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Effects of a combination of Capsicum oleoresin and clove essential oil on metabolic status, lactational performance, and enteric methane emissions in dairy cows

  • T. Silvestre
    Affiliations
    Department of Animal Sciences, The Pennsylvania State University, University Park 16802
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  • Author Footnotes
    * Current address: Department of Agricultural Sciences, University of Helsinki, PO Box 28, FI-00014, Finland.
    S.E. Räisänen
    Footnotes
    * Current address: Department of Agricultural Sciences, University of Helsinki, PO Box 28, FI-00014, Finland.
    Affiliations
    Department of Animal Sciences, The Pennsylvania State University, University Park 16802
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  • S.F. Cueva
    Affiliations
    Department of Animal Sciences, The Pennsylvania State University, University Park 16802
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  • D.E. Wasson
    Affiliations
    Department of Animal Sciences, The Pennsylvania State University, University Park 16802
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  • Author Footnotes
    † Current address: Cornell Cooperative Extension, Cornell Cooperative Extension, Bath, NY 14810.
    C.F.A. Lage
    Footnotes
    † Current address: Cornell Cooperative Extension, Cornell Cooperative Extension, Bath, NY 14810.
    Affiliations
    Department of Animal Sciences, The Pennsylvania State University, University Park 16802
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  • L.F. Martins
    Affiliations
    Department of Animal Sciences, The Pennsylvania State University, University Park 16802
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  • Author Footnotes
    ‡ Current affiliation: Nutreco Exploration, Nutreco, P.O. Box 299, the Netherlands.
    E. Wall
    Footnotes
    ‡ Current affiliation: Nutreco Exploration, Nutreco, P.O. Box 299, the Netherlands.
    Affiliations
    AVT Natural North America, Santa Clara, CA 95054
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  • A.N. Hristov
    Correspondence
    Corresponding author
    Affiliations
    Department of Animal Sciences, The Pennsylvania State University, University Park 16802
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  • Author Footnotes
    * Current address: Department of Agricultural Sciences, University of Helsinki, PO Box 28, FI-00014, Finland.
    † Current address: Cornell Cooperative Extension, Cornell Cooperative Extension, Bath, NY 14810.
    ‡ Current affiliation: Nutreco Exploration, Nutreco, P.O. Box 299, the Netherlands.
Open AccessPublished:October 18, 2022DOI:https://doi.org/10.3168/jds.2022-22203

      ABSTRACT

      Botanical extracts have a potential to modify ruminal fermentation while enhancing metabolism and immunity in dairy cows. The objective of this study was to investigate the effects of a combination of Capsicum oleoresin and clove essential oil (botanicals; BTC) on lactational performance, nutrient utilization, enteric methane (CH4) emissions, and blood parameters in dairy cows. Twenty Holstein cows (12 multiparous and 8 primiparous) averaging (±SD) 77 ± 28 d in milk in the beginning of the study were used in a replicated 4 × 4 Latin square design experiment with 4 periods of 28 d each. Cows were grouped into squares based on parity, milk yield and days in milk, and assigned to 1 of 4 treatments: control (CON), 150, 300, or 600 mg/cow per day of BTC. Cows received the same basal diet and BTC were top-dressed on the total mixed ration once daily. Dry matter intake, milk production, and milk composition were not affected by BTC supplementation, except for milk fat content that tended to be increased in BTC, compared with CON. Daily CH4 emission (measured using the GreenFeed system) was linearly decreased by up to 7.5% with increasing doses of BTC. Treatment decreased CH4 yield (kg of CH4 ÷ kg of DMI) and tended to decrease CH4 intensity (kg of CH4 ÷ kg of milk or energy-corrected milk yields) by 5% in BTC, compared with CON. Supplementation of BTC resulted in a quadratic decrease of serum β-hydroxybutyrate in all cows, and a linear decrease of serum insulin concentration in primiparous but not in multiparous cows. Nutrient utilization and other blood parameters (e.g., blood cells count) were not affected by BTC in the current study. The reduction of enteric CH4 emission demonstrates a moderate mitigation effect on carbon footprint of milk by BTC supplementation. These results must be further investigated and confirmed in longer-term experiments.

      Key words

      INTRODUCTION

      In recent years, research aimed to identify plant-based feed additives that could enhance animal performance while optimizing the health of dairy cows (
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      Host-mediated effects of phytonutrients in ruminants: A review.
      ). For instance, increased productivity (e.g., milk yield, ECM, FCM, or ADG) has been reported in dairy (
      • Wall E.H.
      • Doane P.H.
      • Donkin S.S.
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      The effects of supplementation with a blend of cinnamaldehyde and eugenol on feed intake and milk production of dairy cows.
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      Dietary supplementation with rumen-protected capsicum during the transition period improves the metabolic status of dairy cows.
      ) and beef cattle (
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      Effects of feeding rumen-protected capsicum oleoresin on growth performance, health status, and total tract digestibility of growing beef cattle.
      ) with dietary supplementation of Capsicum oleoresin (CAP), cinnamaldehyde, and clove extracts. Capsicum contains bioactive compounds (i.e., capsaicinoids) with a potential to enhance immune status and decrease oxidative stress responses in LPS-challenged dairy cows (
      • Oh J.
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      Effects of rumen-protected capsicum oleoresin on immune responses in dairy cows intravenously challenged with lipopolysaccharide.
      ). Moreover, lymphocytes percentage (
      • Oh J.
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      Effects of dietary capsicum oleoresin on productivity and immune responses in lactating dairy cows.
      ) and blood monocytes count (
      • Oh J.
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      Effects of phytonutrients alone or in combination with monensin on productivity in lactating dairy cows.
      ) were decreased in clinically healthy cows. Interestingly, supplementation with low doses of CAP resulted in decreased insulin concentration during a glucose tolerance test (
      • Oh J.
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      • Bravo D.M.
      • Wall E.H.
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      Effects of rumen-protected capsicum oleoresin on productivity and responses to a glucose tolerance test in lactating dairy cows.
      ) and an LPS challenge (
      • Oh J.
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      • Wall E.H.
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      Effects of rumen-protected capsicum oleoresin on immune responses in dairy cows intravenously challenged with lipopolysaccharide.
      ) in dairy cows. The implications of these immune and metabolic responses were subsequently explored by
      • Oh J.
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      • Chen X.
      • Nedelkov K.
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      Dietary supplementation with rumen-protected capsicum during the transition period improves the metabolic status of dairy cows.
      who described decreased blood BHB concentration prepartum with CAP supplementation, indicating improved energy status of transition dairy cows. Similar effects of capsaicinoids on the insulin axis, glucose, and energy metabolism have also been observed in rodents and humans (
      • Chaiyasit K.
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      Pharmacokinetic and the effect of capsaicin in capsicum frutescens on decreasing plasma glucose level.
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      A review of the effects of capsicum annuum L. and its constituent, capsaicin, in metabolic syndrome.
      ). Other plant extracts can also immunomodulate and improve intestinal barrier in animals. Eugenol (i.e., a constituent of clove essential oil) supplementation increased resistance of mice to enteric pathogen infection by stimulating the production of the inner mucus layer (
      • Wlodarska M.
      • Willing B.P.
      • Bravo D.M.
      • Finlay B.B.
      Phytonutrient diet supplementation promotes beneficial Clostridia species and intestinal mucus secretion resulting in protection against enteric infection.
      ), which is known as a key mucosal barrier against pathogens in the intestines. Taken together, these observations demonstrate the potential of using botanicals to improve metabolic health and intestinal barrier in dairy cattle.
      As reported by

      IPCC. 2021. IPCC press release AR6. Clim. Chang. 2013 - Phys. Sci. Basis 1–6.

