Effect of vitamin D source and amount on vitamin D status and response to endotoxin challenge

The objectives were to test the effects of dietary vitamin D 3 [cholecalciferol (CHOL)] compared with 25-hydroxyvitamin D 3 [calcidiol (CAL)] on vitamin D status and response to an endotoxin challenge. Forty-five Holstein bull calves (5 ± 2 d of age) were blocked into weekly cohorts, fed a basal diet that provided 0.25 µg/kg body weight (BW) CHOL, and assigned randomly to 1 of 5 treatments: control [(CON) no additional vitamin D], 1.5 µg/kg BW CHOL (CHOL1.5), 3 µg/kg BW CHOL (CHOL3), 1.5 µg/kg BW CAL (CAL1.5), or 3 µg/kg BW CAL (CAL3). Calves were fed milk replacer until weaning at 56 d of age and had ad libitum access to water and starter grain throughout the experiment. Treatments were added daily to the diet of milk replacer until weaning and starter grain after weaning. Measures of growth, dry matter intake, and serum concentrations of vitamin D, Ca, Mg, and P were collected from 0 to 91 d of the experiment. At 91 d of the experiment, calves received an intravenous injection of 0.1 µg/kg BW lipopolysaccharide (LPS). Clinical and physiological responses were measured from 0 to 72 h relative to LPS injection. Data were analyzed with mixed models that included fixed effects of treatment and time, and random effect of block. Orthogonal contrasts evaluated the effects of (1) source (CAL vs. CHOL), (2) dose (1.5 vs. 3.0 µg/ kg BW), (3) interaction between source and dose, and (4) supplementation (CON vs. all other treatments) of vitamin D. From 21 to 91 d of the experiment, mean BW of supplemented calves was less compared with CON calves, but the effect was predominantly a result of the CHOL calves, which tended to weigh less than the CAL calves. Supplementing vitamin D increased concentrations of 25-hydroxyvitamin D in serum compared with CON, but the increment from increasing the dose from 1.5 to 3.0 µg/kg BW was greater for CAL compared with CHOL (CON = 18.9, CHOL = 24.7 and 29.6, CAL = 35.6 and 65.7 ± 3.2 ng/mL, respectively). Feeding CAL also increased serum Ca and P compared with CHOL. An interaction between source and dose of treatment was observed for rectal temperature and derivatives of reactive metabolites after LPS challenge because calves receiving CHOL3 and CAL1.5 had lower rectal temperatures and plasma derivatives of reactive metabolites compared with calves receiving CHOL1.5 and CAL3. Supplementing vitamin D increased plasma P concentrations post-LPS challenge compared with CON, but plasma concentrations of Ca, Mg, fatty acids, glucose, β-hydroxybutyrate, haptoglobin, tumor necrosis factor-α, and antioxidant potential did not differ among treatments post-LPS challenge. Last, supple-menting vitamin D increased granulocytes as a percentage of blood leukocytes post-LPS challenge compared with CON. Supplementing CAL as a source of vitamin D to dairy calves was more effective at increasing serum 25-hydroxyvitamin D, Ca, and P concentrations compared with feeding CHOL. Supplemental source and dose of vitamin D also influenced responses to the LPS challenge.


