JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2008, 59, Suppl 1, 35–54
www.jpp.krakow.pl
R. ZABIELSKI1, M.M. GODLEWSKI1,2, P. GUILLOTEAU3
CONTROL OF DEVELOPMENT OF GASTROINTESTINAL
SYSTEM IN NEONATES
Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University
of Life Sciences, Warsaw, Poland; 2Department of Chemistry and Biomedical Sciences, Faculty
of Environmental and Life Sciences, Macquarie University, Sydney, NSW 2109, Australia;
3INRA, UMR 1079, Systeme d'Elevage, Nutrition Animale et Humaine (SENAH), Domaine
de la Prise, 35590 - Saint.-Gilles, France
1
Our recent studies of structure and function of gastrointestinal tract mucosa revealed
that the domestification of Sus scrofa corresponds with the significant slowing of the
organ development. On top of genetic potential, the nutritional factors (or more
precisely - lack of certain biologically active substances in the feed of pregnant sows)
are responsible. Moreover, feeding neonates with milk replacers instead of mother's
milk further slows down the development. This is manifested by reduced mitotic
activity in the crypts and enhanced apoptosis of enterocytes. The negative effects
consist of slower replacement of fetal type, vacuolated enterocytes to adult type
enterocytes, modified profile of brush border enzymes, alterations in intestinal
mucosa barrier, higher susceptibility to infectious agents, and many others. On the
other hand, farmers in order to intensify the production, shorten the suckling period
imposing the neonatal piglets to be weaned at 3-4 weeks of life and even earlier.
Altogether, it makes the weaning disorders one of the most important problems in pig
husbandry, and the mortality of piglets in the leading pig-producing countries still
reaches 10%. A number of strategies have been developed to counteract the postweaning problems. One of them is to stimulate the development of the
gastrointestinal tract of the neonate by supplementation of the sow diet with certain
biologically active substances and plants. The other idea is to speed up the postnatal
development of the gut mucosa for example by plant lectins. Lessons from pig
studies can be also useful in human nutrition and medicine since the development of
porcine gastrointestinal tract shows a great similarity to that of humans.
K e y w o r d s : enterocyte, maturation, wearing rebuilding, apoptosis, lectin, neonatal pig
36
INTRODUCTION
The postnatal development of the gastrointestinal (GI) system is a very
dynamic process. In the neonatal pig with the mean birth body weight of 1.45 kg,
the small intestine and pancreas weight contribute to ca. 3.1% and 0.14% of the
total body weight, respectively (Table 1). Within the first four postnatal weeks
weight of the piglet is increased >5-fold, with the GI organs growing faster than
many other organs of the body (Table 1). In addition to an intensive growth of the
GI system, during the first month of life an intense rebuilding of the tissues takes
place. The most intensive processes are observed in the epithelium of the small
intestine (1, 2) as discussed further below. Can we suspect the same changes in
human neonates? Presumably yes, but the intensity of the remodeling is not as
dramatic. The development in humans is slower, and the growth rate is slower in
comparison to pigs. For example, in humans the birth weight is doubled within
ca. 170 days. Nevertheless, a number of similarities between pig and human in
the processes of the development can be seen.
Progress in the understanding of digestive physiology and nutrition helped to
find a number of factors limiting the animal performance. The composition of food
was accordingly adjusted to energy and protein (limiting amino acid) requirements
as well as minerals and vitamins in relation to the age and productivity. This
information is easily obtainable in the textbooks, and present milk replacer
formulas provide farm animals and human babies with similar available energy
and protein to maternal milk. More recently, the role of a variety of bioactive
substances from milk and solid food on the GI perinatal development was
elaborated. Most of these substances are ingested in low quantities (micro- to
picomoles) and are of minor relevance as nutrients, but are important as regulators
of local GI and/or general functions in the neonate organism. The list of substances
is as long as complex is the composition of colostrum and milk. Included are: milk
regulatory proteins and peptides with a number of different physiological roles
(e.g., hormones, growth factors, immunostimulators, antibacterial, antiinflammatory and transport proteins; for review see: 3, 4), amino acid derivatives,
Table 1. Contribution of the selected organs to the total body weight in landrace x Pietrain crossbred
piglets. Data obtained in a state livestock farm in Poland in 2004 (Zabielski et al - unpublished data).
Body weight (kg)1
Small intestine2
Pancreas2
Stomach2
Heart2
Brain2
1
mean±SD, 2% of total body weight.
Unsuckling neonates
(0 d old)
Suckling piglets
(28 d old)
1.45±0.22
3.10
0.14
0.48
0.76
2.07
8.17±1.22
4.03
0.15
0.49
0.56
0.59
37
polyunsaturated fatty acids, oligosaccharides and antioxidants. More recently an
important role of plant lectins (regarded as anti-nutritional factors) in stimulating
the maturation of GI mucosa was described (5,6). This review will briefly discuss
the role of several bioactive substances in the control of physiological GI
development, and introduction of this knowledge into practice.