      , it is unequivocal that emissions of CO2, CH4, and N2O have been increasing since the beginning of the industrial era, and they are contributing to climate change across the globe. Considering the rising demand for animal protein by the growing population worldwide, the use of sustainable practices is crucial to decrease the contribution of the livestock sector to global greenhouse gas (GHG) emissions (
      • Beauchemin K.A.
      • Ungerfeld E.M.
      • Eckard R.J.
      • Wang M.
      Review: Fifty years of research on rumen methanogenesis: Lessons learned and future challenges for mitigation.
      ). Additionally, enteric CH4 is not only an environmental concern but also represents a loss of dietary gross energy in ruminants (
      • Johnson K.A.
      • Johnson D.E.
      Methane emissions from cattle.
      ). Although research has identified effective GHG mitigation strategies for ruminants (
      • Hristov A.N.
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      • Gerber P.J.
      • Henderson B.
      • Tricarico J.M.
      Special Topics: Mitigation of methane and nitrous oxide emissions from animal operations: I. A review of enteric methane mitigation options.
      ;
      • Melgar A.
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      • Harper M.T.
      • Oh J.
      • Räisänen S.E.
      • Chen X.
      • Cueva S.F.
      • Duval S.
      • Hristov A.N.
      Dose-response effect of 3-nitrooxypropanol on enteric methane emissions in dairy cows.
      ;
      • Cueva S.F.
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      • Lage C.F.A.
      • Wasson D.E.
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      • Pelaez A.M.
      • Roth G.W.
      • Hristov A.N.
      Lactational performance, rumen fermentation, and enteric methane emission of dairy cows fed an amylase-enabled corn silage.
      ), adoption of these strategies by farmers will only take place if accompanied by improved animal performance and farm profitability, or if incentivized by government policies (
      • Hristov A.N.
      • Oh J.
      • Firkins J.L.
      • Dijkstra J.
      • Kebreab E.
      • Waghorn G.
      • Makkar H.P.S.
      • Adesogan A.T.
      • Yang W.
      • Lee C.
      • Gerber P.J.
      • Henderson B.
      • Tricarico J.M.
      Special Topics: Mitigation of methane and nitrous oxide emissions from animal operations: I. A review of enteric methane mitigation options.
      ).
      Nutritional interventions to mitigate enteric CH4 have been thoroughly investigated and discussed (
      • Hristov A.N.
      • Oh J.
      • Firkins J.L.
      • Dijkstra J.
      • Kebreab E.
      • Waghorn G.
      • Makkar H.P.S.
      • Adesogan A.T.
      • Yang W.
      • Lee C.
      • Gerber P.J.
      • Henderson B.
      • Tricarico J.M.
      Special Topics: Mitigation of methane and nitrous oxide emissions from animal operations: I. A review of enteric methane mitigation options.
      ;
      • Arndt C.
      • Hristov A.N.
      • Price W.J.
      • McClelland S.C.
      • Pelaez A.M.
      • Cueva S.F.
      • Oh J.
      • Bannink A.
      • Bayat A.R.
      • Crompton L.A.
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      • Kebreab E.
      • Kreuzer M.
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      • Martin C.
      • Newbold C.J.
      • Reynolds C.K.
      • Schwarm A.
      • Shingfield K.J.
      • Veneman J.B.
      • Yáñez-Ruiz D.R.
      • Yu Z.
      Strategies to mitigate enteric methane emissions by ruminants – a way to approach the 2.0°C target.
      ;
      • Congio G. F. de S.
      • Bannink A.
      • Mayorga Mogollón O.L.
      Latin America Methane Project Collaborators
      Enteric methane mitigation strategies for ruminant livestock systems in the Latin America and Caribbean region: A meta-analysis.
      ), and it is likely that strategies based on supplementation with plant extracts, such as essential oils (EO), may have a higher acceptance by livestock producers compared with, for example, the use of antibiotics. The main reason is that most of these plant extracts are Generally Recognized as Safe (GRAS) compounds by the Food and Drug Administration (
      • US FDA (Food and Drug Administration)
      CRF - Code of Federal Regulations Title 21- Food and Drugs. 21CFR182.20.
      ) and could have an appeal to the consumer. However, research investigating the effect of EO on ruminal fermentation and enteric CH4 emissions has been mostly based on in vitro batch or continuous culture studies (
      • Tekippe J.A.
      • Hristov A.N.
      • Heyler K.S.
      • Zheljazkov V.D.
      • Ferreira J.F.S.
      • Cantrell C.L.
      • Varga G.A.
      Effects of plants and essential oils on ruminal in vitro batch culture methane production and fermentation.
      ), and results are not always replicated in experiments with dairy cows (
      • Benchaar C.
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      • Chouinard P.Y.
      Digestion, ruminal fermentation, ciliate protozoal populations, and milk production from dairy cows fed cinnamaldehyde, quebracho condensed tannin, or Yucca schidigera saponin extracts.
      ;
      • Hristov A.N.
      • Lee C.
      • Hristova R.A.
      • Huhtanen P.
      • Firkins J.
      A meta-analysis of the variability in continuous culture rumen fermentation and digestibility data.
      ).
      The objective of this study was to investigate the effects of a combination of CAP and clove EO (botanicals; BTC) on the lactational performance, enteric CH4 emission, nutrient excretion, and blood parameters in dairy cows. Our hypothesis was that BTC supplementation would improve ruminal fermentation and metabolic status (e.g., reduced insulin and increased blood glucose concentrations), leading to enhanced nutrient utilization and lactational performance of the cows. We also hypothesized that enhanced animal productivity would reduce intensity of enteric CH4 emission contributing for decreased carbon footprint of milk.

      MATERIALS AND METHODS

      All procedures involving animals carried out in the study were approved by The Pennsylvania State University's Institutional Animal Care and Use Committee.

      Animals, Experimental Design, and Treatments

      The experiment was conducted in the tiestall barn of The Pennsylvania State University's Dairy Teaching and Research Center. A total of 20 lactating Holstein cows (12 multiparous and 8 primiparous) averaging (±SD) 2.5 ± 1.5 lactations, 77 ± 8 DIM, and 49 ± 9 kg/d milk yield (MY) at the beginning of the experiment, were used in a replicated 4 × 4 Latin square design experiment balanced for carryover effects. The experiment consisted of 4 periods with 28 d each, of which 21 d were allowed for adaptation to treatments and 7 d for data and samples collection. Cows were grouped into 5 squares based on parity, DIM, and MY. Cows within square were randomly assigned to 1 of 4 treatments: control (CON), 150, 300, or 600 mg/cow per day of BTC (150, 300, and 600, respectively). The doses were determined based on in vitro studies conducted in our laboratory aimed at evaluating the effect of BTC on CH4 emissions (unpublished data by A. N. Hristov, The Pennsylvania State University). According to the manufacturer's (AVT Natural North America) specifications, the product contained a mixture of Capsicum oleoresin (1.25% total standardized content) and a full-spectrum clove EO, with a total volatile oil content of 5.2% (as-is basis). The remainder of the product (93.5%) was a carrier constituted of fat and used for encapsulation. All cows received the same basal diet (Table 1) once daily at around 0800 h, and the diet was fed ad libitum targeting 10% refusals. The BTC was top-dressed daily, by mixing with approximately 500 g of TMR during feeding. The diet was formulated to meet or exceed the nutrient requirements of a lactating Holstein cow weighing 650 kg BW, producing 48 kg/d MY, with 3.80% fat and 3.20% true protein, at 27 kg/d of DMI (
      • NRC
      Nutrient Requirements of Dairy Cattle.
      ). Cows were milked twice daily at 0600 and 1800 h and always had free access to drinking water.
      Table 1Ingredient and nutrient composition of the basal diet fed in the experiment
      ItemDiet
      Feed ingredients, % of DM
       Corn silage
      Corn silage was 40.0% DM and contained (DM basis) 7.0% CP and 34.6% NDF.
      43.4
       Alfalfa haylage
      Alfalfa haylage was 47.0% DM and contained (DM basis) 17.2% CP and 49.1% NDF.
      10.0
       Straw/hay mix
      Straw/hay mix was 92.0% DM and contained (DM basis) 9.4% CP and 69.0% NDF.
      5.03
       Ground corn8.92
       Whole roasted soybeans3.49
       SoyPLUS
      Heat-treated soybean meal (West Central Cooperative); contained (DM basis) 45.1% CP.
      6.02
       Canola meal12.5
       Whole cottonseed4.47
       Molasses4.92
       Mineral/vitamin mix
      Mineral and vitamin premix (Cargill Animal Nutrition, Cargill Inc.) contained (%, as-is basis) limestone, 36.75; dry corn distillers grains with solubles, 29.00; NaCl, 24.85; MgO (54% Mg), 4.15; Bio-Phos, 2.45 (Rootwise Soil Dynamics); zinc sulfate, 0.96; mineral oil, 0.5; vitamin E, 0.37; manganese sulfate, 0.37; copper sulfate, 0.26; ferrous sulfate, 0.16; selenium, 0.13; vitamin A, 0.03; vitamin D3, 0.013; calcium iodate, 0.008; cobalt carbonate, 0.005.
      1.02
       Urea0.22
      Nutrient composition,
      Unless indicated otherwise, values were calculated using the nutrient analysis (Cumberland Valley Analytical Services Inc., Waynesboro, PA) of individual feed ingredients and their inclusion rates in the diet.
      % of DM (or as indicated)
       CP16.1
       NDF31.4
       ADF20.4
       NFC
      Estimated based on NRC (2001) using actual DMI, milk yield, milk composition, and BW of the cows throughout the experiment.
      45.4
       Starch24.2
       NEL balance,
      Estimated based on NRC (2001) using actual DMI, milk yield, milk composition, and BW of the cows throughout the experiment.
      Mcal/kg
      1.54
       Ash4.50
       Ca0.60
       P0.40
      1 Corn silage was 40.0% DM and contained (DM basis) 7.0% CP and 34.6% NDF.
      2 Alfalfa haylage was 47.0% DM and contained (DM basis) 17.2% CP and 49.1% NDF.
      3 Straw/hay mix was 92.0% DM and contained (DM basis) 9.4% CP and 69.0% NDF.
      4 Heat-treated soybean meal (West Central Cooperative); contained (DM basis) 45.1% CP.
      5 Mineral and vitamin premix (Cargill Animal Nutrition, Cargill Inc.) contained (%, as-is basis) limestone, 36.75; dry corn distillers grains with solubles, 29.00; NaCl, 24.85; MgO (54% Mg), 4.15; Bio-Phos, 2.45 (Rootwise Soil Dynamics); zinc sulfate, 0.96; mineral oil, 0.5; vitamin E, 0.37; manganese sulfate, 0.37; copper sulfate, 0.26; ferrous sulfate, 0.16; selenium, 0.13; vitamin A, 0.03; vitamin D3, 0.013; calcium iodate, 0.008; cobalt carbonate, 0.005.
      6 Unless indicated otherwise, values were calculated using the nutrient analysis (Cumberland Valley Analytical Services Inc., Waynesboro, PA) of individual feed ingredients and their inclusion rates in the diet.
      7 Estimated based on
      • NRC
      Nutrient Requirements of Dairy Cattle.
      using actual DMI, milk yield, milk composition, and BW of the cows throughout the experiment.
      In previous research, we observed that the ruminal escape of CAP was up to 33%, depending on the dose (
      • Oh J.
      • Bravo D.M.
      • Wall E.H.
      • Hristov A.N.
      Rumen disappearance of capsaicin and dihydrocapsaicin in lactating dairy cows.
      ), and that of polyphenols (e.g., eugenol) was up to 73% (
      • Oh J.
      • Harper M.T.
      • Smith P.
      • Wall E.
      • Hristov A.N.
      Rumen disappearance of carvacrol and anethole in lactating dairy cows.
      ). The ruminal escape fraction of the BTC used in the current experiment was estimated to be 23%, based on a 24-h in situ incubation experiment (unpublished data by A. N. Hristov, The Pennsylvania State University). Briefly, an in situ experiment was conducted with 3 ruminally cannulated cows using a modification of the procedure described in
      • Lee C.
      • Hristov A.N.
      • Cassidy T.W.
      • Heyler K.S.
      • Lapierre H.
      • Varga G.A.
      • De Veth M.J.
      • Patton R.A.
      • Parys C.
      Rumen- protected lysine, methionine, and histidine increase milk protein yield in dairy cows fed a metabolizable protein-deficient diet.
      . Cows were maintained on a basal diet containing feed ingredients typically fed at Penn State's Dairy Teaching and Research Center (corn silage, alfalfa haylage, ground corn grain, canola meal, whole roasted soybeans, and others). Approximately 3 g of BTC were weighed in a 5 × 10-cm, 50-μm porosity polyester bag (Ankom Technology Corp.), soaked in warm water (39°C) for 1 min and incubated in the rumen for 0, 6, 12, and 24 h. The incubation time points were based on
      • Lee C.
      • Hristov A.N.
      • Cassidy T.W.
      • Heyler K.S.
      • Lapierre H.
      • Varga G.A.
      • De Veth M.J.
      • Patton R.A.
      • Parys C.
      Rumen- protected lysine, methionine, and histidine increase milk protein yield in dairy cows fed a metabolizable protein-deficient diet.
      and the bags were incubated in triplicate for each time point and cow. Bags were inserted into the rumen simultaneously and removed sequentially. When removed from the rumen, bags were washed with cold tap water until the runoff water was clear. Washed bags were then dried in a forced-air oven at 55°C for 72 h; 0-h bags were processed as for the other time points, except for the rumen incubation. The dried bags were weighed, and DM disappearance was calculated based on the original DM of the product, and the dry weights of the incubated BTC sample.

