Original Paper Biol Neonate 1992;62:155-163
a Department of Animal Science and Laboratories of Molecular and Developmental Biology. The Ohio State University and The Ohio Agricultural Research and Development Center, Columbus. Ohio; b Department of Food Science and Technology. The Ohio State University, Columbus. Ohio. USA
Keywords Colostrum Small intestine Fatty acid binding proteins Pigs Nutrient regulation Development
Intestinal Development and Fatty Acid Binding Protein Activity of Newborn Pigs Fed Colostrum or Milk
Abstract Newborn pigs (n = 20) were gavage-fed sow’s colostrum, defat ted colostrum, milk, defatted milk or a 5% lactose solution over 24 h in order to evaluate effects on growth and functional differentiation of small intestine. Colostrum-fed pigs had greater (p < 0.01) mucosal mass in the proximal half of the small intestine than did the milk- or lactose-fed groups. Total fatty acid binding protein (FABP) activity and FABP activity per mg DNA in proximal intestines of colostrum-fed pigs exceeded that for the lactose group. FABP activities (per g mucosa or mg soluble protein) were greater (p < 0.01) in the proximal segments of small intestines of pigs fed whole versus the corresponding defatted secretion. These results indicate that the feeding of colostrum specifically augments perinatal intestinal growth and differentiation as manifested by in creased cellular hypertrophy and FABP activity. Milk lipid and unidentified factor(s) enriched in colostrum are inducers of intestinal FABP activity.
The gastrointestinal tract undergoes growth and functional maturation during the perinatal period. The molecular mechanisms regulating induction and progression of post natal gastrointestinal tissue development are poorly understood; however, dietary, hor monal and genetic influences are all impor tant [1],
Widdowson [2] reported increases in jeju nal and ileal wet weights during the first 24 h afterbirth in the nursing pig. During this peri od. weight of intestinal mucosa markedly in creased and this was attributed to incorpora tion of milk proteins by enterocytes [3], Fatty acid binding proteins (FABPs) are abundant, low-molecular weight proteins present within
Dr. Gregory A. Reinhart The lams CompanyResearch and Development PO Box 189 Lcwisburg, OH 45338 (USA)
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G.A. Reinhart3 F.A. Sim m ena D.C. M ahana M.E. Whitea K.L. Roehrigb
Table 1. Diet composition
Dry matter, % Nitrogen, % Ash, % Fatty acid, %
Lactose Whole solution1 colostrum
Defatted colostrum
Whole milk
Defatted milk
5.62 0.09 0.60 -
15.81 1.67 0.67 2.21
16.66 0.82 0.79 12.64
11.47 0.87 0.81 2.38
20.23 1.61 0.73 12.41
1 Diet contained 5.00% lactose, 0.39% potassium phosphate, 0.022% citric acid. 0.005% potassium citrate and 0.28% glycine.
the soluble fraction of the enterocyte. FABPs are known to bind long-chain fatty acids and their coenzyme A derivatives [4], The muco sal distribution of FABPs [5, 6] and the quan titative variation of intestinal FABPs in re sponse to dietary lipid intake [5, 7] are consis tent with the role of these proteins as potential mediators of fatty acid absorption by the gas trointestinal tract [5], The possible differential effects, after feed ing, of colostrum, milk and milk lipid on FABP activity in the small intestine of the neonate has not been previously addressed. The present study was undertaken to evaluate the effect of mammary secretions of differing composition on intestinal growth and FABP activity in the newborn pig. Materials and Methods
Tissues At the conclusion of the 24-hour feeding period, pigs were killed and the small intestine from pylorus to ileocecal junction removed, immersed in cold saline and maintained at 0-4 °C. The intestinal tract was linearized by removal of attached mesentery and di vided into proximal and distal halves. A 2-cm segment was removed at the midpoint of the proximal half (25% of length of entire intestine), submerged in fixa tive (0.1 M phosphate buffer, pH 7.4, containing 3% glutaraldehyde, 2% paraformaldehyde and 1.5 % acro lein) and stored at 4 °C until preparation for scanning electron microscopy. The remainder of the intestinal segments were weighed and the mucosa obtained as previously described [10]. Tissues were dehydrated in an ethanol-Freon series [11] and coated with platinum for scanning electron microscopy.
Cytosol Preparation
Twenty colostrum-deprived newborn pigs were ob tained immediately at birth from 2 sows and randomly allotted to one of five dietary treatments (table 1). Intragastric delivery of 20.0 ml lactose/electrolyte so lution, 20.0 ml whole colostrum, 17.8 ml defatted co lostrum, 20.0 ml whole milk or 17.7 ml defatted milk was given every' 2 h over a 24-hour period using 10-F feeding tubes. Prior to the experiment, colostrum and milk were collected during the initial 24 h and day 21 of lactation, respectively, from another group of sows. Portions of pooled colostrum and milk were centri fuged at 20,000 g for 45 min to remove lipid. Dry-mat
Intestinal mucosa was homogenized in two vol umes (w/v) of 0.154 .1/ KCI/0.01 V/ potassium phos phate buffer, pH 7.4. The homogenate was subjected to preparative ultracentrifugation for 2.0 h at 105,000,? in a prechilled Beckman type 55.2 fixedangle rotor at 4 °C. The clear supernatant layer, exclu sive of the lipid layer, was collected. Protein concentra tion of the cytosol was determined by use of the dye binding method of Bradford [12]. DNA content of tis sue homogenates was determined using an indole pro cedure [13] with calf thymus DNA as the standard.