PERINATAL DEVELOPMENT OF THE GASTROINTESTINAL TRACT
The development of the mammalian GI system is preprogrammed, but the
program can be enhanced or lessened during the intrauterine and early postnatal
life. The most critical (and most extensively studied) period for piglets is
weaning, when the switch from liquid (mother's milk) into the solid food takes
place. In the intensive animal production the switch is abrupt (without any or with
inadequate period for the adaptation). Weaning for farm animals occurs in an
early age, when the GI system motility, digestive and absorptive functions are not
yet matured and prepared for food other than milk. In a wild boar, domestic pig
ancestor, the offspring is weaned in much older age and change of the diet is
gradual, therefore weaning disorders are nearly nonexistent. In intensive livestock
production shorter suckling period benefits in increased number of piglets born
per year, but at the negative side is an increased number of weaning disorders.
The total mortality of piglets in majority of pig farms oscillates around 10%, and
weaning disorders are major cause of these high records. A number of strategies
have been proposed to overcome weaning problems, but most of them address
treatment after weaning and concern only the reduction of post-weaning diarrhea.
Unfortunately these strategies do not target the reasons of the problem. We think
that the best strategy lies in early-age conditioning of gastrointestinal tract which
should prepare the piglets long before weaning occurs. As discussed further
below, an induction of precocious maturation in the GI system of newborn piglets
with kidney bean lectins (phytohaemagglutinins) is one of them (5,6).
Program of the GI tract development. Rebuilding of the small intestinal epithelium.
The perinatal development of the GI system can be divided into three phases.
The prenatal phase is characterized by minimal stimulation from the GI lumen,
the neonatal phase is associated with milk suckling, and the post-weaning phase
is associated with the adaptation of the digestive system to utilize solid feed
components. The present review will focus mainly on the neonatal phase of small
intestinal functions. A number of studies published in the recent years illustrate
the progress in our understanding of the role of nutrition and neuro-hormonal
pathways in the regulation of small intestine functions (for references see 3,4,7).
During perinatal development of the mammalian gastrointestinal tract two
opposing processes intertwine. One that allows transient absorption of
immunoglobulins and other biologically active molecules in an intact form
38
through the unique apical canalicular system (ACS) in fetal-type enterocytes. The
other process is associated with fast development of the digestive functions and
increased integrity of the epithelium. The neonatal pig doubles its weight within
the first week of postnatal life, but the weight of the small intestine increases
much quicker, doubling within the first 2-4 days. Such drastic changes rely on
three major mechanisms: (a) the increase of local GI blood flow parallel with a
reduction in basal vascular resistance (8), (b) accumulation of colostrum proteins
in the enterocytes as a result of an open "gut barrier" (9), and finally (c) changes
in epithelial cell turnover, namely, increased mitosis accompanied by the
inhibition of apoptosis which result in a 2-fold increase in the mitosis/apoptosis
ratio within the first 2 postnatal days (Table 2). Currently none artificial feeding
system (milk, artificial milk formula, nor feeding with any other compositions
like lactose, glucose solutions) could reproduce the developmental characteristics
obtained with maternal colostrum feeding. Furthermore, high specificity of
colostrum, especially concerning the composition of hormones and bioactive
compounds prevents utilization of colostrum of other species as the replacement.
After birth, the length of the villi quickly increases by the sheer action of large
volume of blood distributed into the intestinal mucosa. Numerous transverse
furrows enable villi elongation without extra energy cost, mechanical effect
reminiscing the accordion (Fig. 1). Within a few postnatal days the number of
transverse furrows and their depth is dramatically reduced (11). After the first
colostrum intake the volume of enterocytes is markedly increased due to colostral
proteins taken up and stored in large-size vacuoles. The effect is so severe that
vacuoles may occupy a half of the cell volume and sometimes even more
(1,12,13). Observed height of duodenal enterocytes increased from 14,6 µm at
birth to 20.0, 25.7 and even 33.7 µm, respectively, at postnatal day 3, 7 and 14.
Postnatal changes observed in the jejunum and ileum were not that dramatic (13).
Shift in the equilibrium between mitosis and apoptosis in the epithelial cell is
vital for maturation. In the early postnatal period, development of intestinal
mucosa is associated with profound tissue remodeling and modification of gut
digestive and absorptive functions (13,14,15). The process of remodeling of small
intestinal mucosa concerns enlargement of the absorptive capacity and adaptation
of brush border enzymes. Both improve digestion of food and absorption of
Table 2. Remodeling of intestinal epithelium in neonatal piglets; mitotic and apoptotic indexes in
the whole mid-jejunum cross-sections evaluated with laser scanning cytometry. Adapted from
Godlewski et al. (10).
