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Effect of camelina oil or live yeasts (Saccharomyces cerevisiae) on ruminal methane production, rumen fermentation, and milk fatty acid composition in lactating cows fed grass silage diets

Open ArchivePublished:February 25, 2015DOI:https://doi.org/10.3168/jds.2014-7976

      Abstract

      The potential of dietary supplements of 2 live yeast strains (Saccharomyces cerevisiae) or camelina oil to lower ruminal methane (CH4) and carbon dioxide (CO2) production and the associated effects on animal performance, rumen fermentation, rumen microbial populations, nutrient metabolism, and milk fatty acid (FA) composition of cows fed grass silage-based diets were examined. Four Finnish Ayrshire cows (53 ± 7 d in milk) fitted with rumen cannula were used in a 4 × 4 Latin square with four 42-d periods. Cows received a basal total mixed ration (control treatment) with a 50:50 forage-to-concentrate ratio [on a dry matter (DM) basis] containing grass silage, the same basal total mixed ration supplemented with 1 of 2 live yeasts, A or B, administered directly in the rumen at 1010 cfu/d (treatments A and B), or supplements of 60 g of camelina oil/kg of diet DM that replaced concentrate ingredients in the basal total mixed ration (treatment CO). Relative to the control, treatments A and B had no effects on DM intake, rumen fermentation, ruminal gas production, or apparent total-tract nutrient digestibility. In contrast, treatment CO lowered DM intake and ruminal CH4 and CO2 production, responses associated with numerical nonsignificant decreases in total-tract organic matter digestibility, but no alterations in rumen fermentation characteristics or changes in the total numbers of rumen bacteria, methanogens, protozoa, and fungi. Compared with the control, treatment CO decreased the yields of milk, milk fat, lactose, and protein. Relative to treatment B, treatment CO improved nitrogen utilization due to a lower crude protein intake. Treatment A had no influence on milk FA composition, whereas treatment B increased cis-9 10:1 and decreased 11-cyclohexyl 11:0 and 24:0 concentrations. Treatment CO decreased milk fat 8:0 to 16:0 and total saturated FA, and increased 18:0, 18:1, 18:2, conjugated linoleic acid, 18:3n-3, and trans FA concentrations. Decreases in ruminal CH4 production to treatment CO were related, at least in part to lowered DM intake, whereas treatments had no effect on ruminal CH4 emission intensity (g/kg of digestible organic matter intake or milk yield). Results indicated that live yeasts A and B had no influence on animal performance, ruminal gas production, rumen fermentation, or nutrient utilization in cows fed grass silage-based diets. Dietary supplements of camelina oil decreased ruminal CH4 and CO2 production, but also lowered the yields of milk and milk constituents due to an adverse effect on intake.

