ABSTRACT
TYLER, HOWARD DAVID. Regulation of Small Intestinal Development in the
Perinatal Period in Calves and Piglets. (Under the direction of HAROLD A. RAMSEY
AND IAN S. LONGMUIR).
An investigation into potential factors regulating small intestinal development in
the newborn was conducted using several approaches and two species. The first
experiment examined the role of glucose availability to the small intestine on the
cessation of macromolecular transport in the small intestine (closure). Fructose was used
to induce a prolonged period of hypoglycemia in newborn calves. Colostrum feeding
was initiated at 24 h; however, absorption of IgG at this time was minimal suggesting
that closure was not delayed. The possibility that fructose was used as an energy source
by the small intestine led to a different approach for the second study.
The use of insulin to induce hypoglycemia provided a more effective approach to
study the initial hypothesis. Calves were injected with either 1 cc of insulin or 1 cc of
saline at birth. In addition, the effects of luminal glucose availability were examined by
fasting half of the calves for 24 h, while the rest were fed colostrum from birth. Closure
in insulin-treated calves was delayed relative to saline-treated calves, and fasted calves
also had a longer period of IgG absorption when compared to fed calves. Decreased rate
of IgG transport in both fasted and insulin-treated calves prevented these calves from
realizing any benefits in terms of increased total IgG absorbed.
To explore the possibility that the change in oxygen availability to the small
intestine normally occurring at parturition initiates the closure process, a technique was
developed to allow perfusion of the small intestine of the fetal calf with highly
oxygenated blood. This technique requires the implantation of a length of polyethylene
tubing into the cranial mesenteric artery of the fetus. The tubing acts as an artificial
circulatory extension (ACE) and is exteriorized in a chronic preparation. Silastic tubing
is spliced into the polyethylene tubing in the exteriorized section of the ACE. The high
gas permeability of Silastic allows blood flowing through this section of tubing to
equilibrate with ambient gas pressures, thus effectively oxygenating this blood prior to
reentry into the fetus and ultimate perfusion of the fetal small intestine..
The fourth experiment was undertaken to characterize metabolic and hormonal
profiles in fetal, newborn and maternal circulations. Glucose and non-esterified fatty
acids increased at birth while fructose decreased. No differences were seen in lactate
concentrations. Most steroid hormones (including cortisol, aldosterone, estradiol, and
testosterone, but with the exception of progesterone) were not different between fetal and
maternal circulations and decreased in newborns. Progesterone was elevated in maternal
blood relative to fetal or newborn blood. Regulatory hormones (thyroxine, growth
hormone and insulin-like growth factor-I) were elevated in fetal calves relative to
maternal values. Arterial oxygen tension and oxyhemoglobin were much lower in the
fetus than either the newborn or adult. Values for pH were not different at any stage, but
bicarbonate concentrations increased postnatally. Elevated Pco2 values were observed in
perinatal animals relative to maternal animals.
To determine the potential for bombesin and vasoactive intestinal peptide (VIP)
to directly affect small intestinal development and(or) function in the newborn, the final
two studies utilized autoradiographic techniques to characterize binding sites for these
gut neuropeptides in duodenal, jejunal and ileal tissues from piglets. Piglets were
obtained at birth, 1 day of age, 1 week, 3 weeks (weaning) and 4 weeks for this
experiment. Binding sites for VIP were present in all tissues at all ages, suggesting that
VIP may have a critical function throughout this period of development. There was a
transient expression of specific binding sites for bombesin between 1 week and 3 weeks
in proximal duodenum, suggesting that the direct actions of bombesin may be limited to
the suckling period in piglets.
The data suggest that regulation of small intestinal development in the perinatal
period is a complex and dynamic process, the mechanisms of which remain poorly
understood.
REGULATION OF SMALL INTESTINAL DEVELOPMENT IN THE
PERINATAL PERIOD IN CALVES AND PIGLETS
by
Howard David Tyler
A thesis submitted to the Graduate Faculty of
North Carolina State University
in partial fulfillment of the
requirements for the Degree of
Doctor of Philosophy
DEPARTMENT OF ANIMAL SCIENCE
RALEIGH
1991
APPROVED BY:
--------------------------------------------
--------------------------------------------
--------------------------------------------
--------------------------------------------
-------------------------------------------Co-Chairman of Advisory
Committee
-------------------------------------------Co-Chairman of Advisory
Committee
ii
ACKNOWLEDGEMENTS
As is customary, the author would like to thank at least some of the many people
who have provided asistance and(or) guidance throughout the last five years. First and
foremost, I am indebted to Dr. Harold Ramsey. He has been my major advisor
throughout my graduate career and deserves a major portion of the credit for any
successes I may have enjoyed during this period. He has been incredibly supportive and
patient, and I would not be at this point were it not for him. Dr. Ian Longmuir has served
as co-chairman of my committee over the last three years, and has also been a source of
inspiration. Dr. Longmuir has opened doors to opportunities that I would never have
even imagined existed, giving my graduate career an extra dimension that I hope will
carry over into my professional career. Both of these men exemplify the very best in
graduate student training, and I consider them friends as well as mentors.
Other members of my committee have provided support and encouragement in
various ways, and all have had an influence on my training. I would like to especially
thank Dr. Jim Croom and Dr. Terry Coffey for their advice and support over the years.
Dr. Robert Horton, beloved professor of biochemistry, has taught me more about
pathways of metabolism and regulation of these pathways than I thought I was capable of
learning. Dr. Charles Hill has helped me by example; his questions are always thoughtprovoking, and I have enjoyed his presence on my committee.
iii
Although this is the extent of my formal committee, in a very real sense many of
the faculty at NC State have served as my advisors. I would like to thank Dr. Lloyd
(Jock) Tate, Dr. Jack Britt, Dr. Jim Lecce, Dr. Jerry Spears, Dr. Billy Flowers, Dr. Jeff
Armstrong, Dr. Sarah Ash and Dr. George Wise for their helpful suggestions and their
friendship. I would especially like to extend my gratitude to Dr. Leonard Bull, who has
always taken the time to listen and help when needed.
Peer support is every bit as necessary as faculty support during graduate studies.
In particular, I would like to thank Dean Morbeck, Missy Moore, Beth Johnson, Chris
Bowie, Randy Stanko, Friederike Jayes, and Lynn Tiller for their support and their
continuing friendship.
I have been fortunate to have done the majority of my research at Unit 2 Dairy.
In particular, I would like to thank Chris Brown, for he is responsible for the
professional, hard-working atmosphere at the dairy. He has been a tremendous help in
carrying out all my projects and has been a great friend as well. In addition, the rest of
the staff at the dairy (Bob Greene, Pete Fricke, Basil Severt, Wayne McLamb, Craig
Gentry, James Johnson and Arthur Swicegood) have made my time at the dairy
enjoyable. Bob and Pete, in particular, have always been more than willing to do
whatever is necessary to help me out.
I would like to thank my family most of all. Throughout the last five years of
long hours and low pay, they have survived and been my "sanctuary of sanity". My
children, Tracy and John, have been my greatest motivation, for I want to be the best
iv
I can for them first and foremost. Allison, my wife, has kept everything together through
good times and tough times, with love and strength. They have been infinitely patient,
supportive and encouraging. My parents, my in-laws, and my entire extended family
have provided support, both moral and financial, to help make everything possible.
Lastly, I would like to thank Dr. Bud Ewing, Dr. Doug Kenealy, Dr. Jerry Young,
Dr. Don Beitz, Dr. Leo Timms and the rest of the Department of Animal Science at Iowa
State University for providing a tremendous motivational stimulus for the last year of my
graduate career and for the future.
v
TABLE OF CONTENTS
LIST OF TABLES ........................................................................
LIST OF FIGURES .......................................................................
Page
GENERAL INTRODUCTION ..........................................................
viii
CHAPTER 1 - Macromolecular Transport in
the Neonatal Calf: a Review ....................................................
Abstract ....................................................................
Introduction ...............................................................
Passive Immunity: Species Differences ...............................
Intestinal Absorption of Macromolecules..............................
Factors Affecting Transport ............................................
Cessation of Uptake .....................................................
Cessation of Transport...................................................
Gastric and Pancreatic Development .........................
Dietary Factors ..................................................
Endocrine Factors ...............................................
Oxygen ............................................................
Gestational Factors ..............................................
Discussion .................................................................
Literature Cited ...........................................................
ix
1
3
4
4
5
10
14
16
18
21
23
25
33
34
35
36
CHAPTER 2 - Effect of Fructose-Induced Hypoglycemia on Cessation
of Macromolecular Transport in the Neonatal Calf .........................
Abstract ....................................................................
Introduction ...............................................................
Materials and Methods ..................................................
Results and Discussion ..................................................
Literature Cited ...........................................................
59
60
61
62
63
74
Results
Discuss
Conclus
Literatu
TABLE OF CONTENTS (continued)
CHAPTER 3 - Effect of Insulin-Induced Hypoglycemia on Cessation
of Macromolecular Transport in the Neonatal Calf .........................
Abstract ....................................................................
Introduction ...............................................................
Materials and Methods ..................................................
Results and Discussion ..................................................
Summary ..................................................................
Literature Cited ...........................................................
CHAPTER 4 - Development of an In Vivo Perfusion System
for Bovine Fetal Small Intestine ................................................
Abstract ....................................................................
Introduction ...............................................................
Materials and Methods ..................................................
Catheter Preparation ............................................
Surgery ............................................................
Sampling and Analysis .........................................
Results .....................................................................
Discussion .................................................................
Literature Cited ...........................................................
CHAPTER 5 - Comparative Endocrine and Metabolic Profiles
of the Fetal, Neonatal, and Maternal Bovine .................................
Abstract ....................................................................
Introduction ...............................................................
Materials and Methods ..................................................
vi
Page
77
78
79
80
82
94
94
102
103
103
106
106
107
109
109
112
113
114
115
116
117
117
118
120
121
121
164
176
176
Materia
Results
Summa
Literatu
TABLE OF CONTENTS (continued)
CHAPTER 6 - Developmental Appearance of Bombesin Receptors
in the Duodenum, Jejunum and Ileum of Piglets From
Birth Through Four Weeks of Age ............................................
Abstract ....................................................................
Introduction ...............................................................
Materials and Methods ..................................................
Tissue Collection ................................................
Autoradiography .................................................
Results and Discussion ..................................................
Summary ..................................................................
Literature Cited ...........................................................
CHAPTER 7 - Developmental Appearance of Receptors for
Vasoactive Intestinal Peptide in the Duodenum,
Jejunum and Ileum of Piglets From Birth
Through Four Weeks of Age....................................................
Abstract ....................................................................
Introduction ...............................................................
CHAP
TER 8
Genera
l
Summ
ary and
Conclu
sions
............
............
..........
vii
Page
182
183
183
184
184
185
186
189
190
192
193
193
195
195
195
196
211
211
214
LIST
OF
TABLES
viii
Chapter 3
1. Rate constants (g/L⋅h-1) for INS, SAL, FAST, and FED
calves for the first four 6-h time periods (T1,
T2, T3, and T4) following initial colostrum
feeding. Pooled SE = .33 g/L⋅h-1. ...........................................
Page
99
Chapter 4
1. Arterial blood gas and acid base values in fetal calves
for blood flowing through a 1.75m length of
Silastic tubing .....................................................................
111
Chapter 5
1. Complete differential profiles for maternal and fetal
blood at day 268 of gestation....................................................
2. Blood profiles for maternal and fetal blood on day 268
of gestation .........................................................................
159
3. Blood chemistry for maternal and fetal blood on day 268
of gestation .........................................................................
160
4. Enzyme activities for maternal and fetal blood on day 268
of gestation .........................................................................
161
5. Lipid profiles of maternal and fetal blood on day 268
of gestation .........................................................................
162
163
LIST OF FIGURES
Chapter 2
7. Peak
plasma
IgG
concen
tration
s
attaine
d by
INSFED,
SALFED,
1. Concentrations of plasma fructose in FRC and GLC calves through
the first 72 h of life. Pooled SE = 12 mg/dl. ..............................
INS-FAST, and
2. Concentrations of plasma glucose in FRC and GLC calves through
the first 72 h of life. Pooled SE = 23 mg/dl. ..............................
3. Concentrations of plasma insulin in FRC and GLC calves through
the first 72 h of life. Pooled SE = 37 μU/ml. .............................
ix
4. Peak plasma IgG concentrations attained by FRC and GLC calves
following colostrum ingestion at 24 h. ........................................
Page
Chapter 3
1. Concentrations of plasma glucose in INS and SAL calves through the
first 72 h of life. Pooled SE = 8 mg/dl. ....................................
65
2. Concentrations of plasma glucose in FED and FAST calves through
the first 72 h of life. Pooled SE = 8 mg/dl. ................................
67
3. Concentrations of plasma insulin in INS and SAL calves through the
first 72 h of life. Pooled SE = 3.9 μU/ml. .................................
70
4. Concentrations of plasma insulin in FED and FAST calves through
the first 72 h of life. Pooled SE = 3.9 μU/ml. ............................
72
5. Age at closure for INS and SAL calves. ...........................................
6. Age at closure for FED and FAST calves. ........................................
83
5.
Conce
ntratio
ns of
growth
hormo
ne in
matern
al (M),
fetal
(F),
86
88
90
92
newborn (N), a
95
6.
Conce
ntratio
ns of
insulin
-like
growth
factorI (IGFI) in
matern
al
97
(M), fetal (F), n
LIST OF FIGURES (continued)
Chapter 5
1. Concentrations of glucose in maternal (M), fetal (F), newborn (N),
and day-old (D) circulations. ....................................................
2. Concentrations of fructose in maternal (M), fetal (F), newborn (N),
and day-old (D) circulations. ....................................................
3. Concentrations of lactate in maternal (M), fetal (F), newborn (N),
and day-old (D) circulations. ....................................................
7.
Conce
ntratio
ns of
thyroxi
ne in
matern
al (M),
fetal
(F),
newbor
n (N),
and day-old (D
4. Concentrations of non-esterified fatty acids (NEFA) in maternal (M),
fetal (F), newborn (N), and day-old (D) circulations. ......................
8.
Concentrations of cortisol in maternal (M), fetal (F), newborn (N),
and day-old (D) circulations. ...................................................
135
9. Concentrations of aldosterone in maternal (M), fetal (F), newborn
(N), and day-old (D) circulations. .............................................
138
10. Concentrations of testosterone in maternal (M), fetal (F), newborn
(N), and day-old (D) circulations. .............................................
140
11. Concentrations of estradiol in maternal (M), fetal (F), and newborn
(N) circulations. ...................................................................
142
12. Concentrations of progesterone in maternal (M), fetal (F), newborn
(N) circulations. ...................................................................
144
13. Arterial pH in maternal (M), fetal (F), newborn (N), and day-old
(D) circulations. ...................................................................
146
x
148
Page
123
125
127
129
131
133
14.
Arteria
l Pco2
in
matern
al (M),
fetal
(F),
newbor
n (N),
and
dayold
(D) circulations. ...................................................................
Page
15. Arterial HCO3- in maternal (M), fetal (F), newborn (N),
and day-old (D) circulations. ....................................................
150
16. Arterial Po2 in maternal (M), fetal (F), newborn (N), and day-old
(D) circulations
152
17. Arterial O2Hb in maternal (M), fetal (F), newborn (N), and day-old
circulations. ...................................................................
(D)
154
Chapter 6
157
125
1. Localization of specific binding sites for -I-(Tyr-4)-bombesin 14
in the porcine duodenum. a. Dark-field photomicrograph
showing the high concentration of bombesin binding sites in
duodenal tissue of a 7-d old piglet. b. A serially adjacent section
to a illustrating the non-specific binding of 125I-bombesin in the
presence of 1μM nonradioactive bombesin. ..................................
Chapter 7
187
125
1. Localization of specific binding sites for I-VIP in the porcine
duodenum. a. Dark-field photomicrograph showing the high
concentration of VIP binding sites in duodenal tissue of a
newborn piglet. b. A serially adjacent section to a illustrating
the non-specific binding of 125I-VIP in the presence of 1μM
nonradioactive VIP. ..............................................................
2. Localization of specific binding sites for 125I-VIP in the porcine
ileum. a. Dark-field photomicrograph showing the high
concentration of VIP binding sites in ileal tissue of a
newborn piglet. b. A serially adjacent section to a illustrating
the non-specific binding of 125I-VIP in the presence of 1μM
nonradioactive VIP. ..............................................................
197
199
xi
xii
LIST OF FIGURES (continued)
Page
3. Localization of specific binding sites for 125I-VIP in the porcine
duodenum. a. Dark-field photomicrograph showing the high
concentration of VIP binding sites in duodenal tissue of a
1-d old piglet. b. A serially adjacent section to a illustrating
the non-specific binding of 125I-VIP in the presence of 1μM
nonradioactive VIP. ..............................................................
201
125
4. Localization of specific binding sites for I-VIP in the porcine
jejunum. a. Dark-field photomicrograph showing the high
concentration of VIP binding sites in jejunal tissue of a
21-d old piglet. b. A serially adjacent section to a illustrating
the non-specific binding of 125I-VIP in the presence of 1μM
nonradioactive VIP. ..............................................................
203
5. Localization of specific binding sites for 125I-VIP in the porcine
duodenum. a. Dark-field photomicrograph showing the high
concentration of VIP binding sites in duodenal tissue of a
21-d old piglet. b. A serially adjacent section to a illustrating
the non-specific binding of 125I-VIP in the presence of 1μM
nonradioactive VIP. ..............................................................
205
125
6. Localization of specific binding sites for I-VIP in the porcine
ileum. a. Dark-field photomicrograph showing the high
concentration of VIP binding sites in ileal tissue of a
21-d old piglet. b. A serially adjacent section to a illustrating
the non-specific binding of 125I-VIP in the presence of 1μM
nonradioactive VIP. ..............................................................
207
7. Localization of specific binding sites for 125I-VIP in the porcine
ileum. a. Dark-field photomicrograph showing the high
concentration of VIP binding sites in ileal tissue of a
28-d old piglet. b. A serially adjacent section to a illustrating
the non-specific binding of 125I-VIP in the presence of 1μM
nonradioactive VIP. ..............................................................
209
GENERAL INTRODUCTION
The perinatal period is without doubt the most critical period in the life of an
animal. It is a time of tremendous change, first with the rapid growth and development
of many organ systems during the final weeks in utero, and then the transition at birth
from reliance on maternal systems to an independent existence. For the fetus, the
placenta provides a constant source of nutrients, growth factors, other hormones and
oxygen. It also aids in detoxification of potentially toxic metabolites in the fetal
circulation and helps rid the fetus of the end products of metabolism while controlling
electrolyte and fluid balance. The intrauterine environment maintains body temperature
of the fetus and protects it physically. After what is often a difficult and traumatic birth,
all of these functions must be quickly and efficiently assumed by the newly-born animal.
Failure of any system can result in irreparable damage or death.
Development of the small intestine is a critical part of the survival process during
this period. The small intestine assumes primary responsibility for absorption of ingested
nutrients after birth. In addition, in many mammalian species, the small intestine is
critical in attainment of immunity through absorption of immunoglobulins from maternal
colostrum. In farm species in particular (calves, piglets, lambs, foals, and kids), the
placenta is impermeable to circulating maternal antibodies and postnatal transmission via
small intestinal absorption is the sole source of passive immunity. Failure of this process
for any reason results in a mortality rate in excess of 50% and long-term impairments of
health and productivity for the survivors. Mortality rate among most newborn farm
species is approximately 15-20% between birth and weaning, with most deaths directly
attributable to failure to attain adequate levels of maternal antibodies during the first day
of life. Devastating economic losses occur due not only to high mortality, but to the
increased morbidity and decreased productivity of those survivors having inadequate
circulating maternal antibodies. These animals exhibit decreased growth rate throughout
the growing period, increased health problems, and decreased productivity extending into
adult life.
These facts underscore the importance of understanding the mechanisms
regulating small intestinal development in the perinatal period. The factors that mediate
development for the first days after birth also control the potential for attainment of
adequate passive immunity, and therefore can affect the profitability (or lack of
profitability) of an animal throughout its entire productive life. The studies reported
herein were an attempt to determine potential factors that initiate developmental changes
at birth or mediate those changes postnatally. Hopefully, they will serve to further our
understanding of these processes and eventually lead to practical techniques for
facilitating the attainment of optimal passive immunity levels in the newborn.
3
MACROMOLECULAR TRANSPORT
IN THE NEONATAL CALF: A REVIEW
Howard Tyler
Department of Animal Science
College of Agriculture and Life Sciences
North Carolina State University
Raleigh 27695
4
ABSTRACT
There exists a large body of research encompassing various aspects of attainment
of passive immunity in the newborn calf. However, there are no recent articles that
attempt to collate this material into a cohesive review specifically concentrating on the
calf. The objective of this paper, then, is to integrate research pertaining to neonatal
macromolecular transport in general and present it as it relates to the calf.
(Key Words: Calf, Immunoglobulins, Newborn, Immunity.)
INTRODUCTION
Due to the absence of transplacental transfer of antibodies from maternal to fetal
circulation in ungulates, newborn calves are born essentially agammaglobulinemic. The
calf is therefore completely reliant on passive immunization via immunoglobulins
concentrated in colostrum. Antibody absorption in calves is non-selective and dependent
on amount of colostrum ingested, mass of immunoglobulin in the ingested colostrum, and
time elapsed between birth and first feeding. Cessation of intestinal transport of
immunoglobulins ("closure") occurs spontaneously in the calf. It is a gradually
accelerating process that ordinarily is completed between 12 and 36 h postpartum.
Premature closure, i.e., prior to 24 h, renders a high percentage of calves
hypogammaglobulinemic despite colostrum ingestion. Impaired performance and high
mortality among these calves illustrates the importance of elucidating mechanisms
controlling closure with the hope of ultimately manipulating these factors in a beneficial
5
manner.
PASSIVE IMMUNITY: SPECIES DIFFERENCES
Most mammals have a well-developed immune system at birth (Sawyer et al.,
1973; Redman, 1979; Ohmann, 1981; Minor and Riese, 1984). There is, however, a
considerable lag between the time of exposure to pathogens and the production of
specific antibodies (Naylor, 1979; Baintner, 1986). The neonate is therefore reliant on
passive immunization by preformed maternal antibodies for survival.
Transmission of antibodies occurs via several distinct routes. Prenatal
transmission of immunity with limited postnatal transmission occurs in primates, guinea
pigs, and rabbits (Ratner et al., 1927; Brambell, 1966; Mach and Pahud, 1971). The
capacity for intestinal uptake of immunoglobulins is present both prenatally and
postnatally, but significant transfer to the vascular system is limited to the prenatal period
(Clarke and Hardy, 1970; Lecce and Broughton, 1973; Walker and Isselbacher, 1974;
Udall et al., 1984). In primates, prenatal immunization occurs transplacentally, while in
guinea pigs and rabbits, it is by the uterine epithelium-visceral yolk sac route (Brambell,
1966; Baintner, 1986). In rodents and carnivores, limited prenatal immune transmission
(Morphis and Gitlin, 1970) is combined with an extensive postnatal period of intestinal
absorption of colostral immunoglobulins (Bangham and Terry, 1957). It is in ungulates
that passive immunity is limited exclusively to postnatal absorption of colostral
immunoglobulins through the small intestine (Earle, 1935; Smith and Holm, 1948;
Bangham et al., 1958; Brambell, 1966; Mach and Pahud, 1971; Merriman, 1971). Lack
6
of prenatal immunization renders the newborn seriously hypogammaglobulinemic (Earle,
1935; Merriman, 1971). Although antibody production in the fetus may occur during the
final trimester in response to antigenic stimulation (Schultz et al., 1971), serum of
ungulates contains barely detectable levels of immunoglobulins at birth (Howe, 1921;
Jameson et al., 1942; Hansen and Phillips, 1949; Lecce and Matrone, 1960; Payne and
Marsh, 1962a; Brandon and Lascelles, 1971; Merriman, 1971; Bush et al., 1971;
Baumwart et al., 1977; Pauli, 1983).
Postnatal immune transmission is dependent on the amount of colostrum ingested
(Smith and Holm, 1948; Aschaffenburg et al., 1949), the mass of immunoglobulin in
ingested colostrum (Earle, 1935; Smith and Holm, 1948; Kruse, 1970; Bush et al., 1971;
Bush et al., 1973) and the elapsed time between birth and first feeding (Jeffcott, 1974).
Most of the variation in absorption by calves is due to age at first feeding (Kruse, 1970),
although this is not the case in lambs and piglets (Lecce and Morgan, 1962). The
importance of this transmission cannot be overemphasized. Rate of gain over the first 6
mo is influenced by the level of passive immunity aquired the first day (Robison et al.,
1988). Milk production during the first lactation has been positively correlated with
immunoglobulin concentration in the newborn period (Denise et al., 1989). The
incidence of both neonatal mortality and disease is strongly correlated with efficiency of
immune transmission (Smith and Little, 1922; Smith and Orcutt, 1925; Aschaffenburg et
al., 1949; Roy et al., 1955; Selman, 1973; Naylor, 1979; Blecha and Kelly, 1981; Nocek
et al., 1984).
Suboptimal postnatal transmission may occur for a variety of reasons: prepartum
7
milking or colostral leakage (Aschaffenburg et al., 1951b; Rowland et al., 1953; Naylor,
1979; Petrie, 1984), lack of milk letdown or production failure (Halliday, 1976; Broom,
1983), acidosis or weakness of the newborn (Broom, 1983; Eigenmann et al., 1983; Odde
et al., 1986), lower quality colostrum from first-calf heifers (Frerking and Aeikens, 1978;
Boyd and Hogg, 1981; Muggli et al., 1984; Petrie, 1984), excitability of dam or lack of
maternal behavior (Broom, 1983; Muggli et al., 1984), pendulous udder (Naylor, 1979;
Boyd and Hogg, 1981; Broom, 1983; Petrie et al., 1984), maternal plane of nutrition
(Naylor, 1979; Blecha et al., 1981; Petrie et al., 1984; Burton et al., 1984) or extreme
environmental temperatures (Frerking and Aeikens, 1978; Cabello and Levieux, 1978;
Blecha and Kelley, 1981; Donovan et al., 1986). Many researchers have reported breed
differences in absorptive capacity (Kruse, 1970; Selman et al., 1971; Baumwart et al.,
1977; Petrie, 1984). Under intensive management systems used on many dairies, the calf
is often separated from the dam at birth, and extensive delays may occur prior to first
feeding (Petrie, 1984; Baintner, 1986). The act of separation itself may diminish rate of
and capacity for postnatal immunoglobulin absorption (Selman et al., 1971; Naylor,
1979; Stott et al., 1979d; Broom, 1983) since microbial infiltration of the small intestine
is likely to occur if feeding is delayed and this impairs Ig absorption (James and Polan,
1978; James et al., 1981).
The incidence of hypogammaglobulinemia (< 500 mg/dl total Ig) has been
estimated at 25% in both calves and foals (Staley et al., 1971; Naylor, 1979; Gay et al.,
1983; Lopez et al., 1988). No cases of prenatal closure have been reported, although
large variation in the absorptive capacity of individual calves is widely recognized
8
(Halliday, 1976; Cabello and Michel, 1977; Cabello and Levieux, 1981; Westrom et al.,
1984a; Lopez et al., 1988). Low immunoglobulin levels post-closure have been
associated with increased incidence of systemic and enteric colibacillosis (Aschaffenburg
et al., 1951a; Briggs et al., 1951; Aschaffenburg et al., 1953; Roy et al., 1955; Wood,
1955; Ingram et al., 1956; Mensik et al., 1978), arthritis (Smith and Little, 1922; Roberts
et al., 1954), pneumonia (Smith and Orcutt, 1925; Williams et al., 1975; Roy, 1980;
Davidson et al., 1981), rhinitis (Smith and Little, 1922) and certain infectious diseases
(Ragsdale and Brody, 1923; Smith and Orcutt, 1925; Naylor, 1979; Muggli et al., 1984;
Donovan et al., 1986). Conversely, high immunoglobulin levels are associated with
increased complement levels in calves, enhanced serum bactericidal activity (Huddleson
et al., 1945) and more efficient neutrophil phagocytosis (Naylor, 1979). Absorption of
the IgM fraction, in particular, enhances endogenous development of active immunity in
the neonate (Stott and Menefee, 1978).
Endogenous production of IgG in non-colostrum fed calves does not reach 24 h
colostrum-fed levels until 2 to 3 mo of age (Hansen and Phillips, 1947a; Naylor, 1979).
The half-life of maternally-provided IgG is estimated at 18 to 22 d (Houdiniere, 1944;
Smith and Holm, 1948; Porter, 1976; Sasaki et al., 1977; Murakami et al., 1985).
Catabolism of immunoglobulins is relatively slow in newborns, which may be due to lack
of development of the lymphoid system (Brandon, 1976). Catabolism is reported as nonexistent in fetal lambs (Brandon, 1976). High levels of maternal immunoglobulins
stimulate catabolism and adversely affect half-life (McGuire et al., 1976). The presence
of maternal IgG inhibits endogenous production in the newborn (Hoerlein, 1957; Logan
9
et al., 1974; Butler, 1986), although up to 1 g/d is still produced during the first 3 wk in
the calf (Sasaki et al., 1977; Devery et al., 1979). Maternally-provided IgA and IgM
have half-lives of 2 and 4 d, respectively (Porter, 1976) apparently due to a high rate of
intraluminal secretion into the gastrointestinal tract during the first week, which provides
local protection (Porter et al., 1972; Porter, 1976; Butler, 1986).
