INTRODUCTION
Vitamin D is essential for maintaining calcium and phosphorus balance and bone health, as well as supporting oxidative defense and immune function, particularly in weaning pigs [1–4]. Vitamin D exhibits antioxidant effects partly by inducing the synthesis of metallothionein, a protein known for its ability to scavenge reactive oxygen species and thereby reduce oxidative stress [5,6]. It plays a critical role in regulating the immune system and inflammatory responses in pigs by reducing pro-inflammatory cytokine levels and supporting intestinal barrier integrity during immune challenges [7].
Piglets are born with naturally low plasma vitamin D levels, and the confined housing conditions commonly used in swine production limit their sunlight exposure, thereby restricting the production of vitamin D in the skin due to reduced exposure to ultraviolet sunlight [8]. In addition, pigs are weaned with a low level of vitamin D in the blood due to insufficient vitamin D levels in milk [9,10]. This emphasizes the importance of providing enough dietary vitamin D to meet the requirements of young pigs. In the current swine industry, dietary vitamin D3 (VD3) supplementation for nursery pigs typically ranges from 1,600–2,600 IU/kg, which is 8–12 times greater than the NRC [11] recommended levels [12], despite a lack of scientific evidence supporting benefits to growth and health at these high levels. Several studies reported that VD3 supplementation positively influenced immune responses and antioxidant status in pigs [7,13], while no effect was observed in growth performance [9,14]. However, previous studies have used lower levels of VD3 (e.g., 800 IU) [15] or its metabolites such as 25-hydroxycholecalciferol (25-OHD3) [4,16] with varying supplementation methods, including oral administration, drinking water and intramuscular injection [2,10]. Thus, there is still limited information regarding the beneficial effects of high dietary VD3 supplementation beyond NRC requirement levels on growth performance, plasma vitamin D, and antioxidant status in weaning pigs. Therefore, this study aims to evaluate the effects of dietary VD3 supplementation at two levels, NRC recommendation (200–220 IU/kg) and 2,000 IU/kg on growth performance, blood vitamin D status, and antioxidant parameters in weaning pigs.
MATERIALS AND METHODS
Animal care
This experiment and sample collections were carried out in an environmentally controlled room at the University of Georgia Large Animal Research Unit.
Animals, experimental design, and housing
A total of 40 newly weaned pigs (Camborough×PIC337; 6.02±1.17 kg initial body weight; weaned at 17.2±0.95 d of age) were assigned to 2 treatments in 5 replicates with 4 pigs (2 barrows and 2 gilts) per pen based on body weight, breed, and sex in the randomized complete block design. Treatments were: 1) NRC-VD3: VD3 supplementation at NRC [11] recommended levels - 220 IU/kg in Phase 1 (d 0–14 postweaning) and 200 IU/kg in Phase 2 (d 14–28 postweaning), added to basal diets without supplemental VD3, and 2) High-VD3: 2,000 IU/kg of VD3 supplementation added to the same basal diets. All pigs were housed in nursery pens (1.0×2.0 m2) with woven-wire flooring and had ad libitum access to water and feed in an environmentally controlled nursery facility. No creep feed was provided during the suckling period.
Experimental diets
All pigs were fed corn-soybean meal-based diets in mash form that were formulated to meet or exceed nutrient requirement estimates of NRC [11] for 7–11 kg (Phase 1) and 11–25 kg (Phase 2) of pigs (Table 1). To minimize differences in non-treatment components of the diets, a basal diet was first mixed with a VD3-free vitamin premix, divided into two fractions, and each fraction was mixed with respective treatment levels of VD3 (NRC recommended level or 2,000 IU/kg) by replacing corn starch.
Data and sample collection
The pigs were individually weighed at d 0, 7, 14, 21, and 28 postweaning. Pen-based feed disappearance was measured when the pigs were weighed and average daily gain (ADG), average daily feed intake (ADFI), and gain-to-feed (G:F) ratio were calculated.