      Key words

      Introduction

      Ruminal methane (CH4) production occurs due to the metabolic activity of methanogenic archaea capable of utilizing hydrogen (H2) and carbon dioxide (CO2) as substrates (
      • Martin C.
      • Morgavi D.P.
      • Doreau M.
      Methane mitigation in ruminants: From microbe to the farm scale.
      ). Formation of CH4 contributes to the efficiency of ruminal OM degradation by preventing the accumulation of H2, which in high concentrations may inhibit microbial enzyme activity and ruminal fermentation (
      • Morgavi D.P.
      • Forano E.
      • Martin C.
      • Newbold C.J.
      Microbial ecosystem and methanogenesis in ruminants.
      ). Nutritional strategies to mitigate ruminal CH4 to lower greenhouse gas emissions from ruminant livestock production have sought to identify and investigate various feed additives and dietary ingredients to promote alternative pathways to CH4 formation for the dissipation of metabolic H2 in the rumen. Dietary supplements of the medium-chain SFA, 12:0 and 14:0 (
      • Jordan E.
      • Lovett D.K.
      • Monahan F.J.
      • Callan J.
      • Flynn B.
      • O’Mara F.P.
      Effect of refined coconut oil or copra meal on methane output and on intake and performance of beef heifers.
      ;
      • Machmüller A.
      Medium-chain fatty acids and their potential to reduce methanogenesis in domestic ruminants.
      ;
      • Hristov A.N.
      • Vander Pol M.
      • Agle M.
      • Zaman S.
      • Schneider C.
      • Ndegwa P.
      • Vaddella V.K.
      • Johnson K.
      • Shingfield K.J.
      • Karnati S.K.R.
      Effect of lauric acid and coconut oil on ruminal fermentation, digestion, ammonia losses from manure, and milk fatty acid composition in lactating cows.
      ), plant oils, or oilseeds (
      • McGinn S.M.
      • Beauchemin K.A.
      • Coates T.
      • Colombatto D.
      Methane emissions from beef cattle: effects of monensin, sunflower oil, enzymes, yeast, and fumaric acid.
      ;
      • Beauchemin K.A.
      • McGinn S.M.
      Methane emissions from beef cattle: Effects of fumaric acid, essential oil, and canola oil.
      ;
      • Martin C.
      • Rouel J.
      • Jouany J.P.
      • Doreau M.
      • Chilliard Y.
      Methane output and diet digestibility in response to feeding dairy cows crude linseed, extruded linseed, or linseed oil.
      ) are known to lower ruminal CH4 production in lactating or growing cattle. However, the decreases in ruminal CH4 formation to relatively high amounts of lipid (≥50 g/kg of DM) from plant oils or oilseeds in the diet are often accompanied by decreases in DMI, nutrient digestion, and animal performance (
      • Beauchemin K.A.
      • McGinn S.M.
      Methane emissions from beef cattle: Effects of fumaric acid, essential oil, and canola oil.
      ;
      • Jordan E.
      • Lovett D.K.
      • Monahan F.J.
      • Callan J.
      • Flynn B.
      • O’Mara F.P.
      Effect of refined coconut oil or copra meal on methane output and on intake and performance of beef heifers.
      ;
      • Martin C.
      • Rouel J.
      • Jouany J.P.
      • Doreau M.
      • Chilliard Y.
      Methane output and diet digestibility in response to feeding dairy cows crude linseed, extruded linseed, or linseed oil.
      ).
      Camelina (Camelina sativa L.), an ancient summer annual oilseed crop, is tolerant to drought and can adapt to different climatic and soil conditions (
      • Hurtaud C.
      • Peyraud J.L.
      Effects of feeding Camelina (seeds or meal) on milk fatty acid composition and butter spreadability.
      ). Cultivation of camelina has received renewed attention due to the relatively high 18:3n-3 content of camelina oil (
      • Hurtaud C.
      • Peyraud J.L.
      Effects of feeding Camelina (seeds or meal) on milk fatty acid composition and butter spreadability.
      ). In lactating cows, dietary supplements of camelina seeds or camelina oil increased cis-9 18:1, cis-9,trans-11 CLA, and 18:3 n-3 and lowered medium-chain SFA concentrations in milk (
      • Hurtaud C.
      • Peyraud J.L.
      Effects of feeding Camelina (seeds or meal) on milk fatty acid composition and butter spreadability.
      ;
      • Halmemies-Beauchet-Filleau A.
      • Kokkonen T.
      • Lampi A.M.
      • Toivonen V.
      • Shingfield K.J.
      • Vanhatalo A.
      Effect of plant oils and camelina expeller on milk fatty acid composition in lactating cows fed diets based on red clover silage.
      ), changes considered beneficial to human health (
      • Shingfield K.J.
      • Bonnet M.
      • Scollan N.D.
      Recent developments in altering the fatty acid composition of ruminant-derived foods.
      ). However, the effects of camelina oil on ruminal fermentation, CH4 and CO2 production, and microbial communities have not been determined.
      Recent evaluations have concluded that dietary lipid supplements resulted in the most consistent decrease in CH4 output when compared with alterations in forage-to-concentrate ratio, direct-fed microbials, or exogenous enzymes (
      • Martin C.
      • Morgavi D.P.
      • Doreau M.
      Methane mitigation in ruminants: From microbe to the farm scale.
      ;
      • Grainger C.
      • Beauchemin K.A.
      Can enteric methane emissions from ruminants be lowered without lowering their production?.
      ). Furthermore, simply replacing forages with concentrate ingredients also ignores the important role of ruminants in utilizing fibrous feeds not suitable for human consumption (
      • Grainger C.
      • Beauchemin K.A.
      Can enteric methane emissions from ruminants be lowered without lowering their production?.
      ), and that when fed in excess, concentrates increase the risk of ruminal acidosis (
      • Fonty G.
      • Chaucheyras-Durand F.
      Effects and modes of action of live yeasts in the rumen.
      ).
      Live yeast products are used as feed additives for ruminants to improve feed efficiency and prevent ruminal acidosis through the scavenging of oxygen within the rumen, provision of microbial growth factors, or direct competition with autochthonous species in the rumen (
      • Newbold C.J.
      • Wallace R.J.
      • Mcintosh F.M.
      Mode of action of the yeast Saccharomyces cerevisiae as a feed additive for ruminants.
      ;
      • Fonty G.
      • Chaucheyras-Durand F.
      Effects and modes of action of live yeasts in the rumen.
      ). Selection of Saccharomyces cerevisiae strains for specifically lowering ruminal CH4 production has been postulated (
      • McGinn S.M.
      • Beauchemin K.A.
      • Coates T.
      • Colombatto D.
      Methane emissions from beef cattle: effects of monensin, sunflower oil, enzymes, yeast, and fumaric acid.
      ;
      • Chaucheyras-Durand F.
      • Walker N.D.
      • Bach A.
      Effects of active dry yeasts on the rumen microbial ecosystem: past, present and future.
      ;
      • Chung Y.-H.
      • Walker N.D.
      • McGinn S.M.
      • Beauchemin K.A.
      Differing effects of 2 active dried yeast (Saccharomyces cerevisiae) strains on ruminal acidosis and methane production in nonlactating dairy cows.
      ), whereas other strains may indirectly decrease CH4 production per unit of milk or meat through improvements in ruminal fiber degradation and overall feed conversion efficiency.
      The present experiment examined the potential of 2 strains of live yeasts or camelina oil enriched in PUFA to lower ruminal CH4 and CO2 production, as well as the associated effects on rumen fermentation, rumen microbial populations, nutrient digestibility, energy and nitrogen (N) metabolism, milk production, and milk FA composition of lactating cows fed grass silage-based diets. Live yeast strains were selected on the basis of in vitro studies demonstrating positive effects on fiber degradation during incubations with mixed rumen populations or Fibrobacter succinogenes.

