Different vitamin D supplementation strategies impact serum vitamin D concentrations and the mRNA expression of genes related to vitamin D metabolism, mitochondria respiration, redox balance, and immune system in weanling piglets

Abstract

This study compared the effects of different vitamin D supplementation strategies to pre- and postweaning piglets on vitamin D metabolism and health-related parameters. Sixty Yorkshire-Landrace × Duroc suckling piglets were selected at the first day of age and randomly assigned to one of two vitamin D supplementation strategies (n = 30 pigs per treatment): CTR—oral saline at days 2, 8, and 21 of age and, from weaning (day 21), in-feed supplementation with 2,000 IU of vitamin D as cholecalciferol; and VD—oral 25-hydroxycholecalciferol (25(OH)D₃) solution at days 2, 8, and 21 of age plus 15-min exposure to UVB light every second day from day 14 until day 21 and, from weaning, in-feed supplementation with 2,000 IU of vitamin D as 25(OH)D₃. Piglets were slaughtered (n = 10 pigs per treatment/day) at days 21 (before start in-feed experimental diets), and 28 and 35 and blood and tissues samples (jejunum, liver, and kidney) were collected. Body weight (BW), concentrations of serum 25(OH)D₃ and jejunum, liver, and kidney mRNA expression of genes related to vitamin D, antioxidant system, and immune defense were measured. Body weight was not affected by treatments (P ≥ 0.34). Serum 25(OH)D₃ concentrations were greater for VD piglets at day 21, 28, and 35 (P < 0.01). No effect of treatment was detected (P ≥ 0.14) for mRNA expression in the jejunum mucosa. In the liver of VD piglets, mRNA expressions of genes related to the antioxidant system were lower at day 21 (NDUFB2) and at day 28 (BNIP3, GPX4, and MSRA) (P ≤ 0.10). The mRNA analysis in kidney during the overall period detected higher expression of genes related to the mitochondria oxidative phosphorylation (COX17, NDUFB2, and NDUFB6) in VD groups compared with CTR (P ≤ 0.09). The expression of CYP27B1 in kidney was higher at day 28 and CYP24A1 was lower at day 21 but higher at day 35 for VD animals. In conclusion, during the preweaning period, dietary 25(OH)D₃ supplementation combined with UVB exposure was effective in increasing serum 25(OH)D₃ concentrations at weaning, whereas in the postweaning period, dietary 25(OH)D₃ supplementation at 2,000 IU/kg was more efficient then dietary cholecalciferol at similar levels. The overall results indicate that 2,000 IU of vitamin D/kg of diet, independently of source, may be enough to improve the vitamin D status of postweaning piglets. However, the use of dietary 25(OH)D₃ may promote a better modulation of vitamin D metabolism and redox balance.

Weaning stress imposes important challenges to the swine industry, especially in the perspective of the ban on the prophylactic use of antibiotics and the restrictions on the use of pharmacological levels of dietary zinc oxide. Dietary vitamin D may be an important tool to manage the high incidence of intestinal disturbances and metabolic unbalances related to weaning. This study shows that 2,000 IU of vitamin D/kg of diet, independently of source, may fulfills the postweaning piglet requirement for this vitamin. However, the use of 25(OH)D₃ may promote additional benefits in terms of better piglets redox balance.

Introduction

The ban on the prophylactic use of antibiotics and the restrictions on the use of pharmacological levels of dietary zinc oxide bring unprecedented challenges to the pig industry, especially during the postweaning period. Weaning stress is frequently followed by a high incidence of intestinal disturbances, bacterial infections, antioxidant unbalances, and energetic deficiencies that lead to important losses to the industry (Lallès et al., 2007; Lallès and Giulou, 2015; Xiong et al., 2015). In this context, vitamins nutrition can be an important tool considering their redox and/or immune modulatory effects (Lauridsen et al., 2021), which could help to ensure the robustness of newly weaned piglets.