In the final weeks of the prepartal period, the mammary gland selectively
concentrates immunoglobulins (Ragsdale and Brody, 1923; Smith, 1948; Rowland et al.,
1953; Bush et al., 1971; Naylor, 1979; Butler, 1986). In species with prenatal passive
immunization with IgG, the mammary gland accumulates secretory IgA (SIgA) which
becomes the primary colostral Ig (Mach and Pahud, 1971; Walker and Isselbacher, 1974;
Porter, 1976). In species with postnatal immune transmission, IgG is the primary
immunoglobulin in colostrum (Mach and Pahud, 1971; Porter, 1976; Oyeniyi and Hunter,
1978; Butler, 1986), and it can reach levels 3 to 12 times higher than those found in
maternal serum. This is a selective transport phenomenon which is demonstrated as
humorally mediated by estrogen and progesterone (Smith, 1971a). IgA reaches levels
three times higher than maternal serum (Porter, 1976), while IgM is concentrated
ninefold (Naylor, 1979). Colostral immunoglobulin levels, especially IgG, show a
precipitous postpartum decline commencing at parturition (Rowland et al., 1953; Kiddy
et al., 1971; Halliday et al., 1978; Oyeniyi and Hunter, 1978; Lopez et al., 1988).
INTESTINAL ABSORPTION OF MACROMOLECULES
Intestinal absorption and transport of immunoglobulins by the newborn can be
10
either selective or non-selective. Selective absorption occurs in those species that absorb
antibodies throughout the suckling period, e.g., rats and mice (Jones and Waldmann,
1972), while non-selective absorption occurs in those species where closure occurs
prenatally (Walker and Isselbacher, 1974) or within the first few days after birth
(Bangham et al., 1958; Butler, 1986).
Non-selective transport is the primary means of macromolecular transmission in
ungulates (Bangham et al., 1958; Pierce et al., 1964; Hardy, 1964; Fey, 1971, Jeffcott,
1971; Brandon, 1976). Both heterologous and homologous antibodies are transmitted
(Earle, 1935; Hansen and Phillips, 1949; Lecce, 1966a; Al-Jawad and Lees, 1985), and
proportions of serum immunoglobulins post-closure are the same as in ingested
colostrum (Halliday et al., 1978; Stott et al., 1979b). There may be some selective
transport by coated vesicles in proximal small intestine (Pierce and Smith, 1967; Witty et
al., 1969; Burton and Smith, 1977; Leary and Lecce, 1979; Healy and Dinsdale, 1979),
although quantitatively this is considered to be of minor importance. The jejunum
appears to be the most efficient area of the small intestine in terms of macromolecular
transmission (Pierce and Smith, 1967). Distal small intestine has been reported to take
up a greater portion of ingested immunoglobulins (James et al., 1978), but high levels of
hydrolytic enzyme activity in ileal vesicles make them inefficient in terms of transport
(Dinsdale and Healy, 1982). Non-protein macromolecules of similar molecular weight to
immunoglobulins are transported in a kinetically similar manner (Lecce et al., 1961;
Hardy, 1968; Naylor, 1979). Westrom et al. (1984b), however, demonstrated that
polyethyleneglycol (PEG) in solution (20% w/v) with albumin (2% w/v) and ovalbumin
11
(2% w/v), although of similar molecular weight to these molecules, appears to be
transported by a mechanism separate from that for albumins.
The morphology of neonatal small intestine is especially adapted to immune
transmission. The glycocalyx is sparse on the microvillus membrane (Staley et al., 1972;
Bush and Staley, 1980). A preformed organelle, termed the apical canalicular system
(ACS), becomes apparent shortly after feeding (Staley et al., 1969; Murata and Namioka,
1977). Intestinal enterocytes take up colostrum through intermicrovillous pores
(Brambell, 1966; Staley et al., 1972; Healy and Dinsdale, 1979). The ACS acts to
concentrate colostral material into subapical vacuoles (Staley et al., 1972; Murata and
Namioka, 1977; Healy and Dinsdale, 1979). These gradually fill with enough material to
be recognized as eosinophilic droplets (Smith, 1925; Hill and Hardy, 1971; Staley et al.,
1969; Martinsson and Jonsson, 1976; Dinsdale and Healy, 1982). Elevation of
intracellular pressure caused by growing droplets may limit further uptake of colostrum.
The nucleus at this point may be pressed to the base of the cell (Comline et al., 1953).
The inversion process involves translocation of the nucleus and the eosinophilic droplet.
For the first 6 to 8 h after feeding, eosinophilic droplets maintain a supranuclear position,
transposing subnuclearly by 16 to 18 h (Comline et al., 1953). Subnuclear vacuoles
show high levels of alkaline phosphatase activity not present prior to translocation, which
led Healy and Dinsdale (1979) to propose that merger with the Golgi apparatus may
occur during this period. During transport, the vacuolar membrane fuses with the
basolateral membrane (Dinsdale and Healy, 1982) and vacuolar contents are exocytosed
into intercellular spaces (Staley et al., 1971; Logan and Pearson, 1978). Levels of
12
enzymes associated with the brush border membrane rise in the serum of the neonate
concomitantly with macromolecules and are apparently exocytosed along with other
vacuolar contents (Dinsdale and Healy, 1982; Pauli, 1983). These enzymes show a high
activity in intestinal tissue at birth and are depleted within 2 d (Dinsdale and Healy,
1982).
Spaces between and below intestinal enterocytes are especially dilated
immediately after birth, extending up to the terminal bar (Cornell and Padykula, 1969;
Staley et al., 1972; Henriques de Jesus and Smith, 1974). The lamina propria is poorly
developed with few lymphoid cells (Staley et al., 1972; Butler et al., 1981).
Macromolecules are taken up by lymph capillaries (Comline et al., 1951; Payne and
Marsh, 1962a; El-Nageh, 1967; Hardy, 1968) which are highly fenestrated at birth. No
basement membrane is apparent around the lymphatic endothelium at this time (Staley et
al., 1972). Lymph flow increases dramatically after colostrum ingestion (Shannon and
Lascelles, 1968; Brandon and Lascelles, 1971). Absorption and transport to lymph from
the duodenum takes 1 to 2 h (Comline et al., 1951; Balfour and Comline, 1962; Bush and
Staley, 1980), and the maximum concentration is reached by 3 to 4 h after ingestion
(Balfour and Comline, 1962; Brandon and Lascelles, 1971). Since uptake occurs within
15 min, accumulation and transport must be rate-limiting. Rate of transport decreases
with increasing age at first feeding in the calf (Comline et al., 1951; Stott et al., 1979c).
Lymphatics access the general circulation via the ductus thoracicus (Comline et al.,
1951), and access may be promoted by the higher flow rate of blood (Baintner, 1986).
Immunoglobulins first appear in plasma 3 h after feeding, with IgG appearing prior to
13
IgM or IgA (Logan et al., 1978). Peak levels occur 6 to 12 h after feeding (Shannon and
Lascelles, 1968; Staley et al., 1972), and feedback inhibition by high serum antibody
titers apparently does not occur (Payne and Marsh, 1962a).
Reported differences in absorptive efficiencies among immunoglobulin classes
(Penhale et al., 1973; Stott and Menefee, 1978; Besser et al., 1985), can be attributed to
differential rates of equilibration between intravascular and extravascular compartments
(Stott and Menefee, 1978; Cabello and Levieux, 1980) and slower uptake of IgM and IgA
by lymphatics (Stott et al., 1979b; Cabello and Levieux, 1980; Bush and Staley, 1980).
Time of closure may vary between different classes of immunoglobulins (Penhale et al.,
1973; Cabello and Levieux, 1980; Olson et al., 1981a), although there is not complete
agreement on this point (Stott et al., 1979a). Loss of IgG and IgM from serum occurs at
a gradually accelerating rate once threshold levels are attained (Besser et al., 1985).
These complicating factors make comparisons such as relative absorptive efficiencies
difficult to calculate with any degree of accuracy. Linear correlations existing between
concentrations of IgG and IgM in colostral secretions and in the serum of the newborn
(Staley et al., 1971; Brandon and Lascelles, 1971) serve to discount any claims of major
differences in absorptive efficiencies among Ig classes.
FACTORS AFFECTING TRANSPORT
In addition to the presence of immunoglobulins in solution other factors are
necessary for transmission to proceed efficiently. Balfour and Comline (1962)
tentatively identified accelerating factors in colostrum. A low-molecular-weight protein
14
found in the whey fraction, when combined with glucose 6-phosphate and inorganic
phosphate, accounts for most of the accelerating ability of colostrum. These factors were
not independently effective. The protein increases the propensity for immunoglobulins to
enter solution, and may be analogous to surface-active agents in enhancing absorption.
Indirect intracellular functions are also possible. These factors may be colostrumspecific, inasmuch as globulins added to milk are poorly absorbed in comparison to
colostral globulins (Lecce and Matrone, 1960); however, the presence of milk proteins or
even non-protein macromolecules like polyvinylpyrrolidone (PVP) enhance the transport
of IgG to some extent (Leary and Lecce, 1979). Fermentation of colostrum diminishes
transport capacity (Snyder et al., 1974), with pH-buffering partially restoring this ability
(Foley et al., 1978). The high osmolality of colostrum may be important in
immunoglobulin transport, since intraluminal hyperosmolality appears to stimulate
pinocytosis (Cooper et al., 1978). Formation of a curd in the abomasum after colostrum
ingestion is also necessary for optimum absorption (Cruywagen, 1986). The high level of
vitamin A in colostrum (Naylor, 1979) and high serum corticosteroid levels in the
neonate at birth (Grongnet et al., 1986) both affect lysosomal membranes, albeit by
different mechanisms, and thus interfere with normal intracellular digestive processes. In
this manner they enhance the non-specific transport function of neonatal intestine.
Immunoglobulins dissolved in ionically-balanced salt solutions to match
colostrum are capable only of minimal transport (Balfour and Comline, 1962; Hardy,
1964; Grongnet et al., 1986). Addition of glucose or lactose has no effect, but the
addition of short-chain fatty acids, lactate or pyruvate to such solutions accelerated
15
transport despite diminished lymph flow (Hardy, 1968). Potassium isobutyrate appears
to be especially effective in this regard (Hardy,1969a), but, when added to colostrum, has
a deleterious effect on both efficiency of absorption and total Ig absorbed (Baumwart et
al., 1977). This may be caused by a shift in ionic concentration.
Smith (1971b) demonstrated that increasing concentrations of potassium or
decreasing concentrations of sodium in a protein solution inhibits uptake of protein
molecules. Lecce (1966c) found that IgG would not bind to the brush border of newborn
pig enterocytes in the absence of sodium. Brown, Smith and Witty (1968) postulated that
the acceleration of metabolism caused by an increased intracellular concentration of
sodium or movement of sodium down a concentration gradient may provide energy
required for protein transport. Calcium is also required for pinocytotic activity (Smith
and Burton, 1972), and absorptive capacity is lower in calves with low levels of calcium
in their blood at birth (Cabello and Michel, 1977).
Smith and Pierce (1967)
studied the effects of various amino acids on gamma-globulin absorption and transport.
Alanine, which is absorbed via an active transport mechanism, stimulates
immunoglobulin transport in newborn pig intestine. Conversely, leucine, absorbed via
facilitated diffusion, inhibits not only the transport of gamma-globulin but also that of
glucose and fluid. Inhibition of globulin transport may well be a secondary response to
diminished availability of glucose or water to the enterocyte. Polycations, however,
stimulate transport of IgG while simultaneously inhibiting glucose and fluid transfer
(Smith et al., 1968; Smith and Burton, 1972). Concentrations used in these studies may
affect brush border membrane structure, or, alternatively, accelerating factors, especially
16
polycations, may have a direct effect on membrane charge in the brush border (Quinton
and Philpott, 1973). Membrane charge affects macromolecular adsorption to cellular
surfaces (Quinton and Philpott, 1973), and uptake selectivity in neonates appears related
to net charge on the immunoglobulin (Jordan and Morgan, 1968).
CESSATION OF UPTAKE
Uptake of macromolecules by the small intestine continues as long as
vacuolization is present. Vacuolated enterocytes disappear following definite patterns,
with proximal segments of small intestine losing their uptake ability long before the
distal portions (Lecce, 1973; Leary and Lecce, 1976), and cells nearer the crypts before
those at the tips of the villi (Clarke and Hardy, 1969). At birth, uptake occurs along the
entire length of the intestinal villus, but never in the crypts (Staley et al, 1969; Logan and
Pearson, 1978; Butler et al., 1981). Both IgA and IgM are strongly adsorbed to the
luminal surface of the crypt epithelium but not to the villus epithelium (Butler et al.,
1981). These are not absorbed, but instead coat cell surfaces and provide local
protection. El-Nageh (1967) reported absorption along the entire length of jejunal villi at
6 h post-partum, while only the apical third of the villi are capable of uptake in the 2-dayold calf. Additionally, cessation of uptake proceeds caudally in the small intestine
(Clarke and Hardy, 1969). In piglets, the duodenum ceases uptake shortly after birth
(Lecce, 1973; Murata and Namioka, 1977), with the jejunum following at approximately
d 4 to 11 (Leary and Lecce, 1976), and the ileum terminating 2 to 3 wk later (Clarke and
Hardy, 1971; Lecce, 1973; Martinsson and Jonsson, 1976). Duration of uptake is
17
decreased by the presence of digesta (Leary and Lecce, 1976). However, transposition of
an ileal segment to a duodenal position will not affect duration of uptake in the
transposed segment, suggesting that duration of uptake is either genetically or humorally
determined, with the influence of digesta being uniform throughout the intestine (Leary
and Lecce, 1976). Murata and Namioka (1977) and Moog and Yeh (1979) noted certain
distinctive histological changes as cessation of uptake proceeds. The terminal web
appears to develop as pinocytosis ceases. Golgi complexes, mitochondria and rough
endoplasmic reticulum become more prominent. Intermicrovillus pores disappear along
with vacuoles, and density of the glycocalyx increases. New cells formed in the crypts
after birth may develop vacuoles, but require at least 4 d after the last DNA synthesis
(Moon et al., 1973). Therefore, slow cell turnover in the neonate helps prolong the
period of uptake, and cessation is due to a combination of increasing cell turnover rate
coupled with redifferentiation of intestinal enterocytes (Baintner, 1986).
CESSATION OF TRANSPORT
Conversely, cessation of transport (closure) is not necessarily related to cell
replacement. The closure process is the loss of the ability of intestinal enterocytes to
exocytose vacuolar contents, which is a gradual phenomenon, with efficiency of transport
slowly diminishing prior to complete cessation (Matte et al., 1982). Despite potential
continued uptake of macromolecules, most transport ceases (Comline et al., 1953; Bush
and Staley, 1980), although residual, size-dependent transport may continue throughout
the first week (Westrom et al., 1984a). Material taken up by the enterocyte is not
18
released into intercellular spaces and is simply shed along with the cell during normal
cell turnover (Logan and Pearson, 1978). Staley et al. (1969) postulated that a shift in
the position of the Golgi apparatus causes a change in cell polarity which favors luminal
rather than basal secretion. Jordan and Morgan (1968) suggested that a progressive
change in the net charge of cell membranes, presumably basolateral membranes, either
by gradual loss of the inherent positive charge or by development of a negative charge,
may be responsible for the gradual loss of transport capability.
Cell migration from crypts to villus tips define the cell turnover period. At birth,
and through the first 16 d in rat pups, this is a relatively slow process, requiring 6 to 7 d
(Koldovsky et al., 1966; Rundell and Lecce, 1972; Klein and McKenzie, 1980). Starting
at d 16, the process rapidly accelerates, with cell turnover on d 18 estimated at 2 to 3 d
(Clarke and Hardy, 1969; Rundell and Lecce, 1972). Height of villi increases by 40%
between d 15 and 23, with size of the crypts increasing 300% during the same period
(Klein and McKenzie, 1980). The number of crypt cells increases dramatically starting
at d 19 (Klein and McKenzie, 1980), indicating cell proliferation is occurring in addition
to cell hypertrophy. Sympathectomy diminishes mitotic rate somewhat, but acceleration
of proliferation still occurs within the same time frame (Klein and McKenzie, 1980),
suggesting a humoral trigger. The correlation between cell turnover and cessation of
uptake does not carry over to other species, e.g., rabbits, hamsters and guinea pigs
(Rundell and Lecce, 1972). Cell replacement in the neonatal intestine of ungulates is
quite slow, well in excess of 48 h in calves and lambs (Moon and Joel, 1975; Trahair et
al., 1986). In the newborn pig, values between 5 and 19 d are published (Moon, 1971;
19
Smith and Jarvis, 1978), with variation due more to experimental method than actual
individual differences. In rats and mice, cessation of uptake and closure coincide.
Estimates of the time of closure in calves range from 8 to 48 h postpartum, with
the consensus near 24 h (Hansen and Phillips, 1947b; McCarthy and McDougall, 1953;
Bush et al., 1971; Baumwart et al., 1977; Logan et al., 1978; Pauli, 1983). Estimates
vary due to procedure utilized for determining closure, feeding regimen and other
variables. Estimates that take into account the large increase in plasma volume
associated with feeding extend the period of absorption several h (Matte et al., 1982);
however, techniques necessary to estimate plasma volume introduce additional sources of
error. Changes in plasma volume are fairly uniform among calves (Husband, et al.,
1973), even with different feeding regimens (McEwan et al., 1968). Shannon and
Lascelles (1968) reported that transport of gamma-globulin ceases within 24 h of first
colostrum feeding. The work of Stott et al. (1979a) tends to verify these findings. They
estimate time of closure of calves fed at birth near 22 h while for those calves first fed at
24 h closure does not occur until 33 h. These data, however, are biased in that calves
experiencing spontaneous closure prior to feeding were censored. This encompassed
50% of calves fasted for 24 h, indicating that differences in closure times between calves
fed at birth and those fed at 24 h may actually be much less.
In rats and mice, closure accelerates over time starting on d 16 (Clarke and Hardy,
1969). In rats weaned at 21 d, cell replacement with a mature type cell coincides with
closure. Although gastric development appears to hold a primary role in the closure
process, cell replacement is equally important, especially in weaned rats.
20
Gastric and Pancreatic Development
Many hypotheses have been put forth that attempt to explain the process of
closure as a function of increasing digestive capability of the neonate. Hill (1956)
postulated that closure was a function of gastric development and increasing proteolytic
activity. In the newborn guinea pig (no significant postnatal transmission of antibodies),
parietal cells are abundant and gastric pH is 1.0 to 2.0. Similar findings have been
reported in human infants within 2 h of birth (Avery et al., 1966). In species with
postnatal transmission, gastric development parallels closure. Inhibition of gastric
function and(or) the use of trypsin inhibitor enhances macromolecular absorption in
mature animals (Walker and Isselbacher, 1974). Development of digestive function
appears to influence closure in rats and mice. Significant proteolysis occurs in the gut of
suckling rat pups (Jordan and Morgan, 1968; Jones, 1972a). Jordan and Morgan (1968)
postulated that development of selective proteolytic activity could explain the
progressive increase in selectivity of protein transmission through the suckling period.
Potency of gastric secretions continues to develop with parietal cells appearing by the
end of the second week and cell numbers increasing rapidly until d 25 (Hill, 1956).
Gastric pH drops from 4.4 to 2.7 during this period. By the end of the third week, no
antibodies reach the lower gut intact. Normal 24-day-old rats show a fivefold increase in
peptic activity and a 10-fold increase in tryptic activity over levels seen in 12-day-old
rats. Halliday (1956) reported closure at 21 d despite continued suckling and continued
21
presence of antibodies in milk. Morris and Begley (1970) demonstrated that IgG was
still transported in 29-day-old unweaned rats if infused directly in the small intestine;
however, oral administration was ineffective. Duodenal infusion was ineffective in
weaned 29-day-old rats (Morris and Begley, 1970). Some aspect of weaning or the
change in diet affects closure, although premature weaning or diet changes will not
induce closure (Halliday, 1956). However, premature weaning diminishes absorptive
capacity (Halliday, 1959).
Closure in ungulates is independent of gastric and pancreatic development
(Westrom et al., 1984a), despite nearly identical time frames. Balfour and Comline
(1962) and Kruse (1983) reported minimal hydrolysis in the gastrointestinal tract of
calves over the first 2 d, although a somewhat higher rate of proteolysis exists in piglets
(Hardy, 1969b). Abomasal pH is relatively high at birth (near 7.0) and steadily decreases
to a pH of 3.0 at 36 h. Accordingly, there is a rapid, progressive increase in the number
of parietal cells during the first 48 h postpartum (Hill, 1956). Although the cellular basis
for mucus and pepsin secretion is present at birth (Hill, 1956), gastric proteolysis is due
primarily to rennin (Hardy, 1969c). Some excretion of Ig(Fab) fragments occurs in the
newborn period presumably due to this action (Kumano et al., 1976). Despite inhibiting
gastric activity in calves, Deutsch and Smith (1957) could demonstrate no transmission
of globulins at 40 h. Non-protein macromolecules that are resistant to gastric proteolysis
cease transport at the same time as immunoglobulins (Jeffcott, 1974; Westrom et al.,
1984b). Antibodies introduced directly into the duodenum, thus bypassing gastric
proteolysis, are absorbed only during the first day (Hansen and Phillips, 1947b; Smith
22
and Erwin, 1959). Uninhibited tryptic digestion of immunoglobulins occurs at closure
and is hypothesized as being important in initiating closure. High levels of trypsin
inhibitor are present in colostrum and prevent tryptic digestion of susceptible
immunoglobulins. Chamberlain et al. (1965) added trypsin inhibitor to immunoglobulin
solutions and fed this to 3-day-old piglets. Trypsin inhibitor proved ineffective in
stimulating transmission. Deoxyribonuclease activity in pancreatic secretions was also
inhibited postpartum (Deutsch and Smith, 1957). This treatment was similarly without
effect.
Levels of alkaline phosphatase in intestinal tissue rise at the time of closure in
rats. To determine if changes in alkaline phosphatase trigger intestinal maturation,
Clarke and Hardy (1969) added alkaline phosphatase to a gamma-globulin solution and
fed this to suckling rats. The lack of response led to the conclusion that closure and
changes in alkaline phosphatase are both independent consequences of the same process.
The effect of amniotic fluid in the gut was studied by Deutsch and Smith (1957) by
feeding amniotic fluid with milk over the first 36 h. No absorption of immunoglobulins
was noted when colostrum was fed at 40 h.
Dietary Factors
Although mechanisms involved in cessation of uptake are similar among all
ungulates, there are marked differences among species in cessation of transport. In pigs,
closure is a diet-induced phenomena. Fasted pigs continue to take up and transport
macromolecules until death (at about 4 d) (Lecce and Morgan, 1962; Payne and Marsh,
23
1962b; Lecce, 1973; Werhahn et al., 1981), however, spontaneous closure has been
documented in fasted pigs during the second day (Lecce and Morgan, 1962), suggesting
that fasting does not halt the closure process, but greatly delays it. Closure in lambs also
appears to be diet-dependent (Lecce and Morgan, 1962; Halliday, 1976). Fasted calves,
on the other hand, differ very little from fed calves with regard to period of absorption
(Stott et al., 1979a). All ungulates, if fed near birth, will cease macromolecular transport
in a similar fashion (Lecce and Morgan, 1962; Payne and Marsh, 1962b; Lecce, 1973).
Colostrum intake accelerates closure in all ungulates to varying degrees.
The difference between a fasted piglet or lamb and a fasted calf may be related to
the postnatal blood glucose pattern. Newborn pigs and lambs are susceptible to fasting
hypoglycemia. Glycogen reserves at birth are relatively limited in these species
compared to calves (Svendson and Bille, 1981). Blood glucose levels decrease shortly
after birth (Hanawalt and Sampson, 1947; Bassett and Alexander, 1971) and do not
recover without feeding (Goodwin, 1957b). In unstressed fed animals, glucose levels
gradually rise over a 2- to 3-wk period (Dawkins, 1964; Bassett and Alexander, 1971;
Daniels et al., 1974). Neonatal calves and foals present a different picture. Glucose
levels are lower at birth, but they rise to twice adult levels within the first 24 h. This rise
is independent of nutritional status (Goodwin, 1957a; Comline and Edwards, 1968;
Daniels et al., 1974). Levels then gradually decline over the next 6 wk (Kennedy et al.,
1939; Ratcliffe et al., 1958; Young et al., 1970). Thus, the availability of glucose to the
neonatal small intestine may be one factor influencing the closure process.
Dietary induction of closure in the pig supports this scenario. In searching for
24
nutritional factors that initiate closure in pigs, Lecce et al. (1964) found that colostral
proteins, fats, vitamins and minerals are without effect, whereas a fat- and protein-free
colostral whey induces closure in a normal manner. Later studies show that pure
solutions of various sugars induce closure (Lecce, 1966b; Werhahn et al., 1981) and that
at least 300 milliequivalents of glucose are required. Solutions of glycine or inorganic
salts are ineffective (Lecce, 1966b). Direct contact with sugar solutions may not be
necessary. Leary and Lecce (1978) reported that feeding induces closure even in isolated
intestinal segments, suggesting that induction of closure is humorally-regulated and not
dependent on luminal exposure to glucose.
Endocrine Factors
The unique composition of colostrum suggests that some of its constituents may
prolong the absorptive period. Pope and Ray (1953) note that estrogenic activity in
colostrum is similar to that in the serum of the dam but calves maintained on three (250
ml) transfusions of maternal blood were unable to absorb antibodies at 40 h (Deutsch and
Smith, 1957). Neither calves (Deutsch and Smith, 1957), lambs (Dawe et al., 1982) nor
rats (Halliday, 1959) treated with injections of estrogenic compounds appear to
experience any delay in closure. The high content of histamine in colostrum prompted
Patt et al. (1972) to add histamine to gamma-globulin-enriched milk. This combination
is detrimental to absorptive capacity and appears to induce premature closure.
Other hormone treatments studied include effects of progesterone (Deutsch and Smith,
1957; Halliday, 1959; Gillette and Filkins, 1966), progesterone in combination with
25
estrogen (Deutsch and Smith, 1957), testosterone (Halliday, 1959), ACTH (Deutsch and
Smith, 1957; Gillette and Filkins, 1966; Boyd and Hogg, 1981), aldosterone (Halliday,
1959) and somatotropin (Smith et al., 1964). None of these treatments are effective in
extending the absorptive period prior to closure.
The effects of thyroxine on closure are well-documented. Thyroxine can be
characterized as a non-specific metabolic enhancer that increases tissue metabolism and
oxygen consumption, which in turn leads to enhanced cardiac output and ventilation rate
(Genuth, 1983). Thyroxine is trophic to small intestinal tissue and therefore increases its
oxygen consumption directly (Levin, 1969). Activity at the gut level leads to increased
motility (Genuth, 1983). Thyroxine also potentiates the stimulatory effects of
corticosteroids, epinephrine, glucagon and growth hormone (Genuth, 1983). Thyroxine
levels rise in rat pups from birth through weaning (Clos et al., 1974). Chan et al. (1973)
and Malinowska et al. (1974) administered high levels of thyroxine to suckling rats and
reported precocious cessation of immunoglobulin absorption. However, effects of
thyroxine were indistinguishable from those expected from corticosteroids stimulated by
this treatment. Moog and Yeh (1979) report that changes in the terminal ileum of the
suckling rat are abolished by hypophysectomy, and increases in mitotic index associated
with closure in rats are not seen in hypophysectomized animals. They theorize that
inhibition of normal developmental changes is due to prevention of maturation of the
pituitary-adrenal response system. Normal ultrastructural changes can be restored in
such surgically-altered animals by daily injections of either cortisone acetate or
thyroxine, although enzymatic changes associated with each treatment are different (Yeh
26
and Moog, 1975). Since thyroxine does not alter corticosteroid levels under these
conditions, effects either are direct tissue effects or are mediated through a separate,
undetermined mechanism. Investigations utilizing thyroidectomized or adrenalectomized
rat pups have also demonstrated that cortisone and thyroxine are independently capable
of inducing normal maturational changes in the small intestine. Repeated injections of
thyroxine to either fetal rat pups or 14-day-old rats induce a decrease in IgG receptors
and maturation of intestinal enzyme profile. Microvillus membranes also mature as
evidenced by a decreased lipid:protein ratio (Israel et al., 1987). The effect on closure in
rats was not examined, but these changes are consistent with those expected during this
process.
Plasma triiodothyronine and thyroxine levels in ungulates are elevated at birth
and decline through the first week (Khurana and Madan, 1984). Thyroxine is the
predominant form at birth (Khurana and Madan, 1984). Boyd and Hogg (1981) observe
that endogenous concentrations of thyroxine at birth bear no discernable relationship to
subsequent immunoglobulin absorption. Cabello and Levieux report on a series of
experiments (Cabello and Levieux, 1978; Cabello and Levieux, 1980; Cabello et al.,
1980; Cabello and Levieux, 1981; Cabello et al., 1983) on the effect of thyroxine on
passive immunization in lambs. Three of these studies showed a decrease in absorptive
capacity in response to exogenous thyroxine, one an enhancement, and one had no effect.
Two showed precocious closure, while one resulted in a delay in closure.
Triiodothyronine, which is more potent biologically than the tetraiodothyronine utilized
in these studies, had no effect on either absorption of immunoglobulins or time of closure
27
while an increase in levels of thyroid-stimulating hormone are associated with a
shortened period of absorption.
Thyroxine has also been reported to induce adrenal maturation (Moog and Yeh,
1979), which might well be its most important effect in the perinatal period with regard
to closure. In searching for a humoral trigger for closure, most attention has focused on
the role of glucocorticoids, especially in rats. Evidence of several endocrine
interrelationships have evolved from this work, but no conclusive data have been
produced to link corticosteroids directly to closure.
Daniels et al. (1973b) has related changes in plasma corticosterone to changes in
the small intestine that accompany closure. Levels remain near 1 μg/dl until d 18 to 21,
then rapidly rise to 5 to 7 μg/dl and continue to gradually increase to 15 μg/dl on d 28.
