An evaluation of supplemental Vitamin D3 on growth performance of pigs pre- and postweaning, nursery feed preference, and serum 25(OH)D3

Three experiments were conducted to evaluate the effects of supplementing different concentrations and sources of vitamin D3 on pig performance, feed preference, and serum 25(OH)D3. In Exp. 1, a total of 398 barrows from 80 litters (PIC 1050, initially 7 d of age) were used in a 38-d study in a 2 Ã— 2 factorial to determine the effects of vitamin D3 suppleÂ¬mentation from either a single oral dose or from high levels of vitamin D3 in early nursÂ¬ery diets on pig performance and serum 25(OH)D3. On d 7 after birth, matched sets of pigs within litters were allotted to 1 of 2 oral dosages (none or 40,000 IU vitamin D3) in a randomized complete block design. Pigs were weighed at d 7 and at weaning (d 21). Following weaning, a subset of 300 barrows were used from d 21 to 45 to determine the effects of the previously administered oral vitamin D3 and 2 levels of dietary vitamin D3 (625 or 6,250 IU/lb; 0.80% Ca and 0.63% available P) from weaning to d 31 on pig growth and serum 25(OH)D3. A common diet containing 625 IU/lb of vitamin D3 (0.70% Ca and 0.47% available P) was fed from 31 to 45 d of age. No dose Ã— diet interÂ¬actions (P > 0.09) were observed. Serum 25(OH)D3 increased (P < 0.01) on d 21 and tended to increase on d 31 after dosing pigs with oral vitamin D3 prior to weaning. On d 31, serum concentrations increased with increasing dietary vitamin D3 levels (P < 0.01). Weaning weight was not influenced (P > 0.17) by the oral dose of vitamin D3. Supplementing vitamin D3 by either dose or diet did not influence (P > 0.23) nursÂ¬ery performance. In Exp. 2, a total of 864 pigs (PIC TR4 Ã— FAST ADN, initially 21 d of age) were used in a 30-d study to determine the effects of water supplementation of vitamin D3 on nursery growth performance and serum 25(OH)D3. Upon arrival to the nursery (d 0), pigs were allocated to pens and pens were randomly allotted to 1 of 2 water vitamin D3 supplementation treatments (none or 4,000,000 IU/gal). There were 24 pigs/pen and 18 pens/treatment. Pigs were provided the water supplementation treatments from d 0 to 10. From d 10 to 30, pigs were administered water with no supplemental vitamin D3. Common diets were fed throughout the study and were formulated to contain 1,000 IU/lb added vitamin D3. Twelve pigs per treatment were randomly selected to be bled on d 0, 10, 20, and 30 to determine serum 25(OH)D3 concentrations. Water supple-mentation of vitamin D3 increased (P < 0.01) serum 25(OH)D3 concentrations on d 10, 20, and 30 of the study but did not affect (P > 0.15) nursery growth performance. In Exp. 3, 72 pigs (PIC 327 Ã— 1050, initially 28 d of age) were used in 2 14-d feed preference comparisons to determine whether pigs discriminate in their choice of feeds containing different concentrations of vitamin D3. On d 0, pigs were weighed and allotted to pens based on BW with 6 pigs/pen and 6 pens per feed comparison. The first preference comparison was between diets containing either 625 (control) or 6,250 IU/ lb vitamin D3, and the second comparison was between diets containing 625 (control) or 20,000 IU/lb vitamin D3. Total pen feed intake was measured, and intake of each diet was expressed as a percentage of total intake. The percentage of feed intake did not differ (P > 0.14) between the control diet and the diet containing 6,250 IU/lb, but pigs chose to consume a greater percentage (P < 0.01) of the control diet (77%) than the diet containing 20,000 IU/lb of vitamin D3. These experiments demonstrated that providing high levels of vitamin D3 in an oral dosage, in the water, or in feed increased serum 25(OH)D3; however, preweaning and nursery pig growth performance was not influenced by elevating vitamin D3 above normal dietary levels.; Swine Day, Manhattan, KS, November 15, 2012 This Research Report article is available in Kansas Agricultural Experiment Station Research Reports: https://newprairiepress.org/ kaesrr/vol0/iss10/1267