Mitotic index (%)
Apoptotic index (%)
Mitosis/Apoptosis ratio
Unsuckling
neonates
Suckling
24 h after birth
Suckling
7 d old
Weaned
12 w old
4.50
21.8
0.20
6.10*
15.9**
0.38
4.45
21.8
0.20
4.80
25.1
0.19
Different from unsuckling neonates; t-test, * P<0.05, ** P<0.01.
39
nutrients. The remodeling may be achieved by modification of enterocyte
functions and/or by the replacement of old epithelial cells (fetal-type enterocytes)
with a new enterocyte generations (adult-type ones) (13,16). The successor cells
differ significantly in the morphology and protein expression pattern. For
instance, severe modifications occur in the composition of membrane receptors,
brush border enzymes and transporter proteins. Full replacement of one
generation of epithelial cells occurs in a few days (17). In sow-reared piglets the
proliferation of crypt stem cells is significantly increased within first two days
after birth (Table 2). Simultaneously, the decrease in number of cells undergoing
apoptosis was observed, resulting in rapid increase of a total number of epithelial
cells (Table 2). Increase in mitotic index is associated with augmented cell
differentiation into the enterocytes, goblet, endocrine and immune cells (18).
Modification in enterocyte structure and function (i.e., the disappearance of large
lysosomal vacuoles) has a significant impact on the closure of the intestinal
barrier. The kinetics of intestinal epithelium rebuilding depends on a variety of
hormones, growth factors and regulatory peptides which are present in
Fig. 1. The surface of duodenal
villi at birth with characteristic
transverse furrows in neonatal
pig (top). Villus fracture shows
continuity of the epithelial layer
in the transverse furrows.
(bottom). The depth of
transverse furrows in neonates
may achieve 20 µm. Scanning
electron microscopy picture
obtained by the courtesy of dr.
dr. T. & H. Skrzypek, Catholic
University of Lublin, Poland).
40
colostrum/milk and/or are released locally in the GI mucosa. These substances
exert the profound effects on the proliferation, differentiation and programmed
cell death. Among them insulin, leptin, ghrelin, epidermal growth factor (EGF),
insulin-like growth factors (IGFs), tumor necrosis factor-α (TNF-α),
transforming growth factor-β (TGF-β), and glucagon-like peptide-2 (GLP-2)
seem to play the most important role (for references see: 4).
Vacuolated fetal-type enterocytes are observed in mammalian fetuses from the
second trimester of pregnancy (1,19). They appear first in the upper part of villi in
the proximal small intestine, and slowly expand downward to the lower small
intestine. The unique feature of fetal-type enterocytes is the occurrence of
cytoplasmic vacuoles of various size which constitute the ACS (1). The habitual
feature of these cells is the craft ability to transport intact proteins from the gut
lumen across the epithelium into circulation (cell subpopulation with transport
vacuoles) or to digest the gut content inside the cell (cell subpopulation with
digestive vacuoles) (16). The enterocytes producing transport vacuoles are present
in the entire small intestine (Fig. 2) and play a key role in the uptake of colostral
Fig. 2. a - SEM micrographs of the longitudinal section of vacuolated fetal enterocytes from ileum
at birth showing microvilli, AEC, large vacuoles and nucleus traces. b-d - Intestinal villi in the distal
jejunum in 14 d old suckling piglets stained with hematoxiline and eosine (b), alcian blue (c), and
PAS (d) (obj. 60x). Large size lysosomal vacuoles (black arrows) located between the cell apex and
nucleus in the enterocytes can be distinguished from the goblet cells (white arrows) with their
content stained with alcian blue (acid and neutral mucopolysaccharides in blue and red,
respectively) and PAS (neutral mucopolysaccharides in red-violet). Adapted from ref. 13.
41
macromolecules (immunoglobulins, hormones, growth factors, etc). In piglets,
these enterocytes are observed only during the first 2-3 days of postnatal life. The
enterocytes producing digestive vacuoles are present in the lower part of the small
intestine, and support the digestive processes by intracellular enzymatic digestion
of milk protein. The enterocytes with digestive vacuoles disappear gradually from
proximal jejunum to the ileum. In piglets the whole process takes approximately
3-4 weeks. The speed of vacuole disappearance is strictly associated with the gut
maturation, i.e., shift into an adult type of digestion and absorption (5,13). Adulttype enterocytes have no ACS and loose the ability to produce large size vacuoles.
The only mature epithelial cells where the capacity to sample gut content persists,
like in the vacuolated, transport enterocytes, are M cells overlying the Peyer's
patches (19). Vacuolated enterocytes have not been found in the gut of new-born
infants. However, in the fetal gut after the formation of the villi (from the 13th to
14th week of fetal life) vacuolated cells were observed in the middle and distal parts
of the small intestine (20,21). Between the 6th and 7th months (at about 30 weeks)
of gestation vacuolization gradually disappears (21,22). Thus, similarity in the
changes occurring in human fetuses as in the distal gut of 3-week-old rats and pigs
can be found. Following Baintner (16) we may call this process foetal closure, and
the timing appears to be unique among mammals.
