Influence of Deliverable Form of Dietary Vitamin D 3 on the Immune Response in Late-Lactating Dairy Goats

: Mastitis-causing bacteria can establish persistent infections in the mammary glands of commercially important dairy animals despite the presence of strong specific humoral and cellular immune mechanisms. We investigated the effect of vitamin D 3 in the diet at a set level, but in two different forms (i.e., unencapsulated and encapsulated by complex coacervation with sulfur-saturated bovine lactoferrin-alginate using microbial transglutaminase-catalyzed crosslinking) on the immune response in late-lactating dairy goats. Dairy goats (n = 18) were randomly assigned to three experimental groups (n = 6). Dairy goats were orally administered 0.35 mg of vitamin D 3 /day in the unencapsulated form and 0.35 mg of vitamin D 3 /day in the encapsulated powder form. Another group received the basal diet. The experimental period lasted 6 weeks. The blood serum concentrations of 25-hydroxyvitamin D 3 [25-(OH)-D 3 ], lactoferrin, immunoglobulin A (IgA), and interferon-gamma (INF-γ ) were measured. There were major differences in these parameters between dietary groups. However, the delivery of vitamin D 3 in the encapsulated powder form to dairy goats resulted in a marked increase in 25-(OH)-D 3 concentration in serum, while the serum level of lactoferrin also increased. Alternatively, the serum levels of IgA and the immunomodulatory cytokine INF-γ were elevated following supplementation with the encapsulated vitamin D 3 . The observed effects suggest that the deliverable form of dietary vitamin D 3 results in differences in the immune response in late-lactating dairy goats.


Introduction
Vitamin D 3 , which is formed in the skin or absorbed through the gastrointestinal tract, is modified by enzymatic hydroxylation to form first in the liver, 25-(OH)-D 3 , which is a plasma transport form of the vitamin, and then in the kidney to form 1,25-(OH) 2 D 3 , the active hormone, or 24,25-(OH) 2 D 3 form of the vitamin and other derivatives.It appears that vitamin D 3 stimulates the active transport of calcium across mucosal cells of the small intestine [1].In growing dairy goats (Capra hircus) and sheep (Ovis aries), a vitamin D 3 -reduced diet combined with missing ultraviolet B (UVB) exposure leads to a sharp drop in the vitamin D 3 status [2].Such findings reinforce the results of studies that employed vitamin D 3 supplementation and UVB exposure in juvenile bearded dragons (Pogona vitticeps) [3].The study reported by Oonincx et al. [3] showed that 2 h of UVB exposure enables adequate physiological concentrations of plasma vitamin D 3 metabolites to be maintained in growing bearded dragons.Vitamin D 3 also has a precise role in immunoregulation in response to microbial threats [4].The protective effect of vitamin D 3 on infectious diseases such as SARS-CoV-2 (COVID- 19), tuberculosis, and mastitis has been linked to a direct interaction of vitamin D 3 with cells of the immune system [4][5][6].
Vitamin D 3 is often included in foods as dispersions in emulsified lipid carriers (e.g., milk), which provide an effective barrier against environmental stresses (e.g., oxidation).A variety of delivery systems have been developed to encapsulate bioactive compounds, and most are applicable to the encapsulation of lipid-soluble bioactive compounds [7].
Usually, encapsulation allows for an even dispersion of bioactive compounds throughout the products, meaning consumers will receive the same amount of a bioactive compound, such as vitamin D 3 , in each bite of food or sip of a drink [8].Encapsulation also improves the shelf-life of products, thereby preventing vitamin D 3 from degrading over time and keeping the formulation stable [8].There are also concerns about intestinal absorption of liposoluble bioactive compounds.Properly encapsulated liposoluble bioactive compounds such as vitamin D 3 are thought to enter the bloodstream faster and in greater amounts [8] compared to the unencapsulated forms.
Protein-polysaccharide complex coacervates can be used as inexpensive encapsulation carriers for bioactive compounds in food and beverage products [9].The coacervation encapsulation approach involves the processing steps of emulsification, coacervation, and shell formation and/or hardening [10].Coacervation is a chemical method for producing biopolymer droplets in suspension based on the separation of two liquid phases into one concentrated colloidal phase [11].Complex coacervation is the result of electrostatic interaction between two biopolymers with opposite charges, generally a protein and a polysaccharide [10].Complex coacervates between whey proteins (e.g., bovine lactoferrin) and polysaccharides such as carboxymethylcellulose, pectin, and alginate have been recently used to encapsulate liposoluble bioactive compounds [12,13].