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Feeding Regimen
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ter content (table 1) was determined by drying mam mary secretions at 100°C for 24 h. Nitrogen content (table I) was determined by the Kjeldahl method [8] and total fatty acids (table 1) by gas-liquid chromatog raphy as previously described [9],
Table 2. Effect of feeding colostrum or milk on neonatal pig intestinal weights Item
Lactose
Colostrum defatted
Milk whole
defatted
SE whole
Number of pigs
4
4
4
4
4
Intestine, g proximal, g distal, g
24.4 13.1 11.4
37.9 22.6 15.3
38.4 21.2 17.2
29.9 15.9 14.0
28.6 14.6 13.9
3.21-2 1.81-3 1.64
Mucosa, g proximal, g distal, g
18.1 9.7 8.4
27.2 16.8 10.4
29.1 16.1 13.0
24.0 13.0 11.1
21.8 11.5 10.4
2.74 1.41-2 1.6
Nonmucosa, g proximal, g distal, g
9.82 5.39 4.43
7.86 4.34 3.52
5.84 2.94 2.90
6.72 3.13 3.60
1.35 0.78 0.57
Lactose vs. colostrum (defatted and whole), p < 0.01. Colostrum (defatted and whole) vs. milk (defatted and whole), p < 0.05. Colostrum (defatted and whole) vs. milk (defatted and whole), p < 0.01. Lactose vs. colostrum (defatted and whole), p < 0.05.
Quantitation o f FA BP Activity Prior to assay for FABPs, high-molecular weight proteins (> 4 5 kD) were removed from the cytosol fraction by precipitation in 70% ammonium sulfate. pH 7.4. at 4 °C as previously described [14]. Samples were immediately centrifuged at 4,000 g for 10 min. and the supernatants collected and desalted on a PD10 column (Pharmacia, Piscataway, N.J., USA). Bind ing of 14C-oleic acid to desalted, low-molecular weight protein-containing fractions was evaluated using pre viously described procedures [15,16]. Bound and un bound fatty acids were separated using Lipidex 1000 as described [16]. No distinctions could be made between different porcine FABP protein types since the antibodies required to accomplish this are unavail able [16]. Rather, the above procedure was used to quantitate total FABP levels via the binding of ligand.
Statistical Analysis Data were analyzed by one-way analysis of variance using the l.SMLMW PC-1 statistical package [17]. Lit ter and sex effects were not significant and were there fore dropped from the statistical model. Preplanned single degree of freedom linear contrasts were made between treatment groups. The four preplanned con trasts were: ( I) lactose versus colostrum (defatted colos trum + whole colostrum), to evaluate treatment differ-
cnces between lactose and both kinds of colostrum combined; (2) lactose versus milk (defatted milk + whole milk), to evaluate treatment differences between lactose and both kinds of milk combined; (3) colos trum (defatted colostrum + whole colostrum) versus milk (defatted milk + whole milk), to determine differ ences between mammary secretion types, and (4) whole (colostrum + milk) versus defatted (colostrum + milk, to evaluate treatment differences due to lipid.
Results Colostrum-fed pigs had greater total intes tinal weights than did lactose- (p < 0.01) or milk-fed pigs (p < 0.05) (table 2). When the intestine was divided by length into halves, proximal intestine weights were greater (p < 0.01) for colostrum-fed animals compared to lactose- and milk-fed animals. For the distal half of the small intestine, colostrum-fed ani mals had greater weights (p < 0.05) than the lactose group. Total mucosal mass of the en tire small intestine was greater (p < 0.01) in
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1 2 3 4
6.24 3.01 2.60
Table 3. Effect of feeding colostrum or milk on soluble protein and DNA in intestinal mucosa1 Item
Lactose
Colostrum
Milk
SE
defatted
whole
defatted
whole
206 19.5 28.2 3.07 6.4
533 30.6 30.5 1.93 16.9
567 34.0 33.7 2.23 16.2
359 26.4 31.1 2.52 11.2
347 20.1 31.8 2.80 7.4
r**i c-i oc
184 19.8 28.3 3.43 6.1
487 46.2 29.6 2.89 15.9
451 35.3 37.1 3.49 13.8
329 28.0 43.7 4.16 7.2
290 20.4 32.3 3.04 7.4
642-3 4.02.3.7
Proximal intestinal mucosa Total protein, mg Protein, mg/g mucosa Total DNA, mg DNA, mg/g mucosa ProtcimDNA ratio
0.54-5 3.7 0.222-6 2.42-4
Distal intestinal mucosa Total protein. mg Protein, mg/g mucosa Total DNA, mg DNA. mg/g mucosa ProteimDNA ratio
Means represent 4 pigs 24 h of age. Lactose vs. colostrum (defatted and whole), p < 0.01. Colostrum (defatted and whole) vs. milk (defatted and whole), p < 0.01. Colostrum (defatted and whole) vs. milk (defatted and whole), p < 0 .10. Lactose vs. colostrum (defatted and whole), p < 0.05. Colostrum (defatted and whole) vs. milk (defatted and whole), p < 0.05. Whole vs. defatted secretions, p < 0.05.