The bad and good apoptosis
Programmed cell death (PCD) is a process in which organism eliminates
surplus, used, altered or damaged cells in the way safe for surrounding cells.
Unlike in necrosis PCD does not trigger the inflammation, remnants of dead cell
are quickly and efficiently eliminated by macrophages or surrounding cells and
the continuum of the tissue remains intact. PCD consists of two intertwining
processes: apoptosis and autophagy, which are strictly controlled genetically and
on the level of protein interactions. Apoptosis is the process of cell elimination,
while autophagy plays dual role. On early stages it protects cell by reducing the
number of proapoptotic organelles. Only during late, advanced stage it facilitates
cell death via digestion of cytoplasm in autophagolysosomes. In the
gastrointestinal tract PCD together with mitosis are the major driving forces
behind mucosa remodeling and the exchange of enterocytes. In the intestinal
mucosa two major PCD events take place in the early life of mammals. First is
the gradual exchange of fetal-type enterocytes to the fully functional adult-type.
It takes place during first weeks of life. Later, during weaning, the increased
apoptosis is associated with the exchange of diet type and is believed to be the
source of intestinal disorders. These points out the "two faces of apoptosis": on
one hand the process is crucial for the proper growth and maturation of intestinal
mucosa, on the other hand, excessive apoptosis, especially associated with
reduced mitosis (common at weaning), leads to GI disorders and infections. In
42
this chapter we shortly discuss the apoptosis in the intestinal epithelium, and give
indication on when and how to modify the process.
In the intestinal mucosa, especially in early postnatal life, apoptosis is
common, with dying cells present in crypts, along the whole length of the villi,
and culminating in massive apoptosis at villous tip in so called extrusion zone.
Characteristic to young age are groups (packets) of several apoptotic cells dying
together (10,11). This process is facilitated by auto/paracrine factors, such as
TGF-β1 and TNFα cytokines, that transmit death signal along the mucosa layer
and even across the lumen between neighboring villi (10,15). Two major
intracellular pathways of apoptosis are known, and both play a role in the
exchange of enterocytes (10). On the receptor pathway, cells response to
apoptotic signal from the outside. This is the pathway mediated by proapoptotic
cytokines, responsible for apoptotic packets in the intestinal mucosa. TGFs are
the major cytokine involved (10,23). TGF receptors signal is transmitted via
cascade of secondary messengers, the SMAD proteins, to the cell nucleus, where
it facilitates changes in the expression of major regulatory proteins of apoptotic
machinery. The equilibrium shifts to the benefit of apoptosis and cell becomes
sensitized to proapoptotic signaling mediated by other cytokines (15). The second
major pathway of apoptosis, the mitochondrial pathway, generates internally after
disruption of cell homeostasis. This type of cell death is characteristic for
intestinal crypts where it facilitates removal of cells with altered or mutated DNA
(15). Autophagy, the second type of PCD, is controlled by different sets of genes.
During autophagy cellular compartments are surrounded by two-layer lipid
membrane, in which cytosol and organelles are degraded by cathepsines (15).
The ratio between mitotic and apoptotic indexes is the marker of processes of
enterocyte turnover and intestinal mucosa maturation in young mammals. After
first intake of colostrum in suckling piglets mitotic index increased significantly,
while highly significant decrease was observed in number of apoptotic cells. This
resulted in two-fold increase in mitosis to apoptosis ratio (Table 2) highlighting
the phase of intensive growth of intestinal mucosa and the start of the exchange
of enterocytes to adult type. A week after, mitosis to apoptosis ratio returned to
the value observed in unsuckling neonates (Table 2), suggesting the end of
enterocyte exchange and final maturation of the gut mucosa. Consequent with this
data were observations of MAP I LC3 expression, a protein involved in
stabilization of autophagosomes, also associated with formation of ACS vacuole
membranes in fetal-type enterocytes (15). The high expression of MAP I LC3
was observed during first four days of life. In day 7 MAP I LC3 index was
reduced by half (24). A common practice is premature weaning of piglets and
substitution of natural milk by milk formula. This leads to significant alterations
in the process of maturation of intestinal mucosa. The decreased mitotic index
together with increase in apoptosis observed at day 7 of life (10) reduce the
nutrients uptake and deteriorate the gut barrier leaving piglets weak and
susceptible to infections. During weaning the high apoptosis in the intestinal
43
mucosa exceeds the potential of the repair in the gut epithelium. High number of
dying enterocytes leaves open gates for the infections in the mucosa plane understood as the space between neighboring cells left unzipped for a period of
time. This markedly increases chance for microbial infections. In the era when
antibiotic supplements in the diet are restricted, the aim should be prevention of
the GI disorders by alimentary regulation. Our research performed on newborn
piglets indicates, that supplementation of milk formula with leptin lowers
apoptosis index to the level observed in piglets fed colostrum and milk. At the
same time increase in the mitotic index allows quicker mucosa maturation (10).
