The Effect of Different Bone and Analytical Methods on the The Effect of Different Bone and Analytical Methods on the Assessment of Bone Mineralization to Dietary Phosphorus, Assessment of Bone Mineralization to Dietary Phosphorus, Phytase, and Vitamin D in Finishing Pigs Phytase, and Vitamin D in Finishing Pigs

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Introduction
In recent years, the occurrence of lameness in growing pigs has increased, leading to an increased incidence of removals and mortality.Lameness is defined as impaired movement or deviation from normal gait.There are many factors that can contribute to lameness, including infectious disease, genetic and conformational deficiencies, impaired physiological development of articular surfaces within joints, and skeletal mineralization.Metabolic bone disease is a common cause of lameness in swine production and can be caused by inappropriate levels of essential vitamins or minerals.
An important component of lameness evaluation is the histopathological examination of tissues including articular surfaces, synovium, and growth plates.While these tissues can provide an indication of a variety of pathological processes, such as metabolic bone disease induced by vitamin D deficiency (Madson et al., 20128 ), they do not always result in a definitive diagnosis.Ancillary diagnostic tests that can be used in a workup of clinical lameness include measures of bone mineralization such as bone ash and serum concentrations of Ca, P, and vitamin D.
Serum 25(OH)D has been the standard for the determination of vitamin D status within swine; however, there has been recent speculation that the activated form, 1,25(OH) 2 D, may provide additional benefits in assessing vitamin D status (Hurst et al., 2020 9 ).There is limited information regarding serum 1,25(OH) 2 D levels in swine under a variety of feeding conditions.
Bone ash is an established method for measuring bone mineralization.However, questions remain about whether defatted or non-defatted bone ash is the more accurate method.Wensley et al. (2020 10 ) compared defatted and non-defatted processing methods to determine their effects on the ability to detect treatment differences.The authors observed that either non-defatted or defatted bone processing methods can be used to determine the bone ash weight and percentage bone ash to assess bone mineralization in nursery pigs although bone ash percentage is greater for the defatted method compared to the non-defatted method.
A non-invasive technique for evaluating mineralization status is the collection and analysis of urine samples.Urinary Ca and P can be measured, and when put in a ratio to creatinine to standardize for potential differences in urine volume, has been shown to be a promising indicator of mineral status.On their own, the presented assays are

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Kansas State University Agricultural Experiment Station and Cooperative Extension Service limited in their ability to diagnose metabolic bone disease and identify the cause.However, when evaluated collectively, the assays can result in a better diagnosis and can lead to intervention.Therefore, the objective of this study was to evaluate the effect of different bone and analytical methods on the assessment of bone mineralization responses to dietary P, phytase, and vitamin D in finishing pigs.

Procedures
The Kansas State University Institutional Animal Care and Use Committee approved the protocol used in this experiment.The trial was conducted at a commercial research facility owned and operated by New Fashion Pork (Jackson, MN).

Animal and diets
A total of 882 pigs (PIC TR4 × (Fast LW × PIC L02); initially 73.2 ± 0.7 lb) were used in a 112-d trial.Pens of pigs were fed common diets from weaning until the start of the study.Calcium and P levels for all common diets were at or above NRC requirement estimates.Pens of pigs (20 pigs per pen) were then randomized to 1 of 5 dietary treatments in a completely randomized design with 9 pens per treatment.The dietary treatments were: 1) STTD P fed at 80% of the NRC requirement estimate (deficient); 2) STTD P fed at 100% of the requirement (NRC requirement) using monocalcium phosphate; 3) STTD P fed at 100% of the NRC requirement with phytase included; 4) STTD P fed at 128% of the NRC requirement (industry level) including 0.14% release from phytase; and 5) diet 4 with additional 2,000 IU/kg of vitamin D equivalency from 25(OH)D 3 (HyD).All diets were manufactured in meal form at the New Fashion Pork feed mill in Estherville, IA (Table 1 and 2).For treatments 1 and 2, P levels were met by only using monocalcium phosphate.All other treatment diets had 2,000 FYT/kg of phytase included in the diet to meet the desired STTD P levels, with an assumed STTD P release of 0.14%.Vitamin D was included in all diets via the vitamin premix at a rate of 992 IU/kg.Treatment 5 had an additional 2,000 IU/kg of vitamin D equivalency from 25(OH)D 3 HyD, (DSM Nutritional Products, Parsippany, NJ).All diets were formulated to an analyzed Ca:analyzed P ratio of 1.20:1.Experimental diets were fed for 112 d.
Pens of pigs were weighed, and feed disappearance was measured every 14 d to determine ADG, ADFI, and F/G.On d 112, 1 pig per pen (9 pigs per treatment) were euthanized and used for the analysis of bones, blood, and urine.The right and left metacarpal, fibula, 2nd rib, and 10th rib were collected from each pig for a total of 8 bones per pig.All bones were analyzed using dual-energy X-ray absorptiometry (DEXA) scans, then bone density, breaking strength, bone ash, and bone Ca and P were determined.Histologic evaluation of hematoxylin and eosin (H&E) stained sections of the 2nd rib, 10th rib, and fibula was performed by three diagnostic pathologists.Hematoxylin stains the cell nuclei a purplish blue and eosin stains the extracellular matrix and cytoplasm pink, with other structures taking on different shades, hues, and combinations of these colors.Bones were scored for lesions of failure of endochondral ossification of the physis and microscopic fractures (infractions).Medullary trabeculae and cortical bone thickness was measured.Ten mL of blood was collected to measure serum chemistry, and 10 mL of urine was collected directly from the bladder to measure Ca, P, and creatinine.