In addition to the well-known role of vitamin D on calcium, phosphate, and magnesium metabolism (Crenshaw, 2000), in the last decades, many studies have reported other important metabolic functions of this vitamin (Murai et al., 2021; Zhang et al., 2023). For example, vitamin D receptors have been identified in a variety of tissues and are involved in the regulation of inflammatory and phagocytic responses (Baeke et al., 2010). According to Matte and Lauridsen (2022), suboptimal vitamin D provision may compromise the immunoregulatory functions at the sites of inflammation, whereas there are in vitro evidences that vitamin D modulates ATP synthesis in the mitochondria (Chudapongse and Lowchareonkul, 1975). In humans and pigs, studies found an association between low vitamin D levels and increased susceptibility to infections (Roth et al., 2008; Ginde et al. 2009; Untersmayr and Kallay, 2020; Madsen et al., 2023) and prechallenge supplementation was related to lower infection rates, lower severity, and reduced use of antibiotics (Sabetta et al., 2010; Norlin et al., 2016). However, little is known about the homeostasis of this vitamin and the potential effects on robustness in pigs.

According to Matte and Audet (2019), the perinatal transfer (placenta and colostrum) of vitamin D in pigs may be limited. In a recent study, Galiot et al. (2024) reported that oral supplementation with 25-hydroxycholecalciferol (25(OH)D₃) in early life rapidly enhanced serum concentration of 25(OH)D₃ in suckling piglets but failed to keep these high status until weaning. In contrast, the exposure to UVB light was efficient in securing high 25(OH)D₃ concentrations at weaning. Therefore, considering the abovementioned effects of vitamin D on health, it can be hypothesized that ensuring optimal vitamin D nutrition to pre- and postweaning piglets may benefit their immune system, redox balance, and growth performance.

The present study evaluated the impact of different vitamin D supplementation strategies to pre- and postweaning piglets on vitamin D metabolism, selected health markers, and growth.

Materials and Methods

The experimental procedures followed the guidelines of the Canadian Council on Animal Care (2009) and were approved by the Institutional Animal Care Committee of the Sherbrooke Research and Development Center (#598). All animals were cared for according to the recommended code of practice of the National Farm Animal Care Council (2014).

Animals and treatments

A total of 10 Yorkshire–Landrace sows were used in this study. Within each litter, six piglets (three per treatment) were selected based on bodyweight (BW) at the first day of age (1.71 ± 0.03 kg BW) and randomly assigned to one of two vitamin D supplementation strategies (n = 30 pigs per treatment): CTR—oral saline at days 2, 8, and 21 of age and, from weaning (day 21), in-feed supplementation with 2,000 IU of vitamin D as cholecalciferol; and VD—oral 25(OH)D₃ solution at days 2 (0.1 mg), 8 (0.2 mg), and 21 (0.3 mg) of age plus 15-min exposure to UVB light (0.06 uW/cm²) every second day from day 14 until day 21 and, from weaning, in-feed supplementation with 2,000 IU of vitamin D as 25(OH)D₃. For both supplementation strategies, other nutrients levels were higher than the NRC (2012) recommendations (Table 1). During the whole preweaning period pigs had free access to water and sows milk but not to creep-feeding and in the postweaning period pigs had free access to water and to the respective experimental diets. At weaning, piglets were group-housed in pens (1.0 × 1.8 m; three piglets per pen) according to BW and slaughtered (n = 10 pigs per treatment/day) at days 21 (before start in-feed experimental diets), 28, and 35. For euthanasia, piglets received an injection of Azaperone (Stresnil; 2.5 mg/kg) and were then stunned using a captive bolt stunner before bleeding.

Table 1.

Basal one-phase postweaning diet¹

Ingredients	Amount (%)
Corn	44.2
Soybean meal 48%	10.0
Oats	22.1
Plasma protein/oats mix	8.0
Whey protein	6.0
Soy protein concentrate	4.0
Monocalcium phosphate	1.3
Limestone	1.0
Soybean oil	1.0
Salt	0.4
Macro premix	2.0
Mineral and vitamin premix2	0.2

¹Basal diet contained 18.0% of protein, 0.74% of Ca, 0.67% of P, 223.9 ppm of Fe, 206.9 ppm of Zn, 11.0 ppm Cu, 92.6 ppm of Mn (analyzed values), and 3,152 Kcal/kg of ME (calculated value).

