling. Previous data have shown that piglets on gravity-fed liquid diets experience greater weight gain than those maternally suckled. This could also lead to initial higher DNA concentrations, as seen with animals in treatments 1 and 2, with or without supplementation of BLG. Gomez and Phillips (1998) used similar rearing regimens that allowed colostrum-deprived piglets either to suckle normally from a sow or to have access to restricted diets of a certain amount per day or ad libitum access. Weight gains at both 2 days and 2 weeks were higher in ad libitum piglets than those remaining on the sow (Gomez and Phillips,1998). Similar results were obtained early in this study with the suggestion of higher DNA concentrations in those piglets as a result of the increase in colostral factors that would stimulate greater GI growth. Zijlstra et al. (1996) found that early weaned piglets that were given a liquid gravity-fed diet with ad libitum access had greater weight gain, more protein, and more fat than those piglets that remained on the sow until weaning. This greater access to feed and colostrum in those piglets that are colostrum-deprived would provide greater GI exposure to these same nutrients and possibly allow for greater initial DNA concentrations.

Gastrointestinal DNA growth has been shown to be greater after the delivery of colostrum and the appropriate colostral factors. Simmen et al. (1988) demonstrated that piglets fed colostrum had greater gut DNA concentrations than piglets receiving mature milk. The growth-promoting factors in colostrum have also been shown to stimulate GI tract DNA in the first days of life (Widdowson et al.,1976). Colostrum-fed piglets have greater protein synthesis rates in liver, kidney, spleen, and skeletal muscle, suggesting that these same colostral factors that are so effective in the gut have an important role in tissues elsewhere (Burrin et al.,1992). The positive influence that colostrum has on the initial DNA concentration in intestinal tissue is seen in the first 5 days of the first trial of this study (Table 1) from treatments 1 and 2 possibly because of the greater access of colostrum and the mitogenic factors contained within it.

Table 2 illustrates the total intestinal DNA concentration after 28 days. The second trial was lengthened to 28 days to observe any prolonged effects that BLG might have on intestinal activity, most notably the increase of total intestinal DNA. In the first trial, animals in treatments 1 and 2 had the highest intestinal DNA concentrations. However, as noted in Table 2, after weaning and after 4 weeks of life, the intestinal values of total DNA are quite similar among all treatments. No differences were found after 28 days in intestinal DNA in any region. All piglets were weaned at day 20 and were placed onto the same solid starter diet. By weaning, weights of all three treatments were similar, suggesting a more consistent feed intake, which would indicate that by this time gut development was similar and would account for similarities in total intestinal DNA levels.

Table 2. 
 Col +, BLG Treatment 1Col + Treatment 2Sow Group Treatment 3
Duodenum3.76 ± .413.13 ± .814.32 ± .61
Jejunum4.19 ± .264.13 ± .444.80 ± .58
Ileum4.72 ± .625.22 ± .736.34 ± 1.22

Enzymatic Activity

Intestinal enzymatic activity was investigated to determine if BLG had any affect on gut maturation. Enzymes analyzed were lactase (β-galactosidase), maltase, and alkaline phosphatase. The significance of lactase is increased activity within the first few days of life while maltase was chosen for its increased activities after those of lactase have declined. Alkaline phosphatase indicates growth and maturation of the intestine. If BLG were to have modulatory effects within the small intestine, then the activities of these enzymes might be modulated.

Previous research has shown that lactase activity is high at birth and for the first few days of life, up until 1 week of age, when its activity begins to decline. Aumaitre and Corring (1978) demonstrated this principle in piglets up to 8 weeks of age. They followed the development of both lactase and maltase in the small intestine. Their results, similar to this study, found that lactase activity is high at birth and decreases until weaning, whereas maltase is low at birth and rises up until 8 weeks (Aumaitre and Corring,1978). Jejunal activity was highest for both enzymes in Aumaitre and Corring's study (1978) and these results were similar to this study for all three treatments. Duodenal activity was lowest in all three treatments. Results from this study indicate that lactase was higher than maltase activity in trial 1 for all three treatments, and in trial 2 maltase activity exceeded that of lactase by day 28, which is in agreement with the work of Aumaitre and Corring (1978). There were no differences found in the activities of either lactase or maltase as a result of BLG supplementation (Tables 3 and 4).

Table 3. 
TRIAL 1Col +, BLG Treatment 1Col + Treatment 2Sow Group Treatment 3
Duodenum168 ± 16170 ± 13177 ± 19
Jejunum218 ± 11208 ± 15211 ± 16
Ileum178 ± 14191 ± 21185 ± 24
TRIAL 1Col +, BLG Treatment 1Col + Treatment 2Sow Group Treatment 3
Duodenum19 ± 422 ± 928 ± 3
Jejunum51 ± 547 ± 344 ± 6
Ileum44 ± 234 ± 532 ± 3
Table 4. 
TRIAL 1Col +, BLG Treatment 1Col + Treatment 2Sow Group Treatment 3
Duodenum14 ± 313 ± 311 ± 3
Jejunum18 ± 119 ± 521 ± 3
Ileum11 ± 416 ± 217 ± 4
TRIAL 1Col +, BLG Treatment 1Col + Treatment 2Sow Group Treatment 3
Duodenum138 ± 14121 ± 21121 ± 14
Jejunum161 ± 17159 ± 13167 ± 16
Ileum144 ± 12132 ± 15134 ± 23

Alkaline phosphatase has increased activity as the piglet ages and these results are similar to those of Yamashiro et al. (1989), who found that alkaline phosphatase activity was highest in those animals that had aged at least 4 days. As in this study, these researchers found that alkaline phosphatase activities did not differ between piglets reared artificially or sow-reared. For all three treatments, alkaline phosphatase activity was highest in the jejunum and ileum and trial 2 saw an increase in activity as compared to trial 1.

