The Effect of Bone and Analytical Methods on the Assessment of The Effect of Bone and Analytical Methods on the Assessment of Bone Mineralization Response to Dietary Phosphorus, Phytase, Bone Mineralization Response to Dietary Phosphorus, Phytase, and Vitamin D in Nursery Pigs and Vitamin D in Nursery 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 is often caused by inappropriate levels of essential vitamins or minerals.
Appropriate mineral supply is essential for the bone structure to become sufficiently strong and to ensure optimal bone mass accumulation during growth.Currently, it is a common practice to feed slightly higher Ca and P levels in swine diets than recommended by NRC. 6 Multiple experiments have showed improved growth performance and bone mineralization when feeding levels of P greater than NRC 6 recommendations when expressed on a dietary percentage basis.
An important component of lameness evaluations 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, 7 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 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 benefit in assessing vitamin D status. 8 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 whether defatted or non-defatted bone ash is the more accurate method.Wensley et al. 9 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.Urine samples can be collected and analyzed to evaluate the mineralization status.This sampling technique is non-invasive and can be used while the animal is alive.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 status. 10 On their own, the presented assays are limited in their ability to diagnose metabolic bone disease and identify the cause.However, when evaluated collectively, the assays can result in a diagnosis with an identified cause that 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 nursery pigs.

Procedures
The Kansas State University Institutional Animal Care and Use Committee approved the protocol used in this experiment.This experiment was conducted at the Kansas State University Swine Teaching and Research Center in Manhattan, KS.Each pen (4 × 4 ft) was equipped with a 4-hole, dry self-feeder, and a nipple waterer to provide ad libitum access to feed and water.

Animals and diets
A total of 350 pigs (DNA 241 × 600; initially 26.2 ± 1.23 lb) were used in a 28-d trial.
Pigs were weaned at approximately 21 d of age and placed in pens of 5 pigs based on initial weight and gender.Common phase 1 and 2 diets were fed for 24 d after weaning.Dietary vitamin D, calcium, and phosphorus levels during phases 1 and 2 were at or above the NRC recommendations and resulted in mean serum levels of 14.6 ng/mL of 25-hydroxyvitamin D 3 , 10.5 mg/dl of Ca, and 9.3 mg/dl of P at d 24 post-weaning.On d 24 after weaning, which was considered d 0 of the trial, pens of pigs were randomly allotted to 1 of 6 dietary treatments with 10 replications per treatment.The dietary treatments were: 1) 0.19% STTD P (deficient); 2) 0.33% STTD P (NRC requirement) using monocalcium phosphate; 3) 0.33% STTD P including 0.14% release from phytase; 4) 0.44% STTD P (industry level) using monocalcium phosphate, phytase, no vitamin D; 5) diet 4 with vitamin D (1,653 IU/kg); and 6) diet 5 with additional 2,000 IU/kg 25(OH)D 3 (HyD) (DSM Nutritional Products, Parsippany, NJ).All diets were manufactured in meal form at Hubbard Feeds in Beloit, KS (Table 1).Treatment 1 had an STTD P level of 0.19%, which was 57% of the NRC requirement for pigs of this weight range.Treatments 2 and 3 had an STTD P level of 0.33%, which was 100% of the NRC requirement for this weight range.Treatments 4, 5, and 6 had an STTD P level of 0.44%, which was approximately 133% of the NRC requirement for this weight range.For treatments 1 and 2, STTD P levels were met by only using monocalcium phosphate.All other treatments 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 3 was included in the diet for treatments 1, 2, 3, 5, and 6 via the same vitamin premix used during the common phase 1 and 2 periods to provide 1,653 IU/kg.Treatment 4 had a special vitamin premix that had no vitamin D. Treatment 6 had 1,653 IU/kg of vitamin D 3 from the vitamin premix and an additional 2,000 IU/kg of vitamin D 3 from HyD.All diets were formulated to an analyzed Ca:analyzed P ratio of 1.20:1.Experimental diets were fed for 28 d.
Pens of pigs were weighed, and feed disappearance was measured every 7 d to determine ADG, ADFI, and F/G.On d 28, 8 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, bone density, breaking strength, bone ash, and bone Ca and P were determined.Histologic evaluation of hematoxylin and eosin (H&E stained) sections of 2nd rib, 10th rib, and fibula was performed by three blinded 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 trabecular 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
Data were analyzed as a randomized complete block design for one-way ANOVA using the lmer function from the lme4 package in R version 3.5.1 (2018-07-2) with pen considered the experimental unit, body weight as a blocking factor, and treatment as a fixed effect.Linear and quadratic effects between treatments were measured based on STTD P % in the diet.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.

Serum analysis
For serum Ca, there was a tendency for a difference between treatments, but no significant mean separation was observed (P = 0.096).For serum P, pigs fed 0.19% P had lower levels compared to pigs fed 0.33% and 0.44% STTD P (P < 0.05).Pigs fed no vitamin D in the diet had the lowest (P < 0.05) circulating 25-hydroxyvitamin D 3 , and pigs fed HyD in the diet had the greatest (P < 0.05), and pigs fed only 1,653 IU/kg of vitamin D 3 intermediate.The compound 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. Pigs fed no vitamin D in the diet had the lowest circulating level of 1,25-dihydroxyvitamin D 3 but pigs fed 0.19% STTD P had the highest (P < 0.05).Pigs fed 1,653 IU/kg of vitamin D with 0.33% STTD P, 0.44% STTD P, and added HyD were intermediate.Pigs fed P deficient diets had increased Ca:creatinine levels (P < 0.001) in the urine compared to pigs fed diets with industry levels of P, with pigs fed NRC P with no phytase being intermediate.For P:creatinine levels, pigs fed no vitamin D, and excess vitamin D in the diet from HyD had increased P levels in the urine (P = 0.004) compared to pigs fed the deficient P levels, with all other treatments being intermediate.