The use of cross-linking agents provides more resistant structures to capsules produced by colloidal complexation [14] or complex coacervation [15].The coacervation encapsulation process may increase the absorption of bioactive compounds through controlled release [16].A previous study reported that the polysaccharide sodium alginate has promising properties to be used as a carrier with adjuvants and prolong release properties that enhance the immunogenicity of vaccines [17].Thus, guinea pigs immunized with diphtheria toxoid-loaded alginate nanoparticles showed the highest humoral immune response to the conventional vaccine [17].Moreover, bovine lactoferrin may be a potential alternative therapy or may serve as an adjunct to conventional therapies for the treatment of inflammation [18].In this context, bovine lactoferrin has been shown to have good activity against inflammation in vitro, with systemic effects suggested for various inflammatory diseases by stopping the production of pro-inflammatory molecules [18].Recently, more evidence has been rising for the direct effects of bovine lactoferrin on the immune system [19].
The primary aim of this study was to specifically evaluate the immunomodulatory role of vitamin D 3 encapsulated by sulfur-saturated bovine lactoferrin-alginate complex coacervates using microbial transglutaminase as a cross-linking agent in late-lactating dairy goats.Ingestion of vitamin D 3 capsules may result in a marked and rapid increase in immunological responses for the prevention of bacterial invasion (e.g., mastitis) in the mammalian host.The unencapsulated form of vitamin D 3 was used for comparison purposes.

Materials and Methods
Cholecalciferol (vitamin D 3 ), dimethyl sulfone (DMSO 2 ), sodium alginate, and microbial transglutaminase were purchased from Sigma-Aldrich (St. Louis, MO, USA).The 25-hydroxyvitamin D 3 [25-(OH)-D 3 ] was purchased in crystalline form from Cayman Chemical (Ann Arbor, MI, USA).Medium-chain triglycerides (MCTs) oil (Neobee M-5, ≥66% C8:0 and ≥32% C10:0 content) was kindly donated by Stepan Company (Northfield, IL, USA).Bovine lactoferrin-FD (10321412) was a gift from MDV International (Delhi, NY, USA), whose purity was more than 90% and 15.7 mg of iron/100 g of bovine lactoferrin.Bovine lactoferrin (2 g) was dissolved in 0.1 M sodium bicarbonate with dimethyl sulfone (0.5 g) for 24 h at room temperature (20 • C), followed by extensive dialysis against sodium bicarbonate to remove excess sulfur ions, and then the solution was freeze-dried (LAB-CONCO, Kansas City, MO, USA).Sulfur saturation of bovine lactoferrin with dimethyl sulfone was 90.4% as determined by the MACRO Cube Elemental Analyzer (Quantum Analytics, The Woodlands, TX, USA).Sulfur nanoparticles have shown antibacterial activity against Escherichia coli and Staphylococcus aureus [20].Bovine lactoferrin per se is active against mastitis pathogens, but its antimicrobial activity is low enough to constitute a viable treatment against mastitis pathogens [21].However, the efficacy of bovine lactoferrin against pathogenic bacteria and viruses can be increased by the metal saturation of lactoferrin [22].The solvents used for the extraction and analysis of samples were HPLC-grade and purchased from Sigma-Aldrich.All other chemicals and reagents were of analytical-grade (Sigma-Aldrich).Deionized water, prepared by passing distilled water over a mixed-bed cation-anion exchanger, was used throughout this study.
The protein and polysaccharide were prepared to contain a total concentration of 1.0% (w/w) and a biopolymer mixing ratio of 9:1 (sulfur-saturated bovine lactoferrin:sodium alginate) in an aqueous solution at pH 4.0.Vitamin D 3 , previously dispersed in warmed (37 • C) MCTs oil (0.75%, w/w) by a magnetic stirring hotplate (Thermo Fisher Scientific, Waltham, MA, USA), was added to the aqueous solution (74.25%, w/w in the final emulsion) of sulfur-saturated bovine lactoferrin to give a concentration of 540 µg/mL vitamin D 3 in the final emulsion, and pre-emulsified with a hand-held homogenizer (Biospec Products Inc., Bartlesville, OK, USA) at low speed for 3 min at 20 • C. The sodium alginate aqueous solution (25.0%, w/w in the final emulsion), previously acidified to pH 4.0 with acetic acid (20%, w/w), and the coarse emulsion were homogenized twice at 82.74 MPa (12,000 psi) at a temperature of 50 • C through a high-pressure TC5 homogenizer (Stansted Fluid Power, Harlow, UK).The temperature of the oil-in-water emulsion was reduced to 5 • C using an ice-water bath for 60 min.Microbial transglutaminase solution (0.25%, w/w) was added under constant magnetic stirring (Thermo Fisher Scientific) at 400 rpm for 3 h at 25 • C to induce cross-linking.Immediately after treatment, microbial transglutaminase was inactivated by freezing the oil-in-water emulsion for 10 min.The vitamin D 3 capsules were kept at 10 • C for 48 h, and then the supernatant was removed by decantation.The vitamin D 3 capsules were frozen in liquid nitrogen and freeze-dried for 24 h.The quantitative determination of vitamin D 3 -loaded capsules was performed using HPLC-UV [23].