Concentration of cortisol remains unchanged throughout this period (Daniels and Hardy,
1972). Patt (1977) saw no changes in corticosterone in the same time frame, but his
technique was less likely to detect differences in the low levels present at this point. The
ability of pharmacological doses of various corticosteroids (especially cortisone acetate)
to induce precocious closure after d 10 in the rat has been thoroughly studied (Halliday,
1958; Halliday, 1959; Jones, 1972b; Daniels et al., 1973a; Daniels et al., 1973b). Morris
and Morris (1976) showed that exogenous corticosteroids at levels high enough to induce
precocious closure also initiate rapid cell turnover and maturation of intestinal epithelium
in distal small intestine. IgG receptor levels are diminished in proximal intestine,
although the possibility of cytological changes remains. Enzyme activities are also
precociously altered by this treatment (Doell and Kretchmer, 1964; Jones, 1972b;
28
Koldovsky and Herbst, 1973). Transmission of IgG began to decrease on the first day of
treatment (Morris and Morris, 1976). In contrast, bilateral adrenalectomy delays onset of
closure by 4 d, but does not abolish it (Daniels and Hardy, 1972; Daniels et al., 1973a).
When closure does occur, it proceeds at a normal rate (Daniels et al., 1973b). The fact
that closure proceeds with only a transient delay suggests that although corticosteroids
may have a permissive role in closure, they are not essential. Malinowska et al. (1972),
in support of this conclusion, noted that while corticosteroid levels are extremely high at
birth in rat pups and rabbits, and despite an increase at d 14 in rabbits, closure does not
occur until the final corticosteroid surge at the end of the third week. Effects due to
exogenous corticosteroids in rats are more accurately categorized as pharmacological
responses than as physiological effects.
Attempts to reproduce the effects of corticosteroids in ungulates have been
universally unsuccessful in terms of reproducible effects on closure. The prepartum
surge in cortisol acts in a regulatory capacity on small intestinal maturation and
proliferation (Trahair et al., 1984; Trahair et al., 1987a; Trahair et al., 1987b). In fed
calves, corticosteroid levels decreased rapidly during the first 12 h postpartum and
gradually during the next 12 h (Nightengale and Stott, 1981; Grongnet et al., 1986).
Fasted calves show the same initial decline, but levels rise during the second 12 h if
fasting continues (Nightengale and Stott, 1981). Feeding induces a transient
hyperadrenalemia (Nightengale and Stott, 1981). Lambs and piglets present a similar
picture, but the relative magnitude of change is less dramatic (Bassett and Alexander,
1971; Dvorak, 1972). The effect of exogenous corticosteroids imposed on this picture
29
serves to diminish absorptive capacity of macromolecules without affecting the time of
closure (Deutsch and Smith, 1957; Husband et al., 1973; Johnston and Oxender, 1979;
Dawe et al., 1982). In contrast to these studies, various studies have reported increased
absorptive capacity as a result of exogenous corticosteroid treatment (Boyd and Hogg,
1981; Bate and Hacker, 1985a). Administration of drugs at birth to decrease cortisol
levels in lambs induced a precocious closure (Hough et al., 1990). Studies relating
endogenous cortisol concentrations at birth to closure or to absorptive capacity also
produce conflicting results. Stott and Reinhard (1978), looking at dystocial and eustocial
calves, found no variation due to differences in cortisol levels at birth. Cabello and
Levieux (1978, 1980) and Cabello et al. (1983) confirm these findings. Boyd and Hogg
(1981) report a negative correlation between absorptive capacity and endogenous cortisol
concentration at birth with no effect on closure. As long as body core temperature is
unaffected, temperature stresses on newborn animals have little effect on absorptive
capacity and time of closure (Olson et al., 1981a; Kelley et al., 1982; Bate and Hacker,
1985b). Extreme cold decreases the rate of antibody transport without affecting
absorptive capacity (Olson et al., 1980; Blecha and Kelley, 1981), as would be expected
based on reports of transport inhibition in isolated intestinal loops exposed to
hypothermic conditions (Griffen et al., 1960). Stott (1980) stated that heat stress also
diminishes absorptive capacity, and suggested this is a secondary response to concurrent
hyperadrenalemia. Cold stress induces a significant increase in concentration of cortisol
(Stott and Reinhard, 1978; Olson et al., 1981b), which may be responsible for any
adverse absorptive effects.
30
The effects of corticosteroids on closure may be due as much to increasing tissue
metabolism in general as to any direct action on the small intestine. They induce
hyperglycemia and, additionally, may increase the hyperglycemia induced by the high
growth hormone levels present at birth in ungulates (Bassett and Alexander, 1971).
Corticosteroids are important in mobilization and oxidation of lipids and stimulate tissue
glycolysis (Wilson, 1979). They decrease the oxygen affinity of hemoglobin, leading to
increases in oxygen delivery at the tissue level (Bauer and Rathschlag-Schaefer, 1968;
Bartels, 1970).
A potential effect of corticosteroids in relation to closure may result from their
ability to induce gastrin receptors. This phenomenon closely parallels the apparent
effects of corticosteroids on closure previously discussed. A single injection of
corticosterone acetate into 7-day-old rat pups results in the premature appearance of
gastrin receptors by d 10 (Peitsch et al., 1981). The same treatment has been shown to
induce closure by d 11 (Daniels et al., 1973a). Receptors normally appear in the rat pup
between 18 and 20 d of age (Takeuchi et al., 1981). Adrenalectomy delays the normal
appearance of gastrin receptors until d 25 (Peitsch et al., 1981). Adrenalectomy has been
shown to delay closure similarly (Daniels et al., 1973a). Therefore, the reported effects
of corticosteroids on closure may possibly be mediated by gastrin.
Gastrin is secreted from G cells. Although there are G cells scattered throughout
the intestinal tract, the highest concentration of these cells is in the antral portion of the
stomach or abomasum (Walsh, 1987). The actions of gastrin are gut-specific, with the
exception of an apparent mildly trophic effect on the pancreas (Enochs and Johnson,
31
1977).
In suckling rats, serum gastrin levels are high from birth through weaning. Antral
levels are low, indicative of the lack of receptors. Antral levels rise on d 20, reaching
adult levels by d 22. After d 25, antral and serum levels decline to normal basal levels.
Early weaning does not affect the timing of these changes, but does diminish their
magnitude (Lichtenberger and Johnson, 1974; Takeuchi et al., 1981). High gastrin
concentrations in the perinatal period have been reported in other mammals (Euler et al.,
1977). The development of gastric acidity in the neonatal period of all species is due in
part to the interaction of gastrin and its receptor (Passaro et al., 1963; Euler et al., 1977;
Soll and Grossman, 1978). This process, as previously discussed, also parallels closure.
The gastrin/receptor interaction regulates differentiation and proliferation of
epithelial cells in the small intestine (Johnson et al., 1969a; Johnson et al., 1969b;
Lichtenberger et al., 1973; Johnson and Chandler, 1973). Gastrin activity increases ratios
of RNA:body weight, gut weight:body weight, and protein:body weight (Lichtenberger
and Johnson, 1974) as well as DNA synthesis in intestinal tissue (Johnson and Guthrie,
1974; Johnson, 1977; Schwartz and Storozuk, 1985; Pollack and Soloman, 1986; Conteas
and Majumdar, 1987). There is a 50% increase in mRNA synthesis within an hour after
injection of exogenous pentagastrin, with an increase in protein synthesis following 2 h
later and peaking within 6 h. By 16 h, DNA synthesis is maximized (Enochs and
Johnson, 1977).
The action of gastrin on gastrointestinal tissue is accompanied by a sharp increase
32
in oxygen consumption at the cellular level (Soll, 1977; Soll, 1978a; Soll, 1978b).
Oxygen, then, may act as a limiting factor in tissue response to stimulation by gastrin.
This suggests a scenario where initiation of closure is prevented prenatally by lack of
oxygen availability to gastrointestinal tissue. This could be mediated directly or via
formation of gastrin receptors.
Oxygen
Although the placenta functions as the organ of nutrient transport (Edelstone and
Holzman, 1982), fetal intestinal oxygen consumption is fairly high (0.4 ml O2⋅min-1⋅kg-1).
This is primarily due to the rapid growth of this tissue in late gestation. Despite rapid
fetal growth, intestinal tissue as a percentage of fetal weight increases from 6.2 to 7.2%
during late gestation (Reeves et al., 1972). During postnatal development, however,
intestinal tissue at rest consumes 1.4 ml O2⋅min-1⋅kg-1 despite extracting only 28% of
delivered oxygen (Edelstone and Holzman, 1981b). Oxygen consumption increases 65 to
72% during digestion (Brodie et al., 1910; Edelstone and Holzman, 1981a). Energy
requirements also increase due to increased gastrointestinal motility (Ruckebush et al.,
1983; Kvietys et al., 1986) and the increase in energy expended for transport functions
(Brodie et al., 1910). This is accomplished via increased oxygen extraction (Edelstone
and Holzman, 1981a) and increased blood flow to the mucosal-submucosal layer
(Nowicki et al., 1983). Oxygen consumption in suckling rat intestine has been shown to
increase in the presence of gamma-globulin (Bamford, 1966). Uptake of gammaglobulin is an active, energy-coupled process that can be reversibly inhibited by various
33
metabolic antagonists (iodoacetate, arsenate, fluoride, 4,6-dinitro-o-cresol, phlorhizin,
cold and anaerobiosis) (Lecce, 1966c).
If the change in oxygen availability at birth initiates closure in the calf, either
directly or through some secondary mechanism or mechanisms, then maintaining the
arterial Po2 of the newborn calf at fetal levels should delay closure. Mixed results were
obtained in a study examining the effects of hypoxia in the immediate postnatal period on
time of closure (Tyler and Ramsey, 1991). Arterial Po2 was maintained near 25 mm Hg
for a period of 24 h. Time of closure was significantly delayed in hypoxic calves fed
colostrum from birth; however, no differences were noted when colostrum feeding was
delayed until 24 h. This may have been due to other changes occurring at birth that
influence intestinal development.
Gestational Factors
If time of closure is determined by prepartum changes in the fetal serum profile,
changes in gestation length and subsequent maturity of the newborn might be expected to
alter absorptive capacity and time of closure. Extended gestation in lambs diminishes
absorptive capacity and induces precocious closure (Cabello and Levieux, 1981). No
such relationship seems to exist in calves (George et al., 1979). If extending gestation
induces precocious closure, there should be some response to prematurity. Calves
removed by cesarian section 2 to 3 wk prior to due date are able to absorb
immunoglobulins from colostrum fed at birth but not at 38 h (Smith et al., 1964).
Surprisingly, fetal calves are unable to absorb high levels of gamma-globulin introduced
34
into the amniotic fluid during the final trimester of gestation (Smith et al., 1964).
DISCUSSION
The process of closure in newborn calves is an integral factor in determining the
level of passive immunity ultimately attained during the first day of life. This in turn
greatly influences prospects for survival through the first 6 months of life. The high rate
of calf mortality that currently exists is nearly identical with rates reported 50 years ago
(Savage and McCay, 1942). This ultimately translates into increased non-selective
culling that in turn results in decreased herd productivity and profits. Despite the
tremendous economic implications, little attention has been directed during the past 10 yr
toward understanding the mechanics of closure. Although the literature reveals no
conclusive evidence that closure is mediated by any single factor, it provides enough
information to formulate a workable hypothesis that accounts for many of the apparently
ambiguous results described. Despite the fact that there appear to be several humoral
factors that influence the closure process to varying degrees, their effects or lack thereof
become more predictable when closure is viewed as an active, energy-driven process,
occurring at a period in the life of the animal when energy availability to the
gastrointestinal tract is in a state of flux.
LITERATURE CITED
Al-Jawad, A.B. and J.L. Lees. 1985. Effects of ewe's colostrum and various substitutes
on the serum immunoglobulin concentration, gut closure process and growth rate of
lambs. Anim. Prod. 40:123.
35
Aschaffenburg, R., S. Bartlett, S.K. Kon, J.H.B. Roy, H.J.Sears, S.Y. Thompson, P.L.
Ingram, R. Lovell, and P.C. Wood. 1953. The nutritive value of colostrum for the calf.
9. The effects of soya-bean lecithin on the vitamin A absorption and on the growth
rate of calves given small quantities of separated colostrum. Br. J. Nutr. 7:275.
Aschaffenburg, R., S. Bartlett, S.K. Kon, J.H.B. Roy, D.M.Walker, C. Briggs and R.
Lovell. 1951a. The nutritive value of colostrum for the calf. 4. The effect of small
quantities of colostral whey, dialysed whey and `immunelactoglobulins'. Br. J. Nutr.
5:171.
Aschaffenburg, R., S. Bartlett, S.K. Kon, J.H.B. Roy, D.M.Walker, C. Briggs and R.
Lovell. 1951b. The nutritive value of colostrum for the calf. 5. The effect of prepartum
milking. Br. J. Nutr. 5:343.
Aschaffenburg, R., S. Bartlett, S.K. Kon, S.Y. Thompson,D.M. Walker, C. Briggs, E.
Cotchin and R. Lovell. 1949. The nutritive significance of colostrum for the calf.
XIIth Int. Dairy Congress (Stockholm) :90.
Assali, N.S. and J.A. Morris. 1964. Circulatory and metabolic adjustments of the fetus at
birth. Biol. Neonate 7:141.
Avery, G.B., J.G. Randolph and T. Weaver. 1966. Gastric acidity in the first day of life.
Pediatrics 37:1005.
Baintner, K. 1986. Intestinal Absorption of Macromolecules and Immune Transmission
from Mother to Young. CRC Press, Boca Raton.
Balfour, W.E. and R.S. Comline. 1962. Acceleration of the absorption of unchanged
globulin in the new-born calf by factors in colostrum. J. Physiol. 160:234.
Bamford, D.R. 1966. Studies in vitro of the passage of serum proteins across the
intestinal wall of young rats. Proc. Roy. Soc. B 166:30.
Bangham, D.R., P.L. Ingram, J.H.B. Roy, K.W.G. Shillam and R.J. Terry. 1958. The
absorption of 131I-labelled serum and colostral proteins from the gut of the young calf.
Proc. Roy. Soc. B 149:184.
Bangham, D.R. and R.J. Terry. 1957. The absorption of131I-labelled homologous and
heterologous serum proteins fed orally to young rats. Biochem. J. 66:579.
Bartels, H. 1970. Prenatal Respiration. North Holland Publishing Co., Amsterdam.
Bassett, J.M. and G. Alexander. 1971. Insulin, growth hormone and corticosteroids in
neonatal lambs. Biol.Neonate 17:112.
36
Bate, L.A. and R.R. Hacker. 1985a. The influence of the sow's adrenal activity on the
ability of the piglet to absorb IgG from colostrum. Can. J. Anim. Sci. 65:77.
Bate, L.A. and R.R. Hacker. 1985b. Influence of environmental temperature during late
gestation and soon after birth on IgG absorption by newborn piglets. Can. J. Anim.
Sci. 65:87.
Bauer, C. and A.M. Rathschlag-Schaefer. 1968. The influence of aldosterone and cortisol
on oxygen affinity and cation concentration of the blood. Resp. Physiol. 5:360.
Baumwart, A.L., L.J. Bush, M. Mungle and L.D. Corley. 1977. Effect of potassium
isobutyrate on absorption of immunoglobulins from colostrum by calves. J. Dairy Sci.
60:759.
Besser, T.E., A.E. Garmedia, T.C. McGuire and C.C. Gay. 1985. Effect of colostral
immunoglobulin G1 and immunoglobulin M concentrations on immunoglobulin
absorption in calves. J. Dairy Sci. 68:2033.
Blecha, F., R.C. Bull, D.P. Olson, R.H. Ross and S. Curtis. 1981. Effects of prepartum
protein restriction in the beef cow on immunoglobin content in blood and colostral
whey and subsequent immunoglobin absorption by the neonatal calf. J. Anim. Sci.
53:1174.
Blecha, F. and K.W. Kelley. 1981. Cold stress reduces the acquisition of colostral
immunoglobulin in piglets. J. Anim. Sci 52:594.
Boyd, J.W. and R.A. Hogg. 1981. Field investigations on colostrum composition and
serum thyroxine, cortisol and immunoglobulin in naturally suckled dairy calves. J.
Comp. Path. 91:193.
Brambell, F.W.R. 1966. The transmission of immunity from mother to young and the
catabolism of immunoglobulins. Lancet 2:1087.
Brandon, M.R. 1976. Selective transfer and catabolism of IgG in the ruminant. In: W. A.
Hemmings (Ed): Maternofoetal Transmission of Immunoglobulins, pp 437-449.
Cambridge University Press Cambridge.
Brandon, M.R. and A.K. Lascelles. 1971. Relative efficiency of absorption of IgG1, IgG2,
IgA and IgM in the newborn calf. Aust. J. Exp. Biol. Med. Sci. 49:629.
Briggs, C., R. Lovell, R. Aschaffenburg, S. Bartlett, S.K. Kon, J.H.B. Roy, S.Y.
Thompson and D.M. Walker. 1951. The nutritive value of colostrum for the calf. 7.
Observations on the nature of the protective properties of colostrum. Br. J. Nutr.
37
5:356.
Brodie, T.G., W.C. Cullis and W.D. Halliburton. 1910. The gaseous metabolism of the
small intestine. Part II. The gaseous exchanges during the absorption of Witte's
peptone. J. Physiol. 40:173.
Broom, D.M. 1983. Cow-calf and sow-piglet behaviour in relation to colostrum
ingestion. Ann. Rech. Vet. 14:342.
Brown, P., M.W. Smith and R. Witty. 1968. Interdependence of albumin and sodium
transport in the foetal and new-born pig intestine. J. Physiol. 198:365.
Burton, J.H., A.A. Hosein, I. McMillan, D.G. Grieve and B.N. Wilke. 1984.
Immunoglobulin absorption in calves as influenced by dietary protein intakes of their
dams. Can. J. Anim. Sci. 64:185.
Burton, K.A. and M.W. Smith. 1977. Endocytosis and immunoglobulin transport across
the small intestine of the new-born pig. J. Physiol. 270:473.
Bush, L.J., M.A. Aguilera, G.D. Adams and E.W. Jones. 1971. Absorption of colostral
immunoglobulins by newborn dairy calves. J. Dairy Sci. 54:1547.
Bush, L.J., M.B. Mungle, L.D. Corley and G.D. Adams. 1973. Factors affecting
absorption of immunoglobulins by newborn dairy calves. J. Dairy Sci. 56:312.
Bush, L.J. and T.E. Staley. 1980. Absorption of colostral immunoglobulins in newborn
calves. J. Dairy Sci. 63:672.
Butler, J.E. 1986. Biochemistry and biology of ruminant immunoglobulins. Prog. Vet.
Microbiol. Immun. 2:1.
Butler, J.E., F. Klobasa and E. Werhahn. 1981. The differential localization of IgA, IgM
and IgG in the gut of suckled neonatal piglets. Vet. Immunol. Immunopath. 2:53.
Cabello, G. and D. Levieux. 1978. The effects of thyroxine and climatic factors on
colostral gammaglobulin absorption in newborn calves. Ann. Rech. Vet. 9:309.
Cabello, G. and D. Levieux. 1980. Comparative absorption of colostral IgG1 and IgM in
the newborn calf: effects of thyroxine, cortisol, and environmental factors. Ann.Rech.
Vet. 11:1.
Cabello, G. and D. Levieux. 1981. Absorption of colostral IgG1 by the newborn lamb:
influence of the length of gestation, birth weight and thyroid function. Res. Vet. Sci.
31:190.
38
Cabello, G., D. Levieux, J.P. Girardeau and J. Lefaivre. 1983. Intestinal K99+
Escherichia coli adhesion and absorption of colostral IgG1 in the newborn lamb:effect
of fetal infusion of thyroid hormones. Res. Vet.Sci. 35:242.
Cabello, G., D. Levieux and J. Lefaivre. 1980. The effect of intra-amniotic injections of
thyroxine on the absorption of colostral IgG1 by the newborn kid. Br.Vet. J. 136:193.
Cabello, G. and M.C. Michel. 1977. Composition of blood plasma (calcium, phosphorus,
magnesium, proteins) during the neonatal period in the calf. Influence of the state of
health. Ann. Rech. Vet. 8:203.
Chamberlain, A.G., G.C. Perry and R.E. Jones. 1965. Effect of trypsin inhibitor isolated
from sows' colostrum on the absorption of gamma-globulin by piglets. Nature
207:429.
Chan, W.S., V.G. Daniels and A.L. Thomas. 1973. Premature cessation of
macromolecule uptake by the young rat intestine following thyroxine administration.
J. Physiol. 229:112.
Clarke, R.M. and R.N. Hardy. 1969. An analysis of the mechanism of cessation of uptake
of macromolecular substances by the intestine of the young rat (`closure'). J. Physiol.
204:127.
Clarke, R.M. and R.N. Hardy. 1970. Structural changes in the small intestine associated
with the uptake of polyvinyl pyrrolidone by the young ferret, rabbit, guinea-pig, cat
and chicken. J. Physiol. 209:669.
Clarke, R.M. and R.N. Hardy. 1971. Histological changes in the small intestine of the
young pig and their relation to macromolecular uptake. J. Anat. 108:63.
Clos, J., F. Crepel, C. Legrand, J. Legrand, A. Rabie and E. Vigouroux. 1974. Thyroid
physiology during the postnatal period in the rat: a study of the development of
thyroid function and of the morphogenetic effects of thyroxine with special reference
to cerebellar maturation. Gen. Comp. Endocr. 23:178.
Comline, R.S. and A.V. Edwards. 1968. The effects of insulin on the new-born calf. J.
Physiol. 198:383.
Comline, R.S., L.W. Hall, R.B. Lavelle, P.W. Nathanielsz and M. Silver. 1974.
Parturition in the cow: endocrine changes in animals with chronically implanted
catheters in the foetal and maternal circulations. J. Endocr. 63:451.
Comline, R.S., R.W. Pomeroy and D.A. Titchen. 1953. Histological changes in the
39
intestine during colostrum absorption. J. Physiol. 122:24.
Comline, R.S., H.E. Roberts and D.A. Titchen. 1951. Route of absorption of colostrum
globulin in the newborn animal. Nature 167:561.
Conteas, C.N. and A.P.N. Majumdar. 1987. The effects of gastrin, epidermal growth
factor, and somatostatin on DNA synthesis in a small intestinal crypt cell line (IEC-6).
Proc. Soc. Exp. Biol. Med. 184:307.
Cooper, M., S. Teichberg and F. Lifshitz. 1978. Alterations in rat jejunal permeability to
a macromolecular tracer during a hyperosmotic load. Lab. Invest. 38:447.
Cornell, R. and H.A. Padykula. 1969. A cytological study of intestinal absorption in the
suckling rat. Am. J. Anat. 125:291.
Cruywagen, C.W. 1986. The effect of curd forming ability of colostrum on the absorption
of immunoglobulins by calves. J. Dairy Sci. 69(Suppl. 1):128.
Daniels, L.B., J.L. Perkins, D. Krieder, D. Tugwell, and D. Carpenter. 1974. Blood
glucose and fructose in the newborn ruminant. J. Dairy Sci. 57:1196.
Daniels, V.G. and R.N. Hardy. 1972. The role of the adrenal gland in the control of
intestinal absorption of macromolecules by the young rat. Experientia 28:272.
Daniels, V.G., R.N. Hardy and K.W. Malinowska. 1973a. The effect of adrenalectomy or
pharmacological inhibition of adrenocortical function on macromolecule uptake by the
new-born rat intestine. J. Physiol. 229:697.
Daniels, V.G., R.N. Hardy, K.W. Malinowska and P.W. Nathanielsz. 1973b. The
influence of exogenous steroids on macromolecular uptake by the small intestine of
the new-born rat. J. Physiol. 229:681.
Davidson, J.N., S.P. Yancey, S.G. Campbell and R.G. Warner. 1981. Relationship
between serum immunoglobulin values and incidence of respiratory disease in calves.
J. Am. Vet. Med. Ass. 179:708.
Dawe, S.T., A.J. Husband and C.M. Langford. 1982. Effects of induction of parturition in
ewes with dexamethasone or oestrogen on concentrations of immunoglobulins in
colostrum, and absorption of immunoglobulins by lambs. Aust. J. Biol. Sci. 35:223.
Dawkins, M.J.R. 1964. Changes in blood glucose and non-esterified fatty acids in the
foetal and newborn lamb after injection of adrenaline. Biol. Neonate 7:160.
DeNise, S.K., J.D. Robison, G.H. Stott and D.V. Armstrong. 1989. Effects of passive
40
immunity on subsequent production in dairy heifers. J. Dairy Sci. 72:552.
Deutsch, H.F. and V.R. Smith. 1957. Intestinal permeability to proteins in the newborn
herbivore. Am.J. Physiol. 191:271.
Devery, J.E., C.L. Davis and B.L. Larson. 1979. Endogenous production of
immunoglobulin IgG1 in newborn calves. J. Dairy Sci. 62:1814.
Dinsdale, D. and P.J. Healy. 1982. Enzymes involved in protein transmission by the
intestine of the newborn lamb. Histochem. J. 14:811.
Doell, R.G. and N. Kretchmer. 1964. Intestinal invertase: precocious development of
activity after injection of hydrocortisone. Science 143:42.
Donovan, G.A., L. Badinga, R.J. Collier, C.J. Wilcox and R.K. Braun. 1986. Factors
influencing passive transfer in dairy calves. J. Dairy Sci. 69:754.
Dvorak, M. 1972. Adrenocortical function in foetal, neonatal and young pigs. J. Endocr.
54:473.
Earle, I.P. 1935. Influence of the ingestion of colostrum on the proteins of the blood sera
of young foals, kids, lambs, and pigs. J. Agr. Res. 51:479.
Edelstone, D.I. and I.R. Holzman. 1981a. Oxygen consumption by the gastrointestinal
tract and liver in conscious newborn lambs. Am. J. Physiol. 240:G297.
Edelstone, D.I. and I.R. Holzman. 1981b. Gastrointestinal tract O2 uptake and regional
blood flows during digestion in conscious newborn lambs. Am. J. Physiol. 241:G289.
Edelstone, D.I. and I.R. Holzman. 1982. Fetal intestinal oxygen consumption at various
levels of oxygenation. Am. J. Physiol. 242:H50.
Eigenmann, V.U.J.E., W. Zaremba, K. Luetgebrune and E. Grunert. 1983.
Untersuchungen uber die kolostrumaufnahme und die immunglobulin absorption bei
kalbern mit und ohne geburtsazidose. Berl. Munch. Tierarztl. Wschr. 96:109.
El-Nageh, M.M. 1967. Relation entre l'arret de la resorption intestinale des anticorps et le
renouvellement de l'epithelium intestinal. Ann. Med. Vet. 111:400.
Enochs, M.R. and L.R. Johnson. 1977. Trophic effects of gastrointestinal hormones:
physiological implications. Fed. Proc. 36:1942.
Euler, A.R., W.J. Byrne, L.M. Cousins, M.E. Ament, R.D. Leake and J.H. Walsh. 1977.
Increased serum gastrin concentrations and gastric acid hyposecretion in the
41
immediate newborn period. Gastroenterology 72:1271.
Fey, H. 1971. Immunology of the newborn calf: its relationship to colisepticemia. Ann.
NY Acad. Sci. 176:49.
Foley, J.A., A.G. Hunter and D.E. Otterby. 1978. Absorption of colostral proteins by
newborn calves fed unfermented, fermented, or buffered colostrum. J. Dairy Sci.
61:1450.
Frerking, H. and T. Aeikens. 1978. About the importance of colostrum for the newborn
calf. Ann. Rech. Vet. 9:361.
Gay, C.C., T.C. McGuire and S.M. Parish. 1983. Seasonal variation in passive transfer of
immunoglobulin G1 to newborn calves. J. Am. Vet. Med. Ass. 183:566.
Genuth, S.M. 1983. The thyroid gland. In: R. M. Berne and M. N. Levy (Eds):
Physiology, pp 1013-1032. C.V. Mosby Co. St. Louis.
George, M., C.R. Balakrishnan, R.K. Bhargava and C.A.R. Raja. 1979. Effect of
gestation length on immunoglobulin absorption capacity of postnatal bovine calves.
Indian J. Dairy Sci. 32:475.
Gillette, D.D. and M. Filkins. 1966. Factors affecting antibody transfer in the newborn
puppy. Am. J. Physiol. 210:419.
Goodwin, R.F.W. 1957a. The concentration of blood sugar during starvation in the newborn calf and foal. J. Comp. Path. 67:289.
Goodwin, R.F.W. 1957b. The relationship between the concentration of blood sugar and
some vital body functions in the new-born pig. J. Physiol. 136:208.
Griffen, W.O., A. Castaneda, D.M. Nicoloff, N.H. Stone and O.H. Wangensteen. 1960.
Influence of local hypothermia on absorption from isolated intestinal segments. Proc.
Soc. Exp. Biol. Med. 103:757.
Grongnet, J.F., E. Grongnet-Pinchon, D. Levieux, M. Piot and J. Lareynie. 1986.
Newborn calf intestinal absorption of immunoglobulins extracted from colostrum.
Reprod. Nutr. Develop. 26:731.
Halliday, R. 1956. The termination of the capacity of young rats to absorb antibody from
the milk. Proc. R. Soc. Lond. B 145:179.
Halliday, R. 1958. The increase in alkaline phosphatase activity of the duodenum and
decrease in absorption of antibodies by the gut induced in young rats by
42
deoxycorticosterone acetate. J. Physiol. 140:44P.
Halliday, R. 1959. The effect of steroid hormones on the absorption of antibody by the
young rat. J. Endocr. 18:56.
Halliday, R. 1976. Some factors influencing immunoglobulin transfer in sheep. In: W. A.
Hemmings (Ed): Maternofoetal Transmission of Immunoglobulins, pp 409-415.
Cambridge University Press Cambridge.
Halliday, R., A.J.F. Russel, M.R. Williams and J.N. Peart. 1978. Effects of energy intake
during late pregnancy and of genotype on immunoglobulin transfer to calves in
suckler herds. Res. Vet. Sci. 24:26.