On d 0, 14, and 28 postweaning, blood samples (10 mL) were collected from eight pigs per treatment (2 pigs per pen from first 4 replicates) selected based on average body weight in each pen via jugular venipuncture in disposable vacutainer tubes containing the anticoagulant K3 EDTA (Becton Dickinson). Plasma sample was obtained by centrifugation at 2,500×g for 30 min at 4°C and stored at −80°C until analysis.
Chemical analysis
Plasma samples were analyzed for 25-OHD3 using liquid chromatography-mass spectrometry at Heartland Assays and antioxidant parameters including superoxide dismutase (SOD; Catalog No. 706002; detection limit - 0.005–0.05 U/mL) activity, total antioxidant capacity (T-AOC; Catalog No. 709001; detection limit - 0.000–0.495 mM trolox equivalents), and malondialdehyde (MDA; Catalog No. 10009055; detection limit - 0.625–50 μM) levels using colorimetric kits (Cayman Chemical Company) and a spectrophotometer (Multiskan Skyhigh; Thermo Fisher Scientific).
Statistical analysis
All data obtained in the current study were analyzed in accordance with a randomized complete block design using the PROC Mixed procedure of SAS (ver. 9.4; SAS Institute). Pen was used as the experimental unit for the analysis of growth performance data. An individual pig was used as an experimental unit for blood analyses. No pigs were excluded from the data. The models included the treatment as a fixed effect and the replicate as a random effect for growth performance and the replicate within pen and pen as random effects for blood parameters. Pearson correlation coefficients between plasma 25-OHD3 concentrations and plasma SOD, MDA, or T-AOC at d 14 and 28 postweaning were determined using PROC CORR of SAS with individual values. The least square means were separated using the PDIFF option of SAS. Statistical differences were established at p<0.05 and tendencies were established at 0.05≤p<0.10.
RESULTS
There were no significant differences between dietary treatments in body weight, ADG, and G:F during the entire experimental period (p>0.10; Table 2). However, pigs in the High-VD3 treatment had significantly lower ADFI (p<0.05) than those in the NRC-VD3 treatment during d 14 to 28 postweaning, although there were no differences in the other phase and overall period (p>0.10).
The pigs in the High-VD3 treatment had greater plasma 25-OHD3 concentrations than those in the NRC-VD3 treatment at d 14 and 28 postweaning (p<0.05; Table 3). There were no significant differences in plasma T-AOC between dietary treatments (p>0.10; Table 4). Pigs in the High-VD3 treatment tended to have greater plasma SOD activity (p = 0.10) than those in the NRC-VD3 treatment at d 14 postweaning, while showing lower plasma MDA levels with a tendency (p = 0.06) compared to those in the NRC-VD3 treatment. At d 28 postweaning, there were no significant differences in plasma SOD activity or MDA levels between two treatments (p>0.10).
Plasma 25-OHD3 concentrations at d 14 postweaning were positively correlated with plasma SOD activity at d 14 postweaning (p<0.05) and showed a tendency for a positive correlation at d 28 postweaning (p = 0.07). Plasma MDA levels at d 14 postweaning showed a tendency for a negative correlation with plasma 25-OHD3 concentrations at d 14 postweaning (p = 0.06; Table 5).
DISCUSSION
VD3 has gained growing attention as a nutritional strategy, not only for its established role in Ca and P homeostasis but also for its emerging functions in supporting immune responses and antioxidant defenses [3,15,17]. Vitamin D exerts antioxidant effects by inducing metallothionein synthesis to scavenge reactive oxygen species [5,6], while also regulating immune and inflammatory responses in pigs by reducing pro-inflammatory cytokines and supporting intestinal barrier integrity [7]. Although the NRC [11] recommends 200–220 IU/kg of dietary VD3 for nursery pigs, commercial feeding practices often exceed this level, with reported supplementation rates ranging from 1,600 to 2,600 IU/kg [12]. This discrepancy may indicate potential benefits of higher VD3 levels under practical conditions; however, scientific evidence validating such practices remains limited. Therefore, the objective of this study was to evaluate the potential benefits of supplementing 2,000 IU/kg of VD3 on growth performance, vitamin D status, and antioxidant parameters in weaning pigs, in comparison with the NRC-recommended levels.
