Effects of feed restriction and supplementary folic acid and vitamin B12 on immune cell functions and blood cell populations in dairy cows N. Vanacker1,2 , C. L. Girard1, R. Blouin2 and P. Lacasse1† 1Sherbrooke Research and Development Centre, Agriculture and Agri-Food Canada, 2000 College Sherbrooke, QC J1M 0C8, Canada; 2Département de Biologie, Faculté des Sciences, Université de Sherbrooke, 2500 boulevard de l'Université, Sherbrooke, QC J1K 2R1, Canada (Received 22 February 2019; Accepted 23 July 2019; First published online 10 October 2019) Cows undergoing a negative energy balance (NEB) often experience a state of immunosuppression and are at greater risk of infectious diseases. The present study aimed to evaluate the impact of a folic acid and vitamin B12 supplement and feed restriction on several immune parameters. Sixteen cows at 45 ± 3 days in milk were assigned to 8 blocks of 2 cows each according to each cow’s milk production in the previous week, and within each block, the cows randomly received weekly intramuscular injections of either saline or 320 mg of folic acid and 10 mg of vitamin B12 for 5 weeks. During week 5, the cows were fed 75% of their ad libitum intake for 4 days. Blood samples were taken before the beginning of the experiment, just before feed restriction and after 3 days of feed restriction, in order to evaluate blood cell populations, the phagocytosis capacity and oxidative burst of polymorphonuclear leukocytes (PMNs), the proliferation of peripheral blood mononuclear cells (PBMCs) and concentrations of non-esterified fatty acids (NEFAs) and β-hydroxybutyrate. The vitamin supplement did not affect any of the tested variables except milk fat and lactose content. Feed restriction reduced milk production and increased the concentration of NEFAs. Feed restriction did not affect blood cell populations but did reduce the percentage of PMN positive for oxidative burst after stimulation with phorbol 12-myristate 13-acetate. The proliferation of PBMCs was reduced when the cell culture medium was supplemented with sera collected during the feed restriction. In conclusion, feed restriction affected the functions of PMN and PBMC and this effect was not prevented by the folic acid and vitamin B12 supplement. These results support the hypothesis that the greater risk of infectious diseases in cows experiencing a NEB is related to impaired immune cell functions by high circulating concentration of NEFAs. Keywords: negative energy balance, immunosuppression, blood metabolites, oxidative burst, lymphoproliferation Implications Cows undergoing a negative energy balance often experience a state of immunosuppression and are at greater risk of infectious diseases. In the present experiment, we tested the hypothesis that a vitamin supplement decreases the metabolic and immunological disturbances induced by feed restriction. The results indicate that the metabolic and immunological disturbances induced by negative energy balance cannot be prevented by folic acid and vitamin B12 supplementation. Introduction The periparturient period is challenging for high-yielding dairy cows. Cows undergo hormonal, metabolic, physiological, nutritional and immunological changes during that period. According to Leblanc et al. (2006), about 75% of diseases happen in the first month after calving. Most of the metabolic diseases, such as milk fever, ketosis, retained placenta and displacement of placenta, and infectious diseases such as mastitis, Johne’s disease and metritis occur within the first 2 weeks of lactation (Goff and Horst, 1997). During the transition period, the energy required for milk production exceeds the energy provided by the diet, resulting in a negative energy balance (NEB). Body reserves are mobi- lized to supply additional nutrients, leading to an increase in blood concentrations of non-esterified fatty acids (NEFAs) and β-hydroxybutyrate (BHB) and a decrease in glucose. This period is also associated with an immunode- pression. Several studies (Hoeben et al., 2000; Kehrli et al., 1989; Moreira da Silva et al., 1998) have shown that † E-mail: Pierre.Lacasse@canada.ca Animal (2020), 14:2, pp 339–345 © Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada 2019 animal doi:10.1017/S1751731119002301 339 https://orcid.org/0000-0003-1340-7697 https://orcid.org/0000-0002-1882-3262 https://orcid.org/0000-0002-3579-7991 mailto:Pierre.Lacasse@canada.ca https://doi.org/10.1017/S1751731119002301 polymorphonuclear leukocytes (PMN) phagocytosis and oxi- dative burst activity are impaired during the transition period in dairy cows and that the responsiveness of blood lympho- cytes when stimulated with mitogenic agents is also decreased, as is the production