Hanawalt, V.M. and J. Sampson. 1947. Studies on baby pig mortality. V. Relationship
between age and time of onset of acute hypoglycemia in fasting newborn pigs. Am. J.
Vet. Res. 8:235.
Hansen, R.G. and P.H. Phillips. 1947a. Studies on proteins from bovine colostrum. I.
Electrophoretic studies on the blood serum proteins of colostrum-free calves and of
calves fed colostrum at various ages. J. Biol. Chem. 171:223.
Hansen, R.G. and P.H. Phillips. 1947b. Studies on the globulins of bovine colostrum. II.
The absorption of globulins by the young calf. J. Dairy Sci. 30:560.
Hansen, R.G. and P.H. Phillips. 1949. Studies on proteins from bovine colostrum. III.
The homologous and heterologous transfer of ingested protein to the bloodstream of
the young animal. J. Biol. Chem. 179:523.
Hardy, R.N. 1964. Intestinal absorption of macromolecules in the new-born pig. J.
Physiol. 176:19P.
Hardy, R.N. 1968. The acceleration by certain anions of the absorption of
macromolecular substances from the small intestine of the new-born calf. J. Physiol.
194:45P.
Hardy, R.N. 1969a. The influence of specific chemical factors in the solvent on the
absorption of macromolecular substances from the small intestine of the new-born
calf. J. Physiol. 204:607.
Hardy, R.N. 1969b. The break-down of [131I]gamma-globulin in the digestive tract of the
new-born pig. J. Physiol.205:435.
Hardy, R.N. 1969c. Proteolytic activity during the absorption of [131I]gamma-globulin in
the new-born calf. J. Physiol. 205:453.
43
Healy, P.J. and D. Dinsdale. 1979. Protein transmission in the intestine of the newborn
lamb: the involvement of acid and alkaline phosphatase activity. Histochem. J. 11:289.
Henning, S.J. 1981. Postnatal development: coordination of feeding, digestion, and
metabolism. Am. J. Physiol. 241:199.
Henriques de Jesus, C.H. and M.W. Smith. 1974. Protein and glucose-induced changes in
sodium transport across the pig small intestine. J. Physiol. 243:225.
Hill, K.J. 1956. Gastric development and antibody transference in the lamb, with some
observations on the rat and guinea-pig. Quart. J. Exptl. Physiol. 41:421.
Hill, K.J. and W.S. Hardy. 1971. Histological and histochemical observations on the
intestinal cells of lambs and kids absorbing colostrum. Nature 54:1353.
Hoerlein, A.B. 1957. The influence of colostrum on antibody response in baby pigs. J.
Immunol. 78:112.
Houdiniere, A. 1944. Le colostrum de vache. Le Lait 24:313.
Hough, R.L., F.D. McCarthy, C.D. Thatcher, H.D. Kent and D.E. Eversole. 1990.
Influence of glucocorticoids on macromolecular absorption and passive immunity in
neonatal lambs. J. Anim. Sci. 68:2459.
Howe, P.E. 1921. An effect of the ingestion of colostrum upon the composition of the
blood of the calves. J. Biol. Chem. 49:115.
Huddleson, I.F., E. Wood and A. Cressman. 1945. The differential diagnosis of bovine
brucellosis from the bactericidal action of blood plasma. Science 101:358.
Husband, A.J., M.R. Brandon and A.K. Lascelles. 1973. The effect of corticosteroid on
absorption and endogenous production of immunoglobulins in calves. Aust. J. Exp.
Biol. Med. Sci. 51:707.
Ingram, P.L., R. Lovell, P.C. Wood, R. Aschaffenburg, S. Bartlett, S.K. Kon, J. Palmer,
J.H.B. Roy, and K.W.G. Shillam. 1956. Bacterium coli antibodies in colostrum and
their relation to calf survival. J. Path. Bact. 72:561.
Israel, E.J., K.Y. Pang, P.R. Harmatz and W.A. Walker. 1987. Structural and functional
maturation of rat gastrointestinal barrier with thyroxine. Am. J. Physiol. 252:G762.
James, R.E. and C.E. Polan. 1978. Effect of orally administered duodenal fluid on serum
proteins in neonatal calves. J. Dairy Sci. 61:1444.
44
James, R.E., C.E. Polan and K.A. Cummins. 1981. Influence of administered indigenous
microorganisms on uptake of [Iodine-125] gamma-globulin in vivo by intestinal
segments of neonatal calves. J. Dairy Sci. 64:52.
James, R.E., C.E. Polan and W.A. O'Connor. 1978. The distributional uptake of 125Igamma-globulin in the small intestine of neonatal calves. J. Dairy Sci. 61(Suppl.
1):176.
Jameson, E., C. Alvarez-Tostado and H.H. Sortor. 1942. Electrophoretic studies on newborn calf serum. Proc. Soc. Exp. Biol. Med. 51:163.
Jeffcott, L.B. 1971. Duration of permeability of the intestine to macromolecules in the
newly-born foal. Vet. Rec. 88:340.
Jeffcott, L.B. 1974. Studies on passive immunity in the foal. II. The absorption of 125Ilabeled PVP (polyvinylpyrrolidone) by the neonatal intestine. J. Comp. Path. 84:279.
Johnson, L.R. 1977. New aspects of the trophic action of gastrointestinal hormones.
Gastroenterology 72:788.
Johnson, L.R., D. Aures and R. Hakanson. 1969a. Effect of gastrin on the in vivo
incorporation of 14C-leucine into protein of the digestive tract. Proc. Soc. Exp. Biol.
Med. 132:996.
Johnson, L.R., D. Aures and L. Yuen. 1969b. Pentagastrin-induced stimulation of protein
synthesis in the gastrointestinal tract. Am. J. Physiol. 217:251.
Johnson, L.R. and A.M. Chandler. 1973. RNA and DNA of gastric and duodenal mucosa
in antrectomized and gastrin-treated rats. Am. J. Physiol. 224:937.
Johnson, L.R. and P.D. Guthrie. 1974. Secretin inhibition of gastrin-stimulated
deoxyribonucleic acid synthesis. Gastroenterology 67:601.
Johnston, N.E. and W.D. Oxender. 1979. Effect of altered serum glucocorticoid
concentrations on the ability of the newborn calf to absorb colostral immunoglobulin.
Am. J. Vet. Res. 40:32.
Jones, E.A. and T.A. Waldmann. 1972. The mechanism of intestinal uptake and
transcellular transport of IgG in the neonatal rat. J. Clin. Invest. 51:2916.
Jones, R.E. 1972a. Intestinal absorption and degradation of rat and bovine gammaglobulins in the suckling rat. Biochim. Biophys. Acta 255:530.
45
Jones, R.E. 1972b. Intestinal absorption and gastro-intestinal digestion of protein in the
young rat during the normal and cortisone-induced post-closure period. Biochim.
Biophys. Acta 274:412.
Jordan, S.M. and E.H. Morgan. 1968. The development of selectivity of protein
absorption from the intestine during suckling in the rat. Aust. J. Exp. Biol. Med. Sci.
46:465.
Kelley, K.W., F. Blecha and J.A. Regnier. 1982. Cold exposure and absorption of
colostral immunoglobulins by neonatal pigs. J. Anim. Sci. 55:363.
Kennedy, W.L., A.K. Anderson, S.I. Bechdel and J.F. Shigley. 1939. Studies on the
composition of bovine blood as influenced by gestation, lactation, and age. J. Dairy
Sci. 22:251.
Khurana, M.L. and M.L. Madan. 1984. Circulating tri-iodothyronine and thyroxine in
female neonate cattle and buffaloes. Indian J. Anim. Sci. 54:304.
Kiddy, C.A., R. McCann, C. Maxwell, C. Rock, C. Pierce and J.E. Butler. 1971. Changes
in levels of immunoglobulins in serum and other body fluids immediately before and
after parturition. J. Dairy Sci. 54:1325.
Klein, R.M. and J.C. McKenzie. 1980. Pattern of crypt cell proliferation in the pre- and
post-closure ileum of the neonatal rat: effects of sympathectomy. Cell Tissue Res.
206:387.
Koldovsky, O. and J.J. Herbst. 1973. Cell migration and cortisone-evoked decrease of
acid beta-galactosidase in the ileum of suckling rats. Gastroenterology 64:1142.
Koldovsky, O., P. Sunshine and N. Kretchmer. 1966. Cellular migration of intestinal
epithelia in suckling and weaned rats. Nature 212:1389.
Kruse, V. 1970. Absorption of immunoglobulin from colostrum in newborn calves.
Anim. Prod. 12:627.
Kumano, Y., Y. Kanamaru, R. Niki and S. Arima. 1976. Occurrence of bovine IgG1
fragment in feces of newborn calf. Jpn. J. Zootech. Sci. 47:551.
Kvietys, P.R., J.A. Barrowman, S.L. Harper and D.N. Granger. 1986. Relations among
canine intestinal motility, blood flow, and oxygenation. Am. J. Physiol. 251:G25.
Leary, H.L. and J.G. Lecce. 1976. Uptake of macromolecules by enterocytes on
transposed and isolated piglet small intestine. J. Nutr. 106:419.
46
Leary, H.L. and J.G. Lecce. 1978. Effect of feeding on the cessation of transport of
macromolecules by enterocytes of neonatal piglet intestine. Biol. Neonate 34:174.
Leary, H.L. and J.G. Lecce. 1979. The preferential transport of immunoglobulin G by the
small intestine of the neonatal piglet. J. Nutr. 109:458.
Lecce, J.G. 1966a. Absorption of macromolecules by neonatal intestine. Biol. Neonate
9:50.
Lecce, J.G. 1966b. Glucose milliequivalents eaten by the neonatal pig and cessation of
intestinal absorption of large molecules (closure). J. Nutr. 90:240.
Lecce, J.G. 1966c. In vitro absorption of gamma-globulin by neonatal intestinal
epithelium of the pig. J. Physiol. 184:594.
Lecce, J.G. 1972. Selective absorption of macromolecules into intestinal epithelium and
blood by neonatal mice. J. Nutr. 102:69.
Lecce, J.G. 1973. Effect of dietary regimen on cessation of uptake of macromolecules by
piglet intestinal epithelium (closure) and transport to the blood. J. Nutr. 103:751.
Lecce, J.G. and C.W. Broughton. 1973. Cessation of uptake of macromolecules by
neonatal guinea pig, hamster and rabbit intestinal epithelium (closure) and transport
into blood. J. Nutr. 103:744.
Lecce, J.G. and G. Matrone. 1960. Porcine neonatal nutrition: the effect of diet on blood
serum proteins and performance of the baby pig. J. Nutr. 70:13.
Lecce, J.G., G. Matrone and D.O. Morgan. 1961. Porcine neonatal nutrition: absorption
of unaltered non-porcine proteins and polyvinylpyrrolidone from the gut of piglets and
the subsequent effect on the maturation of the serum protein profile. J. Nutr. 73:158.
Lecce, J.G. and D.O. Morgan. 1962. Effect of dietary regimen on cessation of intestinal
absorption of large molecules (closure) in the neonatal pig and lamb. J. Nutr. 78:263.
Lecce, J.G., D.O. Morgan and G. Matrone. 1964. Effect of feeding colostral and milk
components on the cessation of intestinal absorption of large molecules (closure) in
neonatal pigs. J. Nutr. 84:43.
Levin, R.J. 1969. The effects of hormones on the absorptive, metabolic and digestive
functions of the small intestine. J. Endocr. 45:315.
Lichtenberger, L. and L.R. Johnson. 1974. Gastrin in the ontogenic development of the
small intestine. Am. J. Physiol. 227:390.
47
Lichtenberger, L., L.R. Miller, D.N. Erwin and L.R. Johnson. 1973. Effect of
pentagastrin on adult rat duodenal cells in culture. Gastroenterology 65:242.
Logan, E.F., C.H. McMurray, D.G. O'Neill, P.J. McParland and F.J. McRory. 1978.
Absorption of colostral immunoglobulins by the neonatal calf. Br. Vet. J. 134:258.
Logan, E.F. and G.R. Pearson. 1978. The distribution of immunoglobulins in the intestine
of the neonatal calf. Ann. Rech. Vet. 9:319.
Logan, E.F., A. Stenhouse, D.J. Ormrod and W.J. Penhale. 1974. The role of colostral
immunoglobulins in intestinal immunity to enteric colibacillosis in the calf. Res. Vet.
Sci. 17:290.
Lopez, J.W., S.D. Allen, J. Mitchell and M. Quinn. 1988. Rotavirus and cryptosporidium
shedding in dairy calf feces and its relationship to colostral immune transfer. 71:1288.
Mach, J-P. and J-J. Pahud. 1971. Bovine secretory immune system. J. Dairy Sci. 54:1327.
Malinowska, K.W., W.S. Chan, P.W. Nathanielsz and R.N. Hardy. 1974. Plasma
adrenocorticosteroid changes during thyroxine-induced accelerated maturation of the
neonatal rat intestine. Experientia 30:61.
Malinowska, K.W., R.N. Hardy and P.W. Nathanielsz. 1972. Neonatal adrenocortical
function and its possible relation to the uptake of macromolecules by the small
intestine of the guinea-pig and rabbit. J. Endocr. 55:397.
Martinsson, K. and L. Jonsson. 1976. The uptake of macromolecules in the ileum of
piglets after intestinal "closure". Zbl. Vet. Med. A 23:277.
Matte, J.J., C.L. Girard, J.R. Seoane and G.J. Brisson. 1982. Absorption of colostral
immunoglobulin G in the newborn dairy calf. J. Dairy Sci. 65:1765.
McCarthy, E.F. and E.I. McDougall. 1953. Absorption of immune globulin by the young
lamb after ingestion of colostrum. Biochem. J. 55:177.
McEwan, A.D., E.W. Fisher and I.E. Selman. 1968. The effect of colostrum on the
volume and composition of the plasma of calves. Res. Vet. Sci. 9:284.
McGuire, T.C., N.E. Pfeiffer, J.M. Weikel and R.C. Bartsch. 1976. Failure of colostral
immunoglobulin transfer in calves dying from infectious disease. J. Am. Vet. Med.
Ass. 169:713.
Mensik, J., E. Salajka, J. Stepanek, L. Ulmann, Z. Prochaska and J. Dressler. 1978. Use
48
of polyvalent cow colostrum in the prevention of enteric infections in calves and
piglets. Ann. Rech. Vet. 9:255.
Merriman, M.J.G.S. 1971. Serum immunoglobulins in newborn calves before and after
colostrum feeding. Can. J. Comp. Med. 35:269.
Minor, J.T. and R.L. Riese. Neonatal calf care. Iowa St. Vet. 46:17.
Moog, F. and K. Yeh. 1979. Pinocytosis persists in the ileum of hypophysectomized rats
unless closure is induced by thyroxine or cortisone. Develop. Biol. 69:159.
Moon, H.W. 1971. Epithelial cell migration in the alimentary mucosa of the suckling pig.
Proc. Soc. Exp. Biol. Med. 137:151.
Moon, H.W. and D.D. Joel. 1975. Epithelial cell migration in the small intestine of sheep
and calves. Am. J. Vet. Res. 36:187.
Moon, H.W., E.M. Kohler and S.C. Whipp. 1973. Vacuolation: a function of cell age in
porcine ileal absorptive cells. Lab. Invest. 28:23.
Morphis, L.G. and D. Gitlin. 1970. Maturation of the maternofoetal transport system for
human gamma-globulinin the mouse. Nature 228:573.
Morris, B. and D. Begley. 1970. The absorption of antibody by the duodenum and
jejunum in young rats. J. Zool. Lond. 162:453.
Morris, B. and R. Morris. 1976. The effects of corticosterone and cortisone on the uptake
of polyvinylpyrrolidone and the transmission of immunoglobulin G by the small
intestine in young rats. J. Physiol. 254:389.
Muggli, N.E., W.D. Hohenboken, L.V. Cundiff and K.W. Kelley. 1984. Inheritance of
maternal immunoglobulin G1 concentration by the bovine neonate. J. Anim. Sci.
59:39.
Murakami, T., N. Hirano, A. Inoue, K. Chitose, K. Tsuchiya, K. Ono and Y. Naito. 1985.
Transfer of antibodies against viruses of calf diarrhea from cows to their offspring via
colostrum. Jpn. J. Vet. Sci. 47:507.
Murata, H. and S. Namioka. 1977. The duration of colostral immunoglobulin uptake by
the epithelium of the small intestine of neonatal piglets. J. Comp. Path. 87:431.
Naylor, J.M. 1979. Colostral immunity in the calf and the foal. Vet. Clin. N. Am.: Large
Anim. Pract. 1:331.
49
Nightengale, G.T. and G.H. Stott. 1981. Adrenal response of the newborn calf to acute
inanition and colostral feeding. J. Dairy Sci. 64:236.
Nocek, J.E., D.G. Braund and R.G. Warner. 1984. Influence of neonatal colostrum
administration, immunoglobulin, and continued feeding of colostrum on calf gain,
health, and serum protein. J. Dairy Sci. 67:319.
Nowicki, P.T., B.S. Stonestreet, N.B. Hansen, A.C. Yao and W. Oh. 1983.
Gastrointestinal blood flow and oxygen consumption in awake newborn piglets: effect
of feeding. Am. J. Physiol. 245:G697.
Odde, K.G., L.A. Abernathy and G.A. Greathouse. 1986. Effect of body condition and
calving difficulty on calf vigor and serum immunoglobulin concentrations in twoyear-old beef heifers. CSU Beef Program Report :16.
Ohmann, H.B. 1981. Immunoglobulin levels in non-aborted and aborted fetuses from
Danish herds of cattle. Acta Vet. Scand. 22:428.
Olson, D.P., R.C. Bull, L.F. Woodard and K.W. Kelley. 1981a. Effects of maternal
nutritional restriction and cold stress on young calves: absorption of colostral
immunoglobulins. Am. J. Vet. Res. 42:876.
Olson, D.P., C.J. Papasian and R.C. Ritter. 1980. The effects of cold stress on neonatal
calves II. Absorption of colostral immunoglobulins. Can. J. Comp. Med. 44:19.
Olson, D.P., R.C. Ritter, C.J. Papasian and S. Gutenberger. 1981b. Sympathoadrenal and
adrenal hormonal responses of newborn calves to hypothermia. Can. J. Comp. Med.
45:321.
Oyeniyi, O.O. and A.G. Hunter. 1978. Colostral constituents including immunoglobulins
in the first three milkings postpartum. J. Dairy Sci. 61:44.
Passaro, E.P. Jr, I.E. Gillespie and M.I. Grossman. 1963. Potentiation between gastrin
and histamine in stimulation of gastric secretion. Proc. Soc. Exp. Biol. Med. 114:50.
Patt, J.A. 1977. Factors affecting the duration of intestinal permeability to
macromolecules in newborn animals. Biol. Rev. 52:411.
Patt, J.A., A. Zarkower and R.J. Eberhart. 1972. Effect of histamine on intestinal
absorption of gamma globulin in newborn calves. J. Dairy Sci. 55:645.
Pauli, J.V. 1983. Colostral transfer of gamma glutamyltransferase in lambs. N. Z. Vet. J.
31:150.
50
Payne, L.C. and C.L. Marsh. 1962a. Absorption of gammaglobulin by the small intestine.
Fed. Proc. 21:909.
Payne, L.C. and C.L. Marsh. 1962b. Gamma globulin absorption in the baby pig: the
non-selective absorption of heterologous globulins and factors influencing absorption
time. J. Nutr. 76:151.
Peitsch, W., K. Takeuchi and L.R. Johnson. 1981. Mucosal gastrin receptor. VI.
Induction by corticosterone in newborn rats. Am. J. Physiol. 240:G442.
Penhale, W.J., E.F. Logan, I.E. Selman, E.W. Fisher and A.D. McEwan. 1973.
Observations on the absorption of colostral immunoglobulins by the neonatal calf and
their significance in colibacillosis. Ann. Rech. Vet. 4:223.
Petrie, L. 1984. Maximising the absorption of colostral immunoglobulins in the newborn
dairy calf. Vet. Rec. 114:157.
Petrie, L., S.D. Acres and D.H. McCartney. 1984. The yield of colostrum and colostral
gammaglobulins in beef cows and the absorption of colostral gammaglobulins by beef
calves. Can. Vet. J. 25:273.
Pierce, A.E., P.C. Risdall and B. Shaw. 1964. Absorption of orally administered insulin
by the newly born calf. J. Physiol. 171:203.
Pierce, A.E. and M.W. Smith. 1967. The in vitro transfer of bovine immune lactoglobulin
across the intestine of new-born pigs. J. Physiol. 190:19.
Pollack, P.F. and T.E. Solomon. 1986. Trophic effect of secretin and pentagastrin on
small intestine of the developing rat. Gastroenterology 90:1588.
Pope, G.S. and J.H.B. Roy. 1953. The oestrogenic activity of bovine colostrum.
Biochem. J. 53:427.
Porter, P. 1976. Intestinal absorption of colostral IgA anti-E. coli antibodies by the
neonatal piglet and calf. In: W. A. Hemmings (Ed): Maternofoetal Transmission of
Immunoglobulins, pp 397-407. Cambridge University Press Cambridge.
Porter, P., D.E. Noakes and W.D. Allen. 1972. Intestinal secretion of immunoglobulins in
the preruminant calf. Immunol. 23:299.
Quinton, P.M. and C.W. Philpott. 1973. A role for anionic sites in epithelial architecture.
J. Cell Biol. 56:787.
Ragsdale, A. and S. Brody. 1923. The colostrum problem and its solution. J. Dairy Sci.
51
6:137.
Ratcliff, L., N.L. Jacobson and R.S. Allen. 1958. Effect of age and of dietary regime on
hemoglobin and reducing-sugar levels in the blood of dairy calves. J. Dairy Sci.
41:1401.
Ratner, B., H.C. Jackson and H.L. Gruehl. 1927. Transmission of protein
hypersensitiveness from mother to offspring. II. The role of colostrum. J. Immun.
14:267.
Redman, D.R. 1979. Prenatal influence on immunocompetence of the neonate. J. Anim.
Sci. 49:258.
Reeves, J.T., F.S. Daoud and M. Gentry. 1972. Growth of the fetal calf and its arterial
pressure, blood gases,and hematologic data. J. Appl. Physiol. 32:240.
Roberts, H.E., A.N. Worden and E.T.R. Evans. 1954. Observations on some effects of
colostrum deprivation in the calf. J. Comp. Path. 64:283.
Robison, J.D., G.H. Stott and S.K. DeNise. 1988. Effects of passive immunity on growth
and survival in the dairy heifer. J. Dairy Sci. 71:1283.
Rowland, S.J., J.H.B. Roy, H.J. Sears and S.Y. Thompson. 1953. The effect of prepartum
milking on the composition of the prepartum and postpartum secretions of the cow. J.
Dairy Res. 20:16.
Roy, J.H.B. 1980. Factors affecting susceptibility of calves to disease. J. Dairy Sci.
63:650.
Roy, J.H.B., J. Palmer, K.W.G. Shillam, P.L. Ingram and P.C. Wood. 1955. The nutritive
value of colostrum for the calf. 10. The relationship between the period of time that a
calfhouse has been occupied and the incidence of scouring and mortality in young
calves. Br. J. Nutr. 9:11.
Ruckebush, Y., C. Dardillat and P. Guilloteau. 1983. Development of digestive functions
in the newborn ruminant. Ann. Rech. Vet. 14:360.
Rundell, J.O. and J.G. Lecce. 1972. Independence of intestinal epithelial cell turnover
from cessation of absorption of macromolecules (closure) in the neonatal mouse,
rabbit, hamster and guinea pig. Biol. Neonate 20:51.
Sasaki, M., C.L. Davis and B. Larson. 1977. Immunoglobulin IgG1 metabolism in new
born calves. J. Dairy Sci. 60:623.
52
Savage, E.S. and C.M. McCay. 1942. The nutrition of calves: a review. J. Dairy Sci.
25:595.
Sawyer, M., B.I. Osburn, H.D. Knight and J.W. Kendrick. 1973. A quantitative
serological assay for diagnosing congenital infections of cattle. Am. J. Vet. Res.
34:1281.
Schultz, R.D., H.W. Dunne and C.E. Heist. 1971. Ontogeny of the bovine immune
response. J. Dairy Sci. 54:1321.
Schwartz, M.Z. and R.B. Storozuk. 1985. Enhancement of small intestinal function by
gastrin. J. Surg. Res. 38:613.
Selman, I.E. 1973. The absorption of colostral globulins by newborn calves. Ann. Rech.
Vet. 4:213.
Selman, I.E., A.D. McEwan and E.W. Fisher. 1971. Absorption of immune lactoglobulin
by newborn dairy calves. Res. Vet. Sci. 12:205.
Shannon, A.D. and A.K. Lascelles. 1968. Lymph flow and protein composition of
thoracic duct lymph in the newborn calf. Quart. J. Exptl. Physiol. 53:415.
Smith, E.L. 1948. The isolation and properties of the immune proteins of bovine milk and
colostrum and their role in immunity: a review. J. Dairy Sci. 31:127.
Smith, E.L. and A. Holm. 1948. The transfer of immunity to the new-born calf from
colostrum. J. Biol. Chem. 175:349.
Smith, K.L. 1971a. Role of estrogen in the selective transport of IgG1 into the mammary
gland. J. Dairy Sci. 54:1322.
Smith, M.W. 1971b. Ionic dependence of protein transport across the new-born pig
intestine. J. Physiol. 214:349.
Smith, M.W. and K.A. Burton. 1972. Calcium dependence of protein transport by the
small intestine of the new-born pig. Experientia 28:667.
Smith, M.W. and L.G. Jarvis. 1978. Growth and cell replacement in the new-born pig
intestine. Proc. R. Soc. Lond. B 203:69.
Smith, M.W. and A.E. Pierce. 1967. Effect of amino-acids on the transport of bovine
immune lactoglobulin across new-born pig intestine. Nature 213:1150.
Smith, M.W., R. Witty and P. Brown. 1968. Effect of poly-L-arginine on rate of bovine
53
IgG transport by newborn pig intestine. Nature 220:387.
Smith, T. 1925. Hydropic stages in the intestinal epithelium of new-born calves. J. Exp.
Med. 41:81.
Smith, T. and R.B. Little. 1922. The significance of colostrum to the new-born calf. J.
Exp. Med. 36:181.
Smith, T. and M.L. Orcutt. 1925. The bacteriology of the intestinal tract of young calves
with special reference to the early diarrhea ("scours"). J. Exp. Med. 41:89.
Smith, V.R. and E.S. Erwin. 1959. Absorption of colostrum globulins introduced directly
into the duodenum. J.Dairy Sci. 42:364.
Smith, V.R., R.E. Reed and E.S. Erwin. 1964. Relation of physiological age to intestinal
permeability in the bovine. J. Dairy Sci. 47:923.
Snyder, A.C., J.D. Schuh, T.N. Wegner and J.R. Gebert. 1974. Passive immunization of
the newborn dairy calf via fermented colostrum. J. Dairy Sci. 57:641.
Soll, A.H. 1977. Secretagogue stimulation of O2 consumption and 14C-aminopyrine (AP)
uptake by enriched canine parietal cells. Gastroenterology 72:1166.
Soll, A.H. 1978a. The actions of secretagogues on oxygen uptake by isolated mammalian
parietal cells. J. Clin. Invest. 61:370.
Soll, A.H. 1978b. The interaction of histamine with gastrin and carbamylcholine on
oxygen uptake by isolated mammalian parietal cells. J. Clin. Invest. 61:381.
Soll, A.H. and M.I. Grossman. 1978. Cellular mechanisms in acid secretion. Ann. Rev.
Med. 29:495.
Staley, T.E., L.D. Corley, L.J. Bush and E.W. Jones. 1972. The ultrastructure of neonatal
calf intestine and absorption of heterologous proteins. Anat. Rec. 172:559.
Staley, T.E., E.W. Jones and L.J. Bush. 1971. Maternal transport of immunoglobulins to
the calf. J. Dairy Sci. 54:1323.
Staley, T.E., E.W. Jones and L.D. Corley. 1969. Fine structure of duodenal absorptive
cells in the newborn pig before and after feeding of colostrum. Am. J. Vet. Res.
30:567.
Stott, G.H. 1980. Immunoglobulin absorption in calf neonates with special considerations
of stress. J.Dairy Sci. 63:681.
54
Stott, G.H., D.B. Marx, B.E. Menefee and G.T. Nightengale. 1979a. Colostral
immunoglobulin transfer in calves. I. Period of absorption. J. Dairy Sci. 62:1632.
Stott, G.H., D.B. Marx, B.E. Menefee and G.T. Nightengale. 1979b. Colostral
immunoglobulin transfer in calves. II. The rate of absorption. J. Dairy Sci. 62:1766.
Stott, G.H., D.B. Marx, B.E. Menefee and G.T. Nightengale. 1979c. Colostral
immunoglobulin transfer in calves. III. Amount of absorption. J. Dairy Sci. 62:1902.
Stott, G.H., D.B. Marx, B.E. Menefee and G.T. Nightingale. 1979d. Colostral
immunoglobulin transfer in calves. IV. Effect of suckling. J. Dairy Sci. 62:1908.
Stott, G.H. and B.E. Menefee. 1978. Selective absorption of immunoglobulin IgM in the
newborn calf. J. Dairy Sci. 61:461.
Stott, G.H. and E.J. Reinhard. 1978. Adrenal function and passive immunity in the
dystocial calf. J. Dairy Sci. 61:1457.
Svendson, J. and N. Bille. 1981. Reducing baby pig mortality. In: A. D. Leman, R. D.
Glock, W. L. Mengeling, R.H.C. Penny, E. Scholl, and B. Straw (Eds): Diseases of
Swine, pp 729-736. The Iowa State University Press Ames.