the colostrum-fed than in the lactose-fed group. Similarly, mucosal mass in the proxi mal half of the small intestine of colostrumfed pigs was greater than those for lactose (p < 0.01) and milk (p < 0.05) groups. No differences in the distal intestine mucosal mass were detected between treatments. Nonmucosal (underlying tissue) mass was also not different between treatments. Animals fed colostrum (defatted and whole secretions) had greater total cytosolic protein in both the proximal and distal seg ments than did the lactose and milk groups (p < 0.01; table 3). Colostrum-fed animals also had greater protein content per g proxi mal intestinal mucosa than those fed lactose (p < 0.05) or milk (p < 0.10). In the distal intestine, feeding of colostrum resulted in greater intracellular protein per g mucosa
158
than either the lactose (p < 0.01) or milk (p < 0.01) group. Feeding secretions with endoge nous lipid present resulted in lower intracellu lar protein content per g of distal intestinal mucosa as compared to defatted secretions (p < 0.05). No treatment differences in total DNA content were found for the proximal or distal half of the small intestine. However, DNA content per g proximal intestinal mucosa was lower in the colostrum (p < 0.01) and milk (p < 0.05) groups compared to the lactose-fed group. ProteinrDNA ratios, however, were markedly higher in colostrum (p < 0.01, proximal and distal) and milk groups (p < 0.01, distal) compared to the lactose group. Total FABP activity in mucosae of the proximal segments were greater for colos trum-fed pigs (p < 0.10) than for those fed
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1 2 3 4 5 6 7
5.5 0.46 2.52' 3
Table 4. Effect of feeding colostrum or milk on FABP activity in small intestinal mucosa1 nmol Oleic acid bound to FABP
Lactose
Colostrum defatted
Milk
SE
whole
defatted
whole
Proximal intestinal mucosa Total activity Per g wet intestine Per g mucosa Per mg soluble protein Per mg DNA
69.7 5.67 7.60 0.416 2.52
114.1 4.99 6.61 0.208 3.98
153.9 7.83 10.05 0.334 4.70
70.6 4.32 5.35 0.218 2.31
108.4 7.45 9.49 0.461 3.38
28.12 0.923 1.213 0.0602-3 0.712
4.13 3.97 5.21 0.286 1.59
67.3 4.55 6.32 0.147 2.13
72.0 4.62 6.08 0.176 2.27
85.8 6.64 7.58 0.286 1.93
73.2 5.17 7.06 0.355 2.47
12.84 5 0.69 0.83 0.0422-6 0.48
Distal intestinal mucosa Total activity Per g wet intestine Per g mucosa Per mg soluble protein Per mg DNA
Means represent 4 pigs 24 h of age. Lactose vs. colostrum (defatted and whole), p < 0.10. Whole vs. defatted secretions, p < 0.01. Lactose vs. milk (defatted and whole), p < 0.10. Lactose vs. colostrum (defatted and whole), p < 0.10. Colostrum (defatted and whole) vs. milk (defatted and whole), p < 0.01.
lactose (table 4). FABP activity per g intes tine. g mucosa or mg cytosolic protein was greater (p < 0.01) in the proximal halves of small intestines of pigs fed whole mammary secretions compared to those fed the corre sponding defatted secretions. Feeding of co lostrum decreased (p < 0.01) proximal FABP activity per mg soluble protein while increas ing (p < 0.01) FABP activity per mg DNA. Total FABP activity in the mucosa of the dis tal half of the small intestine was greater in colostrum- (p < 0.10) and milk- (p < 0.10) fed groups than in the lactose group. The pres ence or absence of lipid in the mammary secretion did not alter FABP activity in the distal region of the small intestine. Colos trum-fed pigs had lower FABP activity per mg soluble protein in the distal intestine than either the lactose (p < 0.01) or milk (p < 0.01) group.
Villus morphology was affected by dietary treatment (fig. 1). Intestinal villi from colos trum-fed pigs were tongue-shaped and pli cated in appearance. Pigs fed defatted colos trum also had tongue-shaped villi with an irregular surface that was less plicated; des quamation was apparent in some villus tips. In contrast, intestinal villi from the pigs fed milk appeared shorter and were broadly ridged in shape. These had a smooth surface and were more blunted than the tongue shaped structures. Villi from pigs fed the de fatted milk were more plicated than those in the whole milk-fed counterparts. Intestinal villi from pigs in the 5% lactose treatment group were slender, finger-like and also pli cated.
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1 2 3 4 5 6
b
c
d
e
Discussion The data demonstrate that the small in testine. and in particular the mucosal layer, increases in mass within 24 h of enteral feed ing of colostrum or milk. Colostrum feeding increased enteric mucosal growth in newborn beagle puppies [ 18] and rats [ 19] compared to newborn or artificially fed littermates. How ever. in our study, type of diet had no differ ential effect on total mucosal DNA content. Moon [20] reported that rates of intestinal epithelial cell replacement in the newborn pig are significantly lower than in older pigs. Our data suggest that intestinal cell division in the
160
piglet is refractory to dietary influences, at least during the initial 24 h post partum. In gestion of colostrum decreased cell density (mg DNA/g mucosa) and increased protein: DNA ratios in the proximal half of the small intestine. Thus, in agreement with the results of Widdowson et al. [3], changes in mucosal mass induced by colostrum, and to a lesser extent by milk, are due largely to uptake of protein and water by preexisting cells. The lactose/electrolyte formulation used in the present study was designed to provide car bohydrate at levels similar to that of sow's colostrum and milk [21]. Moreover, protein intake by secretion type was equalized since
Reinhart/Simmen/Mahan/White/ Roehrig
Colostrum Stimulates Intestinal FA BP Activity
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Fig. 1 . Effect of feeding colos trum or mature milk on small intestinal villus morphology. Scanning electron microscopy at 25% of small intestinal length in neonatal pigs after feeding of one of the following diets for a 24hour period: whole colostrum (a), defatted colostrum (b). whole day 21 milk (c). defatted day 21 milk (d). 5% lactose/electrolyte solu tion (e). Three pigs per treatment were examined with representa tive views presented (X 150). Pigs fed colostrum (whole or de fatted) had tongue-shaped, pli cated villi. Intestinal villi from pigs fed milk (whole or defatted) appeared more blunted and smoother in appearance. Villi from pigs in the lactose treatment group were slender and fingerlike in shape.
