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Timing of the Intestinal Barrier Closure in Puppies
S Chastant-Maillard
1,2
, L Freyburger
3,
*, E Marcheteau
3
, S Thoumire
4,5
, JF Ravier
6
and K Reynaud
4,5
1
INRA, UMR 1225, IHAP, Toulouse, France;
2
Universite
´de Toulouse, INP-ENVT, UMR 1125, IHAP, Toulouse, France;
3
Universite
´Paris-Est,
Ecole Nationale Ve
´te
´rinaire d’Alfort, Maisons-Alfort, France;
4
INRA, UMR 1198 INRA/ENVA Developmental Biology and Reproduction, Jouy-en
Josas, France;
5
ENVA, UMR 1198 INRA/ENVA Developmental Biology and Reproduction, Maisons-Alfort, France;
6
MERIAL, Lyon, France
Contents
As puppies are born with very low immunoglobulin concen-
trations, they rely on passive immune transfer from ingested
colostrum to acquire a protective immunity during the first few
weeks of life. The purpose of this study was to describe the
timing of gut closure in canine neonates. Twenty-two Beagle
puppies received 3 ml of standardized canine colostrum at 0, 4,
8, 12, 16 or 24 h after birth using a feeding tube. Blood
immunoglobulins G (IgG, M and A) were assayed 0, 4 and
48 h after colostrum ingestion. IgG absorption rate was
significantly affected by the time of colostrum administration,
and the IgG concentrations in puppies serum 48 h after
administration were significantly higher when colostrum was
ingested at 0–4 h of age than at 8–12 h or 16–24 h (1.68 ±0.4,
0.79 ±0.07 and 0.35 ±0.08 g/l, respectively; p <0.001). In the
canine species, gut closure seems thus to begin as early as 4–
8 h after birth and to be complete at 16–24 h. Consequently,
this phenomenon appears to occur earlier in puppies than in
most other species.
Introduction
In puppies, mortality rates from birth to weaning range
from 20% to 40%, with more than half of the cases
occurring during the first 3 weeks after birth. Infection,
especially by E. coli,Bordetella bronchiseptica and Strep-
tococcus sp, is identified as the cause of at least 30% of the
deaths (Nielen et al. 1998; Scha
¨fer-Somi et al. 2005).
As puppies are nearly agammaglobulinaemic at birth
(Bouchard et al. 1992), absorption of colostral antibodies
is crucial for neonatal and paediatric immunity. Puppies
rely on colostrum as the main source of circulating
antibodies during the first 3–6 weeks of their life. In
bovine, equine and porcine species, the passive immune
transfer to the newborn through colostrum is one of the
key elements to control morbidity and mortality rates until
weaning (Levieux 1984; Besser and Gay 1994). Optimiza-
tion of the passive transfer of ingested immunoglobulins
requires that ingestion of colostrum occurs within the first
hours after birth (24 h for calves, for example): the ability
of gut to absorb ingested immunoglobulins decreases with
time elapsed from birth (Stott et al. 1979a). This phenom-
enon, known as intestinal barrier closure (Lecce and
Morgan 1962), is not conclusively timed yet in the canine
species, despite its interest for puppies’ management. The
aim of this study was thus to describe the kinetics of the
intestinal barrier closure in puppies.
Materials and Methods
Bitches
All bitches used in this study were housed in an
experimental kennel. They were routinely vaccinated
against distemper, adenovirus, parvovirus and parain-
fluenza and specifically vaccinated against canine her-
pesvirus 1 within 10 days after insemination and at
30 days after insemination (EURICAN HERPES;
Merial, Lyon, France).
Colostrum
Mammary secretions from five Beagle bitches were
manually collected 1 or 2 days after whelping. They
were pooled, aliquoted (3 ml samples) and frozen (20°C)
until distribution.
Puppies
Four Beagle bitches (aged 20, 22 months, 4 and 5 years,
respectively, and different from those on which colostrum
was collected) were inseminated with the sperm collected
from two Beagle males of proven fertility. Ovulation time
was determined through regular blood progesterone
assays and transabdominal ultrasound examinations.