of immunoglobulin by B cells (Lacetera et al., 2005; Nonnecke et al., 2003). Nonnecke et al. (2003) reported that the peripartum reduction in the functional capacities of blood lymphocytes was abolished by mastec- tomy, which suggests that NEB is causing the periparturient immunodepression. Ster et al. (2012) showed that lymphocyte proliferation and interferon-γ secretion were lower when lym- phocytes were incubated with sera harvested in the postpar- tum period and that this was due to high NEFA concentrations. Therefore, strategies that prevent the increase in NEFAs in a period of NEB may have a positive impact on immunity. A combined supplement of folic acid and vitamin B12 has been reported to change energy partitioning during early lactation. Vitamin B12 is a coenzyme involved in two essential metabolic pathways. First, it allows isomerization by methylmalonyl-CoA mutase into succinyl-CoA which will then be able to enter the Krebs cycle. Secondly, vitamin B12 allows the transfer of a methyl group to 5-methyltetrahydrofolate, which is the methylated form of folic acid. This leads to the formation of homocysteine which can then form methionine that could be used to support the production of proteins (Scott, 1999). Methionine is also one of the amino acids limit- ing milk production (Duplessis et al., 2017). As a consequence, the vitamin supplement increased milk production and plasma glucose and decreased hepatic lipids (Graulet et al., 2007). Preynat et al. (2009) found that vitamin supplementation increased the whole-body rate of appearance of glucose, and Duplessis et al. (2014) observed a reduction in BW losses without a decrease in milk yield. Those results taken together suggest a better energy balance. Accordingly, folic acid supple- mentation tends to reduced (Graulet et al., 2007) or reduced (Duplessis et al., 2017) postpartum NEFA concentration. Therefore, the present study aimed to evaluate the impact of a folic acid and vitamin B12 supplement and feed restriction on blood cell populations, on the phagocytosis capacity and oxidative burst of PMN and proliferation of peripheral blood mononuclear cell (PBMC). In order to discriminate between the effects of energy balance with those related to calving itself (hormonal changes, calving stress, variation in calcium concentration and dystocia), the experiment was carried out in early lactation cows where NEB was induced by feed restriction. Materials and methods Animals and experimental procedures Sixteen multiparous Holstein cows at 45 ± 3 days in milk (DIM) and that have produced 9080 ± 210 kg of milk during previous lactation were housed at Agriculture and Agri-Food Canada’s Sherbrooke Research and Development Centre (Sherbrooke, QC, Canada). The cowswere assigned to 8 blocks of 2 cows each according to the cows’ milk production during the previous week, 45 ± 6 kg/day. Within each block, the cows randomly received weekly intramuscular injections of either 5 ml of saline (0.9% NaCl; control) or 320mg of folic acid (pteroylmonoglutamic acid; MP Biomedicals, Solon, OH, USA) and 10 mg of vitamin B12 (cyanocobalamin, 5000 μg/ml; Vétoquinol, Lavaltrie, QC, Canada) for 5 consecutive weeks. A total mixed ration providing 15.9% CP, 18.3% ADF, 29.1% NDF, 31.1% starch and 6.53 MJ/kg of net energy of lactation was served once daily, at 0800 h. The ingredients were corn and grass silages, chopped grass hay, finely ground corn grain, soybean meal, distillers grain (corn), corn gluten meal, canola meal, micronized soybean, beet pulp and mineral premix calculated to meet the requirements of the National Research Council (2001). During the first 4 weeks of the experiment (treatment period), daily intake and orts for each cow were weighed and the feed offered was adjusted if needed, allowing 10% refusals (Figure 1). The cows had free access to water. In week 5, the cows were fed 75% of their ad libitum intake of the previous week for 4 days (feed restriction period). Milk yield and composition The cows were milked twice a day, between 0700 and 0800 h and between 1900 and 2000 h. Milk production was recorded at each milking throughout the experiment. Milk composition (lactose, protein, fat and SCC) was evaluated by a commercial laboratory (Valacta Inc., Sainte-Anne-de- Bellevue, QC, Canada) using mid-IR reflectance spectrometry with Foss MilkoScan FT 6000 (Foss Electric A/S, Hillerød, Denmark) on samples collected at milking on d 29 and 30. Blood collection Blood samples were collected from the coccygeal vein at 1330 h on the day before the experiment began (d−1) (i.e., before the cows received the experimental diet and the vitamin supplement), at the end of the treatment period (d 26) and after 3 days of