      Materials and Methods

      Animals, Experimental Design, and Diets

      All experimental procedures were approved by the National Ethics Committee (Hämeenlinna, Finland) in accordance with the guidelines established by the European Community Council Directive 86/609/EEC (
      European Union
      Council Directive 86/609/EEC on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes.
      ). Four multiparous Finnish Ayrshire dairy cows fitted with rumen cannulas (#1C, i.d. 100 mm, Bar Diamond Inc., Parma, ID) of (mean ± SD) similar parity (3.8 ± 0.63), DIM (53 ± 7), and BW (711 ± 23 kg) producing 33.8 ± 3.4 kg of milk/d were allocated at random to experimental diets according to a 4 × 4 Latin square with four 42-d periods comprising 23 d of adaptation, 5 d of sample collection, and 14 d of washout.
      Cows were housed in individual tiestalls in a dedicated metabolism unit, had free access to water and salt block, and were milked in situ twice daily at 0700 and 1645 h. Treatments comprised a basal TMR (designated as the control treatment; forage-to-concentrate ratio 50:50 on a DM basis) or a TMR containing camelina oil (CO) based on grass silage with an in vitro OM digestibility of 686 g/kg of DM. Experimental silage was prepared from primary growths of tall fescue (Festuca arundinacea) grown at Jokioinen (60°49′N, 23°28′E), cut with a mower conditioner, and wilted for 5 h. Cut grass was ensiled with a mixture of Pediococcus acidilactici strain MA18/5M, Lactobacillus buchneri strain NCIMB 40788, cellulase, and hemicellulase applied at 5.0 L/tonne of fresh matter (Lalsil Dry, Lallemand Animal Nutrition).
      Cows on treatments A and B received the basal TMR (Table 1) supplemented with 1 of 2 live yeast (Saccharomyces cerevisiae) strains, A or B, supplied as 0.5 g/d of a highly concentrated dried product (Lallemand Animal Nutrition) at 1010 cfu/d. Live yeasts were selected based on in vitro studies demonstrating positive effects on fiber degradation by mixed rumen populations (strain A), or the ability to promote the growth and degradation of fiber by Fibrobacter succinogenes [strain B, a rumen bacterium that does not produce H2; F. Chaucheyras-Durand, A. Ameilbonne (Lallemand Animal Nutrition, Blagnac, France), and E. Forano, unpublished data]. Viable yeast counts were checked before the start of the experiment and both dried products conformed to expectations. Yeast supplements were stored in the dark at 4°C, away from humidity, throughout the experiment. Previous experiments in vitro have demonstrated that each yeast strain survived in the rumen for at least 12 h [F. Chaucheyras-Durand, A. Ameilbonne (Lallemand Animal Nutrition), and E. Forano, unpublished data]. Live yeast supplements were placed directly into the rumen via the cannula at 0600 h, and mixed with rumen solid contents to ensure that all viable cells for treatments A and B were administered. For treatment CO, 60 g of camelina oil/kg of diet DM (Raisio Feed Ltd., Raisio, Finland) replaced concentrate ingredients, which resulted in a higher gross energy (GE) and FA and lower CP and carbohydrate concentration compared with the basal TMR (Table 1). The FA composition of grass silage, concentrates, and camelina oil is presented in Table 2. Diets were offered 4 times daily at 0600, 1000, 1630, and 1900 h as a TMR to avoid selection of dietary components and to maintain the desired forage-to-concentrate ratio. Experimental diets were offered ad libitum to result in 10% refusals and formulated to meet or exceed ME and protein requirements of lactating cows producing 30 kg of milk/d (

      MTT Agrifood Research Finland. 2006. Finnish feed tables and feeding recommendations: 2006. Accessed Nov. 13, 2010. http://www.mtt.fi/mtts/pdf/mtts106.pdf

      ).
      Table 1Formulation and chemical composition of the experimental diets
      ItemTreatment
      Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5g/d of 1 of 2 strains of probiotic live yeast strains A or B or 60g/kg DM of camelina oil (CO).
      ControlABCO
      Ingredient (g/kg of DM)
       Grass silage
      Mean fermentation characteristics of experimental silage: pH 4.55; in DM lactic acid, 37.8g/kg; acetic acid, 84.1g/kg; propionic acid, 16.1g/kg; butyric acid, 0.6g/kg; soluble N, 693g/kg of total N, ammonia-N, 127g/kg of total N; GE, 19.0MJ/kg of DM.
      500500500500
       Barley, rolled225225225197
       Sugar beet pulp, molasses120120120105
       Rapeseed meal, solvent extracted140140140123
       Camelina oil
      Gross energy content 40.2MJ/kg of DM.
      60
       Vitamin and mineral premix
      Vitamin and mineral supplement (Onni-Kivennäinen, Melica Finland Ltd., Vaasa, Finland) declared as containing calcium (205g/kg), magnesium (72g/kg), sodium (85g/kg), phosphorus (27g/kg), zinc (1.46g/kg), manganese (0.35g/kg), copper (0.27g/kg), iodine (39mg/kg), cobalt (27mg/kg), selenium (20mg/kg), retinyl acetate (120 IU/g), cholecalciferol (25 IU/g), and dl-α tocopheryl acetate (0.34 IU/g).
      15.015.015.015.0
      Chemical composition (g/kg of DM, unless otherwise stated)
       DM (g/kg as fed)327327327328
       OM913913913917
       CP163163163152
       NDF401401401387
       Potentially digestible NDF
      Calculated as NDF – indigestible NDF.
      325325325313
       ADF228228228221
       Water-soluble carbohydrate32.732.732.729.0
       Starch120120120106
       FA21.921.921.977.3
      Gross energy (MJ/kg of DM)18.718.718.720.0
      1 Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5 g/d of 1 of 2 strains of probiotic live yeast strains A or B or 60 g/kg DM of camelina oil (CO).
      2 Mean fermentation characteristics of experimental silage: pH 4.55; in DM lactic acid, 37.8 g/kg; acetic acid, 84.1 g/kg; propionic acid, 16.1 g/kg; butyric acid, 0.6 g/kg; soluble N, 693 g/kg of total N, ammonia-N, 127 g/kg of total N; GE, 19.0 MJ/kg of DM.
      3 Gross energy content 40.2 MJ/kg of DM.
      4 Vitamin and mineral supplement (Onni-Kivennäinen, Melica Finland Ltd., Vaasa, Finland) declared as containing calcium (205 g/kg), magnesium (72 g/kg), sodium (85 g/kg), phosphorus (27 g/kg), zinc (1.46 g/kg), manganese (0.35 g/kg), copper (0.27 g/kg), iodine (39 mg/kg), cobalt (27 mg/kg), selenium (20 mg/kg), retinyl acetate (120 IU/g), cholecalciferol (25 IU/g), and dl-α tocopheryl acetate (0.34 IU/g).
      5 Calculated as NDF – indigestible NDF.
      Table 2Fatty acid composition and content of grass silage, concentrate supplements, and camelina oil
      FA