      Sample Collections

      Feed and TMR Sampling

      Weights of feed offered and refusals were recorded daily. Fresh TMR, refusals, and feed ingredient samples were collected twice weekly and stored frozen at −20°C until oven-dried at 55°C for 72 h for further analyses. Dried samples were ground using a Wiley mill (Thomas Scientific) through a 1-mm screen and composited by experimental period (TMR and forage samples) or for the entire experiment (concentrate feeds). Individual feed ingredients were analyzed at Cumberland Valley Analytical Services (Waynesboro, PA) by wet chemistry methods for CP (method 990.03;
      • AOAC International
      Official Methods of Analysis.
      ), amylase-treated NDF (
      • Van Soest P.J.
      • Robertson J.B.
      • Lewis B.A.
      Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition.
      ; not ash-corrected), ADF (method 973.18;
      • AOAC International
      Official Methods of Analysis.
      ; not ash-corrected), ether extract (method 2003.05;
      • AOAC International
      Official Methods of Analysis.
      ), ash (method 942.05;
      • AOAC International
      Official Methods of Analysis.
      ), minerals (method 985.01;
      • AOAC International
      Official Methods of Analysis.
      ), and calculated NFC (NRC, 2001). Composite TMR samples were analyzed for starch according to
      • Hall M.B.
      Determination of starch, including maltooligosaccharides, in animal feeds: Comparison of methods and a method recommended for AOAC collaborative study.
      and indigestible NDF (iNDF) as described in
      • 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.
      and modified by
      • Lee C.
      • Hristov A.N.
      • Cassidy T.W.
      • Heyler K.S.
      • Lapierre H.
      • Varga G.A.
      • De Veth M.J.
      • Patton R.A.
      • Parys C.
      Rumen- protected lysine, methionine, and histidine increase milk protein yield in dairy cows fed a metabolizable protein-deficient diet.
      . Nutrient composition of the basal diet was reconstituted from analyzed values of individual feed ingredients and their inclusion rate in the TMR (Table 1).

      Production Data Collection and Milk Sampling

      Milk yield (Afimilk system) and BW (AfiFarm 3.04E scale system; SAE Afikim) of cows were recorded daily throughout the experiment. Milk samples were collected from 4 consecutive milkings (a.m. and p.m.) over 2 consecutive days during the last week of each experimental period (i.e., a total of 4 samples per cow and period) into 20-mL tubes containing bromo-2-nitropropane-1,3-diol. Samples were analyzed for concentrations of milk fat, true protein, MUN, lactose, TS, and SCC by Dairy One laboratory using Milkoscan models 6000, FT+, or 7 and Fossomatic models 5000 or FC (Foss Electric A/S). Separate unpreserved samples were also collected as described above and stored frozen at −20°C. These samples were composited on an equal-volume basis per cow and period and analyzed for fatty acid (FA) profile as described by
      • Rico D.E.
      • Harvatine K.J.
      Induction of and recovery from milk fat depression occurs progressively in dairy cows switched between diets that differ in fiber and oil concentration.
      .

      Fecal and Urine Sampling

      During the last week of each experimental period, 8 spot fecal samples were collected during 3 consecutive days at intervals staggered in time to cover a 24-h feeding cycle: at 0500, 1100, 1700, and 2300 h (d 1), 0800, 1400, and 2000 h (d 2), and 0200 h (d 3). Fecal samples were oven-dried at 55°C for 72 h, ground through a 1-mm sieve in a Wiley mill (Thomas Scientific) and composited per cow and experimental period. Composite fecal samples were then analyzed for total N using a Costech ECS 4010 elemental analyzer (Costech Analytical Technologies Inc.), ADF and NDF using an Ankom 200 fiber analyzer (Ankom Technology Corp.), and starch (
      • Hall M.B.
      Determination of starch, including maltooligosaccharides, in animal feeds: Comparison of methods and a method recommended for AOAC collaborative study.
      ). Indigestible NDF was used as a total-tract digestibility marker (
      • Schneider B.H.
      • Flatt W.P.
      The Evaluation of Feeds through Digestibility Experiments.
      ). For the iNDF analysis, composited fecal samples were oven-dried at 55°C, ground through a 1-mm screen, and incubated for 12 d in the rumen of a lactating rumen-cannulated cow 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.
      , except 25-µm pore size filter bags (Ankom Technology Corp.) were used for the incubation (
      • Lee C.
      • Hristov A.N.
      • Cassidy T.W.
      • Heyler K.S.
      • Lapierre H.
      • Varga G.A.
      • De Veth M.J.
      • Patton R.A.
      • Parys C.
      Rumen- protected lysine, methionine, and histidine increase milk protein yield in dairy cows fed a metabolizable protein-deficient diet.
      ). The rumen-cannulated cow used for the iNDF incubation was fed a diet containing corn silage, alfalfa haylage, concentrate feeds, and a mineral and vitamin premix.
      Urine samples were collected at the same time points as for fecal samples by perineal stimulation and were filtered through 2 layers of cheesecloth. Aliquots (10 mL) were acidified with 0.6 mL of 2 M sulfuric acid, diluted 1:10 with 90 mL of distilled water, and stored at −20°C until further analyses. Samples were composited by cow and experimental period and analyzed for allantoin (
      • Chen X.B.
      • Chen Y.K.
      • Franklin M.F.
      • Ørskov E.R.
      • Shand W.J.
      The effect of feed intake and body weight on purine derivative excretion and microbial protein supply in sheep.
      ), uric acid (Stanbio Uric acid Kit 1045; Stanbio Laboratory Inc.), urea-N (Stanbio Urea Nitrogen Kit 580; Stanbio Laboratory Inc.), and creatinine (Stanbio Creatinine Kit 420; Stanbio Laboratory Inc.). Composite urine samples were freeze-dried and analyzed for total N using a Costech ECS 4010 elemental analyzer (Costech Analytical Technologies Inc.). Urinary creatinine concentration was used to estimate daily urine output, assuming a creatinine excretion rate of 29 mg/kg of BW determined from total urine collections in lactating Holstein cows (
      • Hristov A.N.
      • Lee C.
      • Cassidy T.
      • Long M.
      • Heyler K.
      • Corl B.
      • Forster R.
      Effects of lauric and myristic acids on ruminal fermentation, production, and milk fatty acid composition in lactating dairy cows.
      ). Daily excretions of total N, urea-N, and purine derivates (allantoin and uric acid) were calculated based on the estimated daily urine output.