Similarly, a cocktail of bioactive compounds (24) supplemented to the sow diet
during late pregnancy and lactation period significantly decreased the apoptotic
index in the small intestine mucosa after first food intake, allowing better
absorption of colostrum in their offspring. In the following days increased mitotic
index was observed, that facilitated quicker exchange of enterocytes and gut
mucosa maturation (24). In young calves supplementation of milk formula and
starter diet with encapsulated butyrate increased significantly the mitotic index in
small intestine epithelium. Accordingly, a significant decrease in apoptosis ratio
was observed (25). Ghrelin, an important GI hormone, contra-partner of the leptin
in the regulation of appetite and food intake, presents an interesting pattern of
action. In newborn piglets and rats it acts as an antimitotic agent and, in higher
dose, induces apoptosis (26). In adult rats, however the observed effect was
opposite. Ghrelin acted as a stimulant of GI mucosa growth presumably through
activation of IGF-1 (27,28). Also a cytoprotective action of these hormones on
gastric mucosa was reported (29,30).
In conclusion, a stimulation of maturation and remodeling of gastrointestinal
mucosa can be efficiently regulated to prevent disorders in early life of mammals.
The use of bioactive compounds and tissue hormones as MF and feed
supplements limits the necessity of antibiotic treatments. After first food intake
the delicate stimulation of apoptosis in the enterocytes quickens the gut
maturation process. On the other hand, during weaning increased apoptosis may
lead to alterations and weakening of intestinal barrier and infections. At this time
action should aim at prevention of the apoptosis.
Neurohormonal regulation
During the prenatal phase, the development of the small intestine largely
depends on the composition of nutrients and biologically active substances
transferred through the placenta. At first the contribution of endogenous neural
and endocrine systems is small, but it gradually increases with fetal
development. The development of the neural system in the GI system is
manifested by neuron density, morphology, and distribution of transmitters and
modulators. Relatively little specific information on this topic is available,
particularly in pigs GI tract. Vagal nerves are formed relatively early in fetal
44
development and quickly begin to release various neuromediators into the
synapses, i.e., acetylcholine, neuropeptide Y (NPY), substance P, and vasoactive
intestinal peptide (VIP). At birth the development of enteric nervous system
(ENS) is not completed. It's development continues in the postnatal life. In
neonatal calves, vagal reflexes seem to be functional at birth. We have found that
vagal-dependent stimulation of pancreatic secretion (cephalic phase) steadily
increased with age and reached its maximum between 2-4 weeks of life
(Zabielski et al., unpublished data). In human infants, Wester et al. (31) studied
post mortem morphology and density of neurons, and the distribution of
myenteric plexus in the small and large intestine. They found an age-related,
significant reduction in the myenteric plexus network, and reduction in the
density of ganglion cells in the myenteric plexus during the first years of life.
This suggests that the postnatal development of the enteric nervous system is a
relatively long-term process. The key feature describing the ENS during its
development is the neuronal plasticity in the expression of neurotransmitters, in
particular VIP, pituitary adenylate cyclase-activating polypeptide (PACAP),
galanin and nitric oxide (NO) (for references see: 31). Natarajan and Pachnis
(32) suggest that a certain number of multipotent cells can be stored in the ENS
and, following the induction, may differentiate into neurons and glial cells in
adult animals. The role of hormones, involving gut regulatory peptides and
growth factors, was studied more intensively than that of the autonomic nervous
system. Still a great number of questions exists since a great number of
differences related to the particular organs, phases of development, and the
species have been found. The GI endocrine system produces some gut regulatory
peptides during the prenatal period (34). The role of these peptides on GI
development of fetus is suggested by alterations in the development of
gastrointestinal mucosa observed when circulating concentrations of gastrin
were decreased by antrectomy (35). However, studies on the role of gastrin
releasing peptide (GRP) and cholecystokinin (CCK) did not confirm their effect
on the fetal pancreas (36,37).