feed restriction (d 29) in uncoated, heparin-coated and EDTA-coated Vacutainer tubes (Becton, Dickinson and Co., Mississauga, ON, Canada; Figure 1). The tubes without additives were left at room temperature for approximately 2 h to allow clotting before centrifugation at 1900×g for 15 min at 4°C. Then, the serum was stored at −20°C until PBMC proliferation assays were performed. The heparin-coated tubes were kept at room temperature until PMN phagocytosis and oxidative burst assays were per- formed (within 2 h after blood collection). The EDTA-coated Vacutainer tubes were sent to the laboratory at the Complexe Blood samples D -1 D29D26D12 Figure 1 (colour online) Flow diagram of the experimental design. Dairy cows were injected weekly with either saline or a mix of folic acid and vita- min B12, and feed-restricted from days 27 to 30. Vanacker, Girard, Blouin and Lacasse 340 de diagnostic et d’épidémiosurveillance vétérinaires du Québec (Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada) for determination of complete blood count. The other blood tubes containing EDTA were placed on ice immediately after collection and centrifuged within 30 min at 1900×g for 15 min at 4°C. Then, the plasma was stored at −20°C until determination of NEFA and BHB concentrations. Blood metabolite assays Plasma BHB was evaluated with a BHB reagent kit (Pointe Scientific Inc., Canton, MI, USA), and absorption was read at 505 nm using a SpectraMax 250 microplate reader (Molecular Devices, Sunnydale, CA, USA). Plasma NEFA concentration was determined by an enzymatic colorimetric method using a Randox kit (Scientifiques ESBE, Saint- Laurent, QC, Canada). Briefly, 100 μl of reagent R1 was added to 5 μl of the plasma sample in a 96-well microplate, which was then incubated at 37°C for 5 min. Next, 200 μl of reagent R2 was added, and the microplate was incubated at 37°C for an additional 5 min. Absorbance was read at 550 nm using a SpectraMax 250 microplate reader (Molecular Devices). Polymorphonuclear leukocytes phagocytosis and oxidative burst assays The phagocytosis assay was performed in whole blood by flow cytometry using BioParticles Conjugated for Phagocytosis (Life Technologies Inc., Burlington, ON, Canada), as described by Vanacker et al. (2017b). The oxidative burst assay was performed in whole blood by flow cytometry, as described by Vanacker et al. (2017b). Peripheral blood mononuclear cell isolation To assess the effect of the serum from the treated cows on PBMC proliferation, PBMCs were isolated from the jugular blood of three healthy lactating cows not included in the experiment. The blood was collected in blood collection bags containing citrate–phosphate–dextrose–adenine anticoagu- lant (Animal Blood Resources International, Dixon, CA, USA). Isolation of PBMC was performed as described by Vanacker et al. (2017b). Peripheral blood mononuclear cell proliferation assay The effect of serum collected from the treated cows on PBMC proliferation was measured. Three assays were performed in 96-well plates using isolated PBMC from 3 healthy cows, as described by Ollier et al. (2016). Briefly, the PBMCs were labeled with 1 μM carboxyfluorescein diacetate succinimidyl ester using the CellTrace CFSE Cell Proliferation Kit (Thermo Fisher Scientific, Burlington, ON, Canada). To assess the effect of the serum from treated cows on PBMC proliferation, labeled PBMCs suspended in RPMI 1640 supplemented with 7.5% sera sampled on d −1, 26 and 29 from each cow were incubated in a 96-well plate (1.5 × 105 cells per well) at 38.5°C with 5% CO2 for 72 h. For each serum tested, the PBMCs of three wells were incubated with the T cell mitogen concanavalin A (Sigma-Aldrich, Oakville, ON, Canada) at 1 μg/ml and the PBMCs of three wells were incubated with- out the mitogen as a negative control. After a 72-h incuba- tion, proliferation was analyzed using the FACSCanto II flow cytometer (BD Biosciences, Mississauga, ON, Canada). This assay was performed three times for each serum, with one assay per healthy cow from which the PBMC had been iso- lated. A control-positive serum obtained by mixing sera from four healthy cows in late lactation and another obtained by mixing sera from three freshly calved cows were included in each plate to determine inter-assay variation. Statistical analysis Data were analyzed using the MIXED procedure of the SAS software package (SAS Institute Inc., Cary, NC, USA). For milk production and DMI, data of the vitamin supplementation and the feed restriction periods were analyzed separately. Time (day) was used as a repeated effect, and cow was used as the subject. For metabolic and immunological parameters, Time (sampling period) was used as a repeated effect