Applied Physiology, Nutrition and Metabolism

Lipid profile, adipose tissue, adipocytokines and body anthropometric in swine

born with Intrauterine Growth Restriction evoke metabolic and development
derangements in postnatal life

INTRODUCTION

In all mammalian species, adequate growth and development during prenatal life
is critical for the survival of the offspring and for the adequate development and function
of all organs (Ashworth 2013). During the prenatal phase, the development of organs
depends largely on the composition of nutrients and biologically active substances
transferred through the placenta (Hongen et al., 2017). Therefore, insults that occur in
intrauterine life may compromise the normal development of an organism and have a
major impact on postnatal life (Desay et al., 2007). This process is known as
‘prenatal/fetal programming’ or developmental origins of health and disease
(Puttabyatappa, et al., 2017).
Intrauterine growth restriction (IUGR) refers to the situation in which the growth
rate of a fetus is less than the normal growth potential for its gestational age and is a result
of genetic, maternal, placental or fetal effects (Wu et al., 2006; Wiyaporn et al., 2013).
The earlier the occurrence of insults during pregnancy, the greater the severity of the
consequences of IUGR in postnatal life (Devaskar et al., 2016). Hence, IUGR
compromises fetal development, resulting in low birth weight. Individuals affected by
IUGR may exhibit gastrointestinal disorders, which may increase the occurrence of
metabolic syndrome, important cause of morbidity and postnatal mortality. Consequently,
the study of changes in adiposity and digestive system in those animals during postnatal
life seems critical.
Studies indicate that IUGR individuals may present catch-up growth in the first
weeks of postnatal life, however this has been associated with an increased risk of
metabolic diseases in later life such as central obesity, insulin resistance, glucose
intolerance, dyslipidemia and hypertension (Seferovic et al., 2015; Vliet et al., 2013; Xu
et al., 2013). There is evidence that IUGR compromises small intestine function and it is
speculated that these disorders are the main causes of low postnatal growth and high
mortality rate in these animals, since nutrient utilization is inadequate. In this sense, it is
imperative to identify, qualify, and quantify the factors that affect nutrient utilization and
intestinal function. (Wang et al., 2010).
Due to physiological and genomic similarities between pigs and humans, the pig
was recognized as an ideal experimental model for the study of IUGR. In addition, as it
is a species that bears litters, the pig exhibits IUGR in a natural and severe manner due to
uteroplacental failure. In this sense, this study was carried out to investigate possible
alterations in growth performance, lipid profile, adiposity and morphology of the small
intestine in newborn, weaned, 70 days (juvenile) and 150day-old (adult) pigs, subjected
or not to IUGR.

MATERIALS AND METHODS

Experimental design

Three hundred newborn male piglets (DanBred " PIC terminal line), were selected
according to birth weight from females of 3rd–6th-parity, and from litters of 15 to 22 pigs,
and allocated to the following experimental groups: IUGR (birth weight range: 0.7 g and
1.0 kg, n = 175) and high birth weight (HW) (birth weight range:1.6 kg and 1.9 kg, n =
175). To define the birth weight range of each experimental group, 1000 piglets from the
same establishment were weighed immediately after birth and the mean weight (μ) and
standard deviation (ϭ) were then calculated (μ = 1.3; ϭ = 0,3). The birth weight bands
ranges of each group were determined as: μ + ϭ a μ + 2ϭ for HW and μ - ϭ a μ - 2ϭ for
IUGR.
Only two piglets from each treatment (2 high weight piglets and 2 IUGR piglets)
were selected from each litter. During maternity, after identification, the piglets were
gathered by the experimental group and kept together (± 13 piglets per sow) in typical
stalls of maternity sheds. The piglets were castrated at seven days and were weaning in
to average 24 days of age. After weaning, his were transferred to the nursery, where they
remained in groups of ± 40 piglets per bay / treatment (3 bays HW piglet and 3 bay IUGR
piglets) up to 70 days of age. After this period the piglets were transferred to the rearing
stage and then transferred to the termination phase. The animals that died during the
production phases were removed from the bay, and this information was recorded for the
calculation of mortality rates. Among the 300 piglets, 60 were randomly selected for
euthanasia and collection of biological material, which were performed at different stages
of development of the animals: 48 hours after weaning (26 days), 70 days and 150 days,
totaling 20 piglets per age (10 HW and 10 IUGR). The remaining animals were followed
up to 150 days of age to collect performance data (Figure 1).