observed that defatted secretions yielded a compact, firm curd while whole secretions resulted in a softer, flocculant material within the lumen of the gastrointestinal tract. It is therefore possible that curd formation re sulted in' a greater proportion of protein reaching the distal region. Colostrum feeding enhanced FABP activ ity in the proximal region mucosae, although this was not proportional to the increase in soluble protein concentration. However, in testinal FABP activity per mg DNA was greater in colostrum- than in lactose-fed pigs, indicating increased FABP activity per cell. FABP activity in mucosa of the proximal intestine was also affected by lipid intake, with intact secretions increasing FABP activ ity over that for defatted secretions. This is consistent with work done on other tissues in which lipids are a major stimulus for cytosolic FABP levels [7, 33, 34], However, in the distal half of the small intestine, differences in FABP activities due to dietary lipid content were not found. Results suggest a substrate induction of FABP(s) only in the proximal portion of the small intestine. Work with rats [5] and chicks [35] also showed an effect of high lipid-containing diets to increase intesti nal FABP activity, but only in the mid and distal small intestinal segments. The 24-hour feeding period used in the present study is considerably shorter than the feeding periods used previously [5, 35], and this, as well as species differences, may have contributed to these discrepancies. Ingestion of the different mammary secre tions resulted in variation in villus morpholo gy. Colostrum, and to a lesser extent milk, favored the formation of tongue-shaped villi. This phenomenon was not attributed to lac tose or lipid and is presumably due to fac tors) in the protein component. The relation ships between villus structure and nutrientabsorptive capacity are at present unknown.
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protein restriction decreases somatic growth [22] , However, colostrum has a greater nitro gen content than the mature milk (table 1) which may account for differences in intesti nal mass between the colostrum- and milk-fed groups. The differing nitrogen content of co lostrum and milk is attributed largely to varia tion in concentration of immunoglobulin [23] . Like piglets, newborn rabbits allowed to suckle have greater small-intestinal weights compared to water-fed controls [24]. How ever, unlike piglets, rabbits obtain immuno globulins totally in utero, thus their colostrum may provide other types of proteins which contribute to growth of the small intestine. Pig colostrum contains high levels of poly peptide growth factors [25-27], It has been speculated that colostral growth factors stim ulate the preferential organ growth of gas trointestinal tissue in the neonatal pig [2528]. In newborn rats, epidermal growth factor when supplemented to artificial formula stim ulated intestinal growth while the addition of epidermal growth factor antibody to natural milk inhibited intestinal growth [29], Injec tion of recombinant epidermal growth factor to 3-day-old pigs increased sucrase and maitase activities in mid and distal regions of the small intestine [30], Thus, the enhanced mu cosal FABP activity of pigs fed colostrum may reflect, in part, growth factor-stimulated syn thesis of FABP messenger RNAs and pro teins. In addition, enteral feeding is known to cause postnatal surges in regulatory peptides and hormones that alter the endocrine milieu [31] , These events may contribute to the ini tiation of developmental changes in the gut [32] , The amount of soluble protein in the intes tinal mucosa tended to reflect dietary protein intake. It is interesting to note that the feeding of defatted secretions resulted in increased soluble protein content in the mucosa of the distal half of intestine. In this regard, it was
In summary, colostrum preferentially stimulated perinatal differentiation of the small intestine as indicated by increased FABP activity. The response of intestinal FABPs to colostrum involves lipid as well as an unidentified factor(s) other than lipid or lactose, presumably in the protein compart ment. The molecular mechanism(s) by which ingested colostrum and milk affect intestinal growth and differentiation remain unknown. However, our results identify FABPs as po tentially useful markers for unraveling the
physiologic linkages of maternal mammary gland secretions and perinatal gut develop ment. Acknowledgments The authors thank Rosalia C.M. Simmen and C. Young Lee for critically reading the manuscript and Jorge Marin for assistance with animals. Salaries and research support were provided by State and Federal funds appropriated to The Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript No. 194-91.
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7 Dempsey ME. Hargis PS. McGuire DM. McMahon A, Olson CD. Salati I.M. Clarke SD. Towle HC: Role of sterol carrier protein in cholesterol metabolism. Chem Phvs Lipids 1985:38:223-237. 8 Association of Official Analytical Chemists. Official Method of Analy sis, ed 14. Washington, Association of Official Analytical Chemists. 1984. 9 Cera KR, Mahan DC, Reinhart GA: Weekly digestibilities of diets sup plemented with corn oil. lard or tal low by weanling swine. J Anim Sci J988:66:1430-1437. 10 Reinhart GA, Simmen FA. Mahan DC, Simmen RCM. White ME, Trulzsch DV: Perinatal ontogeny of fatty acid binding protein activity in porcine small intestine. Nutr Res. in press. 11 Liepens A. DeHaven E: A rapid method of cell dry ing for scanning electron microscopy. Scanning Elec tron Microsc 1978:2:37. 12 Bradford MM: A rapid and sensitive method for the quantitation of mi crogram quantities of protein utiliz ing the principle of protein-dye binding. Anal Biochem 1976:72: 248-254. 13 Hubbard RW. Matthew WT.Dubowik DA: Factors influencing the de termination of DNA with indole. Anal Biochem 1970:38:190-201.