      (g/100 g of FA, unless

      otherwise noted)
      Ingredient
      SilageConcentrateCamelina

      oil
      12:00.300.030.01
      14:00.640.280.06
      16:023.314.95.62
      trans-3 16:12.18
      cis-9 16:10.180.620.08
      18:01.621.402.39
      cis-9 18:14.2326.911.6
      cis-11 18:10.505.460.71
      18:2n-618.039.415.7
      18:3n-335.66.5437.0
      20:00.700.361.51
      cis-11 20:10.330.7615.1
      20:2n-60.000.092.15
      22:01.100.320.32
      cis-13 22:10.210.185.25
      22:2n-60.000.000.16
      24:00.970.350.17
      26:01.140.07
      28:00.910.01
      30:00.610.01
      Unidentified4.490.19
      Other
      Includes the sum of 15:0 anteiso, 15:0, 16:1 (n=5 isomers), 16:2 (n=2), 17:0, 17:1 (double bond position and geometry indeterminant), 18:1 (n=9), 18:0 iso, 19:0, 18:2 (n=6), 18:3 (n=2), 20:1 (n=4), 21:0, 18:4n-3, cis-15 22:1, 20:4n-6, 23:0, cis-14 23:1, cis-15 24:1, 25:0, cis-17 26:1, 29:0, and 10-oxo-18:0.
      2.922.162.21
      Σ SFA32.818.310.2
      Σ MUFA6.9835.134.6
      Σ PUFA55.746.555.2
      Total FA (g/kg of DM)13.830.0954
      1 Includes the sum of 15:0 anteiso, 15:0, 16:1 (n = 5 isomers), 16:2 (n = 2), 17:0, 17:1 (double bond position and geometry indeterminant), 18:1 (n = 9), 18:0 iso, 19:0, 18:2 (n = 6), 18:3 (n = 2), 20:1 (n = 4), 21:0, 18:4n-3, cis-15 22:1, 20:4n-6, 23:0, cis-14 23:1, cis-15 24:1, 25:0, cis-17 26:1, 29:0, and 10-oxo-18:0.

