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Monaco et al. J Environ Expo Assess 2024;3:18 https://dx.doi.org/10.20517/jeea.2024.10 Page 3 of 18

DEHP in milk replacer formula would alter intestinal morphology, digestive enzyme activity, and
microbiota composition relative to animals not exposed to DEHP.

EXPERIMENTAL
Animal care and housing
[19]
The study design and animal protocol were described by Lee et al. . Briefly, two-day-old piglets (n = 24)
(12 males and 12 females from four different litters) were obtained from the Swine Research Center at the
University of Illinois in Urbana-Champaign and transferred to the AAALAC-approved animal facilities in
the Edward R. Madigan Laboratory on the University of Illinois in Urbana-Champaign campus. Upon
arrival at the animal facility, piglets were randomized by initial BW and sex to three dietary treatments,
where there were eight piglets (four females and four males in each treatment group). All animal and
experimental procedures were in accordance with the National Research Council Guide for the Care and
[20]
Use of Laboratory Animals and approved by the University of Illinois in Urbana-Champaign Institutional
Animal Care and Use Committee (protocol # 18112).

Phthalate exposures
Piglets were assigned to receive DEHP at dose levels of 0 (CON), 20 (DEHP20), or 200 (DEHP200) mg/kg
BW/day. The DEHP (Sigma-Aldrich, St. Louis, MO) was dissolved in tocopherol-stripped corn oil and
added to the feeding bowl prior to the first feeding of the day from postnatal day (PND) 3-23. The CON
group received a daily dose of DEHP-free tocopherol-stripped corn oil at the volume of the DEHP200. The
DEHP doses reflect those previously reported in the literature [21-23] . The 20 mg/kg/day dose aligns with
exposure levels from medical devices [21,22] . The 200 mg/kg/day dose of DEHP has been used in previous
[23]
studies by our group . Piglets were fed a nutritionally adequate sow-milk replacer formula reconstituted at
18.3% w/v (Milk Specialties Global, Eden Prairie, Minnesota). Piglet BW was measured daily, and the
volume of the formula provided was based on 300 mL/kg BW/day (PND3-7) and 325 mL/kg BW/day
(PND8-23). Formula intake was calculated by subtracting daily formula waste from the formula provided.

Sample collection
On PND23, animals were sedated with an intramuscular injection of Telazol® (Tiletamine HCl and
Zolazepam HCl, 3.5 mg/kg BW each, Pfizer Animal Health, Fort Dodge, IA) followed by an intravenous
injection of 86 mg/kg BW sodium pentobarbital (Euthasol; Virbac AH, Inc., Fort Worth, TX). The spleen,
brain, kidneys, heart, lungs, intestine, and liver were excised and weighed. Samples from the small and large
intestines were fixed in 10% buffered formalin for morphological analysis. The small intestine mucosa was
scraped using a microscope slide and snapped-frozen to assess disaccharidase activity. Ascending colon
(AC) and rectal contents (RC) were collected for microbiota analysis. In addition, urine samples were
collected via cystocentesis (urine collected directed from the bladder using a sterile needle and syringe) for
urinary DEHP metabolites assessment by liquid chromatography/mass spectrometry (LC/MS/MS).

Histomorphological analyses
Formalin-fixed sections of duodenum, jejunum, ileum, and AC were processed into paraffin blocks,
sectioned into 5-μm slices, and placed on glass slides. Digital images (10× magnification) of hematoxylin-
eosin–stained tissues were captured with the use of the NanoZoomer Slider Scanner (Hamamatsu
Corporation; Institute for Genomic Biology Core Microscopy and Imaging Facility, UI). Small intestinal
villus height (μm), crypt depth (μm), and colon cuff depth (μm), and villus, crypt, and colon cuff areas
(mm ) were measured from 10-15 well-oriented crypts-villi with AxioVision 4.8 Digital Image Processing
2
Software (Carl Zeiss MicroImaging, Inc.).