Body conformation measurements and feed intake in the pigs

The animals were weighed at birth, weaning (26 days), at70 days, 110 days and
150 days of age to evaluate the average daily weight gain (ADG) in each experimental
phase. For evaluation of offspring early-postnatal development, during the milk-feeding
phase (from birth to 26 days old), body weight, biparietal diameter, crown-to-rump-
length, shoulder height, thorax weight and abdominal circumference (AC) was measured
at least twice weekly. After until the end of the study, these measurements were made in
day care leave (70 days), and final finishing phase (150 days).
Recent evidence suggests that body shape, rather than birth weight alone, may be
a more important diagnostic indicator of future growth and development (1). For this,
data obtained from body weight, AC and crown-to-rump length were used to calculate
relationship between AC and body weight (AC/Body weight), body mass index (BMI),
tri-ponderal mass index (TMI) and body adiposity index (BAI) off all pigs at each age
point. In human studies, these parameters are a common indicator for obesity, however,
the calculation in pigs must be corrected, in which the height corresponds to the crown-
to-rump-length. Recent studies indicate that the tri-ponderal mass index can estimate
body fat levels more accurately than BMI and this index was calculated, according to a
study (Peterson et al., 2017; Ramírez-Velez, 2018).
In order to calculate the adiposity percentage estimate with greater precision, in
humans, the body adiposity index (BAI) was calculated, using the formula: abdominal
circumference / (height) 1.5. However, Bergmanet al. (2011) defined a new formula to
make this estimate in pigs, where height is replaced by length. All animals were received
a diet specific for each stage of the production cycle, In addition, the supply of feed and
water was ad libitum throughout the experimental period.
Blood sample collection

The blood, collected before slaughter by electric stunning followed by bleeding,

was drawn from the jugular vein after 12 hours of fasting, in piglets at weaning, 70 and
150 days of age (n=10 piglets IUGR and 10 RW per age). The sample was conditioning
into tubes containing sodium heparin and immediately placed on ice until they arrived at
the Laboratory of Structural Biology and Reproduction – UFMG. The blood was
centrifuged at 3.000 rpm for 10 min and the isolated plasma samples were stored at -80ºC
for further analysis.

Biometric parameters collection

Immediately after euthanasia, an incision was made in the midline of the animals,
when the organs were removed. The heart, liver and small intestine of each piglet were
weighed using a digital scale. The small intestine was removed from the pylorus
(stomach-duodenum junction) to the ileocecal valve and removed the luminal contents
before measurement of the length with the aid of a tape measure. Subsequently, the
relative weight of the organs (relation between organ weight: body weight) was calculated
to investigate in a more detailed way the organ development pattern, since the
experimental treatments consisted of values of birth weights with variation of 700 to 1900
g.

Tissue sample collection

Samples of small intestine and adipose tissue were removed for histological and
biochemistry analysis. The samples of the duodenum and jejunum were taken from the
cranial flexure and from mid-jejunum respectively. The visceral adipose tissue was
removed from perirenal region and subcutaneous backfat between the 12th 13th ribs.
Approximately 2 cm in length of each tissue were fixed by immersion in 4% in
paraformaldehyde solution at 24 hours and in 0.05 M sodium phosphate buffer for
histological analyzes. Samples of same tissues of approximately 4 cm were frozen in
liquid N2 and then stored at -80° C for biochemistry analyzes.
Histology of adipose tissue

Adipose tissue samples were prepared using standard paraffin embedding

procedures (n=5 by group) by crescent dehydration in ethanol, clarification in xylol and
embedding in paraffin (Paraplast, Merck). Three transverse sections of 8 μm thickness
were cut along the adipose tissue fragments and all sections were stained with
hematoxylin and eosin. The histomorphometrical studies at subcutaneous and visceral
were performed in digital images obtained in an Olympus BX51 photomicroscope
equipped with a Q-Color 3 (Olympus) digital camera. Using the Image-Pro Express
program (Media Cybernetics), from the digital images were determined the adipocyte size
and adipocyte density by mm2. For each animal, ten whole regions of the visceral and
subcutaneous adipose tissue were selected randomly, and in each section, their area was
determined in mm2 and were counted the number of adipocytes by area. The diameter
filled by each adipocyte was determined using Feret's diameter (Fig.4). To stratify the
diameters of adipocytes, they were allocated in 5 classes (μm), according to the size of
these cells, as follows: 1-20; 21-40; 41-60; 61-80; 81-100; >100.

Glycemic and lipid profile

6.9.1. Glucose

The glycemia was determined by enzymatic assay (Labtest kit, Brazil), whose
principle consists in the oxidation of glucose to gluconic acid by the action of glucose
oxidase enzyme, with the release of hydrogen peroxide. This, by the action of peroxidase
in the presence of phenol and 4-aminoantipyrine, forms a red anti-pilquinonimine whose
color intensity is proportional to the concentration of glucose in the sample. For glucose
determination, the blood was collected in tube with fluoride, for being antiglycolytic.
Aliquots of 10 μL of plasma was transferred to test tubes containing 1000 μL of reagent
1 and after incubation in a water bath for 10 minutes at 37 ° C, the absorbance was read
at 520 nm in photo colorimeter, setting the zero with the blank sample. For the calculation
of glycemia in mg / dL, the following formula (indicated in the Labtest kit) was used:
Mean of the absorbance of the sample / mean of the absorbance of the standard x 100.
6.9.2. Total cholesterol

