Weaning is one of the most stressful periods separating piglets from their sow as it can negatively affect the immune system, intestinal function, and cause oxidative stress in pigs [1–3]. Vitamin D, a fat-soluble vitamin, plays an important role in bone development, mineralization, and other biological functions, including immune and antioxidant systems [4,5]. Therefore, supplementing diets with vitamin D is critical for health, growth and development of weaning pigs as a previous study reported that an increased vitamin D status for weaning pigs may promote intestinal robustness against disease challenge [6]. Generally, vitamin D₃ (VD₃) has been supplemented to swine diets as a traditional form because pigs are housed in confinement housing, thus limiting vitamin D synthesis in the body. The U.S. swine industry typically supplemented diets with VD₃ at the levels ranging from 1,600 to 2,600 IU/kg for nursery pigs from weaning to 25 kg body weight [7]. Recently, 25-hydroxycholecalciferol (25-OHD₃) has been increasingly used in swine diets as a circulating form of VD₃ that is hydroxylated in the liver after absorption, which bypasses the metabolic process of VD₃ in the liver [8,9]. The 25-OHD₃ is known to have stronger biological activity than VD₃ [10]. The intestine more easily absorbs 25-OHD₃ than VD₃ as its absorption is less dependent on bile acids and chylomicrons than VD₃ [11]. In addition, vitamin D-binding proteins exhibit a 500 times stronger affinity for 25-OHD₃ than VD₃ [12] and greater daily excretion of metabolites was observed in VD₃ than 25-OHD₃ [13]. Previous studies reported that dietary 25-OHD₃ supplementation improved growth performance, immunity, and antioxidant status in nursery pigs when supplemented to diets with certain levels (2,000–2,500 IU/kg) of supplemental VD₃ [10,14]. While these studies investigated the efficacy of 25-OHD₃ in increasing plasma 25-OHD₃ levels and improving antioxidant capacity of weaned pigs, it is also important to clearly demonstrate the effect of dietary 25-OHD₃ supplementation in diets without supplemental VD₃ and its potential relationships with antioxidant capacity and oxidative stress in weaning pigs, as they are susceptible to oxidative stress after weaning, which can affect postweaning growth performance [15]. Therefore, this study was conducted to evaluate the effects of dietary VD₃ and 25-OHD₃ supplementation on growth performance, blood vitamin D status, and antioxidant parameters of weaned pigs.

VD₃ has been supplemented in nursery diets from 1,600 to 2,600 IU/kg [6] as VD₃ supplementation may improve bone health, immune system, and antioxidant capacity of weaning pigs [10,18]. The 25-OHD₃ has been considered as an alternative form of VD₃ due to its relatively greater bioavailability than the VD₃ in pigs, that can improve vitamin D status more efficiently than VD₃ [9,14]. However, there is limited information on the direct comparison between VD₃ and 25-OHD₃ as sources of vitamin D, as several previous studies have focused on the effect of 25-OHD₃ supplementation in diets that already contained VD₃ at levels ranging from 2,000 to 2,500 IU/kg [10,14,19]. Therefore, the current study evaluated the effect of dietary 25-OHD₃ supplementation in nursery pig diets containing no supplemental VD₃ on growth performance, plasma 25-OHD₃ concentrations, and antioxidant parameters compared with conventional VD₃ supplementation.