INTRODUCTION
Vitamin D is critical for the proper growth and development of calves (Bechdel et al., 1937).The vitamin D receptor is present in most tissues of the body and contributes to multiple physiological processes beyond mineral homeostasis and skeletal development, such as immune signals, antioxidant systems, and cellular differentiation (Norman, 2008).Consequently, it is critical to manage and feed calves so that vitamin D supplies are adequate to support the multiple physiological actions of the vitamin D hormone, 1,25-dihyroxyvitamin D 3 [1,25(OH) Cattle naturally acquire most of their vitamin D from conversion of 7-dehydrocholesterol to vitamin D 3 , also known as cholecalciferol (CHOL), in skin exposed to UV B light (Hymøller and Jensen, 2010).Cholecalciferol is readily converted to 25-hydroxyvitamin D 3 [25(OH) D 3 ] by hepatic 25-hydroxylases, and the 25(OH)D 3 is subsequently converted to the active metabolite 1,25(OH) 2 D 3 by renal and extrarenal 1α-hydroxylases in a tightly controlled manner (Sommerfeldt et al., 1983).The 25(OH)D 3 is distributed via the circulatory system, bound predominantly to vitamin D binding proteins to supply the endocrine and intracrine vitamin D signaling systems of the body (Hymøller and Jensen, 2017).The concentration of 25-hydroxyvitamin D {[25(OH)D] the combination of 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3 } in serum or plasma is the best indicator of vitamin D status of an animal (Horst and Littledike, 1982).Serum or plasma 25(OH) D concentrations <20 ng/mL are considered deficient for proper mineral homeostasis and bone development; however, scientists have reasoned that a serum 25(OH) D level >30 ng/mL is necessary for optimal immunity and overall health (Hollis, 2005;Holick, 2011).
Most dairy calves receive supplemental CHOL because season or housing may prevent adequate sun exposure for vitamin D synthesis (Nelson et al., 2016a).The NRC Nutrient Requirements for Dairy Cattle (NRC, 2001) recommended dairy calves receive 15 µg CHOL/kg DM milk replacer (1 µg CHOL = 40 IU vitamin D), which meets the requirement to prevent rickets in growing calves (Bechdel et al., 1937).The National Academy for Science, Engineering and Medicine (NAS-EM, 2021) increased the vitamin D recommendation for dairy calves to 80 µg CHOL/kg DM milk replacer (0.8 µg/kg BW) with the intent of maintaining a serum 25(OH)D level >30 ng/mL.Despite recommendations, serum 25(OH)D concentrations of dairy calves are quite variable under current dairy management practices.For example, calves housed indoors and fed pasteurized waste milk without supplemental vitamin D had a serum 25(OH)D level <10 ng/mL (Krueger et al., 2014;Blakely et al., 2019).Cholecalciferol is commonly supplemented in calf milk replacers at 125 to 250 µg/ kg DM, and commercial dairy calves fed milk replacer had 40 to 100 ng/mL serum 25(OH)D (Nelson et al., 2016a).In comparison, serum 25(OH)D concentrations of calves outdoors in summer are typically between 30 and 60 ng/mL (Nelson et al., 2016b).Although current recommendations and practices are deemed adequate for maintaining sufficient vitamin D status, evidence in support of current recommendations and practices is scarce.
Recent reports have indicated that dietary 25(OH) D 3 , also known as calcidiol (CAL), is more effective at increasing concentrations of 25(OH)D in serum compared with equal amounts of dietary CHOL (Rodney et al., 2018;Poindexter et al., 2020).Feeding CAL also had positive immune effects in dairy cows (Vieira-Neto et al., 2021).Because vitamin D signaling contributes to antiinflammatory and antioxidant systems (Chen et al., 2015b;Xu et al., 2015), we hypothesized that supplementing diets of dairy calves with CAL would be more effective at elevating serum 25(OH)D levels compared with supplemental CHOL and, consequently, would attenuate the inflammatory response to an endotoxin challenge.The main objectives of the experiment were to compare effects of vitamin D source (CHOL vs. CAL) and amount (1.5 vs. 3.0 µg/kg BW) on serum 25(OH)D levels and responses to intravenous LPS injection as a model of an acute endotoxin challenge.

Sample Size Calculation
A sample size was calculated using the POWER procedure of SAS/STAT version 9.4 (SAS Institute Inc.) to detect a difference in serum concentrations of 25(OH)D of 40 ng/mL, with a standard deviation (SD) of 15 ng/ mL.A 2-sided sample size was calculated, adjusting α to 0.01 by dividing the critical threshold of 0.05 by 5. Therefore, the sample size calculation (α = 0.01, β = 0.80) resulted in a minimum of 6 calves per treatment.For the LPS challenge model, the sample size was calculated considering an increase in rectal temperature of 1.6°C, with an SD of 0.2°C, in response to an LPS challenge (Carroll et al., 2009).The sample size anticipated 7 calves/group would allow us to detect a difference in rectal temperature of 0.27°C after an LPS challenge (2-sided test; α = 0.01, β = 0.80) among treatments.

Diets and Management of Calves
Care and treatment of calves was approved by the University of Florida Institutional Animal Care and Use Committee (Protocol No. 201709749).Forty-five Holstein bull calves born between September and December 2017 at the University of Florida Dairy Research Unit were enrolled in the experiment (Table 1).Calves were removed from the dam and fed 3.8 L colostrum (mean Brix score, 24 ± 3%) within 4 h of birth.Calves were kept in individual wire pens bedded with sand bottoms in a shed with shadecloth curtains until 63 d of age.From birth until 14 d of age, calves were fed 0.34 kg DM of milk replacer (Land O Lakes Amplifier Max, Purina Animal Nutrition LLC) without supplemental vitamin D (Table 2) in 2 L twice daily (0600 and 1500 h) for a total of 0.68 kg of DM milk replacer per day.
Calves were fed 0.45 kg DM of milk replacer in 3 L twice daily for a total of 0.9 kg DM milk replacer per day from 14 to 49 d of age and once daily from 49 to 56 d of age.The milk replacer was mixed in 46°C water using an electric drill with a whisk and was cooled to 38 ± 1°C for feeding.Ad libitum access to starter pellets (Purina; 22% CP, 4% fat, no vitamin D; Table 2) and fresh water were provided via bucket, with offers and refusals of grain measured daily.
After weaning, calves were moved to a pen equipped with a Calan Broadbent Feeding System (American Calan) inside a shed with shadecloth curtains, and calves continued to receive ad libitum starter grain and water.At 96 ± 2 d of age, calves were relocated to a facility with individual tiestalls equipped with rubber mats and bedded with wood shavings to perform the intravenous LPS injection.
Calves were vaccinated and treated by farm personnel according to protocols established by the herd veterinarian.Diarrhea was treated with electrolytes (Gener-Lyte, Bio-Vet Inc.) and bismuth subsalicylate (Bismusol, First Priority Inc.).Respiratory disease and otitis were treated with florfenicol (Nuflor, Merck Animal Health).Calves were vaccinated with Bovi-Shield Gold (Zoetis) at 3 and 6 wk of age, and Ultrabac (Zoetis) at 5 and 7 wk of age.Calves were dehorned at 3 wk of age.