The encapsulation efficiency (EE%) of the vitamin D 3 capsules was calculated as follows: EE% = (loaded vitamin D 3 /total vitamin D 3 ) × 100 The stability of vitamin D 3 -loaded capsules was evaluated during storage at 24 • C under nitrogen gas for 6 months.Vitamin D 3 content was measured at the 1st, 5th, 10th, 15th, 30th, 60th, 90th, and 180th days.The content of vitamin D 3 was determined by HPLC-UV [23].
In a 6-week feeding trial, eighteen American-bred, French-Alpine dairy goats (mean age 2.5 years, mean BW 114.2 kg) were selected from the milking herd of the International Goat Research Center (IGRC) at Prairie View A&M University, Prairie View, TX, USA.All experimental procedures with the goats were in strict compliance with the current guidelines and legal requirements established in the United States for the proper use and care of animals and approved by the Institutional Animal Care and Use Committee at Prairie View A&M University (Protocol # 2023-051).A total of six goats per experimental group (n = 18) were used.Our sample size also follows the recommendations of the Institutional Animal Care and Use Committee at Prairie View A&M University for discovery experiments where at least 4 biological replicates are needed.
All dairy goats were housed indoors at the IGRC.The temperature in the house was approximately 25 • C. The selected dairy goats were randomly assigned to three experimental groups: control (n = 6), vitamin D 3 supplementation (0.35 mg vitamin D 3 /day) in the unencapsulated form previously dispersed in MCTs oil (n = 6), and vitamin D 3 supplementation (0.35 mg vitamin D 3 /day) in the encapsulated powder form (n = 6).After the initial 2 weeks of adjustment period, the experiment continued for 6 weeks.All goats were in late lactation [172 DIM; 1.06 L/day; level of α s1 -casein (high: 18.8% of total protein)] and randomly assigned to an individual feeding gate on the day of enrollment.
Goat's α s1 -casein milks produced by animals of Alpine breed in the same stage of lactation used in this study were confirmed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS), and densitometry was used to assess the relative concentration of the α s1 -casein component [24].
The treatment of dairy goats fed the encapsulated vitamin D 3 consisted of 1 g of vitamin D 3 capsules per goat (0.35 mg vitamin D 3 /day) in addition to the base ration that provided 0.05 mg of vitamin D 3 daily.As a functional protein, sulfur-saturated bovine lactoferrin is an interesting biopolymeric wall system used to encapsulate vitamin D 3 .Thus, the treatment of the group of dairy goats fed the encapsulated vitamin D 3 consisted of 1 g of the vitamin D 3 capsules that provided sulfur-saturated bovine lactoferrin (900 mg sulfursaturated bovine lactoferrin/day).It should be noted here that the treatment group of dairy goats fed the encapsulated vitamin D 3 consisted of 1 g of vitamin D 3 and also provided a polysaccharide, sodium alginate (100 mg sodium alginate/day), used to encapsulate vitamin D 3 .A top-dress supplement for the treatment with the encapsulated vitamin D 3 was prepared by mixing 1 g of the vitamin D 3 capsules with cornmeal to provide 0.35 mg of vitamin D 3 , 900 mg of sulfur-saturated bovine lactoferrin, and 100 mg of sodium alginate in 100 g of the total mixture.A top-dress supplement for the treatment with the unencapsulated vitamin D 3 was prepared by combining vitamin D 3 in MCTs oil (0.35 mg of vitamin D 3 /5 mL of MCTs oil), homogenized for adequate dispersal, and mixed with cornmeal to provide 0.35 mg of vitamin D 3 in 100 g of the total mixture.The two topdress supplements were not mixed into the ration and were consumed readily by all goats upon delivery.