In the current study, individual 25-OHD₃ treatments showed no significant difference in growth performance from the VD₃ treatment except that 1,000 or 2,000 IU/kg of 25-OHD₃ supplementation increased feed intake in d 21–28 postweaning compared with 2,000 IU/kg of VD₃ supplementation. A greater growth rate was observed in the late nursery and overall periods when the combined 25-OHD₃ treatments were compared to the VD₃ treatment. This result indicates that dietary 25-OHD₃ supplementation had the potential to increase postweaning growth rate of piglets compared with dietary VD₃ supplementation and this could be attributed to increased feed intake in the late nursery period by dietary 25-OHD₃ supplementation. Although various factors affect postweaning feed consumption in pigs, one possible explanation for the increased feed intake could be due to the reduction in plasma MDA levels observed at d 28 postweaning in the current study when pigs consumed diets supplemented with 25-OHD₃. This suggests that dietary 25-OHD₃ supplementation may help reduce postweaning oxidative stress in pigs leading to increases in feed intake and growth rate, as oxidative stress could reduce postweaning feed intake and slow down weight gain of piglets [20]. Zhou et al [10] reported that 2,000 IU/kg of 25-OHD₃ supplementation in the basal diets containing 2,000 IU/kg of VD₃ had a greater growth rate and feed intake in the late nursery periods compared with 2,000 IU/kg VD₃ supplementation in the basal diets. Zhang et al [14] also reported that additional 2,000 IU/kg of 25-OHD₃ supplementation in the low Ca and P basal diets containing 2,500 IU/kg of VD₃ for weaning pigs could increase growth rate in late nursery and overall periods compared with the basal diets with only 2,500 IU/kg of VD₃. These findings are consistent with the current study, showing the potential of dietary 25-OHD₃ supplementation to enhance feed intake and growth rate during the late nursery period. However, the current study lacked a significant difference in growth performance among the individual treatments with increasing levels of 25-OHD₃ supplementation, which contrasts to the findings of the previous studies [10,14,18]. Although these previous studies utilized different breeds of pigs, diets with low Ca and P content, and varying levels of basal VD₃ content, the reason remains unclear. However, possible explanations for this discrepancy could be that those previous studies used weaned piglets with higher initial weaning weights, ranging from 6.3 to 8.4 kg [10,14,18], compared to the 5.3 kg average in the current study and observed the effect of dietary 25-OHD₃ supplementation only becoming apparent during the late nursery period. Therefore, with the results of the current study, it can be suggested that the growth response to 25-OHD₃ supplementation may not become evident until the late nursery phase. In addition, Sandoval et al [21] reported no significant difference in growth performance when 1,000 or 2,000 IU/kg of 25-OHD₃ was supplemented to the diets containing 44 IU/kg of VD₃ for the pigs weighing from 10 to 38 kg. Therefore, the difference in initial weight could have influenced the response of pigs to dietary supplementation, potentially due to variations in body growth and development at different weaning weights [22]. Additionally, a sufficient duration of feeding 25-OHD₃ may be necessary to observe more pronounced improvements in pig growth performance, as the effect was only seen during the late nursery period.

In the current study, there was no significant difference in feed efficiency among dietary treatments. This result agrees with previous studies [10,14,18] reporting that dietary 25-OHD₃ supplementation up to 2,000 IU/kg did not affect feed conversion ratio. Therefore, this result indicates that dietary 25-OHD₃ supplementation in nursery diets had potential to increase postweaning feed intake, thus growth rate in the nursery period instead of improving feed efficiency. However, further research may be needed to investigate the effect of dietary 25-OHD₃ supplementation in nursery pigs under various conditions, such as different weaning weights, diet regimes, and feeding durations, in larger scale settings to explore the potential mechanisms through which it could enhance postweaning growth.

The 25-OHD₃ is a circulating form of VD₃ in the body, which has been commonly used as the best biomarker of body vitamin D status [23]. Moreover, concentrations of 25-OHD₃ in different tissues are highly correlated with the blood 25-OHD₃ concentrations [21,24]. In the current study, when pigs were fed the diets containing increasing levels of 25-OHD₃ (1,000 and 2,000 IU/kg diet), plasma 25-OHD₃ levels increased significantly compared with those fed diets with VD₃. This result agreed with previous studies [14,19] reporting that dietary 25-OHD₃ supplementation in nursery diets increased blood 25-OHD₃ concentrations. Zhang et al [14] reported that the 2,000 IU/kg 25-OHD₃ supplementation in low Ca and P nursery diets containing 2,500 IU/kg VD₃ increased blood 25-OHD₃ concentrations compared with the basal diets containing 2,500 IU/kg VD₃ only. von Rosenberg et al [19] reported that plasma 25-OHD₃ concentrations of pigs fed diets with 2,000 IU/kg of VD₃ was 16.5 ng/mL after feeding treatment diets for 42 d, whereas the same level (2,000 IU/kg diet) of 25-OHD₃ supplementation resulted in plasma 25-OHD₃ concentrations 4 times greater (64.1 ng/mL) than the 2,000 IU/kg of VD₃ diet, which was comparable to the results observed at d 28 postweaning in the current study. These results indicate that dietary 25-OHD₃ supplementation is more effective in improving blood vitamin D status than the VD₃ supplementation as 25-OHD₃ is absorbed faster and to a larger extent than VD₃ in the small intestine due to polarity and solubility [12,25] and VD₃ should undergo the metabolic process to be converted to the circulating form (i.e., 25-OHD₃) in the liver by 25-hydroxylase [9,26]. Although the current study only measured blood 25-OHD₃ levels, von Rosenberg et al [19] also reported that increasing supplementation levels of 25-OHD₃ increased tissue vitamin D status (liver, skin, and muscle) measured by tissue 25-OHD₃ content. Similarly, Burild et al [24] reported that increasing supplementation levels of 25-OHD₃ from 200 to 2,000 IU/kg diet increased tissue (liver, fat, and muscle) 25-OHD₃ content in finishing pigs. Therefore, further study may be needed to accurately compare the bioavailability of these two VD₃ sources by measuring both blood and tissue 25-OHD₃ concentrations and finding a correlation between blood and tissue 25-OHD₃ concentrations.