Summary
Three experiments were conducted to evaluate the effects of supplementing different concentrations and sources of vitamin D 3 on pig performance, feed preference, and serum 25(OH)D 3 .
In Exp. 1, a total of 398 barrows from 80 litters (PIC 1050, initially 7 d of age) were used in a 38-d study in a 2 × 2 factorial to determine the effects of vitamin D 3 supplementation from either a single oral dose or from high levels of vitamin D 3 in early nursery diets on pig performance and serum 25(OH)D 3 .On d 7 after birth, matched sets of pigs within litters were allotted to 1 of 2 oral dosages (none or 40,000 IU vitamin D 3 ) in a randomized complete block design.Pigs were weighed at d 7 and at weaning (d 21).Following weaning, a subset of 300 barrows were used from d 21 to 45 to determine the effects of the previously administered oral vitamin D 3 and 2 levels of dietary vitamin D 3 (625 or 6,250 IU/lb; 0.80% Ca and 0.63% available P) from weaning to d 31 on pig growth and serum 25(OH)D 3 .A common diet containing 625 IU/lb of vitamin D 3 (0.70% Ca and 0.47% available P) was fed from 31 to 45 d of age.No dose × diet interactions (P > 0.09) were observed.Serum 25(OH)D 3 increased (P < 0.01) on d 21 and tended to increase on d 31 after dosing pigs with oral vitamin D 3 prior to weaning.On d 31, serum concentrations increased with increasing dietary vitamin D 3 levels (P < 0.01).Weaning weight was not influenced (P > 0.17) by the oral dose of vitamin D 3 .Supplementing vitamin D 3 by either dose or diet did not influence (P > 0.23) nursery performance.
In Exp. 2, a total of 864 pigs (PIC TR4 × FAST ADN, initially 21 d of age) were used in a 30-d study to determine the effects of water supplementation of vitamin D 3 on nursery growth performance and serum 25(OH)D 3 .Upon arrival to the nursery (d 0), pigs were allocated to pens and pens were randomly allotted to 1 of 2 water vitamin D 3

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supplementation treatments (none or 4,000,000 IU/gal).There were 24 pigs/pen and 18 pens/treatment.Pigs were provided the water supplementation treatments from d 0 to 10. From d 10 to 30, pigs were administered water with no supplemental vitamin D 3 .Common diets were fed throughout the study and were formulated to contain 1,000 IU/lb added vitamin D 3 .Twelve pigs per treatment were randomly selected to be bled on d 0, 10, 20, and 30 to determine serum 25(OH)D 3 concentrations.Water supplementation of vitamin D 3 increased (P < 0.01) serum 25(OH)D 3 concentrations on d 10, 20, and 30 of the study but did not affect (P > 0.15) nursery growth performance.
In Exp. 3, 72 pigs (PIC 327 × 1050, initially 28 d of age) were used in 2 14-d feed preference comparisons to determine whether pigs discriminate in their choice of feeds containing different concentrations of vitamin D 3 .On d 0, pigs were weighed and allotted to pens based on BW with 6 pigs/pen and 6 pens per feed comparison.The first preference comparison was between diets containing either 625 (control) or 6,250 IU/ lb vitamin D 3 , and the second comparison was between diets containing 625 (control) or 20,000 IU/lb vitamin D 3 .Total pen feed intake was measured, and intake of each diet was expressed as a percentage of total intake.The percentage of feed intake did not differ (P > 0.14) between the control diet and the diet containing 6,250 IU/lb, but pigs chose to consume a greater percentage (P < 0.01) of the control diet (77%) than the diet containing 20,000 IU/lb of vitamin D 3 .

Introduction
Vitamin D is a fat-soluble steroid known for its role in the absorption and homeostasis of Ca and P in the body.The two main forms of vitamin D are vitamin D 2 (ergocalciferol) and vitamin D 3 (cholecalciferol).Previous research has shown that pigs discriminate in the metabolism of these two forms and more readily convert vitamin D 3 to its circulating metabolite 25(OH)D 3 .This metabolite of vitamin D is the main circulating form in the blood and acts as a clinically useful marker for vitamin D status.In recent years, more focus has been placed on vitamin D because of documented cases where it has been absent from premixes fed to pigs.In these cases, large percentages of pigs have reportedly developed metabolic bone disease, which is categorized as disturbances related to bone formation and remodeling and can lead to bone breakages and clinical symptoms of rickets.Previous work conducted at Kansas State University (Flohr et al.,  2011 7 ) has shown that supplementation of vitamin D 3 (40,000 or 80,000 IU) given in a single oral dose after birth can increase serum 25(OH)D 3 concentrations of pigs up to 10 d after weaning, but no benefit in growth performance or bone mineralization was observed.Further research is needed to determine whether pig performance can be influenced by oral supplementation of high levels of vitamin D 3 .