The control animals received the basal diet without supplementation.The basal diet was composed of 69.3% oats, 1.35% soybean meal, 2.05% corn, 26.04% alfalfa meal, 0.93% cottonseed hulls, 0.31% dicalcium phosphate, and 0.06% vitamin A, D 3 , and E supplements.The experimental and control animals were fed twice a day, in the morning and the evening.The animals were given 2.07 kg of either the control or the experimental diet twice a day, in the morning and evening, and the leftovers were measured.The unencapsulated vitamin D 3 and the encapsulated vitamin D 3 were only added to the morning feeding as a topdress.The feeding duration of this experiment was 6 weeks.Hay and water were available to animals ad libitum.Housing and care of the animals conformed to the approved institutional animal care and use committee practices and standards of the university.
Blood samples (5 mL) were collected by puncture of the jugular vein using siliconized needles (21 G × 1 in) with a vacuum system.The blood samples taken at 0 and 6 weeks of treatment were transferred into a 10-mLvacuum tube (serum separator tube) (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA).Tubes were centrifuged at 3500× g for 5 min in a refrigerated centrifuge (4 • C) for serum separation within 30 min of sample collection.Serum samples were transferred into microtubes using Pasteur pipettes.The samples were maintained at 2-8 • C and immediately analyzed, avoiding freeze-thaw cycles because this is detrimental to many serum components.
All serum analyses were performed in triplicate.Serum 25-(OH)-D 3 concentrations were quantified using a CDC-certified LC-MS/MS method [25].Serum lactoferrin, IgA, and INF-γ concentrations were quantified using commercially available ELISA kits (Sigma-Aldrich), according to the manufacturer's directions.

Statistical Analysis
Quantitative data for the combination of response and treatment variables are summarized with a mean ± standard error.A paired t-test under the pre-posttest design was performed to compare the difference between before supplementation (week 0) and after supplementation (week 6) with animals assigned to different dietary groups.The outcome variables [25-(OH)-D 3 , lactoferrin, IgA, and INF-γ serum concentrations] were measured before and after the supplementation.The 5% significance level was applied to the paired t-test.Experimental data were analyzed using SAS software (version 9.4, SAS Institute, Cary, NC, USA).

Results and Discussion
The encapsulation of vitamin D 3 attained a high encapsulation efficiency of 91.2%.The capsules contained vitamin D 3 at a concentration of 0.35 mg per gram (0.035%) as determined by HPLC-UV [23].As to the storage stability of the vitamin D 3 capsules, there was little change in concentration after 3 months of storage at 24 • C, with a decrease in concentration of about 0.2%.Six months later, the storage stability decreased by 1.3% at 24 • C, which was not significant.These results suggest that vitamin D 3 loading in the complex coacervates of sulfur-saturated lactoferrin and sodium alginate with transglutaminase, as an effective protein cross-linker, has good stability.
Studies involving different species of experimental animals and using deficient as well as supradietary levels of vitamin D 3 indicate that the vitamin D 3 metabolite 25-(OH)-D 3 is involved in the maintenance of immune function [26,27].Activated T lymphocytes and macrophages, among others, have been found to express vitamin D receptors [28].Several clinical trials have demonstrated the potential benefit of increased vitamin D status on immune function [29].In these clinical trials, the supplemented groups showed significant improvement in several parameters of immune responsiveness and a marked reduction in illness due to infections; moreover, supplemented subjects presenting with illness were observed to require shorter antibiotic treatment regimens.
The in vivo test performed on dairy goats confirmed the efficiency of the intake of encapsulated vitamin D 3 (0.35 mg vitamin D 3 /day) in increasing the concentration of circulating 25-(OH)-D 3 during late lactation (Table 1).Charoenngam and Holick [30] noted that enhancing 25-(OH)-D 3 status contributes to increased immunological vigor in humans.Merriman et al. [31] found that intramammary 25-(OH)-D 3 treatment directly influences immune cells of the mammary gland of lactating dairy cows in response to endotoxininduced mastitis by increasing vitamin D signaling in mammary immune cells.Moreover, Poindexter et al. [32] observed that feeding 25-(OH)-D 3 to lactating dairy cows increased circulating 25-(OH)-D 3 , which appears to improve immune responses to the intramammary Streptococcus uberis challenge, thereby protecting the mammary glands from mastitis.These nutritionally induced changes observed by Poindexter et al. [32] also occurred via vitamin D signaling.Comparison of 25-hydroxyvitamin D 3 [25-(OH)-D 3 ] serum levels between baseline and supplementation (unencapsulated vitamin D 3 vs.encapsulated vitamin D 3 ) indicated significant (p < 0.0001) improvements in both supplemented groups (Table 1), but we observed a higher (p < 0.0001) 25-(OH)-D 3 serum concentration at 6 weeks in dairy goats fed the encapsulated form of vitamin D 3 , compared with the serum concentration of dairy goats fed the unencapsulated form of vitamin D 3 at 6 weeks (Table 1).This may be attributable to the efficacy of the delivery system.