In the current study, pigs fed diets supplemented with 2,000 IU/kg of VD3 had no difference in body weight, ADG, or G:F throughout the nursery period compared to those fed diets with the NRC-recommended VD3 level. This agrees with previous findings [9,14] suggesting that dietary VD3 supplementation does not significantly affect growth performance in nursery pigs. However, pigs fed diets with the NRC-recommended VD3 level had a greater feed intake in the late nursery period compared to those fed diets with 2,000 IU/kg of VD3, although there was no difference in overall feed intake. The underlying cause of this increase is unclear. In the current study, however, pigs fed diets with the NRC-recommended VD3 level had relatively lower antioxidant status, as evidenced by greater plasma MDA levels and lower plasma SOD activity observed at d 14 postweaning compared to those fed diets supplemented with 2,000 IU/kg of VD3. Hao et al [18] reported that oxidative stress impairs energy homeostasis by increasing the demand for metabolic resources required for cellular repair and antioxidant defense. Thus, it is possible that pigs experiencing elevated oxidative stress potentially due to low VD3 intake may divert energy from growth toward maintaining physiological balance, which possibly results in a greater nutritional need. This pattern was evident in the NRC-VD3 group, where elevated ADFI, likely reflecting greater metabolic demands, did not result in enhanced growth performance. Instead, both growth rate and feed efficiency in the late nursery period were numerically lower when pigs were fed diets with the NRC-recommended VD3 level, suggesting potentially inefficient nutrient utilization in those pigs.
In the current study, plasma 25-OHD3 levels clearly increased in pigs fed diets with 2,000 IU/kg of VD3 compared to those fed diets with the NRC-recommended VD3 level on both d 14 and 28 postweaning. Since 25-OHD3 is the main circulating form of vitamin D in the blood, it is widely used as a marker for blood vitamin D status [19]. Our findings align with previous studies that reported greater VD3 intake increases blood 25-OHD3 levels in weaning pigs [2,9,20]. Although we only measured blood 25-OHD3 levels, Burild et al [20] reported that increasing dietary VD3 levels enhanced tissue 25-OHD3 levels, suggesting that high dietary VD3 intake can also improve vitamin D retention throughout the body.
Although previous studies have consistently shown that supplementation of vitamin D using 25-OHD3 resulted in dose-dependent increases in circulating blood 25-OHD3 concentrations in pigs [16,21,22], the current study found that increasing dietary VD3 by tenfold increased plasma 25-OHD3 concentrations by only about 40%. This result agrees with Burild et al [20] reporting that pigs fed diets with VD3 from 200 to 2,000 IU/kg had about 2.5 times less increase in plasma 25-OHD3 concentrations compared to those fed diets with 25-OHD3. Thus, this result indicates the lower efficiency of VD3 in elevating circulating 25-OHD3 compared to its hydroxylated form (25-OHD3), likely due to differences in intestinal absorption, hepatic conversion, and systemic utilization. The VD3, being highly lipophilic, relies on micelle formation and lymphatic transport and is more prone to sequestration in adipose tissues, whereas 25-OHD3, with greater hydrophilicity, is absorbed more efficiently via both portal and lymphatic routes and enters circulation more directly [20,23–25].