The total cholesterol was determined by enzymatic assay (Labtest kit, Brazil),
whose principle consists in the hydrolysis of cholesterol esters by the enzyme cholesterol
esterase. Free cholesterol is oxidized by the enzyme cholesterol oxidase, forming cholest-
4-em-one and hydrogen peroxide. After oxidation, the anti-pyrilquinonimine is formed,
in which the intensity of the red color formed in the reaction is directly proportional to
the concentration of cholesterol in the sample. Aliquots of 10 μL of plasma was
transferred to test tubes containing 1000 μL of reagent 1 and after incubation in a water
bath for 10 minutes at 37 °C, the absorbance was read at 500 nm in photocolorimeter,
setting the zero with the blank sample. For the calculation of total cholesterol in mg / dL,
the following formula (indicated in the Labtest kit) was used: Mean of the absorbance of
the sample / mean of the absorbance of the standard x 200.

6.9.3. HDL-cholesterol

The HDL-cholesterol was determined by enzymatic assay (Bioclin kit, Brazil),

whose principle consists in the selective dosage of HDL. Reagents composed of modified
enzymes stabilize the fractions of low density lipoprotein (LDL), very low density
(VLDL) and chylomicrons. HDL, however, is solubilized by the action of a detergent,
allowing the enzymatic action on the cholesterol attached to it. In this way, the intensity
of the staining is proportional to the HDL cholesterol concentration in the sample.
Aliquots of 3 μL of plasma was transferred to test tubes containing 300 μL of reagent 1
and after incubation in a water bath for 5 minutes at 37 ° C. 300 μL of reagent 2 was
added and the solution was incubated in a water bath for 5 minutes at 37 °C. The
absorbance was read at 500 nm in photo colorimeter, setting the zero with the blank
sample. For the calculation of HDL-cholesterol in mg / dL, the following formula
(indicated in the Bioclin kit) was used: Mean of the absorbance of the sample / mean of
the absorbance of the standard x calibrator concentration.

6.9.4. LDL-cholesterol

The LDL-cholesterol was determined by enzymatic assay (Bioclin kit, Brazil),

whose principle consists in the detergent present in the reagent solubilizes only non-LDL-
C lipoproteins. The cholesterol released from these lipoproteins is then consumed by the
enzymes: cholesterol esterase, cholesterol oxidase and peroxidase. A second detergent
solubilizes the LDL lipoprotein, allowing its consumption by the previous enzymes,
leading to the formation of hydrogen peroxide. The color intensity formed is directly
proportional to the concentration of LDL-C in the sample. Aliquots of 3 μL of plasma
was transferred to test tubes containing 300 μL of reagent 1 and after incubation in a water
bath for 5 minutes at 37 ° C. Subsequently, 300 μL of reagent 2 was added and the solution
was incubated in a water bath for 5 minutes at 37 ° C. Aliquots of 5000 μl of reagent 2
was added and the solution was incubated in a water bath for 10 minutes at 37 ° C. The
absorbance was read at 580 nm in photo colorimeter, setting the zero with the blank
sample. For the calculation of alkaline phosphatase in mg / dL, the following formula
(indicated in the Bioclin kit) was used: Mean of the absorbance of the sample / mean of
the absorbance of the standard x 100.

6.9.5. Triglycerides

The triglycerides levels were determined by enzymatic assay (Bioclin kit, Brazil),
whose principle consists in the hydrolysis of triglycerides by lipoprotein lipase, releasing
glycerol, which is converted to glycerol-3-phosphate. This molecule is oxidized to
dihydroxyacetone and hydrogen peroxide and then a coupling reaction occurs between
hydrogen peroxide, 4-aminoantipyrine and 4-chlorophenol. There will be production of
quinoneimine, whose red color intensity is proportional to the concentration of the
triglycerides in the sample. Aliquots of 10 μL of plasma was transferred to test tubes
containing 1000 μL of reagent 1 and 10 μL of reagent 2 and after incubation in a water
bath for 10 minutes at 37 ° C. The absorbance was read at 540 nm in a photo colorimeter,
setting the zero with the blank sample. For the calculation of triglycerides in mg / dL, the
following formula (indicated in the Bioclin kit) was used: Mean of the sample’s
absorbance e / mean of the standard’s absorbance x 100.

6.9.6. Calculation of risk of cardiovascular disease

Serum lipid profile allows the evaluation of the risk of cardiovascular disease
through the calculation of indexes considering plasma concentration of HDL, LDL and
triglycerides. The higher the level of LDL and the lower the HDL, the greater the risk
which can be quantified through the Castelli indexes:

Total cholesterol LDL

𝐂𝐚𝐬𝐭𝐞𝐥𝐥𝐢 𝐈: ; 𝐂𝐚𝐬𝐭𝐞𝐥𝐥𝐢 𝐈𝐈:
HDL HDL
Furthermore, the ratio of triglycerides to HDL-cholesterol is used as an indicator
of dyslipidemia, which increases the cardiovascular risk.