The prenatal phase of development affects the postnatal function of the GI
tract, particularly during the first few postnatal days. The regulation of small
intestine development (especially the tissue growth) is in a positive feed-back
loop to colostrum and milk intake (38). After birth in pigs the underdeveloped
hormonal and neural systems controlling the gut functions undergo the period of
intensive development and expansion. For example, in 28 d old milk fed piglet,
the level of plasma CCK is 4 fold higher than at day 5. At 7 days of age, gastrin
concentration in pig mucosa is 4.6 fold higher than at birth (39). Similar pattern
as for CCK was observed also for glucagon-like peptide-2 (GLP-2) concentration
(Guilloteau et al, unpublished data). The development of control mechanisms in
pigs seems to be accelerated when compared with rodents and carnivores. The
relative immaturity is manifested by dynamic changes in the expression of gut
regulatory peptides, tissue hormones and their receptors in the gut during the
45
early postnatal period. Van Ginneken and Weyns (40) quantified secretin and
gastric inhibitory peptide (GIP) immunoreactivity (IR) in the duodenum, jejunum
and ileum of fetal and neonatal piglets. In addition, sections were processed for
GLP-1. The volume density of the tunica mucosa increased after birth, giving rise
to a decreased volume density of the tela submucosa and tunica muscularis.
Generally known region-specific morphological distinctions were reflected in
different volume densities of the various layers. The highest volume density of
GIP-IR epithelial cells was observed in the jejunum of the neonate. In contrast,
the volume density of secretin-IR epithelial cells was highest in the duodenum of
both fetal and neonatal piglets. The volume occupied by GIP-IR and secretin-IR
epithelial cells increased in the jejunum after birth. Additionally, ileal secretin-IR
epithelial cells were more numerous in the neonatal piglet.
At birth, a functional immaturity of hormonal and nervous systems is
compensated by the presence of gut regulatory peptides in colostrum and milk.
That induces the postnatal development of the gut as an endocrine organ and
helps to control digestive functions until endogenous regulatory systems are
adequately developed (41,42). In calf, ingestion of colostrum caused a marked
rise in plasma concentration of the most of gut regulatory peptides, including
gastrin, CCK, VIP and pancreatic polypeptide (PP) and a decrease of
somatostatin concentration (43). These peptide levels could stimulate GI growth
and digestive functions. Indeed, duodenal morphology and stem cell proliferation
are modified by feeding high amounts of first colostrum, which enhances the
survival rate of mature mucosal epithelial cells (44).
A particular attention must be given to the endocrine L-cells product, GLP-2,
since it is considered as a potent growth factor of small intestinal epithelium
which stimulates stem cell proliferation and enterocyte regeneration, and
simultaneous reduction of apoptosis. This peptide also seems to enhance
epithelial barrier and reduce gut permeability (45,46). Maturation effects of GLP2 were manifested by modifications in the brush border enzyme activities and
membrane protein transporters (47,48). In re-fed mice physiological doses of
GLP-2 regulated the dynamic adaptation of the gut mucosal epithelium in
response to luminal nutrients (49). Exogenous GLP-2, besides direct effects on
the intestinal mucosa has been shown to stimulate the blood flow rate in the
proximal small intestine and pancreas (50). Leptin (51) was found to control the
small intestine development in neonatal pigs. The contribution at physiological
level is assured by the fact that it is produced in the mammary glands and secreted
into the colostrum and milk in a number of species including pig (52-56).
Exogenous leptin increased length of the small intestine and mitotic index, and
subsequently enhanced the disappearance of vacuolated enterocytes - sign of
increased cell turnover. The profile of brush border proteases and lactase
activities was changed to the levels observed in older, more mature small
intestine. The intestinal integrity was also enhanced as demonstrated by lower
absorption of marker macromolecules (56). The mechanism of leptin action is
46
associated with the abundance of specific receptors in the mucosa (57,58).
Ghrelin, a growth-hormone-releasing acylated peptide, was isolated from rat and
human stomachs (59,60). Kotunia et al. (61) found a significant reduction in the
body weight and small intestine length following intragastric administration of
ghrelin in pig neonates. Morphologically, a reduction in the length of intestinal
villi, increase in crypt depth as well as enlargement of enterocyte lysosomal
vacuoles were observed in ghrelin-treated piglets. This lead to a conclusion of
retarded intestinal mucosa development. These results, though somehow
paradoxically, fit with the recent data on the development of rat pancreas and
stomach presented by Dembinski et al. and Warzecha et al. (27,28). In their study,
administration of ghrelin in suckling rats did not affect the body weight gain,
whereas in young adults (7 w old rats) it stimulated it. In suckling rats, ghrelin
decreased the pancreatic and stomach weights, pancreatic amylase content,
pancreatic and stomach DNA synthesis and DNA contents. In contrast, ghrelin
increased all these factors in weaned and 7 w old rats. Ghrelin increased serum
level of growth hormone in all rat groups. The effect was weak in suckling rats,
higher in weaned and the highest in 7-w old animals. Ghrelin did not affect serum
level of IGF-I in suckling rats whilst in the older rats it caused an increase.
Authors concluded that the biphasic effect of ghrelin in young rats on the
pancreas and stomach growth seems to be related to age-dependent changes of the
release of anabolic IGF-I.