Takeuchi, K., W. Peitsch and L.R. Johnson. 1981. Mucosal gastrin receptor. V.
Development in newborn rats. Am. J. Physiol. 240:G163.
Tenore, A., J.S. Parks, M. Gasparo and O. Koldovsky. 1980. Thyroidal response to
peroral TSH in suckling and weaned rats. Am. J. Physiol. 238:E428.
Trahair, J.F., R.A. Perry, M. Silver and P.M. Robinson. 1986. Enterocyte migration in the
foetal sheep small intestine. Biol. Neonate 50:214.
Trahair, J.F., R.A. Perry, M. Silver and P.M. Robinson. 1987a. Studies on the maturation
of the small intestine of the fetal sheep. I. The effects of bilateral adrenalectomy.
Quart. J. Exptl. Physiol. 72:61.
Trahair, J.F., R.A. Perry, M. Silver and P.M. Robinson. 1987b. Studies on the maturation
of the small intestine in the fetal sheep. II. The effects of exogenous cortisol. Quart. J.
Exptl. Physiol. 72:71.
Trahair, J.F., P.M. Robinson and M. Silver. 1984. The development of the late-term
ovine fetal small intestine and the effect of adrenalectomy. Can. J. Anim. Sci. (Suppl.)
64:259.
55
Tyler, H.D. and H.A. Ramsey. 1991. Hypoxia in neonatal calves: effect on intestinal
transport of immunoglobulins. J. Dairy Sci. 74:316.
Udall, J.N., K.J. Bloch, G. Vachino, P. Feldman and W.A. Walker. 1984. Development of
the gastrointestinal mucosal barrier. IV. The effect of inhibition of proteolysis on the
uptake of macromolecules by the intestine of the newborn rabbit before and after
weaning. Biol. Neonate 45:289.
Walker, W.A. and K.J. Isselbacher. 1974. Uptake and transport of macromolecules by the
intestine: possible role in clinical disorders. Gastroenterology 67:531.
Wallace, K.H. and A.R. Rees. 1980. Studies on the immunoglobulin-G Fc-fragment
receptor from neonatal rat small intestine. Biochem. J. 188:9.
Walsh, J.H. 1987. Gastrointestinal hormones. In: L.R. Johnson (Ed): Physiology of the
Gastrointestinal Tract, pp 181-253. Raven Press New York.
Werhahn, E., F. Klobasa and J.E. Butler. 1981. Investigation of some factors which
influence the absorption of IgG by the neonatal piglet. Vet. Immunol. Immunopath.
2:35.
Westrom, B.R., J. Svendson and C. Tagesson. 1984a. Intestinal permeability to
polyethyleneglycol 600 in relation to macromolecular `closure' in the neonatal pig.
Gut 25: 520.
Westrom, B.R., J. Svendsen, B.G. Ohlsson, C. Tagesson and B.W. Karlsson. 1984b.
Intestinal transmission of macromolecules (BSA and FITC-labelled dextrans) in the
neonatal pig. Biol. Neonate 46:20.
Williams, M.R., R.L. Spooner and L.H. Thomas. 1975. Quantitative studies on bovine
immunoglobulins. Vet. Rec. 96:81.
Wilson, J.A. 1979. Principles of Animal Physiology. Macmillan Publishing Co., New
York.
Witty R., P. Brown and M.W. Smith. 1969. The transport of various immune globulins
by the new-born pig intestine. Experientia 25:310.
Wood, P.C. 1955. The epidemiology of white scours among calves kept under
experimental conditions. J. Path. Bact. 70:179.
Yeh, K. and F. Moog. 1975. Development of the small intestine in the
hypophysectomized rat. II. Influence of cortisone, thyroxine, growth hormone, and
prolactin. Develop. Biol. 47:173.
56
Young, J.W., E.O. Otchere, A. Trenkle and N.L. Jacobson. 1970. Effect of age on
glucose, reducing sugars and plasma insulin in blood of milk-fed calves. J. Nutr.
100:1267.
57
EFFECT OF FRUCTOSE-INDUCED HYPOGLYCEMIA ON
CESSATION OF MACROMOLECULAR TRANSPORT
IN THE NEONATAL CALF
Howard Tyler and Harold Ramsey
Department of Animal Science
College of Agriculture and Life Sciences
North Carolina State University
Raleigh 27695-7621
58
ABSTRACT
Three colostrum-deprived calves were assigned at birth to each of two treatments,
GLUCOSE (GLC) and FRUCTOSE (FRC), to determine if availability of glucose during
early life mediates the cessation of intestinal transport of immunoglobulins (closure).
Glucose and fructose (100 g in 2 L of electrolyte solution) were fed to GLC and FRC
calves, respectively, at 3, 6, 9, 12, 15, 18, and 21 h postpartum. Colostrum (1 kg) was fed
to all calves at 24, 36, and 48 h postpartum. Venous blood was sampled and plasma
harvested for the measurement of glucose, fructose, insulin and IgG. During the first 24
h, mean values for plasma glucose from GLC and FRC calves, respectively, were 282
and 24 mg/dl (p < .01). The latter value reflects the degree of hypoglycemia induced by
FRC. During the same period, mean concentrations of plasma fructose from GLC and
FRC calves, respectively, were 4 and 230 mg/dl (p < .01). Plasma insulin concentrations
for precolostral calves were 101 and 11 μU/ml for GLC and FRC calves, respectively (p
< .01). Peak IgG levels were extremely low for both groups of calves, indicating that
intestinal transport of immunoglobulins had virtually ceased by the end of the 24-h
treatment period. Thus, fructose-induced hypoglycemia had no effect on the time of
intestinal closure in the newborn calf.
(Key Words: Calf, Hypoglycemia, Immunoglobulins, Newborn, Immunity.)
59
INTRODUCTION
Postnatal immune transmission in calves is dependent on the amount of colostrum
ingested (Smith and Holm, 1948; Aschaffenburg et al., 1949), the mass of
immunoglobulin in the ingested colostrum (Earle, 1935; Smith and Holm, 1948; Kruse,
1970; Bush et al., 1971; Bush et al., 1973) and the elapsed time between birth and first
feeding (Jeffcott, 1974). Most of the variation in absorption by calves is due to age at
first feeding (Kruse, 1970), although this is not the case in lambs and piglets (Lecce and
Morgan, 1962). In piglets, fasting delays closure to a much greater extent than in calves..
While investigating nutritional factors that initiate closure in piglets, Lecce et al.
(1964) found that colostral proteins, fats, vitamins and minerals are without effect,
whereas a fat- and protein-free colostral whey induces closure in a normal manner. Later
studies showed that pure solutions of various sugars induce closure (Lecce, 1966;
Werhahn et al., 1981) and that at least 300 milliequivalents of glucose are required. The
possibility that closure could also be mediated by the same mechanism in neonatal calves
has never been explored.
The primary objective of this study was to explore the relationship between
availability of glucose to the small intestine and timing of closure. Fructose has been
purported to induce hypoglycemia in newborn calves without evoking an insulin
response (Edwards and Powers, 1967). By using fructose in this study, the effects of
hypoglycemia should be removed from those of hyperinsulinemia.
MATERIALS AND METHODS
60
Six Holstein calves were obtained at birth and assigned alternately to treatment
groups. FRC calves were fed 100 g of fructose at each feeding in an electrolyte solution
with a final osmolarity of 996 mOsm. To balance for the effects of osmolarity, GLC
calves were treated identically except for the substitution of glucose for fructose. All
calves were fed 2 L of the appropriate solution at 3, 6, 12, 15, 18, and 21 h postpartum.
One kg of pooled colostrum was fed at 24, 36, and 48 h. Two kg of milk were fed at 60
and 72 h.
Blood samples were obtained via jugular venipuncture every 3 h through the first day,
every 6 h through the second day, and every 12 h through the third day postpartum.
Plasma was separated by centrifugation at 1286 x g for 15 min and stored at -20° C.
Plasma samples were analyzed for glucose, fructose, insulin, and IgG. Fructose was
determined spectrophotometrically by the method of Roe (1934). Glucose was
determined by the oxygen rate method using a Beckman oxygen electrode (Beckman
Instruments, Inc., Brea, CA). Quantification of IgG was by radial immunodiffusion with
commercial gels (ICN Biomedical, Inc., Costa Mesa, CA). Insulin concentrations were
determined via radioimmunoassay. Intraassay coefficient of variation was 3.8% and
sensitivity of the assay was .16 μU/ml.
Time of closure was estimated by calculating the join point (J) as described by
Hudson (1966) and subsequently modified by Stott et al. (1979a). This procedure relies
on the gradual decline in plasma IgG following cessation of transport, which reflects both
catabolism of IgG and its transfer to extravascular pools (Bush et al., 1971). Thus, the
resulting IgG concentration pattern in colostrum-fed calves over the first 72 h describes a
61
parabolic curve. By eliminating data points not occuring during the linear phase of
absorption, the resulting observations can be fitted to two first-order regression lines.
The intersection of these lines defines J, the estimate for time of closure. Sampling of
blood, therefore, must be continued post-closure to satisfy requirements for both
regression equations.
All data were analyzed using the General Linear Models Procedure of SAS (SAS,
1985). The statistical model included treatment and calf effects. The data were sorted by
time to evaluate treatment effects at each sampling period. In addition, data were sorted
by period (day 1, 2, or 3) to evaluate treatment effects throughout the entire treatment
period and post-treatment periods. Significance of difference between means were
determined by the method of least squares means using ANOVA. The data for time of
closure (J) were analyzed separately using the General Linear Models Procedure of SAS
(SAS Institute, Cary, NC). The model included treatment and calf effects. In all cases,
probabilities greater than .05 were not considered significant and are reported
accordingly.
RESULTS AND DISCUSSION
The inert nature of fructose in fetal and newborn lambs and piglets is well documented
(Alexander et al., 1970; Scott et al., 1967; Ballard and Oliver, 1965; Warnes et al.,
1982). However, Setchell et al. (1972) found that fructose contributed carbon to
glycogen synthesis in fetal lambs, and White et al. (1982) reported that labeled carbon
(14C-fructose) from fructose in fetal pigs was used for nucleic acid synthesis. In both
62
these studies, less than 5% of infused fructose was metabolized, still supporting the
hypothesis that fructose is relatively metabolically inert.
Fructose feeding in the present study induced a hyperfructosemic condition in FRC
calves (Figure 1) well in excess of the concentrations reported by Edwards and Powers
(1964) as necessary for inducing hypoglycemia. By 6 h, fructose had risen to 168 mg/dl
in FRC calves vs 11 mg/dl in GLC calves (p < .01). Concentrations of fructose peaked at
289 mg/dl at 21 h in FRC calves, whereas fructose was undetectable in GLC calves by 12
h. During day 1, mean concentrations of plasma fructose from GLC and FRC calves,
respectively, were 4 and 230 mg/dl (p < .01).
Glucose concentrations in FRC calves decreased to 20 mg/dl by 6 h (Figure 2).
Glucose concentrations in GLC calves were 283 mg/dl at this time (p < .01). Glucose
concentrations were significantly different between treatment groups for the period
between 6 and 30 h. Glucose concentrations in GLC calves peaked at 427 mg/dl at 18 h,
and declined rapidly following cessation of treatment. During the first 24 h, mean values
for plasma glucose from GLC and FRC calves, respectively, were 282 and 24 mg/dl (p <
.01). The latter value reflects the degree of hypoglycemia induced by FRC. Glucose
concentrations in FRC calves decreased to levels similar to GLC calves by 42 h, closely
paralleling the loss of plasma fructose in these calves. Fructose concentrations decreased
linearly from 24 to 42 h, at which time fructose was undetectable in FRC calves.
63
64
65
66
67
Contrary to the reports of Edwards and Powers (1967), hyperfructosemia was
associated with an increase in insulin concentrations in this experiment (Figure 3). This
response was somewhat obscured by the hyperinsulinemia induced in GLC calves.
Insulin concentrations for day 1 were 11 μU/ml and 101 μU/ml in FRC and GLC calves,
respectively (p < .01). Concentrations in FRC calves were similar to those seen previous
studies in colostrum-fed newborn calves, but higher than expected in fasted calves (Tyler
and Ramsey, unpublished observations). Whether this increase is responsible for the
hypoglycemic condition of these calves, and more importantly, whether it influenced
closure, cannot be determined from this study. Insulin concentrations continued to be
different during day 2 (8 vs 26 μU/ml in FRC and GLC calves, respectively) (p < .01).
By day 3, concentrations of insulin in both groups were similar (2 μU/ml for both
groups).
The primary objective of this experiment was to determine the effects of
hypoglycemia on immunoglobulin (Ig) transport and timing of cessation of this transport
in newborn calves. Timing of closure was not determined due to the fact that absorption
occurred only during the first bleeding interval following initial ingestion of colostrum.
Accurate determination of age at closure is not possible using the join point method
under these conditions. Minimal concentrations of IgG attained by FRC and GLC calves
in this study suggest that closure was virtually complete by 24 h in both groups (Figure
4). GLC calves actually tended to reach higher peak IgG concentrations (68 mg/dl) than
FRC calves (29 mg/dl) (p = .06).
68
69
70
71
72
The possibility exists that the high concentrations of fructose attained by FRC calves
in this study coupled with their hypoglycemic condition may have stimulated utilization
of fructose as an energy source. Recent research has suggested that pathways for
conversion of fructose to glucose in newborn calves are active to a greater extent than
previously reported (Kurz, 1990). Therefore, the question of whether glucose availability
may mediate closure has not been fully resolved.
LITERATURE CITED
Alexander, D.P., H.G. Britton and D.A. Nixon. 1970. The metabolism of fructose and
glucose by the sheep foetus: studies on the isolated perfused preparation with
radioactively labelled sugars. Quart. J. Exp. Physiol. 55:346.
Aschaffenburg, R., S. Bartlett, S.K. Kon, S.Y. Thompson, D.M. Walker, C. Briggs, E.
Cotchin and R. Lovell. 1949. The nutritive significance of colostrum for the calf.
XIIth Int. Dairy Congress (Stockholm) :90.
Ballard, F.J. and I.T. Oliver. 1965. Carbohydrate metabolism in liver from foetal and
neonatal sheep. Biochem. J. 95:191.
Bush, L.J., M.A. Aguilera, G.D. Adams and E.W. Jones. 1971. Absorption of colostral
immunoglobulins by newborn dairy calves. J. Dairy Sci. 54:1547.
Bush, L.J., M.B. Mungle, L.D. Corley and G.D. Adams. 1973. Factors affecting
absorption of immunoglobulins by newborn dairy calves. J. Dairy Sci. 56:312.
Earle, I.P. 1935. Influence of the ingestion of colostrum on the proteins of the blood sera
of young foals, kids, lambs, and pigs. J. Agr. Res. 51:479.
Edwards, A.V. and N. Powers. 1967. Effect of intravenous of fructose in newborn
calves. Nature. 214:728.
Hudson, D.J. 1966. Fitting segmented curves whose join points have to be estimated.
Am. Stat. Ass. J. 61:1097.
73
Jeffcott, L.B. 1974. Studies on passive immunity in the foal. II. The absorption of 125Ilabeled PVP (polyvinylpyrrolidone) by the neonatal intestine. J. Comp. Path. 84:279.
Kruse, V. 1970. Absorption of immunoglobulin from colostrum in newborn calves.
Anim. Prod. 12:627.
Kurz, M. 1990. Studies on the dynamics and benefits of fructose and sorbitol as
supplements for neonatal and growing calves. PhD Dissertation. The Ohio State
University, Columbus, Ohio.
Lecce, J.G. 1966. Glucose milliequivalents eaten by the neonatal pig and cessation of
intestinal absorption of large molecules (closure). J. Nutr. 90:240.
Lecce, J.G. and D.O. Morgan. 1962. Effect of dietary regimen on cessation of intestinal
absorption of large molecules (closure) in the neonatal pig and lamb. J. Nutr. 78:263.
Lecce, J.G., D.O. Morgan and G. Matrone. 1964. Effect of feeding colostral and milk
components on the cessation of intestinal absorption of large molecules (closure) in
neonatal pigs. J. Nutr. 84:43.
Roe, J.H. 1934. A colorimetric method for the determination of fructose in blood and
urine. J. Biol. Chem. 107:15.
SAS. 1985. SAS User's Guide: Statistics. SAS Institute, Inc. Cary, NC.
Setchell, B.P., J.M. Bassett, N.T. Hinks and N.M. Graham. 1972. The importance of
glucose in the oxidative metabolism of the pregnant uterus and its contents in
conscious sheep and with some preliminary observations on the oxidation of fructose
and glucose by fetal sheep. Quart. J. Exp. Physiol. 57:257.
Smith, E.L. and A. Holm. 1948. The transfer of immunity to the new-born calf from
colostrum. J. Biol. Chem. 175:349.
Stott, G.H., D.B. Marx, B.E. Menefee and G.T. Nightengale. 1979a. Colostral
immunoglobulin transfer in calves. I. Period of absorption. J. Dairy Sci. 62:1632.
Warnes, D.M., R.F. Seamark and F.J. Ballard. 1977. Metabolism of glucose, fructose and
lactate in vivo in chronically cannulated foetuses and in suckling lambs. Biochem. J.
162:617.
Werhahn, E., F. Klobasa and J.E. Butler. 1981. Investigation of some factors which
influence the absorption of IgG by the neonatal piglet. Vet. Immunol. Immunopath.
2:35.
74
White, C.E., E.L. Piper, P.R. Noland and L.B. Daniels. 1982. Fructose utilization for
nucleic acid synthesis in the fetal pig. 55:73.
75
EFFECT OF INSULIN-INDUCED HYPOGLYCEMIA ON
CESSATION OF MACROMOLECULAR TRANSPORT
IN THE NEONATAL CALF
Howard Tyler and Harold Ramsey
Department of Animal Science
College of Agriculture and Life Sciences
North Carolina State University
Raleigh 27695-7621
76
ABSTRACT
The effect of hypoglycemia on the absorption of colostral immunoglobulins was studied
in twelve neonatal calves. Hypoglycemic calves received 1 cc (100 units) of insulin
(INS) at birth, whereas control calves received 1 cc of saline (SAL). Two dietary
regimens were imposed, with colostrum-feeding being initiated at birth (FED) or at 24 h
(FAST). Venous blood was sampled and plasma harvested for the measurement of
glucose, insulin, and IgG. Insulin induced decreases (P < .01) in circulating glucose in
INS calves from 12 to 42 h postnatally, with insulin values being significantly higher (p <
.05) from 12 through 24 h. Concentrations of glucose were also significantly decreased
(p < .05) when compared to FED calves over the same span of time, although insulin
values were not different. Time of closure was delayed in INS calves relative to SAL
calves (40 and 29 h, respectively) and in FAST calves relative to FED calves (45 and 23
h, respectively). Peak IgG levels were not different between INS-FED and SAL-FED
calves or between INS-FAST and SAL-FAST calves, primarily due to decreased rate of
IgG transport in INS calves relative to SAL calves. The results suggest that glucose
availability influences timing of closure in the calf, although the decreased rate of
absorption of IgG in hypoglycemic calves prevents them from realizing any benefit in
terms of higher peak IgG levels than their normoglycemic counterparts.
(Key Words: Calf, Hypoglycemia, Immunoglobulins, Newborn, Immunity.)
77
INTRODUCTION
Cessation of macromolecular transport by the small intestine of the neonatal calf
(closure) is a gradual phenomenon, with efficiency of transport slowly diminishing prior
to complete cessation (Matte, et al., 1982). Shannon and Lascelles (1968) reported that
transport of gamma-globulin ceases within 24 h of first colostrum feeding. The work of
Stott et al. (1979a) tends to verify this finding. They estimated time of closure of calves
fed at birth to be 22 h while for those calves first fed at 24 h closure did not occur until
33 h. The ability of colostrum to accelerate closure suggests that some factor in
colostrum may be acting to stimulate closure either luminally or humorally. Colostrum
intake accelerates closure in all ungulates to varying degrees.
In piglets, fasting delays closure to a much greater extent than in calves. The
difference between a fasted piglet or lamb and a fasted calf may be related to the
postnatal blood glucose pattern. Newborn pigs and lambs are susceptible to fasting
hypoglycemia. Glycogen reserves at birth are relatively limited in these species as
compared to those of calves (Svendson and Bille, 1981). Blood glucose concentrations
decrease shortly after birth (Hanawalt and Sampson, 1947; Bassett and Alexander, 1971)
and do not recover without feeding (Goodwin, 1957b). In unstressed fed piglets, glucose
levels gradually rise over a 2- to 3-wk period (Dawkins, 1964; Bassett and Alexander,
1971; Daniels et al., 1974). Neonatal calves and foals present a different scenario.
Glucose concentrations are lower at birth, but they rise to twice adult levels within the
first 24 h. This rise is generally considered to be independent of nutritional status
(Goodwin, 1957a; Comline and Edwards, 1968; Daniels et al., 1974). Following the
78
initial 24-h rise in blood glucose, concentrations gradually decline over the next 6 wk
(Kennedy et al., 1939; Ratcliffe et al., 1958; Young et al., 1970).
Dietary induction of closure in the piglet supports this hypothesis. The stimulating
action of feeding in the piglet has been shown to be due to the glucose or lactose ingested
(Lecce, 1966; Werhahn et al., 1981). Pure solutions of various sugars induce closure and
at least 300 milliequivalents of glucose are required. Solutions of glycine or inorganic
salts are ineffective. Leary and Lecce (1976) reported that feeding induces closure even
in surgically isolated intestinal segments, suggesting that induction of closure is
humorally regulated and not dependent on luminal exposure to glucose. The primary
objective of this study, then, is to explore the relationship between availability of glucose
to the small intestine and timing of closure both luminally and humorally through either
dietary regimen, exogenous insulin treatment, or a combination thereof.
MATERIALS AND METHODS
Twelve Holstein calves were obtained at birth and assigned alternately to treatment
groups. Calves were treated at birth with either 1 cc (100 units) of insulin (INS) or 1 cc
of saline (SAL). Calves within each treatment group were either fed colostrum at birth (2
kg) and 12 h (1 kg) (FED) or fasted for the first 24 h (FAST). FED calves also received 1
kg colostrum at 24 h, followed by 2 kg whole milk every 12 h to 72 h. FAST calves were
fed 2 kg colostrum at 24 h, 1 kg colostrum at 36 and 48 h, and 2 kg whole milk at 60 and
72 h. All colostrum feedings were from a pooled source of colostrum.
Blood samples were obtained every 6 h from birth through the second day, and every
79
12 h through the third day postpartum. Blood was obtained via jugular venipuncture into
sterile evacuated tubes containing potassium oxalate and sodium fluoride (Becton
Dickinson, Rutherford, NJ). Plasma was immediately separated by centrifugation at
1286 x g for 15 min and stored at -20° C. Plasma samples were analyzed for glucose,
insulin, and IgG. Glucose was determined by the oxygen rate method using a Beckman
oxygen electrode (Beckman Instruments, Inc., Brea, CA). Quantification of IgG was by
the radial immunodiffusion method of Mancini et al. (1965) as modified by Fahey and
McKelvey (1965) using commercial gels (ICN Biomedical, Inc., Costa Mesa, CA).
Insulin concentrations were determined via radioimmunoassay. Intraassay coefficient of
variation was 3.7% and the sensitivity of the assay was .16 μU/ml.
Time of closure was estimated by calculating the join point as described by Hudson
(1966) and subsequently modified by Stott et al. (1979a). This procedure relies on the
gradual decline in plasma IgG following cessation of transport, which reflects both
catabolism of IgG and its transfer to extravascular pools (Bush et al., 1971). Thus, the
resulting IgG concentration pattern in colostrum-fed calves over the first 72 h describes a
parabolic curve. By eliminating data points not occuring during the linear phase of
absorption, the resulting observations can be fitted to two first-order regression lines.
The intersection of these lines defines J, the estimate for time of closure. Sampling of
blood, therefore, must be continued post-closure to satisfy requirements for both
regression equations.
Rate of absorption was determined by the increase in plasma IgG observed during
bleeding intervals (Stott et al., 1979b) for the 4 consecutive sampling periods following
80
the initial feeding. Periods were designated T1, T2, T3, and T4.
The experiment was designed as a simple factorial with diet and treatment as
dependent variables. All data were analyzed using the General Linear Models Procedure
of SAS (SAS, 1985). The statistical model included treatment, diet, and calf effects. The
data were sorted by time to evaluate treatment and diet effects and treatment by diet
interactions. Significance of difference between means was determined by the method of
least squares means using ANOVA. The data for time of closure and rate of absorption
were analyzed separately using the General Linear Models Procedure of SAS (SAS,
1985). The model included treatment and diet and diet by treatment interactions.
Significance of difference between means for rate of absorption at different time intervals
was determined by the method of least squares means using ANOVA.
RESULTS AND DISCUSSION
A hypoglycemic condition was induced in INS calves during the first 2 d (Figure 1).
By 6 h, glucose concentrations tended to be lower in INS calves relative to SAL calves (p
= .07). From 12 to 42 h, mean glucose concentrations were significantly
81
82
83
lower in INS calves than in SAL calves (p < .01). Differences were still apparent at 48 h
(p = .08), but by 60 h, glucose concentrations in INS calves were only slightly lower than
in SAL calves (62 vs 70 mg/dl, respectively). INS treatment at birth may induce a
compensatory response in circulating glucose concentrations, as 72 h values tended to be
higher in INS than SAL calves (p = .08). This was most apparent in INS-FAST calves,
with glucose values in these calves significantly higher than INS-FED, SAL-FAST and
SAL-FED calves (p < .05).
Glucose was also affected by diet (Figure 2), with differences beginning to become
apparent as early as 6 h (p = .06). Glucose concentrations were lower in FAST calves
than in FED calves through 42 h (p < .05), but returned to normal by 48 h. No
differences in glucose due to diet were apparent during day 3.
As expected, insulin concentrations were elevated in INS calves relative to SAL
calves, beginning at 12 h (Figure 3) (p < .05). Insulin in INS calves remained
significantly higher than in SAL calves through 24 h (p < .01). By 30 h, peaking values
in SAL calves and decreasing concentrations in INS calves left all calves with similar
insulin levels. Insulin decreased for all calves after this time, but a lower rate of
clearance was apparent in INS calves, and insulin values were again significantly higher
in this group at 42 h (p < .05). Both groups had similar insulin concentrations throughout
the remainder of the experiment. No significant differences were detectable between
FED and FAST calves (Figure 4).
Time of closure was delayed in INS calves relative to SAL calves (40 and 29 h,
respectively)(Figure 5) and in FAST calves relative to FED calves (45 and 23 h,
84
85
86
87
88
89
90
91
92
respectively) (Figure 6). Peak IgG levels were not different between INS-FED and SALFED calves or between INS-FAST and SAL-FAST calves (Figure 7), apparently due to
decreased rate of IgG transport in INS calves relative to SAL calves (Table 1). Rate of
absorption for INS calves was non-significantly lower for periods T1, T2, and T3 when
compared to SAL calves.
SUMMARY
The results of this study suggest that mediation of closure is similar in calves, pigs and
lambs. Availability of glucose, either through endogenous or exogenous sources, is
critical to the closure process in calves, piglets and lambs, and deprivation of either
luminal or humoral glucose sources will delay closure in all these species.
In conclusion, insulin-induced hypoglycemia prolonged the absorptive period in
newborn calves. Glucose availability influences timing of closure in the calf, although
the decreased rate of absorption of IgG in hypoglycemic calves prevents them from
realizing any benefit in terms of higher peak IgG levels than those found in their
normoglycemic counterparts.
LITERATURE CITED
Bassett, J.M. and G. Alexander. 1971. Insulin, growth hormone and corticosteroids in
neonatal lambs. Biol.Neonate 17:112.
Bush, L.J., M.A. Aguilera, G.D. Adams and E.W. Jones. 1971. Absorption of colostral
immunoglobulins by newborn dairy calves. J. Dairy Sci. 54:1547.
Comline, R.S. and A.V. Edwards. 1968. The effects of insulin on the new-born calf. J.
Physiol. 198:383.
93
94
95
96
97
Table 1. Rate constants (g/L⋅h-1) for INS, SAL, FAST and FED calves
for the first four 6-h time periods following initial colostrum
feeding (T1, T2, T3, and T4). Pooled SE = .33 g/L⋅h-1.
INS
SAL
FAST
FED
T1
.77
.85
.48
1.13
T2
.29
.67
.23
.73
T3
.18
.47
.13
.52
T4
.19
.22
.02
.39
98
Daniels, L.B., J.L. Perkins, D. Krieder, D. Tugwell, and D. Carpenter. 1974. Blood
glucose and fructose in the newborn ruminant. J. Dairy Sci. 57:1196.
Dawkins, M.J.R. 1964. Changes in blood glucose and non-esterified fatty acids in the
foetal and newborn lamb after injection of adrenaline. Biol. Neonate 7:160.
Fahey, J.L. and E.M. McKelvey. 1965. Quantitative determination of serum
immunoglobulins in antibody agar plates. J. Immunol. 94:84.
Goodwin, R.F.W. 1957a. The concentration of blood sugar during starvation in the newborn calf and foal. J. Comp. Path. 67:289.
Goodwin, R.F.W. 1957b. The relationship between the concentration of blood sugar and
some vital body functions in the new-born pig. J. Physiol. 136:208.
Hanawalt, V.M. and J. Sampson. 1947. Studies on baby pig mortality. V. Relationship
between age and time of onset of acute hypoglycemia in fasting newborn pigs. Am. J.
Vet. Res. 8:235.
Hudson, D.J. 1966. Fitting segmented curves whose join points have to be estimated.
Am. Stat. Ass. J. 61:1097.
Kennedy, W.L., A.K. Anderson, S.I. Bechdel and J.F. Shigley. 1939. Studies on the
composition of bovine blood as influenced by gestation, lactation, and age. J. Dairy
Sci. 22:251.