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These experiments were designed to: (1) evaluate the effects of supplementing vitamin D 3 in a single oral dose on growth performance of suckling pigs in a commercial facility, (2) evaluate the effect of supplementing additional vitamin D 3 in early nursery diets or by water supplementation on pig growth performance and serum 25(OH)D 3 concentrations in the nursery, and (3) determine any potential preferences of young pigs to consume diets with different concentrations of supplemental vitamin D 3 .

Procedures
The protocols in these experiments were approved by the Kansas State University Institutional Animal Care and Use Committee.The preweaning portion of Exp. 1 was conducted at Innovative Swine Solutions in Carthage, IL, and the nursery portion was performed at the K-State Segregated Early Weaning Facility in Manhattan, KS.Experiment 2 was conducted at New Fashion Pork in Buffalo Center, IN.Experiment 3 was conducted at the K-State Swine Teaching and Research Center in Manhattan, KS.
For Exp. 1, a total of 398 barrows from 80 litters (PIC 1050, initially 7 d of age) were used in a 38-d study in a 2 × 2 factorial to determine the effects of supplementing vitamin D 3 from either a single oral dose or from high concentrations in early nursery diets on pig growth performance and serum 25(OH)D 3 .On d 7 after birth, matched pairs of pigs within litters were allotted to 1 of 2 oral dosage treatments (none or 40,000 IU vitamin D 3 ) in a randomized complete block design.Pigs were weighed on d 7 and at weaning (d 21).Following weaning, a subset of 300 barrows were used from d 21 to 45 to determine the effects of the previously administered vitamin D 3 dose and 2 levels of dietary vitamin D 3 (625 or 6,250 IU/lb vitamin D 3 , 0.80% Ca, and 0.63% available P; Table 1) from weaning through d 31 on pig performance and serum 25(OH)D 3 .Common diets (625 IU/lb vitamin D 3 , 0.70% Ca, and 0.47% available P) were fed from d 31 to 45. Barrows were allotted to pens based on their previously administered oral vitamin D 3 dose, then pens were randomly assigned to dietary treatments.All pens contained a 4-hole dry self-feeder and a cup waterer to allow for ad libitum access to feed and water.
Pigs and feeders were weighed on d 21, 26, 31, 38, and 45 to determine ADG, ADFI, and F/G.Serum was collected from 12 pigs per treatment via jugular venipuncture at weaning (d 21), d 31, and d 45.To select pigs bled for serum, the average weight pig from each of 12 pens/treatment were used.All blood samples were collected in serum separator tubes and were refrigerated for at least 6 h after collection.Blood was centrifuged at 1,600 × g for 25 min.Serum was extracted and stored in 2-mL vials and frozen in a freezer at -4ºF.All 25(OH)D 3 testing was performed by Heartland Assays Inc. (Ames, IA).Additionally, barrows were vaccinated for porcine circovirus type 2 (PCV2) and Mycoplasma hyopnuemoniae (M.hyo).A 1-dose product, Ingelvac Circo-FLEX (CircoFLEX; Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO) was given for PCV2.For the Mycoplasma hyopneumoniae (M.hyo) vaccine, Respisure (Pfizer Animal Health, New York, NY), a 2-dose product was used.Serum samples collected at weaning and on d 64 (5 wk postvaccination) were analyzed for PCV antibody titers to distinguish potential effects of supplemental vitamin D 3 on acquired immunity.Serum was analyzed at the K-State Veterinary Diagnostic Laboratory using indirect fluorescent assays (IFA).Titration endpoints were calculated as the reciprocal of the last serum dilution that gave a positive fluorescence result.Prior to analysis, all IFA titers were SWINE DAY 2012 log 2 -transformed to approximate a normal distribution of titers.Log 2 -transformed antibody titers were used to quantify the change in antibody titers from weaning (d 21) through d 64 based on supplemental vitamin D 3 treatments.
In Exp. 2, 864 pigs (PIC TR4 × FAST ADN; initially 21 d of age) were used in a 30-d nursery study to determine the effects of water supplementation of vitamin D 3 on nursery pig growth performance and serum 25(OH)D 3 concentrations.Pigs were placed in pens upon arrival in the nursery facility with 24 pigs/pen.Pens were randomly allotted to 1 of 2 water vitamin D 3 supplementation treatments (none or 4,000,000 IU/gal).The 4,000,000 IU dose was provided by mixing Hi-D 2X (Alpharma, Inc.) at 2 oz/gal.Each treatment comprised 18 pens.Pens contained a 5-hole dry self-feeder and nipple waterer to allow for ad libitum access to feed and water.Pigs and feeders were weighed on d 0, 10, 20, and 30 to determine ADG, ADFI, and F/G.Twelve pigs/treatment were bled via jugular venipuncture on d 0, 10, 20, and 30 to determine 25(OH)D 3 concentrations.All blood samples were collected in serum separator tubes and refrigerated for at least 6 h after collection.Blood was centrifuged at 1,600 × g for 25 min.Serum was extracted and stored in 2-mL vials and frozen in a freezer at -4ºF.All 25(OH)D 3 testing was performed by Heartland Assays Inc. (Ames, IA).
In Exp. 3, 72 pigs (PIC 327 × 1050, initially 28 d of age) were used in 2 14-d feed preference comparisons to evaluate if pigs differentiate between feeds containing different levels of vitamin D 3 .All pigs received a common Phase 1 diet for 7 d prior to the start of the study.On d 0 (7 d postweaning), pigs were weighed and allotted to pens based on BW.There were 6 pigs/pen and 6 pens per treatment, and pens were randomly assigned to 1 of the 2 feed comparisons between Phase 2 nursery diets (Table 1).The first preference comparison was between diets containing 625 (control) or 6,250 IU/ lb vitamin D 3 , and the second comparison was between diets containing 625 (control) or 20,000 IU/lb vitamin D 3 .Pens contained two 4-hole dry self-feeders and a nipple waterer to allow for ad libitum access to feed and water.Diets were placed in the separate feeders and feeders were positioned adjacent to each other.Every morning, feeders were weighed and switched in pen location to discourage any location bias by the pig.Total pen feed intake was calculated, and intake of each diet for both comparisons was expressed as a percentage of total intake.
Vitamin premixes and feed samples used in Exp. 1 and 3 were collected and sent to DSM Nutritional Products Laboratory, Inc. (Parsippany, NJ) for vitamin D 3 analysis (Table 2).Accepted analytical errors associated with complete feed vitamin D 3 assays is ± 25% of target level.All diets were within ±25% of their formulated level.
Statistical analysis conducted for each experiment was performed using the PROC MIXED procedure of SAS (SAS Institute, Inc., Cary, NC).In Exp. 1, for the preweaning period, the growth data were analyzed as a randomized complete block design.Individual pig was the experimental unit, initial weight on the day of dosing was used as a covariate, and sow was used as a random effect.Only pigs that completed the full lactation period (d 7 to 21) were used in this analysis.Nursery growth performance data were analyzed as a completely randomized design using pen as the experimental unit and barn as a random effect.Serum 25(OH)D 3 and PCV antibody titer results were analyzed using the repeated measures function to determine the effect of dosage or diet on response criteria over time and the treatment × time interactions.For Exp. 2,