In the current study, 2,000 IU/kg of dietary VD3 supplementation tended to reduce plasma MDA concentrations at d 14 postweaning, suggesting that high dietary VD3 may help alleviate oxidative stress in weaning pigs. Similarly, previous studies using the hydroxylated form of VD3 (25-OHD3) have demonstrated reductions in plasma MDA concentrations in weaning pigs, supporting the role of improving vitamin D status in mitigating oxidative stress [16,26]. One proposed mechanism involves the active form of vitamin D, 1,25-dihydroxycholecalciferol, enhancing the expression of metallothionein, a protein that scavenges reactive oxygen species and supports redox balance [5,6,27]. In addition, a tendency toward increased plasma SOD activity in pigs fed diets supplemented with 2,000 IU/kg of VD3 at d 14 postweaning indicates a potential stimulatory effect of VD3 on enzymatic antioxidant defenses. This observation aligns with previous findings where 25-OHD3 supplementation significantly increased serum SOD activity in weaning pigs [26]. Therefore, the reduction in plasma MDA, along with increased plasma SOD activity, indicates that high VD3 supplementation at 2,000 IU/kg can result in improved overall pig health by reducing oxidative stress and improving antioxidant capacity.
On the other hand, T-AOC activity was not significantly affected by high VD3 supplementation in the current study. This result is consistent with a recent study in broilers, where dietary supplementation with 4,000 IU/kg of VD3 did not affect serum T-AOC, SOD activity, and MDA levels compared to the control group [28]. Although species differences may exist, these findings suggest that high VD3 supplementation does not always enhance systemic antioxidant enzyme activity. Kwon et al [16] also reported that the effects of dietary vitamin D sources on antioxidant enzyme responses may vary depending on multiple factors such as the animal’s oxidative stress level, health status, diet composition, and environmental conditions. Therefore, further research is needed to demonstrate how VD3 supplementation can effectively enhance enzymatic antioxidant defenses in weaning pigs.
Based on the observed increases in plasma 25-OHD3 and improvements in antioxidant status following 2,000 IU/kg of VD3 supplementation, the current correlation analysis revealed relationships between blood vitamin D status and antioxidant parameters in weaning pigs. At d 14 postweaning, plasma 25-OHD3 concentrations were positively correlated with plasma SOD activity and tended to be negatively correlated with plasma MDA levels, suggesting that improved vitamin D status can result in enhanced enzymatic antioxidant defense and potentially reduced oxidative stress in early nursery period. Additionally, a trend for a positive correlation was observed between plasma 25-OHD3 concentrations at d 14 postweaning and plasma SOD activity at d 28 postweaning, indicating that early improvements in vitamin D status may have prolonged effects on antioxidant responses. Kwon et al [16] reported that plasma MDA levels reached their minimum when plasma 25-OHD3 concentrations exceeded approximately 23.7 ng/mL by d 28 post-weaning based on the broken-line analysis. In the current study, however, plasma 25-OHD3 levels did not reach this threshold with VD3 supplementation at 2,000 IU/kg. This result indicates that increasing blood vitamin D status beyond the level achieved with this dosage may further improve the antioxidant protection system in pigs. In addition, together with our previous work [16], which observed a similar correlation between plasma 25-OHD3 and MDA levels, the correlations found in the current study between plasma 25-OHD3 and antioxidant markers support that vitamin D requirement in pigs may need to be expressed based on plasma vitamin D status, using 25-OHD3 as a biomarker. Additionally, the relative bioefficacy of VD3 compared to 25-OHD3 may need to be more precisely evaluated.
CONCLUSION
Dietary supplementation with 2,000 IU/kg of VD3 in weaning pigs improved vitamin D status, as evidenced by elevated plasma 25-OHD3 concentrations, without affecting growth performance compared to NRC [11] recommended levels. High VD3 supplementation at 2,000 IU/kg also reduced oxidative stress and enhanced antioxidant enzyme activity in the early nursery period. In addition, plasma vitamin D status was positively correlated with antioxidant enzyme activity and negatively correlated with an oxidative stress marker. These findings support higher-than-recommended levels of VD3 may be a beneficial nutritional strategy to improve antioxidant status and overall health in weaning pigs. Further research is needed to refine dosing strategies and explore the long-term effects of improved vitamin D status on immune function, gut health, and performance outcomes.
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