The form in which vitamin D 3 is delivered can have a great influence on whether that vitamin D 3 is consumed and could influence the immunomodulatory effects of vitamin D 3 .One must consider dosage factors to equate for body weight, which leads to different serum levels of vitamin D 3 metabolites.One must also consider the effects of milk production during late lactation and milk quality in terms of technological properties, where high-type α s1 -casein milk seems more suitable for cheesemaking because of its firmer curd and higher casein content.Given all these variables, and perhaps others as yet unknown, it is not surprising that the effects of vitamin D 3 in the unencapsulated and encapsulated forms are seen as highly individual.Vitamin D 3, in unencapsulated form, at 0.35 mg/day was provided for 6 weeks, followed by testing.Although, surprisingly, the dose did not result in significant differences in serum levels of lactoferrin, IgA, and INF-γ, a clearer dose response was seen in serum levels of 25-(OH)-D 3 in the unencapsulated group of dairy goats (Table 1).We also observed that supplementation with the encapsulated vitamin D 3 significantly (p < 0.0001) increased the serum levels of lactoferrin compared to the group of dairy goats that were fed the unencapsulated vitamin D 3 (Table 1).The sulfur-saturated bovine lactoferrin, was used as a biopolymeric wall system to encapsulate vitamin D 3 , which contributes to immune regulation in lactating dairy goats by maintaining the circulation of IgA in late lactation at a high concentration compared to the experimental group of dairy goats fed the unencapsulated vitamin D 3 (Table 1).Lactoferrin should, in theory, exert an advantageous immunomodulatory effect because it can pass through the blood/milk barrier, thereby protecting the mammary gland from bacterial invasion [33].In the experimental group of dairy goats that were fed the encapsulated vitamin D 3 , the serum levels of IgA were significantly (p < 0.0001) increased (Table 1).Lactoferrin has been identified as a critical nutrient, and the combination of lactoferrin and IgA is particularly important for infants, whose immune systems are still developing.Breast milk contains high levels of both lactoferrin and IgA, which help to protect infants against infections [34].
As noted, 25-(OH)-D 3 is a precursor to the active form of vitamin D 3 , which is a hormone that plays a vital role in many bodily functions, including immune regulation [35].The oral intake of vitamin D 3 or preformed vitamin D 3 [25-(OH)-D 3 ] has been shown to work together with lactoferrin to boost immune function even more effectively than either one alone among those individuals who were affected by exposure to SARS-CoV-2 infection [36].Moreover, high serum levels of INF-γ, an immunomodulatory cytokine, are associated with a stronger immune response and improved protection against infection [37].INF-γ production was markedly higher (p < 0.0001) in blood serums obtained from the dairy goats fed the encapsulated vitamin D 3 (0.35 mg vitamin D 3 /day) than from the group of dairy goats fed the unencapsulated vitamin D 3 (0.35 mg vitamin D 3 /day) (Table 1).These results suggest that the encapsulated vitamin D 3 exhibits sustained release and enhanced absorption through complex coacervation technology.

Conclusions
These vitamin D 3 capsules can be used as dietary supplements for their beneficial effects on the host and its immune system.

Table 1 .
Serum concentrations showing significant differences in 25-hydroxyvitamin D 3 [25-(OH)-D 3 ], lactoferrin, immunoglobulin A (IgA), and interferon-gamma (INF-γ) at baseline (0 week) and at feeding period (6 weeks) between dietary groups1,2.Dairy goats in late lactation fed a basal diet (control group), a basal diet supplemented with the unencapsulated form of vitamin D 3 (treatment group U), and a basal diet supplemented with the encapsulated form of vitamin D 3 (treatment group E).2Data are presented as means ± standard error; n = 18.