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pen was the experimental unit, and initial BW on d 0 was used as a covariate.Serum 25(OH)D 3 was analyzed using the repeated measures function to determine the effect of water vitamin D 3 supplementation on serum over time and the treatment × time interactions.For Exp. 3, pen was again the experimental unit, and differences associated with the main effect of diet on the percentage of total feed intake were determined in both comparisons.Results were considered significant at P ≤ 0.05 and considered a trend at P ≤ 0.10.

Results and Discussion
In Exp. 1, no differences were observed (P > 0.17) for weaning weight (Table 3), but weaning weights were 0.2 lb/pig numerically heavier for pigs supplemented with the oral dosage of 40,000 IU of vitamin D 3 .During the nursery phase (d 21 to 45), neither previously administered oral vitamin D 3 dose nor dietary level of vitamin D 3 in the diet affected (P > 0.23) ADG, ADFI, or F/G (Table 4).No dose × diet interactions were observed for any criteria in Exp. 1 except for a tendency (P = 0.06) for F/G from d 21 to 31.Here, F/G worsened with increasing dietary vitamin D 3 for pigs initially dosed on d 7 with 40,000 IU, but for pigs not orally dosed with vitamin D 3 , F/G improved with increasing dietary vitamin D 3 .
At weaning (d 21), serum 25(OH)D 3 concentrations (Table 5) increased (P < 0.01) in pigs that received an oral dose of 40,000 IU vitamin D 3 .On d 31, a tendency (P = 0.08) for an increase in serum 25(OH)D 3 was observed for pigs dosed with vitamin D 3 prior to weaning.Also on d 31, increased serum 25(OH)D 3 concentrations were observed (P < 0.01) in pigs fed increased levels of vitamin D 3 (Figure 1).PCV antibody titer results showed no dose × diet interaction (P = 0.74; Table 6) and no main effects of either dose or diet (P > 0.59) associated with the change in log 2 reciprocal dilutions from d 21 to d 64.
In Exp. 2, supplementation of vitamin D 3 through the water did not affect (P > 0.15) overall ADG, ADFI, or F/G (Table 7), but F/G improved (P = 0.05) during the first phase (d 0 through 10) in pigs supplemented with 4,000,000 IU/ gallon of vitamin D 3 .On the other hand, from d 10 to 30, ADG decreased significantly (P = 0.03) and F/G worsened (P = 0.05) in pigs supplemented 4,000,000 IU vitamin D 3 during the first phase.
For serum 25(OH)D 3 concentrations (Table 8), supplementing 4,000,000 IU vitamin D 3 /gallon from d 0 to 10 increased (P < 0.01) serum 25(OH)D 3 concentrations in pigs on d 10, 20, and 30 (Figure 2).In Exp. 3, there was no difference in preference between diets containing 625 or 6,250 IU of vitamin D 3 (Table 9), but when pigs were offered a choice between diets containing 625 and 20,000 IU of vitamin D 3 , they consumed a greater portion (P < 0.01) of the diet containing 625 IU of vitamin D 3 .
Results from Exp. 1 and Exp. 2 agree with our previous research (Flohr et al., 2011).Supplementation of vitamin D 3 appeared to significantly increase serum 25(OH)D 3 concentrations in the weaned pig but did not lead to increases in growth performance.