Statistical analysis
Growth performance, blood, and urine analysis were analyzed as a completely randomized design for one-way ANOVA using the lm function from the lm package in R version 3.5.1 (07-02-2018) with pen considered as the experimental unit and treatment as a fixed effect.A Tukey multiple comparison adjustment was used when appropriate.Differences between treatments were considered significant at P ≤ 0.05 and marginally significant at 0.05 < P ≤ 0.10.
For the statistical analysis for bones, data were analyzed as a completely randomized design for two-way ANOVA using the lmer function from the lme4 package in R (version 3.5.1 (2018-07-02), R Foundation for Statistical Computing, Vienna, Austria), with pen considered as the experimental unit, pen as a random effect, and treatment, bone, and the associated interaction included as fixed effects.Results were considered significant at P ≤ 0.05 and marginally significant at 0.05 < P ≤ 0.10.

Growth performance
For final body weight and overall growth performance, there were no differences between the dietary treatments (P > 0.10; Table 3).

Serum analysis
For serum Ca, pigs fed 80% of the NRC requirement for P had increased circulating Ca compared to pigs fed NRC P levels with phytase (P < 0.05), with pigs fed industry levels of P, and NRC levels of P with no phytase intermediate.Pigs fed NRC levels of P with no phytase had increased serum P levels compared to pigs fed industry levels of P with HyD included in the diet (P < 0.05), with the other 3 treatments intermediate.Pigs fed the diets containing 25-hydroxyvitamin D 3 from HyD had increased (P < 0.05) circulating 25-hydroxyvitamin D 3 compared to pigs fed industry levels of vitamin D in the diet.25-hydroxyvitamin D 3 is the precursor to the active form of vitamin D and undergoes a bioconversion process within the kidney to form the active metabolite, 1,25-dihydroxyvitamin D 3.There was no difference between treatments for circulating 1,25-dihydroxyvitamin D 3 (P = 0.147).For urine Ca:creatinine, there was a tendency for a difference between treatments (P = 0.081), but no significant mean separation was observed between treatments (P > 0.05).For urine P:creatinine levels, there was no difference between treatments (P = 0.378).

Bone analysis
There were no treatment differences for defatted bone ash percent, percentage non-defatted bone ash, non-defatted bone ash grams, or percentage and grams of bone ash Ca and P (P > 0.10).For defatted bone ash grams, pigs fed industry levels of P had numerically increased bone ash grams compared to pigs fed the other diets, but no significant mean separation was observed (P > 0.05).The fibula had greater percentage defatted bone ash compared to the 2nd and 10th ribs (P < 0.05), while the metacarpal had the lowest (P < 0.05) defatted bone ash percentage (Table 4).In non-defatted bones, percentage bone ash of the fibulas were greater compared to the 2nd rib and metacarpal, and the 2nd rib and 10th rib both had greater (P < 0.05) percentage bone ash than the metacarpal.