6.10. Determination of proinflammatory cytokines IL12p70, TNF, IL10, IL6, IL1 β

e IL-8
Serum cytokine assays were performed by the Cytometric Bead Array (CBA)
method using the Human Inflammatory Cytokine Kit: IL12p70, TNF, IL10, IL6, IL1β
and IL-8 (BD Pharmigen, CA, USA). This method consists of the use of labeled
polystyrene beads with different degrees of fluorescence, coated with specific antibodies,
in which cytokines bound to the antibodies are detected by the use of antibodies labeled
with phycoerythrin. The fluorescence intensity measured with phycoerythrin is
proportional to the concentration of cytokines in the sample is quantified from a
calibration curve. Aliquots of 25 mL of sample were used and 25 mL of cytokine
standards were submitted to serial dilution with diluent G of kit CBA. Samples and
standards were transferred to 5 mL polystyrene tubes and 15 mL of the capture bead
mixture, conjugated to anti-cytokine monoclonal antibodies IL12p70, TNF, IL10, IL6,
IL1β and IL-8 were added. Subsequently, 18 µL of the cocktail of labeled human
monoclonal antibodies phycoerythrin were added and incubated for three hours at room
temperature and protected from light. Then, 18 μL of the antibody cocktail were
conjugated with phycoerythrin-PE and incubated for 3 hours at room temperature. After
incubation, the beads were washed and centrifuged at 1300 revolutions per minute (rpm)
for seven minutes at 18 ° C. The supernatant was discarded, leaving approximately 100
μL remaining in each tube. The samples were evaluated with a flow cytometer.

6.11. Histology of small intestine

Duodenal and jejunal samples were prepared using standard paraffin embedding
procedures (n=5 per group) by crescent dehydration in ethanol, clarification in xylol and
embedding in paraffin (Paraplast, Merck). Three transverse sections of 5 μm thickness
were cut along the intestine fragments and all sections were stained with hematoxylin and
eosin. A total of 10 intact, well-oriented crypt-villus units were selected in triplicate for
each intestine of piglets. Sections were evaluated through a light microscope at a
magnification of 20X (Model CX21; Olympus, Tokyo, Japan) equipped with an ruler
inside an ocular that allowed measurement without taking pictures. Measurements (in
micrometer) of the duodenum wall were determined as follows: (a) height of duodenal
mucosa (HM), from the muscularis mucosae up to the apex of the villus; (b) height of the
villi (HV), from the base up to the apex of the villus; (c) diameter of the villus (DV); (d)
depth of the intestinal crypt (D), from the muscularis mucosa to the base of the villus; (e)
diameter of the crypt (DC) and (e) height of the enterocytes (HE) (Figure3). To determine
if the effects of IUGR could functionally affect the intestinal absorption capacity, it was
determined the relationship between villi and intestinal crypt, and villus surface area (S).
The calculation of villus surface area as performed using the formula described by Dong
et al. (2016):

𝑣𝑖𝑙𝑙𝑢𝑠 𝑤𝑖𝑑𝑡ℎ 𝑣𝑖𝑙𝑙𝑢𝑠 𝑤𝑖𝑑𝑡ℎ 2

𝑺: 𝜋 √( ) + 𝑣𝑖𝑙𝑙𝑢𝑠 𝑙𝑒𝑛𝑔ℎ𝑡 2
2 2

Figure 3: Histological parameters analyzed in the small intestine: (A) height of villus, (B)
depth of the crypt, (C) height of the enterocytes, (D) diameter of the villus, (E) diameter
of the crypt, (F) height of mucosa.
6.12. Statistical analyses

The experimental design was a completely randomized design, with two

treatments (groups of birth weight), with one animal per experimental unit. Data were
submitted to analysis of variance (ANOVA) and means, compared by the Student- T test.
Important associations between performance parameters and biochemical parameters
were evaluated by correlation analysis. The statistical program used was SAS (Statistical
Analysis System Institute Inc., Cary, NC, 2003), with level of significance considered as
P < 0.05.

7.0 RESULTS

7.1. Postnatal growth performance

Body weight changes and average daily weight gain at the end of each production
phase are shown in Tables 1 and Figure 5. IUGR piglets presented compromised growth
potential during all stages of development, as they showed lower body weights and daily
gains compared to their HW counterparts (Table 1; Figure 5; P<0.01).

Table 1. Growth performance data (kg) from high birth weight (HW) and IUGR pigs
Parameter Treatments s.e.m P-value
HW (n=145) IUGR(n=126)
Birth weight 1.70a 0.92b 0.01 b -
Body weight at 26 days 6.60a 4.80b 0.11 b <0.001 b
Body weight at 70 days 25.00a 18.50b 0.04 b <0.001 b
Body weight at 105 days 62.60a 53.50b 1.00 b <0.001 b
Body weight at 150 days 105.80a 89.82b 1.30 b <0.001 b
ADG suckling 0.206a 0.163b 0.004 b <0.001 b
ADG 26-70 days 0.433a 0.330b 0.007 b <0.001 b
ADG 70-105 days 0.962a 0.884b 0.018 b <0.001 b
ADG 105-150 days 1.235 a 1.106b 0.025 b <0.001 b
ADG, average daily gain; NS, not significant.ab Within a row, least-square means with
different superscripts differ (P < 0.05).