Bone analysis
The response to treatment for bone density and ash was dependent upon the bone that was analyzed (density × bone interaction, P = 0.044; non-defatted bone ash × bone interaction, P = 0.060; defatted bone ash × bone interaction, P = 0.068; Table 4).Pigs fed 0.19% STTD P had decreased (P < 0.05) bone density and ash (non-defatted and defatted) for all bones compared to 0.44% STTD P, with 0.33% STTD P generally intermediate or similar to 0.44% STTD P. Pigs fed 0.44% STTD P with no vitamin D had the greatest (P < 0.05) non-defatted fibula ash compared to all treatments other than 0.44% STTD P with added HyD.Pigs fed the three diets with 0.44% STTD P had greater (P < 0.05) defatted 2nd rib ash compared to pigs fed 0.19% STTD P or 0.33% STTD P with no phytase.
For bone ash content, there was no difference between treatments for the percentage of Ca and P in bone ash regardless of the level of P, phytase, and vitamin D in the diet (P > 0.10).Pigs fed 0.19% STTD P had reduced grams of P and Ca in the bone ash compared to pigs fed 0.33% and 0.44% STTD P (P < 0.05).This means the bone increases in mineralization when adequate levels of Ca and P are fed, but the ratio of Ca and P in the bone mineral remains unchanged, because the bone will only increase in size when adequate or excess levels of Ca and P are fed.
For histopathology, the 10th rib had more lesions of endochondral ossification and infraction than the 2nd rib or fibula (P < 0.001; Table 3).Pigs fed a P deficient diet had significantly higher scores indicating failure of endochondral ossification, more infractions, and thinner medullary trabecular bone compared to other treatment groups.
For defatted bone ash percent, the 2nd rib had the lowest, fibulas highest, and the 10th rib and metacarpal were intermediate (P < 0.001).For non-defatted bone ash percent, the bone ash percent changed by bone as the metacarpal was the lowest, then increased for the fibula, 2nd rib, and 10th rib, respectively (P < 0.001).For histopathology, the 10th rib had the highest physeal score, infraction score, fibrosis score, and lowest trabeculae bone thickness compared to the fibula and 2nd rib (P < 0.001).
In summary, bone density and ash responses varied depending on the bone analyzed.Differences in bone density and ash in response to P and vitamin D were most apparent with fibulas and 2nd ribs.The difference between bone ash procedures was more apparent than the differences between treatments.For histopathology, 10th ribs were more sensitive than 2nd ribs or fibulas for detection of lesions.

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.Eight 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.Eight 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 dried at 221°F for 7 days and then ashed in a muffle furnace at 1,112°F for 24 h.

Swine Day 2022
Kansas State University Agricultural Experiment Station and Cooperative Extension Service One pig per pen (8 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 (8 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.

2 BW 5 Serum
= body weight; ADG = average daily gain; ADFI = average daily feed intake; F/G = feed efficiency.3STTD P, quadratic, P < 0.05. 4 STTD P, linear, P < 0.05.Ca and P were measured at the Iowa State University College of Veterinary Medicine Veterinary Diagnostic Laboratory (Ames, IA) as a part of the large animal complete profile.The vitamin D serum analysis was conducted at Heartland Assays (Ames, IA).

Table 1 .
Diet composition (as-fed basis) 1 of each diet was collected from each feeder at 3 different time points throughout the study.Samples were stored at -4°F until they were homogenized, subsampled, and submitted to the K-State Research and Extension Soil Test Laboratory (Manhattan, KS) for proximate analysis and Ca and P.
Kansas State University Agricultural Experiment Station and Cooperative Extension Service1 Diets were fed to pigs from approximately 26 to 65 lb BW. 2 Vitamin premix contained 1,653 IU/kg of vitamin D 3 in the diet when the premix was included in the diet at 0.25%.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. 4 HyD provided an additional 2,000 IU/kg 25(OH)D 3 to the diet.5 A representative sample Kansas State University Agricultural Experiment Station and Cooperative Extension Service

Table 2 .
Effect of STTD P, phytase, and vitamin D on growth performance, serum, and bone analysis of nursery pigs 1 Kansas State University Agricultural Experiment Station and Cooperative Extension Service

Table 2 .
Effect of STTD P, phytase, and vitamin D on growth performance, serum, and bone analysis of nursery pigs 1 abcMeans within a row with different superscripts differ (P < 0.05). 1 A total of 350 pigs were used in a 28-d nursery trial with 5 pigs per pen and 10 pens per treatment.Pigs were placed on experimental diets 24 days postweaning (~19 d of age).

Table 3 .
Effect of STTD P, phytase, and vitamin D on bone analysis of nursery pigs 1 2

Table 4 .
Interactive effects of STTD P, phytase, and vitamin D on bone analysis Kansas State University Agricultural Experiment Station and Cooperative Extension Service

Table 4 .
Interactive effects of STTD P, phytase, and vitamin D on bone analysis Kansas State University Agricultural Experiment Station and Cooperative Extension Service

Table 4 .
Interactive effects of STTD P, phytase, and vitamin D on bone analysis abcMeans within a row with different superscripts differ (P < 0.05).1