      Blood Sampling

      During the last week of each experimental period, blood samples were collected from the coccygeal vein or artery of cows 4 times in 2 consecutive days at 1100 and 1900 h (d 1) and 0700 and 1500 h (d 2). Samples (approximately 10 mL) were collected into serum-separating vacuum tubes (BD Biosciences). Serum was separated by centrifugation at 1,800 × g at 20°C for 30 min and stored at −80°C until further analyses. Serum samples were composited per cow and experimental period on an equal-volume basis and enzymatic colorimetric methods were used for total FA [total FA-HR(2), kit no. 999-34691, intra- and inter-assay CV: 0.75 and 4.91%, respectively; Wako Diagnostics] and BHB (Autokit 3-HB, kit no. 417-73501, intra- and inter-assay CV: 5 and 10%, respectively; Wako Diagnostics) analyses. The minimum detectable levels of the methods were 0.0014 μmol/L (oleic acid equivalent) and 3 μmol/L for total FA and BHB, respectively. Serum insulin was analyzed using an enzyme-linked immunoassay kit (Bovine Insulin ELISA, kit no. 10-1201-01, Mercodia AB). The inter- and intra-assay CV were both <10%. The minimum detection level for insulin was 0.05 μg/L. Blood chemistry (glucose, creatinine, BUN, total protein, albumin, globulin, alanine transaminase, and alkaline phosphatase) were analyzed using a Catalyst One Chemistry Analyzer (Idexx Laboratories Inc.). A second set of blood samples was collected from the coccygeal vein or artery at the same time points as indicated above for hematology analysis. Samples (approximately 10 mL) were collected into tubes containing EDTA (BD Biosciences), kept refrigerated (4°C), and analyzed on the same day. The analysis included red blood cell count, hemoglobin, hematocrit, and total white blood cell count, including total count of neutrophils, eosinophils, lymphocytes, monocytes, and basophils using an automated hematology analyzer (HemaVet; Drew Scientific).

      Enteric Gas Emissions

      Enteric gas (CH4, CO2, and H2) emissions were measured using the GreenFeed system (C-Lock Inc.) One GreenFeed unit was used and calibrated at the beginning of each measurement period based on manufacturer's recommendations (https://globalresearchalliance.org/wp-content/uploads/2018/08/GreenFeeds-SOP-_final.pdf; accessed Apr. 11, 2022). Cows were properly trained to use the system before the beginning of the experiment. Cows were identified with a unique radio-frequency identification ear tag for recognition in the GreenFeed unit. Gas measurements were taken 8 times over 3 consecutive days during the last week of each experimental period at 0900, 1500, and 2100 h (d 1), 0300, 1200, and 1500 h (d 2), and 0000 and 0300 h (d 3), following the procedure described by
      • Hristov A.N.
      • Oh J.
      • Giallongo F.
      • Frederick T.
      • Weeks H.
      • Zimmerman P.R.
      • Harper M.T.
      • Hristova R.A.
      • Zimmerman R.S.
      • Branco A.F.
      The use of an automated system (GreenFeed) to monitor enteric methane and carbon dioxide emissions from ruminant animals.
      . Individual breath samples were collected during 5 min sampling events, followed by 2-min intervals for background air collection between cows. Gas samples were collected following the sequence of cows in the tiestall barn, and the sequence of sampling was maintained throughout the entire experiment. A pelletized bait feed (Stocker Grower 14, Purina Animal Nutrition LLC) was used to attract cows to the GreenFeed unit during gas sampling events. The intake of pellets was calculated based on the weight of pellets in each drop (32 g) and the number of drops during a sampling event. Intake of pellets was included in the daily DMI calculation during sampling days. Dry matter intake, digestible OM intake, MY, and ECM yield during sampling week were used to calculate CH4 emission yield (i.e., g CH4 ÷ kg DMI and digestible OM intake) and intensity (i.e., g CH4 ÷ kg MY and ECM yield).

      Statistical Analysis

      All data were analyzed with SAS (release 9.4, SAS Institute Inc.). Two cows were removed from the experiment after they were diagnosed with severe mastitis and coxofemoral luxation during experimental period 3. Data collected from these cows on periods 1 and 2 remained in the analysis. Data were analyzed for outliers using the REG procedure and outliers were removed based on an absolute studentized residue value >3. Statistical analyses were performed using the MIXED procedure. Milk yield, DMI, BW, and feed efficiency, from the last 7 d of each experimental period, were analyzed as repeated measures using the AR(1) covariance structure. Statistical models included experimental period, treatment, parity, and treatment × parity interaction as fixed effects, and day as the repeated term. Square and cow within square were random effects. When nonsignificant (P > 0.05), treatment × parity interaction was removed from the final models. Milk composition, blood chemistry and hematology, nutrient intake and digestibility, urinary excretions, and enteric gas emission data were analyzed without the repeated term in the models. Gas emissions data were averaged across all sampling time points and the averaged values were used for statistical analysis. Orthogonal and polynomial contrasts were used to evaluate BTC treatment versus control and to test for linear and quadratic effects of BTC dose. Statistical differences were considered significant at P ≤ 0.05 and tendencies were declared at 0.05 < P ≤ 0.10. Data are presented as least squares means.

      RESULTS AND DISCUSSION

      Production Data

      Dry matter intake and MY were not affected by BTC supplementation (Table 2). Milk fat and MUN concentrations tended to be greater (P = 0.06 and P = 0.08, respectively) for BTC compared with CON. Botanicals had no effects on the other production variables evaluated in the current study.
      Table 2Dry matter intake, milk production and composition, and BW of dairy cows fed botanicals combination (BTC)
      ItemCONTreatment
      Treatments were as follows: CON = control, 0 mg/cow per day of BTC; 150 = 150 mg/cow per day of BTC; 300 = 300 mg/cow per day of BTC; 600 = 600 mg/cow per day of BTC.
      SEM
      Largest SEM published in table; n = 76 (n represents the number of observations used in the statistical analysis).
      P-value
      Contrasts: C vs. T = control vs. BTC treatments; L = linear effect of BTC; Q = quadratic effect of BTC. There was no effect of parity or a parity × treatment interaction for any variable (P ≥ 0.25).
      150300600C vs. TLQ
      DMI, kg/d27.928.227.627.31.250.720.210.76
      Milk yield, kg/d38.939.038.838.41.330.790.480.80
      Feed efficiency,
      Milk yield ÷ DMI.
      kg/kg
      1.411.401.431.420.0620.950.730.93
      Milk fat, %3.803.953.913.980.1860.060.110.47
      Milk fat yield, kg/d1.471.531.511.500.0790.290.790.35
      Milk true protein, %3.012.952.972.970.0600.110.450.28
      Milk true protein yield, kg/d1.171.141.151.130.0310.240.180.89
      Lactose, %4.824.844.824.810.0540.910.400.51
      Milk lactose yield, kg/d1.881.891.871.850.0700.790.400.77
      Total solids, %12.512.612.612.60.230.150.220.58
      MUN, mg/dL10.611.310.811.00.330.080.450.44
      Log SCC × 103 cells/mL1.631.611.591.580.0970.490.410.81
      ECM,
      Energy-corrected milk (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
      37.137.737.436.71.490.830.480.40
      ECM feed efficiency,
      ECM yield ÷ DMI.
      kg/kg
      1.341.351.371.350.0530.560.710.47
      BW, kg62562762562120.20.590.131.00
      1 Treatments were as follows: CON = control, 0 mg/cow per day of BTC; 150 = 150 mg/cow per day of BTC; 300 = 300 mg/cow per day of BTC; 600 = 600 mg/cow per day of BTC.
      2 Largest SEM published in table; n = 76 (n represents the number of observations used in the statistical analysis).
      3 Contrasts: C vs. T = control vs. BTC treatments; L = linear effect of BTC; Q = quadratic effect of BTC. There was no effect of parity or a parity × treatment interaction for any variable (P ≥ 0.25).
      4 Milk yield ÷ DMI.
      5 Energy-corrected milk (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.
      ).
      6 ECM yield ÷ DMI.
      Contrary to our hypothesis and to results from previous studies evaluating CAP (
      • Oh J.
      • Giallongo F.
      • Frederick T.
      • Pate J.
      • Walusimbi S.
      • Elias R.J.
      • Wall E.H.
      • Bravo D.
      • Hristov A.N.
      Effects of dietary capsicum oleoresin on productivity and immune responses in lactating dairy cows.
      ,
      • Oh J.
      • Harper M.T.
      • Melgar A.
      • Räisänen S.
      • Chen X.
      • Nedelkov K.
      • Fetter M.
      • Ott T.
      • Wall E.H.
      • Hristov A.N.
      Dietary supplementation with rumen-protected capsicum during the transition period improves the metabolic status of dairy cows.
      ) or botanical extracts containing eugenol (
      • Wall E.H.
      • Doane P.H.
      • Donkin S.S.
      • Bravo D.
      The effects of supplementation with a blend of cinnamaldehyde and eugenol on feed intake and milk production of dairy cows.
      ;
      • Benchaar C.
      • Hassanat F.
      • Petit H.V.
      Dose-response to eugenol supplementation to dairy cow diets: Methane production, N excretion, ruminal fermentation, nutrient digestibility, milk production, and milk fatty acid profile.
      ), BTC did not improve lactational performance of dairy cows in the present study. It is noted, however, that comparisons with the above studies should be done with caution as the treatment in the current experiment contained both CAP and eugenol and these bioactive compounds were not studied individually. Similar to previous reports (
      • Wall E.H.
      • Doane P.H.
      • Donkin S.S.
      • Bravo D.
      The effects of supplementation with a blend of cinnamaldehyde and eugenol on feed intake and milk production of dairy cows.
      ;
      • Oh J.
      • Giallongo F.
      • Frederick T.
      • Pate J.
      • Walusimbi S.
      • Elias R.J.
      • Wall E.H.
      • Bravo D.
      • Hristov A.N.
      Effects of dietary capsicum oleoresin on productivity and immune responses in lactating dairy cows.
      ,
      • Oh J.
      • Harper M.T.
      • Melgar A.
      • Räisänen S.
      • Chen X.
      • Nedelkov K.
      • Fetter M.
      • Ott T.
      • Wall E.H.
      • Hristov A.N.
      Dietary supplementation with rumen-protected capsicum during the transition period improves the metabolic status of dairy cows.
      ), DMI was not affected by BTC in the current study. The tendency for increased milk fat content in response to BTC supplementation may be related to the decreased BHB concentration and increased uptake of ketone bodies by the mammary gland (see later discussion), which could reflect a differential metabolic adaptation and energy partitioning in cows fed BTC.
      In agreement with the current data,
      • da Silva R.B.
      • Pereira M.N.
      • Araujo R.C.D.
      • Silva W.D.R.
      • Pereira R.A.N.
      A blend of essential oils improved feed efficiency and affected ruminal and systemic variables of dairy cows.
      reported that dairy cows fed a blend of EO containing carvacrol, eugenol, and capsaicin tended to increase milk fat concentration by 0.11 percentage units. Energy status of the animal is an important factor to be considered when evaluating milk fat regulation. Increased milk fat synthesis as a consequence of increased BHB uptake by the mammary gland in cows fed a diet supplemented with BTC has not been described yet and needs further investigation. Alternatively, the tendency for increased milk fat could be associated with increased ruminal acetate and BHB produced from butyrate absorbed in the rumen because these compounds are the major precursors supporting milk fat synthesis (
      • Bauman D.E.
      • Griinari J.M.
      Nutritional regulation of milk fat synthesis.
      ). However, it is important to point out that ruminal VFA concentrations were not analyzed in the current study. The effect on milk fat in the current experiment was counteracted by a slightly numerical decrease in milk true protein concentration, which resulted in lack of treatment effect on ECM yield.

