Published November 9, 2016

Characterization of a novel encapsulated oral iron supplement

to prevent iron deficiency anemia in neonatal piglets1
R. Antileo, J. Figueroa, and C. Valenzuela2
Departamento de Fomento de la Producción Animal, Facultad de Ciencias Veterinarias y Pecuarias,
Universidad de Chile, La Pintana 8820000, Santiago, Chile

ABSTRACT: The aim of this study was to char- with 126 mg oral iron supplement on d 2 and 8 (oral
acterize nonheme iron (NHI) and heme iron (HI) I; n = 22), or with 84 mg oral iron supplement on d
microparticles to elaborate a novel oral iron supple- 2, 8, and 14 (oral II; n = 22). Red blood cells (RBC),
ment, determining its effect on the iron nutritional hemoglobin (Hb), hematocrit (Ht), mean corpuscular
status of neonatal piglets. Nonheme iron (ferrous sul- volume (MCV), mean corpuscular hemoglobin con-
fate) was dissolved at 20, 30, and 40% wt/vol, and HI centration (MCHC), free erythrocyte protoporphyrin
(porcine blood cells) was dissolved at 30% wt/vol in (FEP), and serum ferritin (SF) were determined. The
a maltodextrin solution (40% wt/vol). Solutions were Hb content of the oral I group was lower (8.3  1.3
spray-dried, obtaining NHI microparticles (NHIM) g/dL) than the parenteral (10.1  1.7 g/dL) and oral
A, B, and C, respectively, and HI microparticles II (10.2  1.4 g/dL) groups (P < 0.05). Moreover,
(HIM). Microparticles were characterized by iron RBC, Ht, MCV, MCHC, and SF were altered in the
content and release profile at gastrointestinal in vitro oral I group, indicating iron deficiency anemia. Iron
pigs’ conditions. NHIM-B was selected and blended deficiency anemia in piglets may be prevented by
with HIM (oral iron supplement). Neonatal pigs (n the administration of an oral encapsulated NHI/HI
= 66) were supplemented with 200 mg of injectable supplement (3 doses) presenting the same benefits as
dextran iron on d 2 of life (parenteral group; n = 22), classical parenteral supplementation.

Key words: anemia, encapsulation, heme iron, nonheme iron, piglets

© 2016 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2016.94:157–160
doi:10.2527/jas2015-9698

INTRODUCTION There are few examples of the use of heme iron (HI)
supplement for pigs that have shown a greater iron
Iron deficiency anemia is a serious problem in bioavailability than NHI (Quintero-Gutiérrez et al.,
neonatal piglets that is usually solved by a parenteral 2008; Lipiński et al., 2013). The emerging technol-
supplementation of 200 mg of dextran iron at d 1 to ogy of encapsulation in animal nutrition could im-
3 postpartum. Several authors have investigated oral prove the bioavailability of NHI and HI, preventing
iron supplementation in piglets; however, results have iron deficiency in humans and rodents (Zimmermann,
not been as expected compared to a parenteral sin- 2004; Xu et al., 2014). However, there is no informa-
gle-dose supplementation (Zimmerman et al., 1959; tion about the effects of NHI or HI encapsulation in
Svoboda and Drábek, 2002), which is explained by a pigs. Therefore, the aim of this study was to charac-
high variation of nonheme iron (NHI) bioavailabil- terize NHI and HI microparticles to elaborate a novel
ity (Lipiński et al., 2013) and by a low expression of oral iron supplement and to determine its effect on
NHI receptors and transporters (Lipiński et al., 2010). the iron nutritional status of neonatal piglets.

1This work was supported by the following research projects:

CONICYT Atracción e Inserción de Capital Humano Avanzado en

la Academia 7912010043 and FONDECYT Iniciación 11140249.
2Corresponding author: cvalenzuelav@u.uchile.cl

157
158 Antileo et al.