Experimental Design and Treatments
The experiment was a randomized complete block design.Nine cohorts of 5 calves were enrolled weekly at 4.4 ± 3.1 d of age.Within each cohort, calves were supplied 0.25 µg/kg BW CHOL as a base amount of vitamin D and were assigned randomly by research personnel to 1 of 5 treatments of additional vitamin D supplement: control (CON; no additional vitamin D), 1.5 µg/kg BW CHOL (CHOL1.5;n = 9), 3 µg/ kg BW CHOL (CHOL3; n = 9), 1.5 µg/kg BW CAL (CAL1.5;n = 9), or 3 µg/kg BW CAL (CAL3; n = 9).Randomization was accomplished using a random number generator in Excel (Microsoft Corp.).The 0.25 µg/kg BW CHOL treatment was added daily to the milk replacer (preweaning) or starter (postweaning) grain for all calves to meet minimum vitamin D requirement to prevent rickets in calves.The additional CHOL and CAL were added daily by research personnel to the milk replacer or starter grain.Research personnel were not blinded to treatments.The vitamin D was prepared from dry powder concentrates containing 1.25% CHOL (Rovimix D3 500, DSM Nutritional Products, Inc.) or 1.25% CAL (Hy-D 1.25%, DSM Nutritional Products) by adding 0.5 g of the concentrate to 50 mL distilled water.The supplements were prepared fresh weekly and stored at 4°C.The amount of supplement provided to each calf was determined based on the weekly average BW of the cohort.

Growth and Health Measurements
Body weight and wither height were collected at birth, weekly from 0 to 91 d of the experiment and every 2 wk from 91 d until the end of the experiment.Calves were evaluated daily for nasal and eye discharge, cough, ear score, and fecal consistency after the morning feeding by research personnel using a 4-point scale (0 = normal to 3 = severe condition) according to the University of Wisconsin's Calf Health Score Chart (McGuirk and Peek, 2014).Incidence and treatment of clinical diseases (i.e., diarrhea, score ≥2; respiratory disease, cough + nasal + temperature score ≥5) diagnosed by farm staff who were blinded to the experimental treatments, were also recorded daily.

Sample Collection Before LPS Challenge
Blood was sampled via jugular venipuncture into evacuated 10-mL serum tubes with a clot activator (Becton Dickinson) weekly from 0 to 35 d of the experi-ment, and every 2 wk from 35 to 91 d of the experiment.Samples were centrifuged at 1,500 × g for 15 min at 4°C and stored in 1-mL aliquots at −20°C until analysis.
3 Body weight analyzed as repeated measures of BW collected every 7 d from enrollment to 91 d of experiment with LSM for BW every 14 d starting at d 21 shown.Model included fixed effects of day (P < 0.001), and interactions between treatment and day (P = 0.99) and BW.Body weight at enrollment was used as a covariate in the models. 4Starter intake was analyzed as repeated measures for weekly averages calculated from 21 to 91 d of the experiment.Model included fixed effects of day (P < 0.001), and interactions between treatment and day (P = 0.99).The LSM for treatment for 21 to 91 d and treatment by day LSM every 14 d are shown.Calves were transitioned from individual pens to a group pen with individual feeding gates at 63 to 77 d of age. 5 Total amount of vitamin D supplied calculated using weekly BW of individual calves.
Plasma sampled at 0, 1, 2, 4, 6, 8, 12, 24, 48, and 72 h relative to LPS challenge was analyzed for total Ca, Mg, P, FA, BHB, and glucose using an automated biochemical analyzer (RX Daytona, Randox Laboratories Ltd.) according to the manufacturer's instructions.Plasma haptoglobin concentrations during the LPS challenge were measured from the peroxidase activity of haptoglobin-hemoglobin complexing (Makimura and Suzuki, 1982).Absolute absorbance values were set at 450 nm and then multiplied by 100 for statistical analysis.Intra-and interassay coefficients of variation were 0.97% and 0.82%, respectively.Plasma tumor necrosis factor (TNF)-α was measured at 0, 1, 2, 4, and 6 h relative to the LPS challenge using a commercially available ELISA kit (Vet Sets ELISA Development Kit, Kingfisher Biotech Inc.).
Derivatives of reactive oxygen metabolites (dROM) and the antioxidant potential (AOP) of plasma samples collected at −12, −0.5, 0, 1, 2, 4, 6, 8, 12, 24, 48, and 72 h relative to the LPS challenge were measured using the FRAS-5 system (H&D S.r.l.) with kits for dROM and AOP tests according to manufacturer instructions.The dROM measure concentrations of hydrogen peroxides in plasma by photometric measurement of oxidized diethyl-para-phenylenediamine absorption at 505 nm.Briefly, 10 µL of plasma was added to dROM kit reagent containing diethyl-para-phenylenediamine in duplicate, mixed gently for 10 s, and measured with the FRAS-5 photometer.Likewise, the AOP assay measures capacity to reduce ferric iron in plasma by photometric absorption at 505 nm.Ten microliters plasma was added to the AOP reagent, mixed gently for 10 s, and measured with the FRAS-5 photometer.The dROM and AOP values are reported in equivalents of H 2 O 2 and ascorbic acid, respectively.All assays for each block of calves were performed on the same day.Plasma samples were stored at −20°C immediately after collection and not thawed until time of assay.