14 Dutta-Roy AK. Trinh MV. Sullivan TF. Trulzsch DV: Choline-deficient diet increases Z-protein concentra tion in rat liver. J Nutr 1988,118: 1116-1119. 15 Glatz JFC, Veerkamp Jll: A radio chemical procedure for the assay of fatty acid binding by proteins. Anal Biochem 1983:132:89-95. 16 Reinhart GA. Simmen FA, Mahan DC, Simmen RCM. White ME: Iso lation. characterization, and devel opmental expression of pig intesti nal fattv-acid binding proteins. J Nutr Biochem 1990:1:592-598. 17 Harvey WR: Mixed model leastsquares and maximum likelihood computer program (LSMLMW). The Ohio State Univ, Columbus, Ohio. 1985. 18 Heird WC, Schwarz SM. Hansen 1H: Colostrum-induced enteric mu cosal growth in beagle puppies. Pediatr Res 1984:18:512-515. 19 Berselh CL. Lichtenberger LM. Morriss FH: Comparison of the gas trointestinal growth-promoting ef fects of rat colostrum and mature milk in newborn rats in vivo. Am J Clin Nutr 1983:37:52-60. 20 Moon HW: Epithelial cell migration in the alimentary' mucosa of the suckling pig. Proc Soc Exp Biol Med 1971;137:151-154.
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References
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a Department of Animal Science and Laboratories of Molecular and Developmental Biology. The Ohio State University and The Ohio Agricultural Research and Development Center, Columbus. Ohio; b Department of Food Science and Technology. The Ohio State University, Columbus. Ohio. USA
Keywords Colostrum Small intestine Fatty acid binding proteins Pigs Nutrient regulation Development
Intestinal Development and Fatty Acid Binding Protein Activity of Newborn Pigs Fed Colostrum or Milk
Abstract Newborn pigs (n = 20) were gavage-fed sow’s colostrum, defat ted colostrum, milk, defatted milk or a 5% lactose solution over 24 h in order to evaluate effects on growth and functional differentiation of small intestine. Colostrum-fed pigs had greater (p < 0.01) mucosal mass in the proximal half of the small intestine than did the milk- or lactose-fed groups. Total fatty acid binding protein (FABP) activity and FABP activity per mg DNA in proximal intestines of colostrum-fed pigs exceeded that for the lactose group. FABP activities (per g mucosa or mg soluble protein) were greater (p < 0.01) in the proximal segments of small intestines of pigs fed whole versus the corresponding defatted secretion. These results indicate that the feeding of colostrum specifically augments perinatal intestinal growth and differentiation as manifested by in creased cellular hypertrophy and FABP activity. Milk lipid and unidentified factor(s) enriched in colostrum are inducers of intestinal FABP activity.
The gastrointestinal tract undergoes growth and functional maturation during the perinatal period. The molecular mechanisms regulating induction and progression of post natal gastrointestinal tissue development are poorly understood; however, dietary, hor monal and genetic influences are all impor tant [1],
Widdowson [2] reported increases in jeju nal and ileal wet weights during the first 24 h afterbirth in the nursing pig. During this peri od. weight of intestinal mucosa markedly in creased and this was attributed to incorpora tion of milk proteins by enterocytes [3], Fatty acid binding proteins (FABPs) are abundant, low-molecular weight proteins present within
Dr. Gregory A. Reinhart The lams CompanyResearch and Development PO Box 189 Lcwisburg, OH 45338 (USA)
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G.A. Reinhart3 F.A. Sim m ena D.C. M ahana M.E. Whitea K.L. Roehrigb
Table 1. Diet composition
Dry matter, % Nitrogen, % Ash, % Fatty acid, %
Lactose Whole solution1 colostrum
Defatted colostrum
Whole milk
Defatted milk
5.62 0.09 0.60 -
15.81 1.67 0.67 2.21
16.66 0.82 0.79 12.64
11.47 0.87 0.81 2.38
20.23 1.61 0.73 12.41
1 Diet contained 5.00% lactose, 0.39% potassium phosphate, 0.022% citric acid. 0.005% potassium citrate and 0.28% glycine.
the soluble fraction of the enterocyte. FABPs are known to bind long-chain fatty acids and their coenzyme A derivatives [4], The muco sal distribution of FABPs [5, 6] and the quan titative variation of intestinal FABPs in re sponse to dietary lipid intake [5, 7] are consis tent with the role of these proteins as potential mediators of fatty acid absorption by the gas trointestinal tract [5], The possible differential effects, after feed ing, of colostrum, milk and milk lipid on FABP activity in the small intestine of the neonate has not been previously addressed. The present study was undertaken to evaluate the effect of mammary secretions of differing composition on intestinal growth and FABP activity in the newborn pig. Materials and Methods
Tissues At the conclusion of the 24-hour feeding period, pigs were killed and the small intestine from pylorus to ileocecal junction removed, immersed in cold saline and maintained at 0-4 °C. The intestinal tract was linearized by removal of attached mesentery and di vided into proximal and distal halves. A 2-cm segment was removed at the midpoint of the proximal half (25% of length of entire intestine), submerged in fixa tive (0.1 M phosphate buffer, pH 7.4, containing 3% glutaraldehyde, 2% paraformaldehyde and 1.5 % acro lein) and stored at 4 °C until preparation for scanning electron microscopy. The remainder of the intestinal segments were weighed and the mucosa obtained as previously described [10]. Tissues were dehydrated in an ethanol-Freon series [11] and coated with platinum for scanning electron microscopy.