Elimination of lactose, lipids, proteins and peptides other than
immunoglobulins from the milk diet considerably changes the profile of plasma
gut regulatory peptides, including plasma leptin. This may result in an
unfavorable effect on gut motility, gastric and pancreatic secretion and absorptive
function of the GI tract (42). In a similar way, additives applied to artificial milk
formula may result in either partial or total deprivation of regulatory substances
affecting the GI development. Thus, in neonatal piglet Biernat et al. (62) have
demonstrated numerous detrimental "early effects" of formula feeding on the
development of intestinal mucosa (reduction of crypt depth, villous size and the
thickness of tunica mucosa).
Consequences of development program disturbances
Disturbances in the development program observed in offspring during
prenatal and/or neonatal periods, may result in a predisposition to nutritional
diseases in adult life (visceral adiposity) and complications linked to an
overweight, i.e., hypertension, cardiovascular diseases, diabetes, obesity, and
growth of certain cancers (63-69). As an example, in rodents, obesity could be
programmed following a prenatal exposure to low (70) or high (71) level
protein diet ingested by the mother according to the fact that several types of
imbalance in the mother diet could result in the same response in the offspring.
However, data on the GI function in this aspect are missing. The only data
47
available so far come from the endocrine pancreas. It is known that the
intrauterine growth retardation leads to impaired insulin secretion and in longer
term (in adolescent or adult age) resulting in insulin resistance. Recently, an
extensive study was conducted on pregnant sows fed unbalanced protein diets.
Preliminary data in their offspring showed a number of anatomical
modifications of GI organs as well as in the activity of intestinal brush border
and pancreatic enzymes (Guilloteau et al. - unpublished data). Further studies
aiming in measurement of enterocyte rebuilding will give more light on the role
of disturbed developmental program in the neonatal period on the susceptibility
to diseases in the adolescent and adult age.
PERINATAL DEVELOPMENT AND DOMESTIFICATION
The average birth weight of wild boar piglet is 500-700 g. Within first month
their body weight is increased >5-fold, ratio similar to that found in livestock
production. However, recent studies using scanning electron microscope suggested
that the domestification of Sus scrofa was associated with significant slowing of GI
tract mucosa development. Skrzypek et al. (72) showed that no major differences
in mucosa structure was observed at birth, but the wild boar crossbreed neonatal
piglets showed more dynamic development of small intestinal mucosa when
compared to Landrace x Pietrain piglets. The most spectacular differences
concerned intestinal villi architecture, including more abundant transverse furrows,
larger extrusion zones, and higher number of goblet cells in wild boar crossbreed
piglets. The number of apoptotic enterocytes in wild boar crossbreed neonates was
also higher suggesting faster process of epithelium rebuilding.
Previously, we have found that feeding pig neonates with milk replacers
instead of mother's milk further slows down the development. This was
manifested by significantly less mitotic activity in the crypts and enhanced
apoptosis of enterocytes, slower replacement of fetal type vacuolated enterocytes
with adult type enterocytes (10). These findings corroborated with slower kinetic
changes in the profile of brush border enzymes, and mucosa more permeable to
macromolecules (73). On the other hand, farmers in order to intensify the
production shorten the suckling period imposing the neonatal piglets to be
weaned at 3-4 weeks of life or earlier. Altogether, it makes the weaning disorders
one of the most important problems in pig husbandry, and the mortality of piglets
in the leading pig-producing countries is still near 10%.
WAYS TO MANIPULATE THE GI TRACT DEVELOPMENT
The perinatal development of the GI system seems to be sensitive to
nutritional manipulation. In our recent studies pregnant and lactating sows were
supplemented with a blend of biologically active substances (taurine, L-carnitine,
48
polyunsaturated fatty acids, flavonoids, antioxidants, and vitamins C, A and E).
The blend of bioactive substances was established following extensive literature
survey in a way to construct ideal placental supply in a late fetal life as well as
ideal milk composition during lactation period. By the way, one of conditions for
blend composition was relatively low cost of the ingredients allowing further use
in livestock production. Transfer of supplemented molecules into the foetal
tissues, colostrum, milk and neonatal tissues was observed (2,74) as well as the
urinary excretion of supplemented vitamins C, E and A by pig neonates (75).