Flow Cytometry
Blood samples from 0, 1, 2, 4, 6, 8, 12, 24, 48, and 72 h after LPS infection were analyzed by flow cytometry for leukocyte count and CD14, CD11b, and CD62L expression.Aliquots of heparinized blood (100 µL) were placed into 12 × 75-mm culture tubes, and red blood cells were removed by incubation in hypotonic buffer.Cells were then incubated in PBS with antibodies for CD11b (MM10A, Washington State University) conjugated to allophycocyanin (Bio-Rad), CD14 (Tuk4, Thermo Fisher Scientific) conjugated to phycoerythrin-Cy5.5 (Thermo Fisher Scientific), and CD62L (BAQ92A, Washington State University) conjugated to phycoerythrin (Abcam) for 15 min on ice.Cells were analyzed on the basis of size, granularity, and fluorescence intensity of allophycocyanin, phycoerythrin, and phycoerythrin-Cy5.5 using an Accuri C6 flow cytometer (BD Biosciences) equipped with 488and 640-nm lasers.Flow cytometry data were analyzed with FCS Express 6.0 (De Novo Software).Granulocyte and mononuclear cell populations were gated on the basis of size and granularity.Within mononuclear cells, monocytes and B cells were identified by the presence of CD14 and CD21, respectively.Expression of CD11b and CD62L on granulocytes, monocytes, B cells, and CD21 − lymphocytes was indicated by the median fluorescence intensity of respective markers.
Neutrophil oxidative burst and phagocytosis activities were measured at 0, 8, and 24 h relative to the LPS challenge using methods described previously (Martinez et al., 2012).Briefly, 100 µL of whole blood was incubated with 10 µL of 50 µM dihydrorhodamine 123 solution (Sigma-Aldrich) for 10 min at 37°C.Heat-killed E. coli 08:H19 (strain KCJ852) labeled with propidium iodide were added to 100 µL of the sample (in duplicates) to achieve a bacterium-to-neutrophil ratio of approximately 40:1.Samples without dihydrorhodamine 123 and without E. coli were used as negative controls.Samples were incubated for 30 min at 37°C with continuous mixing.Cells were placed on ice to stop neutrophil phagocytosis and oxidative burst activities.Red blood cells were removed using an automated lysing system (Q-Prep Epics Immunology Workstation, Coulter Corp.).Samples were analyzed using an Accuri C6 flow cytometer (Becton Dickinson).Neutrophils were gated on the basis of size and granularity.Median fluorescence intensities of rhodamine 123 and propidium iodide as indicators of neutrophil oxidative burst and phagocytosis activities were determined with FCS Express 6.0 (De Novo Software).