Alkaline phosphatase, as its name suggests, is optimally functional in an alkaline, rather than acidic, pH. It acts to cleave phosphorous from larger molecules. The immature gastric functions of young piglets until weaning create a less acidic pH than normal, hence the investigation of alkaline phosphatase as opposed to acid phosphatase. No differences were recorded among all three treatments from either trial demonstrating that BLG did not influence the tested enzymatic activities of the small intestine of the piglet during the time period studied (Table 5).

Table 5. 
TRIAL 1Col +, BLG Treatment 1Col + Treatment 2Sow Group Treatment 3
Duodenum19 ± 221 ± 517 ± 2
Jejunum23 ± 629 ± 219 ± 3
Ileum28 ± 423 ± 625 ± 4
TRIAL 1Col +, BLG Treatment 1Col + Treatment 2Sow Group Treatment 3
Duodenum109 ± 10116 ± 18102 ± 8
Jejunum124 ± 21121 ± 12122 ± 12
Ileum132 ± 25128 ± 17126 ± 13

Intestinal Morphology

Morphometric data of piglet intestine could allow speculation into the possible role that BLG may play in increasing villi height or crypt depth. An overall increase in mucosal mass that would in turn lead to increased surface area of the developing intestine might also illustrate a possible role for BLG.

In this present study, piglets that remained on the sow until weaning (day 20) had the greatest villi heights (P < 0.05). Villi heights were similar between the two experimental groups fed artificially ad libitum, with or without BLG supplementation, but reduced when compared to those of sow-reared piglets. These results are in agreement with previous morphometric data that have investigated ad libitum feeding regimens versus naturally reared piglets. Gomez and Phillips (1998) found that both jejunal and ileal villi heights were reduced in those piglets that had unlimited access to milk in comparison to those piglets that were naturally reared on the sow (Table 6).

Table 6. 
TRIAL 1Treatment 1 Col +, BLGTreatment 2 Col +Treatment 3 Sow Group
Duodenal388 ± 12341 ± 11461 ± 27
Jejunal523 ± 31441 ± 21761* ± 36
Ileal528 ± 22687 ± 25836* ± 17

Morphometric data from the present study illustrate that jejunal villi heights were reduced in comparison to ileal heights among all three treatments regardless of diet (Table 6). Control piglets as well as piglets on both experimental diets had ileal heights that were greater than jejunal villi heights. Control piglets, however, had significantly greater ileal and jejunal heights as compared to those of treatments 1 and 2 (P < 0.05). Burrin et al. (1996) illustrated that piglets consuming a formula diet had greater ileal villus heights than jejunal heights and this research was further validated when Houle et al. (1997) showed that piglets consuming a formula diet had greater ileal heights than jejunal heights. The morphometric data from the present study are in agreement with previous work on intestinal villi heights and the addition of BLG did not affect villus height or crypt depth, further suggesting that BLG does not affect intestinal morphology. Figures 2 and 3 compare jejunal (Fig. 2) and ileal (Fig. 3) morphology. Ileal heights are greatest among all three treatments as compared to jejunal heights of all three treatments.

Figure 2.

Jejunal morphology of hemotoxylin and eosin-stained representative sections of treatments 1–3. Measurements are in μm. Treatment 3 is at 4× magnification. Treatments 1 and 2 are at 10× magnification. Significance is denoted by asterisk and was found in treatment 3 as compared to that of treatments 1 and 2 (P < 0.05).

Figure 3.

Ileal morphology of hemotoxylin and eosin-stained representative sections of treatments 1–3. Measurements are in μm. Magnification is 10× for all three treatments. Significance is denoted by asterisk and was found in treatment 3 as compared to treatments 1 and 2.

At day 28, there were no differences among treatments in the depth of the crypts. Enterocyte differentiation arises from these crypt cells and all animals, regardless of treatment, had similar crypt depths (Table 7). Table 8 shows the ratio of villus height and crypt depth. Control animals that remained on the sow until weaning had the highest ratio of villus height to crypt depth, with the greatest ratio occurring in the region of the ileum (P < 0.05; Table 8).

Table 7. 
TRIAL 1Treatment 1 Col +, BLGTreatment 2 Col +Treatment 3 Sow Group
Duodenal106 ± 1198 ± 5102 ± 8
Jejunal114 ± 9118 ± 3116 ± 5
Ileal118 ± 17129 ± 18112 ± 9
Table 8. 
TRIAL 1Treatment 1 Col +, BLGTreatment 2 Col +Treatment 3 Sow Group
Duodenal3.6:13.4:14.5:1
Jejunal4.5:13.7:16.5:1*
Ileal4.4:15.3:17.4:1*

Delivery of nutrients to an organism is usually done orally with digestion and absorption occurring in the small intestines. In the first hours of life, piglets share in this delivery system. Nutrient-rich milk is initially consumed in the form of colostrum and this colostrum is responsible for establishing proper growth and maturation of the intestinal tissue. From the initial to the subsequent decline of activity seen in lactase to the rise of activity with gut maturation seen with maltase and alkaline phosphatase, the gut is very responsive to the growth factors and nutrients present in colostrum and milk. Whether artificially reared or naturally reared, piglets receive the necessary nutrients also to stimulate DNA activity and morphological growth. The presence of BLG in colostrum could be thought to stimulate or even modulate these early intestinal changes. These initial changes are crucial to the success of the piglet but it seems that BLG does not play a major role in the development of these changes.

Ancillary