²Added per kilogram of feed: 10,000 IU vitamin A, 100 IU vitamin E, 5-mg vitamin K, 3-mg thiamine, 10-mg riboflavin, 35-mg niacin, 25-mg pantothenic acid, 6-mg pyridoxine, 0.3-mg biotin, 1.5-mg folic acid, 0.04-mg vitamin B₁₂, 250-mg choline, 1-mg iodine, 0.3-mg selenium.

Sampling

Blood samples were collected by venipuncture from the jugular vein prior to slaughter. After at least 4 hours standing at room temperature, whole blood samples were centrifuged at 1,800 × g for 10 min at 4 °C, and serum was collected, split into aliquots, and frozen at −20 °C. Body weight was individually recorded at day 1, 14 and at slaughter (days 21, 28, or 35). At slaughter, samples from the left lateral lobe of the liver and the left kidney were collected along with intestinal mucosal scrapings from the jejunum section. Samples were then rinsed in phosphate-buffered saline, snap-frozen in liquid nitrogen, and stored at −80 °C.

Vitamin D determination

Concentrations of 25(OH)D₃ were measured in samples of serum using an high performance liquid chromatography (HPLC) method adapted from Horst et al. (1981). Briefly, 500 µL of plasma were mixed with 200 µL of ethanol (100%) and 400 µL of isopropanol. Hexane (800 µL) was added before centrifuging at 1,800 × g for 5 min at 4 ^oC. After transferring the hexane-phase to another tube, 500 µL of methane was added followed by another round of centrifugation (1,800 × g for 5 min at 4 ^oC). The methanol-phase was transferred to another tube and evaporated using nitrogen. Finally, 125 µL of acetonitrile 65% was added, mixed, and transferred to a vial. Each sample was injected (125 µL) into an HPLC (LC 1260 System; Agilent Technologies; pre-column: MetaGuard Pursuit 5 C18 10 × 4.6 mm, Agilent; column: Agilent Pursuit 5 C18 150 × 4.6 mm, Agilent) with a isocratic mobile phase of 65% acetonitrile and 35% of water. All reagents were laboratory grade 99% pure, except if otherwise indicated in the text. The vitamin D standards (50-500 μg/mL) were prepared using 25-hydroxycholecalciferol (H4014; Sigma) diluted in anhydrous ethanol. Detection of vitamins D was performed at 264 nm and retention time was 7-10 min. The average intra- and interassay CV were 4.3% and 5.5%, respectively. Only CVs ≤ 10% between duplicates were accepted for assessing the average value of a sample.

RNA extraction, cDNA synthesis, and quantitative RT-PCR

The Qiagen columns Kit (RNeasy Qiagen Canada, Mississauga, ON) was used for total RNA extraction from 50-mg samples of jejunum mucosa, liver, and kidney. RNA extraction and cDNA synthesis were performed as previously described in Novais et al. (2021). Primers were designed (Supplementary Table 1) according to the National Center for Biotechnology Information database. Quantitative real-time PCR were performed in a 10-µL reaction volume using Power SYBR-Green Master Mix (Applied Biosystems) with the following cycling conditions: 10 min at 95 °C followed by 40 cycles at 95 °C for 15 s and at 60 °C for 45 s. Specificity of amplified fragments was verified with the melting curve analysis (Design and Analysis Software 2.6; Applied Biosystems). Amplifications were performed in triplicate. The cycle threshold (Ct; the number of cycles required for the fluorescent signal to cross the threshold) was determined for all studied and reference genes. The relative expression of each gene was then calculated using 2^−ΔCt, where ΔCt is the difference between the Ct of the genes of interest and the reference genes which were the least affected by treatment according to the NormFinder statistical algorithm (Andersen et al., 2004). The selected references genes were UBC for jejunum mucosa samples, RPL32 for liver samples, and RPL32 and H3F3A for kidney samples (Supplementary Table 1). Mean values from triplicates were used to perform the statistical analyses.