Insemination was performed with fresh sperm 48 and
72 h after ovulation. Sixty or 61 days after ovulation, 22
puppies were delivered by elective caesarean section with
no sign of anoxia. Puppies were weighted at birth. Every
4 h, they were fed with artificial milk using a baby bottle
(Mixol, Laboratoire Moureau, Luzarches, France) pre-
viously assayed for canine immunoglobulins (containing
no detectable canine IgG, IgM or IgA following the
method below), except for one meal when they were given
3 ml frozen/thawed colostrum via an orogastric tube.
Depending on the experimental group, colostrum was
given at birth (H0 group; n =4), four (H4; n =3), eight
(H8; n =3), 12 (H12; n =4), 16 (H16; n =3) or 24 (H24;
n=5) hours after birth. Blood (1 ml) was collected from
the jugular vein into plain tubes immediately before
colostrum administration and then at 4 and 48 h after it.
After the second blood sampling, puppies were allowed to
suck from their dam.
Immunoglobulins (Ig) assay
Canine IgG, IgM and IgA were assayed in duplicate on
sera, on artificial milk and on colostrum (Dog IgG-,
IgM-, IgA-Quantitation Kits; Bethyl Lab, Montgomery,
AL, USA). IgG absorption rates were calculated for
each group as the ratio between the amount of IgG
contained in 3 ml colostrum (IgG concentration in
colostrum 93/1000 g) and the amount of IgG in the
puppies bloodstream 48 h after colostrum administra-
tion (blood volume 9(1%PCV) 9serum IgG con-
centration).
©2012 Blackwell Verlag GmbH
Reprod Dom Anim 47 (Suppl. 6), 190–193 (2012); doi: 10.1111/rda.12008
ISSN 0936–6768
Statistical analysis
Data were pooled (H0 +H4: group H0–4n=7;
H8 +H12: group H8–12 n =7; H16 +H24: group
H16–24 n =8) and analysed through the non-paramet-
ric Kruskal–Wallis and Mann–Whitney tests. Results
are expressed as mean ±SEM, and differences were
considered significant when p <0.05.
Results
Weights at birth were not significantly different between
groups (mean ±SEM: 272 ±8.7 g, n =22). Before
colostral administration, circulating immunoglobulin
concentrations were low (0.3 ±0.01 g/l IgG,
0.1 ±0.01 g/l IgM, non-detectable IgA) and not differ-
ent between groups. The colostrum fed contained
17.8 g/l IgG, 1.1 g/l IgM and 20.6 g/l IgA.
Four hours after administration, a significant increase
in blood IgG concentration was observed for group H0–
4 and H8–12, but not for H16–24 (Fig. 1). IgG
concentrations at 4 h after administration were affected
by the age at colostrum ingestion (p <0.001). IgG
concentrations were significantly higher in group H0–4
than in group H8–12 (1.68 ±0.4 and 0.79 ±0.07 g/l,
respectively; p =0.007), and higher in group H8–12
than in group H16–24 (0.35 ±0.08 g/l; p =0.006).
Within groups, IgG concentrations were not signifi-
cantly different between 4 and 48 h after administration,
and the same effect of the age at colostrum administra-
tion was noticed (p <0.001; Fig. 2). Similarly, the IgG
absorption rate steadily decreased with age at colostral
administration (p <0.001), being higher in group H0–4
than in group H8–12 (29.6% ±8.2% vs 10.7% ±1.2%;
p=0.01) and higher in group H8–12 than in group H16
–24 (2.1% ±1.1%; p =0.001; Table 1).
The same differences were observed between groups
for IgA, both at 4 and 48 h after colostrum adminis-
tration. At 4 h after administration, IgA concentrations
were 0.7 ±0.3 g/l for group H0–4, 0.4 ±0.2 g/l for
group H8–12 and 0.1 ±0.0 g/l for group H16–24
(p <0.05). Serum IgA concentrations decreased
between 4 and 48 h after colostrum administration.