Cytosol Preparation
Twenty colostrum-deprived newborn pigs were ob tained immediately at birth from 2 sows and randomly allotted to one of five dietary treatments (table 1). Intragastric delivery of 20.0 ml lactose/electrolyte so lution, 20.0 ml whole colostrum, 17.8 ml defatted co lostrum, 20.0 ml whole milk or 17.7 ml defatted milk was given every' 2 h over a 24-hour period using 10-F feeding tubes. Prior to the experiment, colostrum and milk were collected during the initial 24 h and day 21 of lactation, respectively, from another group of sows. Portions of pooled colostrum and milk were centri fuged at 20,000 g for 45 min to remove lipid. Dry-mat
Intestinal mucosa was homogenized in two vol umes (w/v) of 0.154 .1/ KCI/0.01 V/ potassium phos phate buffer, pH 7.4. The homogenate was subjected to preparative ultracentrifugation for 2.0 h at 105,000,? in a prechilled Beckman type 55.2 fixedangle rotor at 4 °C. The clear supernatant layer, exclu sive of the lipid layer, was collected. Protein concentra tion of the cytosol was determined by use of the dye binding method of Bradford [12]. DNA content of tis sue homogenates was determined using an indole pro cedure [13] with calf thymus DNA as the standard.
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Feeding Regimen
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ter content (table 1) was determined by drying mam mary secretions at 100°C for 24 h. Nitrogen content (table I) was determined by the Kjeldahl method [8] and total fatty acids (table 1) by gas-liquid chromatog raphy as previously described [9],
Table 2. Effect of feeding colostrum or milk on neonatal pig intestinal weights Item
Lactose
Colostrum defatted
Milk whole
defatted
SE whole
Number of pigs
4
4
4
4
4
Intestine, g proximal, g distal, g
24.4 13.1 11.4
37.9 22.6 15.3
38.4 21.2 17.2
29.9 15.9 14.0
28.6 14.6 13.9
3.21-2 1.81-3 1.64
Mucosa, g proximal, g distal, g
18.1 9.7 8.4
27.2 16.8 10.4
29.1 16.1 13.0
24.0 13.0 11.1
21.8 11.5 10.4
2.74 1.41-2 1.6
Nonmucosa, g proximal, g distal, g
9.82 5.39 4.43
7.86 4.34 3.52
5.84 2.94 2.90
6.72 3.13 3.60
1.35 0.78 0.57
Lactose vs. colostrum (defatted and whole), p < 0.01. Colostrum (defatted and whole) vs. milk (defatted and whole), p < 0.05. Colostrum (defatted and whole) vs. milk (defatted and whole), p < 0.01. Lactose vs. colostrum (defatted and whole), p < 0.05.
Quantitation o f FA BP Activity Prior to assay for FABPs, high-molecular weight proteins (> 4 5 kD) were removed from the cytosol fraction by precipitation in 70% ammonium sulfate. pH 7.4. at 4 °C as previously described [14]. Samples were immediately centrifuged at 4,000 g for 10 min. and the supernatants collected and desalted on a PD10 column (Pharmacia, Piscataway, N.J., USA). Bind ing of 14C-oleic acid to desalted, low-molecular weight protein-containing fractions was evaluated using pre viously described procedures [15,16]. Bound and un bound fatty acids were separated using Lipidex 1000 as described [16]. No distinctions could be made between different porcine FABP protein types since the antibodies required to accomplish this are unavail able [16]. Rather, the above procedure was used to quantitate total FABP levels via the binding of ligand.
Statistical Analysis Data were analyzed by one-way analysis of variance using the l.SMLMW PC-1 statistical package [17]. Lit ter and sex effects were not significant and were there fore dropped from the statistical model. Preplanned single degree of freedom linear contrasts were made between treatment groups. The four preplanned con trasts were: ( I) lactose versus colostrum (defatted colos trum + whole colostrum), to evaluate treatment differ-
cnces between lactose and both kinds of colostrum combined; (2) lactose versus milk (defatted milk + whole milk), to evaluate treatment differences between lactose and both kinds of milk combined; (3) colos trum (defatted colostrum + whole colostrum) versus milk (defatted milk + whole milk), to determine differ ences between mammary secretion types, and (4) whole (colostrum + milk) versus defatted (colostrum + milk, to evaluate treatment differences due to lipid.
Results Colostrum-fed pigs had greater total intes tinal weights than did lactose- (p < 0.01) or milk-fed pigs (p < 0.05) (table 2). When the intestine was divided by length into halves, proximal intestine weights were greater (p < 0.01) for colostrum-fed animals compared to lactose- and milk-fed animals. For the distal half of the small intestine, colostrum-fed ani mals had greater weights (p < 0.05) than the lactose group. Total mucosal mass of the en tire small intestine was greater (p < 0.01) in
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1 2 3 4
6.24 3.01 2.60
Table 3. Effect of feeding colostrum or milk on soluble protein and DNA in intestinal mucosa1 Item
Lactose
Colostrum
Milk
SE
defatted
whole
defatted
whole
206 19.5 28.2 3.07 6.4
533 30.6 30.5 1.93 16.9
567 34.0 33.7 2.23 16.2
359 26.4 31.1 2.52 11.2
347 20.1 31.8 2.80 7.4
r**i c-i oc
184 19.8 28.3 3.43 6.1
487 46.2 29.6 2.89 15.9
451 35.3 37.1 3.49 13.8
329 28.0 43.7 4.16 7.2
290 20.4 32.3 3.04 7.4
642-3 4.02.3.7
Proximal intestinal mucosa Total protein, mg Protein, mg/g mucosa Total DNA, mg DNA, mg/g mucosa ProtcimDNA ratio
0.54-5 3.7 0.222-6 2.42-4
Distal intestinal mucosa Total protein. mg Protein, mg/g mucosa Total DNA, mg DNA. mg/g mucosa ProteimDNA ratio
Means represent 4 pigs 24 h of age. Lactose vs. colostrum (defatted and whole), p < 0.01. Colostrum (defatted and whole) vs. milk (defatted and whole), p < 0.01. Colostrum (defatted and whole) vs. milk (defatted and whole), p < 0 .10. Lactose vs. colostrum (defatted and whole), p < 0.05. Colostrum (defatted and whole) vs. milk (defatted and whole), p < 0.05. Whole vs. defatted secretions, p < 0.05.