Leary, H.L. and J.G. Lecce. 1976. Uptake of macromolecules by enterocytes on
transposed and isolated piglet small intestine. J. Nutr. 106:419.
Lecce, J.G. 1966b. Glucose milliequivalents eaten by the neonatal pig and cessation of
intestinal absorption of large molecules (closure). J. Nutr. 90:240.
Mancini, G., A.O. Carbonara and J.F. Heremans. 1965. Immunochemical quantitations of
antigens by single radial immunodiffusion. Immunochem. 2:235.
Matte, J.J., C.L. Girard, J.R. Seoane and G.J. Brisson. 1982. Absorption of colostral
immunoglobulin G in the newborn dairy calf. J. Dairy Sci. 65:1765.
Ratcliff, L., N.L. Jacobson and R.S. Allen. 1958. Effect of age and of dietary regime on
hemoglobin and reducing-sugar levels in the blood of dairy calves. J. Dairy Sci.
41:1401.
SAS. 1985. SAS User's Guide: Statistics. SAS Institute, Inc. Cary, NC.
99
Shannon, A.D. and A.K. Lascelles. 1968. Lymph flow and protein composition of
thoracic duct lymph in the newborn calf. Quart. J. Exptl. Physiol. 53:415.
Stott, G.H., D.B. Marx, B.E. Menefee and G.T. Nightengale. 1979a. Colostral
immunoglobulin transfer in calves. I. Period of absorption. J. Dairy Sci. 62:1632.
Stott, G.H., D.B. Marx, B.E. Menefee and G.T. Nightengale. 1979b. Colostral
immunoglobulin transfer in calves. II. The rate of absorption. J. Dairy Sci. 62:1766.
Svendson, J. and N. Bille. 1981. Reducing baby pig mortality. In: A. D. Leman, R. D.
Glock, W. L.Mengeling, R.H.C. Penny, E. Scholl, and B. Straw (Eds): Diseases of
Swine, pp 729-736. The Iowa State University Press Ames.
Werhahn, E., F. Klobasa and J.E. Butler. 1981. Investigation of some factors which
influence the absorption of IgG by the neonatal piglet. Vet. Immunol. Immunopath.
2:35.
Young, J.W., E.O. Otchere, A. Trenkle and N.L. Jacobson. 1970. Effect of age on
glucose, reducing sugars and plasma insulin in blood of milk-fed calves. J. Nutr.
100:1267.
100
DEVELOPMENT OF AN IN VIVO PERFUSION
SYSTEM FOR BOVINE FETAL SMALL INTESTINE
Howard Tyler
Department of Animal Science
North Carolina State University
Raleigh, NC 27695
Lloyd Tate, Jr.
Department of Food Animal and Equine Medicine
College of Veterinary Medicine
North Carolina State University
Raleigh, NC 27695
Harold Ramsey
Department of Animal Science
College of Agriculture and Life Sciences
North Carolina State University
Raleigh, NC 27695
Ian Longmuir
Department of Biochemistry
College of Agriculture and Life Sciences
North Carolina State University
Raleigh 27695
101
ABSTRACT
Oxygen availability has been shown to be involved in the cessation of macromolecular
transport in the small intestine of the newborn calf. To further investigate this
phenomenon, a technique is described which allows control of the oxygen tension in the
blood perfusing the small intestine of the fetal calf. Briefly, an artificial circulatory
extension is surgically inserted in the superior mesenteric artery of the fetus.
Exteriorization of a silastic section of this extension allows equilibration of the blood
flowing in the cannula with ambient gas pressures, and the length of the silastic section
determines the extent of oxygenation of the blood. Advantages and drawbacks of this
technique are discussed.
(Key Words: Calf, Oxygen Tension, Small Intestine.)
INTRODUCTION
Previous work at this institution has focused on the role of oxygen availability on the
development of bovine small intestine during the perinatal period. In particular, the
change in oxygen tension associated with the conversion from placental to pulmonary
respiration at birth was hypothesized to initiate an alteration in macromolecular
permeability during the first 24 h of life that is characteristic of the bovine neonate. The
first model to test this hypothesis was the hypoxic postnatal calf (Tyler and Ramsey,
1991). By providing the newborn calf with a 90:10 mixture of N2:O2, arterial Po2 was
maintained at a level similar to that of the fetal calf. The results of this study were
inconclusive, however, which may have been due to other changes occurring at birth that
102
influence intestinal development.
In a postnatal in vivo model, there appears to be no acceptable way to isolate the
neonatal small intestinal system. Moreover, the potential for an in vitro model is limited
by the fragility of the newborn small intestinal tissue, thus giving a high degree of
inaccuracy in absorptive studies lasting more than a few hours. A better approach,
therefore, may be to increase oxygen availability to the small intestine of the fetus,
thereby initiating oxygen-induced changes which normally occur at birth. This approach
has several distinct advantages over a postnatal model. Fetal calves are relatively stable
metabolically and endocrinologically compared to newborns. Additionally, changes that
normally occur in the prepartum period, while they may have a role in the cessation of
macromolecular permeability of the small intestine in the postnatal period, do not
normally induce this change prenatally. Although a surgical approach is required, the
intestinal tissue itself is not involved, and any effect of surgical manipulation would be
reflected in the control animals.
Chronic catheterization of fetal vessels as a technique for studying fetal metabolism
has become a relatively common procedure. There are, however, some inherent
limitations in this technique. Catheters are subject to blockage and prone to inaccuracies,
such as dislodging of position, which can be difficult to detect and impractical to correct.
Additionally, chronic alteration of arterial oxygen tension in the fetus to any significant
extent is difficult to accomplish by any previously established techniques. To further
complicate matters, mixing of oxygenated and deoxygenated blood occurs at several
locations in the fetus between the point where oxygenated blood enters the fetus in the
103
umbilical vein and the cranial mesenteric artery. This effectively eliminates the
possibility of manipulating either umbilical blood or maternal blood to control Po2 of the
blood supply of the small intestine. Also, increasing oxygen tension in the entire fetus
may introduce additional sources of error into the experimental design.
The development of our technique was intended to overcome these drawbacks in an
effective manner and allow manipulation of a single organ or system while minimizing
effects on the fetus as a whole. The approach involves the implantation of a circulatory
extension in the cranial mesenteric artery of the bovine fetus at approximately day 268 of
gestation. The goals of the technique were fourfold: 1) to implant an extension in the
cranial mesenteric artery and maintain continuous blood flow through the extension postoperatively; 2) to obtain complete post-operative recovery for the dam and her fetus
culminating in parturition at the appropriate time; 3) to maintain catheter patency through
parturition and into the postnatal period; and 4) to generate accurate and meaningful data
throughout the experimental period.
The purpose of this discourse is to discuss the model under development, the
application of this model in terms of oxygen manipulation, and potential applications
under consideration for later study. With respect to the current study that necessitated
development of the model, the objective was to test the hypothesis that increasing
mesenteric arterial oxygen availability stimulates development of small intestinal tissue
prior to parturition.
MATERIALS AND METHODS
104
Catheter Preparation
The goal of treatment is to alter oxygen tension in flowing blood with minimal effects
on peripheral resistance and flow rate. Our approach is to utilize the gas permeability
characteristics of Silastic brand tubing (Dow Corning Corporation, Midland, MI) as an
oxygenator. However, the same characteristic that makes Silastic ideal as a treatment
catheter in this model makes it unacceptable elsewhere in the system. Although
equilibration with the air occurs in the exteriorized section of the tubing, equilibration
with tissue would be occurring in other parts of the catheter, thereby creating significant
inaccuracies. Silastic is also a pliable tubing with a propensity for kinking and(or)
collapsing under external pressure. Therefore, polyethylene tubing (Intramedic tubing,
Becton Dickinson and Company, Parsippany, NJ) is used internally as it is relatively gas
impermeable and rigid.
For control animals, a single length of polyethylene tubing (3.17mm x 3.99mm x
6.75m) is used. Spliced junctions are created in the tubing 2.5m from either end using
Silastic cuffs (2.94mm x 4.08mm x 5cm). For treated animals, a 1.75m piece of Silastic
brand tubing (2.94mm x 4.08mm) is spliced between two sections of polyethylene
(3.17mm x 3.99mm x 2.5m). Polyethylene catheters are encased within a double layer of
Silastic tubing (4.76mm x 7.94mm inside; 6.35mm x 9.53mm outside) for improved
suturing characteristics at exteriorization sites and to facilitate healing. Sheathing in this
manner also minimizes kinking of the catheter during normal fetal movements. Silastic
sheaths and cuffs are prepared by swelling in toluene for 1 h prior to slipping over the
polyethylene. Evaporation of the toluene shrinks the Silastic, providing a tight-fitting
105
sheath (Huntington et al., 1989). Polyethylene cannulas are all pretreated with 2%
tridodecylmethylammonium heparinate (TDMAC-heparin) (Polysciences, Inc.,
Warrington, PA) complex to minimize clotting problems. All catheters are sterilized
with ethylene oxide prior to surgery.
Surgery
Pregnant cows are obtained on approximately day 268 of gestation. Feed and water
are withheld for 24 h prior to surgery. Anesthesia is induced with 50 g guafenisen and
4.5 g thiamylal intravenously and maintained with halothane. The cow is placed in
dorsal recumbency, clipped and prepared in a routine manner. The abdomen is opened
via a mid-ventral incision immediately ventral to the mammary vein and extending from
the umbilicus to the cranial edge of the mammary gland (approximately 25 cm). Skin,
subcutaneous tissues, and linea alba are all incised. Hemmorhage is controlled with a
combination of electrocautery and ligation. The pregnant uterine horn is located and a
ventral incision is made over the greater curvature of the uterus in a hypovascular area.
The fetus is delivered caudally to cranially, exposing only the hindlegs and abdomen as
far as the sternum. The fetal abdomen is opened with a midline incision approximately
from 5 cm above to 5 cm below the umbilicus. The subcutaneous tissue and linea alba
are likewise incised in this area. The cranial mesenteric artery is isolated, and, if
necessary, the pancreas bluntly dissected away to expose a 2-cm length of free artery.
Vascular clamps on either end of the exposed length of artery control blood flow. The
artery is clamped, severed in the middle, and the proximal end of the catheter tubing is
106
inserted upstream about 1.5 cm. This tubing is secured by 2-0 Ethicon as blood flows to
the distal end of the tubing. Then the distal end of the tubing is inserted downstream in
the artery as the clamp is removed, reestablishing blood flow to the small intestine. This
end of the tubing is likewise secured. The fetal abdomen is closed with 2-0 Ethicon in a
simple continuous pattern combining linea alba, subcutaneous tissue and skin together.
The catheter exits the caudal end of the incision, and is secured to the skin of the fetal
abdomen and right hind leg. The fetus is repositioned in the uterus and the uterus is
closed with #2 Dexon using a modified Lembert pattern over an inverted Connell pattern.
The catheters exit the incision at the tip of the uterus and are secured using a purse string
suture and liberal amounts of tissue. Subcutaneous tissues are undermined and the
catheters exit the body wall through a 2-cm stab wound on the right flank. This incision
is closed with #2 Vetafil in a simple continuous pattern. The linea alba in the primary
incision is closed with #2 Dexon in a simple continuous pattern. The superficial fascia
and subcutaneous tissues are closed with 2-0 catgut in a simple continuous pattern, while
#2 Vetafil is used in a Ford interlocking pattern to close the skin. The catheters are fixed
to the skin using 2-0 Ethalon. The tubing now serves as an artificial circulatory extension
(ACE).
Sampling and Analysis
Heparinized 1-ml blood samples are drawn twice daily from both the upstream and
downstream ends of the Silastic tubing, reflecting pre- and post-treatment values,
107
respectively. Samples are analyzed on an Instrumentation Laboratories System 1302
Blood Gas System and subsequently on an Instrumentation Laboratories 482 Cooximeter System. Values for pH, Po2, Pco2, and [HCO3-] are presented.
RESULTS
Exteriorization of the ACE as described allows direct measurements of blood flow,
access for both manipulation of both blood flow and blood constituents, and ease of
sampling. The use of Silastic tubing allows manipulation of oxygen tension in the blood.
Mixing of this highly oxygenated blood with the less oxygenated blood from the
umbilical vein at the ductus venosus should result in only small increases in the Po2 of
the blood supplying the rest of the fetus. Thus, this system effectively isolates treatment
effects to small intestinal tissue in an in vivo system.
Prenatal perfusion of fetal intestinal tissue with highly oxygenated blood should allow
developmental changes to occur prenatally similar to those occurring in newborn calves.
If cessation of immunoglobulin transport in the small intestine is initiated by the change
in oxygen tension associated with birth, treated calves should absorb no
immunoglobulins after colostrum ingestion postnatally while control calves should
absorb immunoglobulins in a normal manner.
In the four surgeries that have been performed, the first goal of the four outlined has
been achieved. In the first surgery, kinking of the ACE ended the experiment the day
after surgery. The ACE was subsequently modified by the addition of the Silastic sheaths
previously described, and no further kinking problems have been encountered. The
108
second attempt ended when the dam was euthanized due to extensive peritonitis. The
peritonitis appears to have been alleviated by having the catheters exit the skin at the
flank stab wound at a different point than they exit the subcutaneous tissues, thus
eliminating a direct access for microorganisms to the body cavity. In addition, particular
attention is paid to keeping the exit wound clean and as sterile as possible. The third
surgery ended with the calf succumbing to the anesthesia during the operation. It should
be noted that all three animals were relatively old (>7 y) and either over- or underweight.
The last surgery was performed on a 3-y old Holstein heifer in excellent condition. The
surgery and recovery both went well, with none of the problems that had plagued our
previous attempts. However, the ACE was apparently not well secured within the cranial
mesenteric artery and it pulled loose. The calf died and the dam was subjected to
euthanasia. The development of the procedure appears to be progressing well and should
require only minor modifications in technique and(or) postsurgical procedures to achieve
success in this project.
One successful aspect of this project has been the use of Silastic as an oxygen
exchanger (Table 1). It should be noted that these values are from a longer length
ofSilastic than is proposed in the protocol. The Po2 values obtained after equilibration
with air are elevated beyond anticipated values for ambient oxygen
109
Table 1. Arterial blood gas and acid-base values in fetal calves for
blood flowing through a 1.75 m length of Silastic tubing.
pH
Po2
(mm Hg)
Pco2
[HCO3-]
(mm Hg)
(mEq/L)
initial values
7.176
27
45.8
17.1
post-treatment
7.713
179
6.6
8.5
110
tension. This may have been due to slight acidification of the blood samples by heparin
within the sealed syringes. The pH of the heparin was 5.8, which may have altered the
pH of the blood enough to dissociate some of the oxygen from hemoglobin, especially at
100% saturation. Even a small increase in unbound oxygen would translate into
relatively large increases in oxygen tension. For future samples, heparin pH will be
adjusted to 7.4 to alleviate these inaccuracies.
The altered acid-base status of the fetus due to decreased Pco2 is an obvious concern.
If shortening the Silastic used does not improve the pH and Pco2 values to acceptable
levels, a method for increasing the CO2 content of the air around the catheter will be
necessary.
DISCUSSION
The results from four attempts to create an exteriorized arterial extension in fetal
calves have been described. All attempts were only partially successful, as frequently
happens in the development of any new procedure. The major technical problems
associated with this procedure appear to be post-surgical peritonitis and preventing the
catheter from kinking due to movement of the fetus. Techniques to alleviate these
problems have been developed but remain to be proven in the course of repeated
successful procedures.
The results indicate that blood flowing through a short length of exteriorized Silastic
tubing equilibrates rapidly with air. The exact length of tubing required for adequate
equilibration will depend on diameter of the tubing and velocity of the blood. Therefore,
111
the length recommended in the protocol is an estimate based on preliminary observations
and will doubtless require further adjustment.
Other research applications for this technique include metabolite and mesenteric blood
flow response to different hormones and(or) metabolite and hormonal responses to
restricted mesenteric blood flow. Implantation of an additional ACE into the portal vein
of the fetus would allow direct measurement of substrate utilization and(or) production
by small intestinal tissue in utero. However, this greatly increases the complexity of the
surgery and the potential for post-surgical complications. Therefore, the perfusion
technique will need to be perfected prior to attempting any additional cannulations.
LITERATURE CITED
Huntington, G.B., C.K. Reynolds and B.H. Stroud. 1989. Techniques for measuring
blood flow in splanchnic tissues of cattle. J. Dairy Sci. 72:1583
Tyler, H.D. and H.A. Ramsey. 1991. Hypoxia in neonatal calves: effects on intestinal
transport of immunoglobulins. J. Dairy Sci. 74:1954
112
COMPARATIVE ENDOCRINE AND METABOLIC
PROFILES OF THE FETAL, NEONATAL
AND MATERNAL BOVINE
Howard Tyler
Department of Animal Science
College of Agriculture and Life Sciences
North Carolina State University
Raleigh 27695-7621
Lloyd Tate, Jr.
Department of Food Animal and Equine Medicine
College of Veterinary Medicine
North Carolina State University
Raleigh, NC 27695
Harold Ramsey
Department of Animal Science
College of Agriculture and Life Sciences
North Carolina State University
Raleigh, NC 27695
113
ABSTRACT
Three fetuses, three dams and two neonates were bled and concentrations of twelve
metabolites and hormones, along with five parameters delineating blood gas and acidbase status, were determined. In addition, complete blood chemistries were performed on
blood samples from a single cow and her fetus. Glucose and non-esterified fatty acids
(NEFA) were significantly lower (p < .05) while fructose was higher (p < .05) in fetal
calves than in maternal circulation. Glucose and NEFA increased at birth while fructose
decreased. Levels of steroid hormones (except progesterone) were not different between
maternal and fetal circulations and concentrations in the neonate decreased postnatally.
Progesterone was higher in maternal blood than either fetal or newborn samples. Peptide
hormone levels were elevated in fetal blood relative to maternal values (p < .05), but
postnatal changes differed among individual peptides. Growth hormone decreased
postnatally, while IGF-I increased, as did thyroxine. Oxygen tension and oxyhemoglobin
values were much lower in the fetal calf than in any other age examined. Arterial pH and
[HCO3-] were not different between maternal and fetal circulations. These results serve
to emphasize some of the differences in metabolism and regulation of development
between fetal, newborn and adult cattle.
(Key Words: Calves, Metabolites, Hormones, Growth Factors, Fetal, Maternal,
Newborn.)
INTRODUCTION
114
The transition from life in utero to extrauterine life requires dramatic and abrupt
changes in metabolism of the calf. The transition from neonate to adult is less abrupt but
no less dramatic. The fetal animal relies on the placenta and the intrauterine environment
for nutrient supply, gas exchange, waste disposal, detoxification and temperature
regulation. Parturition transfers all of these demands to the calf, and the ability to
perform all of these functions efficiently immediately after birth greatly influences
opportunities for survival for the calf. This transitional existence may bear little
resemblance to the relatively stable regulatory and metabolic patterns of the mature
animal. Unfortunately, few reports are available documenting the dynamics of these
changes in cattle, with most major research emphases on laboratory rats and mice and
fewer reports on sheep, pigs and humans. Interspecies differences in regulation of
metabolism and development appear to be especially large during the perinatal period,
and the importance of having accurate species-specific information is crucial for
developing reasonable hypotheses regarding regulation of developmental processes in the
perinatal period. The primary objective of this project, then, was to establish normal
values for dynamics of several blood constituents in the perinatal period and of the
bovine to determine the differences in these constituents, if any, between the fetus,
neonate and adult. A secondary objective was to test the efficacy of a new design for an
in vivo fetal blood oxygenator, and the subsequent effects of altered oxygen tension on
metabolites, hormones and their interrelationships in the prenatal period.
MATERIALS AND METHODS
115
Catheter preparation
Two separate catheters were prepared; one for collection of fetal arterial blood and the
second for altering fetal arterial Po2. The blood collection catheter was a 4.5-m length of
tridodecylmethylammonium heparinate (TDMAC-heparin)(Polysciences, Inc.,
Warrington, PA) treated polyethylene tubing (1.67mm x 2.42mm)(Intramedic tubing,
Becton Dickinson and Company). The oxygenation catheter was a triple catheter. The
inside catheter was a 4.5 m length of polyethylene tubing (1.67mm x 2.42mm) and served
as a gas release catheter. The middle catheter was a 4.5-m length of polyethylene tubing
(3.17mm x 3.99mm). It was open on one end and adapted to fit to a gas regulator and
tank. The other end was sealed with a melted polyethylene plug. The inside catheter
exited the second catheter 25 cm prior to the open end through a small hole cut in the side
of the second catheter. Air leaks were prevented by sealing with Silastic brand medical
adhesive (Dow Corning Corporation, Midland, MI). The last 25 cm of the closed end of
the second catheter was perforated by cutting small holes with a scalpel. This perforated
area was covered with the third (outside) catheter, a length of Silastic tubing (2.94 cm x
4.08 mm). Since this length of tubing was meant to act as an oxygen exchanger, the
tubing was stretched tightly over the polyethylene to minimize the diffusion coefficient
for oxygen. This was accomplished by swelling a 20-cm length of Silastic in toluene for
1 h. This increased both diameter and length of the tubing. The Silastic was then pulled
over the perforated area of polyethylene and superglued to either end. The toluene
evaporated, shrinking the Silastic and providing a thin-walled, tight-fitting length of
tubing which will act as an efficient oxygen exchanger. The plugged end of this oxygen
116
exchanger was filed down to a blunt point for easier insertion into the artery. When the
complete catheter was in place in the aorta of the calf, pure oxygen could be pumped
through the outside polyethylene catheter. Oxygen should diffuse readily through the
Silastic tubing at the end of the catheter, increasing the oxygen tension of the blood
flowing past. The inside tube prevents pressure from rising too high within the catheter.
Surgery
Three pregnant cows were obtained on approximately day 268 of gestation. Feed and
water were withheld for 24 h prior to surgery. Anesthesia was induced with 50 g
guafenisen and 4.5 g thiamylal intravenously and maintained with halothane. The cow
was placed in dorsal recumbency, clipped and prepared in a routine manner. The
abdomen was opened via a mid-ventral incision immediately lateral to the mammary vein
and extending from the umbilicus to the cranial edge of the mammary gland
(approximately 25 cm). The incision included the skin, subcutaneous tissues, and linea
alba. Hemorrhage was controlled with a combination of electrocautery and ligation. The
pregnant uterine horn was located and the ventral incision was made over the greater
curvature of the uterus. The fetus was partially extracted and the hindquarters exposed.
The right femoral artery was located by palpation and a 5-cm cutdown was made in the
inguinal region of the calf. A 3-cm length of artery was exposed by blunt finger
dissection, and two strands of 2-0 silk were run under the artery 2 cm apart to control
blood flow and retract the artery. A 3-mm incision into the artery allowed insertion of
the oxygenation catheter. The catheter was extended approximately 45 cm to ultimately
117
stop in the descending aorta near the heart. The artery was tied down over the catheter
using 2-0 silk and the incision was closed using 2-0 silk in a simple continuous pattern.
The left femoral artery was prepared in the same manner, and the collection catheter was
inserted approximately 25 cm to the point where the cranial mesenteric artery leaves the
abdominal aorta. The catheter was secured and the incision closed in the manner
previously described. The fetus was repositioned in the uterus and the uterus was closed
with #2 Dexon using a modified Lembert pattern over an inverted Connell pattern. The
catheters exited the uterus and were secured using a purse string suture and liberal
amounts of tissue. Subcutaneous tissues were undermined and the catheters exited the
body wall through a 2-cm stab wound on the right flank. This incision was closed with
#2 Vetafil in a simple continuous pattern. The linea alba in the primary incision was
closed with #2 Dexon in a simple continuous pattern. The superficial fascia and
subcutaneous tissues were closed with 2-0 catgut in a simple continuous pattern, while #2
Vetafil was used in a Ford interlocking pattern to close the skin. The catheters were
fixed to the skin using 2-0 Ethicon.
Sampling and Analysis
Samples were taken 5 h postsurgically simultaneously from the dam and the fetus. In
one case, a second sample was obtained 12 h after the first. Plasma was separated
immediately by centrifugation at 1286 x g for 15 min and stored at -20° for later analysis.
118
Other samples were stored at 5° overnight, centrifuged at 1700 x g for 20 min and the
supernatant fractions were stored at -20° for later analysis. Samples for blood gas
analysis were stored on ice in sealed heparinized syringes and analyzed within 5 min of
collection on an Instrumentation Laboratories 1302 Blood Gas System and subsequently
on an Instrumentation Laboratories 482 Co-Oximeter System.
Plasma samples were analyzed for glucose, lactate, fructose and non-esterified fatty
acids (NEFA). Glucose was determined by the oxygen rate method using a Beckman
oxygen electrode (Beckman Instruments, Inc., Brea, CA). Lactate was determined
enzymatically using a commercial kit (Boehringer Mannheim, Indianapolis, IN).
Fructose was determined spectrophotometrically by the method of Roe (1934).
Concentrations of NEFA were determined spectrophotometrically using a commercially
available kit (Biochemical Diagnostics, Inc., Edgewood, NY).
Serum samples were assayed for growth hormone (GH), insulin-like growth factor-I
(IGF-I), estradiol, thyroxine, testosterone, progesterone, cortisol, and aldosterone. All
hormone concentrations were determined by radioimmunoassay. Intraassay coefficients
of variation for all assays were less than 9%.
For one maternal-fetal pair, blood samples were sent to a commercial lab for analysis.
Parameters assayed included total protein, albumin, total bilirubin, alkaline phosphatase,
glutamic oxaloacetic transaminase, creatinine, lactate dehydrogenase, glutamic pyruvic
transaminase, gamma-glutamyl transpeptidase, uric acid, inorganic phosphate, ßcreatinine, globulin, cholesterol, triglycerides, high density lipoproteins, low density
lipoproteins, very low density lipoproteins, thyroxine, sodium, potassium, chloride, blood
119
urea nitrogen, calcium, iron, red blood cells, hemoglobin, hematocrit, mean corpuscular
volume, red cell distribution width, segmented neutrophils, banded neutrophils,
lymphocytes, monocytes, eosinophils, basophils, atypical lymphocytes, metamyelocytes,
myelocytes, progranulocytes, blastocytes, and nucleated red blood cells.
Statistics
All data were analyzed using the General Linear Models Procedure of SAS (SAS,
1985). The statistical model included status effects (maternal, fetal or neonatal), and data
were sorted by status prior to analysis. Significance of difference between means was
determined by the method of least squares means using ANOVA. In all cases,
probabilities greater than .05 were not considered significant and are reported
accordingly.
RESULTS
Post-surgical complications in these animals precluded the opportunity for utilization
of the oxygenation catheter for manipulation of fetal arterial oxygen tension, therefore,
only pre-treatment values are presented. Three surgeries were performed using the
procedures outlined above, and all three required euthanasia of the dam within 24 h of
surgery. Therefore, all fetal and maternal values presented here are from a single
sampling time 5 h post-surgery. Newborn and 1-day-old values are from untreated fed
calves being utilized in a separate experiment.
Mean glucose values (Figure 1) for fetal calves were 22.3 mg/dl, significantly lower (p
120
< .01) than maternal values (49.7 mg/dl), which were significantly lower (p < .01) than
newborns (76.5 mg/dl), which were significantly lower (p < .01) than 1-day old calves
(104 mg/dl). Fetal and newborn fructose concentrations were nearly identical (52.3 and
52.0 mg/dl, respectively), and both were significantly higher (p < .05) than 1-day old and
maternal values (6.0 and 0.0 mg/dl, respectively) (Figure 2). Concentrations of lactate
were not different for any status animal (Figure 3). Fetal calves had lower (p < .05)
NEFA values (133 mEq/L) than either newborns or adult cows (1077 and 1267 mEq/L,
respectively)(Figure 4). Day-old calves had intermediate concentrations of NEFA (742
mEq/L).
Growth factors assayed included GH, IGF-I and thyroxine. Fetal values for GH (78
ng/ml) were greatly increased (p < .001) when compared to all other status animals.
Concentrations of GH in day-old calves (20 ng/ml) were higher (p < .05) than either
newborns or adults (10 ng/ml for both groups)(Figure 5). Conversely, IGF-I
concentrations (Figure 6) increased from fetal values of 76 ng/ml to birth values of 149
ng/ml (p<.01), then tended to decrease (p = .08) by 1 day of age (107 ng/ml). Adult
cattle had the lowest values (21 ng/ml) of any status examined (p < .05). Thyroxine
concentrations (Figure 7) were similar for fetal calves, newborns and
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
1-day old animals (225, 355 and 391 ng/ml, respectively); however, all these values were
significantly higher than to maternal concentrations (84 ng/ml).
Steroid hormones assayed included cortisol, aldosterone, testosterone, estradiol and
progesterone. Cortisol concentrations (Figure 8) were similar for fetal and maternal
samples (44 and 31 ng/ml, respectively), and were elevated (p < .05) postnatally (121 and
107 ng/ml for newborn and 1-day old calves, respectively). Aldosterone concentrations
(Figure 9) were similar in fetal and maternal samples (6314 and 7449 ng/dl) and
decreased non-significantly after birth (1235 and 781 ng/dl in newborn and 1-day old
calves, respectively). Testosterone levels (Figure 10) were similar for fetal and maternal
samples (313 and 283 pg/ml, respectively), were decreased at birth (55 mg/dl) and
became non-detectable by 1 day of age. Estradiol values are presented in Figure 11.
Concentrations of estradiol in fetal and maternal circulations were 86 and 157 pg/ml,
respectively, while newborn concentrations were 21 pg/ml. As expected, progesterone
(Figure 12) was elevated (p < .01) in maternal blood (9.5 ng/ml) relative to fetal (1.7
ng/ml) and newborn values (1.3 ng/ml).
Blood gas values were obtained for two animals of each status. Arterial pH (Figure
13) was not different for any status, although mean values did increase numerically with
age. Values for Pco2 were significantly lower (p <.05) in maternal blood than in fetal,
newborn or day-old calves (Figure 14), with newborn calves having the highest values.