      Measurements and Chemical Analysis

      Daily feed intake was determined as the amount of TMR offered minus refusals and milk yield were recorded throughout the experiment. Measurements of intake between d 24 and 28 were used for statistical analysis. During this period, representative samples of silage and concentrates were collected daily, composited by period, and submitted for chemical composition determinations (
      • Shingfield K.J.
      • Jaakkola S.
      • Huhtanen P.
      Effect of forage conservation method, concentrate level and propylene glycol on diet digestibility, rumen fermentation, blood metabolite concentrations and nutrient utilization of dairy cows.
      ). The chemical composition of TMR was calculated based on the chemical composition of silage, concentrates, and camelina oil, assuming no selection of dietary ingredients as indicated by negligible differences in the DM content of experimental TMR offered and feed refusals determined for each cow in all experimental periods. Concentrations of GE in samples of silage, concentrates, camelina oil, feces, and urine were determined by bomb calorimetry (1108 Oxygen bomb, Parr Instrument, Moline, IL). Indigestible NDF concentration of silage, concentrates, and feces was determined in duplicate by incubation of 1.0 g of sample DM in nylon bags (60 × 120 mm, pore size 0.017 mm) within the rumen of 2 cows fed a grass silage-based diet (forage-to-concentrate ratio = 70:30 on a DM basis) for 12 d. Once removed from the rumen, bags were rinsed in cold water for 25 min using a household washing machine, incubated for 1 h in boiling neutral detergent solution, rinsed, and dried to a constant weight at 60°C. Potentially digestible NDF (pdNDF) concentration was calculated as NDF – indigestible NDF. Samples of rumen fluid (n = 8) were collected on d 28 of each period from each cow at 1.5-h intervals from 0600 to 1630 h. Following the removal of rumen fluid, pH was measured and samples were filtered through 2 layers of cheesecloth and stored at −20°C until analyzed for VFA and ammonia-N (
      • Shingfield K.J.
      • Jaakkola S.
      • Huhtanen P.
      Effect of forage conservation method, concentrate level and propylene glycol on diet digestibility, rumen fermentation, blood metabolite concentrations and nutrient utilization of dairy cows.
      ).
      For microbial assessments, 100-g samples of digesta were collected from each region of the rumen (anterior dorsal, anterior ventral, posterior dorsal, and posterior ventral) immediately after the collection of rumen fluid and just before morning feeding on d 28 of each experimental period. Samples of ruminal digesta were combined and mixed thoroughly. Subsamples (50 g) of composite digesta were mixed with 100 mL of RNAlater solution (Fisher Scientific, Illkirch, France), split into smaller aliquots, and stored at −80°C until quantitative PCR (qPCR) analysis.
      Ruminal gas production was estimated using the sulfur hexafluoride (SF6) tracer gas technique (
      • Boadi D.A.
      • Wittenberg K.M.
      • Kennedy A.D.
      Validation of the sulphur hexafluoride (SF6) tracer gas technique for measurement of methane and carbon dioxide production by cattle.
      ) with modifications. Measurements of CO2 and CH4 were made over a 4-d interval starting at 0600 h on d 24 of each experimental period. Thin wafer permeation tubes containing SF6 (i.d. 16.4 × 46.5 mm, Fine Permeation Tubes, Spadafora, Italy) with a declared rate of release of 1.0 mg/d were placed into the rumen of each cow on d 16 of the experiment. Actual SF6 release rates (1.12 ± 0.17 mg/d) for each tube \ determined by weight difference over the course of the experiment, from d 16 through 168, were used in the calculations. Gases in the rumen headspace were withdrawn continuously (1.7 mL/min) over 4 consecutive 24-h periods into evacuated 5.5-L air-tight canisters via a 100-mm length of capillary tubing (PEEK 1.6 × 0.13 mm i.d., VICI Valcro Instruments Co, Houston, TX). Gas collection tubes were fitted with a water lock (50 mL) and a filter (0.2-µm pore size) to prevent rumen liquor entering the collection tube and gas canisters. The tubes were anchored securely to the neck of the rumen cannula allowing gas to be collected at approximately 5 cm above the rumen mat. The end of each tube was covered with nylon (17-µm pore size) that prevented the entrance of rumen particulates. Each canister was replaced after 24 h, pressurized with N2 gas to 110 kPa, and left for 2 h to ensure a thorough mixing of N2 with the collected gases. Gases were released slowly from the canister, subsampled in triplicate, transferred into evacuated 10-mL glass tubes equipped with a rubber stopper, and analyzed for CH4, CO2, and SF6 concentrations.
      Apparent total-tract digestibility coefficients were determined by total fecal collection over a 96-h interval starting at 1800 h on d 24 of each experimental period. Feces excreted was weighed, thoroughly mixed, subsampled (5% wt/wt), and stored at –20°C before chemical analysis. Urine was separated from feces by means of a light harness and flexible tubing attached to the vulva and collected in plastic canisters containing 500 mL of 5 mol/L sulfuric acid. Collection vessels were changed at 12-h intervals.
      Samples of milk were collected over 4 consecutive milking starting at 1645 h on d 24. Milk samples treated with preservative (Bronopol, Valio Ltd., Helsinki, Finland) and analyzed for milk fat, CP, and lactose by infrared analysis (MilkoScan FT6000, Foss Electric, Hillerød, Denmark). Milk composition was calculated based on weighted average milk yield. Unpreserved milk samples were also collected, composited according to yield, and stored at −20°C until analyzed for FA composition.

      Gas Analysis

      Samples of gases collected from the rumen were analyzed in triplicate for CH4, CO2, and SF6 concentrations using a gas chromatograph (Agilent 6890N, Agilent Technologies, Santa Clara, CA) equipped with flame ionization (FID) and electron-capture detectors, autosampler, and a nickel catalyst for converting CO2 to CH4 (
      • Regina K.
      • Alakukku L.
      Greenhouse gas fluxes in varying soils types under conventional and no-tillage practices.
      ). Concentration of gases was determined based on calibration curves constructed using authentic standards (AGA Ltd., Espoo, Finland) over a range of 2.5 to 25% for CO2 and CH4 and 0.00625 to 0.125 µL/L for SF6. Peaks were identified by retention time comparisons with authentic standards. Daily CH4 and CO2 production was calculated based on known amount of SF6 released in the rumen. No correction was made for background CH4 and CO2 concentrations because cows were housed in a well-ventilated facility (72 m3/min) and fitted with custom-made sponges placed between the outer edge of the cannula flange and the abdominal wall to minimize the exchange of surrounding air with ruminal contents.