      Apparent Total-Tract Digestibility and Nitrogen Excretions

      Intake of dietary nutrients during the digestibility measurement period was not affected by BTC supplementation (Table 3). Apparent total-tract digestibility of DM was decreased (P = 0.04) by less than 2 percentage units, and starch digestibility tended to be decreased (P = 0.09) by BTC, compared with CON. Additionally, digestibility of other nutrients was not affected by BTC supplementation. The small effect of BTC on DM and starch digestibility, and the lack of effect on digestibility of other nutrients, corroborates with previous studies that supplemented a similar composition of botanical products to dairy (
      • Benchaar C.
      • Hassanat F.
      • Petit H.V.
      Dose-response to eugenol supplementation to dairy cow diets: Methane production, N excretion, ruminal fermentation, nutrient digestibility, milk production, and milk fatty acid profile.
      ;
      • Oh J.
      • Giallongo F.
      • Frederick T.
      • Pate J.
      • Walusimbi S.
      • Elias R.J.
      • Wall E.H.
      • Bravo D.
      • Hristov A.N.
      Effects of dietary capsicum oleoresin on productivity and immune responses in lactating dairy cows.
      ) and beef cattle (
      • Westphalen M.F.
      • Carvalho P.H.V.
      • Oh J.
      • Hristov A.N.
      • Staniar W.B.
      • Felix T.L.
      Effects of feeding rumen-protected capsicum oleoresin on growth performance, health status, and total tract digestibility of growing beef cattle.
      ).
      • Yang W.Z.
      • Benchaar C.
      • Ametaj B.N.
      • Beauchemin K.A.
      Dose response to eugenol supplementation in growing beef cattle: Ruminal fermentation and intestinal digestion.
      reported that total-tract digestibility of starch in beef heifers tended to decrease linearly with increasing dietary inclusion of eugenol (0, 400, 800, and 1,600 mg/d) in their diet. However, effect of BTC on starch digestibility, and perhaps on that of DM, was biologically insignificant in the current study.
      Table 3Intake and apparent total-tract digestibility of nutrients in lactating dairy cows fed botanicals combination (BTC)
      ItemCONTreatment
      Treatments were as follows: CON = control, 0 mg/cow per day of BTC; 150 = 150 mg/cow per day of BTC; 300 = 300 mg/cow per day of BTC; 600 = 600 mg/cow per day of BTC.
      SEM
      Largest SEM published in table; n = 76 for total-tract digestibility of DM, CP, and starch; n = 75 for total-tract digestibility of ADF; n = 77 for all other variables (n represents the number of observations used in the statistical analysis).
      P-value
      Contrasts: C vs. T = control vs. BTC treatments; L = linear effect of BTC; Q = quadratic effect of BTC. There was no effect of parity or a parity × treatment interaction for any variable (P ≥ 0.10).
      P-values for intake of dietary nutrients are the same as for DMI.
      150300600C vs. TLQ
      Intake,
      Dry matter intake during the fecal collection, last 3 d of each experimental period.
      kg/d
       DM27.727.927.327.11.180.670.230.83
       OM25.926.225.625.41.11
       NDF9.719.809.609.520.414
       ADF4.904.954.854.800.209
       CP4.554.594.504.460.194
       Starch6.616.676.536.480.282
      Apparent digestibility, %
       DM69.268.668.268.40.430.040.110.11
       OM69.869.369.169.40.570.330.640.35
       NDF49.449.949.950.00.690.320.370.65
       ADF36.935.735.936.41.460.580.900.58
       CP69.769.969.169.60.810.780.770.61
       Starch98.798.598.598.50.080.090.210.49
      1 Treatments were as follows: CON = control, 0 mg/cow per day of BTC; 150 = 150 mg/cow per day of BTC; 300 = 300 mg/cow per day of BTC; 600 = 600 mg/cow per day of BTC.
      2 Largest SEM published in table; n = 76 for total-tract digestibility of DM, CP, and starch; n = 75 for total-tract digestibility of ADF; n = 77 for all other variables (n represents the number of observations used in the statistical analysis).
      3 Contrasts: C vs. T = control vs. BTC treatments; L = linear effect of BTC; Q = quadratic effect of BTC. There was no effect of parity or a parity × treatment interaction for any variable (P ≥ 0.10).
      4 P-values for intake of dietary nutrients are the same as for DMI.
      5 Dry matter intake during the fecal collection, last 3 d of each experimental period.
      Considering the potential of botanical compounds to modify ruminal fermentation (
      • Calsamiglia S.
      • Busquet M.
      • Cardozo P.W.
      • Castillejos L.
      • Ferret A.
      Invited review: Essential oils as modifiers of rumen microbial fermentation.
      ;
      • Oh J.
      • Harper M.
      • Lang C.H.
      • Wall E.H.
      • Hristov A.N.
      Effects of phytonutrients alone or in combination with monensin on productivity in lactating dairy cows.
      ), cows supplemented with BTC may have enhanced ruminal fermentation and potentially microbial protein synthesis, which would lead to decreased urinary N excretion. However, excretions of fecal or urinary N, urinary urea, total excreta N, and urinary purine derivates were not affected by BTC supplementation (Table 4). These results may be a consequence of a similar pattern of protein and carbohydrates fermentation in the rumen of cows fed CON or BTC, which is in line with the lack of differences in milk N secretion, N use efficiency, and BUN concentration observed in the current study. Digestibility and N excretion data from the current study should be interpreted with caution because spot sampling and markers were used to estimate total fecal and urinary excretions. The spot sampling technique has been validated but results are inherently more variable than total fecal and urine collections (
      • Lee C.
      • Hristov A.N.
      Short communication: evaluation of acid-insoluble ash and indigestible neutral detergent fiber as total-tract digestibility markers in dairy cows fed corn silage-based diets.
      ;
      • Lee C.
      • Morris D.L.
      • Dieter P.A.
      Validating and optimizing spot sampling of urine to estimate urine output with creatinine as a marker in dairy cows.
      ). The latter technique can also produce erroneous results, particularly related to N excretions and balance, if not correctly applied. A recent meta-analysis of 86 dairy cow experiments described that N retention is typically overestimated, mainly due to unaccounted volatilization losses of N during collection and processing of fecal and urine samples (
      • Spanghero M.
      • Kowalski Z.M.
      Updating analysis of nitrogen balance experiments in dairy cows.
      ); these authors reported no statistical difference in estimated N balance between total collection and spot sampling techniques (63 vs. 23 studies, respectively).
      Table 4Nitrogen excretion and secretion, and urinary purine derivates excretion in lactating dairy cows fed botanicals combination (BTC)
      ItemCONTreatment
      Treatments were as follows: CON = control, 0 mg/cow per day of BTC; 150 = 150 mg/cow per day of BTC; 300 = 300 mg/cow per day of BTC; 600 = 600 mg/cow per day of BTC.
      SEM
      Largest SEM published in table; n = 77 (n represents the number of observations used in the statistical analysis).
      P-value
      Contrasts: C vs. T = control vs. BTC treatments; L = linear effect of BTC; Q = quadratic effect of BTC. There was no effect of parity or a parity × treatment interaction for any variable (P ≥ 0.10).
      150300600C vs. TLQ
      N intake, g/d72873472071331.00.670.230.83
      N excretion or secretion, g/d
       Urine N17517216417716.80.800.920.53
       Urinary urea-N1351361321399.520.870.600.58
       Fecal N2212202222209.890.880.870.92
       Total excreta N39739338838421.60.600.510.91
       Milk N1831821801764.630.370.110.79
      As % of N intake
       Urine N23.823.022.325.11.880.870.590.36
       Urinary urea-N18.318.718.419.50.990.570.300.65
       Fecal N30.330.130.930.40.810.780.780.61
       Total excreta N54.153.052.954.51.810.780.810.52
       Milk N25.224.425.024.70.870.270.680.56
       Unaccounted N
      Unaccounted N = N intake − (Urinary N + Fecal N + Milk N).
      20.721.621.120.42.000.890.860.76
      Purine derivates, mmol/d
       Allantoin49653846755465.00.680.550.59
       Uric acid45.944.739.642.43.380.250.290.27
       Total PD
      Purine derivatives.
      54458350759666.50.760.610.53
      1 Treatments were as follows: CON = control, 0 mg/cow per day of BTC; 150 = 150 mg/cow per day of BTC; 300 = 300 mg/cow per day of BTC; 600 = 600 mg/cow per day of BTC.
      2 Largest SEM published in table; n = 77 (n represents the number of observations used in the statistical analysis).
      3 Contrasts: C vs. T = control vs. BTC treatments; L = linear effect of BTC; Q = quadratic effect of BTC. There was no effect of parity or a parity × treatment interaction for any variable (P ≥ 0.10).
      4 Unaccounted N = N intake − (Urinary N + Fecal N + Milk N).
      5 Purine derivatives.