Carnitine was the only supplement that did not change in colostrum, milk and
tissues of the offspring (2). Extensive examination of the GI organs showed a
number of signs of accelerated maturation. In the gastric mucosa, the proteolytic
activity was increased (2). In the small intestinal mucosa an enhanced capacity to
absorb colostral proteins was found in the first postnatal days, leading to higher
body weight gains and enhanced bone functional development (74). In the
following days histology examination of intestinal mucosa showed enhanced
maturation in piglets from supplemented sows as indicated by increased crypt
depth and faster disappearance of fetal-type enterocytes expressing lisosomal
vacuoles as compared to control piglets from non-supplemented sows
(Grabowska, Zabielski et al. - unpublished data). The corresponding study by
Strza³kowski and co-workers (24) indicated that the mitotic index in the mid
jejunum crypts in offspring of the supplemented sows was significantly increased
during the first postnatal week. Interestingly, p53 expression was extremely low
in supplemented group presumably due to the abundance of antioxidants, omega3 acids and taurine supplemented with mother's milk. Supplementation might
diminish oxygen free radicals in the mucosa resulting in lower number of
defectively divided crypt cells that have to be eliminated and in consequence
increased pool of fully functional epithelial cells (24). Overall, the mitosis to
programmed cell death ratio was increased and the maturation of epithelium was
substantially quickened which confirmed histology and functional studies. This
study indicates that manipulation with bioactive substances supply of the diet of
pregnant and lactating sow may accelerate maturation of the small intestinal
epithelium in their offspring and prepare better for transition into solid food at
weaning.
Among the substances which could be used in practice (76) to accelerate the
program of GI development rebuilding is lectin extracted form kidney bean
(Phaseolus vulgaris, L). This glycoprotein is constituted from a mixture of
erythro- and leucoagglutinating isolectins (phytohaemagglutinins, PH-A): PHAE4, PHA-E3L, PHA-E2L2, PHA-EL3, and PHA-L4. A number of studies
demonstrated that kidney bean lectin given at specific age to neonatal piglets (1014 d of life) can accelerate the process of intestinal mucosa maturation. In such
pre-conditioned gut weaning results in a significantly smaller frequency and
intensity of disorders of GI tract functions (5,6). Knowledge about red kidney
bean lectin is relatively plentiful, since the kidney beans are one of the most
49
commonly used leguminous plants in human and farm animal nutrition. Problems
of poisoning by consumption of improperly cooked kidney beans were
recognized well over a half of the century ago (77,78) and prompted a number of
extensive studies in animals. The target organs of kidney bean lectin seem to be
the digestive tract, in particular the small intestine, and the pancreas (5,79,80).
The effect is associated with lectin binding to gut epithelial cells (80) and
stimulation of crypt stem cell mitoses (5). The effect on the pancreas is driven by
CCK (81). Other tissues are less affected and seem to be influenced indirectly,
since the absorption of kidney bean lectin is limited according to Pusztai (82) or
even nonexistent according to immunohistochemical studies of Linderoth and coworkers (80). Intragastric administration of kidney bean lectin to neonatal
suckling piglets at 10, 11 and 12 days of life resulted in significant reduction of
villi length and significant increase of crypt depth, mitotic index, and cell
apoptosis as compared with controls (5). A number of functional changes were
observed, which suggest accelerated maturation of intestinal mucosa, such as
faster disappearance of vacuolated enterocytes, kinetic changes in brush border
disaccharidase activities and reduction in intestinal tissue permeability for marker
molecules (5). The p53 protein expression was found only in the intestinal crypt
region in both the control and lectin-treated groups of piglets, however, its
expression was markedly reduced in the lectin-treated group (Fig. 3). The results
of laboratory studies have been verified in standard pig farm conditions in a study
involving total of 298 piglets in 4 different farms in Eastern Poland. Kidney bean
lectin extract (Suilectin®, BIOLEK) given between day 10 and 14 of life increased
Fig. 3. Localization of p53 (FITC - green fluorescence) in the middle part of the jejunum of weaned
38 d old piglets that were treated with kidney bean lectin (1) or saline (2) when they were 11 d old.
The control slices show a greater abundance of p53 suggesting more intense DNA alterations. Lens
magn. 20x (Zabielski et al. - unpublished data).
50
the daily body weight gain measured at 49 or 63 days of life, improved the feed
conversion ratio and reduced weaning diarrhoea as compared with controls (6).
CONCLUSIONS
Concluding, there are a number of strategies developed to counteract the postweaning problems in livestock. But only a few consider the physiology of the GI
development. One of them is to stimulate the development of the gastrointestinal
tract of the neonate by supplementation of the sow diet with certain biologically
active substances and plants. The other is to speed up the postnatal development
of the gut mucosa for example by plant lectins. Lessons from pig studies can be
also useful for humans since the development of porcine gastrointestinal tract
shows great similarities to that of humans.
Acknowledgements: Grant no. PBZ-KBN-083/P06/2003 from Ministry for Research and Higher
Education, Poland, and bilateral program POLONIUM (nr 7068/R07/R08 Poland, and 13968PE
France) are acknowledged.
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R e c e i v e d : July 5, 2008
A c c e p t e d : July 24, 2008
Author’s address: Prof. Romuald Zabielski, DVM, PhD, Department of Physiological Sciences,
Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Nowoursynowska 159, 02-766
Warsaw, Poland. Tel.: +48603757933; e-mail: [email protected]