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Research Article| Volume 98, ISSUE 9, P6327-6339, September 2015

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Effects of dietary Capsicum oleoresin on productivity and immune responses in lactating dairy cows

Open ArchivePublished:July 15, 2015DOI:https://doi.org/10.3168/jds.2014-9294
Effects of dietary Capsicum oleoresin on productivity and immune responses in lactating dairy cows
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      Abstract

      This study investigated the effect of Capsicum oleoresin in granular form (CAP) on nutrient digestibility, immune responses, oxidative stress markers, blood chemistry, rumen fermentation, rumen bacterial populations, and productivity of lactating dairy cows. Eight multiparous Holstein cows, including 3 ruminally cannulated, were used in a replicated 4 × 4 Latin square design experiment. Experimental periods were 25 d in duration, including a 14-d adaptation and an 11-d data collection and sampling period. Treatments included control (no CAP) and daily supplementation of 250, 500, or 1,000 mg of CAP/cow. Dry matter intake was not affected by CAP (average 27.0 ± 0.64 kg/d), but milk yield tended to quadratically increase with CAP supplementation (50.3 to 51.9 ± 0.86 kg/d). Capsicum oleoresin quadratically increased energy-corrected milk yield, but had no effect on milk fat concentration. Rumen fermentation variables, apparent total-tract digestibility of nutrients, and N excretion in feces and urine were not affected by CAP. Blood serum β-hydroxybutyrate was quadratically increased by CAP, whereas the concentration of nonesterified fatty acids was similar among treatments. Rumen populations of Bacteroidales, Prevotella, and Roseburia decreased and Butyrivibrio increased quadratically with CAP supplementation. T cell phenotypes were not affected by treatment. Mean fluorescence intensity for phagocytic activity of neutrophils tended to be quadratically increased by CAP. Numbers of neutrophils and eosinophils and the ratio of neutrophils to lymphocytes in peripheral blood linearly increased with increasing CAP. Oxidative stress markers were not affected by CAP. Overall, in the conditions of this experiment, CAP did not affect feed intake, rumen fermentation, nutrient digestibility, T cell phenotypes, and oxidative stress markers. However, energy-corrected milk yield was quadratically increased by CAP, possibly as a result of enhanced mobilization of body fat reserves. In addition, CAP increased neutrophil activity and immune cells related to acute phase immune response.

      Key words

      Introduction

      Capsicum is a genus of flowering plants containing capsaicinoids as its active compounds. Original interest in Capsicum as a feed additive in beef and dairy production systems was based on its potential as modifier of rumen fermentation and its pungency to increase feed intake (
      • Calsamiglia S.
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      • Ferret A.
      Invited review: Essential oils as modifiers of rumen microbial fermentation.
      J. Dairy Sci. 2007; 90: 2580-2595
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      • Gonzalez L.
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      • Calsamiglia S.
      Effects of dietary addition of capsicum extract on intake, water consumption, and rumen fermentation of fattening heifers fed a high-concentrate diet.
      J. Anim. Sci. 2012; 90: 1879-1884
      ). Recent findings suggested, however, that the improvement in animal performance may be due to a host response (physiological and immunological) rather than direct antimicrobial or sensory effects. For example, Capsicum oleoresin or its mixture with other plant extracts have reduced oxidative stress (
      • Karadas F.
      • Pirgozliev V.
      • Rose S.P.
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      • Oduguwa O.
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      Dietary essential oils improve the hepatic antioxidative status of broiler chickens.
      Br. Poult. Sci. 2014; 55: 329-334
      ), prevented disease symptoms (
      • Lee S.H.
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      • Lee K.W.
      • Bravo D.
      • Lillehoj E.P.
      Effects of dietary supplementation with phytonutrients on vaccine-stimulated immunity against infection with Eimeria tenella.
      Vet. Parasitol. 2011; 181: 97-105
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      • Lee S.H.
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      • Jang S.I.
      • Lillehoj E.P.
      • Min W.
      • Bravo D.M.
      Dietary supplementation of young broiler chickens with Capsicum and turmeric oleoresins increases resistance to necrotic enteritis.
      Br. J. Nutr. 2013; 110: 840-847
      ;
      • Liu Y.
      • Song M.
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      • Pettigrew J.E.
      Anti-inflammatory effects of several plant extracts on porcine alveolar macrophages in vitro.
      J. Anim. Sci. 2012; 90: 2774-2783
      ), and improved gut health during normal or disease conditions (
      • Liu Y.
      • Song M.
      • Che T.M.
      • Bravo D.
      • Maddox C.W.
      • Pettigrew J.E.
      Effects of capsicum oleoresin, garlic botanical, and turmeric oleoresin on gene expression profile of ileal mucosa in weaned pigs.
      J. Anim. Sci. 2014; 92 (a): 3426-3440
      ,
      • Liu Y.
      • Song M.
      • Che T.M.
      • Lee J.J.
      • Bravo D.
      • Maddox C.W.
      • Pettigrew J.E.
      Dietary plant extracts modulate gene expression profiles in ileal mucosa of weaned pigs after an Escherichia coli infection.
      J. Anim. Sci. 2014; 92 (b): 2050-2062
      ) in both poultry and swine. In addition, studies using rats showed that capsaicinoids, active compounds in Capsicum, had modulatory effects on immune cells such as neutrophils and T cells (
      • Franco-Penteado C.F.
      • De Souza I.A.
      • Lima C.S.
      • Teixeira S.A.
      • Muscara M.N.
      • De Nucci G.
      • Antunes E.
      Effects of neonatal capsaicin treatment in the neutrophil production, and expression of preprotachykinin-I and tachykinin receptors in the rat bone marrow.
      Neurosci. Lett. 2006; 407: 70-73
      ;
      • Takano F.
      • Yamaguchi M.
      • Takada S.
      • Shoda S.
      • Yahagi N.
      • Takahashi T.
      • Ohta T.
      Capsicum ethanol extracts and capsaicin enhance interleukin-2 and interferon-gamma production in cultured murine Peyer's patch cells ex vivo.
      Life Sci. 2007; 80: 1553-1563
      ), and decreased oxidative stress markers and induced negative energy balance (
      • Yoshioka M.
      • Matsuo T.
      • Lim K.
      • Tremblay A.
      • Suzuki M.
      Effects of capsaicin on abdominal fat and serum free-fatty acids in exercise-trained rats.
      Nutr. Res. 2000; 20: 1041-1045
      ;
      • Abdel-Salam O.M.
      • Abdel-Rahman R.F.
      • Sleem A.A.
      • Farrag A.R.
      Modulation of lipopolysaccharide-induced oxidative stress by capsaicin.
      Inflammopharmacology. 2012; 20: 207-217
      ). With respect to ruminants,
      • Oh J.
      • Hristov A.N.
      • Lee C.
      • Cassidy T.
      • Heyler K.
      • Varga G.A.
      • Pate J.
      • Walusimbi S.
      • Brzezicka E.
      • Toyokawa K.
      • Werner J.
      • Donkin S.S.
      • Elias R.
      • Dowd S.
      • Bravo D.
      Immune and production responses of dairy cows to postruminal supplementation with phytonutrients.
      J. Dairy Sci. 2013; 96: 7830-7843
      reported that a short-term (9-d) abomasal infusion of Capsicum oleoresin increased lymphocyte proportion and CD4+ T cells and did not affect oxidative stress markers in blood of dairy cows. However, it was unknown if such host-based physiological effects of Capsicum would be observed when supplementation is through the feed versus postruminal delivery.
      Based on the existing data, we hypothesized that dietary supplementation of Capsicum oleoresin in unprotected granular form (CAP) could potentially modify rumen fermentation, facilitate immune response, and reduce oxidative stress in dairy cows. The CAP product could also stimulate feed intake and animal productivity. Thus, the objective of the experiment was to investigate the physiological effects of CAP in relation to feed intake, rumen function, blood chemistry, hematology, immune responses, oxidative stress markers, and productivity of lactating dairy cows.