      Enumeration of Rumen Microbial Populations by qPCR

      Microbial DNA was extracted from 250 mg of centrifuged rumen contents in quadruplicate with the Nucleospin for soil kit (Macherey-Nagel, Hoerdt, France). Samples were homogenized by bead beating in the presence of 700 µL of lysis buffer SL1 and 15 µL of Enhancer SX buffer using the Fast Prep-24 instrument (MP Biomedicals, Illkirch, France). All steps were performed according to the instructions provided by the manufacturer. The DNA extracts were assessed for purity and quantity using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Illkirch, France), and stored at −20°C until molecular analysis.
      Rumen microbes were quantified by qPCR targeting of the 16S rRNA gene for bacteria and methanogenic archaea, the 18S rRNA gene for protozoa, and the ITS1 region for fungi (Table 3). The qPCR was performed using SYBR-green chemistry on a MasterCycler ep Realplex thermal cycler (Eppendorf, Le Pecq, France). Primer sets and PCR conditions used were the same as reported in the literature for methanogenic archaea (
      • Ohene-Adjei S.
      • Teather R.M.
      • Ivan M.
      • Forster R.J.
      Postinoculation protozoan establishment and association patterns of methanogenic archaea in the ovine rumen.
      ), protozoa (
      • Sylvester J.T.
      • Karnati S.K.R.
      • Yu Z.
      • Newbold C.J.
      • Firkins J.L.
      Evaluation of a real-time PCR assay quantifying the ruminal pool size and duodenal flow of protozoal nitrogen.
      ), Ruminococcus flavefaciens, Fibrobacter succinogenes, and fungi (
      • Denman S.E.
      • McSweeney C.S.
      Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen.
      ). Total bacteria were quantified using 520F and 799R2 primers (
      • Edwards J.E.
      • Huws S.A.
      • Kim E.J.
      • Kingston-Smith A.H.
      Characterization of the dynamics of initial bacterial colonization of nonconserved forage in the bovine rumen.
      ). The qPCR was carried out in a total volume of 20 μL containing 2× Takara SYBR Premix Ex Taq kit (Lonza, Levallois-Perret, France), 40 ng of DNA template, and 0.5 μM of each forward and reverse primer. Each reaction was run in triplicate in 96-well plates (BioRad, Marnes-la-Coquette, France). Standards were used to determine the absolute abundance of microbial groups, expressed as the log number of DNA copies per microgram of DNA. For total bacteria, cellulolytic bacteria, and methanogenic archaea, the standard curves were prepared according to
      • Mosoni P.
      • Martin C.
      • Forano E.
      • Morgavi D.P.
      Long-term defaunation increases the abundance of cellulolytic ruminococci and methanogens but does not affect the bacterial and methanogen diversity in the rumen of sheep.
      ). For protozoa, the standard curve was developed using pSC-A-amp/kan plasmids (Strataclone PCR cloning kit, Agilent Technologies) containing the near full 18S rRNA gene from Polyplastron multivesiculatum, Eudiplodinium maggii, and Ostracodinium dentatum mixed in equal amounts. For fungi, the ITS1 fragment from Piromyces spp. was amplified using qPCR primers (
      • Lwin K.O.
      • Hayakawa M.
      • Ban-Tokuda T.
      • Matsui H.
      Real-time PCR assays for monitoring anaerobic fungal biomass and population size in the rumen.
      ) and cloned using the pCR2.1 Topo TA Cloning kit (Invitrogen, Life Technologies, Saint Aubin, France). The number of gene copies present in each plasmid was calculated using the plasmid DNA concentration and the molecular mass of the vector and the insert. For each target, a standard curve was prepared from 102 to 109 copies by serial dilution. Efficiency of the qPCR for each target varied between 97 and 102% with a slope from −3.0 to −3.4 and a regression coefficient above 0.95 in accordance with the MIQE guidelines (
      • Bustin S.A.
      • Benes V.
      • Garson J.A.
      • Hellemans J.
      • Huggett J.
      • Kubista M.
      • Mueller R.
      • Nolan T.
      • Pfaffl M.W.
      • Shipley G.L.
      • Vandesompele J.
      • Wittwer C.T.
      The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments.
      ).
      Table 3Oligonucleotide primers used for real-time quantitative PCR
      Microbial groupPrimer setFragment

      length

      (bp)
      Reference
      Total bacteria5′-AGCAGCCGCGGTAAT-3′

      5′-CAGGGTATCTAATCCTGTT-3′
      280
      • Edwards J.E.
      • Huws S.A.
      • Kim E.J.
      • Kingston-Smith A.H.
      Characterization of the dynamics of initial bacterial colonization of nonconserved forage in the bovine rumen.
      Fibrobacter succinogenes5′-GTTCGGAATTACTGGGCGTAAA-3′

      5′-CGCCTGCCCCTGAACTATC-3′
      121
      • Denman S.E.
      • McSweeney C.S.
      Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen.
      Ruminococcus flavefaciens5′-CGAACGGAGATAATTTGAGTTTACTTAGG-3′

      5′-CGGTCTCTGTATGTTATGAGGTATTACC-3′
      132
      • Denman S.E.
      • McSweeney C.S.
      Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen.
      Methanogenic archaea5′-GAGGAAGGAGTGGACGACGGTA-3′

      5′-ACGGGCGGTGTGTGCAAG-3′
      233
      • Ohene-Adjei S.
      • Teather R.M.
      • Ivan M.
      • Forster R.J.
      Postinoculation protozoan establishment and association patterns of methanogenic archaea in the ovine rumen.
      Fungi5′-GAGGAAGTAAAAGTCGTAACAAGGTTTC-3′

      5′-CAAATTCACAAAGGGTAGGATGATT-3′
      120
      • Denman S.E.
      • McSweeney C.S.
      Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen.
      Protozoa5′-GCTTTCGWTGGTAGTGTATT-3′

      5′-CTTGCCCTCYAATCGTWCT-3′
      223
      • Sylvester J.T.
      • Karnati S.K.R.
      • Yu Z.
      • Newbold C.J.
      • Firkins J.L.
      Evaluation of a real-time PCR assay quantifying the ruminal pool size and duodenal flow of protozoal nitrogen.