Statistical analysis

Data were analyzed using the SAS procedure for mixed models (SAS Inst. Inc., Cary, NC) (Littell et al., 1996) according to a 2 × 3 factorial arrangement of treatments with two dietary treatments (CTR and VD) and three ages at slaughter (21, 28, and 35 days of age) in the experimental period as the main independent variables. The piglet was considered as the experimental unit. The residual error term was used to test treatment effects. When appropriate, analyses with heterogeneous variances were performed. Differences were considered significant at P ≤ 0.05 and tendencies at 0.05 < P ≤ 0.10. All results are expressed as adjusted means ± SEM.

Results

Body weight was not affected by treatments at days 1, 14, 21, 28, or 35 (P ≥ 0.34). Average values were 1.71 ± 0.03, 5.52 ± 0.11, 7.50 ± 0.24, 8.74 ± 0.24, and 11.26 ± 0.24 kg at days 1, 14, 21, 28, and 35, respectively.

Serum 25(OH)D₃ concentrations were affected by treatments (treatment × age interaction; P < 0.01) (Figure 1) in which VD piglets had greater serum 25(OH)D₃ concentrations at days 21, 28, and 35.

Figure 1.

Serum 25(OH)D₃ concentrations (ng/mL) in weanling piglets according to dietary treatments and age at slaughter, presented as LS means ± SEM. Serum 25(OH)D₃ concentrations were greatest for VD groups at days 21, 28, and 35 (treatment × age interaction; P < 0.01). CTR—oral saline at days 2, 8, and 21 of age and, from weaning (day 21), in-feed supplementation with 2,000 IU of vitamin D as cholecalciferol; and VD—oral 25(OH)D₃ solution at days 2 (0.1 mg), 8 (0.2 mg), and 21 (0.3 mg) of age plus 15-min exposure to UVB light every second day from day 14 until day 21 and, from weaning, in-feed supplementation with 2,000 IU of vitamin D as 25(OH)D₃.

For mRNA expression analysis in jejunum mucosa, there was no effect of treatment or treatment × age interaction (P ≥ 0.14; Supplementary Table 2). The expression of most genes (31 out of 38) was affected by age at slaughter (P ≤ 0.03) (Supplementary Table 2). For vitamin D metabolism, the expression of CYP2R1 (related to the synthesis of 25(OH)D₃) and CYP27A1 (related to the degradation of cholecalciferol) were higher at days 28 and 35 compared with day 21. However, the mRNA expression of CYP27B1 (related to the synthesis of 1,25(OH)₂D₃) was lower at days 28 and 35 compared with day 21. From the 11 studied genes related to mitochondria respiration and antioxidant system, 8 genes had lower expression levels at day 28 and/or 35 compared with day 21. In terms of immune defense and inflammation, the expression of 14 out of the 21 studied genes was lower at days 28 and/or 35 compared with day 21.

No effect of treatment was detected (P ≥ 0.14; Supplementary Table 3) for the hepatic mRNA expression of the selected genes. Treatment × age interactions were detected for BNIP3 (modulates the permeability of the outer mitochondrial membrane; P = 0.09), GPX4 (protects cells against membrane lipid peroxidation; P = 0.10), MSRA (repairs oxidative damage to proteins; P = 0.05), and NDUFB2 (subunit of NADH dehydrogenase of the mitochondria respiratory chain; P = 0.10) in which the expressions of BNIP3, GPX4, and MSRA were lower for VD piglets at day 28, whereas for NDUFB2, it was lower at day 21 (Table 2). The expression of most genes (21 out of 26) was affected by age at slaughter (P ≤ 0.04) (Supplementary Table 3). For vitamin D metabolism, the expression of CYP2R1 was higher at day 28 and lower at day 35 compared with day 21, whereas CYP27A1 was higher at days 28 and 35 compared with day 21. From the 12 studied genes related to mitochondria respiration and antioxidant system, 10 had higher expression levels at days 28 and/or 35 compared with day 21. In terms of immune defense and inflammation, the expression of 5 genes was higher and of 6 genes was lower at days 28 and/or 35 compared with day 21.

Table 2.