and cow was used as the subject. The different sampling periods were compared using the Tukey–Kramer adjustment. For immunological parameters, data from d −1 were used as a covariable. The correlations between the blood concentra- tion of metabolites and the percentages of phagocytic PMN, oxidative-burst-positive PMN and proliferative PBMC were evaluated using the CORR procedure of SAS. Differences were considered statistically significant when P< 0.05. Results Milk production, milk composition, DM intake and BW There was no effect of vitamin supplementation onmilk produc- tion during the treatment period (P= 0.96) and the feed restric- tion period (P= 0.87; Figure 2). Milk production decreased slightly throughout the treatment period (P< 0.01). During feed restriction, milk production decreased abruptly (P< 0.01). Milk from the cows receiving the vitamin supplement tended to have a lower concentration of fat (P= 0.06) and had a greater con- centration of lactose (P< 0.01) than the milk from the control cows did during the restriction period (Table 1). Dry matter intake was not affected by vitamin supplementation during the treatment period (P= 0.52; Supplementary Figure S1). On d 29, all the cows were in a NEB, averaging −19.8± 1.26Mcal/day. Accordingly, BW was not affected by vitamin supplementation during the treatment period (P= 0.27; Supplementary Figure S2) or during feed restriction. However, cows lost BW during feed restriction (P< 0.001). Blood metabolic marker concentrations Vitamin supplementation (P= 0.69) and feed restriction (P= 0.16) did not affect the blood concentration of BHB (Figure 3a). Blood BHB concentration tended to be higher at the beginning of the experiment (d −1) than at the end of the treatment period (P= 0.06). As was the case for BHB, vitamin supplementation did not affect the blood concentration of NEFA during the treatment Vitamins and feed restriction effect on immunity 341 https://doi.org/10.1017/S1751731119002301 https://doi.org/10.1017/S1751731119002301 period (P= 0.86) and the feed restriction period (P= 0.94; Figure 3b). The concentration of NEFA was higher before the treatment period than at the end of it (P= 0.04). Blood NEFA concentration was greater during feed restriction than on d −1 and 26 (P< 0.001). Plasma concentration of glucose was analyzed and is presented in a companion paper (Girard et al., 2019). It was not affected by treatments or feed restriction. Immunological parameters Leukocyte populations were not affected by vitamin supple- mentation (Table 2). However, we observed a time effect for leukocyte (P< 0.01), lymphocyte (P= 0.01) and basophil (P= 0.03) concentrations and a trend (P= 0.06) for eosinophil concentration. Leukocyte concentration was higher at the end of the treatment period than on d−1 (P< 0.01) or during feed restriction (P< 0.01). Lymphocyte (P= 0.01) and basophil (P= 0.03) concentrations were lower before the treatment period than at the end of it. Nevertheless, all values were within the normal physiological ranges (Roland et al., 2014). Vitamin supplementation and feed restriction did not affect the percentage of PMN positive for phagocytosis (P= 0.50; Figure 4). The percentage of PMN that were positive for oxidative burst after stimulation with phorbol 12-myristate 13-acetate was not affected by vitamin supplementation (P= 0.47) but was reduced by feed restriction (P= 0.03; Figure 5). The proliferation of PBMC was lower when the cell culture medium was supplemented with sera collected during the feed restriction (P< 0.001; Figure 6). Vitamin supplemen- tation did not affect PBMC proliferation (P= 0.11). There was a negative correlation between blood NEFA concentration and lymphocyte proliferation (r=−0.42; P< 0.01). Discussion Several studies (Carbonneau et al., 2012; Esposito et al., 2014; Hoeben et al., 2000; Kehrli et al., 1989; Leblanc 2010; Moreira da Silva et al., 1998) have looked at the immune response dur- ing the transition period. Although a NEB is an important cause of suppression of the immune response during the transition period, it is difficult to isolate the effect of a NEB from those of the physiological and endocrine changes that occur. From a more practical point of view, conducting experiments to alleviate periparturient immunosuppression are complicated by the fact that the exact calving time cannot be predicted and by the high incidence of health problems. In the present study, even though milk production decreased when the feed restriction was applied, all the cows were in Table 1 Milk composition of samples collected at milking on d 29 and 30 from cows injected weekly with either saline (control, C) or a mix of folic acid and vitamin B12 (V) Treatments