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These studies clearly demonstrate that increasing supplementation of vitamin D 3 through an oral dose, diet, or water can increase serum 25(OH)D 3 .
Porcine circovirus type 2 antibody titer results from Exp. 1 suggest that vitamin D 3 supplementation has no effect on acquired immunity of the nursery pig, but to truly quantify vitamin D's role in immune function, disease challenge or studies with additional vaccines should be conducted.
In Exp. 3, the studies suggest that young pigs have a truly wide range of acceptance for different dietary vitamin D 3 concentrations; however, when diets contain extremely high concentrations (20,000 IU/lb) of vitamin D 3 , pigs will reduce intake of the diet, which could potentially have negative effects on growth performance.If additional supplementation of vitamin D 3 is utilized in an operation, dietary levels should be monitored to reduce the risk of negative effects in growth performance or potential for vitamin D toxicity.
Multiple studies conducted at K-State associated with the supplementation of additional vitamin D 3 to the nursery pig have consistently shown that serum 25(OH)D 3 concentrations can be increased without influence on growth performance of pigs preor postweaning.Future research needs to better quantify the relationship of circulating 25(OH)D 3 to proper bone mineralization and ideal Ca and P absorption to determine optimal circulating concentrations of 25(OH)D 3.More information is needed to determine whether another metabolite or other related protein may better quantify vitamin D status.

Figure 1 .Figure 2 .
Figure 1.Effects of supplemental vitamin D 3 by either an oral dose or in early nursery diets on serum 25(OH) D 3 concentrations as determined by jugular venipuncture of 12 pigs/ treatment on d 21 (weaning), 31, and 45 (Exp.1).

Table 1 .
Diet composition (as-fed basis) 1 A total of 300 barrows were used from d 21 to 45 of age to determine the effects of supplemental vitamin D 3 on nursery growth performance from either a single oral dose or in early nursery diets.There were 5 barrows per pen and 15 pens per treatment.
1 2 Oral dosage treatments were administered at d 7 of age. 3 Dietary vitamin D 3 levels were fed in Phase 1 diets (d 21 to 31), then pigs were fed common diets containing 625 IU/lb vitamin D 3 from d 31 to 45.

Table 5 .
Effects of supplemental vitamin D 3 from either an oral dose or in early nursery diets on serum 25(OH) D 3

Table 6 .
Effects of supplemental vitamin D 3 by an oral dose or in early nursery diets on PCV2 antibody titers1,2 1 Serum collected on d 21 (weaning) and 5 wk postvaccination (d 64) was sent to the K-State Veterinary Diagnostic Laboratory for indirect fluorescent assays.There were 12 samples/treatment. 2Endpoint antibody titers determined by indirect fluorescent antibody (IFA) assay were log 2 -transformed.

Table 7 .
Effects of water supplemented vitamin D 3 on nursery growth performance

Table 9 .
Evaluation of nursery pig feed preference for diets formulated to varying levels of vitamin D 3 (Exp.3) 1 A total of 72 pigs (PIC 327 × 1050; initially 28 d of age) were used in a 14-d feed comparison to evaluate nursery pig preference to diets containing varying levels of vitamin D 3 . 2There were 6 pigs/pen and 6 pens/feed comparison. 1