Interestingly, the 2,000 IU/kg of 25-OHD₃ supplementation resulted in approximately 2 times greater plasma 25-OHD₃ concentrations than the 1,000 IU/kg of 25-OHD₃ supplementation, which follows a pattern consistent with a two-fold difference in their dietary supplementation levels. This result agreed with previous studies reporting that dietary 25-OHD₃ supplementation in pig diets improved vitamin D status as determined by plasma 25-OHD₃ concentrations in a dose-dependent manner [19,21]. von Rosenberg et al [19] reported that the pigs fed increasing supplementation levels of 25-OHD₃ from 2,000 to 20,000 IU/kg diet for 42 d showed greater plasma 25-OHD₃ concentrations than those fed the diet with VD₃ at 2,000 IU/kg and the levels increased in a dose-dependent manner ranging from 64.1 to 360 ng/mL. Sandoval et al [21] also reported greater plasma 25-OHD₃ concentrations in pigs fed diets with increasing levels of 25-OHD₃ from 1,000 to 10,000 IU/kg diet for 39 d than the diet with 44 IU/kg VD₃. As mentioned above, it is known that small intestine can absorb 25-OHD₃ more efficiently and to a larger extent than VD₃ due to its lesser dependence on bile acids and chylomicrons compared to VD₃ [25,26] and it is also absorbed directly into portal blood as well as via chylomicrons into the lymph due to its polarity [26]. Therefore, these results indicate that plasma 25-OHD₃ concentrations increase when 25-OHD₃ is supplemented to diets in a dose-dependent manner.

Regarding antioxidant capacity and oxidative stress, the 2,000 IU/kg of 25-OHD₃ supplementation reduced plasma MDA levels significantly compared with the 2,000 IU/kg of VD₃ supplementation with intermediate levels in the 1,000 IU/kg of 25-OHD₃ supplementation. This result agreed with a previous study that increasing 25-OHD₃ supplementation levels in nursery diets decreased plasma MDA concentrations [10]. The plasma MDA level has been widely used as a marker of oxidative stress and lipid peroxidation to measure pigs’ health status. Weaning stress could decrease plasma SOD activity but increase plasma MDA levels in piglets, which may impact their postweaning feed intake, thus growth [15]. Vitamin D is thought to have antioxidant mechanisms related to inducing metallothionein synthesis which is known to have reactive oxygen species scavenger activities that can reduce oxidative stress [27,28] as 1,25-dihydroxycholecalciferol, a hormonally active form of VD₃ that was activated from 25-OHD₃ in kidney is known to increase mRNA expression of metallothionein gene [29]. Therefore, the result of the current study indicates that dietary 25-OHD₃ supplementation could alleviate oxidative stress in weaning pigs and a higher level, which is 2,000 IU/kg diet, may potentially be more effective. In the broken-line analysis, although there is a relatively large variation observed due to individual pig values used in the analysis, plasma MDA level was minimized when the plasma 25-OHD₃ concentrations reached 23.7 ng/mL, which could be achieved by approximately 1,600 IU/kg 25-OHD₃ supplementation level when it is estimated by plasma 25-OHD₃ concentrations in 1,000 and 2,000 IU/kg 25-OHD₃ treatments at d 28 postweaning in the current study. However, because the 2,000 IU/kg of VD₃ supplementation could not achieve this plasma 25-OHD₃ level in nursery period, it may be beneficial to use 25-OHD₃ as a vitamin D source in nursery diets due to its high bioavailability and effectiveness. Interestingly, although dietary 25-OHD₃ supplementation increased plasma 25-OHD₃ levels in a dose-dependent manner and reduced oxidative stress, greater supplementation level (2,000 IU/kg diet) did not result in further increase of growth rate and feed intake. Zhang et al. [18] reported that pigs fed diets with increasing levels of 25-OHD₃ supplementation from 0 to 4,000 IU/kg showed quadratic responses in growth rate and feed intake with the greatest performance at the 2,000 IU/kg of 25-OHD₃ supplementation level. However, in the current study, over 1,000 IU/kg 25-OHD₃ may be needed to maximize its benefits for not only pig growth but also health including oxidative stress as the further reduction in plasma MDA levels was observed when the higher level of 25-OHD₃ was supplemented to the nursery diets. Additionally, it should be noted that a further increase in dietary 25-OHD₃ supplementation levels over 2,000 IU/kg might not result in additional benefits for growth and antioxidant status, as supplementing 25-OHD₃ in nursery diets over 2,000 IU/kg may decrease growth rate, feed intake, and serum T-AOC activity of pigs [18]. Further studies are needed to identify the optimal plasma 25-OHD₃ levels that maximize health benefits in pigs, particularly in terms of antioxidant capacity and oxidative stress, through varying levels of dietary 25-OHD₃ supplementation. In addition, research should explore how baseline blood 25-OHD₃ levels affect the effectiveness of 25-OHD₃ supplementation in pigs.