Swine Day 2022 Kansas State University Agricultural Experiment Station and Cooperative Extension Service
The response to treatment for bone density and bone mineral content was dependent upon the bone analyzed (density interaction, P = 0.053; mineral interaction, P = 0.078; Table 5).There were no treatment differences for bone density and bone mineral content for metacarpals, fibulas, and 2nd rib (P > 0.05).For 10th rib bone density, pigs fed industry levels of P and vitamin D had increased (P < 0.05) bone density compared to pigs fed NRC levels with phytase, with pigs fed deficient P, NRC P with no phytase, and extra vitamin D from HyD intermediate.Pigs fed extra vitamin D from 25-hydroxyvitamin D 3 (from HyD) had increased bone mineral content compared to pigs fed deficient P and NRC levels of P with phytase, with pigs fed industry P and vitamin D, and NRC P with monocalcium intermediate.
For histopathological analysis of bones, there were no treatment differences for infraction score, trabeculae bone thickness, and cortical bone thickness (P > 0.10).For physeal score, there was a significant difference between treatments (P = 0.040), but no significant mean separation was observed.Numerically, pigs fed 80% of the NRC P requirement and those fed industry levels of P with industry levels of vitamin D had an increased physeal score, representing more lesions of endochondral ossification.
In summary, bone density and bone mineral content responses varied depending on the bone.The difference between bone ash procedures was more apparent than the differences between treatments.Differences in bone density and mineral content in response to P and vitamin D were most apparent with the 10th ribs.

Brand names appearing in this publication are for product identification purposes only.
No endorsement is intended, nor is criticism implied of similar products not mentioned.Persons using such products assume responsibility for their use in accordance with current label directions of the manufacturer.Vitamin premix contained 992 IU/kg of vitamin D 3 in the diet when the premix was included in the diet at 0.15% and 827 IU/kg of vitamin D 3 in the diet when the premix was included in the diet at 0.13%.
3 Ronozyme HiPhos 2700 (DSM Nutritional Products, Parsippany, NJ) was included at 2,000 FYT/kg with an assumed release of 0.14 STTD P when included in the diet at 0.08%.When phytase was provided in the diet at 0.03% it included 770 FYT/kg with an assumed release of 0.12 STTD P.  Vitamin premix contained 661 IU/kg of vitamin D 3 in the diet when the premix was included in the diet at 0.10% and 496 IU/kg of vitamin D 3 in the diet when the premix was included in the diet at 0.08%.
3 Ronozyme HiPhos 2700 (DSM Nutritional Products, Parsippany, NJ) was included at 2,000 FYT/kg with an assumed release of 0.14 STTD P when included in the diet at 0.08%.When phytase was provided in the diet at 0.03% it included 770 FYT/kg with an assumed release of 0.12 STTD P. When phytase was provided in the diet at 0.02% it included 490 FYT/kg with an assumed release of 0.10 STTD P.

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Kansas State University Agricultural Experiment Station and Cooperative Extension Service  HyD provided 2,000 IU/kg of vitamin D equivalency from 25(OH)D 3 to the diet (DSM Nutritional Products, Parsippany, NJ).
5 Serum Ca and P were measured at the Iowa State University Veterinary Diagnostic Lab (Ames, IA) as a part of the large animal complete profile.The vitamin D serum analysis was conducted at Heartland Assays (Ames, IA). 6 One pig per pen (9 pigs per treatment) were euthanized and the left and right metacarpal, fibula, 2nd rib, and 10th rib were collected to determine bone ash weight and percentage bone ash.All bones were cleaned of tissue and then placed in Soxhlet extractors containing petroleum ether for 7 days as a means of removing water and fat.Bones were then dried at 221°F for 7 days and then ashed in a muffle furnace at 1,112°F for 24 h.

7
One pig per pen (9 pens per treatment) were euthanized and the left and right metacarpal, fibula, 2nd rib, and 10th rib were collected to determine bone ash weight and percentage bone ash.All bones were cleaned of tissue and then dried at 221°F for 7 days and then ashed in a muffle furnace at 1,112°F for 24 h.

8
After bone ash was completed, the ash was digested and sent to the K-State Research and Extension Soil Testing Laboratory, Manhattan, KS, for analysis of Ca and P via ICP.9 The left fibula, 2nd rib, and 10th rib were taken to Iowa State University VDL for analysis of histopathology.Each bone was scored on a scale of 0 to 3 on the severity of fractures on the physis of each growth plate.