Item C V SEM P-value Milk composition (g/kg) Fat 43.2 39.6 1.20 0.06 Protein 30.4 31.0 0.71 0.60 Lactose 45.2 46.4 0.27 0.005 Total solids 118.7 116.9 1.83 0.51 Milk yield (kg/day) Fat 1.68 1.43 0.114 0.14 Protein 1.14 1.13 0.054 0.84 Lactose 1.69 1.70 0.073 0.92 Total solids 4.52 4.26 0.228 0.43 Feed restriction Figure 2 Milk production of cows injected weekly with either saline (▴) or a mix of folic acid and vitamin B12 (▪). All cows were feed-restricted from days 27 to 30. Milk production was not affected by treatments but was decreased by feed restriction (P< 0.01). (a) (b) Figure 3 Serum concentrations of β-hydroxybutyrate (BHB) (a) and non- esterified fatty acids (NEFAs) (b) in cows injected weekly with either saline (white bars) or a mix of folic acid and vitamin B12 (gray bars), as measured before the experiment (d −1), just before feed restriction (d 26) and after 3 days of feed restriction (d 29). NEFA concentrations were greater on d 29 than on d −1 and d 26 (P< 0.001). Vanacker, Girard, Blouin and Lacasse 342 a NEB, as indicated by the blood metabolite concentrations. Although all the cows were in a state of NEB, none of them became ill. The energy balance after 3 days of the feed restriction averaged −83.07 Mcal/day, which is comparable to the energy balance found at the beginning of lactation in high-producing cows (McGuireet al., 2004). Therefore, the experimental design that was chosen to induce a state of energy balance similar to the one experienced by cows after parturition seems to be adequate. Injections of folic acid and vitamin B12 did not affect energy balance or plasma concentrations of NEFA and BHB. In pre- vious experiments, similar vitamin supplements given during the peripartum period and in early lactation changed energy partitioning either by increasing milk production without increasing DMI and losses of BW or BCS (Gagnon et al., 2015; Graulet et al., 2007; Preynat et al., 2009) or by decreas- ing BW or body condition score losses without affecting DMI and milk component yields (Duplessis et al., 2017). However, the effects of these supplements on plasma concentrations of NEFA and BHB differed among those studies. Moreover, in the present experiment, it cannot be ruled out that the duration of the NEB (4 days) was too short for an effect of the vitamin supplement on energy partitioning to be detected. Table 2 Concentrations of the leukocyte populations of cows injected weekly with either saline (control, C) or a mix of folic acid and vitamin B12 (V), as measured before the experiment (d −1), just before feed restriction (d 26), and after 3 d of feed restriction (d 29) Cells (×109/l) d −1 d 26 d 29 P-value C V C V C V SEM T1 P2 T*P Normal Range Leukocytes 6.67 7.4 7.63 8.23 6.45 7.7 0.49 0.117 0.0016 0.47 5 to 1.77 Neutrophils 2.53 3.03 2.6 2.27 1.91 2.54 0.38 0.44 0.23 0.20 1.5 to 5.2 Lymphocytes 3.65 3.72 4.23 5.17 3.98 4.41 0.47 0.31 0.014 0.39 1.9 to 6.4 Monocytes 0.30 0.33 0.38 0.31 0.28 0.26 0.069 0.76 0.44 0.69 0 to 0.6 Eosinophils 0.18 0.24 0.39 0.36 0.27 0.43 0.10 0.57 0.06 0.32 0 to 3.6 Basophils 0.05 0.03 0.06 0.11 0.04 0.06 0.017 0.22 0.03 0.22 0 to 0.2 1Treatment. 2Period. Figure 4 Percentages of polymorphonuclear leukocytes (PMN) positive for phagocytosis in cows injected weekly with either saline (white bars) or a mix of folic acid and vitamin B12 (gray bars), as measured just before feed restriction (d 26) and after 3 days of feed restriction (d 29). Figure 5 Percentages of polymorphonuclear leukocytes (PMN) stimulated with phorbol 12-myristate 13-acetate that were positive for oxidative burst in cows injected weekly with either saline (white bars) or a mix of folic acid and vitamin B12 (gray bars), as measured just before feed restriction (d 26) and after 3 days of feed restriction (d 29). **The percentages of PMN that were positive for oxidative burst after stimulation with phorbol 12-myris- tate 13-acetate were reduced by the feed restriction (P= 0.03). Figure 6 Percentages of proliferative peripheral blood mononuclear cells (PBMCs) in cows injected weekly with either saline (white bars) or a mix of folic acid and vitamin B12 (gray bars), as measured just before feed restric- tion (d 26) and after 3 d of feed restriction (d 29). ***The proliferation of PBMC was lower when the cell culture medium was supplemented with sera collected during the feed restriction (d 29) (P< 0.001). Vitamins and feed restriction effect on immunity 343 As expected, feed restriction resulted in a NEB and an increase in blood NEFA concentration. Blood BHB concentra- tion was not increased by feed restriction. In previous studies, our research team observed that a NEB induced by feed restriction (Ollier et al., 2016) or by