Interestingly, the reduction of plasma MDA levels by dietary 25-OHD₃ supplementation was only observed at d 28 postweaning but not at d 14 postweaning, although the increase in plasma 25-OHD₃ concentrations was observed from d 14 postweaning. In previous studies [14,18], there were also inconsistent responses in these antioxidant parameters in pigs during the nursery period when they were fed diets supplemented with 25-OHD₃. Zhang et al [18] reported an increase in serum T-AOC of nursery pigs at d 28 postweaning, but not at d 14 postweaning although the glutathione peroxidase (GSH-Px) activities and 25-OHD₃ levels in serum increased at both d 14 and 28 postweaning. In addition, Zhang et al [14] also reported that serum SOD and catalase activities only increased at d 28 postweaning but not at d 14 postweaning when pigs were fed low Ca-P diets supplemented with 25-OHD₃. Although there is no clear explanation in these responses, it can be noted that sufficient duration of feeding diets supplemented with 25-OHD₃ may be needed to observe its effects on antioxidant status.

Although there was no effect of dietary 25-OHD₃ supplementation on plasma SOD activity and T-AOC in the current study, previous studies reported that dietary 25-OHD₃ supplementation at 2,000 IU/kg diet increased plasma T-AOC, SOD, and GSH-Px activities of weaned pigs [10,18]. Sauvé et al [30] reported that dietary 25-OHD₃ supplementation to mycotoxin-contaminated diets for weaning pigs after lipopolysaccharide challenge did not improve catalase and SOD activities and MDA levels in blood, while increasing GSH-Px activity although there were different responses in mRNA expression of these antioxidant parameters in liver and jejunum. Therefore, the response of pigs to dietary 25-OHD₃ supplementation in antioxidant parameters may vary depending on multiple factors such as status of oxidative stress, health challenge, environment, diets, etc. Due to these inconsistent results, further studies are also needed to demonstrate the efficacy of dietary 25-OHD₃ supplementation on antioxidant capacity and oxidative stress markers in various tissues and blood when weaning pigs have increased oxidative stress after weaning.

Dietary 25-OHD₃ supplementation as a vitamin D source in nursery diets at 1,000 and 2,000 IU/kg diet showed the potential to improve feed intake and growth rate in the late nursery period, increased plasma 25-OHD₃ concentrations in a dose-dependent manner, and decreased plasma MDA levels in the late nursery period compared with VD₃ supplementation. The plasma MDA level was correlated with plasma vitamin D status and minimized when the plasma 25-OHD₃ level reached 23.7 ng/mL, that could be achieved by approximately 1,600 IU/kg 25-OHD₃ supplementation level. Because the current study only used two 25-OHD₃ supplementation levels with limited number of pigs, further studies are needed to demonstrate the optimal 25-OHD₃ supplementation levels with various supplementation levels in a larger scale research setting.