A B
 Figure 5: (A) Growth curve and (B) average daily gain from birth up to 150 days of
age.* Values statistically different (p<0.001).

The percentage of body weight gain in early life in HW and IUGR is represented
in Figure 6. Although IUGR piglets presented lower body weight in all ages studied
evaluated, a catch-up-growth was observed in IUGR piglets in early postnatal life (from
birth to weaning).

Figure 6: Percentage of body weight gain in early life in HW and IUGR piglets.* Values
statistically different (p<0.05).

The average daily feed intake and feed conversion data are showed in Table 3.
Throughout postnatal development, the IUGR animals from the growth phase consumed
more feed than their HW counterparts, which negatively affected feed conversion rate.
Additionally, the mortality rate reported throughout the postnatal period was 18% and 8%
respectively for the IUGR and HW groups.

Table 2. Average daily feed intake and feed conversion rate throughout postnatal
development
Production Feed intake (kg) Weight gain (kg) Feed conversion
phase HW IUGR HW IUGR HW IUGR
Nursery 24.00 23.00 18.26 13.70 1.31 1.67
Grower 93.00 97.00 37.31 35.05 2.49 2.76
Finishing 120.00 133.00 46.00 36.00 2.60 2.89
Sample number: Nursery: HW (132), IUGR (113); Grower HW (84), IUGR (75); Finishing HW (95), IUGR (79).
7.2. Organs’ weight

As shown in Table 2, liver, heart and small intestine (SI) weights were lower in
IUGR piglets (P < 0.05). On the other hand, the difference not was notable in all ages
analyzed. At birth and 70 days, there was a reduction on liver and small intestine and the
heart was smaller from 70 days to 150 days. The length of SI organ was also affected by
IUGR in piglets from weaning to the exit phase of day care (70 days) but at 150 days, this
parameter was similar between the two groups. The results showed at relative weight of
the small intestine seems to be related to prenatal programming, since was statistically
lower in IUGR piglets at all ages evaluated. However, relative length of small intestine
was similar between 2 groups in all ages available. Were found the positive correlations
between body weight and ID weight (r = 0.82, P < 0.01), body weight and ID length (r =
0.62, P < 0.05) and body weight and abdominal circumference (r = 0.59, P < 0.05), infers
that the smaller the animal weight, the lower the development of the organs.

Table 3-Organs biometrical parameters from high weigh (HW) and IUGR
piglets during postnatal development
Parameter Treatment s.e.m P-value
HW(n=10) IUGR(n=10)
Liver weight (kg)
26 days a 0.14a 0.10a 0.05a <0.01a
70 days a 0.91a 0.62a 0.03a <0.01 a
150 days a 1.84a 1.86a 0.09a NSa
Heart weight (kg)
26 days a 0.03a 0.02a 0.003a NSa
70 days a 0.20a 0.14a 0.08 a <0.01a
150 days a 0.43a 0.38b 0.01a <0.05a
SI weight (kg)
26 days a 0.24a 0.17b 0.02 a <0.05a
70 days a 1.60a 1.27b 0.70 a <0.01 a
150 days a 3.16a 3.07a 0.20 a NS a
SI weight/BW
26 days a 4.30a 4.00a 0.40 b NSb
70 days a 5.77a 7.54b 0.60 b <0.05b
150 days a 2.77a 3.27a 0.20 b NSb
SI lenght (m)
26 days a 7.86a 6.77b 0.28 a <0.05 a
70 days a 15.33a 13.65b 0.54 a <0.01 a
150 days a 18.50a 18.00a 0.60 a NS a
SI lenght/BW
26 days a 0.164a 0.196b 0.01 a <0.05 a
70 days a 0.550a 0.820b 0.06 a <0.05 a
150 days a 0.164a 0.196b 0.01 a <0.05 a
NS, not significant. ab Within a row, least square means with different superscripts
differ (P < 0.05).
7.3. Body measurements

Birth weight affected crown-rump-length, biparietal diameter, thorax

circumference, abdominal circumference, shoulder height and thorax height at birth (P
<0.001) showing a large difference in size between littermate piglets. At weaning, this
same pattern was observed, except for abdominal circumference, which was similar
between both experimental groups. In a similar way, as development progressed, body
biometric measurements were similar in HW and IUGR pig, apart from shoulder height,
which was shorter in IUGR piglets at all ages evaluated (Table 4).

