Over the years, nutritionists have continued to evaluate the vitamin and mineral requirements of swine. Recently, it was documented that US swine nutritionists feed a margin of safety above the 2012 NRC recommendations to offset any potential vitamin degradation or manufacturing challenges.^1,2 Little information has been compiled over the last 15 years to document current vitamin and mineral concentrations present in healthy swine of modern genetics. A widely used publication for mineral and vitamin reference values was published in 1994.^3,4 Modern hog production has changed greatly in the last 20 years particularly in reference to intensively raised, indoor swine as well as genetics and growth rate. In addition, vitamin D levels of hogs raised indoors have noticeably different levels compared to outdoor raised hogs.⁵

Therefore, sampling healthy swine being raised indoors would be important to establish reference values for vitamins and minerals to assist diagnostic laboratories, veterinarians, and nutritionists in discerning potential nutritional differences when assessing modern day pigs. However, the process of creating new reference values is costly. The objective of this study was to survey the vitamin and mineral levels in various tissues from healthy swine of modern genetics in different production phases to assess if new reference values need to be generated.

Animal care and use

The study was conducted on 6 different farms located across the United States. All animal care practices were conducted by following the routine farm management procedures and Pork Quality Assurance guidelines.⁶ Additionally, the trial was approved by the Iowa State University Animal Care Committee (IACUC #19-340).

Materials and methods

Samples

The 6 farms used in this study were selected based on voluntary participation from written communication with companies identified within the top 25 largest production systems and with individual producers based on timeframe available for study personal to collect the samples. Selected farms verified that the animals were fed vitamins and minerals at levels that met or exceeded the 2012 NRC recommendations.² The farms had to verify that the pigs used for sample collection were free of acute illness. Animals selected for sample collection were identified as animals with a physical abnormality (eg, hernia or prolapse) that would prevent the animal from completing the production life cycle, were scheduled for euthanasia (eg, growth study sampling), or were being harvested. The number of animals selected from each farm varied due to the number of animals available on the day that sampling personnel were present on the farm. Seven sows and a minimum of 10 animals from each phase of production (suckling, nursery, and finishing) were selected for sampling. The suckling phase was defined as day 1 through 21 of age. The nursery and finisher phases were defined as day 22 to 64 of age and 65 to 165 days of age, respectively. Euthanasia was conducted using methods approved for swine by the American Veterinary Medical Association.⁷ Injectable euthanasia agents were not used in this study. After euthanasia, the diaphragm and liver were collected and placed into a sterile bag and a blood sample was collected using sterile methods. Samples were placed on ice and transported to the Iowa State University Veterinary College and submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISUVDL) to be held in a -20°C freezer until analysis.

Sample analysis

Samples were analyzed for vitamin and mineral concentrations using procedures outlined by ISUVDL (Vitamin A and E in serum – ISUVDL 9.833; Vitamin A in tissue – ISUVDL 9.2429; Vitamin E in tissue – ISUVDL 9.2430; Trace mineral in tissue – ISUVDL 9.2420). Serum and tissue samples were stored at -80°C. Vitamin A and E analyses of both serum and tissues were conducted following the established standard operating procedure (SOP) using internally validated methods. A 0.5 mL aliquot of serum was placed in a 15 mL screw-top tube. Two milliliters of 95% ethanol and 4 mL of 95/5 hexane/chloroform were added. Samples were gently shaken to mix and then centrifuged for 5 minutes at 2000 rpm. Following centrifugation, 2 mL of the hexane/chloroform was transferred to a 7 mL glass vial encased in foil.

One gram of fresh liver for each vitamin A and E analysis was weighed into 50 mL polypropylene tubes and 0.2 g of celite was added. For vitamin A, 5 mL of 0.01% butylated hydroxy toluene in 95% ethanol was added, followed by 1 mL of 50% sodium hydroxide. Samples were placed in an oven at 60°C for 30 minutes, and then chilled for 10 minutes at -20°C. Samples were vortexed at 2000 rpm for 10 minutes, and then centrifuged for 5 minutes at 2000 rpm. Following centrifugation, 1 mL of the hexane/chloroform was transferred to a 7 mL glass vial encased in foil. For vitamin E, 5 mL of 0.01% butylated hydroxytoluene in 95% ethanol was added, followed by 10 mL of 95/5 hexane/chloroform. The sample was vortexed at 2000 rpm for 10 minutes and then centrifuged for 5 minutes at 2000 rpm. Following centrifugation, 5 mL of the hexane/chloroform was transferred to a 7 mL glass vial encased in foil.