      Milk Fatty Acid Composition

      Only minor changes in milk FA profile were observed in the current study (Table 5). Concentrations of 17:0 increased (P = 0.02) and trans-5 18:1 tended to decrease (P = 0.08) by BTC, compared with CON. Supplementation with BTC decreased (P = 0.05) the concentrations of cis-6,cis-9,cis-12 18:3 and cis-9,trans-11 CLA (P = 0.05), compared with CON. Additionally, concentration of 20:2n6 was lower (P = 0.002) for BTC, compared with CON, and tended to be quadratically decreased (P = 0.009) by BTC dose. Concentration of 20:3n6 also tended to be lower (P = 0.10) in BTC, compared with CON.
      Table 5Fatty acid composition of milk fat (g/100 g of total fatty acids) in lactating dairy cows fed botanicals combination (BTC)
      ItemCONTreatment
      Treatments were as follows: CON = control, 0 mg/cow per day of BTC; 150 = 150 mg/cow per day of BTC; 300 = 300 mg/cow per day of BTC; 600 = 600 mg/cow per day of BTC.
      SEM
      Largest SEM published in table; n = 77 (n represents the number of observations used in the statistical analysis).
      P-value
      Contrasts: C vs. T = control vs. BTC treatments; L = linear effect of BTC; Q = quadratic effect of BTC. There was no effect of parity or a parity × treatment interaction for any variable (P ≥ 0.10).
      150300600C vs. TLQ
      4:04.954.844.784.710.2200.370.300.78
      6:02.432.452.432.400.0910.890.560.76
      8:01.311.311.321.290.0490.820.620.73
      10:02.972.963.002.960.1080.980.930.76
      12:03.383.333.423.380.1180.990.820.90
      14:010.910.911.010.90.240.800.990.56
      cis-9 14:10.870.780.870.840.0530.350.980.66
      15:00.991.011.041.010.0460.380.610.33
      16:026.827.227.127.20.570.240.360.65
      cis-9 16:11.211.151.261.240.0870.840.280.92
      17:00.480.510.500.500.0130.020.190.15
      18:011.611.911.611.70.710.640.950.78
      trans-4 18:10.020.020.020.030.0020.820.710.70
      trans-5 18:10.0170.0150.0140.0130.00130.080.080.46
      trans-6,8 18:10.360.340.340.340.0150.120.250.41
      trans-9 18:10.300.290.290.290.0130.130.360.30
      trans-10 18:10.840.800.760.900.1570.790.440.16
      trans-11 18:11.131.061.101.070.0530.170.380.57
      trans-12 18:10.580.570.570.560.0170.230.240.84
      cis-9 18:119.419.419.419.60.500.960.770.86
      cis-11 18:11.191.241.231.220.6670.310.770.39
      cis-12 18:10.470.450.460.440.0120.250.160.85
      cis-9,cis-12 18:22.882.872.802.930.0630.830.610.26
      cis-6,cis-9,cis-12 18:30.0310.0250.0300.0250.00180.050.090.96
      20:00.130.130.130.130.0060.830.720.90
      cis-9,trans-11 CLA0.570.520.550.530.0280.050.240.39
      cis-12,trans-10 CLA0.0010.0040.0010.0020.0020.510.970.91
      20:2n60.0340.0220.0230.0260.0030.0020.150.009
      20:3n60.0210.0150.0120.0100.0050.100.110.45
      Total trans-fatty acids3.823.623.653.740.2310.190.780.17
      Σ SFA67.868.468.268.00.590.340.850.36
      Σ MUFA28.025.826.026.20.580.930.600.68
      Σ PUFA3.753.623.723.730.0720.430.810.44
      Σ De novo
      De novo fatty acids (FA; <C16) are synthesized by the mammary gland; preformed FA (>C16) originate primarily from extraction from plasma; and mixed fatty acids (C16) originate from both sources.
      28.928.629.028.50.550.710.700.86
      Σ Mixed
      De novo fatty acids (FA; <C16) are synthesized by the mammary gland; preformed FA (>C16) originate primarily from extraction from plasma; and mixed fatty acids (C16) originate from both sources.
      28.128.528.528.60.640.270.320.67
      Σ Preformed
      De novo fatty acids (FA; <C16) are synthesized by the mammary gland; preformed FA (>C16) originate primarily from extraction from plasma; and mixed fatty acids (C16) originate from both sources.
      41.741.841.741.90.810.870.780.90
      Σ OBCFA
      Odd- and branched-chain FA; sum of C11:0, iso C13:0, ante-iso C13:0, C13:0, iso C14:0, iso C15:0, ante-iso C15:0, C15:0, iso C16:0, iso C17:0, ante-iso C17:0, C17:0, and C17:1 cis-9.
      3.283.303.363.290.0710.360.760.16
      1 Treatments were as follows: CON = control, 0 mg/cow per day of BTC; 150 = 150 mg/cow per day of BTC; 300 = 300 mg/cow per day of BTC; 600 = 600 mg/cow per day of BTC.
      2 Largest SEM published in table; n = 77 (n represents the number of observations used in the statistical analysis).
      3 Contrasts: C vs. T = control vs. BTC treatments; L = linear effect of BTC; Q = quadratic effect of BTC. There was no effect of parity or a parity × treatment interaction for any variable (P ≥ 0.10).
      4 De novo fatty acids (FA; <C16) are synthesized by the mammary gland; preformed FA (>C16) originate primarily from extraction from plasma; and mixed fatty acids (C16) originate from both sources.
      5 Odd- and branched-chain FA; sum of C11:0, iso C13:0, ante-iso C13:0, C13:0, iso C14:0, iso C15:0, ante-iso C15:0, C15:0, iso C16:0, iso C17:0, ante-iso C17:0, C17:0, and C17:1 cis-9.
      The evaluation of milk FA profile can be used as a proxy to understand ruminal fermentation and metabolism in dairy cows. For instance, impaired ruminal fermentation is related to increased biohydrogenation intermediates with a potential to induce milk fat depression (
      • Rico D.E.
      • Harvatine K.J.
      Induction of and recovery from milk fat depression occurs progressively in dairy cows switched between diets that differ in fiber and oil concentration.
      ). Increased concentration of preformed FA in milk, in contrast, can indicate excessive mobilization of adipose tissue and greater negative energy balance (
      • Dórea J.R.R.
      • French E.A.
      • Armentano L.E.
      Use of milk fatty acids to estimate plasma nonesterified fatty acid concentrations as an indicator of animal energy balance.
      ). None of these conditions, however, were observed in the current study. Few studies have evaluated the effects of CAP and eugenol on milk FA profile of dairy cows. A study by
      • Oh J.
      • Giallongo F.
      • Frederick T.
      • Pate J.
      • Walusimbi S.
      • Elias R.J.
      • Wall E.H.
      • Bravo D.
      • Hristov A.N.
      Effects of dietary capsicum oleoresin on productivity and immune responses in lactating dairy cows.
      only reported minor changes in milk FA profile (e.g., decreased concentration of cis-9,trans-11 CLA) and increased milk fat yield in cows fed with CAP, which is in line with the tendency for increased milk fat content described in the current study. Similarly,
      • Benchaar C.
      • Hassanat F.
      • Petit H.V.
      Dose-response to eugenol supplementation to dairy cow diets: Methane production, N excretion, ruminal fermentation, nutrient digestibility, milk production, and milk fatty acid profile.
      also described minor changes in milk FA profile of cows fed increasing doses of eugenol (0, 25, 50, and 75 mg/kg of feed DM). It is interesting to note that, from all FA herein described, decreased concentration of cis-9,trans-11 CLA in milk fat has been also reported by others (
      • Oh J.
      • Giallongo F.
      • Frederick T.
      • Pate J.
      • Walusimbi S.
      • Elias R.J.
      • Wall E.H.
      • Bravo D.
      • Hristov A.N.
      Effects of dietary capsicum oleoresin on productivity and immune responses in lactating dairy cows.
      ). The enzyme Δ9-desaturase plays an important role in the endogenous synthesis of cis-9,trans-11 CLA, which is the major CLA in dairy products. The substantial differences among individual cows in Δ9-desaturase activity and its contribution to milk fat CLA reported by
      • Peterson D.G.
      • Kelsey J.A.
      • Bauman D.E.
      Analysis of variation in cis-9, trans-11 conjugated linoleic acid (CLA) in milk fat of dairy cows.
      may help explain differences between treatments in the current experiment. Additionally, vaccenic acid (trans-11 C18:1) is described as the main ruminal precursor for cis-9,trans-11 CLA synthesis in the mammary gland (
      • Kay J.K.
      • Mackle T.R.
      • Auldist M.J.
      • Thomson N.A.
      • Bauman D.E.
      Endogenous synthesis of cis-9, trans-11 conjugated linoleic acid in dairy cows fed fresh pasture.
      ) and it was numerically decreased (P = 0.17) by BTC in the current study.
      • da Silva R.B.
      • Pereira M.N.
      • Araujo R.C.D.
      • Silva W.D.R.
      • Pereira R.A.N.
      A blend of essential oils improved feed efficiency and affected ruminal and systemic variables of dairy cows.
      also reported a numerical decrease in milk concentration of vaccenic acid in dairy cows fed a blend of botanicals including CAP and eugenol, which is in line with our data. The mechanisms by which BTC may reduce Δ9-desaturase activity was not investigated in the current study and should be addressed in future research. Although we speculate that the tendency for increased milk fat content in BTC cows would be associated with an increased uptake of BHB by the mammary gland, no differences for de novo-synthesized FA were observed between BTC and CON.