Relative mRNA expression of selected hepatic genes affected by treatments in postweaning piglets, according to age at slaughter and dietary treatments

		Dietary treatment (mg/kg)			P-value
Gene	Age (days)	CTR	VD	SEM	Treatment effect	Age effect	Interaction effect
BNIP31	21	4.12	4.28	0.10	0.85	<0.01	0.09
	28	4.96a	4.73b	0.08
	35	4.85	4.88	0.07
GPX42	21	3.04	3.03	0.05	0.33	<0.01	0.10
	28	3.30a	3.10b	0.06
	35	2.94	3.00	0.07
MSRA1	21	4.59	4.55	0.07	0.82	0.04	0.05
	28	4.82a	4.64b	0.07
	35	4.47	4.66	0.07
NDUFB21	21	5.05a	4.96b	0.04	0.43	<0.01	0.10
	28	5.32	5.20	0.06
	35	5.07	5.17	0.06

CTR—oral saline at days 2, 8, and 21 of age and, from weaning (day 21), in-feed supplementation with 2,000 IU of vitamin D as cholecalciferol; and VD—oral 25(OH)D₃ solution at days 2 (0.1 mg), 8 (0.2 mg), and 21 (0.3 mg) of age plus 15-min exposure to UVB light every second day from day 14 until day 21 and, from weaning, in-feed supplementation with 2,000 IU of vitamin D as 25(OH)D₃.

¹For each specific age, different superscript letters within a row indicate differences between treatments using a Tukey adjustment (0.05 < P ≤ 0.10).

²For each specific age, different superscript letters within a row indicate differences between treatments using a Tukey adjustment (P ≤ 0.05).

For mRNA expression analysis in kidney, treatment effects were detected (P ≤ 0.09; Table 3) for COX17, NDUFB2, and NDUFB6, all genes related to the mitochondria respiratory chain, in which VD groups has higher mRNA expression compared with CTR groups. Treatment × age interactions were detected for CYP27B1 (related to the synthesis of 1,25(OH)₂D₃; P = 0.09) and CYP24A1 (related to the degradation of 1,25(OH)₂D₃; P < 0.01) in which the expression of CYP27B1 was higher for VD animals only at day 28, whereas for CYP24A1, it was lower for VD groups at day 21 and higher at day 35 (Table 3). The expression of most genes (22 out of 26) was affected by age at slaughter (P ≤ 0.10) (Supplementary Table 4). For vitamin D metabolism, the expression of FGFR1 (supresses vitamin D activation and stimulates degradation) was higher at day 35 compared with days 21 and 28 and that of KL (co‐receptor for FGF23 signaling) was higher at day 35 compared with day 28, whereas VDR (vitamin D receptor) was lower at days 28 and 35 compared with day 21. From the 9 studied genes related to mitochondria respiration and antioxidant system, 6 genes had lower expression levels at days 28 and/or 35 compared with days 21 and 3 were not affected by age at slaughter. In terms of immune defense and inflammation, the expression of 8 out of the 11 studied genes was lower at days 28 and/or 35 compared with day 21 (Supplementary Table 4).

Table 3.

Relative mRNA expression of selected genes affected by treatments in the kidney of postweaning piglets, according to age at slaughter and dietary treatments

		Dietary treatment (mg/kg)			P-value
Gene	Age (days)	CTR	VD	SEM	Treatment effect	Age effect	Interaction effect
COX17	21	2.55	2.62	0.06	0.09	<0.01	0.59
	28	2.57	2.60	0.06
	35	2.17	2.31	0.04
CYP24A11	21	6.26a	4.66b	0.31	0.06	0.03	<0.01
	28	5.83	4.88	0.42
	35	4.12b	5.02a	0.34
CYP27B11	21	6.49	6.48	0.19	0.09	0.10	0.09
	28	5.78b	6.60a	0.25
	35	6.61	6.65	0.17
NDUFB2	21	3.96	3.97	0.07	0.04	<0.01	0.37
	28	3.97	4.07	0.05
	35	3.54	3.70	0.04
NDUFB6	21	3.84	3.94	0.08	0.06	<0.01	0.70
	28	3.80	3.87	0.09
	35	3.39	3.58	0.06

CTR—oral saline at days 2, 8, and 21 of age and, from weaning (day 21), in-feed supplementation with 2,000 IU of vitamin D as cholecalciferol; and VD—oral 25(OH)D₃ solution at days 2 (0.1 mg), 8 (0.2 mg), and 21 (0.3 mg) of age plus 15-min exposure to UVB light every second day from day 14 until day 21 and, from weaning, in-feed supplementation with 2,000 IU of vitamin D as 25(OH)D₃.

