2.1. Animal Care and Use
All animal and experimental procedures were in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals and approved by the University of Illinois at Urbana-Champaign Institutional Animal Care and Use Committee. Forty-two naturally-farrowed, intact male pigs were obtained from a commercial swine farm in one of two replicates, and transferred to the University of Illinois Piglet Nutrition and Cognition Laboratory (PNCL) at PND 2. Per standard agricultural protocol, pigs were provided an intramuscular injection of a prophylactic antibiotic (0.1 mL of ceftiofur crystalline free acid as Excede, Zoetis, Parsippany, NJ, USA) within 24 h of birth. Contrary to typical agricultural procedures, pigs on this study were never provided supplemental iron (i.e., injectable iron dextran), because iron is the nutrient of interest. Recent pig studies observed hippocampal transcriptome changes [16
] and possible effects of iron overload [9
] after iron dextran administration in the first few days of life, which further justify our decision to not provide iron dextran to any pigs. Upon arrival to PNCL on PND 2, pigs were stratified into one of two experimental diets, described below. Pigs were provided experimental milk replacer treatments from PND 2 until PND 32 or 33 (phase 1), at which point, both treatment groups were transitioned onto a common series of industry-standard diets from PND 32 or 33 until PND 61 or 62 (phase 2).
For phase 1 of this study, 42 pigs were housed individually in custom pig rearing units (87.6 cm long, 88.9 cm wide, 50.8 cm high), which were composed of three acrylic walls, one stainless steel wall, and vinyl-coated, expanded-metal flooring. This caging environment was designed for pigs to see, hear, and smell, but not touch, neighboring pigs. Pigs were allowed to physically interact with one another for approximately 15 min each day, and each pig was provided a toy for enrichment in their home-cage throughout the study. Facility lighting was maintained on a 12 h light and dark cycle from 0800 to 2000 h, with ambient temperature set at 26.6 °C for the first 21 days of the study, and gradually lowered to 22 °C during the last seven days of phase 1.
For phase 2 of this study, 20 pigs from phase 1 were transferred to the University of Illinois Veterinary Medicine Research Farm at PND 32 or 33, and housed there until the end of the study. While in this facility, pigs were housed individually in floor pens (1.5 m2), and the rearing environment was maintained on a 12 h light and dark cycle from 0800 to 2000, with ambient temperature set at 22 °C.
All animal and experimental procedures were in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals, and approved by the University of Illinois at Urbana-Champaign Institutional Animal Care and Use Committee. Approval for this research project was confirmed on 3 March 2015 under the title Nutrition and Brain Development in Young Pigs, and was terminated on 7 February 2018.
2.2. Dietary Treatments
For phase 1 of this study, pigs (n = 21 per diet) were provided one of two milk replacer treatments with varying iron content. The CONT diet was formulated to meet all of the nutrient requirements of the growing pig and was formulated to contain 106.3 mg Fe/kg milk replacer powder. The ID diet was identical to the CONT diet, with the exception that ferrous sulfate (i.e., the predominant iron source in CONT) was removed to provide only 13.6 mg Fe/kg milk replacer powder. Additionally, both diets were formulated to contain ARA (2.08 g ARA/kg milk replacer powder) and DHA (1.04 g DHA/kg milk replacer powder). Milk replacer was reconstituted fresh daily with 200 g of milk replacer powder per 800 g water. Thus, formulated iron concentrations in reconstituted pig milk replacers were 21.3 and 2.72 mg Fe/L milk replacer for the CONT and ID treatments, respectively. All pigs were provided ad libitum access to liquid milk replacer treatments from PND 2 until PND 32 or 33.
For phase 2 of this study, all pigs (n
= 10 per diet) were transitioned onto the same common series of industry-relevant, iron-adequate diets (containing 180–300 mg Fe/kg of diet), regardless of their phase 1 dietary iron treatment group. Pigs were provided ad libitum
access to standard complex diets (major ingredients including corn, whey, and soybean meal) and standard agricultural feeding practices were followed by sequentially switching to stage 1, 2, and 3 diets on PND 32, 41, and 50, respectively. During phase 2 of the study, all diets were formulated to meet all nutrient requirements of growing pigs [17
], including iron. No zinc oxide, copper sulfate, or in-feed antibiotics were included in any diets. Analyzed values of iron in diets can be found in Figure 1
Porcine milk was collected as part of a previous study [18
]. Samples were then analyzed for mineral profiles by using standardized procedures (Mead Johnson Nutrition, Evansville, IN, USA) to establish iron content. Specifically, porcine milk samples were digested using a combination of concentrated nitric acid and 30% hydrogen peroxide at 220 °C for 10 min in a microwave digestion system (UltraWAVE; Milestone Inc., Shelton, CT, USA). After digestion, the samples were diluted to volume and quantified by inductively-coupled plasma mass spectrometry (ICP-MS; NexION 300D; Perkin Elmer, Waltham, MA, USA). The instrument was operated in kinetic energy discrimination mode using helium to reduce polyatomic interferences. All samples were analyzed in duplicate.