Statistical Analysis
The experiment followed a randomized block design, and calf was the experimental unit.Continuous data were analyzed with mixed models with the MIXED procedure of SAS version 9.4 (SAS Institute Inc.).The models included fixed effects of treatment, and random effect of block.For repeated measures, models included fixed effects of time and interaction of treatment and time, and random effects of block and calf nested within treatment.Residuals of each continuous dependent variable were examined for normality after fitting the statistical models.Age and BW at the start of treatment were included as covariates in the initial models, along with measurements collected at d 0 for a given variable (i.e., serum Ca), and were retained in the final models when P < 0.1.The covariance structure with the least Akaike's information criterion was chosen, and most analyses used the first-order autoregressive structure for equally spaced measurements, or spatial power for unequally spaced measurements.The Kenward-Roger method was used to compute the approxi-mate denominator degrees of freedom for the F tests in the statistical models.Preplanned contrasts were performed to test the effects of (1) source (CAL vs. CHOL), ( 2) dose (1.5 vs. 3.0 µg/kg BW), (3) interaction between source and dose, and (4) supplementation (CON vs. all other treatments).Treatment differences with P ≤ 0.05 were considered significant, whereas tendencies for differences were reported if 0.05 < P ≤ 0.10.Simple linear regression analyses were performed post hoc for the relationship between serum 25(OH)D and other blood measures when trends in the data were apparent [i.e., the relationship between serum 25(OH) D and plasma AOP].

RESULTS
Of the 45 bulls enrolled, data from 42 were used for analyses before the LPS challenge.Three calves, one each in the CHOL1.5,CHOL3.0, and CAL 1.5 treatments, died within the first week of the experiment because of diarrhea and were excluded from all analyses.Data from 1 calf in the CHOL1.5 group that was euthanized at 72 d because of chronic illness were included in analyses of data collected from 0 to 72 d of the experiment.

Growth, Feed Intake, and Health
The BW of supplemented calves was less (P = 0.04) compared with CON calves from 21 to 91 d, but the effect was predominantly a result of CHOL calves, which tended to weigh less (P = 0.06) than CAL calves (Table 3).The effect of treatment on BW gain was most apparent preweaning, from 0 to 49 d of the experiment, when supplemented calves had lower (P = 0.02) weight gains compared with CON calves (Table 3); supplementing CHOL calves tended to result in lower (P = 0.06) weight gains compared with CAL calves.The lower BW gains corresponded to less (P = 0.006) starter intake for supplemented calves compared with CON calves (Table 3).Moreover, feeding CHOL resulted in lower (P = 0.02) intakes compared with CAL (Table 3).
The number of days with fecal consistency and eye discharge scores of 2 or more did not differ among groups (Supplemental Table S1, https: / / original -ufdc .uflib.ufl.edu// IR00011930/ 00001).The number of days with nasal discharge, cough, and ear scores of 2 or more was <3 d on average.Seventy-nine percent of calves were treated for diarrhea within the first 28 d of the experiment (Supplemental Table S1).Two calves were treated for respiratory infection and 3 calves were treated for otitis.

Serum 25(OH)D, Minerals, and Metabolites Before LPS Challenge
Serum 25(OH)D levels of CON calves decreased from 18.0 ± 5.0 ng/mL at the start of the experiment to 8.3 ± 9.9 ng/mL at 91 d (Figure 1A).In contrast, serum 25(OH)D remained constant or increased (P < 0.001) over time in CHOL-and CAL-supplemented calves (Figure 1A).Moreover, the increment in serum 25(OH)D from increasing the dose from 1.5 to 3.0 µg/kg BW was greater (P < 0.001) for CAL compared with CHOL (Table 4).Serum 25(OH)D levels increased 5.2 ± 1.3 ng/mL for each microgram per kilogram BW of total vitamin D for calves receiving CHOL, whereas serum 25(OH)D levels increased 19.5 ± 1.9 ng/mL for each µg/kg BW of total vitamin D for calves receiving CAL (Figure 1B).Concentrations of 25(OH)D 3 in serum measured by liquid chromatography-MS/MS were similar to total 25(OH)D levels measured by ELISA (Table 4).Feeding CAL increased (P < 0.05) serum Ca and P concentrations compared with CHOL (Table 4).Concentrations of Mg, BHB, glucose FA, IgG1, and IgG2 in serum did not differ among treatments (Table 4).