      Enteric Gas Emissions

      Daily CH4 emission and CH4 yield were decreased (P ≤ 0.05) by BTC, compared with CON (Table 6). As BTC supplementation level increased, daily CH4 production decreased linearly (P = 0.04) by up to 7.5%. The CH4 emission per kilogram of digestible OM intake was not affected by treatments, although a numerical decrease of 4% was observed. Methane emission intensity (per MY or ECM) tended to be decreased (P = 0.08) by BTC, compared with CON. Supplementation with BTC had no effect on CO2 emission and yield and daily H2 emission.
      Table 6Enteric gas emissions
      Enteric gas emissions were measured using the GreenFeed system (C-Lock Inc.).
      in lactating dairy cows fed botanicals combination (BTC)
      ItemCONTreatment
      Treatments were as follows: CON = control, 0 mg/cow per day of BTC; 150 = 150 mg/cow per day of BTC; 300 = 300 mg/cow per day of BTC; 600 = 600 mg/cow per day of BTC.
      SEM
      Largest SEM published in table; n = 76 (n represents the number of observations used in the statistical analysis).
      P-value
      Contrasts: C vs. T = control vs. BTC treatments; L = linear effect of BTC; Q = quadratic effect of BTC. There was no effect of parity or a parity × treatment interaction for any variable (P ≥ 0.57).
      150300600C vs. TLQ
      CH4, g/d38936536736011.80.010.040.22
       CH4 per DMI, g/kg14.013.113.413.30.530.050.270.24
       CH4 per dOM,
      dOM = intake of digestible OM, calculated as dOM = OM intake, kg/d × OM digestibility.
      g/kg
      21.520.820.720.81.050.390.480.66
       CH4 per milk yield, g/kg10.19.549.609.670.4940.080.350.18
       CH4 per ECM yield, g/kg10.59.949.9310.00.570.080.290.19
      CO2, g/d12,16611,99612,26412,0462410.710.780.71
       CO2 per DMI, g/kg44243245044919.80.870.210.91
      H2, g/d1.061.120.980.990.0690.690.230.96
      1 Enteric gas emissions were measured using the GreenFeed system (C-Lock Inc.).
      2 Treatments were as follows: CON = control, 0 mg/cow per day of BTC; 150 = 150 mg/cow per day of BTC; 300 = 300 mg/cow per day of BTC; 600 = 600 mg/cow per day of BTC.
      3 Largest SEM published in table; n = 76 (n represents the number of observations used in the statistical analysis).
      4 Contrasts: C vs. T = control vs. BTC treatments; L = linear effect of BTC; Q = quadratic effect of BTC. There was no effect of parity or a parity × treatment interaction for any variable (P ≥ 0.57).
      5 dOM = intake of digestible OM, calculated as dOM = OM intake, kg/d × OM digestibility.
      To the best of our knowledge, the current experiment is the first study to report enteric CH4 emissions of lactating dairy cows fed a blend of CAP and clove EO. The effects of BTC on CH4 yield and intensities in the current study were a result of decreased daily CH4 production (g/d) and similar DMI, MY, and ECM among treatments. It is important to note that the decrease in DM digestibility in the current study (less than 2%) was smaller than the decrease in CH4 yield (5 to 6%), which indicates that CH4 inhibition was not only a result of decreased digestibility. The lack of statistical significance for CH4 emission per kilogram of digestible OM intake was likely a result of intrinsic variability associated with the marker methodology used for estimation of digestibility of nutrients.
      The effects of individual or combinations of EO on enteric CH4 emissions in ruminants have been studied (
      • Hristov A.N.
      • Oh J.
      • Firkins J.L.
      • Dijkstra J.
      • Kebreab E.
      • Waghorn G.
      • Makkar H.P.S.
      • Adesogan A.T.
      • Yang W.
      • Lee C.
      • Gerber P.J.
      • Henderson B.
      • Tricarico J.M.
      Special Topics: Mitigation of methane and nitrous oxide emissions from animal operations: I. A review of enteric methane mitigation options.
      ;
      • Benchaar C.
      • Hassanat F.
      • Petit H.V.
      Dose-response to eugenol supplementation to dairy cow diets: Methane production, N excretion, ruminal fermentation, nutrient digestibility, milk production, and milk fatty acid profile.
      ;
      • Beauchemin K.A.
      • Ungerfeld E.M.
      • Eckard R.J.
      • Wang M.
      Review: Fifty years of research on rumen methanogenesis: Lessons learned and future challenges for mitigation.
      ), and several in vitro studies have investigated the CH4 mitigation potential of EO and their bioactive compounds (
      • Calsamiglia S.
      • Busquet M.
      • Cardozo P.W.
      • Castillejos L.
      • Ferret A.
      Invited review: Essential oils as modifiers of rumen microbial fermentation.
      ;
      • Benchaar C.
      • Greathead H.
      Essential oils and opportunities to mitigate enteric methane emissions from ruminants.
      ;
      • Tekippe J.A.
      • Hristov A.N.
      • Heyler K.S.
      • Zheljazkov V.D.
      • Ferreira J.F.S.
      • Cantrell C.L.
      • Varga G.A.
      Effects of plants and essential oils on ruminal in vitro batch culture methane production and fermentation.
      ). For instance,
      • Chaves A.V.
      • He M.L.
      • Yang W.Z.
      • Hristov A.N.
      • McAllister T.A.
      • Benchaar C.
      Effects of essential oils on proteolytic, deaminative and methanogenic activities of mixed ruminal bacteria.
      reported that cinnamon leaf oil (containing 760 g/kg eugenol) decreased CH4 production by 72% while increasing the molar proportion of butyrate (but decreasing propionate) in vitro. These authors suggested that cinnamon oil could be used as an alternative to ionophores due to its inhibition of ruminal methanogens. It must be pointed out, however, that in vitro data are rarely representative of in vivo responses (
      • Hristov A.N.
      • Lee C.
      • Hristova R.A.
      • Huhtanen P.
      • Firkins J.
      A meta-analysis of the variability in continuous culture rumen fermentation and digestibility data.
      ). For instance, in an experiment with mid-lactation dairy cows,
      • Benchaar C.
      • Hassanat F.
      • Petit H.V.
      Dose-response to eugenol supplementation to dairy cow diets: Methane production, N excretion, ruminal fermentation, nutrient digestibility, milk production, and milk fatty acid profile.
      did not find any effects of eugenol (inclusion up to 75 mg/kg of feed DMI) on daily CH4 emission or emission yield. Although the effect of BTC on CH4 in the current experiment is encouraging, it needs to be confirmed in following-up in vivo studies across a range of diets and inclusion rates.