      Materials and Methods

      Animals and Treatments

      The Pennsylvania State University Animal Care and Use committee approved all procedures in this experiment. The design of the experiment was a replicated 4 × 4 Latin square with 8 multiparous Holstein cows averaging 590 ± 32.6 kg of BW, 50 ± 9.6 DIM, and 52 ± 2.4 kg/d milk yield at the beginning of the experiment. Three cows were fitted with soft plastic rumen cannulas (10 cm internal diameter; Bar Diamond Inc., Parma, ID) and constituted 1 incomplete square of the experiment. Experimental periods were 25 d, including a 14-d adaptation and an 11-d data collection period. Treatments were control (0 mg/d of CAP per cow) and 3 application rates of CAP: 250 (C250), 500 (C500), and 1,000 mg/d per cow (C1000). The CAP product used in the experiment was CapsXL (X60–7035; 20% Capsicum oleoresin; 1.2% capsaicinoids; Pancosma S. A., Geneva, Switzerland). The product was top-dressed daily mixed with a small amount of TMR during feeding. Cows were housed in a tiestall barn, fed once daily at approximately 0800 h, and had free access to fresh water. The basal diet (Table 1) was fed ad libitum as a TMR, targeting approximately 10% refusals. The diet was formulated to meet or exceed the nutrient requirements of a lactating Holstein cow yielding 50 kg of milk/d with 3.80% fat and 3.20% true protein at 27 kg/d of DMI and 650 kg of BW (
      NRC
      Nutrient Requirements of Dairy Cattle. 7th rev. Natl. Acad. Sci., Washington, DC2001
      ). Cows were milked twice daily at 0500 and 1700 h and treated with recombinant bST (rbST; 500 mg, i.m., Posilac; Elanco Co., Greenfield, IN) on d 1 and 13 of each experimental period.
      Table 1Ingredient and chemical composition of the diet fed during the experiment
      ItemMeasurement
      Ingredient, % of diet DM
       Corn silage
      Corn silage was 46.5% DM and contained (DM basis) 8.5% CP, 32.1% NDF, and 41.4% starch.
      		44.0
       Grass hay
      Grass hay was 90.7% DM and contained (DM basis) 7.0% CP and 73.8% NDF.
      		7.0
       Cottonseed, hulls5.5
       Corn grain, ground9.5
       Candy by-product meal
      Candy by-product meal (Graybill Processing, Elizabethtown, PA) contained (DM basis) 16.6% CP and 27.8% NDF.
      		1.8
       Soybean seeds, whole heated
      Soybean seeds contained (DM basis) 40.0% CP.
      		8.0
       Canola meal
      Canola meal contained (DM basis) 41.8% CP.
      		12.0
       Molasses
      Molasses (Westway Feed Products, Tomball, TX) contained (DM basis) 3.9% CP and 66% total sugar.
      		5.4
       Vitamin and mineral premix
      The premix (Cargill Animal Nutrition, Cargill Inc., Roaring Spring, PA) contained (%, as-is basis) trace mineral mix, 0.86; MgO (56% Mg), 8.0; NaCl, 6.4; vitamins A, D, and E premix (Cargill Animal Nutrition), 0.48; limestone, 37.2; selenium premix (Cargill Animal Nutrition), 0.07; and dry corn distillers grains with solubles, 46.7; Ca, 14.1%; P, 0.39%; Mg, 4.59%; K, 0.44%; S, 0.39%; Se, 6.91 mg/kg; Cu, 362 mg/kg; Zn, 1,085 mg/kg; Fe, 186 mg/kg, vitamin A, 276,717 IU/kg; vitamin D, 75,000 IU/kg; and vitamin E, 1,983 IU/kg.
      		3.0
       SoyPLUS
      SoyPLUS (West Central Cooperative, Ralston, IA) contained (DM basis) 46.6% CP.
      		3.4
       Optigen
      Optigen is a slow-release urea (Alltech Inc., Nicholasville, KY).
      		0.4
      Composition,
      Values calculated using the chemical analysis (Cumberland Valley Analytical Services Inc., Maugansville, MD) of the ingredients of the diet.
      % of DM

       (unless otherwise noted)
       CP
      Values calculated using the chemical analysis (Cumberland Valley Analytical Services Inc., Maugansville, MD) of the ingredients of the diet.
      		15.8
       RDP
      Estimated by NRC (2001).
      		10.2
       RUP
      Estimated by NRC (2001).
      		7.0
       NDF
      Values calculated using the chemical analysis (Cumberland Valley Analytical Services Inc., Maugansville, MD) of the ingredients of the diet.
      		30.7
       ADF
      Values calculated using the chemical analysis (Cumberland Valley Analytical Services Inc., Maugansville, MD) of the ingredients of the diet.
      		18.8
       NEL, Mcal/kg
      Values calculated using the chemical analysis (Cumberland Valley Analytical Services Inc., Maugansville, MD) of the ingredients of the diet.
      		1.58
       Ca
      Values calculated using the chemical analysis (Cumberland Valley Analytical Services Inc., Maugansville, MD) of the ingredients of the diet.
      		0.70
       P
      Values calculated using the chemical analysis (Cumberland Valley Analytical Services Inc., Maugansville, MD) of the ingredients of the diet.
      		0.40
       NFC
      Estimated by NRC (2001).
      		43.2
       Average NEL balance,
      Estimated based on NRC (2001) using actual DMI, milk yield, milk composition, and BW of the cows throughout the experiment (Control, C250, C500, and C1000, respectively).
      Mcal/d      		0.2, −0.9, −0.9, and 0.4
       Average MP balance,
      Estimated based on NRC (2001) using actual DMI, milk yield, milk composition, and BW of the cows throughout the experiment (Control, C250, C500, and C1000, respectively).
      g/d      		−140, −137, −163, and −57
      1 Corn silage was 46.5% DM and contained (DM basis) 8.5% CP, 32.1% NDF, and 41.4% starch.
      2 Grass hay was 90.7% DM and contained (DM basis) 7.0% CP and 73.8% NDF.
      3 Candy by-product meal (Graybill Processing, Elizabethtown, PA) contained (DM basis) 16.6% CP and 27.8% NDF.
      4 Soybean seeds contained (DM basis) 40.0% CP.
      5 Canola meal contained (DM basis) 41.8% CP.
      6 Molasses (Westway Feed Products, Tomball, TX) contained (DM basis) 3.9% CP and 66% total sugar.
      7 The premix (Cargill Animal Nutrition, Cargill Inc., Roaring Spring, PA) contained (%, as-is basis) trace mineral mix, 0.86; MgO (56% Mg), 8.0; NaCl, 6.4; vitamins A, D, and E premix (Cargill Animal Nutrition), 0.48; limestone, 37.2; selenium premix (Cargill Animal Nutrition), 0.07; and dry corn distillers grains with solubles, 46.7; Ca, 14.1%; P, 0.39%; Mg, 4.59%; K, 0.44%; S, 0.39%; Se, 6.91 mg/kg; Cu, 362 mg/kg; Zn, 1,085 mg/kg; Fe, 186 mg/kg, vitamin A, 276,717 IU/kg; vitamin D, 75,000 IU/kg; and vitamin E, 1,983 IU/kg.
      8 SoyPLUS (West Central Cooperative, Ralston, IA) contained (DM basis) 46.6% CP.
      9 Optigen is a slow-release urea (Alltech Inc., Nicholasville, KY).
      10 Values calculated using the chemical analysis (Cumberland Valley Analytical Services Inc., Maugansville, MD) of the ingredients of the diet.
      11 Estimated by
      NRC
      Nutrient Requirements of Dairy Cattle. 7th rev. Natl. Acad. Sci., Washington, DC2001
      .
      12 Estimated based on
      NRC
      Nutrient Requirements of Dairy Cattle. 7th rev. Natl. Acad. Sci., Washington, DC2001
      using actual DMI, milk yield, milk composition, and BW of the cows throughout the experiment (Control, C250, C500, and C1000, respectively).