Table 4- Body measurements of high birth weight (HW) and IUGR pigs during
postnatal development
Parameter Treatment s.e.m P-value
HW IUGR
Crown-rump-length (cm)
Birth a 30.01a 25.01b 0.01 b <0.001 b
26 days a 43.07a 41.07b 0.50 b <0.05 b
70 days a 72.30a 71.00a 1.00 b NS b
150 days a 109.70a 110.20a 1.30 b NS b
Biparietal diameter (cm)
Birtha 4.30a 3.60b 0.02 b <0.001 b
26 days a 6.00a 5.60b 0.05 b <0.001 b
70 days a 9.60a 10.00a 0.20 b NS b
150 days a 12.30a 9.70b 0.30 b <0.001 b
Thorax circunference (cm)
Birth 27.70a 22.20b 0.01 b <0.001 b
26 days a 42.00a 40.00a 0.50 b NS b
70 days a 63.60a 64.20a 1.00 b NS b
150 days a 101.40a 107.80a 2.40 b NS b
Abdominal circumference (cm)
Birth 27.60a 22.20b 0.10 b <0.001 b
26 days a 43.70a 42.40a 0.60 b NS b
70 days a 73.00a 71.40a 0.80 b NS b
150 days a 111.00a 103.00a 0.20b <0.05 b
Shoulder height (cm)
Birtha 8.20a 6.90b 0.30 b <0.001 b
26 days a 14.00a 12.00b 0.30 b <0.001 b
70 days a 27.00a 24.40b 0.40 b <0.001 b
150 days a 38.70a 34.00b 0.90 b <0.001 b
Thorax height (cm)
Birtha 22.60a 18.00b 0.10 b <0.001 b
26 days a 29.50a 28.00b 0.40 b <0.05 b
70 days a 43.00a 42.00a 0.50 b NS b
150 days a 66.30a 68.00a 1.00 b NS b
NS, not significant. ab Within a row, least square means with different superscripts
differ (P < 0.05).
7.4. Anthropometric index of adiposity

Figure 7 showed the anthropometric index to evaluate adiposity in pigs during

postnatal development. Apart from body mass index, which was different between IUGR
and HW piglets (P < 0.05; Figure 7B), all other indices were different between
experimental groups at specific time points. At birth, piglets showed higher abdominal
circumference and body weight ratio (Figure 7A). On the other hand, this ratio became
similar between groups throughout postnatal development, but over the age, it was
expressively smaller. Regarding body mass index, IUGR piglets presented lower values
when compared to HW animals (P < 0.05), a pattern that was repeated in all ages studied.
Moreover, tri-ponderal mass index was lower at birth and at 150 days of age (P < 0.05;
Figure 7C). At 26 and 70 days, the values were similar between the two groups, however
at 70 days, the tri-ponderal mass index presented values around 5 times higher than the
other ages.

A B

C D
Figure 7: Anthropometric index for evaluate adiposity in pigs during postnatal development.
(A) Relationship between abdominal circumference (AC) and body weight; (B) Body mass
index; (C) Tri-ponderal mass index, (D) Body adiposity index.*Values within the same age
differ (P <0.05).

7.5. Serum glucose, lipid profile and the risk of cardiovascular disease

Regarding the lipid profile, cholesterol levels at 70 days of age were higher in
IUGR pigs compared to their HW counterparts (Table 5; P < 0.05). Levels of triglycerides
and HDL fractions were similar between the two experimental groups at all ages
evaluated. Notwithstanding the lack of effect on triglycerides and HDL fractions, LDL
fractions were higher in IUGR pigs at 150 days of age.
Additionally, body weight at 70 day was negatively correlated to blood glucose (r
= -0.56, P < 0.05), and at 150 days to cholesterol (r = -0.59, P < 0.01) and a trend between
body weight and triglycerides levels (r = -0.52, P < 0.08) was observed (Figure 8). These
results suggest that in IUGR individuals, the low body weight is associated to higher
glucose, cholesterol and triglycerides levels. Moreover, the risk of cardiovascular
diseases calculated by the Castelli I and II indexes and the ratio between triglyceride and
HDL were not affected by fetal growth restriction, remaining similar between the
experimental groups.
 Table 5-Lipid profile and blood glucose from high weigh (HW) and IUGR
piglets during postnatal development
Parameter (mg / Treatment s.e.m P-value
dL) HW(n=10) IUGR (n=10)
Total Cholesterol
26 days a 84.48a 87.46a 7.87 a NS a
70 days a 66.50a 78.78b 3.60 a <0.05 a
150 days a 78.90a 81.30a 6.96 a NS a
HDL
26 days a 55.71a 59.51a 4.70 b NS a
70 days a 48.05a 44.30a 2.70 b NS a
150 days a 39.72a 46.11a 3.40 b NS a
LDL
26 days a 50.22a 46.25a 5.87 a NS a
70 days a 43.76a 48.21a 2.80 a NS a
150 days a 27.24a 33.54b 2.01 a <0.05 a
Triglycerides
26 days a 72.23a 105.88a 20.52 a NS a
70 days a 74.44a 87.97a 7.04 a NS a
150 days a 45.08a 50.87a 5.66 a NS a
TC/HDL (Castelli I)
26 days a 1.55a 1.53a 0.19 a NS a
70 days a 1.41a 1.82b 0.11 a <0.05a
150 days a 2.05a 1.82a 0.18 a NS a
LDL/HDL (Castelli II)
26 days a 0.90a 0.80a 0.2a NS
70 days a 0.90a 1.10a 0.1a NS
150 days a 0.90a 1.10a 0.1 NS
TG/HDL
26 days a 1,28a 1.81a 0.33 a NS a
70 days a 1.61a 2.02a 0.15 a NS a
150 days a 1.17a 1.13a 0.15 a NS a
Glucose
26 days a 112,36 a 112,53a 4.48 a NS a
70 days a 108.53a 95.70a 7.76 a NS a
150 days a 90.63a 96.27a 5.51 a NS a
NS, not significant. ab Within a row, least square means with different superscripts
differ (P < 0.05).