Responses to the LPS Challenge
Data from 38 calves (n = 6 CHOL1.5, n = 8 for each of the other treatments) were used for analyses of data collected from the LPS challenge.Three calves were withheld from the LPS challenge because they were diagnosed with respiratory infections just before the challenge.
The LPS challenge induced a rapid response with increased rectal temperature and respiration rate (Figure 2).The change in rectal temperature over time tended (P = 0.06) to be affected by vitamin D treatment (Figure 2A).Moreover, rectal temperatures after the LPS challenge were affected by the interaction (P = 0.008) of source and dose, because CHOL3 and CAL1.5 were less than CHOL1.5 and CAL3 (Table 5).Feed intake and BW gain during the LPS challenge did not differ among vitamin D treatments (Table 5).Plasma Ca, Mg, and P concentrations decreased (P < 0.01) during the first 8 h after challenge and then returned to normal values by 24 h (Figure 3).Plasma Ca and Mg did not differ among vitamin D treatments; however, plasma P was less (P = 0.01) for CON compared with other treatments during the LPS challenge (Table 6, Figure 4C).Plasma BHB, FA, and glucose did not differ among vitamin D treatments during the LPS challenge (Table 6).
Plasma TNF-α increased (P < 0.001) rapidly after the LPS challenge, but did not differ among treatments during the LPS challenge (Table 6, Figure 4).Plasma haptoglobin and AOP also did not differ among treatments (Table 6).Plasma dROM was affected by the interaction of source by dose (P = 0.01) during the LPS challenge, because plasma dROM of CHOL1.5 and CAL3 were less compared with CHOL3 and CAL1.5 (Table 6).However, the interaction was influenced largely by increased plasma dROM of CHOL1.5 calves.
The concentration of leukocytes in blood was the least at 4 h (3,022 ± 1,040 cells/µL) and greatest at 48 h (10,388 ± 1,141 cells/µL; effect of time, P < 0.001), but leukocyte concentrations did not differ among treatments during the LPS challenge (Table 7).Monocytes and granulocytes as percentages of total leukocytes decreased after the LPS challenge (monocytes, 3.4 ± 1.5% at 1 h; granulocytes, 23.5 ± 3.2% at 8 h), but then rebounded within a few hours.In contrast, B cells as a percentage of leukocytes decreased (P < 0.001) over time to a minimum of 22.5 ± 5.9% at 48 h (Figure 5C).The percentage of monocytes did not differ among treatments during the LPS challenge (Table 7).However, the percentage of granulocytes during the LPS challenge was less (P = 0.03) for CON compared with other treatments (Table 7, Figure 5B).In contrast to granulocytes, the percentages of B cells and CD21 − lymphocytes were greater (P < 0.07) for CON compared with other treatments (Table 7).