Arterial bicarbonate concentrations (Figure 15) were higher (p < .05) in newborn and
day-old calves than in either maternal cows or fetal calves. Oxygen tension in arterial
blood from fetal and newborn calves (Figure 16)
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
was lower than maternal arterial oxygen tension (p < .05), and day-old calves tended to
be lower than adults (p=.09). Oxyhemoglobin values were lower (p < .001) in fetal
calves than in newborns, day-old calves or cows (Figure 17). The high levels of
oxyhemoglobin in all other animals show that oxygen tension during the first day is
adequate to maintain nearly complete oxygenation of hemoglobin.
Those blood constituents that were determined for a single fetal-maternal pair are
presented in Tables 1, 2, 3, 4, and 5. Complete differentials are presented in Table 1.
There was a slight increase in the number of immature cell types in fetal circulation vs
maternal circulation and a decreased level of circulating lymphocytes in fetal blood.
Table 2 compares blood profiles for fetal and maternal samples. Hemoglobin,
hematocrit, red blood cells and mean corpuscular volume were all numerically lower in
fetal blood than in maternal. Table 3 is a summary of clinical chemistry values for these
animals. Levels of minerals were not different between fetal and maternal blood.
However, concentrations of total protein were decreased in fetal calves due to decreases
in both globulin and albumin. Creatinine was fivefold higher in fetal blood than in
maternal, while BUN:creatinine was fivefold lower. Table 4 summarizes activities of
selected enzymes. Fetal serum enzyme activities were substantially lower than maternal
activities for all enzymes with the notable exception of alkaline phosphatase, which was
fivefold higher in fetal blood. Lipid profiles are presented in Table 5. All lipids were
numerically lower in the fetal calf than in the dam, with cholesterol and high-density
lipoproteins, in particular, being fourfold and fivefold lower, respectively.
155
156
157
Table 1. Complete differential profiles for maternal and fetal blood
at day 268 of gestation.
Cell Type (%)
Maternal
Fetal
Segmented Neutrophils
33
35
Banded Neutrophils
20
25
Lymphocytes
42
29
Monocytes
0
0
Eosinophils
2
0
Basophils
2
1
Atypical Lymphocytes
1
2
Metamyelocytes
0
5
Myelocytes
0
3
Progranulocytes
0
0
Blastocytes
0
0
Nucleated Red Blood Cells
0
0
158
Table 2. Blood profiles for maternal and fetal blood on day 268
of gestation.
Parameter
Maternal
Fetal
Red Blood Cells (10-6/μL)
8.46
7.40
Hemoglobin (g/dl)
14.9
11.3
HematocrMw (%)
41
34
Mean Corpuscular Volume (fL)
49
46
Red Cell Distribution Width (%)
34
37
159
Table 3. Blood chemistry for maternal and fetal blood on day 268 of gestation.
Constituent
Maternal
Fetal
Sodium (mmol/L)
147
142
Potassium (mmol/L)
4.3
4.4
Chloride (mmol/L)
108
95
Blood Urea Nitrogen (mg/dl)
15
14
Calcium (mg/dl)
8.2
12.2
Total Protein (g/dl)
7.5
4.1
Albumin (g/dl)
3.7
2.2
Total Bilirubin (mg/dl)
.7
.6
Creatinine (mg/dl)
2.4
12.0
Uric Acid (mg/dl)
1.0
.9
Phosphate (mg/dl)
4.7
6.6
BUN:Creatinine
6.1
1.2
Globulin (g/dl)
6.0
3.4
160
Table 4. Enzyme activities in fetal and maternal blood samples on day
268 of gestation.
Enzyme (IU/L)
Maternal
Fetal
Alkaline Phospatase (EC 3.1.3.1)
32
154
Glutamic Oxaloacetic Transaminase (EC 2.6.1.1)
124
29
Lactate Dehydrogenase (EC 1.1.1.27)
931
459
Glutamic Pyruvic Transaminase (EC 2.6.1.2)
19
5
Gamma-Glutamyl Transpeptidase (EC 2.3.2.2)
22
11
161
Table 5. Lipid profiles of maternal and fetal blood on day 268 of gestation.
Lipid (mg/dl)
Maternal
Fetal
Cholesterol
70
17
Triglycerides
36
29
High Density Lipoproteins
62.0
11.6
Low Density Lipoproteins
0.8
0
Very Low Density Lipoproteins
7.2
6.0
162
DISCUSSION
The failure of these animals to survive past 24 h following surgery prevented the
attainment of the objectives outlined in the introduction. All three animals were
euthanized for different reasons. During the first surgery, there was a breach in sterile
procedure resulting in massive infection and death of the fetus within 12 h of recovery.
Acute peritonitis of the cow terminated the second attempt, and the third cow never
recovered from anesthesia. It should be noted that the final surgery appeared to be
successful in terms of correcting the problems of the first two surgeries. The cow was
partially paralyzed, apparently due to an adverse reaction to the anesthesia, and
eventually pulmonary edema became apparent and euthanasia became necessary.
Glucose
Low concentrations of glucose in fetal circulations are well-documented, but
extremely low levels appear to be a peculiarity of those species with high fetal fructose.
Low blood glucose concentration prior to birth may be influenced by the hypoglycemic
effect of fructose (Sasaki et al., 1971; Fayet et al., 1977; Edwards and Powers, 1967).
Fetal levels in this study were about half the concentrations in maternal blood. Levels
rise abruptly postnatally, with concentrations at birth threefold higher than fetal levels.
Levels continue to increase throughout the first day, with levels at 1 d of age reaching
twice adult levels. The postnatal increase in blood glucose has been alternately explained
by spiking cortisol concentrations commonly seen at birth (Massip, 1980), decreasing
concentrations of fructose (Pettigrew et al., 1971), and increasing activities of
gluconeogenic enzymes. Glucose 6-phosphatase, hexose bisphosphatase, and pyruvate
163
carboxylase are all present in fetal rat liver, but exhibit an increased activity postnatally
(Yeung et al., 1967). Phosphopyruvate carboxylase activity does not appear until
immediately postnatally, and rapidly increases during the first day (Yeung et al., 1967).
Maternal glucose concentrations in this study are much lower than levels seen in
monogastric animals (85-100 mg/dl). In ruminants, there is a transition in energy
metabolism from glucose to volatile fatty acids serving as the major energy source.
Dietary carbohydrate in adult ruminants is fermented to volatile fatty acids in the rumen
rather than being digested to yield hexoses. Ruminants then utilize volatile fatty acids in
a manner that spares glucose.
Fructose
The placenta is the site of fructose synthesis for fetal calves (Cole and Hitchcock,
1946; Alexander et al., 1955; Britton et al., 1963). Setchell et al. (1972) demonstrated
that fructose contributed carbon to glycogen synthesis in fetal lambs. White et al. (1982)
reported that labeled carbon from fructose in fetal pigs was used for nucleic acid
synthesis. Both of these conversions were found to occur at minimal rates, and fructose
utilization by fetal and neonatal sheep and piglets is minimal.
Fructose has been shown to be present in fetal sheep throughout pregnancy and to
disappear several hours after birth (Shelly and Dawes, 1962). Plasma fructose was
nondetectable by 24 hours of life in piglets, lambs and calves (Shelly and Dawes, 1962;
Daniels et al., 1974; Curtis et al., 1966; Pettigrew et al., 1971). In this study, over 90%
of the high levels of fructose in the fetal and newborn calves had disappeared by 24 h.
164
Fructose is apparently excreted after birth without being utilized. The amount of fructose
excreted in urine is more than sufficient to account for the apparent loss of fructose from
blood in lambs (Shelly and Dawes, 1962; Aherne et al., 1969) and in piglets (Talbot,
1964).
Lactate
Lactate levels remain stable throughout fetal life (Comline and Silver, 1972; Burd et
al., 1975), rising sharply at parturition in direct proportion to degree of dystocia (Comline
and Silver, 1972; Vermorel et al., 1983). Lactate is produced primarily by the placenta
(Burd et al., 1975; Warnes et al., 1977a) and provides an energy source for oxidative
metabolism in the fetus (Barker and Britton, 1958) and in the neonate (Warnes et al.,
1977a). Gluconeogenesis from lactate does not occur in fetal liver, but is initiated by the
change in oxygen tension at birth (Warnes et al., 1977b). The increase in lactate at birth
in calves from this study was minimal, and concentrations in day-old calves were near
adult levels.
NEFA
Grigsby et al. (1974) reported that free fatty acids decreased in fetal calves throughout
gestation while maternal levels increased. Levels found in fetuses and in their dams at
day 260 of gestation were similar to concentrations in fetal calves and in their dams
observed in this study. The dramatic increase at birth may be due to increasing cortisol
concentrations at this time.
165
Growth Hormone and IGF-I
Growth hormone can be detected in fetal blood by 10 wk of gestation in the human,
reaching a peak by 24 wk (Kaplan et al, 1972). High levels of fetal GH in sheep are due
to a pituitary insensitivity to the suppressant effects of somatostatin (Silverman et al.,
1989). The lack of GH response in the face of high fetal GH levels is due to lack of GH
receptors in fetal tissues (Gluckman, 1986). Only fetal liver tissue has receptors for GH
in humans (Handwerger and Freemark, 1987; Hill et al., 1988), and no fetal hepatic GH
receptors are present in sheep (Gluckman, 1986). In mature rats, GH receptors appear to
be present throughout the gastrointestinal tract, suggesting a direct role for GH in gut
growth and(or) differentiation (Lobie et al., 1990).
GH-dependent growth appears somewhere between 3 and 12 mo in man, between 1
and 12 wk in sheep, at about 3 wk in rats and at 15 wk in rabbits (Gluckman, 1986).
Growth hormone is more effective in stimulating tissue growth than skeletal growth
during the neonatal period (Glasscock et al., 1991), although it may stimulate cartilage
growth and differentiation during this time (Maor et al., 1989), possibly due to a
stimulation of local IGF-I production.
Concentrations of growth hormone in this study were similar to those seen by Oxender
et al. (1972) in fetal calves and their dams at day 260 of gestation, as well as in newborn
calves. The dramatic decrease in GH at birth suggests that negative feedback
mechanisms at the level of the hypothalamus become functional immediately following
parturition.
166
Growth hormone exerts somatotrophic effects through IGF's, at least in mature
animals. IGF-I, IGF-II, relaxin and ß-nerve growth factor are a family of mitogenic
peptides structurally homologous to proinsulin (Daughaday and Heath, 1984). IGF's
have been detected in human fetal blood from 13 weeks of gestation (Bennett et al.,
1983; Gluckman, 1986; Ashton et al., 1984; D'Ercole et al., 1986). IGF-I levels in the
fetus are thought to be independent of GH control in lambs and rabbits (Gluckman,
1986), although more recent evidence suggests that this may be an artifact of the acidethanol extraction technique used in most assays, and that IGF-I concentrations in the
sheep fetus are under a combination of pituitary and thyroid control, whereas IGF-II is
not (Mesiano et al., 1989). Most adult IGF-I is secreted from the liver; however several
tissues contribute significantly to fetal IGF-I production, including both liver and lung
tissue (Gluckman, 1986). IGF's act by endocrine, paracrine and autocrine mechanisms to
increase growth and differentiation in a number of organs (D'Ercole et al., 1984).
Density of receptors for both IGF-I and IGF-II is increased in suckling rat small intestine
relative to adult tissues (Young et al., 1990). Density decreases progressively during the
suckling period for IGF-II receptors; however, IGF-I receptor density remains high
(Young et al., 1990). Neither IGF-I nor IGF-II appears to influence intestinal growth in
the suckling rat, but development of jejunal brush border enzymes is apparently
stimulated by both growth factors (Young et al., 1990). In addition, IGF's are potent
mitogens for the fetal musculoskeletal system at low concentrations (Hill et al., 1985).
Concentrations of IGF-I are similar in ovine fetal and maternal circulations, but
dramatically rise at birth, whereas IGF-II is highest during fetal life, intermediate in
167
maternal blood, and lowest in the neonate (Mesiano et al., 1989). In the present study,
fetal levels were considerably higher than maternal, with the postnatal increase evident
even in newborn calves. The postnatal rise in IGF-I is suggested to be due to a
combination of maturation of the somatotropic axis and the appearance of GH receptors
in hepatic tissue (Gluckman, 1986).
Contradictory reports may be the result of assay variability. IGF-BPs are known to
produce rather large artifacts in IGF assays (Mesiano et al., 1988), and techniques for
preventing these artifactual responses may have been ineffectual in some cases.
Thyroxine
The most important function of the thyroid gland is regulation of metabolic rate in
different tissues (Evans et al., 1960). The secretory products of the thyroid gland are
iodothyronines. These are a series of compounds resulting from the coupling of two
iodinated tyrosine molecules. The three hormones secreted are thyroxine (T4; 3,5,3',5'tetraiodothyronine), triiodothyronine (T3; 3,5,3'-triiodothyronine), and reverse T3 (rt3;
3,5',3'-triiodothyronine). T4 is monodeiodinated to T3 in peripheral tissues at a rate
inversely related to the T4 production rate (Larson et al., 1955).
The pituitary-thyroid axis is active from 10-12 wk gestation in humans (Chard, 1989).
Reverse T3 (rT3) is produced in excess of T3, although the function of rT3 is not known
(Chard, 1989). Fetal serum T3 concentrations increase while T4 and rT3 concentrations
decrease during the 4-6 d preceding birth in lambs (Klein et al., 1978). Serum T3
continues to increase following delivery, and this increase may be mediated by the
168
cortisol-induced increase in T4 to T3 interconversion (Klein et al., 1978).
Hernandez et al. (1972), reported that serum levels of T4 at birth (170 ng/ml) were
approximately twice the concentrations in the mature bovine. These levels declined
exponentially and approached adult bovine values (70 ng/ml) by 6 days of age. Newborn
levels in this study were approximately twice the levels reported by Hernandez, although
maternal levels were similar.
Cortisol and Aldosterone
The two major zones of the adrenal gland are the cortex and medulla.
The two primary groups of hormones secreted by the adrenal cortex are the glucocorticoids (e.g. cortisone and cortisol) and mineralocorticoids (e.g. desoxycorticosterone
and aldosterone). Mineralocorticoids are important in water and electrolyte metabolism
and in the ability to reabsorb sodium from the glomerular filtrate, whereas glucocorticoids are associated with carbohydrate and protein metabolism.
A large portion of fetal cortisol is of maternal origin (Chard, 1989). High
corticosteroid levels present on the day of birth in piglets are due to endogenous
biosynthesis (rather than placental transfer) (Dvorak, 1986). These high concentrations
of corticosteroids are also capable of providing a negative feedback mechanism against
further endogenous production, presumably by suppressing ACTH production (Dvorak,
1986). High glucocorticoid concentrations in calves are associated with stress at calving,
and are highly correlated with the degree of acidosis in calves at birth (Hoyer et al., 1990;
Szenci and Taverne, 1988). Cortisol concentrations at birth are 2.5-fold higher in the calf
169
than in the dam (121 ng/ml vs 50 ng/ml), and levels decrease rapidly to 49 ng/ml by 12 h
and then more slowly to 11 ng/ml by 12 d (Eberhart and Patt, 1971). Concentrations in
this study for the newborn calf and dam were nearly identical to those reported by
Eberhart and Patt, although cortisol levels in day-old calves in this study remained
similar to newborn values. Fetal levels (44 ng/ml) were much closer to maternal values
than newborn values.
Most of the aldosterone in fetal blood is of fetal origin (Wintour et al., 1980). Rouffet
et al. (1990) reported that fetal aldosterone near term was 40 pg/ml, rising to 85 pg/ml at
birth. Maternal levels were near 50 pg/ml at parturition. Other reports for newborn
calves have given values from 15 to 185 pg/ml (Cabello, 1979; Itoh et al., 1985).
Concentrations of aldosterone determined in this study were much higher than those
reported previously, possibly due to the utilization of an assay validated for newborn
bovine plasma in this study. Previous studies have used adult human standards for
determination of aldosterone concentrations in bovine plasma.
Other Steroids
Concentrations of testosterone in fetal calves in this study were somewhat higher than
those reported previously (Kim et al., 1972), but values in newborn calves agreed with
those reported by Rawlings et al. (1972). The decrease in estradiol at birth may be due to
an alteration in the rate of interconversion of estradiol to estrone. In human fetal tissues
reductive pathways of estrogen interconversion (e.g. estrone to estradiol) are prevalent in
placenta, lung, and the fetal zone of the adrenal gland while oxidative pathways are
170
favored in liver, intestine, stomach, kidney, brain, and heart (Milewich et al., 1989).
Activity of 17-hydroxysteroid oxidoreductase (the enzyme facilitating the
interconversion of estrone and estradiol) in vitro is unaffected by insulin, glucagon or
dexamethasone. The loss of the major reductive pathways at birth could result in a
considerable alteration in estradiol:estrone ratio.
The high levels of maternal progesterone in this study were compatible with
maintenance of pregnancy, and the levels in the fetus and newborn are comparable to
concentrations expected in non-cycling, non-pregnant mature cattle.
Blood Gases and Acid-Base Status
The low pH of fetal and newborn blood relative to maternal blood has been reported
elsewhere (Gahlenbeck et al., 1968; Reeves et al., 1972; Moore, 1969). Increases in pH
during the first day correlate with decreases in lactate during the same period, although
this is not the only factor involved. Respiratory compensation is apparent in the
decreased Pco2 values for day-old calves relative to birth values. The high Pco2 and low
[HCO3-] in fetal calves infers the presence of relatively high concentrations of carbonic
acid in the fetus. Bicarbonate levels increase in postnatal calves, an additional factor
correlating with the steadily rising pH values following birth.
Previous estimates for oxygen tension in fetal calves have ranged from 19.4 mm Hg
(Gahlenbeck et al., 1968) to 29.5 mm Hg (Reeves et al., 1972), depending on technique,
sampling site and use of anesthesia. Values from this experiment (19 mm Hg) tend to
confirm the former values. The high variation in newborn and day-old calves all
171
occurred near the upper plateau of the oxygen dissociation curve, as evidenced by high
values for oxyhemoglobin in these calves.
Cellular Constituents
Calves at birth exhibit leukocytosis, eosinopenia, and a preponderance of neutrophils
over lymphocytes (Eberhart and Patt, 1971). The differential profiles reported in this
study show low levels of circulating lymphocytes, slight increases in neutrophils, and
increased numbers of immature cells (metamyelocytes and myelocytes) in fetal
circulation relative to adult values, while numbers of red blood cells in fetal blood were
lower than in the dam. This resulted in decreased hemoglobin concentrations and lower
hematocrit in the fetus. Red cell distribution width, an index usually used to characterize
regenerative conditions in the animal, was high in both fetal and maternal blood, no
doubt a consequence of the rapid growth of the fetus and rapidly increasing blood
volumes of both fetus and dam at this time.
Blood Chemistry
Values for most clinical chemistry components of fetal blood were similar to those of
maternal blood; however, differences in protein components, creatinine and ß-creatinine
were apparent. Serum creatinine concentration and an endogenous creatinine clearance
are often used to assess glomerular filtration rate (Mitch et al., 1976), in that an increase
in serum creatinine is associated with a decrease in glomerular filtration rate (Goldston,
1981). Increased fetal creatinine values may reflect limited efficiency in the placental
172
removal of fetal waste.
Total serum protein is an important reflection of protein metabolism and transport of
nutrients, hormones and waste products to various organs. Total proteins can be divided
into two classifications: albumin and globulin, both of which were lower in concentration
in fetal blood than in maternal blood.
Enzyme Activities
Serum enzymes are routinely used to indicate cellular damage. Glutamic-oxaloacetic
transaminase (GOT) is predominately found in cardiac, liver, and muscle tissue (Laird,
1972). This is a cytosolic enzyme which is released into blood after cellular damage.
The normal serum GOT activity in older calves is 48 IU/L and in mature cows it is 68
IU/L (Jenkins et al., 1982). The relatively high value in maternal blood might be
anticipated in a post-surgical sample, while the low value in the fetus suggests a lack of
major trauma during surgery.
Glutamic-pyruvic transaminase (GPT) is found in heart and muscle tissue. Normal
serum GPT activity for calves between 4 and 8 wk of age is 9 IU/L and for adult bovines,
34 IU/L (Jenkins et al., 1982). The low values for both maternal and fetal blood again
suggest lack of major heart and muscle trauma during the surgical procedure.
Another enzyme which is beneficial in hepatic diagnostics is gamma-glutamyl
transferase or transpeptidase (GGT). This enzyme is a cytomembranous enzyme
involved in glutathione metabolism and glomerular filtration. The activity of this enzyme
was similar for young (23 IU/L) and mature bovines (27 IU/L) (Jenkins et al., 1982).
173
Again, these values are comparable to those found in this study.
Damage to muscle, bone repair, or liver anomalies (Laird, 1972) may result in an
increase in the activity of alkaline phosphatase (ALP). Alkaline phosphatase is a
cytomembranous enzyme found in bone, cells of the kidney renal tubules, liver, intestine,
and placenta. Serum ALP activity decreases with age. The activity of ALP for calves 4
to 8 wk of age was approximately 367 IU/L, and for mature bovines it was 52 IU/L
(Jenkins et al., 1982). Fetal values in this experiment are lower than those reported for
calves, presumably due to the suckling-induced increases occurring postnatally. ALP is
associated with the epithelial lining of the gastrointestinal tract and increases in neonatal
serum may be due to intestinal enterocyte exocytosis of this enzyme along with colostral
constituents.
Lipids
Triglyceride and cholesterol form the major components of plasma lipids which circulate
as lipoprotein particles. Plasma triglycerides increase during the first 2 d after birth.
Cholesterol esters increase more rapidly than free cholesterol contributing to a decrease
in the free:total cholesterol ratio between birth and 15 days of age (Shannon and
Lascelles, 1966). Fetal calves in this study had very low levels of cholesterol and highdensity lipoproteins relative to maternal values.
CONCLUSIONS
The high degree of variation in values for circulating blood constituents for fetal,
174
newborn, day-old, and maternal animals in this study emphasizes the dynamic nature of
these periods and suggests differences in metabolism and development. Variations in
regulation of development are seen during different stages of life, and findings in adult
animals will not necessarily apply to fetal, newborn or growing animals.
LITERATURE CITED
Aherne, F.X., V.W. Hayes, R.C. Kwan and V.C. Speer. 1969. Glucose and fructose in the
fetal and newborn pig. J. Anim. Sci. 29:906.
Alexander, D.P, A.G. Huggett, D.A. Nixon and W.F. Widdas. 1955. The placental
transfer of sugars in the sheep. The influence of concentration gradient upon the rates
of hexose formation as shown in umbilical perfusion of the placenta. J. Physiol.
129:367.
Ashton, I.K., J. Zapf, I. Einschenk and I.Z. MacKenzie. 1985. Insulin-like growth factors
(IGF)I and II in human foetal plasma and relationship to gestational age and foetal
size during mid-pregnancy. Acta Endocr. 10:558.
Barker, J.H., And H.G. Britton. 1958. Lactate and pyruvate metabolism in foetal sheep. J.
Physiol. 143:50P.
Bennett, A., D.M. Wilson, F. Liu, R. Nagashimi, R.G. Rosenfeld and R.I. Hintz. 1983.
Levels of insulin-like growth factors I and II in human cord blood. J. Clin. Endocr.
Metabol. 57:609.
Britton, H.G., A.G. Huggett and D.A. Nixon. 1963. Carbohydrate metabolism in the
perfused sheep placenta studied with radioactive sugars. J. Physiol. 166:21P.
Burd, L.I., M.D. Jones, M.A. Simmons, E.L. Makowski, G. Meschia and F.C. Battaglia.
1975. Placental production and foetal utilisation of lactate and pyruvate. Nature
254:710.
Cabello, G. 1979. Neonatal changes in the plasma levels of cortisol, cortisone and
aldosterone in the calf. Biol. Neonate 36:35.
Chard, T. 1989. Hormonal control of growth in the human fetus. J. Endocr. 123:3.
Cole, S.W., and M.W.S. Hitchcock. 1946. Sugars in the foetal and maternal bloods of
175
sheep. Biochem. J. 40:1i.
Colvin, H.W., J.T. Attebery and L.B. Daniels. 1967. Effect of diet on glucose tolerance
of dairy calves ont to thirteen weeks old. J. Dairy Sci. 50:362.
Comline, R.S. and M. Silver. 1972. The composition of foetal and maternal blood during
parturition in the ewe. J. Physiol. 222:223.
Corley, L.D., T.E. Staley, L.J. Bush, and E.W. Jones. 1977. Influence of colostrum on
transepithelial movement of Escherichia coli 055. J. Dairy Sci. 60:1416.
Curtis, S.E., C.H. Heidenreich and C.W. Foley. 1966. Carbohydrate assimilation and
utilization by newborn pigs. J. Anim. Sci. 25:655.
Daniels, L.B., J.L. Perkins, D. Kreider, D. Tugwell and D. Carpenter. 1974. Blood
glucose and fructose in the newborn ruminant. J. Dairy Sci. 57:1196.
Daughaday, W.H. and E. Heath. 1984. Physiological and possible clinical significance of
epidermal and nerve growth factors. J. Clin. Endocr. Metabol. 13:207.
D'Ercole, A.J., A.D. Stiles and L.E. Underwood. 1984. Tissue concentration of
somatomedin C: further evidence for multiple sites of synthesis and
paracrine/autocrine mechanisms of action. Proc. Natl. Acad. Sci. USA. 81:935.
D'Ercole, D., D.J. Hill, A.J. Strain and L.E. Underwood. 1986. Tissue and plasma
somatomedin-C/insulin-like growth factor I concentrations in the human fetus during
the first half of gestation. Pediatr. Res. 20:253.
Dvorak, M. 1986. Inhibition of adrenocortical activity by dexamethasone in newborn
piglets. Acta Vet. Brno. 55:155.
Eberhart, R.J., and J.A. Patt. 1971. Plasma cortisol concentrations in newborn calves.
Am. J. Vet. Res. 32:1921.
Edwards, A.V. and N. Powers. 1967. Effect of intravenous infusion of fructose in
newborn calves. Nature 214:728.
Evans, E.S., L.L. Rosenberg and M.E. Simpson. 1960. Relative sensitivity of different
biological responses to thyroxine. Endocr. 66:433.
Fayet, J.C., C. Renouf, M.C. Michel and J. Overwater. 1977. Effect of intravenous
infusion of glucose and/or fructose on the composition of blood plasma and the
clinical response of the calf. Ann. Rech. Vet. 8:171.
176
Gahlenbeck, H., H. Frerking, A.M. Rathschlag-Schaefer and H. Bartels. 1968. Oxygen
and carbon dioxide exchange across the cow placenta during the second part of
pregnancy. Resp. Physiol. 4:119.
Glasscock, G.F., K.K.L. Gin, J.D. Kim, R.L. Hintz and R.G. Rosenfeld. 1991. Ontogeny
of pituitary regulation of growth in the developing rat: comparison of effects of
hypophysectomy and hormone replacement on somatic and organ growth, serum
insulin-like growth factor-I (IGF-I) and IGF-II levels, and IGF-binding protein levels
in the neonatal and juvenile rat. Endocr. 128:1036.
Gluckman, P.D. 1986. The regulation of fetal growth. In: Control and Manipulation of
Animal Growth. Butterworths. London.
Goldston, R.T., R.D. Wilkes and I.M. Seybold. 1981. Evaluation of renal function. 1.
Blood urea nitrogen and creatinine determinations. Vet. Med. 2:157.
Grigsby, J.S., W.D. Oxender, H.D. Hafs, D.G. Britt and R.A. Merkel. 1974. Serum
insulin, glucose, and free fatty acids in the cow and fetus during gestation. Proc. Soc.
Exp. Biol. Med. 1974 147:830.
Handwerger, S. and M. Freemark. 1987. Role of placental lactogen and prolactin in
human pregnancy. Adv. Exp. Med. Biol. 219:399.
Hernandez, M.V., K.M. Etta, E.P. Reineke, W.D. Oxender, and H.D. Haps. 1972.
Thyroid function in the prenatal and neonatal bovine. J. Anim. Sci. 34:780.
Hill, D.J., C.J. Crace, S.P. Nissley, D. Morrell, A. Holder and R.D.G. Milner. 1985. Fetal
rat myoblasts release both rat somatomedin-C (SM-C)/insulin-like growth factor (IGFI) and multiplication-stimulating activity in vitro: partial characterization and
biological activity of myoblast-derived SM-C/IGF-I. Endocr. 117:2061.
Hill, D.J., M. Freemark, A.J. Strain, S. Handwerger and R.D.G. Milner. 1988. Placental
lactogen and growth hormone receptors in fetal tissues: relationship to fetal plasma
human placental lactogen concentrations and fetal growth. J. Clin. Endocr. Metabol.
66:1283.
Hoyer, C., E. Grunert and W. Jochle. 1990. Plasma glucocorticoid concentrations in
calves as an indicator of stress during parturition. Am. J. Vet. Res. 51:1990
Itoh, N., D. Murakami, Y. Naitoh and R. Satoh. 1985. Plasma aldosterone levels and
plasma renin activity during the neonatal period in calves. Jpn. J. Vet. Sci. 47:397.
Jenkins, S.J., S.A. Green, and P.A. Clark. 1982. Clinical chemistry reference values of
normal domestic animals in various age groups - As determined on the ABA-100R.
177
Cornell Vet. 72:403.
Kaplan, S.L., M.M. Grumbach and T.H. Shepard. 1972. The ontogenesis of human fetal
hormones. I. Growth hormone and insulin. J. Clin. Invest. 51:3080.
Kim, C.K., S.S.C. Yen and K. Benirschke. 1972. Serum testosterone in fetal cattle. Gen.
Comp. Endocr. 18:404.