the colostrum-fed than in the lactose-fed group. Similarly, mucosal mass in the proxi mal half of the small intestine of colostrumfed pigs was greater than those for lactose (p < 0.01) and milk (p < 0.05) groups. No differences in the distal intestine mucosal mass were detected between treatments. Nonmucosal (underlying tissue) mass was also not different between treatments. Animals fed colostrum (defatted and whole secretions) had greater total cytosolic protein in both the proximal and distal seg ments than did the lactose and milk groups (p < 0.01; table 3). Colostrum-fed animals also had greater protein content per g proxi mal intestinal mucosa than those fed lactose (p < 0.05) or milk (p < 0.10). In the distal intestine, feeding of colostrum resulted in greater intracellular protein per g mucosa
158
than either the lactose (p < 0.01) or milk (p < 0.01) group. Feeding secretions with endoge nous lipid present resulted in lower intracellu lar protein content per g of distal intestinal mucosa as compared to defatted secretions (p < 0.05). No treatment differences in total DNA content were found for the proximal or distal half of the small intestine. However, DNA content per g proximal intestinal mucosa was lower in the colostrum (p < 0.01) and milk (p < 0.05) groups compared to the lactose-fed group. ProteinrDNA ratios, however, were markedly higher in colostrum (p < 0.01, proximal and distal) and milk groups (p < 0.01, distal) compared to the lactose group. Total FABP activity in mucosae of the proximal segments were greater for colos trum-fed pigs (p < 0.10) than for those fed
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1 2 3 4 5 6 7
5.5 0.46 2.52' 3
Table 4. Effect of feeding colostrum or milk on FABP activity in small intestinal mucosa1 nmol Oleic acid bound to FABP
Lactose
Colostrum defatted
Milk
SE
whole
defatted
whole
Proximal intestinal mucosa Total activity Per g wet intestine Per g mucosa Per mg soluble protein Per mg DNA
69.7 5.67 7.60 0.416 2.52
114.1 4.99 6.61 0.208 3.98
153.9 7.83 10.05 0.334 4.70
70.6 4.32 5.35 0.218 2.31
108.4 7.45 9.49 0.461 3.38
28.12 0.923 1.213 0.0602-3 0.712
4.13 3.97 5.21 0.286 1.59
67.3 4.55 6.32 0.147 2.13
72.0 4.62 6.08 0.176 2.27
85.8 6.64 7.58 0.286 1.93
73.2 5.17 7.06 0.355 2.47
12.84 5 0.69 0.83 0.0422-6 0.48
Distal intestinal mucosa Total activity Per g wet intestine Per g mucosa Per mg soluble protein Per mg DNA
Means represent 4 pigs 24 h of age. Lactose vs. colostrum (defatted and whole), p < 0.10. Whole vs. defatted secretions, p < 0.01. Lactose vs. milk (defatted and whole), p < 0.10. Lactose vs. colostrum (defatted and whole), p < 0.10. Colostrum (defatted and whole) vs. milk (defatted and whole), p < 0.01.
lactose (table 4). FABP activity per g intes tine. g mucosa or mg cytosolic protein was greater (p < 0.01) in the proximal halves of small intestines of pigs fed whole mammary secretions compared to those fed the corre sponding defatted secretions. Feeding of co lostrum decreased (p < 0.01) proximal FABP activity per mg soluble protein while increas ing (p < 0.01) FABP activity per mg DNA. Total FABP activity in the mucosa of the dis tal half of the small intestine was greater in colostrum- (p < 0.10) and milk- (p < 0.10) fed groups than in the lactose group. The pres ence or absence of lipid in the mammary secretion did not alter FABP activity in the distal region of the small intestine. Colos trum-fed pigs had lower FABP activity per mg soluble protein in the distal intestine than either the lactose (p < 0.01) or milk (p < 0.01) group.
Villus morphology was affected by dietary treatment (fig. 1). Intestinal villi from colos trum-fed pigs were tongue-shaped and pli cated in appearance. Pigs fed defatted colos trum also had tongue-shaped villi with an irregular surface that was less plicated; des quamation was apparent in some villus tips. In contrast, intestinal villi from the pigs fed milk appeared shorter and were broadly ridged in shape. These had a smooth surface and were more blunted than the tongue shaped structures. Villi from pigs fed the de fatted milk were more plicated than those in the whole milk-fed counterparts. Intestinal villi from pigs in the 5% lactose treatment group were slender, finger-like and also pli cated.