DISCUSSION
The objectives of this experiment were to test 2 sources of vitamin D (CHOL vs. CAL) at 2 levels (1.5 vs. 3 µg/kg BW + 0.25 µg CHOL/kg BW) compared with CON on vitamin D status and immune responses to the LPS challenge of calves.The data support the hypotheses that supplemental vitamin D source and amount influence vitamin D status and immune responses of calves, albeit the effect on immune responses was relatively small.In addition, although effects of vitamin D supplementation on growth were not a main objective of this experiment, supplemental vitamin D decreased BW gain and starter intake before weaning compared with CON.
Feeding CAL increased serum 25(OH)D levels more effectively than CHOL.The concentration of 25(OH)D in serum or plasma is the best indicator of vitamin D status.The 25(OH)D metabolites also supply vitamin D signaling processes of the various tissues (Hewison, 2012).Several 25-hydroxylases readily convert vitamin D to 25(OH)D in the liver, and the 25(OH)D metabolites circulate in blood with a half-life of approximately 2 to 4 wk (Wilkens et al., 2013).Similarly, feeding CAL was more effective than CHOL at increasing concentrations of 25(OH)D in serum in dairy cows (Guo et al., 2018;Rodney et al., 2018;Poindexter et al., 2020) and other species (Lauridsen et al., 2010;Han et al., 2016).The improved efficacy of CAL compared with CHOL was possibly the result of improved intestinal absorption (Soares et al., 1995).Cholecalciferol is primarily incorporated into micelles taken up by chylomicrons in the intestine; but CAL, a more polar metabolite, does not depend on the presence micelles in the intestine (Nechama et al., 1978).Alternatively, inefficient hepatic conversion of CHOL to CAL may explain the difference because supplemental CHOL increased serum vitamin D 3 concentrations but did not affect serum 25(OH)D 3 concentrations in dairy cows (Poindexter et al. 2020).Regardless of the reason, CAL increased serum 25(OH) D of calves more effectively compared with CHOL and thereby more supplied effectively the vitamin D signaling processes involved in growth and immunity.The data from our experiment provided further evidence to guide recommendations and practices for vitamin D supplementation of calves.The NRC (2001) recommendation of 15 µg CHOL/kg of DM milk replacer supplied enough vitamin D to prevent rickets in calves (Bechdel et al., 1937).The NASEM (2021) increased vitamin D recommendations for calves to 0.8 µg/kg BW (80 µg/kg DM milk replacer), with the intent of maintaining serum 25(OH)D levels >30 ng/ mL.In a previous experiment (Nelson et al., 2016a) with male Holstein calves, the change in serum 25(OH) D concentration as a function of supplemental CHOL in milk replacer was estimated to be 6 to 7 ng/mL of 25(OH)D for every 25 µg of CHOL/kg of milk replacer, which equates to an increase of approximately 16 ng/mL serum 25(OH)D per 1 µg CHOL/kg BW for a 60-kg calf.Here, the change in serum 25(OH)D as a function of supplemental CHOL was estimated to be approximately 5 ng/mL of 25(OH)D per 1 µg CHOL/ kg BW.Accordingly, the NASEM (2021) recommendation still may not be adequate to achieve serum 25(OH)D concentrations >30 ng/mL using CHOL as the source of vitamin D. Feeding 250 µg CHOL/kg DM milk replacer did not yield serum 25(OH)D levels >30 ng/mL in spring-born dairy calves in Ireland (Flores-Villalva et al., 2021).In contrast, our data indicate approximately 1.1 µg/kg BW CAL is necessary to achieve serum 25(OH)D levels >30 ng/mL in Holstein male calves.A note of caution is warranted, however, as male calves have lower serum 25(OH)D concentrations than female calves (Nelson et al., 2016b), and optimal serum 25(OH)D values for health and growth of calves are not established.
A second objective of this experiment was to explore the effect of supplemental vitamin D on the immunity of calves.Vitamin D signaling in immune cells dampens proinflammatory responses and stimulates antioxidant pathways (Chen et al., 2015b;Xu et al., 2015;Kweh et al., 2021).The intracrine vitamin D signaling in immune cells is influenced by a supply of 25(OH)D.Before the LPS challenge, we observed a positive linear relationship between serum 25(OH)D and the AOP of plasma, and a negative linear relationship between serum 25(OH)D and percentage of neutrophils with oxidative burst after ex vivo E. coli stimulation.Although neutrophil oxidative bursts contribute to the elimination of pathogens, excessive oxidation leads to lipid peroxidation (Lauridsen, 2019).We speculate  that vitamin D-mediated antioxidant activity limited the degree of oxidative burst in neutrophils in calves with greater serum 25(OH)D concentrations.In support of this notion, plasma AOP correlated positively with serum 25(OH)D in dairy cows (Strickland et al., 2021).Supplemental vitamin D also increased plasma glutathione peroxidase and catalase activity, and decreased plasma malondialdehyde in weaned calves (Xu et al., 2021) and pigs (Yang et al., 2019).The data from our experiment and elsewhere collectively support the theory that vitamin D contributes to redox balance.The effects of supplemental vitamin D on inflammatory responses to the LPS challenge were somewhat mixed.The intravenous LPS challenge serves as a model of acute infection, with rapidly elevated body temperature, respiration rate, and cytokine production (Carroll et al., 2009).A robust response to microbial-associated molecular patterns such as LPS supports clearance of infections; however, an excessive response can be detrimental to the host, as observed with the cytokine storm in severe coronavirus disease (Fajgenbaum and June, 2020).Vitamin D signaling in immune cells counteracts nuclear factor-κB signaling activation in the acute response to LPS (Chen et al., 2015a).As postulated with coronavirus disease, vitamin D lessens severity by attenuating the cytokine response and preventing the cytokine storm associated with severe disease (Kalia et al., 2021).Supplemental vitamin D attenuated the response to LPS in our experiment, but it was not consistent because the amount of vitamin D increased.On one hand, the greater rectal temperatures of CON and CHOL1.5 calves, which had the lowest serum 25(OH) D concentrations, agrees with the notion that low vitamin D is associated with inflammation and redox imbalance (Cannell et al., 2014;Celi et al., 2020).On the other hand, CAL3 calves had the greatest serum 25(OH)D levels, but similar responses to CON calves and CHOL1.5 calves.In a somewhat similar fashion, the quadratic effects of dietary CAL on measures of antioxidant activity, immunity, and growth also were observed in weaned piglets, with intermediate amounts of CAL (25 to 50 µg/kg feed) yielding more positive results compared with 5.5 or 105.5 µg CAL/kg feed (Zhang et al., 2021).Overall, the relationship of inflammatory indices with supplemental vitamin D resembles the U-shaped association between serum 25(OH)D and all-cause mortality in the human population (Melamed et al., 2008).Based on data from our experiment, which show that amount and source of vitamin D influence immunity, the effect of supplemental vitamin D on calf morbidity and mortality should be examined.Some results from our experiment indicate the CON treatment, which was a similar rate as the NRC (2001) vitamin D recommendation, was inadequate for calves.During the LPS response, CON calves had the lower plasma P level and lower percentage of granulocytes compared with calves supplemented with CHOL or CAL.Plasma minerals decrease rapidly during the acute-phase response, but typically rebound within a few hours.The vitamin D endocrine system contributes to maintenance of blood Ca and P concentrations (Wilkens and Muscher-Banse, 2020).The lower plasma P levels of CON calves in comparison with other calves is an indication that vitamin D was not adequate to maintain P homeostasis during the LPS challenge.Regarding granulocytes, acute infections stimulate granulopoiesis to increase the supply of neutrophils to combat the infection (Lawrence et al., 2018).We are not aware of a direct role for vitamin D in granulopoiesis, but similar to plasma P, the fewer granulocytes seem to indicate that CON calves did not have adequate vitamin D to maintain granulocytes when challenged.These findings are relevant to calves fed diets of whole milk or pasteurized waste milk as the vitamin D content of milk is low [i.e., <10 µg/kg DM (NRC, 2001)].
An unexpected effect of supplemental vitamin D on BW gain and starter intake was observed during the preweaning period.Our experiment was not designed to test the effect of supplemental vitamin D on the growth of calves, and 20 calves per treatment are recommended to detect a meaningful difference in BW gain of dairy calves (Kertz and Chester-Jones, 2004).Nonetheless, negative effects of supplemental vitamin D should be examined because of the toxicity potential of vitamin D. Cholecalciferol reduced weight gain and feed intake in feedlot steers, but at much greater rates (i.e., >25 µg/kg BW) than those supplied in our experiment.Moreover, Celi et al. (2018) reported that feeding calves up to 8.5 µg/kg BW CAL for 90 d did not have any adverse effects on growth, gross pathology, and histological examination; so, depressed feed intake as a consequence of vitamin D toxicity was unlikely in our experiment.In contrast, Xu et al. (2021) reported a positive effect of supplemental vitamin D on BW gain of Holstein calves, but again the sample size was small.The effects of supplemental vitamin D on growth should be considered in future research, particularly as milk replacers supply CHOL at a rate of 125 to 250 µg/ kg DM.