Klein, A.H., T.H. Oddie and D.A. Fisher. 1978. Effect of parturition on serum
iodothyronine concentrations in fetal sheep. Endocr. 103:1453.
Laird, C.W. 1972. Representative values for animal and veterinary populations and their
clinical significances. Hycel, Inc., Houston Texas.
Lobie, P.E., W. Breipohl and M.J. Waters. 1990. Growth hormone receptor expression in
the rat gastrointestinal tract. Endocr. 126:299.
Maor, G, Z. Hochberg, M. Silbermann. 1989. Growth hormone stimulates the growth of
mouse neonatal condylar cartilage in vitro. Acta Endocr. 120:526.
Massip, A. 1980. Relationship between pH, plasma cortisol and glucose concentrations in
the calf at birth. Br. Vet. J. 136:597.
Mesiano, S., I.R. Young, A.W. Hey, C.A. Browne and G.D. Thorburn. 1989.
Hypophysectomy of the fetal lamb leads to a fall in the plasma concentration of
insulin-like growth factor I (IGF-I), but not IGF-II. Endocr. 124:1485.
Mesiano, S., I.R. Young, C.A. Browne and G.D. Thorburn. 1988. Failure of acid-ethanol
treatment to prevent interferance by binding proteins in radioligand studies for the
insulin-like growth factors. J. Endocr. 119:453.
Milewich, L., P.C. MacDonald and B.R. Carr. 1989. Activity of 17ß-hydroxysteroid
oxidoreductase in tissues of the human fetus. J. Endocr. 123:509.
Mitch, W.E., M. Walser and G.A. Buffington. 1976. A simple method of estimating
progression of chronic renal failure. Lancet 2:1326.
Moore, W.E. 1969. Acid-base and electrolyte changes in normal calves during the
neonatal period. Am. J. Vet. Res. 30:1133.
Oxender, W.D., H.D. Hafs and L.A. Edgerton. Serum growth hormone, LH and prolactin
in the pregnant cow. J. Anim. Sci. 35:1972.
Pettigrew, J.E., D.R. Zimmerman and R.C. Ewan. 1971. Plasma carbohydrate levels in
178
the neonatal pig. J. Anim. Sci. 32:895.
Rawlings, N.C., H.D.Hafs and L.V. Swanson. 1972. Testicular and blood plasma
androgens in Holstein bulls from birth through puberty. J. Anim. Sci. 34:435.
Reeves, J.T., F.S. Daoud and M. Gentry. 1972. Growth of the fetal calf and its arterial
pressure, blood gases, and hematologic data. J. Appl. Physiol. 32:240.
Roe, J.H. 1934. A colorimetric method for the determination of fructose in blood and
urine. J. Biol. Chem. 107:15.
Rouffet, J., M. Dalle, C. Tournaire, J.-P. Barlet and P. Delost. 1990. Sodium intake by
pregnant cows and plasma aldosterone and cortisol concentrations in the fetus during
late pregnancy. J. Dairy Sci. 73:1762.
Sasaki, Y. and K. Takeshita. 1971. Effect of fructose injection on plasma glucose
concentration in newborn calves. Jap. J. Zootech. Sci. 42:421.
SAS. 1985. SAS User's Guide: Statistics. SAS Institute, Inc. Cary, NC.
Setchell, B.P., J.M. Bassett, N.T. Hinks, and N. McC. Graham. 1972. The importance of
glucose in the oxidative metabolism of the pregnant uterus and its contents in
conscious sheep and with some preliminary observations on the oxidation of fructose
and glucose by fetal sheep. Quart. J. Exp. Physiol. 57:257.
Shannon, A.D. and A.K. Lascelles. 1966. Changes in the concentration of lipids and
some other constituents in the blood plasma of calves from birth to 6 months of age.
Austrialian J. Biol. Sci. 19:831.
Shelly, H.G. and G.S. Dawes. 1962. Fate of fructose in the newly delivered lamb. Nature
194:296.
Silverman, B.L., M. Bettendorf, S. L. Kaplan, M.M. Grumbach and W.L. Miller. 1989.
Regulation of growth hormone (GH) secretion by GH-releasing factor, somatostatin,
and insulin-like growth factor I in ovine fetal and neonatal pituitary cells in vitro.
Endocr. 124:84.
Szenci, O. and M.A.M. Taverne. 1988. Perinatal blood gas and acid-base status of
caesarian-derived calves. J. Vet. Med. 35:572
Talbot, R.B. 1964. Erythocyte, plasma and total blood volume of pigs from birth through
six weeks of age. Ph.D. Thesis. Iowa State University of Science and Technology,
Ames, Iowa.
179
Vermorel, M., C. Dardillat, J. Vernet, Saido and C. Demigne. 1983. Energy metabolism
and thermoregulation in the newborn calf. Ann. Rech. Vet. 14:382.
Warnes, D.M., R.F. Seamark and F.J. Ballard. 1977a. Metabolism of glucose, fructose
and lactate in vivo in chronically cannulated foetuses and in suckling lambs. Biochem.
J. 162:617.
Warnes, D.M., R.F. Seamark and F.J. Ballard. 1977b. The appearance of gluconeogenesis
at birth in sheep. Biochem J. 162:627.
White, C.E., E.L. Piper, P.R. Noland, and L.B. Daniels. 1982. Fructose utilization for
nucleic acid synthesis in the fetal pig. J. Anim. Sci. 55:73.
Wintour, E.M., J.P. Coghlan, K.J. Hardy, B.E. Lingwood, M. Rayner and B.A. Scoggins.
1980. Placental transfer of aldosterone in the sheep. J. Endocr. 86:305.
Yeung, D., R.S. Stanley and I.T. Oliver. Development of gluconeogenesis in neonatal rat
liver. Biochem. J. 105:1219.
Young, G.P., T.M. Taranto, H.A. Jonas, A.J. Cox, A. Hogg and G.A. Werther. 1990.
Insulin-like growth factors and the developing and mature rat small intestine:
receptors and biological actions. Digestion 46(suppl 2):240.
DEVELOPMENTAL APPEARANCE OF BOMBESIN
RECEPTORS IN THE DUODENUM, JEJUNUM
AND ILEUM OF PIGLETS FROM BIRTH
THROUGH FOUR WEEKS OF AGE
Howard Tyler
Department of Animal Science
College of Agriculture and Life Sciences
North Carolina State University
180
Raleigh, NC 27695
Steve Vigna and Douglas McVey
Department of Cell Biology
Duke University Medical Center
Durham, NC 27710
Warren J. Croom, Jr.
Department of Animal Science
College of Agriculture and Life Sciences
North Carolina State University
Raleigh, NC 27695
ABSTRACT
The objective of this study was to determine the age at which different segments of small
intestine express specific binding sites for bombesin. Piglets were sacrificed at birth, 1 d,
1 wk, 3 wk and 4 wk and samples of duodenal, jejunal and ileal tissue were harvested for
autoradiographic analysis. Monoiodinated, biologically active (Tyr-4)-bombesin 14 (100
pM) was applied to slide-mounted sections of piglet gut tissue and localized using
quantitative autoradiography. Specific binding sites first appeared in duodenal tissue of
7-d old piglets with evidence for continued presence in this tissue through 21 d. No
evidence for specific binding of 125I-(Tyr-4)-bombesin 14 was found in gut sections from
either newborn, 1-d old or 4-wk old piglets. These results suggest sites of direct action of
181
bombesin in 1-wk old and 3-wk old piglets.
(Key Words: Bombesin, Piglet, Small Intestine, Newborn, Weaning.)
INTRODUCTION
Bombesin is a tetradecapeptide isolated from amphibian skin extracts (Anastasi et al.,
1972). It acts to regulate several specific functions and to stimulate epithelial growth in
the adult mammalian gastrointestinal tract (Bertaccini et al., 1974; Taylor et al., 1979;
Poitras et al., 1983). However, regulation of adult gut function and growth is in many
cases different than regulation of the same tissues in newborn animals. Gastrin,
cholecystokinin, and caerulin, with well established functions on mature gastric mucosa
and pancreas, have no activity on these tissues in suckling rats (Majumdar and Johnson,
1982; Brants and Morisset, 1976; Zahavi et al., 1984; Morisset, 1980).
The presence of bombesin-like immunoreactivity in milk from several species (Jahnke
and Lazarus, 1984; Ekman et al., 1985) suggests a potential role for this peptide in the
developing gut. The primary objective of this study was to determine if specific binding
sites for bombesin are present in newborn or suckling porcine small intestine. In
addition, we sought to determine the timing of the appearance of these sites and the
specific areas of the small intestine where binding sites are present. Such determinations
should further our understanding of the importance of this peptide during this critical
developmental period.
MATERIALS AND METHODS
182
Tissue Collection
Three piglets at each of 5 different ages were sacrificed by intracardial barbiturate
overdose. Animals from different litters born on the same day were obtained at birth, and
animals from these same litters were obtained at 1 d, 1 wk, 3 wk (weaning), and 4 wk.
Blocks of tissue were obtained from three different sites from the small intestine of each
animal. A section from the cranial 5% of the small intestine represented proximal
duodenal tissue, a section from the middle 5% represented mid-jejunal tissue, and a
section from the caudal 5% just prior to the cecum represented terminal ileal tissue.
Sections were thoroughly cleaned with saline, embedded in cryoform and stored at -80°C
until all tissues were obtained. Blocks of tissue were then serially sectioned at 20 μm on
a cryostat at -20°C, thaw-mounted on gelatin-coated microscope slides, and stored in
boxes with desiccant at -80°C until use.
Autoradiography
Monoiodinated, biologically active (Tyr-4)-bombesin 14 (Bachem, Torrence, CA) was
prepared using iodogen, purified by reverse-phase high-performance liquid
chromatography, reduced using dithiothreitol, and repurified by high-performance liquid
chromatography as before (Vigna et al., 1987).
The quantitative autoradiographic receptor binding technique (Young and Kuhar,
1979) was used to identify the specific bombesin binding sites in porcine gastrointestinal
tissues. The incubation conditions were modified from those used by Wolf et al. (1983).
The slide-mounted tissue sections were first preincubated in 10 mM N-2-
183
hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES), pH 7.4, for 5 min at room
temperature. They were then incubated in 10 mM HEPES, 130 mM NaCl, 4.7 mM KCl,
5 mM MgCl2, 1 mM ethyleneglycol-bis(ß-aminoethylether)-N-N'-tetraacetic acid, 0.1%
bovine serum albumin, 100 μg/ml bacitracin (pH 7.4), and 100 pM 125I-(Tyr-4)-bombesin
14 for 1 h at room temperature. To estimate non-specific binding, paired serial sections
were incubated as described above except that unlabeled bombesin was added to the
incubation solution to a final concentration of 1 μM. The slide-mounted tissue sections
were then washed four times for 2 min each in 10 mM HEPES (pH 7.4) at 4°C. Finally,
the slides were rinsed twice for 5 sec each at 4°C in distilled water. The slides were dried
at 4°C under a stream of cold air and stored at room temperature overnight in boxes
containing desiccant. The slides were placed in apposition to LKB Ultrofilm (Bromma,
Sweden) for 14 d and developed in Kodak D-19 (Eastman Kodak, Rochester, NY), fixed,
and washed. The autoradiograms in the figures are enlargements of the LKB Ultrofilm
negatives.
RESULTS AND DISCUSSION
No evidence for specific binding of 125I-(Tyr-4)-bombesin 14 was found in any
sampled tissues from newborn or 1-d old piglets. This is somewhat surprising in light of
research by Lehy et al. (1986), suggesting a role for bombesin in hypertrophy of neonatal
rat small intestine. However, species-specific actions of gut peptides and neuropeptides
in the neonatal period appear to be the rule rather than the exception. In particular,
regulation of small intestinal development in rats and mice appears to bear little
184
resemblance to such development in farm species.
By 7 d, specific binding sites for 125I-(Tyr-4)-bombesin 14 were observable in
proximal duodenal tissue of piglets (Figure 1), although no such binding was observed in
either mid-jejunal or distal ileal tissues. This is in contrast to adult dogs, in which
binding sites are evenly distributed throughout the small intestine (Vigna et al., 1987).
Binding sites were still present in proximal duodenal tissue obtained from 3 wk old
piglets, with no evidence of binding in other areas of the small intestine.
Bombesin has been shown to decrease intraluminal duodenal pressure in humans
(Corrazziari et al., 1974) and to stimulate secretion of neurotensin (Barber et al.,
185
186
a.
b.
187
1986) and cholecystokinin (Jansen and Lamers, 1983) from duodenal tissue in dogs and
humans, respectively. Similar functions may be involved in suckling piglets.
No specific binding sites for 125I-(Tyr-4)-bombesin 14 were observed in either
duodenal, jejunal or ileal tissues from 4-wk old piglets. The apparent disappearance of
binding sites following weaning in the piglet is intriguing. The switch from liquid to
solid food may necessitate alterations in regulation of duodenal function. Alternatively,
the presence of bombesin has been reported in the milk of several species (Jahnke and
Lazarus, 1984; Ekman et al., 1985), and loss of this exogenous source of the peptide
downregulate receptor levels. It remains for future studies to resolve these questions.
SUMMARY
The results from this study reinforce the importance of species-specific developmental
research. Regulation of development and function in the small intestine of newborn and
suckling animals not only differs substantially from the same processes in adult animals,
but additionally may vary within this time span as well. The transient appearance of
specific binding sites for bombesin in duodenal tissues from suckling pigs during a short
window of time suggests that bombesin may be important in duodenal function and(or)
development only during this time frame. Either the function of bombesin in small
intestinal development is complete at weaning, or other gut peptides may assume this
function upon weaning. Differential responses of gut tissues to gut peptides at different
ages have been demonstrated for several hormones (Majundar and Johnson, 1982; Brants
and Morisset, 1976; Zahavi et al., 1984; Morisset, 1980).
188
In conclusion, transient expression of specific binding sites for bombesin were
observed in proximal duodenum from 7 d to 3 wk in suckling piglets. No evidence was
seen supporting the presence of receptors in mid-jejunal or distal ileal tissues in these
animals. Additionally no evidence was seen for the presence of specific binding sites for
this peptide in newborn, 1 d old or 4 wk old piglets.
LITERATURE CITED
Anastasi, A., V. Ersamer and M. Bacci. 1972. Isolation and amino acid sequences of
alytesin and bombesin, two analogous active tetradecapeptides from the skin of
European discolossid frogs. Arch. Biochem. Biophys. 148:443.
Barber, D.L., A.M.J. Buchan, J.H. Walsh, A.H. Soll. 1986. Isolated canine ileal mucosal
cells in short-term culture: a model for study of neurotensin release. Am. J. Physiol.
250:G374.
Bertaccini, G., V. Erspamer, P. Melchiorri and N. Sopranzi. 1974. Gastrin release by
bombesin in the dog. Br. J. Pharmacol. 52:219.
Brants, F. and J. Morisset. 1976. Trophic effect of cholecystokinin-pancreozymin on
pancreatic acinar cells from rats of different ages. Proc. Soc. Exp. Biol. Med. 153:523.
Corrazziari, E., A. Torsoli, G.F. Delle Fave, P. Melchiorri and F.J. Habib. 1974. Effects
of bombesin on the mechanical activity of the human duodenum and jejunum. Rend.
Gastroenterol. 6:55.
Ekman, R., S. Ivarsson and L. Jansson. 1985. Bombesin, neurotensin and pro-τmelanotropin immunoreactants in human milk. Regul. Pept. 10:99.
Jahnke, G.D. and L.H. Lazarus. 1984. A bombesin immunoreactive peptide in milk. Proc.
Natl. Acad. Sci. USA 81:578.
Jansen J.B.M.J. and C.B.H.W. Lamers. 1983. Molecular forms of cholecystokinin in
human plasma during infusion of bombesin. Life Sci. 33:2197.
Lehy, T., F. Puccio, J. Chariot and D. LaBeille. 1986. Stimulating effect of bombesin on
the growth of gastrointestinal tract and pancreas in suckling rats. Gastroenterol.
90:1942.
189
Majumdar, A.P.N. and L.R. Johnson. 1982. Gastric mucosal cell proliferation during
development in rats and effects of pentagastrin. Am. J. Physiol. 242:G135.
Morisset, J. 1980. Stimulation of pancreatic growyh by secretin and caerulein in suckling
rats. Biomed. Res. 1:405.
Poitras, P., D. Tasse and P. Laprise. 1983. Stimulation of motilin release by bombesin in
dogs. Am. J. Physiol. 245:G249.
Taylor, I.L., J.H. Walsh, D. Carter, J. Wood and M.I. Grossman. 1979. Effect of atropine
and bethanecol on bombesin-stimulated release of pancreatic polypeptide and gastrin
in dog. Gastroenterol. 77:714.
Vigna, S.R., C.R. Mantyh, A.S. Giraud, A.H. Soll, J.H. Walsh and P.W. Mantyh. 1987.
Localization of specific binding sites for bombesin in the canine gastrointestinal tract.
Gastroenterol. 93:1287.
Wolf, S.S., T.W. Moody, T.L. O'Donohue, M.A. Zarbin and M.J. Kuhar. 1983.
Autoradiographic visualization of rat brain binding sites for bombesin-like peptides.
Eur. J. Pharmacol. 87:163.
Young, W.S. III and M.J. Kuhar. 1979. A new method for receptor autoradiography: 3Hopiod receptors in rat brain. Brain Res. 179:255.
Zahavi, I., J. Kelly and D.G. Gall. 1984. Role of gastrin and cholecystokinin in the
ontogenic development of the gastrointestinal tract. Biol. Neonate 45:95.
190
DEVELOPMENTAL APPEARANCE OF RECEPTORS FOR
VASOACTIVE INTESTINAL PEPTIDE IN THE
DUODENUM, JEJUNUM AND ILEUM OF
PIGLETS FROM BIRTH THROUGH
FOUR WEEKS OF AGE
Howard Tyler
Department of Animal Science
College of Agriculture and Life Sciences
North Carolina State University
Raleigh, NC 27695
Steve Vigna and Douglas McVey
Department of Cell Biology
Duke University Medical Center
Durham, NC 27710
Warren J. Croom, Jr.
Department of Animal Science
College of Agriculture and Life Sciences
North Carolina State University
Raleigh, NC 27695
191
ABSTRACT
The objective of this study was to determine the age at which different segments of small
intestine express specific binding sites for vasoactive intestinal peptide (VIP). The
actions of VIP in gut tissues of developing animals is poorly documented, and definition
of potential target tissues during this period should help further our understanding of the
importance of this peptide during this period. Piglets were sacrificed at birth, 1 d, 1 wk,
3 wk and 4 wk and samples of duodenal, jejunal and ileal tissue were harvested for
autoradiographic analysis. Monoiodinated, biologically active VIP (100 pM) was applied
to slide-mounted sections of piglet gut tissue and localized using quantitative
autoradiography. Specific binding sites were present in duodenal, jejunal and ileal
tissues of piglets of all ages studied. These results suggest potential sites of direct action
of VIP in newborn, suckling and newly weaned piglets.
(Key Words: Vasoactive Intestinal Peptide, Piglet, Newborn, Weaning, Small Intestine,
Suckling.)
INTRODUCTION
Vasoactive intestinal peptide (VIP) is a linear polypeptide of 28 amino acid residues
that is structurally related to both secretin and glucagon (Said and Mutt, 1972).
Physiological and pharmacological studies have suggested a variety of functions for VIP
in the gastrointestinal tract. Stimulation of the peripheral ends of the thoracic vagi results
in release of this neuropeptide in small intestinal tissue (Edwards et al., 1978). VIP in
192
adult mammalian intestine is important in water and electrolyte secretion by intestinal
epithelium (Barbezat and Grossman, 1971; Krejs et al., 1980; Krejs and Fordtran, 1980;
Albuquerque et al., 1979), is a potent vasodilator (Said and Mutt, 1972), regulates
gastrointestinal motility (Anuras and Cooke, 1978; Bennet et al., 1984; Fontaine et al.,
1986; Jaffer et al., 1974), and stimulates bicarbonate secretion and epidermal growth
factor release from Brunner's glands in rat duodenum (Kirkegaard et al., 1984). Increases
in VIP are also associated with short-term downregulation of glycolysis by small
intestinal enterocytes (Rossi et al., 1989). However, regulation of adult gut function is in
many cases different than regulation of the same tissues in newborn animals. Gastrin,
cholecystokinin and caerulin, with well established functions on mature gastric mucosa
and pancreas, have no activity on these tissues in suckling rats (Majumdar and Johnson,
1982; Brants and Morisset, 1976; Zahavi et al., 1984; Morisset, 1980).
The primary
objective of this study was to determine if specific binding sites for VIP are present in
newborn or suckling porcine small intestine. In addition, we sought to determine the
timing of the appearance of these sites and the specific areas of the small intestine where
binding sites are present. Such determinations should further our understanding of the
importance of VIP during this critical developmental period.
MATERIALS AND METHODS
193
Tissue Collection
Three piglets at each of 5 different ages were sacrificed by intracardial barbiturate
overdose. Animals from different litters born on the same day were obtained at birth, and
animals from these same litters were obtained at 1 d, 1 wk, 3 wk (weaning), and 4 wk.
Blocks of tissue were obtained from three different sites from the small intestine of each
animal. A section from the cranial 5% of the small intestine represented proximal
duodenal tissue, a section from the middle 5% represented mid-jejunal tissue, and a
section from the caudal 5% just prior to the cecum represented terminal ileal tissue.
Sections were thoroughly cleaned with saline, embedded in cryoform and stored at -80°C
until all tissues were obtained. Blocks of tissue were then serially sectioned at 20 μm on
a cryostat at -20°C, thaw-mounted on gelatin-coated microscope slides, and stored in
boxes with desiccant at -80°C until use.
Autoradiography
Monoiodinated, biologically active VIP (Amersham Corp., Arlington Heights, IL) was
prepared using chloramine T. The quantitative autoradiographic receptor-binding
technique (Young and Kuhar, 1979) was used to identify the specific VIP-binding sites in
porcine gastrointestinal tissues. The incubation conditions were modified from those
used by Zimmerman et al. (1989). The slide-mounted tissue sections were first
preincubated in 10 mM N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES),
pH 7.4, for 5 min at room temperature. They were then incubated in 10 mM HEPES, 130
mM NaCl, 4.7 mM KCl, 5 mM MnCl2, 1 mM ethyleneglycol-bis(ß-aminoethylether)-N-
194
N'-tetraacetic acid, 1% bovine serum albumin, 1 mg/ml bacitracin (pH 7.4), and 100 pM
125
I-VIP for 2 h at room temperature. To estimate non-specific binding, paired serial
sections were incubated as described above except that unlabeled VIP was added to the
incubation solution to a final concentration of 1 μM. The slide-mounted tissue sections
were then washed two times for 15 min each in the incubation solution without VIP (pH
7.4). Finally, the slides were rinsed four times for 5 sec each at 4°C in distilled water.
The slides were dried at 4°C under a stream of cold air for 1 h, then stored at room
temperature overnight in boxes containing desiccant. The slides were placed in
apposition to LKB Ultrofilm (Bromma, Sweden) for 14 d and developed in Kodak D-19
(Eastman Kodak, Rochester, NY), fixed and washed. The autoradiograms in the figures
are enlargements of the LKB Ultrofilm negatives.
RESULTS AND DISCUSSION
Specific binding sites for VIP were observed in all tissues studied. The
autoradiograms verifying these sites are presented in Figures 1 through 7. The presence
of binding sites for VIP in proximal duodenum, mid-jejunum, and terminal ileum in
piglets from birth through the suckling period and into the post-weaning period suggests
that VIP may be critical to small intestinal function throughout development. The
presence of these binding sites in animals at birth suggests that
195
196
a.
b.
197
198
a.
b.
199
200
a.
b.
201
202
a.
b.
203
204
a.
b.
205
206
a.
b.
207
208
a.
b.
209
VIP may have a function prior to that time, as it is highly likely that these receptors were
present during late gestation.
The apparent irreplaceable nature of VIP in these tissues is in contrast to many other
gut peptides with age-specific roles in gut function. The role of VIP in regulation of
water and electrolyte secretion by intestinal epithelium (Barbezat and Grossman, 1971;
Krejs et al., 1980; Krejs and Fordtran, 1980) would appear to be such a function. Water
and electrolyte secretion are developmentally stable phenomena; presumably they are as
critical to the fetus as to the adult. In this sense, the presence of binding sites for VIP
throughout the small intestine and throughout development of the animal is not
surprising.
SUMMARY
In conclusion, the results from this study demonstrate the presence of binding sites for
VIP throughout the porcine small intestine at birth, throughout the suckling period, and
into the post-weaning period. These findings confirm the importance of VIP to small
intestinal function for piglets throughout development.
LITERATURE CITED
Anuras, S. and A.R. Cooke. 1978. Effects of some gastrointestinal hormones on two
muscle layers of duodenum. Am. J. Physiol. 234:E60.
Barbezat, G.O. and M.I. Grossman. 1971. Intestinal secretion: stimulation by peptides.
Science 174:422.
210
Bennet, A., S.R. Bloom, J. Ch'ng, N.D. Christofides, L.E. Peacock and J.A. Rennie. 1984.
Is vasoactive intestinal peptide an inhibitory transmitter in circular muscle but not
longitudinal muscle in guinea pig colon. J. Pharm. Pharmacol. 36:787.
Brants, F. and J. Morisset. 1976. Trophic effect of cholecystokinin-pancreozymin on
pancreatic acinar cells from rats of different ages. Proc. Soc. Exp. Biol. Med. 153:523.
Edwards, A.V., P.M.M. Bircham, S.J. Mitchell and S.R. Bloom. 1978. Changes in the
concentration of vasoactive intestinal peptide in intestinal lymph in response to vagal
stimulation in the calf. Experientia. 34:1186.
Fontaine, J., A.R. Grivegnee and P. Robberecht. 1986. Evidence against VIP as the
inhibitory transmitter in non-adrenergic, non-cholinergic nerves supplying the
longitucinal muscle of the mouse colon. Br. J. Pharmacol. 89:599.
Jaffer, S.S., J.T. Farrar, W.M. Yau and G.M. Makhlouf. 1974. Mode of action and
interplay of vasoactive intestinal peptide (VIP), secretin and octapeptide of
cholecystokinin (OCTA-CCK) on duodenal and ileal muscle in vitro. Gastroenterol.
66:716.
Krejs, G.J., R.M. Barkley, N.W. Read and J.S. Fordtran. 1980. Intestinal secretion
induced by vasoactive intestinal peptide: a comparison with cholera toxin in the
canine jejunum in vivo. J. Clin. Invest. 61:1337.
Krejs, G.J. and J.S. Fordtran. 1980. Effect of VIP infusion on water and ion transport in
human jejunum. Gastroenterol. 78:722.
Kirkegaard, P., P.S. Olsen, E. Nexo, J.J. Holst and S.S. Poulsen. 1984. Effect of
vasoactive intestinal peptide and somatostatin on secretion of epidermal growth factor
and bicarbonate from Brunner's glands. Gut 25:1225.
Majumdar, A.P.N. and L.R. Johnson. 1982. Gastric mucosal cell proliferation during
development in rats and effects of pentagastrin. Am. J. Physiol. 242:G135.
Morisset, J. 1980. Stimulation of pancreatic growyh by secretin and caerulein in suckling
rats. Biomed. Res. 1:405.
Rossi, I., L Monge and J.E. Feliu. 1989. Short-term regulation of glycolysis by
vasoactive intestinal peptide in epithelial cells isolated from rat small intestine.
Biochem. J. 262:397.
Said, S.I. and V. Mutt. 1972. Isolation from porcine intestinal wall of a vasoactive
octacosapeptide related to secretin and glucagon. Eur. J. Biochem. 28:199.
211
Young, W.S. III and M.J. Kuhar. 1979. A new method for receptor autoradiography: 3Hopiod receptors in rat brain. Brain Res. 179:255.
Zahavi, I., J. Kelly and D.G. Gall. 1984. Role of gastrin and cholecystokinin in the
ontogenic development of the gastrointestinal tract. Biol. Neonate 45:95.
Zimmerman, R.P., T.S. Gates, C.R. Mantyh, S.R. Vigna, M.L. Welton, E.P. Passaro, Jr.
and P.W. Mantyh. 1989. Vasoactive intestinal polypeptide receptor binding sites in the
human gastrointestinal tract: localization by autoradiography. Neurosci. 31:771.
212
GENERAL CONCLUSIONS
This research is the extension of a continuing effort to understand the regulation of
small intestinal development in the newborn. The developmental changes occurring in
the gastrointestinal tract during the perinatal period must be coordinated and
accomplished with precise timing. Losses associated with the increased morbidity and
mortality of newborns not attaining adequate levels of passive immunity emphasize the
importance of this research.
The findings regarding the relationship between glucose availability and closure not
only extend basic knowledge of the closure process in calves, but begin to allow a more
unified concept of closure in ungulates. The mediation of closure in pigs, calves, and
lambs may be accomplished in a similar manner.
Glucose availability is undoubtedly one factor influencing the timing of closure, but
this does not preclude the possibility of other factors playing a role in this process.
Endocrine involvement is a strong possibility and has been well documented in rats and
mice. The development of a reasonable hypothesis for endocrine involvement in closure
in calves is hampered by the paucity of information regarding perinatal changes in
metabolites and hormones in calves. The fourth study attempted to provide some of this
information, although it is far from complete. The last two studies were a continuation of
this same process; aquisition of basic knowledge from untreated animals. The
determination of the presence or absence of receptors for growth factors and gut peptides
is critical to understanding regulation of gut development and function. Again, the
results obtained from these studies represent a fraction of the knowledge needed to
213
understand these processes well enough to manipulate them in a beneficial manner.
New techniques, such as the development of the in vivo perfusion technique for fetal
small intestine, should provide a tool for furthering our understanding of the role of
oxygen availability for intestinal development. In addition, variations on this procedure
should allow the study of other potential factors regulating this process.