      Lipid Analysis

      Fatty acid methyl esters in freeze-dried feed samples were prepared in a one-step extraction-transesterification procedure using chloroform and 2% (vol/vol) sulfuric acid in methanol (
      • Halmemies-Beauchet-Filleau A.
      • Kokkonen T.
      • Lampi A.M.
      • Toivonen V.
      • Shingfield K.J.
      • Vanhatalo A.
      Effect of plant oils and camelina expeller on milk fatty acid composition in lactating cows fed diets based on red clover silage.
      ). Lipid in a 1-mL milk sample was extracted in triplicate using a mixture of ammonia, ethanol, diethylether, and hexane (0.2:1.0:2.5:2.5 by vol). Extracts were combined and evaporated to dryness at 40°C under oxygen-free N2. Samples were dissolved in hexane and methyl acetate and transesterified to FAME using freshly prepared methanolic sodium methoxide (
      • Halmemies-Beauchet-Filleau A.
      • Kokkonen T.
      • Lampi A.M.
      • Toivonen V.
      • Shingfield K.J.
      • Vanhatalo A.
      Effect of plant oils and camelina expeller on milk fatty acid composition in lactating cows fed diets based on red clover silage.
      ).
      The FAME prepared from samples of feeds and milk fat were quantified using a gas chromatograph (model 6890N, Agilent Technologies) equipped with an FID, automatic injector, split injection port, and a 100-m fused silica capillary column (i.d. = 0.25 mm) coated with a 0.2-μm film of cyanopropyl polysiloxane (CP-Sil 88, Agilent Technologies;
      • Halmemies-Beauchet-Filleau A.
      • Kokkonen T.
      • Lampi A.M.
      • Toivonen V.
      • Shingfield K.J.
      • Vanhatalo A.
      Effect of plant oils and camelina expeller on milk fatty acid composition in lactating cows fed diets based on red clover silage.
      ). For methyl esters not available as commercial standards, peaks were identified based on GC-MS analysis of 4,4-dimethyloxoline derivatives prepared from FAME (
      • Halmemies-Beauchet-Filleau A.
      • Kokkonen T.
      • Lampi A.M.
      • Toivonen V.
      • Shingfield K.J.
      • Vanhatalo A.
      Effect of plant oils and camelina expeller on milk fatty acid composition in lactating cows fed diets based on red clover silage.
      ). The distribution of CLA isomers in milk fat FAME was determined by Ag+ HPLC (Model 1090, Agilent Technologies;
      • Halmemies-Beauchet-Filleau A.
      • Kokkonen T.
      • Lampi A.M.
      • Toivonen V.
      • Shingfield K.J.
      • Vanhatalo A.
      Effect of plant oils and camelina expeller on milk fatty acid composition in lactating cows fed diets based on red clover silage.
      ).
      Milk FA composition was expressed as a weight percentage of total FA using theoretical relative response factors to account for the carbon deficiency in the FID response for FAME containing 4- to 10-carbon atoms (
      • Halmemies-Beauchet-Filleau A.
      • Kokkonen T.
      • Lampi A.M.
      • Toivonen V.
      • Shingfield K.J.
      • Vanhatalo A.
      Effect of plant oils and camelina expeller on milk fatty acid composition in lactating cows fed diets based on red clover silage.
      ). Concentrations of CLA isomers were calculated based on proportionate peak area responses determined by HPLC and the sum of trans-7,cis-9 CLA, trans-8,cis-10 CLA, and cis-9,trans-11 CLA weight percentage determined by GC analysis.

      Calculations

      Digestible and metabolizable energy (MJ/d) were calculated as
      Digestibleenergy=energyintakefecalenergy,andME=digestibleenergyurinaryenergymethaneenergy.


      RetainedN(g/d)wascalculatedasRetainedN=NintakefecalNurinaryNmilkN.


      Daily ECM yield (kg/d) was calculated according to
      • Sjaunja L.O.
      • Baevre L.
      • Junkkarinen L.
      • Pedersen J.
      • Setälä J.
      ):
      ECM=milk(kg/d)×[38.3×fat(g/kg)+24.2×protein(g/kg)+16.54×lactose(g/kg)+20.7]/3,140.


      Statistical Analysis

      Experimental data were analyzed by ANOVA for a 4 × 4 Latin square using the Mixed procedure of SAS (version 9.2, SAS institute, Cary, NC) with a model that included the fixed effects of period and treatment and random effect of cow. Measurements of rumen fermentation characteristics were analyzed by ANOVA for repeated measures using a model that included the fixed effects of period, treatment, time, and their interaction and random effect of cow assuming an autoregressive order-one covariance structure fitted on the basis of Akaike information and Schwarz Bayesian model-fit criteria. Least squares means ± SEM are reported. Treatment effects were declared significant at P ≤ 0.05, and a trend was assumed for probabilities <0.1 and >0.05. When the overall effect of treatment was significant, differences among means were further evaluated using the Fisher’s least significant difference test.

      Results

      Nutrient Intake

      Treatment CO lowered (P = 0.05) DMI and tended (P = 0.06) to lower OM intake compared with the control and treatment B (Table 4). Relative to the control and treatments A and B, treatment CO decreased (P < 0.05) the intake of CP, NDF, pdNDF, ADF, water-soluble carbohydrate, and starch, but increased (P < 0.05) the intake of all FA other than 12:0, 26:0, 28:0, and 30:0 (Table 4).
      Table 4Effect of dietary supplements of 2 live yeast strains or camelina oil on nutrient intake of lactating cows
      Intake