Conversely, IgM concentrations increased over the
same period. The time elapsed from birth and colostrum
ingestion influenced IgM only at 4 h post-ingestion
(p =0.01) and not at 48 h.
Over the 22 puppies studied, 19 reached the age of
2 months without any morbidity.
Discussion
Because of the endothelial structure of the canine
placenta, circulating Ig concentrations are quite low in
the neonates at birth. In this study, serum IgG concen-
tration before colostral ingestion was approximately
0.3 g/l, that is, only 1.5% of the IgG concentration 48 h
after ingestion in the optimal conditions (H0–4). In the
literature, the placental transfer was found to account
for 1–7% of the total Ig concentration in the canine
neonate, immunoglobulins being acquired by colostrum
ingestion (Poffenbarger et al. 1991; Bouchard et al.
1992). Transfer of colostral Ig from the colostrum to
the blood is the result of a transient, non-selective
macromolecular transport across the small intestinal
absorptive epithelium, involving uptake by apical
tubules and micropinocytotic vesicles and secretion at
the basement membrane. The absorbed Ig molecules
enter the bloodstream with the intestinal lymph via the
thoracic duct. Absorption rates vary among species and
are 5–25% in piglets and 8–90% in calves, depending on
the calculation method (Levieux 1984). In the present
study, the maximal absorption rate at birth was
approximately 40%. Our calculation method may have
resulted in a lower absorption rate than actual as we
presumed that the circulating volume was not modified
by colostrum/milk administration, and we neglected any
eventual extravascular transfer of Ig.
The intestinal epithelium of the newborn retains the
ability to absorb macromolecules for only a few hours.
This ‘gut closure’ phenomenon, defined as ‘the cessation
of absorption of macromolecules from gut to blood in
neonates’ (Lecce and Morgan 1962), seems to occur
earlier in puppies than in calves or in piglets. In piglets,
it is described at 24–36 h of age (Lecce and Morgan
1962). In calves, the reduction in the absorption to half
its efficacy at birth is observed to be between 8 and 20 h,
generally approximately 12 h (Stott et al. 1979a; Levi-
Hours aŌer colostrum administraƟon
Blood IgG concentraƟon (g/L)
0.00
H+0 H+4 H+48
0.50
1.00
1.50
2.00
2.50
3.00
H0
H4
H8
H12
H16
H24
Fig. 1. Blood IgG concentration in puppies according to the age at
colostrum administration (H0 n =4; H4 n =3; H8 n =3; H12 n =4;
H16 n =3; H24 n =5). IgG were assayed 0, 4 and 48 h after
administration
H0-4
n = 6
Blood IgG
concentraƟon (g/L)
Age at colostrum administraƟon
H8-12
n = 7
H16-24
n = 8
0
0.5
1
1.5
2
2.5
3
3.5
4
Q1
min
median
max
Q3
Fig. 2. IgG concentration at 48 h after administration according to
the age at colostrum ingestion. Box plot analysis. Q1: upper quartile;
Q3: lower quartile; min: minimum; max: maximum
Intestinal Barrier Closure in Puppies 191
©2012 Blackwell Verlag GmbH
eux 1984); in comparison, in our study, the same
reduction by 50% was obtained already at 4 h after
birth in puppies (Table 1). In cats, seems to be approx-
imately 16 h after birth, that is, also earlier than in most
other domestic species (Casal et al. 1996).
In calves, mean closure time was approximately 25–
26 h and similar for IgG, IgM and IgA (Stott et al.
1979a). In our experiment, as IgM concentrations
continued to increase between 4 and 48 h after birth,
it was not possible to conclude about the time of closure
for this Ig class, but it seems to be later than 24 h after
birth. This finding is similar to what was observed in
kittens, in which serum IgM concentration steadily
increased to plateau only at approximately day 60 of life
(Casal et al. 1996). Closure for IgA occurs approxi-
mately 16–24 h after birth, as administration at that
time was followed by no increase in serum IgA
concentration. In puppies as in kittens (Casal et al.
1996), IgA levels peak at colostrum ingestion and
gradually decline. IgA have been shown to transudate
reversely from blood through the epithelium of the
respiratory tract (Salmon et al. 2009).