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1 2 3 4 5 6
b
c
d
e
Discussion The data demonstrate that the small in testine. and in particular the mucosal layer, increases in mass within 24 h of enteral feed ing of colostrum or milk. Colostrum feeding increased enteric mucosal growth in newborn beagle puppies [ 18] and rats [ 19] compared to newborn or artificially fed littermates. How ever. in our study, type of diet had no differ ential effect on total mucosal DNA content. Moon [20] reported that rates of intestinal epithelial cell replacement in the newborn pig are significantly lower than in older pigs. Our data suggest that intestinal cell division in the
160
piglet is refractory to dietary influences, at least during the initial 24 h post partum. In gestion of colostrum decreased cell density (mg DNA/g mucosa) and increased protein: DNA ratios in the proximal half of the small intestine. Thus, in agreement with the results of Widdowson et al. [3], changes in mucosal mass induced by colostrum, and to a lesser extent by milk, are due largely to uptake of protein and water by preexisting cells. The lactose/electrolyte formulation used in the present study was designed to provide car bohydrate at levels similar to that of sow's colostrum and milk [21]. Moreover, protein intake by secretion type was equalized since
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Fig. 1 . Effect of feeding colos trum or mature milk on small intestinal villus morphology. Scanning electron microscopy at 25% of small intestinal length in neonatal pigs after feeding of one of the following diets for a 24hour period: whole colostrum (a), defatted colostrum (b). whole day 21 milk (c). defatted day 21 milk (d). 5% lactose/electrolyte solu tion (e). Three pigs per treatment were examined with representa tive views presented (X 150). Pigs fed colostrum (whole or de fatted) had tongue-shaped, pli cated villi. Intestinal villi from pigs fed milk (whole or defatted) appeared more blunted and smoother in appearance. Villi from pigs in the lactose treatment group were slender and fingerlike in shape.
observed that defatted secretions yielded a compact, firm curd while whole secretions resulted in a softer, flocculant material within the lumen of the gastrointestinal tract. It is therefore possible that curd formation re sulted in' a greater proportion of protein reaching the distal region. Colostrum feeding enhanced FABP activ ity in the proximal region mucosae, although this was not proportional to the increase in soluble protein concentration. However, in testinal FABP activity per mg DNA was greater in colostrum- than in lactose-fed pigs, indicating increased FABP activity per cell. FABP activity in mucosa of the proximal intestine was also affected by lipid intake, with intact secretions increasing FABP activ ity over that for defatted secretions. This is consistent with work done on other tissues in which lipids are a major stimulus for cytosolic FABP levels [7, 33, 34], However, in the distal half of the small intestine, differences in FABP activities due to dietary lipid content were not found. Results suggest a substrate induction of FABP(s) only in the proximal portion of the small intestine. Work with rats [5] and chicks [35] also showed an effect of high lipid-containing diets to increase intesti nal FABP activity, but only in the mid and distal small intestinal segments. The 24-hour feeding period used in the present study is considerably shorter than the feeding periods used previously [5, 35], and this, as well as species differences, may have contributed to these discrepancies. Ingestion of the different mammary secre tions resulted in variation in villus morpholo gy. Colostrum, and to a lesser extent milk, favored the formation of tongue-shaped villi. This phenomenon was not attributed to lac tose or lipid and is presumably due to fac tors) in the protein component. The relation ships between villus structure and nutrientabsorptive capacity are at present unknown.
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protein restriction decreases somatic growth [22] , However, colostrum has a greater nitro gen content than the mature milk (table 1) which may account for differences in intesti nal mass between the colostrum- and milk-fed groups. The differing nitrogen content of co lostrum and milk is attributed largely to varia tion in concentration of immunoglobulin [23] . Like piglets, newborn rabbits allowed to suckle have greater small-intestinal weights compared to water-fed controls [24]. How ever, unlike piglets, rabbits obtain immuno globulins totally in utero, thus their colostrum may provide other types of proteins which contribute to growth of the small intestine. Pig colostrum contains high levels of poly peptide growth factors [25-27], It has been speculated that colostral growth factors stim ulate the preferential organ growth of gas trointestinal tissue in the neonatal pig [2528]. In newborn rats, epidermal growth factor when supplemented to artificial formula stim ulated intestinal growth while the addition of epidermal growth factor antibody to natural milk inhibited intestinal growth [29], Injec tion of recombinant epidermal growth factor to 3-day-old pigs increased sucrase and maitase activities in mid and distal regions of the small intestine [30], Thus, the enhanced mu cosal FABP activity of pigs fed colostrum may reflect, in part, growth factor-stimulated syn thesis of FABP messenger RNAs and pro teins. In addition, enteral feeding is known to cause postnatal surges in regulatory peptides and hormones that alter the endocrine milieu [31] , These events may contribute to the ini tiation of developmental changes in the gut [32] , The amount of soluble protein in the intes tinal mucosa tended to reflect dietary protein intake. It is interesting to note that the feeding of defatted secretions resulted in increased soluble protein content in the mucosa of the distal half of intestine. In this regard, it was
In summary, colostrum preferentially stimulated perinatal differentiation of the small intestine as indicated by increased FABP activity. The response of intestinal FABPs to colostrum involves lipid as well as an unidentified factor(s) other than lipid or lactose, presumably in the protein compart ment. The molecular mechanism(s) by which ingested colostrum and milk affect intestinal growth and differentiation remain unknown. However, our results identify FABPs as po tentially useful markers for unraveling the
physiologic linkages of maternal mammary gland secretions and perinatal gut develop ment. Acknowledgments The authors thank Rosalia C.M. Simmen and C. Young Lee for critically reading the manuscript and Jorge Marin for assistance with animals. Salaries and research support were provided by State and Federal funds appropriated to The Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript No. 194-91.
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