MATERIALS AND METHODS Table 1. Microparticle characteristics of nonheme iron

(NHIM) and heme iron (HIM).
Encapsulation and Characterization of
Characteristic NHIM-A NHIM-B NHIM-C HIM
Iron Microparticles
Total iron (mg/g) 74  6a 86  8b 94  12c 0.85  0.12d
Maltodextrin (Prinal S.A., Santiago, Chile) solu- HI (mg/g) – – – 0.77  0.15
tion (40% wt/vol) in deionized water was prepared. Iron release GF (%) 26  8a 31  10a 38  5b 22  4c
Nonheme iron (iron sulfate heptahydrate; Merck S.A.) Iron release IF (%) 72  8 a 66  13 a 61  10 a 76  5a
a–dMeans  SD with different superscript letters are statistically
was suspended in maltodextrin solution at 20, 30, and
different (P < 0.05).
40% (wt/vol) and spray-dried (Buchi Mini Spray Dryer
B-290; Buchi, Flawil, Switzerland), obtaining type A,
B, and C NHI microparticles (NHIM), respectively. muscles on d 2 of life (parenteral group, n = 22), 2)
Porcine blood cells (Licán Alimentos S.A., Santiago, piglets supplemented with 2 oral doses of the iron sup-
Chile) were suspended in maltodextrin solution at 30% plement on d 2 and 8 (oral I group, n = 22), or 3) piglets
wt/vol and were spray-dried obtaining HI microparti- supplemented with 3 oral doses of the iron supplement
cles (HIM). The total iron content of NHIM and HIM on d 2, 8, and 14 (oral II group; n = 22). The oral sup-
was determined using an atomic absorption spectropho- plement was given by using a blunt-tipped applicator
tometer (905AA; GBC, Braeside, Australia) ( = 248.3 within their snouts (2 mL liquid paste). Blood samples
nm). Heme iron content was determined according to (2 mL) were taken from the piglets’ jugular vein at d 1
Valenzuela et al. (2014). To simulate the pig digestive and 21 by venipuncture to determine iron status. Red
tract, 2 conditions were used: gastric fluid (GF; 2 g/L blood cell (RBC), hemoglobin (Hb), hematocrit (Ht),
of NaCl containing 10 g/L of pepsin with pH adjusted mean corpuscular volume (MCV), mean corpuscu-
to 2.0 with HCl 1 N) and intestinal fluid (IF; 50 g/L lar hemoglobin concentration (MCHC) (Model ZBI;
of pancreatin and 31.2 g/L of bile extract in an intesti- Coulter, Hialeah, FL and Cell-Dyn 1700; Abbott Di-
nal solution of 8.76 g/L NaCl and phosphate-buffered agnostic, Abbott Park, IL, USA), free erythrocyte pro-
saline at 0.1 M, pH 7.4; the pH of IF was adjusted to toporphyrin (FEP) (Hematofluorimeter model 206D;
6.0 with HCl 1 N). The NHIM and HIM (0.5 g) were AVIV, Lakewood, NJ, USA), and serum ferritin (SF)
mixed in 100 mL of GF and IF and were incubated for (Pig ferritin, FE ELISA Kit; Cusabio) were determined
60 and 200 min, respectively, at 37°C with constant agi- at d 1 and 21. Animals were weighted at the beginning
tation at 150 rpm. The released pattern of total iron was (d 1) and end (d 21) of the experiment.
measured from aliquots of 3 mL by atomic absorption
spectroscopy at the end of each digestion. Statistical Analysis

Oral Iron Supplement Statistix 8 software was used for all of the statistical
analyses. The assays were processed by ANOVA, and
The NHIM-B were blended with HIM at 10:2 means comparisons were adjusted by Tukey (P < 0.05).
and 8:2 ratios wt/wt for 2 and 3 doses, respectively
and were suspended into distilled water (2 mL). The RESULTS AND DISCUSSION
supplement was prepared according pigs requirements
Encapsulated Oral Iron Supplement
(10 mg per day) (NRC, 2012).
Characteristics of NHIM and HIM are presented
Animal and Experimental Design in Table 1. Nonheme iron microparticles showed a
high total iron content that increased in relation with
Experimental procedures were approved by the the NHI concentration. The iron released in GF was
Bioethical Committee on Animal Experimentation higher for NHIM-C, discarding this type of micropar-
of the Facultad de Ciencias Veterinarias y Pecuarias, ticle, since the maximum release was expected at the
Universidad de Chile (certificate 05-2015). The experi- duodenum level where the iron is absorbed (Lipiński
ment was conducted in a commercial pig farm (Región et al., 2013). A similar iron release was observed in
Metropolitana, Chile). A total of 66 male and female all NHIM types in IF, where the maltodextrin matrix
1-d-old piglets, weighing 1.61  0.09 kg were selected was disintegrated due to pancreatic amylase. Finally,
and allocated with 6 sows (11 piglets per litter) of the NHIM-B was selected from the in vitro study due to
same parity number. Piglets were randomly assigned its high iron content and adequate release of iron at the
inside each litter to 3 experimental groups: 1) piglets gastrointestinal tract. The total iron content for HIM
supplemented with 200 mg of injectable dextran iron was lower than that for NHIM despite the fact that HI
(Veterquímica, San Bernardo, Chile) into the thigh presents a higher bioavailability than NHI (Quintero-
 Oral iron supplementation in piglets 159