120
160
Cholesterol (mg / dL)

Glucose (mg / dL)

100
120
80
80
60

40 R2 = 0.59 40 R2 = 0.56
P < 0.05 P < 0.05
20 0
0 10 20 30 40 50 70 90 110 130 150
Body weight (kg) Body weight (kg)

Figure 8 – Correlation between (A) body weight (kg) and serum cholesterol level in
piglets at 70 days of age (r = -0.59, P < 0.01) and = (B) body weight (kg) and serum
glucose level at 150 days of age (r = -0.56, P < 0.05).
7.6. Histomorphometrical analysis of subcutaneous and visceral adipose tissue

The histomorphometrical evaluations of the visceral and subcutaneous adipose

tissues are shown in the Figures 9 and 10. IUGR affected the density of adipocytes/mm2.
In juvenile males (70 days of age), adipocytes number of the visceral adipose tissue was
similar between both experimental groups. However, in adult IUGR pigs (150 days of
age) the number of adipocytes was increased (P<0.05). On the other hand, when it comes
to the subcutaneous adipose tissue, adipocytes density was similar between both
experimental groups at all ages evaluated.

Subcutaneous Visceral
A B

Figure 9: Number of adipocytes / mm2 in piglets at 70 and 150 days of age in high birth
weight (HW) and IUGR experimental groups. (A) Subcutaneous adipose tissue, (B)
Visceral adipose tissue.*Within the same age, values differ (P <0.05).

The adipocyte size frequency of subcutaneous adipose tissue was similar between
HW and IUGR piglets at the two ages studied. However, it was observed that at 150 days
of age there was a higher number of adipocytes among the larger size ranges, compared
to 26 and 70 days of age. Regarding visceral adipose tissue, cell size frequencies were
also similar between the two experimental groups. In addition, while at 70 days of age
about 80% of the adipocytes had up to 40 nm, at 150 days, 60% presented sizes larger
than 40nm, showing adipocytes hypertrophy throughout the animals’postnatal
development along the development of the pig.
 Subcutaneous Subcutaneous
26 days 70 days
A B

Subcutaneous
150 days
C

Visceral Visceral
70 days 150 days
D E

Figure 11: Frequency of adipocyte size (% total) from different experimental groups (HW and
IUGR) in piglets at 26, 70 and 150 days of age. (A) Subcutaneous adipose tissue from 26 days
of age, (B) Subcutaneous adipose tissue from 70 days of age, C) Subcutaneous adipose tissue
from 150 days of age.*Within the same age, are statistically different (P <0.05).
7.7. Levels of proinflammatory serum cytokines

Figure 11 shows the serum level of proinflammatory cytokines in HW and IUGR

pigs at 150 days of age. Serum quantification of those cytokines demonstrated similar
pattern between the two groups, suggesting that IUGR appears to be unrelated to
inflammatory processes in adulthood.

A B

C D

E F
Figure 11: Evaluation of the expression of proinflammatory serum cytokines in high-
birth-weight piglets and IUGR. The horizontal line represents the median. A) TNF, B)
IL-8, C) IL-12p70, D) IL-1β, E) IL-6 and F) IL-10. There was no significant difference
between groups.