feeding only dry hay (Bernier-Dodier et al., 2011; Ollier et al., 2014) induces a sharp increase in blood NEFA but a limited increase in blood BHB. Therefore, the feed restriction period in the present was probably too short for an increase in BHB to be seen. We observed a decrease in the ability of PBMC to proliferate after 3 days of feed restriction. Similarly, a decrease in lympho- proliferation was previously observed when PBMCs were incu- bated with sera from early-postpartum or feed-restricted cows (Ster et al., 2012; Ollier et al., 2014, 2016). In all those studies, there was a negative correlation between blood NEFA concen- tration and PBMC proliferation, suggesting that circulating NEFAs impair PBMC function. Ster et al. (2012) reported that serum harvested at 61 DIM but supplemented with enough NEFA to reach the level present in serum harvested at 5 DIM resulted in PBMC proliferation similar to that obtained with the latter serum. Consequently, the lack of effect of vita- min injections on blood NEFA concentration in the present study likely explains their lack of effect on PBMC proliferation. We observed a decrease in the ability of PMN to enter into oxidative burst after 3 days of feed restriction. This effect was not observed in previous studies (Hoeben et al., 2000; Moreira da Silva et al., 1998). Nevertheless, using a dose-effect assay, Ster et al. (2012) reported that concentrations of NEFA above 500 μMwere able to inhibit oxidative burst, which suggests that although PMNare less sensitive to NEFAs than PBMCs are, PMN may still be affected by a high NEFA level. In the assays carried out by Ster et al. (2012) and Carbonneau et al. (2012), tested serum was added to PMN from healthy cows at a low concen- tration, such that the final concentration was low owing to dilution. In the present study, PMN functions were evaluated in whole-blood assays where the dilution factor was much lower. This likely resulted in greater sensitivity for the assays. In addition,whole-blood assayswould also detect if the immune cells themselves were affected by the treatments. Those results support the finding that a NEB affects PMN functions. In conclusion, feed restriction affected the functions of PMN and PBMC, supporting the hypothesis that the greater risk of infectious diseases in cows experiencing a NEB is related to the impairment of PMN and PBMC functions by NEFAs or other components of blood that are still unknown. These results suggest that a severe feed restriction could be used as a model in order to study the effect of the NEB on the immune system of dairy cows. In the tested conditions, supplementation with folic acid and vitamin B12 did not affect the increase in NEFA or BHB concentration and did not reduce the impairment of immune cell functions. Acknowledgements The authors would like to thank the following people from Agriculture and Agri-Food Canada (Sherbrooke, QC, Canada): Valérie Beaudet, Catherine Thibault, and Véronique Roy for providing technical assistance, and the dairy barn staff for taking care of the animals. The authors are grateful to Mary Varcoe, from the Translation Bureau, Public Services and Procurement Canada, for her careful editing of this manuscript. This research was financially supported by Agriculture and Agri-Food Canada (Ottawa, ON, Canada) and by the Natural Sciences and Engineering Research Council of Canada (Ottawa, ON, Canada). Preliminary results have been published in an abstract form (Vanacker et al., 2017a). N. Vanacker 0000-0003-1340-7697 R. Blouin 0000-0002-1882-3262 P. Lacasse 0000-0002-3579-7991 Declaration of interest The authors declare no conflict of interest. Ethics statement The study was conducted in accordance with the guidelines of the Canadian Council on Animal Care (1993). Software and data repository resources Data were not deposited in an official repository. 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Vitamins and feed restriction effect on immunity 345 Effects of feed restriction and supplementary folic acid and vitamin B12 on immune cell functions and blood cell populations in dairy cows Implications Introduction Materials and methods Animals and experimental procedures Milk yield and composition Blood collection Blood metabolite assays Polymorphonuclear leukocytes phagocytosis and oxidative burst assays Peripheral blood mononuclear cell isolation Peripheral blood mononuclear cell proliferation assay Statistical analysis Results Milk production, milk composition, DM intake and BW Blood metabolic marker concentrations Immunological parameters Discussion Acknowledgements Declaration of interest Ethics statement Software and data repository resources Supplementary material References