      Blood Chemistry

      In the current study, serum BHB concentration tended to be decreased (P = 0.06) by BTC compared with CON, and it was decreased by the low and medium BTC doses but not affected at the high BTC dose (P = 0.05, a quadratic effect). There was no effect of treatment on blood, total FA and glucose concentrations (Table 7). A treatment × parity interaction (P = 0.02) was detected for serum insulin concentration. Insulin was linearly decreased (P = 0.01) in BTC primiparous cows, but not in multiparous cows. Decreased insulin concentration in primiparous cows receiving BTC corroborates with
      • Oh J.
      • Harper M.
      • Giallongo F.
      • Bravo D.M.
      • Wall E.H.
      • Hristov A.N.
      Effects of rumen-protected capsicum oleoresin on productivity and responses to a glucose tolerance test in lactating dairy cows.
      and may be explained by lower glucose uptake by insulin-dependent tissues such as skeletal muscle and adipose tissue, as a result of insulin resistance (
      • De Koster J.D.
      • Opsomer G.
      Insulin resistance in dairy cows.
      ). Contrary, another explanation for decreased insulin levels in the current experiment is that insulin sensitivity of insulin-dependent tissues might be increased by BTC; however, insulin sensitivity was not determined in the current study. If the later hypothesis is true, it could also explain the lower concentration of BHB in BTC cows (i.e., reduced lipolysis and increased lipogenesis). Circulating concentrations of total FA and BHB can be used as energy balance markers, with total FA being related to fat mobilization and BHB related to liver oxidation and ketone bodies formation (
      • Duffield T.F.
      • Lissemore K.D.
      • McBride B.W.
      • Leslie K.E.
      Impact of hyperketonemia in early lactation dairy cows on health and production.
      ;
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Evaluation of nonesterified fatty acids and β-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases.
      ). These data are in line with a previous study conducted in our laboratory (
      • Oh J.
      • Harper M.T.
      • Melgar A.
      • Räisänen S.
      • Chen X.
      • Nedelkov K.
      • Fetter M.
      • Ott T.
      • Wall E.H.
      • Hristov A.N.
      Dietary supplementation with rumen-protected capsicum during the transition period improves the metabolic status of dairy cows.
      ), which reported improved energetic status of transition cows supplemented with CAP during the prepartum period (i.e., reduced BHB concentration). Despite the physiological difference in lactation stage, mid-lactation versus transition period in the study by
      • Oh J.
      • Harper M.T.
      • Melgar A.
      • Räisänen S.
      • Chen X.
      • Nedelkov K.
      • Fetter M.
      • Ott T.
      • Wall E.H.
      • Hristov A.N.
      Dietary supplementation with rumen-protected capsicum during the transition period improves the metabolic status of dairy cows.
      , decreased BHB concentration in both studies indicates that BTC supplementation (specifically CAP) has a potential to enhance the metabolic status of dairy cows mediated by changes in insulin and, perhaps, the somatotropic axis. The mechanisms behind the changes in metabolic markers remain unclear; however, they may be linked to the capsaicin intestinal receptor TRPV1 stimulating the release of neuropeptides such as calcitonin gene-related peptide, which is known to directly influence the insulin axis in other species (
      • Oh J.
      • Wall E.H.
      • Bravo D.M.
      • Hristov A.N.
      Host-mediated effects of phytonutrients in ruminants: A review.
      ).
      Table 7Blood chemistry of lactating dairy cows fed botanicals combination (BTC)
      ItemCONTreatment
      Treatments were as follows: CON = control, 0 mg/cow per day of BTC; 150 = 150 mg/cow per day of BTC; 300 = 300 mg/cow per day of BTC; 600 = 600 mg/cow per day of BTC.
      SEM
      Largest SEM published in table; n = 74 for insulin, n = 76 for alkaline phosphatase, n = 77 for all other variables (n represents the number of observations used in the statistical analysis).
      P-value
      Contrasts: C vs. T = control vs. BTC treatments; L = linear effect of BTC; Q = quadratic effect of BTC. There was no effect of parity or a parity × treatment interaction for any variable (P ≥ 0.17), except for insulin (P = 0.02).
      150300600C vs. TLQ
      BHB, μM99588485892851.50.060.480.05
      Total FA, μM1301201301600.0170.740.160.38
      Insulin (overall), pmol/L17819518617019.70.770.570.42
       Primiparous23221918214226.70.090.010.99
       Multiparous14117919318426.30.060.160.17
      Glucose, mg/dL59.260.258.960.62.170.740.670.82
      BUN, mg/dL11.211.411.511.20.470.620.940.39
      Creatinine, mg/dL0.620.630.630.650.0300.650.490.91
      Total protein, g/dL7.777.697.607.750.2400.640.940.48
      Albumin, g/dL2.722.732.712.760.0460.740.530.72
      Globulin, g/dL5.044.944.874.980.2190.520.790.45
      Alanine transferase, U/L63.365.764.363.92.350.480.980.49
      Alkaline phosphatase, U/L46.646.345.145.33.600.640.580.78
      1 Treatments were as follows: CON = control, 0 mg/cow per day of BTC; 150 = 150 mg/cow per day of BTC; 300 = 300 mg/cow per day of BTC; 600 = 600 mg/cow per day of BTC.
      2 Largest SEM published in table; n = 74 for insulin, n = 76 for alkaline phosphatase, n = 77 for all other variables (n represents the number of observations used in the statistical analysis).
      3 Contrasts: C vs. T = control vs. BTC treatments; L = linear effect of BTC; Q = quadratic effect of BTC. There was no effect of parity or a parity × treatment interaction for any variable (P ≥ 0.17), except for insulin (P = 0.02).
      It is interesting to note that, in the current study, responses to BTC supplementation appeared to be both gut-mediated responses (changes in blood metabolites) and rumen-mediated responses (changes in enteric CH4 emissions). This might be possible due to the relatively high level of ruminal escape of the active compounds in BTC allowing the supplement to induce both ruminal and postruminal effects. Future research in our laboratory will further explore these responses and evaluate their consistency.

      Blood Cell Count

      Blood cell count, hemoglobin concentration, and hematocrit percentage were not affected by BTC supplementation (Table 8), and the count of total white and red blood cells were within normal levels for cattle (
      • Roland L.
      • Drillich M.
      • Iwersen M.
      Hematology as a diagnostic tool in bovine medicine.
      ). Although some studies reported decreased lymphocytes percentage (
      • Oh J.
      • Giallongo F.
      • Frederick T.
      • Pate J.
      • Walusimbi S.
      • Elias R.J.
      • Wall E.H.
      • Bravo D.
      • Hristov A.N.
      Effects of dietary capsicum oleoresin on productivity and immune responses in lactating dairy cows.
      ) and decreased blood monocytes count (
      • Oh J.
      • Harper M.
      • Lang C.H.
      • Wall E.H.
      • Hristov A.N.
      Effects of phytonutrients alone or in combination with monensin on productivity in lactating dairy cows.
      ) by EO supplementation, the lack of effect of BTC on blood cells count is in line with a previous study that supplemented the diet of lactating dairy cows with a rumen-protected CAP (
      • Oh J.
      • Harper M.
      • Giallongo F.
      • Bravo D.M.
      • Wall E.H.
      • Hristov A.N.
      Effects of rumen-protected capsicum oleoresin on immune responses in dairy cows intravenously challenged with lipopolysaccharide.
      ). Similarly, rumen-protected CAP supplementation did not affect blood cell count in beef cattle (
      • Westphalen M.F.
      • Carvalho P.H.V.
      • Oh J.
      • Hristov A.N.
      • Staniar W.B.
      • Felix T.L.
      Effects of feeding rumen-protected capsicum oleoresin on growth performance, health status, and total tract digestibility of growing beef cattle.
      ). Capsicum and bioactive compounds extracted from clove EO have been described as potential molecules to modulate the immune system by leading to anti-inflammatory, antioxidant, or immune enhancement properties mediated by the connection with ion channels (i.e., TRPV1, TRPV3, and TRPA1;
      • Vennekens R.
      • Vriens J.
      • Nilius B.
      Herbal compounds and toxins modulating TRP channels.
      ;
      • Oh J.
      • Harper M.
      • Giallongo F.
      • Bravo D.M.
      • Wall E.H.
      • Hristov A.N.
      Effects of rumen-protected capsicum oleoresin on immune responses in dairy cows intravenously challenged with lipopolysaccharide.
      ); however, effects of BTC on immune cells were not observed in the current study.
      Table 8Blood cell counts in lactating dairy cows fed botanicals combination (BTC)
      ItemCONTreatment
      Treatments were as follows: CON = control, 0 mg/cow per day of BTC; 150 = 150 mg/cow per day of BTC; 300 = 300 mg/cow per day of BTC; 600 = 600 mg/cow per day of BTC.
      SEM
      Largest SEM published in table; n = 72 for white blood cells, n = 73 for lymphocytes counts, n = 76 for all other variables (n represents the number of observations used in the statistical analysis).
      P-value
      Contrasts: C vs. T = control vs. BTC treatments; L = linear effect of BTC; Q = quadratic effect of BTC. There was no effect of parity or a parity × treatment interaction for any variable (P ≥ 0.12).
      150300600C vs. TLQ
      White blood cells, 103/μL7.878.047.817.870.4390.910.860.96
       Neutrophils3.203.213.483.170.3000.740.990.42
       Lymphocytes3.764.113.783.860.4850.330.940.54
       Monocytes1.111.301.121.100.1270.490.560.40
       Eosinophils0.330.320.280.300.0390.270.300.31
      As % of total
       Neutrophils39.036.339.439.42.310.770.610.69
       Lymphocytes43.745.543.743.82.120.740.840.75
       Monocytes13.214.213.513.10.850.670.620.47
       Eosinophils4.024.003.483.690.4580.320.260.40
       Neutrophil:lymphocyte0.990.860.990.970.0980.590.810.69
       Red blood cells, 106/μL5.835.765.945.760.1700.910.680.20
       Hemoglobin, g/dL9.409.349.579.310.1630.950.610.11
       Hematocrit, %27.427.227.927.10.4830.960.570.13
      1 Treatments were as follows: CON = control, 0 mg/cow per day of BTC; 150 = 150 mg/cow per day of BTC; 300 = 300 mg/cow per day of BTC; 600 = 600 mg/cow per day of BTC.
      2 Largest SEM published in table; n = 72 for white blood cells, n = 73 for lymphocytes counts, n = 76 for all other variables (n represents the number of observations used in the statistical analysis).
      3 Contrasts: C vs. T = control vs. BTC treatments; L = linear effect of BTC; Q = quadratic effect of BTC. There was no effect of parity or a parity × treatment interaction for any variable (P ≥ 0.12).

      CONCLUSIONS

      Contrary to our hypothesis, a combination of Capsicum oleoresin and clove EO did not affect lactational performance of mid-lactation dairy cows, despite some indications of improved metabolic status illustrated by tendencies for decreased serum BHB and insulin (primiparous cows only). Although nutrient utilization was not affected by BTC supplementation, enteric CH4 production was linearly decreased by up to 7.5% with increasing doses of BTC. The moderate decrease in enteric CH4 yield suggests a potential of BTC to reduce the carbon footprint of milk, but this effect needs to be confirmed.

      ACKNOWLEDGMENTS

      This work was supported by the USDA (Washington, DC) National Institute of Food and Agriculture Federal Appropriations under Project PEN 04539 and Accession Number 1000803. The authors thank AVT Natural North America (Santa Clara, CA) for providing partial financial support and the BTC product for this project. The authors also thank the staff of The Pennsylvania State University Dairy Teaching and Research Center (University Park) for their conscientious care and management of the experimental cows and for technical assistance during the study. The authors have not stated any conflicts of interest.

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