¹For each specific age, different superscript letters within a row indicate differences between treatments using a Tukey adjustment (P ≤ 0.05).

Discussion

Vitamin D can be synthesized in the skin or absorbed from the diet. Ultraviolet B irradiation (UVB, 290–315 nm) catalyzes the synthesis of previtamin D₃ from 7-dehydrocholesterol (provitamin D₃) in the skin. In a heat-dependent reaction, previtamin D₃ is converted into cholecalciferol, which is transported to the liver bound to vitamin D-binding protein (Haddad et al., 1993). For dietary cholecalciferol, after uptake in the small intestine, it is incorporated into chylomicrons and transported in the lymph to the liver. In the liver, both dietary and endogenously synthesized cholecalciferol are hydroxylated to 25(OH)D₃, the circulatory form of the vitamin, that is further converted to 1,25(OH) ₂D₃, the active form of the vitamin, in the kidney (Adams et al., 1982; Bender, 2003). The efficiency of UVB light exposure to maintain a high concentration of 25(OH)D₃ and 1,25(OH)₂D₃ has been demonstrated before in pigs (Barnkob et al., 2016; Maruf et al., 2024; Galliot et al., 2024). Among the sources of dietary vitamin D, dietary 25(OH)D₃ was reported to be better absorbed at the intestinal level and to have greater bioavailability (Quesada-Gómez and Bouillon, 2018; Cesareo et al., 2019).

According to Galliot et al. (2024), oral supplementation with 25(OH)D₃ at days 2 and 8 after birth rapidly enhanced serum concentration of 25(OH)D₃ in suckling piglets, but these levels were not maintained until weaning. However, piglets exposed to UVB light during 15 min every second day from day 2 after birth had the highest serum concentration of 25(OH)D₃ at weaning. Therefore, in the present study in which the preweaning vitamin D supplementation aimed to enhance the piglets’ vitamin D status at weaning as a strategy to improve postweaning robustness, both dietary 25(OH)D₃ and UVB exposure were combined. Indeed, this combined vitamin D supplementation strategy during the suckling period was effective in increasing (by 2.8-fold) the serum 25(OH)D₃ concentration of piglets at weaning, compared with nonsupplemented piglets. After the first week postweaning (day 28), this difference was amplified to 3.6-fold but decreased to 2.2-fold at day 35. The greater difference at day 28 could be due to the better absorption/bioavailability of dietary 25(OH)D₃ compared with cholecalciferol fed during the postweaning period. However, it cannot be ruled out that this effect at day 28 is at least partially due to a potential residual effect of UVB exposure during the suckling period because, differently from dietary vitamin D sources that are rapidly metabolized in the liver, vitamin D synthesized in the skin after UVB light exposure is bound to plasma vitamin D-binding protein and is metabolized more gradually (Bender, 2003). Although this residual effect may have faded out at day 35, the 2.2-fold difference between treatments supports the better absorption/bioavailability of dietary 25(OH)D₃ compared with cholecalciferol, as discussed above.

According to the present results for genes related to vitamin D metabolism, these differences in serum 25(OH)D3 concentrations between treatments would not be modulated by the synthesis of 25(OH)D₃ in the liver but by the utilization of this circulatory form for the synthesis and/or degradation of the active form (1,25(OH)₂D₃) of the vitamin in the kidney. In fact, at day 21, the degradation of 1,25(OH)₂D₃ was down-regulated in VD piglets, whereas at day 28, its synthesis was up-regulated. If the residual effect of UVB exposure was indeed present at day 28, it could suggest that this alternative supplementation strategy may establish a better balance between utilization and degradation of 1,25(OH)₂D₃ because at day 35, when this eventual residual effect have apparently faded out, the mRNA expression of the gene VDR was reduced and that of genes FGFR1 and KL was enhanced in VD groups leading to a lower utilization and a greater degradation of the active vitamin D form, which was not detected at day 21 or 28.