      Sampling and Analyses

      Feed intake was measured daily, TMR samples were collected twice weekly, and individual ingredients of forage and concentrate were sampled once weekly. Composite samples of the TMR, forages, and concentrate feeds were processed and analyzed for OM, CP, NDF, ADF, Ca, P, indigestible NDF (iNDF), and NEL as described in
      • Oh J.
      • Hristov A.N.
      • Lee C.
      • Cassidy T.
      • Heyler K.
      • Varga G.A.
      • Pate J.
      • Walusimbi S.
      • Brzezicka E.
      • Toyokawa K.
      • Werner J.
      • Donkin S.S.
      • Elias R.
      • Dowd S.
      • Bravo D.
      Immune and production responses of dairy cows to postruminal supplementation with phytonutrients.
      J. Dairy Sci. 2013; 96: 7830-7843
      .
      Milk production of the cows was recorded daily and samples for milk composition were collected on d 20 and 24 of each experimental period from evening and morning milkings, preserved with 2-bromo-2-nitropropane-1,3 diol, and submitted to Dairy One laboratory for analysis (Pennsylvania DHIA, University Park, PA). Milk was analyzed for fat, true protein, lactose, and MUN using infrared spectroscopy (MilkoScan 4000; Foss Electric, Hillerød, Denmark). Another aliquot was collected in a tube containing no preservative, kept at −20°C, and analyzed for milk FA composition as described in
      • Hristov A.N.
      • Varga G.
      • Cassidy T.
      • Long M.
      • Heyler K.
      • Karnati S.K.R.
      • Corl B.
      • Hovde C.J.
      • Yoon I.
      Effect of Saccharomyces cerevisiae fermentation product on ruminal fermentation and nutrient utilization in dairy cows.
      J. Dairy Sci. 2010; 93: 682-692
      ).
      Samples of whole ruminal contents were collected from the cannulated cows on d 24 and 25 of each experimental period at 2, 4, and 6 h after feeding, processed as described elsewhere (
      • Hristov A.N.
      • Lee C.
      • Cassidy T.
      • Long M.
      • Corl B.
      • Forster R.
      Effects of lauric and myristic acids on ruminal fermentation, production, and milk fatty acid composition in lactating dairy cows.
      J. Dairy Sci. 2011; 94: 382-395
      ), and analyzed for pH (59000–60 pH Tester, Cole-Parmer Instrument Company, Vernon Hills, IL), VFA (
      • Yang C.M.
      • Varga G.A.
      Effect of three concentrate feeding frequencies on rumen protozoa, rumen digesta kinetics, and milk yield in dairy cows.
      J. Dairy Sci. 1989; 72: 950-957
      ), and ammonia concentration (
      • Chaney A.L.
      • Marbach E.P.
      Modified reagents for determination of urea and ammonia.
      Clin. Chem. 1962; 8: 130-132
      ). Aliquots of whole ruminal contents were composited (on an equal wet weight basis) per cow and period and stored frozen at −80°C for bacterial population analysis. The 16S rRNA gene V4 variable region PCR primers 515/806 (
      • Caporaso J.G.
      • Lauber C.L.
      • Walters W.A.
      • Berg-Lyons D.
      • Lozupone C.A.
      • Turnbaugh P.J.
      • Fierer N.
      • Knight R.
      Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample.
      Proc. Natl. Acad. Sci. USA. 2011; 108: 4516-4522
      ) were used in a single-step 30-cycle PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, Germantown, MD) under the following conditions: 94°C for 3 min, followed by 28 cycles (5 cycles used on PCR products) of 94°C for 30 s, 53°C for 40 s, and 72°C for 1 min, after which a final elongation step at 72°C for 5 min was completed. Sequencing was performed at Molecular Research DNA (www.mrdnalab.com; Shallowater, TX) on an Ion Torrent PGM (Life Technologies, Carlsbad, CA) following the manufacturer’s guidelines. Sequence data were processed using a proprietary analysis pipeline (Molecular Research DNA). In summary, sequences were depleted of barcodes and primers, then sequences <150 bp were removed, and sequences with ambiguous base calls and with homopolymer runs exceeding 6 bp were also removed. Sequences were denoised, operational taxonomic units generated, and chimeras removed. Operational taxonomic units were defined by clustering at 3% divergence (97% similarity). Final operational taxonomic units were taxonomically classified using BLASTn against a curated database derived from GreenGenes, RDPII, and NCBI (www.ncbi.nlm.nih.gov;
      • DeSantis T.Z.
      • Hugenholtz P.
      • Larsen N.
      • Rojas M.
      • Brodie E.L.
      • Keller K.
      • Huber T.
      • Dalevi D.
      • Hu P.
      • Andersen G.L.
      Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB.
      Appl. Environ. Microbiol. 2006; 72: 5069-5072
      , http://rdp.cme.msu.edu; accessed Dec. 24, 2014).
      Spot fecal and urine samples were collected by stimulating defecation or from the rectum and by massaging the vulva, respectively, at 1000, 1600, and 2200 h on d 22; 0400, 1300, and 1900 h on d 23; and 0100 and 0700 h on d 24 of each experimental period. Fecal and urine samples were collected, processed and analyzed for OM, CP, NDF, and ADF (fecal samples) and total N, urinary urea N, creatinine, and the purine derivatives (PD) allantoin and uric acid (urine samples) as described in
      • Oh J.
      • Hristov A.N.
      • Lee C.
      • Cassidy T.
      • Heyler K.
      • Varga G.A.
      • Pate J.
      • Walusimbi S.
      • Brzezicka E.
      • Toyokawa K.
      • Werner J.
      • Donkin S.S.
      • Elias R.
      • Dowd S.
      • Bravo D.
      Immune and production responses of dairy cows to postruminal supplementation with phytonutrients.
      J. Dairy Sci. 2013; 96: 7830-7843
      . Daily volume of excreted urine was estimated based on urinary creatinine concentration, assuming a creatinine excretion rate of 29 mg/kg of BW (determined based on total urine collection samples from
      • Hristov A.N.
      • Lee C.
      • Cassidy T.
      • Long M.
      • Corl B.
      • Forster R.
      Effects of lauric and myristic acids on ruminal fermentation, production, and milk fatty acid composition in lactating dairy cows.
      J. Dairy Sci. 2011; 94: 382-395
      ). Estimated urine output was used to calculate daily total N and urinary urea N excretions. Apparent total-tract digestibility of nutrients was estimated using iNDF as an intrinsic digestibility marker (
      • Schneider B.H.
      • Flatt W.P.
      The Evaluation of Feeds Through Digestibility Experiments. University of Georgia Press, Athens, GA1975
      ). Fecal and TMR samples were analyzed for iNDF according to
      • Huhtanen P.
      • Kaustell K.
      • Jaakkola S.
      The use of internal markers to predict total digestibility and duodenal flow of nutrients in cattle given six different diets.
      Anim. Feed Sci. Technol. 1994; 48: 211-227
      , with the exception that 25-µm pore size Ankom filter bags (Ankom Technology, Macedon, NY) were used for the rumen incubation.
      Blood samples were collected from the coccygeal vein or artery at 2 and 4 h after feeding on d 22 of each period for hematology analyses. Samples (approximately 10 mL) were collected into vacuumed tubes containing EDTA (BD Biosciences, Franklin Lakes, NJ), kept refrigerated (4°C), and analyzed the same day. The analysis included red blood cell count, hemoglobin, hematocrit, platelet count, mean platelet volume, and total white blood cell count, including total count for neutrophils, eosinophils, lymphocytes, monocytes, and basophils using an automated hematology analyzer (HemaVet; Drew Scientific, Oxford, CT). A separate set of blood samples was collected into vacuumed tubes containing silica clot activator (SST Tube; BD Biosciences) at 2 and 4 h after feeding on d 22 and 23 of each experimental period. Blood serum was separated (after clotting) through centrifugation at 3,000 × g at room temperature for 15 min, composited on an equal volume basis per cow, period, and sampling time point and stored at −20°C. Serum was analyzed for albumin, amylase, BUN, Ca, cholesterol, Cl, creatinine, globulin, glucose, K, Na, P, and total protein (Idexx VetTest and VetLyte Chemistry and Electrolyte Analyzers, Idexx Laboratories Inc., Westbrook, ME). Serum samples were also analyzed for BHBA and NEFA using biochemistry analyzer (Cobas 6000; Roche, Berlin, Germany) and for insulin using RIA (Coat-a-count insulin kit TKIN5; Siemens Healthcare Diagnostics, Los Angeles, CA). A third set of blood samples was collected into vacuumed tubes containing EDTA (BD Biosciences) at 2 and 4 h after feeding on d 22 and 23 of each experimental period for oxidative stress markers. Plasma was obtained by centrifugation at 1,500 × g at 4°C for 10 min, composited on an equal volume basis per cow, period, and sampling time point, and stored frozen at −80°C. Samples were analyzed for thiobarbituric acid reactive substances using colorimetric assay kits (Cayman Chemical, Ann Arbor, MI) and 8-isoprostane using ELISA kits (Cayman Chemical) and for oxygen radical absorbance capacity, as described elsewhere (
      • Cao G.
      • Prior R.L.
      Measurement of oxygen radical absorbance capacity in biological samples.
      Methods Enzymol. 1999; 299: 50-62
      ).
      Whole-blood (approximately 50 mL) samples were collected via tail vein or artery before feeding from 1 experimental square (4 cows) on d 24 and from the other square (4 cows) on d 25 of each period for T cell phenotype analysis. Whole blood was transferred to borosilicate glass tubes and centrifuged at 1,500 × g at 4°C for 10 min to obtain a lymphocyte-rich white blood cell layer. Peripheral blood mononuclear cells were isolated by centrifugation at 650 × g at 25°C for 30 min over Ficoll-Paque PLUS (GE Healthcare Bio-Sciences, Piscataway, NJ). A flow cytometer (GuavaEasyCyte Plus; Millipore, Billerica, MA) was used for counting and analysis of T lymphocytes. Peripheral blood mononuclear cells (1 × 106) were directly labeled with fluor-conjugated primary antibodies against T lymphocyte-surface antigens following a protocol described by
      • Poole D.H.
      • Pate J.L.
      Luteal microenvironment directs resident T lymphocyte function in cows.
      Biol. Reprod. 2012; 86: 29
      . The antibodies used were against cluster of differentiation antigen (CD) 4 (MCA1653F; AbD Serotech, Raleigh, NC), CD25α (CACT116A; VMRD, Pullman, WA), CD8α (MCA837F; AbD Serotech), and δ T cell receptor (δTCR; CACT61A, VMRD). The primary antibodies for CD25α and δTCR were conjugated to phycoerythrin using IgG1 and IgM goat anti-mouse antibodies [IgG1 (STAR132P) and IgM (102009), respectively; AbD Serotech] following the manufacturers guidelines. The following antibodies were used as controls: IgG2a negative control (MCA929F; AbD Serotech), IgG1 negative control (MCA928PE and MCA928F; AbD Serotech), and IgM negative control (MCA692; AbD Serotech). Samples were analyzed in duplicate, CD4 was paired with CD25α and CD8 was paired with δTCR. Flow cytometer (GuavaEasyCyte Plus; Millipore) was used to determine the proportion of single- and dual-stained cells.
      The phagocytic capacity of neutrophils was measured by a flow cytometric assay (
      • Smits E.
      • Burvenich C.
      • Heyneman R.
      Simultaneous flow cytometric measurement of phagocytotic and oxidative burst activity of polymorphonuclear leukocytes in whole bovine blood.
      Vet. Immunol. Immunopathol. 1997; 56: 259-269
      ). Killed bacteria, Streptococcus uberis, were provided by the Animal Diagnostic Laboratory at the Pennsylvania State University, suspended in 1 mL of PBS, and labeled with propidium iodide (PI) for 60 min at room temperature. The PI-labeled bacteria were washed 3 times with PBS to remove excess PI, suspended in RPMI-1640 medium (Gibco Laboratories, Grand Island, NY), and counted using a flow cytometer (GuavaEasyCyte Plus, Millipore). Aliquots were stored at −80°C and thawed immediately before use. Whole blood (10 mL) was collected from the coccygeal vein or artery into heparinized-vacutainers (BD Biosciences) on d 23 of each experimental period. The blood was centrifuged at 4°C and 1,500 × g for 10 min, the supernatant and buffy coat layers were aspirated and discarded, and the packed red blood cells were lysed by the addition of 2 volumes of red blood cell lysis buffer (0.14 M NH4Cl, 10 mM KHCO3). Lysis was stopped by adding a volume of Hanks’ balanced salt solution after 1 min. The lysate was centrifuged at 400 × g at 4°C for 10 min with no brake, washed with Hanks’ balanced salt solution once at 300 × g at 4°C for 10 min, and finally suspended in RPMI-1640 medium. Total neutrophils were counted by flow cytometer (GuavaEasyCyte Plus, Millipore). Bacteria labeled with PI were added into 96-well plates at a ratio of the bacteria to neutrophils of 15:1 (bacteria:neutrophils = 15 × 105:1 × 105). After 20 min of incubation at room temperature, the plates were read on a flow cytometer (GuavaEasyCyte Plus, Millipore) and the proportion of neutrophils that had phagocytized bacteria was determined. Also, histogram analysis for mean fluorescence intensity of PI was conducted to estimate mean phagocytic activity of total gated neutrophils, which indicated mean number of engulfed bacteria per neutrophil (
      • Silvestre F.T.
      • Carvalho T.S.
      • Crawford P.C.
      • Santos J.E.
      • Staples C.R.
      • Jenkins T.
      • Thatcher W.W.
      Effects of differential supplementation of fatty acids during the peripartum and breeding periods of Holstein cows: II. Neutrophil fatty acids and function, and acute phase proteins.
      J. Dairy Sci. 2011; 94: 2285-2301
      ). The assay was run in duplicate.