Table 2. Iron status biomarkers of 1 (T1) and 21-d-old (T21) piglets subjected to different iron supplementation
protocols.
Parenteral Oral I Oral II
Cutoff
Biomarkers1 T1 T21 T1 T21 T1 T21 value
RBC (106  mm3) 6.0  0.8aA 6.0  0.5aA 5.9  0.9aA 5.6  0.6bB 6.0  1.0aA 6.3  0.8aB <5.32
Hb (g/dL) 11.5  1.7aA 10.2  1.4aA 11.3  1.9aA 8.3  1.3bB 11.7  1.4aA 10.1  1.7aA <9.02
Ht (%) 37.1  5.4aA 34.1  3.5aA 36.3  5.8aA 30.2  3.0bB 37.9  4.9aA 35.1  4.8aA <322
MCV (fL) 62.1  3.4aA 57.0  4.6aA 61.4  3.0aA 53.7  2.9bB 63.7  3.1aA 56.2  3.3aB <502
MCHC (%) 31.1  0.7aA 29.7  1.6aA 31.2  0.8aA 27.3  2.3bB 31.0  0.8aA 28.7  1.5aA n.d.3
FEP (g/dL RBC) 88  19aA 133  37aB 105  30aA 153  29aB 94  18aA 140  31aB n.d.
SF (g/L) 13.5  4.4aA 9.9  4.4aB 25.5  4.8bA 4.1  1.3bB 17.8  4.8aA 8.7  3.9aB <12.14
a,bMeans  SD with different superscript lowercase letters are statistically different (P < 0.05) to compare different treatments.
A,BMeans  SD with different superscript uppercase letters are statistically different (P < 0.05) within the same treatment for different days of sampling.
1RBC, red blood cell; Hb, hemoglobin; Ht, hematocrit; MCV, mean corpuscular volume: MCHC, mean corpuscular hemoglobin concentration; FEP, free
erythrocyte protoporphyrin; SF, serum ferritin.
2Zimmerman (1980).
3n.d., Not determined for nursery piglets in the literature.
4Adams et al. (1988).

Gutiérrez et al., 2008; Lipiński et al., 2013). The 91% of functional iron (decreased Hb). For parenteral and
of total iron of HIM corresponded to HI, which was oral II groups, the biomarkers most affected between 1
expected by the origin of this product. The iron re- and 21 d were FEP and SF. FEP increase in both groups,
lease pattern of HIM was lower in GF compared with which could indicate a lower availability of iron for the
NHIM, which is possibly due to the high gelling ca- synthesis of Hb. Although there is no established cutoff
pacity of the iron source, retarding its release from the for FEP in suckling pigs, Espinoza et al. (2014) deter-
matrix (Valenzuela et al., 2014). mined a normal value of 106  23 g/dL RBC; thus,
The oral iron encapsulated supplement contained all of the groups presented this parameter altered at d
252 mg of the total iron, distributed in 2 or 3 doses of 21. The SF was also lower at d 21, indicating a state of
126 or 84 mg of iron each, respectively. depletion of iron deposits in these animals. However,
the SF values of these groups are higher than the ones
Effects of Oral Iron Encapsulated Supplement from the oral I group, although these animals presented
on Iron Status of Neonatal Piglets the highest values of SF at baseline.
Other authors also observed anemia with oral
No differences were found on the body weight of iron supplementation in neonatal piglets. However, to
piglets at d 21 between groups: 6.27  0.91 kg (parenter- achieve a similar hematinic effect to parenteral supple-
al), 6.28  1.01 kg (oral I), and 6.27  0.93 kg (oral II). mentation, they used several doses (8 doses for 4 wk)
Iron status biomarkers are presented in Table 2. (Zimmerman et al., 1959), making this management im-
At d 1, groups did not differ on RBC, Hb, Ht, MCV, practicable in the current conditions of pig farms. Svo-
MCHC, and FEP (baseline), presenting a similar iron boda and Drábek (2002) prevented piglet anemia at 21 d
nutritional status. However, the SF was higher in the by delivering only 2 doses of 200 mg of ferrous fuma-
oral I group, indicating better iron deposits in these rate; nevertheless, this value exceeds at least twice suck-
animals. At d 21 parenteral and oral II animals in- ling piglets requirements of this mineral (NRC, 2012).
creased their levels of RBC, Hb, Ht, MCV, MCHC, Iron deficiency anemia of neonatal piglets could be
and SF due to supplementation compared with oral I prevented with an encapsulated oral iron supplement
piglets that presented Hb, Ht, MCV, MCHC, and SF divided into 3 doses of 84 mg of iron each, probably by
below the cut-off points. the greater bioavailability of the NHI and HI sources
As iron deficiency anemia is defined as low Hb and due to the encapsulation process (Xu et al., 2014; Zim-
2 or more of any of the biomarkers altered (see cut-off mermann, 2004) and/or a synergic effect of NHI/HI
in Table 2) (Zimmerman, 1980), both protocols (paren- combinations on iron absorption.
teral and oral II) prevented the development of iron de-
ficiency anemia in the piglets of this study. In the oral I LITERATURE CITED
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