Gut closure seems to occur earlier in puppies than in
other species, except for kittens. Because of ethical
considerations, puppies were not starved until colos-
trum administration but fed with milk devoid of
canine Ig. Nevertheless, it cannot be ruled out that
feeding may have hastened gut closure by a few hours,
as demonstrated in piglets, lambs and calves (Stott et al.
1979a). In calves, feeding at birth shortens the Ig
absorption period by 12 h compared with calves fed for
the first time at 24 h (21–24 h vs 31–33 h) (Stott et al.
1979a). Therefore, our experimental design does not
exactly fit the situation encountered by puppies starved
from colostrum because of their mother’s death, in
which the gut closure may be delayed by complete
starvation. The effect of mammary suckling versus
feeding with baby bottle or feeding tube on absorption
has also to be examined, as suckled calves have higher
absorption rates (Stott et al. 1979b). Presence of dam or
occurrence of stressors may also influence the timing of
gut closure (Selman et al. 1971). The exact mechanism
of gut closure has yet to be elucidated, but it probably
reflects a combination of exhaustion of pinocytotic
capability and enterocyte replacement by a mature
population of epithelial cells, together with development
of intestinal enzymes, increased stomach acidity and
installation of digestive flora. What determines the shift
from cells capable of pinocytosis to cells with microvilli
and enzymes is also unknown; this may relate to a role
of some hormones such as insulin, corticosteroids and
thyroxine or contact of cells with glucose at the time of
colostral ingestion (Levieux 1984).
Conclusion
IgG was the predominant isotype during the first days of
life of puppies as reported by Bouchard et al. (1992) and
Poffenbarger et al. (1991). IgG are key elements of the
immune protection during the early weeks of life.
Crucial for systemic protection, they also transudate
reversely into the intestinal lumen and probably
decrease intestinal virus replication (Salmon et al.
2009). This study demonstrates that the canine intestinal
barrier remains permeable to immunoglobulins mainly
during the first 12 hours after birth, but with a sharp
decrease in absorption as early as after 4 h. Therefore,
attention to maternal suckling has to be given very early
after birth for the optimization of the passive immune
transfer in puppies. Nevertheless, the minimal quantity
and quality for colostrum required to limit morbidity
and mortality remain to be determined in puppies.
Acknowledgements
The authors acknowledge Dr Alexandre Feugier (Royal Canin,
France) for his help in statistical analysis. This work was partially
funded by Merial, Lyon, France.
Conflicts of interests
All the authors disclose any financial or personal relationships with
people or organisations that could have inappropriately biased or
influenced this work.
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Table 1. Efficacy of IgG absorption according to the time elapsed from birth. Results are expressed as mean ±SEM. Percentage of IgG absorbed
is calculated at 48 h after colostrum administration
Age at colostral administration (hours after birth) 0 4 8 12 16 24
Number of puppies 3 3 3 4 3 5
IgG concentration in serum at 48 h post-colostrum administration (g/l) 2.2 ±0.7 1.2 ±0.2 0.9 ±0.1 0.7 ±0.1 0.6 ±0.1 0.2 ±0.0
Percentage of IgG absorption (%) 39.0 ±14.8 20.2 ±5.3 14.1 ±0.8 8.7 ±0.3 4.9 ±1.3 0.0 ±0.0
192 S Chastant-Maillard, L Freyburger, E Marcheteau, S Thoumire, JF Ravier and K Reynaud
©2012 Blackwell Verlag GmbH
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Submitted: 29 Jun 2012; Accepted: 6 Jul 2012
Author’s address (for correspondence): S
Chastant, Reproduction, Ecole Nationale
Ve
´te
´rinaire de Toulouse, 23 Chemin des
Capelles, 31076 Toulouse Cedex 03, France.
E-mail: s.chastant@envt.fr
*Present address: Vetagro-Sup Campus
Ve
´te
´rinaire, Marcy L’ Etoile, France
Intestinal Barrier Closure in Puppies 193
©2012 Blackwell Verlag GmbH