      Statistical Analysis

      All data were analyzed using the MIXED procedure of SAS (version 9.4; SAS Institute Inc., Cary, NC). Milk yield, DMI, and estimated feed efficiency data for the last 11 d and milk composition data for the 2 sampling days of each experimental period were averaged and the average values were used in the statistical analysis. The averaged milk yield, DMI, and milk composition data were used to calculate yields of milk fat, protein, lactose, 4.0% FCM, and ECM.
      Nutrient intake, digestibility, rumen microbial population, urinary and fecal N excretions, milk composition, hematology, blood chemistry, BHBA, NEFA, T cell phenotypes, and neutrophil phagocytosis data were analyzed by ANOVA Latin square. The model used was as follows:
      Yijkl=μ+Si+C(S)ij+Pk+Tl+eijkl,


      where Yijkl is the dependent variable, μ is the overall mean, Si is the square, C(S)ij is the cow within square, Pk is the kth period, and Tl is the lth treatment with the error term eijkl. Square and cow within square were random effects and all others were fixed.
      Rumen fermentation data (pH and ammonia and VFA concentrations), DMI, milk yield, SCC, and feed efficiency data were analyzed as repeated measures assuming an AR(1) covariance structure. The model used was as follows:
      Yijklm=μ+Si+C(S)ij+Pk+Tl+Dm+TDlm+eijklm,


      where Yijklm is the dependent variable, μ is the overall mean, Si is the square, C(S)ij is the cow within square, Pk is the kth period, Tl is the lth treatment, Dm is the time effect, and TDlm is the treatment × time of sampling interaction, with the error term eijklm. Square and cow within square were random effects and all others were fixed.
      Data were tested for normality using the UNIVARIATE procedure of SAS. Log-transformed data were analyzed when the W statistic of the Shapiro-Wilk test was less than 0.05. Orthogonal contrasts were used to evaluate CAP treatments versus control, linear, and quadratic effects of CAP supplementation. Statistical differences were considered significant at P ≤ 0.05 and trends at 0.05 < P ≤ 0.10.