Despite the greater vitamin D status, no beneficial effect of the VD treatment during the suckling period was detected on growth performance of healthy piglets at weaning, as also reported in recent studies (Flohr et al., 2016; Yang and Ma, 2021; Hasan et al., 2023; Galliot et al., 2024). In fact, not the direct supplementation of suckling piglets but maternal vitamin D supplementation appears to be more efficient in impacting piglets growth performance (Weber et al., 2014; Thayer et al., 2019; Zhang et al., 2019; Upadhaya et al., 2021, 2022), which could explain the present results as the same maternal diet was used for all sows. Regarding the postweaning period, the greater vitamin D status in VD piglets did not improve growth performance compared with piglets fed similar levels of cholecalciferol. According to the NRC (2012), the vitamin D requirement for 7-25 kg piglets would be 200-220 IU/kg, whereas typical vitamin D levels range between 2,200 and 3,500 IU/kg in commercial diets (Dalto et al., 2020; Yang et al., 2021; Faccin et al., 2023). Despite the higher vitamin status in VD piglets, the serum 25(OH)D₃ concentrations in CTR animals increased with time, indicating that the use of 2,000 IU/kg of vitamin D, independently of source, enhances the piglets serum 25(OH)D₃ concentrations in healthy postweaning piglets.

The most known function of the active form of vitamin D is the maintenance of Ca homeostasis, but in the last decades, many studies have reported other important metabolic functions (Dittmer and Thompson, 2011). In the present study, the mRNA expression results suggest a better redox balance in VD piglets. Despite the absence of VD treatment effect on genes expression in jejunum, the hepatic genes BNIP3, GPX4, and MSRA (all related to redox balance) were less expressed in VD compared with CTR piglets, suggesting that these animals had lower levels of oxidative stress. Additionally, in kidney, the higher expression of COX17, NDUFB2, and NDUFB6 in VD animals, all related to the mitochondria respiratory chain, suggest a stimulus to the mitochondria oxidative phosphorylation for the synthesis of ATP. These results are in line with Zhang et al. (2023) who reported the effects of 25(OH)D₃ in modulating the antioxidant system of pigs and with many studies in humans (Manson et al., 2019; Murai et al., 2021) that have shown the antioxidant mechanisms of vitamin D to be related to its membrane and lipoprotein scavenger activities. To the best of our knowledge, this is the first study reporting effects of vitamin D on the regulation of the mitochondria respiratory chain in pigs. According to Chudapongse and Lowchareonkul (1975), cholecalciferol may inhibit ATP synthesis in rats, which apparently contrasts with the present results. Therefore, more studies are needed to better characterize these effects.

Although no effect of vitamin D supplementation on the mRNA expression of genes related to the immune response was detected in the present study, many studies in pigs and other species have described effects of vitamin D, specially in the form of 25(OH)D₃, on the humoral and innate immune response, gut immunity, and modulation of systemic and mucosal antimicrobial reactions (Tanaka et al., 2014; Fields et al., 2019; Upadhaya et al., 2022, Madsen et al., 2023). As discussed above, it cannot be ruled out that the absence of VD effects in the present study may be due to the levels of vitamin D in CTR diet (2,000 IU/kg) that have likely been sufficient to potentiate the metabolism of this vitamin in healthy postweaning piglets. In fact, Wierzbicka et al. (2024) did not detect any difference in miRNA expression in the lungs of pigs supplemented with 2,000 IU/kg of cholecalciferol or 25(OH)D₃. In a recent review, Hasan et al. (2023) described that, in pigs supplemented with 2,000 IU/kg of both cholecalciferol or 25(OH)D₃, most effects were observed on the concentrations of immunoglobulins, leucocytes, monocytes, granulocytes, and interleukins in serum.