2.5. Blood and Tissue Collection, Processing, and Analysis
At PND 32 (CONT, n = 6; ID, n = 7) and PND 61 and 62 (n = 20; n = 10 per phase 1 diet), pigs were euthanized for tissue and blood collection. All animals were euthanized in a food-deprived state, with no access to dietary treatments for at least 6 h prior to euthanasia. Pigs were anesthetized using an intramuscular injection of telazol/ketamine/xylazine administered at 0.022 mL/kg bodyweight (50.0 mg tiletamine plus 50.0 mg of zolazepam reconstituted with 2.50 mL ketamine (100 g/L) and 2.50 mL xylazine (100 g/L); Fort Dodge Animal Health, Overland Park, KS, USA). To ensure pigs were properly anesthetized prior to euthanasia, all pigs were tested for reflex via the eye blink response. Pigs were euthanized using a 390 mg/mL sodium pentobarbital solution (Patterson Veterinary Supply, Columbus, OH, USA) at 1 mL/5 kg body weight by intracardiac injection. Blood was collected from the jugular vein immediately after euthanasia on PND 32 and 61, and collected into evacuated serum and EDTA-containing tubes (Becton, Dickenson and Company, Franklin Lakes, NJ, USA). Serum was left at room temperature to clot for at least thirty minutes. Plasma was collected into EDTA tubes, gently inverted, and stored on ice for up to four hours until processing. Serum and plasma were processed by spinning blood down utilizing an Allegra 6R centrifuge (Beckman Coulter Life Sciences, Indianapolis, IN, USA), aliquoted, and stored at −80 °C. Serum was analyzed for hepcidin and ferritin concentration via validated porcine enzyme-linked immunosorbent assay (ELISA) kits (Elabscience, Hongshan, Hubei Province, China), with a detection range of 1.56–100 ng/mL and 4.69–300 ng/mL, for hepcidin and ferritin, respectively.
Duplicate aliquots of liver tissue (~0.5 g each) were collected immediately following euthanasia on PND 32 or 61, rinsed with ice-cold phosphate buffered saline (0.01 mM), snap frozen in liquid nitrogen, and stored at −80 °C until processing. Liver samples were processed and analyzed via the same validated porcine ELISA kits mentioned above for hepcidin and ferritin concentrations. Proximal duodenum scrapings were collected in duplicate, snap frozen, and stored at −80 °C until processing, to analyze divalent metal transporter 1 (DMT1) concentration via validated porcine ELISA kits (DL Develop, Wuxi, Jiangsu Province, China), with a detection range of 0.156–10 ng/mL. All measures analyzed by ELISA kit were assessed in duplicate, and run according to the manufacturer’s instructions. Samples with a result above the upper limit of the kit’s detection range were diluted and re-analyzed.
Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to quantify gene expression of ferritin and hepcidin in liver samples, and DMT1 in proximal duodenum scrapings. Frozen liver and proximal duodenum scrapings aliquots (50 to 100 mg) were placed into 2 mL microcentrifuge tubes containing a 5 mm stainless steel bead and one mL of TRIzol reagent (Invitrogen, Carlsbad, CA, USA), to enable tissue disruption for two minutes at 30 Hz (TissueLyser II, Qiagen, Valencia, CA, USA). Ribonucleic acid (RNA) extraction was carried out according to manufacturer recommendations for TRIzol reagent, and total extracted RNA was quantified using a spectrophotometer (NanoDrop ND-1000, NanoDrop Technologies, Wilmington, DE, USA). Complimentary DNA (cDNA) was transcribed from the isolated RNA using a high capacity cDNA Reverse Transcriptase kit (Thermo Fisher Scientific Inc., Waltham, MA, USA), with samples placed in a thermocycler (Bio-Rad, Hercules, CA, USA) set to run at 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min, and then cooled at 4 °C, and held at 4 °C overnight. Samples were then removed and kept at −20 °C until plating. The TaqMan Gene Expression Assay (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to perform qRT-PCR to quantify relative gene expression of porcine target genes hepcidin, ferritin, and DMT1 (HAMP, FTH1, and SLC11A2, respectively) and the reference gene β-actin (Applied Biosystems, Carlsbad, CA, USA) [20
]. Sample cDNA was amplified using TaqMan (Thermo Fisher Scientific Inc., Waltham, MA, USA) oligonucleotide probes containing 5′ fluorescent reporter dye (6-FAM) and 3′ non-fluorescent quencher dye, and fluorescence was determined using a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). To normalize gene expression, parallel amplification of endogenous β-actin was performed in triplicate for each sample. Relative gene expression was then calculated using the comparative threshold cycle method [21
] and results are expressed as fold-change relative to CONT pigs.
Following sample collection procedures at the end of phase 1, nine pigs per treatment group were used to perform a carcass composition analysis at the University of Illinois Meat Science Laboratory (Urbana, IL, USA) using standardized procedures. Specifically, carcasses were skinned by hand, and an air skinner was utilized to leave subcutaneous fat with the carcass. The head, feet, testicles, and heart were then removed, leaving only muscle, fat, and associated connective tissue (i.e., total soft tissue), which was used to collect a standardized final carcass weight. All bones were separated from soft tissue and knife-scraped to remove residual tissue. Dissected carcasses were divided and weighed, with categories including skin, bone, and soft tissue. Soft tissue was prepared for proximate composition analysis by grinding and homogenizing all soft tissue through a commercial bowl chopper. A 10 g sample of soft tissue was oven-dried at 110 °C for approximately 24 h to determine percentage moisture. The dried sample was then washed multiple times in an azeotropic mixture of warm chloroform/methanol, as described by Swensen et al. [22
], to determine the fat content of the soft tissue. Finally, the percentage of carcass weight, categorized as fat vs. fat-free lean, was calculated for each pig.