      Results

      The basal diet used in this experiment was formulated to meet NEL and MP requirements of cows milking 50 kg/d. Due to higher than expected milk yield and BW deposition during the experiment, however, the diet provided NEL slightly below
      NRC
      Nutrient Requirements of Dairy Cattle. 7th rev. Natl. Acad. Sci., Washington, DC2001
      requirements (−2.0 and −2.1% for C250 and C500, respectively) and MP from −1.8 to −5.0% below the requirements (Table 1). Cows gained, on average, 34 ± 10.3 kg of BW during the experiment (P = 0.26).
      Dry matter intake was not affected by CAP supplementations (Table 2). Milk yield tended to quadratically increase (P = 0.09) with CAP. Cows in this experiment were experiencing milk fat depression. Despite numerical differences, milk fat concentration was not affected by treatments, but milk fat yield was higher (P = 0.05) for CAP than the control and tended to be quadratically increased (P = 0.09) by CAP supplementation. Compared with the control, 4% FCM and ECM were increased (P = 0.04 and P = 0.06, respectively) by CAP and quadratically increased (P = 0.04) with increasing CAP supplementation rate. No effect of CAP supplementation on milk true protein content and yield was observed. Concentration of lactose linearly decreased (P < 0.01), but concentrations of TS and MUN were not affected by CAP. Milk NEL increased quadratically (P = 0.05) with CAP supplementation. As dietary NEL intake remained constant, milk NEL efficiency was higher (P = 0.05) for CAP than the control. Treatment did not affect milk SCC and BW of the cows.
      Table 2Effect of dietary Capsicum oleoresin (CAP) on DMI, milk yield and composition, and BW in dairy cows
      ItemTreatment
      Control=0 mg/d of CAP; C250=250 mg/d of CAP; C500=500 mg/d of CAP; C1000=1,000 mg/d of CAP.
      		SEM
      Highest SEM shown; n=359 for DMI and dietary NEL intake, n=353 for milk yield and feed efficiency, n=339 for BW, n=32 for all other variables (n represents number of observations used in the statistical analysis).
      		P-value
      Con vs. T=control vs. treatment; L=linear effect of CAP; Q=quadratic effect of CAP.
      ControlC250C500C1000Con vs. TLQ
      DMI, kg/d27.027.527.026.50.640.750.270.84
      Milk yield, kg/d50.551.951.550.30.860.370.540.09
      Feed efficiency
      Milk yield ÷ DMI.
      , kg/kg      		1.901.932.021.960.0470.160.270.25
      Milk fat, %2.893.123.033.070.0100.120.360.33
      Yield, kg/d1.461.621.551.560.0540.050.600.09
      4% FCM, kg/d42.345.444.243.41.010.040.790.04
      ECM,
      ECM (kg/d)=kg of milk × [(38.3 × % fat × 10 + 24.2 × % true protein × 10 + 16.54 × % lactose × 10 + 20.7) ÷ 3,140] (Sjaunja et al., 1990).
      kg/d      		46.649.548.347.31.020.060.980.04
      Milk true protein, %3.013.002.982.980.0360.430.340.73
      Yield, kg/d1.531.561.541.500.0380.850.300.26
      Milk lactose, %4.864.824.784.780.0160.01<0.010.13
      Yield, kg/d2.462.502.492.410.0460.960.290.29
      TS, %11.812.011.811.80.130.480.990.65
      MUN, mg/100 mL16.616.116.715.80.690.340.210.63
      Milk NEL,
      Milk NEL (Mcal/d)=kg of milk × (0.0929 × % fat + 0.0563 × % true protein + 0.0395 × % lactose) (NRC, 2001).
      Mcal/d      		32.033.933.032.30.780.090.830.05
      Dietary NEL intake, Mcal/d42.743.142.441.71.020.760.240.65
      Milk NEL efficiency,
      Milk NEL ÷ NEL intake × 100.
      %      		74.979.077.877.61.860.050.300.13
      SCC, ×10
      Con vs. T=control vs. treatment; L=linear effect of CAP; Q=quadratic effect of CAP.
      cells/mL      		17213377.213886.50.400.670.33
      BW, kg62362161461410.30.260.160.57
      1 Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP.
      2 Highest SEM shown; n = 359 for DMI and dietary NEL intake, n = 353 for milk yield and feed efficiency, n = 339 for BW, n = 32 for all other variables (n represents number of observations used in the statistical analysis).
      3 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP.
      4 Milk yield ÷ DMI.
      5 ECM (kg/d) = kg of milk × [(38.3 × % fat × 10 + 24.2 × % true protein × 10 + 16.54 × % lactose × 10 + 20.7) ÷ 3,140] (
      • Sjaunja L.O.
      • Baevre L.
      • Junkkarinen L.
      • Pedersen J.
      • Setälä J.
      A Nordic proposal for an energy corrected milk (ECM) formula.
      27th Session of the International Commission for Breeding and Productivity of Milk Animals, Paris, France. Wageningen Academic Publishers, Wageningen, the Netherlands1990
      ).
      6 Milk NEL (Mcal/d) = kg of milk × (0.0929 × % fat + 0.0563 × % true protein + 0.0395 × % lactose) (
      NRC
      Nutrient Requirements of Dairy Cattle. 7th rev. Natl. Acad. Sci., Washington, DC2001
      ).
      7 Milk NEL ÷ NEL intake × 100.
      The milk FA data from this experiment are shown in Table 3. Saturated FA in milk were not affected by CAP, except 18:0 and 20:0. Concentration of 18:0 tended to linearly increase (P = 0.09) and that of 20:0 was linearly increased (P = 0.03) by CAP compared with the control. Milk 18:2 cis-9,cis-12, and 18:1 trans-11 tended to linearly decrease (P = 0.10 and 0.07, respectively), whereas other 18:1 trans and cis FA were not affected by CAP. Supplementation with CAP linearly increased (P = 0.04) concentration of 18:3. Compared with the control, concentration of CLA cis-9,trans-11 was decreased (P = 0.03) by CAP. The sum of saturated FA, total trans FA, and MUFA were not affected by CAP. Concentration of PUFA was linearly decreased (P = 0.05) by CAP.
      Table 3Effect of dietary Capsicum oleoresin (CAP) on milk FA in dairy cows (% of milk fat)
      ItemTreatment
      Control=0 mg/d of CAP; C250=250 mg/d of CAP; C500=500 mg/d of CAP; C1000=1,000 mg/d of CAP.
      		SEM
      n=32 for all variables (n represents number of observations used in the statistical analysis).
      		P-value
      Con vs. T=control vs. treatment; L=linear effect of CAP; Q=quadratic effect of CAP.
      ControlC250C500C1000Con vs. TLQ
      4:03.313.383.543.470.2110.620.240.19
      6:01.761.851.841.840.1050.730.210.30
      8:01.031.091.061.060.0470.820.310.50
      10:02.422.572.452.470.0980.970.380.61
      12:02.852.952.802.860.0830.990.610.90
      14:09.139.268.909.060.3100.770.520.62
      14:11.181.050.991.070.1080.790.300.25
      15:00.950.960.860.880.0770.200.411.00
      16:022.723.222.923.11.690.450.690.70
      16:11.691.521.501.580.1160.740.300.36
      17:00.420.430.420.420.0270.570.950.57
      18:09.409.549.8510.00.3780.210.090.77
      18:1 trans-6–80.560.570.570.580.0350.860.860.56
      18:1 trans-90.450.440.420.460.0240.580.850.18
      18:1 trans-102.322.452.192.530.4030.480.820.19
      18:1 trans-111.331.261.121.090.1810.780.070.50
      18:1 trans-120.610.580.540.580.0370.940.390.15
      18:1 cis-924.924.325.524.51.840.120.870.54
      18:1 cis-111.911.871.851.850.0620.110.250.46
      18:2 cis-9,cis-124.554.494.424.340.0990.810.100.84
      18:30.430.430.420.410.0090.220.040.73
      20:00.100.100.100.110.0040.210.030.88
      CLA cis-9,trans-110.610.580.540.510.0770.030.080.76
      CLA trans-10,cis-120.010.010.010.010.0020.200.730.60
      Σ unidentified5.295.265.115.120.1510.070.110.44
      Σ SFA54.155.354.855.32.290.570.460.67
      Σ trans FA5.335.164.905.310.3370.930.520.25
      Σ MUFA35.033.934.734.32.000.300.730.72
      Σ PUFA5.605.515.405.270.1730.620.050.80
      1 Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP.
      2 n = 32 for all variables (n represents number of observations used in the statistical analysis).
      3 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP.
      Rumen ammonia and VFA concentrations were not affected by CAP supplementation (Table 4). Rumen pH tended to decrease (P = 0.08) with CAP compared with the control. Intake and apparent total-tract digestibility of dietary nutrients were not affected by treatment (Table 5). Supplementation with CAP also did not affect N excretion in feces or urine, urine volume, urinary urea N excretion, total excreta N, milk N secretion, and urinary PD excretion (Table 6).
      Table 4Effect of dietary Capsicum oleoresin (CAP) on rumen fermentation in dairy cows
      ItemTreatment
      Control=0 mg/d of CAP; C250=250 mg/d of CAP; C500=500 mg/d of CAP; C1000=1,000 mg/d of CAP.
      		SEM
      Highest SEM shown; n=71 for VFA, n=72 for all other variables (n represents number of observations used in the statistical analysis).
      		P-value
      Con vs. T=control vs. treatment; L=linear effect of CAP; Q=quadratic effect of CAP.
      ControlC250C500C1000Con vs. TLQ
      pH5.865.785.765.780.0750.080.220.23
      Ammonia, mM2.222.222.212.030.2840.700.320.60
      Total VFA, mM1401391471386.30.860.880.32
      Total VFA, %
       Acetate50.851.350.549.80.810.590.100.53
       Propionate31.130.431.431.91.160.850.340.74
       Butyrate12.612.912.312.50.580.930.750.88
       Isobutyrate0.640.630.610.630.0160.420.770.17
       Valerate3.353.373.563.480.4740.520.490.58
       Isovalerate1.471.561.561.540.0420.130.390.23
       Acetate:propionate1.661.701.621.600.0830.710.220.76
      1 Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP.
      2 Highest SEM shown; n = 71 for VFA, n = 72 for all other variables (n represents number of observations used in the statistical analysis).
      3 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP.
      Table 5Effect of dietary Capsicum oleoresin (CAP) on intake and apparent total-tract digestibility of dietary nutrients in dairy cows
      ItemTreatment
      Control=0 mg/d of CAP; C250=250 mg/d of CAP; C500=500 mg/d of CAP; C1000=1,000 mg/d of CAP.
      		SEM
      n=32 for all variables (n represents number of observations used in the statistical analysis) except starch (n=31).
      		P-value
      Con vs. T=control vs. treatment; L=linear effect of CAP; Q=quadratic effect of CAP.
      ControlC250C500C1000Con vs. TLQ
      Intake, kg/d
       DM
      DMI data are for the fecal collection period.
      		27.928.027.427.00.660.540.240.94
       OM26.326.425.825.40.620.540.230.93
       CP4.394.424.314.240.1020.540.210.89
       NDF9.329.379.159.020.2190.570.240.90
       ADF6.526.456.326.200.1470.250.100.86
       Starch6.116.136.005.910.1430.540.240.91
      Apparent digestibility, %
       DM66.366.166.967.20.490.520.160.91
       OM67.367.067.968.10.470.600.180.88
       CP65.765.767.267.02.110.530.360.72
       NDF42.841.242.541.30.800.280.410.89
       ADF38.138.237.737.11.150.790.540.89
       Starch95.895.496.396.40.390.680.140.82
      1 Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP.
      2 n = 32 for all variables (n represents number of observations used in the statistical analysis) except starch (n = 31).
      3 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP.
      4 DMI data are for the fecal collection period.
      Table 6Effect of dietary Capsicum oleoresin (CAP) on nitrogen utilization and urinary purine derivatives excretion in dairy cows
      ItemTreatment
      Control=0 mg/d of CAP; C250=250 mg/d of CAP; C500=500 mg/d of CAP; C1000=1,000 mg/d of CAP.
      		SEM
      n=32 for all variables (n represents number of observations used in the statistical analysis).
      		P-value
      Con vs. T=control vs. treatment; L=linear effect of CAP; Q=quadratic effect of CAP.
      ControlC250C500C1000Con vs. TLQ
      N intake, g/d70370768967916.30.540.210.89
      N excretion or secretion, g/d
       Urine N1942041972016.70.300.530.69
       UUN
      UUN=urinary urea nitrogen.
      , g/d      		14715815015811.00.130.220.83
       Fecal N24324522922616.90.360.140.82
       Total excreta N43844842642813.20.760.291.00
       Milk N2452502472406.10.850.310.27
      N intake, %
       Urine N28.128.929.829.91.750.350.300.66
       Fecal N34.334.332.833.02.110.530.360.72
       Total excreta N62.463.262.663.01.440.770.870.93
       Milk N35.335.737.336.00.960.430.580.32
      Urine output, kg/d16.116.816.316.40.330.380.930.55
      Urinary PD
      PD=purine derivatives.
      excretion, mmol/d
       Allantoin42243140738927.60.630.250.80
       Uric acid69.768.374.864.42.880.880.290.11
       Total PD49249948245327.80.640.230.67
      1 Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP.
      2 n = 32 for all variables (n represents number of observations used in the statistical analysis).
      3 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP.
      4 UUN = urinary urea nitrogen.
      5 PD = purine derivatives.