In addition to the effects of treatments on the studied parameters, the detected time effects provide important insights about the development of the vitamin D metabolism in healthy weanling piglets. In jejunum, there was a potential increase in 25(OH)D₃ and decrease in 1,25(OH)₂D₃ synthesis, whereas the hepatic synthesis of 25(OH)D₃ was apparently reduced. In kidney, the results suggest higher synthesis but lower utilization of 1,25(OH)₂D₃. Considering that the intestinal tissue is not known as an important site for the synthesis of 25(OH)D₃ and 1,25(OH)₂D₃, the relevance of these results remains to be further explored. In liver, that is the main site for the synthesis of 25(OH)D₃, the present results suggest that vitamin D requirements may decrease over time during the postweaning period or, as discussed above and supported by kidney results, that the dietary vitamin D levels used in the present study were higher than the piglets requirements.

Time effects were also detected on the modulation of the antioxidant and immune systems with an improved antioxidant balance and reduced inflammation from weaning. It has been demonstrated that oxidative stress conditions occur in weaned piglets independently of weaning age (Zhu et al., 2012; Buchet et al., 2017; Cao et al., 2018). Novais et al. (2020; 2021) reported that mitochondrial dysfunction, cellular oxidative stress, and inflammation are induced in the first days postweaning in piglets of 21 days of age and lasted for at least 2 weeks, which is in contrast to the present better redox balance and reduced inflammation. Considering that in Novais et al. (2020; 2021), no treatment was used to modulate those functions, whereas in the present study, high levels of vitamin D, which are known to affect these parameters, were fed to piglets in both treatments, these results are in line with the above reasoning that 2,000 IU vitamin D/kg, independently of source, enhance the piglets serum 25(OH)D₃ concentrations in healthy postweaning piglets.

Conclusions

During the preweaning period, oral 25(OH)D₃ supplementation combined with UVB exposure was effective in increasing serum 25(OH)D₃ concentrations at weaning, whereas in the postweaning period, the dietary 25(OH)D₃ supplementation at 2,000 UI/kg was more efficient than dietary cholecalciferol at similar levels. The greater vitamin D status in VD piglets apparently improved the antioxidant response of these animals but had limited effects on piglets growth and immune system at weaning and in the postweaning period.

Glossary

Abbreviations

25(OH)D₃

25-hydroxycholecalciferol

BNIP3

BCL2 Interacting Protein 3

BW

body weight

COX17

Cytochrome C Oxidase Copper Chaperone COX17

Ct

cycle threshold

CV

coefficient of variation

CYP24A1

Cytochrome P450 Family 24 Subfamily A Member 1

CYP27A1

Cytochrome P450 Family 27 Subfamily A Member 1

CYP27B1

Cytochrome P450 Family 27 Subfamily B Member 1

CYP2R1

Cytochrome P450 Family 2 Subfamily R Member 1

FGFR1

Fibroblast Growth Factor Receptor 1

GPX4

glutathione peroxidase 4

H3F3A

Histone H3, family 3A

KL

klotho

MSRA

Methionine Sulfoxide Reductase A

NDUFB2 - NADH

Ubiquinone Oxidoreductase Subunit B2

NDUFB6 - NADH

Ubiquinone Oxidoreductase Subunit B6

NRC

National Research Council

PBS

phosphate-buffered saline

RPL32

60S ribosomal protein L32

UBC

ubiquitin C

VDR

vitamin D receptor

Vitamin D₃

cholecalciferol

Author contributions

Danyel Dalto (Conceptualization, Data curation, Investigation, Methodology, Project administration, Supervision, Writing—original draft), Danyel Dalto (Conceptualization, Data curation, Investigation, Methodology, Project administration, Supervision, Writing—original draft, Writing—review & editing), Caroline Roy (Data curation, Formal analysis, Writing—review & editing), Geneviève Villeneuve (Data curation, Formal analysis, Writing—review & editing), Isabelle Audet (Data curation, Formal analysis, Writing—review & editing), J. Jacques Matte (Conceptualization, Funding acquisition, Investigation, Methodology, Writing—review & editing), and Jerome Lapointe (Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Writing—review & editing)

Funding

This research was funded by Swine Innovation Porc grant number 1795, DSM-Firmenich, Lallemand, Centre de Recherche en Sciences Animales de Deschambault, Agri-Marché, and Agriculture and Agri-Food Canada.

Conflict of interest statement

There is no conflict of interest for all authors that could be perceived as prejudicing the impartiality of the research reported.