Serum and tissue extracts were dried using a nitrogen stream. Serum extracts were dissolved in 250 µL high-performance liquid chromatography (HPLC)-grade methanol while tissue extracts for vitamins A and E were dissolved in 1 mL of 0.09% hydrochloric acid in methanol and 500 µL HPLC-grade methanol, respectively. Following the extraction process, both serum and tissue extracts were analyzed using ultra HPLC. Serum vitamin D was analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS) through Heartland Assays. Samples were processed and analyzed for mineral content following the established SOP on a wet weight basis. A National Institute of Standards and Technology liver standard was included in the run. An in-house laboratory control liver was also used to ensure quality control and to verify instrument accuracy. Serum samples were analyzed for calcium, copper, iron, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, and zinc using inductively coupled plasma mass spectrometry (ICP-MS; Analytik Jena Inc) in CRI mode with hydrogen as the skimmer gas. Analysis of tissues was performed by the same instrument but also included cadmium, cobalt, chromium, and sodium per laboratory method. Standards for elemental analyses were obtained from Inorganic Ventures while 15 mL centrifuge tubes, 50 mL digestion vessels, trace mineral grade nitric acid, and hydrochloric acid were obtained from Fisher Scientific. Serum samples were diluted in 1% nitric acid. Serum samples were transferred to 15 mL tubes in 0.25 mL portions and 4.75 mL of 1% nitric acid was added and then analyzed by ICP-MS. Tissue samples were digested using a microwave digestor by placing 0.5 g samples into 50 mL digestion tubes and adding 10 mL of 70% nitric acid. After digestion, all samples were diluted to 25 mL using 1% nitric acid with 0.5% hydrochloric acid. An additional 1:10 dilution using 1% nitric acid was made and then analyzed by ICP-MS. For quality control, bismuth, scandium, indium, lithium, yttrium, and terbium were used as internal standards for the ICP-MS.

Data analysis

Data were analyzed using SAS (version 9.4; SAS Institute Inc) and were presented as minimum and maximum concentrations with standard error. If the element of analysis was below the detectable limit, the lower limit threshold was divided by 2 to provide a value.⁸ The experimental unit was the animal. Tables were generated to demonstrate the different concentrations of each vitamin and mineral by sample type along with phase of growth.

Results

Vitamins and minerals are stored in different locations of the body and dictates which locations are more ideal for analysis (Table 1). Liver tissue levels of vitamin A, vitamin E, copper, zinc, selenium, and iron were higher than those in serum and diaphragm tissue (Table 2). Vitamin A and E levels were not detectable in the diaphragm tissue at any phase of production (Tables 2, 3, and 4). Most mineral concentrations tended to be higher in tissues (diaphragm and liver) compared to serum. Serum had similar levels of calcium as the liver tissue (Table 4). Median data were provided for each sampling location in Tables 5, 6, and 7. Data from previously published references were compiled for further evaluation of current findings (Table 8).

Discussion

Vitamin and mineral concentrations do differ across production phases and sample types. Some of this variation can be associated with dietary ingredients or immune status, which can influence antioxidant status. In addition, vitamin and mineral analysis conducted in tissues or serum which do not adequately reflect common stores can result in misinterpretation of results. Understanding where vitamins and minerals are stored within the body is important when determining the appropriate sample to assess for concentration status. Iron, copper, manganese, selenium, zinc, and vitamins A, D, and E are stored in the liver. Although predominately stored in adipose tissue, vitamin E is stored in the liver in a limited capacity. Lastly, minerals such as magnesium, phosphorus, and calcium are typically found in the bone. These macrominerals are tightly regulated within the body as evidenced by the maintenance of serum concentrations.