      Relative to the control, plasma BHBA concentration was quadratically increased (P = 0.02) by CAP, but no effect of CAP on NEFA and insulin concentrations was noted (Table 7). Plasma BHBA concentration in our experiment was below the level for subclinical ketosis (1,200 to 2,900 µM;
      • Oetzel G.R.
      Monitoring and testing dairy herds for metabolic disease.
      Vet. Clin. North Am. Food Anim. Pract. 2004; 20: 651-674
      ). Supplementation of the diet with CAP increased (P = 0.02) BUN compared with the control. Concentrations of glucose, creatinine, total protein, albumin, globulin, amylase, Na, and Cl in blood plasma were not affected by CAP except cholesterol that tended to quadratically increase (P = 0.07). Plasma K concentration linearly decreased (P < 0.01) with CAP supplementation.
      Table 7Effect of dietary Capsicum oleoresin (CAP) on blood chemistry in dairy cows
      ItemTreatment
      Control=0 mg/d of CAP; C250=250 mg/d of CAP; C500=500 mg/d of CAP; C1000=1,000 mg/d CAP.
      		SEM
      Highest SEM shown; n=57 for amylase, n=64 for all other variables (n represents number of observations used in the statistical analysis).
      		P-value
      Con vs. T=control vs. treatment; L=linear effect of CAP; Q=quadratic effect of CAP.
      ControlC250C500C1000Con vs. TLQ
      BHBA, µM76786394882784.90.070.490.02
      NEFA, mM0.140.130.160.130.0090.710.900.55
      Insulin, µIU/mL10.813.510.011.01.230.570.590.78
      Glucose, mg/dL62.160.359.661.01.940.230.650.17
      BUN, mg/dL16.117.417.117.30.700.020.110.17
      Creatinine, mg/dL0.680.690.660.670.0180.590.540.66
      Total protein, g/dL7.837.887.907.690.1070.970.210.18
      Albumin, g/dL3.083.093.113.020.0370.920.250.21
      Globulin, g/dL4.744.784.784.660.0850.960.360.31
      Cholesterol, mg/dL2822852882725.60.690.240.07
      Amylase, U/L47.155.048.041.84.810.810.130.18
      P, mg/dL6.145.845.915.980.1190.090.550.13
      Ca, mg/dL10.110.110.19.940.1010.370.130.65
      Na, mM1471461491441.60.910.240.11
      K, mM4.384.184.164.010.132<0.01<0.010.39
      Cl, mM1041031041011.320.420.130.58
      1 Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d CAP.
      2 Highest SEM shown; n = 57 for amylase, n = 64 for all other variables (n represents number of observations used in the statistical analysis).
      3 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP.
      As revealed by the sequencing results, the predominant bacterial orders in whole ruminal contents were Clostridiales and Bacteroidales (Table 8). Bacteroidales decreased quadratically (P = 0.03) and Bifidobacteriales tended to be linearly increased (P = 0.10) by CAP. Relative to the control, Synergistales increased quadratically (P = 0.03) and Spirochaetales tended to be quadratically increased (P = 0.09) by CAP. The relative abundance of Ruminococcaceae, which was the most predominant genus in the rumen, was not affected by treatment (Table 9). Prevotella and Roseburia were quadratically decreased (P ≤ 0.04) and the proportion of Butyrivibrio was increased quadratically (P < 0.01) by CAP supplementation. Capsicum quadratically increased (P ≤ 0.04) the abundance of Fecalibacterium, Syntrophococcus, and Dorea.
      Table 8Effect of dietary Capsicum oleoresin (CAP) on relative abundance (as percentage
      The percentage represents the percentage of the total sequences analyzed within the sample.
      of total sequences) of rumen major bacterial order in dairy cows
      OrderTreatment
      Control=0 mg/d of CAP; C250=250 mg/d of CAP; C500=500 mg/d of CAP; C1000=1,000 mg/d of CAP.
      		SEM
      n=12 for all variables (n represents number of observations used in the statistical analysis).
      		P-value
      Con vs. T=control vs. treatment; L=linear effect of CAP; Q=quadratic effect of CAP.
      ControlC250C500C1000Con vs. TLQ
      Clostridiales60.160.865.962.82.510.170.200.15
      Bacteroidales27.625.017.021.41.730.020.030.03
      Erysipelotrichales2.413.083.652.950.4390.150.410.12
      Bifidobacteriales1.011.713.763.351.3930.120.100.27
      Coriobacteriales1.491.271.982.100.5600.500.191.00
      Fibrobacterales1.171.311.351.470.3000.370.320.81
      Methanobacteriales1.020.911.561.220.2350.540.450.45
      Synergistales0.971.221.391.080.0700.060.510.03
      Chromatiales1.331.370.831.020.7690.600.510.66
      Spirochaetales0.901.380.920.830.1500.200.110.09
      Bacillales0.360.350.270.310.0900.670.660.69
      Desulfovibrionales0.200.210.060.090.0550.190.100.34
      Sphingobacteriales0.090.110.160.110.0450.570.790.44
      Planctomycetales0.150.100.090.070.0460.280.290.63
      Aeromonadales0.100.100.090.080.0500.980.830.91
      Lactobacillales0.080.090.110.060.0210.780.550.22
      1 The percentage represents the percentage of the total sequences analyzed within the sample.
      2 Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP.
      3 n = 12 for all variables (n represents number of observations used in the statistical analysis).
      4 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP.
      Table 9Effect of dietary Capsicum oleoresin (CAP) on relative abundance (as percentage
      The percentage represents the percentage of the total sequences analyzed within the sample.
      of total sequences) of rumen major bacterial genera in dairy cows
      GenusTreatment
      Control=0 mg/d of CAP; C250=250 mg/d of CAP; C500=500 mg/d of CAP; C1000=1,000 mg/d of CAP.
      		SEM
      n=12 for all variables (n represents number of observations used in the statistical analysis).
      		P-value
      Con vs. T=control vs. treatment; L=linear effect of CAP; Q=quadratic effect of CAP.
      ControlC250C500C1000Con vs. TLQ
      Ruminococcaceae21.422.222.422.62.830.740.770.87
      Prevotella18.816.411.315.01.280.040.080.04
      Acetitomaculum6.456.519.986.892.7930.680.820.47
      Butyrivibrio5.076.726.976.201.080<0.010.03<0.01
      Prevotellaceae6.126.093.774.350.8340.170.110.32
      Ruminococcus3.993.363.853.290.3520.300.340.98
      Roseburia4.582.482.733.490.5900.040.380.05
      Blautia3.283.413.103.150.2040.680.280.85
      Saccharofermentans2.623.522.342.990.5360.650.960.94
      Bifidobacterium0.661.473.142.701.2490.150.150.28
      Coprococcus1.561.892.011.740.3690.450.790.39
      Fecalibacterium1.481.741.961.420.1340.210.590.04
      Syntrophococcus1.601.191.342.060.5080.800.130.10
      Fibrobacter1.171.311.351.470.2500.370.320.81
      Synergistaceae0.951.201.381.060.0700.050.480.02
      Dorea0.891.171.411.000.1230.100.640.04
      Methanobrevibacter0.890.821.451.120.2230.460.400.40
      Erysipelotrichaceae0.850.961.181.250.2840.130.080.49
      Thioalkalibacter1.221.270.770.960.7160.610.520.65
      Olsenella1.000.621.161.400.4430.870.280.61
      Treponema0.901.370.920.830.1490.210.110.09
      1 The percentage represents the percentage of the total sequences analyzed within the sample.
      2 Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP.
      3 n = 12 for all variables (n represents number of observations used in the statistical analysis).
      4 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP.
      T cell phenotypes were not affected by CAP supplementation (Table 10). Although CAP did not affect numbers of neutrophils positive for phagocytosis, mean fluorescence intensity tended to be quadratically increased (P = 0.08). Supplementation with CAP linearly increased total white blood cells, neutrophils, and eosinophils (P ≤ 0.04; Table 11). The proportion of lymphocytes in total white blood cells decreased linearly (P < 0.01) and that of neutrophils increased linearly (P < 0.01) with increasing CAP supplementation; therefore, the ratio of neutrophils to lymphocytes was linearly increased (P < 0.01) by CAP. Treatment did not affect concentrations of monocytes and basophils in blood. Red blood cells and hemoglobin concentration quadratically increased (P ≤ 0.04) and platelets linearly decreased (P = 0.04) with CAP, even though mean platelet volume was not affected. Hematocrit percentage tended to be increased (P = 0.07) by CAP. Oxidative stress markers were not affected by CAP supplementation (Table 12).
      Table 10Effect of dietary Capsicum oleoresin (CAP) on T cell phenotypes and phagocytosis of neutrophils in dairy cows
      ItemTreatment
      Control=0 mg/d of CAP; C250=250 mg/d of CAP; C500=500 mg/d of CAP; C1000=1,000 mg/d of CAP.
      		SEM
      n=32 for all variables (n represents number of observations used in the statistical analysis).
      		P-value
      Con vs. T=control vs. treatment; L=linear effect of CAP; Q=quadratic effect of CAP.
      ControlC250C500C1000Con vs. TLQ
      T-cell phenotypes, %
       CD4+CD25-9.508.036.987.831.2970.150.320.21
       CD4-CD25+8.008.418.407.910.7570.750.830.51
       CD4+CD25+10.812.511.911.31.320.190.930.15
       Total CD420.320.518.919.21.240.640.460.79
       Total CD2518.820.920.319.21.950.310.950.20
       CD8+γδ-6.084.975.815.400.8010.410.720.72
       CD8-γδ+8.629.349.388.730.5890.490.950.31
       CD8+γδ+2.692.252.362.180.2580.160.250.57
       Total CD88.777.228.177.570.8560.290.530.65
       Total γδ11.311.611.710.90.700.910.640.47
      Neutrophil phagocytosis
       Positive cells, %77.881.282.375.94.090.480.780.20
       MFI
      MFI=mean fluorescence intensity, arbitrary units.
      		14317718114019.90.310.840.08
      1 Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP.
      2 n = 32 for all variables (n represents number of observations used in the statistical analysis).
      3 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP.
      4 MFI = mean fluorescence intensity, arbitrary units.
      Table 11Effect of dietary Capsicum oleoresin (CAP) on blood cell counts in dairy cows
      ItemTreatment
      Control=0 mg/d of CAP; C250=250 mg/d of CAP; C500=500 mg/d of CAP; C1000=1,000 mg/d of CAP.
      		SEM
      n=64 for all variables (n represents number of observations used in the statistical analysis).
      		P-value
      Con vs. T=control vs. treatment; L=linear effect of CAP; Q=quadratic effect of CAP.
      ControlC250C500C1000Con vs. TLQ
      White blood cells, 10
      Con vs. T=control vs. treatment; L=linear effect of CAP; Q=quadratic effect of CAP.
      /μL      		7.067.297.068.010.2980.290.040.36
       Neutrophils3.593.753.624.560.2520.190.010.33
       Lymphocytes2.993.002.912.850.0760.470.160.89
       Monocytes0.220.210.230.220.0190.920.940.68
       Eosinophils0.250.310.290.370.0380.020.010.46
       Basophils0.010.020.010.020.0050.270.300.99
      Total, %
       Neutrophils51.151.151.755.11.310.14<0.010.43
       Lymphocytes42.341.641.037.01.200.04<0.010.30
       Monocytes3.072.813.162.820.200.540.570.72
       Eosinophils3.414.254.024.780.4960.030.040.34
       Basophils0.150.230.170.240.0480.220.270.96
       Neutrophil:lymphocyte1.261.281.291.730.0840.06<0.010.24
       Red blood cells, 106/μL5.665.705.855.660.2540.300.900.04
       Hemoglobin, g/dL8.598.848.968.770.106<0.010.13<0.01
       Hematocrit, %24.724.925.524.70.670.360.970.07
       Platelets, 10
      Con vs. T=control vs. treatment; L=linear effect of CAP; Q=quadratic effect of CAP.
      /μL      		40537138035248.90.050.040.72
       Mean platelet volume, fL6.216.206.186.140.1930.760.590.94
      1 Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP.
      2 n = 64 for all variables (n represents number of observations used in the statistical analysis).
      3 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP.
      Table 12Effect of dietary Capsicum oleoresin (CAP) on oxidative stress markers in dairy cows
      ItemTreatment
      Control=0 mg/d of CAP; C250=250 mg/d of CAP; C500=500 mg/d of CAP; C1000=1,000 mg/d of CAP.
      		SEM
      n=64 for all variables (n represents number of observations used in the statistical analysis).
      		P-value
      Con vs. T=control vs. treatment; L=linear effect of CAP; Q=quadratic effect of CAP.
      ControlC250C500C1000Con vs. TLQ
      8-Isoprostane, pg/mL16.514.215.314.63.720.250.440.99
      TBARS,
      TBARS=thiobarbituric acid reactive substances; trolox equivalents.
      µM      		3.113.403.304.290.640.310.450.69
      ORAC,
      ORAC=oxygen radical absorbance capacity.
      µM      		8,9679,3369,4119,414322.00.310.420.52
      1 Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP.
      2 n = 64 for all variables (n represents number of observations used in the statistical analysis).
      3 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP.
      4 TBARS = thiobarbituric acid reactive substances; trolox equivalents.
      5 ORAC = oxygen radical absorbance capacity.