CONCLUSIONS
Supplemental CAL increased serum 25(OH)D concentrations more effectively than CHOL and is an alternative vitamin D source to achieve vitamin D sufficiency in dairy calves.Although it was not a main objective of our experiment, supplementing vitamin D decreased BW gain and starter grain intakes before weaning compared with CON calves, but calves fed CAL seemed to fair better compared with calves fed CHOL.The source and amount of vitamin D influenced inflammatory responses to LPS consistent with previous reports and modes of action of vitamin D. An optimal amount of supplemental vitamin D for calves remains unknown, but supplementing CHOL at 0.25 µg/kg BW did not maintain plasma P during an acute endotoxin challenge, nor did it support the circulating granulocyte population compared with supplementing CHOL or CAL at an additional 1.5 or 3 µg/kg BW.

Table 1 .
Blakely et al.: SUPPLEMENTAL VITAMIN D FOR CALVES Age at enrollment, quality of colostrum fed, and BW at birth and enrollment of calves used in the experiment

Table 3 .
Effects of vitamin D supplementation on growth and DMI of calves

Table 4 .
Blakely et al.: SUPPLEMENTAL VITAMIN D FOR CALVES Concentrations of 25-hydroxyvitamin D, minerals, metabolites, and immunoglobulins in serum before LPS challenge Serum 25-hydroxyvitamin D [25(OH)D, indiscriminate of 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3 ] as measured by ELISA at 7, 21, 35, 49, 63, 77, and 91 d of the experiment in all calves.Serum 25-hydroxyvitamin D 3 [25(OH)D 3 ] as measured by liquid chromatography-MS/MS at 35, 49, 63, 77, and 91 d in a subset (n = 5/treatment) of calves.Minerals, energy metabolites, and immunoglobulins in serum were measured at 35, 63, and 91 d.Concentrations of respective analytes measured at d 0 of the experiment were used as covariates in the analyses.

Table 5 .
Blakely et al.: SUPPLEMENTAL VITAMIN D FOR CALVES Effects of vitamin D supplementation on body temperature, respiration, feed intake, and growth during LPS challenge

Table 6 .
Blakely et al.:SUPPLEMENTAL VITAMIN D FOR CALVES Effects of vitamin D supplementation on plasma mineral, energy substrate, and acute-phase markers during LPS challenge CON) at 0.25 µg/kg BW of cholecalciferol (CHOL), CHOL at 1.5 or 3 µg/kg BW, or calcidiol (CAL) at 1.5 or 3 µg/kg BW in addition to 0.25 µg/kg BW of CHOL.Calves were challenged with LPS (0.1 µg/kg BW) via intravenous infusion at 91 d of the experiment.Effect of treatment (Trt) and contrasts of source (CHOL vs. CAL), dose (1.5 vs. 3 µg/kg BW), and CON vs. all other treatments (Supp).

Table 7 .
Blakely et al.: SUPPLEMENTAL VITAMIN D FOR CALVES Effects of vitamin D supplementation on blood leukocytes in response to LPS challenge 1Leukocytes in blood sampled at 1, 2, 4, 8, 12, 24, 48, and 72 h relative to LPS challenge were analyzed by flow cytometry.