8.0. DISCUSSION

To establish the association between fetal growth restriction and the risk of
metabolic disorders and cardiovascular diseases in adulthood, IUGR piglets were
followed up in the first days of life to investigate catch-up growth, and during 5 months
to establish lipid profile, adiposity and morphology of the small intestine. To the best of
our knowledge, this is the first report showing adiposity and small intestine morphology
alterations in IUGR individuals from birth until adulthood, using the pig as an
experimental model.
Body weight changes during the pre-weaning period showed that IUGR piglets
gained more weight at that stage, providing evidence of catch-up growth. The occurrence
of catch-up growth associated to higher abdominal circumference to body weight ratio
have been associated to higher percentages of visceral fat later in life (Myrie et al., 2017).
In turn, high percentage of visceral fat is a predisposing factor to metabolic syndrome and
cardiovascular diseases (Tzschoppe et al. 2017).
In this study, all biometric parameters were lower in IUGR piglets at birth. These
results are in agreement with He et al. (2011), Alvarenga et al. (2013), Tzschoppe et al.
(2017), which also identified a body growth impairment in IUGR individuals. Moreover,
correlations between body weight and parameters as small intestine weight, small
intestine length and abdominal circumference suggest that lighter animals present lighter
organs and, consequently, small abdominal circumference. The smaller organs size may
be due to the "brain sparing" effect, usually found in IUGR individuals, representing an
asymmetric pattern of organ growth while the brain is spared (Ashworth et al., 2001, Wu
et al., 2006; Alvarenga et al., 2013).
The current data suggest that the risk of an obesogenic profile later in life would
be low if the prediction was based on the actual results of the anthropometric measures
evaluated, such as BMI, TMI and BAI, which are important predictors of obesity in
adolescence and adulthood (De Lorenzo, 2018). Furthermore, the positive correlations
between body weight and cholesterol, triglycerides and glucose levels in juvenile pigs
suggest that growth restriction in utero may lead to higher levels of those parameters in
adulthood, which negatively affects glycemic and lipid profiles. According to Zhou et al.,
2015, the ratio between total cholesterol and HDL levels is a more sensitive indicator of
cardiovascular disease than individual lipid levels, thus, suggesting that IUGR juvenile
individuals may be more susceptible to those diseases.
The present data indicate that the number of adipocytes between the IUGR and
HW piglets remain similar at all ages evaluated. However, the adipocyte density of the
visceral adipose tissue in the IUGR group was higher at 150 days of age. On the other
hand, when evaluating the adipocytes’ size, the results revealed that it remained similar
at weaning and at 70 days, but at 150 days, IUGR piglets presented a low frequency of
larger cells (81-120 mm). In this regard, two processes related to adipose tissue growth
may be involved: hyperplasia (proliferation and differentiation of preadipocyte), and
hypertrophy (increased cell volume by the accumulation of triglycerides). Considering
this scenario, the predominance of larger adipocytes in the HW group at 150 days may be
related to lipid incorporation, as those animals presented higher feed intake and body
weight gain was accentuated compared to their IUGR counterparts. Previous studies have
reported that the accumulation of hypertrophic adipocytes generates hypoxia,
inflammation and macrophages infiltration (Queiroz, et al., 2009). Furthermore, IUGR
individuals presented a hyperplastic adipocytes profile, a condition that can increase the
synthesis of fatty acids, both predictive of a greater propensity to fat storage. In fact,
considering that cell recruitment for the formation of new adipocytes is a slow process
and decreases with age, the higher density of visceral adipocytes in IUGR in juvenile
animals may predict that they mature over time and assume the opposite characteristic
(hypertrophy) in adulthood.
Regarding subcutaneous adipose tissue, adipocyte density as well as cell size were
similar between experimental groups at all ages investigated in the present study. Indeed,
the measurement of backfat thickness at the time of euthanasia has already shown absence
of IUGR effect on this parameter. A study by Schinckel et al. (2010) revealed that the
lower the birth weight, the greater the thickness of adipose tissue in adult life. However,
in the present study, the commercial genetic line used has a high potential for muscle
accretion and, consequently, low body fat deposition.
In juvenile animals (70 days of age), some parameters evaluated had major
alterations in the two experimental groups, among them high body mass index, high level
of cholesterol and cardiovascular risk, as well as the number of adipocytes. All these
results seem to be associated to the different feeding management at this stage,
considering that the experiment was carried out at a commercial pig farm, whose purpose
is to increase weight gain in a shorter period. Due to the abrupt changes in environment
and feed to piglets after weaning, and the limitations in the development of the piglet
digestive system, the diets offered at this age are highly digestible and have high energy
content, which may explain such changes in adiposity at this specific age.
The negative effects of maternal prenatal stress on postnatal immune responses
have been studied in pigs with effects on pro-inflammatory cytokines. However, the
characterization of immune responses in IUGR pigs is incomplete. Our data show that in
both groups, the levels of proinflammatory cytokines investigated were similar. Although
these peptides’ levels have not changed under any conditions, a negative relation between
body weight and IL6 concentration was found (r = -0.52; P < 0.05) suggesting that low
weight is associated with the increase in the concentration of this cytokine in IUGR
individuals. Certainly, a detailed approach of IL-6 deserves further investigation, since
higher concentrations of IL-6 may induce insulin resistance (Smerieri et al., 2011).

5.0. CONCLUSION

Taken together, the results obtained in the present study provide evidence that
growth restriction in fetal life contributes in various ways to developmental impairment
from birth to adulthood, affecting lipid profile and glucose levels, which are associated
with the risk of metabolic and cardiovascular diseases in adulthood. In this regard,
structural alterations of the small intestine absorptive may contribute to the impairment
in nutrient utilization, affecting postnatal growth development.