      Discussion

      Supplementation on the diet with Capsicum oleoresin increased feed intake in beef cattle (
      • Cardozo P.W.
      • Calsamiglia S.
      • Ferret A.
      • Kamel C.
      Effects of alfalfa extract, anise, capsicum, and a mixture of cinnamaldehyde and eugenol on ruminal fermentation and protein degradation in beef heifers fed a high-concentrate diet.
      J. Anim. Sci. 2006; 84: 2801-2808
      ;
      • Rodríguez-Prado M.
      • Ferret A.
      • Zwieten J.
      • Gonzalez L.
      • Bravo D.
      • Calsamiglia S.
      Effects of dietary addition of capsicum extract on intake, water consumption, and rumen fermentation of fattening heifers fed a high-concentrate diet.
      J. Anim. Sci. 2012; 90: 1879-1884
      ). In these experiments, the pungent property of Capsicum led to high consumption of water followed by increased feed intake. However, CAP in the current experiment had no effect on DMI. The CAP product contained fat to reduce its pungent taste, which may have resulted in lack of effect on DMI. The application rate was also somewhat higher in the beef studies compared with the current experiment. For example, the amount of Capsicum oleoresin used in the study of
      • Rodríguez-Prado M.
      • Ferret A.
      • Zwieten J.
      • Gonzalez L.
      • Bravo D.
      • Calsamiglia S.
      Effects of dietary addition of capsicum extract on intake, water consumption, and rumen fermentation of fattening heifers fed a high-concentrate diet.
      J. Anim. Sci. 2012; 90: 1879-1884
      was 2.5 to 10.6 mg/kg of DMI, whereas application rate of Capsicum oleoresin was 1.8 to 7.5 mg/kg of DMI in the current study.
      • Tager L.R.
      • Krause K.M.
      Effects of essential oils on rumen fermentation, milk production, and feeding behavior in lactating dairy cows.
      J. Dairy Sci. 2011; 94: 2455-2464
      also reported no effect of Capsicum on DMI in dairy cows.
      The combination of dietary nutrient supplies and quadratic increases of milk, 4% FCM, and ECM yields with CAP supplementation in the current experiment resulted in negative NEL balances (−0.9 Mcal/d) for both C250 and C500 treatment. The negative energy balance may have led to mobilization of body fat reserves, which may be responsible for the observed quadratic increase in serum BHBA concentration with the CAP treatments (
      • McArt J.A.A.
      • Nydam D.V.
      • Oetzel G.R.
      Epidemiology of subclinical ketosis in early lactation dairy cattle.
      J. Dairy Sci. 2012; 95: 5056-5066
      ). This potentially enhanced fat mobilization in the CAP-treated cows may explain the numerical trend for increased milk fat concentration and the quadratic increase in 4% FCM and ECM, compared with the control cows. This hypothesis, however, was not supported by the similar serum NEFA concentrations between the CAP-treated and control cows.
      Milk fat concentration was, on average, 3.03% in our experiment, which is lower than typical values for Holstein cows (

      CDCB (Council on Dairy Cattle Breeding). 2013. USDA summary of 2013 herd averages DHI Report K-3. Reynoldsburg, OH. Accessed Dec. 24. 2014. https://www.cdcb.us/publish/dhi/current/hax.html

      ) and is indicative of milk fat depression. This may have been caused by the high starch content of the corn silage (41.4%) and high proportion of corn silage in the basal diet. Diets rich in starch are known to cause milk fat depression by decreasing mammary lipogenesis (
      • Shingfield K.J.
      • Bernard L.
      • Leroux C.
      • Chilliard Y.
      Role of trans fatty acids in the nutritional regulation of mammary lipogenesis in ruminants.
      Animal. 2010; 4: 1140-1166
      ) and an increase of milk fat 18:1 trans-10 has been consistently associated with milk fat depression (
      • Shingfield K.J.
      • Griinari J.M.
      Role of biohydrogenation intermediates in milk fat depression.
      Eur. J. Lipid Sci. Technol. 2007; 109: 799-816
      ). Concentrations of milk fat 18:1 trans-10 in the current study were 2.19 to 2.53% for all treatment, which could cause 20 to 22% reduction in milk fat in lactating cows according to
      • Shingfield K.J.
      • Bernard L.
      • Leroux C.
      • Chilliard Y.
      Role of trans fatty acids in the nutritional regulation of mammary lipogenesis in ruminants.
      Animal. 2010; 4: 1140-1166
      . The relatively low rumen pH for all treatments is also reflective of high level of starch fermentation in the rumen. A decrease by CAP in cis-9,trans-11 CLA is consistent with the results for 18:1 trans-11 in the current experiment because cis-9,trans-11 CLA is synthesized in the mammary gland from 18:1 trans-11 by Δ9-desaturase (
      • Griinari J.M.
      • Corl B.A.
      • Lacy S.H.
      • Chouinard P.Y.
      • Nurmela K.V.V.
      • Bauman D.E.
      Conjugated linoleic acid is synthesized endogenously in lactating dairy cows by Δ9-desaturase.
      J. Nutr. 2000; 130: 2285-2291
      ).
      Overall, rumen fermentation parameters were not affected by CAP in the current experiment, although a slight decrease (P = 0.06) in pH was observed. These results are consistent with those reported by
      • Tager L.R.
      • Krause K.M.
      Effects of essential oils on rumen fermentation, milk production, and feeding behavior in lactating dairy cows.
      J. Dairy Sci. 2011; 94: 2455-2464
      , in which ruminal ammonia and VFA concentrations were not affected by 250 mg/d of a Capsicum product in lactating dairy cows. Others, however, have reported decreased pH and acetate concentration and increased ammonia concentration in the rumen of beef cattle supplemented with encapsulated Capsicum oleoresin (
      • Rodríguez-Prado M.
      • Ferret A.
      • Zwieten J.
      • Gonzalez L.
      • Bravo D.
      • Calsamiglia S.
      Effects of dietary addition of capsicum extract on intake, water consumption, and rumen fermentation of fattening heifers fed a high-concentrate diet.
      J. Anim. Sci. 2012; 90: 1879-1884
      ).
      Explanation of the increased BUN concentration by CAP in the current experiment remains uncertain because ruminal ammonia concentration and MUN were not different from the control. None of the other blood chemistry variables analyzed, with the exception of BHBA, were affected by CAP. The BUN results were not consistent with previous data from experiments with nonruminant species. For example, capsaicin or Capsicum extract were reported to decrease BUN concentration in rats (
      • Monsereenusorn Y.
      Subchronic toxicity studies of capsaicin and capsicum in rats.
      Res. Commun. Chem. Pathol. Pharmacol. 1983; 41: 95-110
      ;
      • Jung S.H.
      • Kim H.J.
      • Oh G.S.
      • Shen A.
      • Lee S.
      • Choe S.K.
      • Park R.
      • So H.S.
      Capsaicin ameliorates cisplatin-induced renal injury through induction of heme oxygenase-1.
      Mol. Cells. 2014; 37: 234-240
      ).
      Antimicrobial properties of capsaicin or Capsicum oleoresin against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Streptococcus pyogenes, Helicobacter pylori, Botrytis cinerea, and Aspergillus niger were reported in vitro (
      • al-Delaimy K.S.
      • Ali S.H.
      Antibacterial action of vegetable extracts on the growth of pathogenic bacteria.
      J. Sci. Food Agric. 1970; 21: 110-112
      ;
      • Cichewicz R.H.
      • Thorpe P.A.
      The antimicrobial properties of chile peppers (Capsicum species) and their uses in Mayan medicine.
      J. Ethnopharmacol. 1996; 52: 61-70
      ;
      • Jones N.L.
      • Shabib S.
      • Sherman P.M.
      Capsaicin as an inhibitor of the growth of the gastric pathogen Helicobacter pylori.
      FEMS Microbiol. Lett. 1997; 146: 223-227
      ;
      • Xing F.
      • Cheng G.
      • Yi K.
      Study on the antimicrobial activities of the capsaicin microcapsules.
      J. Appl. Polym. Sci. 2006; 102: 1318-1321
      ). Therefore, some antimicrobial effects of Capsicum in the rumen were expected in the current experiment. Supplementation of CAP decreased Prevotella species in the rumen, gram-negative bacteria that degrade proteins and are involved in the uptake and fermentation of peptides (

      Stewart, C. S., H. J. Flint, and M. P. Bryant. 1997. The rumen bacteria. Page 14 in The Rumen Microbial Ecosystem. P. N. Hobson and C. S. Stewart, ed. Chapman & Hall, New York, NY.