Samples derived from the liver had higher concentrations of certain vitamins and minerals compared to other samples. For example, most of the body’s vitamin A is stored in the liver as retinyl esters and therefore, the liver would be the primary sample site when testing for a vitamin A deficiency.⁹ When sampling, personnel must not only understand the correct sample type to collect, but also the health status of the animal and the manner and condition in which samples are collected to allow for adequate interpretation. For example, minerals such as iron and zinc may be sequestered in the liver during inflammatory or infectious processes resulting in elevated concentrations. Conversely in serum samples, the degree of hemolysis may result in elevated concentrations of iron and potassium but decreased vitamin E concentrations resulting from degradation. Furthermore, some vitamin and mineral concentration ranges are different from the values presented in Puls.^3,4 Serum vitamin A and selenium levels from the current study are lower than previously published values. Previously reported vitamin A ranges were 0.4 to 0.5 ppm in suckling and nursery pigs and 0.25 to 0.40 ppm in sows compared to the current ranges of 0.01 to 0.39 ppm and 0.03 to 0.32 ppm, respectively.⁴ Serum selenium was reported to be 0.14 to 0.30 ppm with no specific age, while the current study documented serum selenium levels to be 0.080 to 0.194 ppm for the suckling/nursery pig and 0.133 to 0.355 ppm for the sow.³ In addition, vitamin D3 concentrations in the current study were lower in the suckling and nursery pigs compared to the published values of 8 to 23 ng/mL and 25 to 30 ng/mL, respectively. Furthermore, more recent work conducted by Flohr et al¹⁰ reported serum vitamin D3 levels in suckling age pigs were between 0.0 and 5.7 ng/mL depending upon maternal dietary consumption and nursery pig serum levels were 22.7 to 30.8 ng/mL. However, the levels in this study were slightly lower than those documented by Flohr et al.¹⁰ Other vitamins and minerals were slightly higher than the referenced values, such as calcium and zinc in the liver. Elevated zinc levels may be associated with feeding higher levels of zinc in the nursery to aid in controlling pathogenic organisms.

This study demonstrates that while some vitamin and mineral concentrations in modern commercial swine are not different than previously published ranges, concentrations in other samples are either higher or lower than previously published work. In addition, previously published reference values did not completely identify the different phases of production. This study demonstrates the need for additional studies focused on the analysis of multiple biological samples from healthy pigs to best determine the appropriate vitamin and mineral ranges for the modern pig.

Implications

Under the conditions of this study:

• Select sample tissue type based on vitamin or mineral of interest.
• Vitamin and mineral levels vary based on age of the animal.
• Further sampling of both healthy and acutely ill animals is needed.

Acknowledgements

The authors would like to acknowledge Iowa Pork Producers Association for funding this project. The authors would also like to thank Olha Ivanytska, Angela Jensen, and Thomas Olsen for analysis of samples.

Conflict of interest

None reported

Disclaimer

Scientific manuscripts published in the Journal of Swine Health and Production are peer reviewed. However, information on medications, feed, and management techniques may be specific to the research or commercial situation presented in the manuscript. It is the responsibility of the reader to use information responsibly and in accordance with the rules and regulations governing research or the practice of veterinary medicine in their country or region.

References

1. Flohr JR, DeRouchey JM, Woodworth JC, Tokach MD, Goodband RD, Dritz SS. A survey of current feeding regimens for vitamins and trace minerals in the US swine industry. J Swine Health Prod. 2016;24(6):290-303.

2. National Research Council. Nutrient Requirements of Swine. 11^th ed. National Academy Press; 2012.

3. Puls R. Mineral Levels in Animal Health: Diagnostic Data. 2^nd ed. Sherpa International; 1994.

4. Puls, R. Vitamin Levels in Animal Health. 2^nd ed. Sherpa International; 1994.

5. Arnold J, Madson DM, Ensley SM, Goff JP, Sparks, C, Stevenson GW, Crenshaw T, Wang C, Horst RL. Survey of serum vitamin D status across stages of swine production and evaluation of supplemental bulk vitamin D premixes used in swine diets. J Swine Health Prod. 2015;23(1):28-34.

*6. National Pork Board. Pork Quality Assurance Plus Handbook. Version 4.0. National Pork Board; 2019.

*7. American Veterinary Medicine Association. Guidelines for Euthanasia of Animals: 2020 Edition. American Veterinary Medical Association; 2020. Accessed January 5, 2022. https://www.avma.org/sites/default/files/2020-01/2020-Euthanasia-Final-1-17-20.pdf

8. Cohen MA, Ryan PB. Observations less than the analytical limit of detection: A new approach. JAPCA. 1989;39(3):328-329. https://doi.org/10.1080/08940630.1989.10466534

9. Thorbjarnarson T, Drummond JC. Conditions influencing the storage of vitamin A in the liver. Biochem J. 1928;32(1):5-9. https://doi.org/10.1042/bj0320005

10. Flohr JR, Woodworth JC, Bergstrom JR, Tokach MD, Dritz SS, Goodband RD, DeRouchey JM. Evaluating the impact of maternal vitamin D supplementation: I. Sow performance, serum vitamin metabolites, and neonatal muscle characteristics. J Anim Sci. 2016;94:4629-4642. https://doi.org/10.2527/jas.2016-0409

* Non-refereed references.