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Kansas State University Agricultural Experiment Station and Cooperative Extension Service One pig per pen (9 pigs per treatment) were euthanized and the left and right metacarpal, fibula, 2nd rib, and 10th rib were collected to determine bone ash weight and percentage bone ash.All bones were cleaned of tissue and then placed in Soxhlet extractors containing petroleum ether for 7 days as a means of removing water and fat.Bones were then dried at 221°F for 7 days and then ashed in a muffle furnace at 1,112°F for 24 h.
3 One pig per pen (9 pens per treatment) were euthanized and the left and right metacarpal, fibula, 2nd rib, and 10th rib were collected to determine bone ash weight and percentage bone ash.All bones were cleaned of tissue and then dried at 221°F for 7 days and then ashed in a muffle furnace at 1,112°F for 24 h.
After bone ash was completed, the ash was digested and sent to the K-State Research and Extension Soil Testing Laboratory (Manhattan, KS) for analysis of Ca and P via ICP.

5
The left fibula, 2nd rib, and 10th rib were taken to the Iowa State University Veterinary Diagnostic Lab (Ames, IA) for analysis of histopathology.Each bone was scored on a scale of 0 to 3 on the severity of fractures on the physis of each growth plate.One pig per pen (9 pens per treatment) were euthanized and the left and right metacarpal, fibula, 2nd rib, and 10th rib were collected to determine bone ash weight and percentage bone ash.All bones were cleaned of tissue and then dried at 221°F for 7 days and then ashed in a muffle furnace at 1,112°F for 24 h.Bone × treatment, P = 0.542.5 Bone × treatment, P > 0.10.

6
One pig per pen (9 pigs per treatment) were euthanized and the left and right metacarpal, fibula, 2nd rib, and 10th rib were collected to determine bone ash weight and percentage bone ash.All bones were cleaned of tissue and then placed in Soxhlet extractors containing petroleum ether for 7 days as a means of removing water and fat.Bones were then dried at 221°F for 7 days, and then ashed in a muffle furnace at 1,112°F for 24 h.Bone × treatment, P = 0.702.

7
The left fibula, 2nd rib, and 10th rib were taken to the Iowa State University Veterinary Diagnostic Lab (Ames, IA) for analysis of histopathology.Each bone was scored on a scale of 0 to 3 on the severity of fractures on the physis of each growth plate.Bone × treatment, P = 0.174.

4
HyD provided 2,000 IU/kg of vitamin D equivalency from 25(OH)D 3 to the diet (DSM Nutritional Products, Parsippany, NJ).Swine Day 2022Kansas State University Agricultural Experiment Station and Cooperative Extension Service

2
HyD provided 2,000 IU/kg of vitamin D equivalency from 25(OH)D 3 to the diet (DSM Nutritional Products, Parsippany, NJ).3 Bone density was measured on each bone based on Archimedes' principle.Bone × treatment, P = 0.053. 4

Table 1 .
Diet composition, phases 1 and 2 (as-fed basis) 1 Kansas State University Agricultural Experiment Station and Cooperative Extension Service1 Phase 1 was fed from 80 to 120 lb.Phase 2 was fed from 120 to 160 lb.2

Table 3 .
Effect of STTD P, phytase, and vitamin D on growth performance, serum, and bone analysis of finishing pigs1,2 Kansas State University Agricultural Experiment Station and Cooperative Extension Service

Table 3 .
Effect of STTD P, phytase, and vitamin D on growth performance, serum, and bone analysis of finishing pigs1,2 abc Means within a row with different superscripts differ (P < 0.05). 1 A total of 882 pigs were used in a 112-d finishing trial with 20 pigs per pen and 9 pens per treatment.Pigs were placed on experimental diets at approximately 73 lb. 2 BW = body weight.ADG = average daily gain.ADFI = average daily feed intake; F/G = feed efficiency.3National Research Council.2012.Nutrient Requirements of Swine: Eleventh Revised Edition.Washington, DC: The National Academies Press.https://doi.org/10.17226/13298.4

Table 4 .
Effect of STTD P, phytase, and vitamin D on bone analysis of finishing pigs 1 2

Table 5 .
Interactive effects of STTD P, phytase, and vitamin D on bone analysis Kansas State University Agricultural Experiment Station and Cooperative Extension Service

Table 5 .
Interactive effects of STTD P, phytase, and vitamin D on bone analysis National Research Council.2012.Nutrient Requirements of Swine: Eleventh Revised Edition.Washington, DC: The National Academies Press.https://doi.org/10.17226/13298.
abc Means within a row with different superscripts differ (P < 0.05). 1