      ). Ruminal ammonia concentration, however, was not affected by CAP in the current experiment. Butyrivibrio species are associated with biohydrogenation of FA in the rumen (
      • Jenkins T.C.
      • Wallace R.J.
      • Moate P.J.
      • Mosley E.E.
      Board-invited review: Recent advances in biohydrogenation of unsaturated fatty acids within the rumen microbial ecosystem.
      J. Anim. Sci. 2008; 86: 397-412
      ). It is known that milk fat 18:1 trans-11 and cis-9,trans-11 CLA are formed from linoleic acid by Butyrivibrio fibrisolvens. The observed quadratic increase in the abundance of Butyrivibrio species and B. fibrisolvens (P = 0.08; 0.01, 0.03, 0.03, and 0.02% for control, C250, C500, and C1000, respectively; data not shown in tables) is consistent with the decrease in PUFA, but did not result in increased milk fat 18:1 trans-11 and cis-9,trans-11 CLA.
      Capsaicinoids have been shown to have immunomodulatory effects in vitro and in vivo in rats (
      • Sancho R.
      • Lucena C.
      • Macho A.
      • Calzado M.
      • Blanco-Molina M.
      • Minassi A.
      • Appendino G.
      • Muñoz E.
      Immunosuppressive activity of capsaicinoids: capsiate derived from sweet peppers inhibits NF-κB activation and is a potent antiinflammatory compound in vivo.
      Eur. J. Immunol. 2002; 32: 1753-1763
      ;
      • Park J.Y.
      • Kawada T.
      • Han I.S.
      • Kim B.S.
      • Goto T.
      • Takahashi N.
      • Fushiki T.
      • Kurata T.
      • Yu R.
      Capsaicin inhibits the production of tumor necrosis factor alpha by LPS-stimulated murine macrophages, RAW 264.7: A PPARgamma ligand-like action as a novel mechanism.
      FEBS Lett. 2004; 572: 266-270
      ;
      • Liu Y.
      • Song M.
      • Che T.M.
      • Bravo D.
      • Pettigrew J.E.
      Anti-inflammatory effects of several plant extracts on porcine alveolar macrophages in vitro.
      J. Anim. Sci. 2012; 90: 2774-2783
      ). Capsaicin and Capsicum oleoresin reduced IL-2 and IFN-γ production in murine Peyer’s patches, lymphoid tissues where systemic immune responses are induced in the intestine (
      • Takano F.
      • Yamaguchi M.
      • Takada S.
      • Shoda S.
      • Yahagi N.
      • Takahashi T.
      • Ohta T.
      Capsicum ethanol extracts and capsaicin enhance interleukin-2 and interferon-gamma production in cultured murine Peyer's patch cells ex vivo.
      Life Sci. 2007; 80: 1553-1563
      ).
      • Takano F.
      • Yamaguchi M.
      • Takada S.
      • Shoda S.
      • Yahagi N.
      • Takahashi T.
      • Ohta T.
      Capsicum ethanol extracts and capsaicin enhance interleukin-2 and interferon-gamma production in cultured murine Peyer's patch cells ex vivo.
      Life Sci. 2007; 80: 1553-1563
      also reported that capsaicin and Capsicum oleoresin decreased proportion of CD4+ cells in Peyer’s patches. These anti-inflammatory effects of capsaicin were shown by
      • Park J.Y.
      • Kawada T.
      • Han I.S.
      • Kim B.S.
      • Goto T.
      • Takahashi N.
      • Fushiki T.
      • Kurata T.
      • Yu R.
      Capsaicin inhibits the production of tumor necrosis factor alpha by LPS-stimulated murine macrophages, RAW 264.7: A PPARgamma ligand-like action as a novel mechanism.
      FEBS Lett. 2004; 572: 266-270
      , who reported that capsaicin suppressed tumor necrosis factor (TNF) production in murine macrophage. In food animals, Capsicum oleoresin linearly decreased TNF, IL-1B, and transforming growth factor production of LPS-induced porcine macrophages in vitro (
      • Liu Y.
      • Song M.
      • Che T.M.
      • Bravo D.
      • Pettigrew J.E.
      Anti-inflammatory effects of several plant extracts on porcine alveolar macrophages in vitro.
      J. Anim. Sci. 2012; 90: 2774-2783
      ), suggesting an anti-inflammatory effect of capsaicin on macrophages in pigs.
      • Oh J.
      • Hristov A.N.
      • Lee C.
      • Cassidy T.
      • Heyler K.
      • Varga G.A.
      • Pate J.
      • Walusimbi S.
      • Brzezicka E.
      • Toyokawa K.
      • Werner J.
      • Donkin S.S.
      • Elias R.
      • Dowd S.
      • Bravo D.
      Immune and production responses of dairy cows to postruminal supplementation with phytonutrients.
      J. Dairy Sci. 2013; 96: 7830-7843
      infused Capsicum oleoresin (2 g/d) into the abomasum of lactating dairy cows and measured inflammatory cytokines production of LPS-induced peripheral blood mononuclear cells ex vivo and subpopulations of T cells in blood. In that experiment, the abomasal infusion of Capsicum oleoresin did not affect inflammatory cytokines such as IFN-γ, IL-6, and TNF, but increased the proportion of CD4+ cells in blood. In the current experiment, however, we did not observe any effect of dietary CAP on populations of T cells. It is possible that the different doses used in the 2 experiments influenced the T cell response. In addition, the mode of supplementation (abomasal infusion vs. TMR top-dressing) might have influenced the effects of CAP.
      Neutrophils are the most abundant type of white blood cell and play critical roles in innate immunity by killing bacteria and inducing an adaptive immune response (
      • Witko-Sarsat V.
      • Rieu P.
      • Descamps-Latscha B.
      • Lesavre P.
      • Halbwachs-Mecarelli L.
      Neutrophils: molecules, functions and pathophysiological aspects.
      Lab. Invest. 2000; 80: 617-653
      ). Supplementation of the diet with CAP in the current experiment increased neutrophil counts in blood and neutrophil activity in phagocytosis. Neutrophils may be directly affected by CAP because they have a transient receptor potential cation channel subfamily V member 1 (TRPV1) channel (
      • Heiner I.
      • Eisfeld J.
      • Lückhoff A.
      Role and regulation of TRP channels in neutrophil granulocytes.
      Cell Calcium. 2003; 33: 533-540
      ), or indirectly affected via release of neuronal peptides (
      • Zimmerman B.J.
      • Anderson D.C.
      • Granger D.N.
      Neuropeptides promote neutrophil adherence to endothelial cell monolayers.
      Am. J. Physiol. 1992; 263: G678-G682
      ). In the current study, it is possible that neurons activated by CAP facilitated neutrophil function through secretion of neuropeptides.
      • Zhukova E.M.
      • Makarova O.P.
      Effect of capsaicin on dynamics of neutrophil functional activity in Wistar rat venous blood.
      Bull. Exp. Biol. Med. 2002; 134: 233-235
      indicated that neuropeptides regulated by capsaicin increased count and activity of neutrophils in rats.
      • Franco-Penteado C.F.
      • De Souza I.A.
      • Lima C.S.
      • Teixeira S.A.
      • Muscara M.N.
      • De Nucci G.
      • Antunes E.
      Effects of neonatal capsaicin treatment in the neutrophil production, and expression of preprotachykinin-I and tachykinin receptors in the rat bone marrow.
      Neurosci. Lett. 2006; 407: 70-73
      reported that capsaicin increased neutrophil production in rat bone marrow. An increase of the neutrophil-to-lymphocyte ratio was suggested as an indicator of an acute stress response (
      • Weiss D.J.
      • Wardrop K.J.
      Schalm's Veterinary Hematology. 6th. Blackwell Publishing, Ames, IA2010
      ). In the current experiment, CAP increased the ratio of neutrophils to lymphocytes as well as the counts of neutrophils and eosinophils. Eosinophils are granulocytes and are involved in acute phase responses, such as host protection against parasites (
      • Rothenberg M.E.
      • Hogan S.P.
      The eosinophil.
      Annu. Rev. Immunol. 2006; 24: 147-174
      ).
      In our previous study, we reported that abomasal infusion of Capsicum oleoresin for 5 d did not affect 8-isoprostane and oxygen radical absorbance capacity in blood of dairy cows (
      • Oh J.
      • Hristov A.N.
      • Lee C.
      • Cassidy T.
      • Heyler K.
      • Varga G.A.
      • Pate J.
      • Walusimbi S.
      • Brzezicka E.
      • Toyokawa K.
      • Werner J.
      • Donkin S.S.
      • Elias R.
      • Dowd S.
      • Bravo D.
      Immune and production responses of dairy cows to postruminal supplementation with phytonutrients.
      J. Dairy Sci. 2013; 96: 7830-7843
      ). This is consistent with data from the current experiment, although CAP was supplied to the cows for longer period (21 d) and through the TMR. In studies with rats, however, capsaicin reduced oxidative stress markers such as thiobarbituric acid reactive substances or malondialdehyde in serum and the animals’ liver, lung, kidney, and muscle (
      • Lee C.Y.
      • Kim M.
      • Yoon S.W.
      • Lee C.H.
      Short-term control of capsaicin on blood and oxidative stress of rats in vivo.
      Phytother. Res. 2003; 17: 454-458
      ;
      • Manjunatha H.
      • Srinivasan K.
      Protective effect of dietary curcumin and capsaicin on induced oxidation of low-density lipoprotein, iron-induced hepatotoxicity and carrageenan-induced inflammation in experimental rats.
      FEBS J. 2006; 273: 4528-4537
      ).

      Conclusions

      Our results suggest that dietary supplementation of CAP has subtle or no effects on feed intake, rumen fermentation, nutrients digestibility, blood chemistry, T cell phenotypes, and antioxidant status in lactating dairy cows. However, CAP appeared to have a positive effect, perhaps through enhanced mobilization of body fat reserves, on the energy balance of the cows, resulting in increased ECM yield. Capsicum facilitated neutrophil activity and immune cells related to the acute phase response.

      Acknowledgments

      The authors thank Pancosma S.A. (Geneva, Switzerland) for providing partial financial support for this project and the CAP used in the experiment, Pennsylvania State University’s Animal Diagnostic Laboratory and Dona Wijetunge for providing S. ubris, Ling Tao and Yufan Zhang in the Department of Food Science (Pennsylvania State University) for analysis of oxidative stress markers, and the staff of the Department of Animal Science Dairy Center (Pennsylvania State University) for their conscientious care of the experimental cows.

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      Published online: July 15, 2015
      Accepted: June 5, 2015
      Received: December 30, 2014

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      DOI: https://doi.org/10.3168/jds.2014-9294

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