      (kg/d, unless otherwise stated)
      Treatment
      Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5g/d of 1 of 2 strains of live yeasts A or B or 60g/kg DM of camelina oil (CO).
      SEMP-value
      ControlABCO
      DM19.0
      Within a row, means without a common superscript differ (P<0.05).
      18.4ab19.2
      Within a row, means without a common superscript differ (P<0.05).
      16.7
      Within a row, means without a common superscript differ (P<0.05).
      0.910.05
      OM17.216.717.415.20.830.06
      CP3.09
      Within a row, means without a common superscript differ (P<0.05).
      3.01
      Within a row, means without a common superscript differ (P<0.05).
      3.13
      Within a row, means without a common superscript differ (P<0.05).
      2.53
      Within a row, means without a common superscript differ (P<0.05).
      0.1430.004
      NDF7.61
      Within a row, means without a common superscript differ (P<0.05).
      7.39
      Within a row, means without a common superscript differ (P<0.05).
      7.68
      Within a row, means without a common superscript differ (P<0.05).
      6.45
      Within a row, means without a common superscript differ (P<0.05).
      0.3590.02
      Potentially digestible NDF
      Calculated as NDF – indigestible NDF.
      6.16
      Within a row, means without a common superscript differ (P<0.05).
      5.99
      Within a row, means without a common superscript differ (P<0.05).
      6.22
      Within a row, means without a common superscript differ (P<0.05).
      5.23
      Within a row, means without a common superscript differ (P<0.05).
      0.2910.02
      ADF4.32
      Within a row, means without a common superscript differ (P<0.05).
      4.20
      Within a row, means without a common superscript differ (P<0.05).
      4.37
      Within a row, means without a common superscript differ (P<0.05).
      3.68
      Within a row, means without a common superscript differ (P<0.05).
      0.2050.02
      Water-soluble carbohydrate0.62
      Within a row, means without a common superscript differ (P<0.05).
      0.60
      Within a row, means without a common superscript differ (P<0.05).
      0.63
      Within a row, means without a common superscript differ (P<0.05).
      0.49
      Within a row, means without a common superscript differ (P<0.05).
      0.0030.002
      Starch2.28
      Within a row, means without a common superscript differ (P<0.05).
      2.22
      Within a row, means without a common superscript differ (P<0.05).
      2.30
      Within a row, means without a common superscript differ (P<0.05).
      1.77
      Within a row, means without a common superscript differ (P<0.05).
      0.1050.002
      Gross energy (MJ/d)35534736033017.60.35
      FA (g/d)
       12:00.500.490.500.480.0240.52
       14:01.61
      Within a row, means without a common superscript differ (P<0.05).
      1.57
      Within a row, means without a common superscript differ (P<0.05).
      1.63
      Within a row, means without a common superscript differ (P<0.05).
      1.88
      Within a row, means without a common superscript differ (P<0.05).
      0.0920.04
       16:071.7
      Within a row, means without a common superscript differ (P<0.05).
      69.8
      Within a row, means without a common superscript differ (P<0.05).
      72.6
      Within a row, means without a common superscript differ (P<0.05).
      109
      Within a row, means without a common superscript differ (P<0.05).
      4.90<0.001
      trans-3 16:13.05
      Within a row, means without a common superscript differ (P<0.05).
      2.97ab3.09
      Within a row, means without a common superscript differ (P<0.05).
      2.68
      Within a row, means without a common superscript differ (P<0.05).
      0.1470.05
      cis-9 16:11.87
      Within a row, means without a common superscript differ (P<0.05).
      1.82
      Within a row, means without a common superscript differ (P<0.05).
      1.89
      Within a row, means without a common superscript differ (P<0.05).
      2.19
      Within a row, means without a common superscript differ (P<0.05).
      0.1070.04
       18:05.94
      Within a row, means without a common superscript differ (P<0.05).
      5.791
      Within a row, means without a common superscript differ (P<0.05).
      6.02
      Within a row, means without a common superscript differ (P<0.05).
      26.0
      Within a row, means without a common superscript differ (P<0.05).
      1.03<0.001
      cis-9 18:176.5
      Within a row, means without a common superscript differ (P<0.05).
      74.5
      Within a row, means without a common superscript differ (P<0.05).
      77.6
      Within a row, means without a common superscript differ (P<0.05).
      163
      Within a row, means without a common superscript differ (P<0.05).
      6.82<0.001
      cis-11 18:115.1
      Within a row, means without a common superscript differ (P<0.05).
      14.7
      Within a row, means without a common superscript differ (P<0.05).
      15.3
      Within a row, means without a common superscript differ (P<0.05).
      18.0
      Within a row, means without a common superscript differ (P<0.05).
      0.880.02
       18:2n-6129
      Within a row, means without a common superscript differ (P<0.05).
      126
      Within a row, means without a common superscript differ (P<0.05).
      131
      Within a row, means without a common superscript differ (P<0.05).
      242
      Within a row, means without a common superscript differ (P<0.05).
      10.3<0.001
       18:3n-367.1
      Within a row, means without a common superscript differ (P<0.05).
      65.3
      Within a row, means without a common superscript differ (P<0.05).
      67.9
      Within a row, means without a common superscript differ (P<0.05).
      385
      Within a row, means without a common superscript differ (P<0.05).
      14.9<0.001
       20:01.93
      Within a row, means without a common superscript differ (P<0.05).
      1.88
      Within a row, means without a common superscript differ (P<0.05).
      1.95
      Within a row, means without a common superscript differ (P<0.05).
      15.0
      Within a row, means without a common superscript differ (P<0.05).
      0.58<0.001
      cis-11 20:12.48
      Within a row, means without a common superscript differ (P<0.05).
      2.41
      Within a row, means without a common superscript differ (P<0.05).
      2.51
      Within a row, means without a common superscript differ (P<0.05).
      135
      Within a row, means without a common superscript differ (P<0.05).
      5.14<0.001
       20:2n-60.25
      Within a row, means without a common superscript differ (P<0.05).
      0.24
      Within a row, means without a common superscript differ (P<0.05).
      0.25
      Within a row, means without a common superscript differ (P<0.05).
      19.2
      Within a row, means without a common superscript differ (P<0.05).
      0.73<0.001
       22:02.39
      Within a row, means without a common superscript differ (P<0.05).
      2.32
      Within a row, means without a common superscript differ (P<0.05).
      2.42
      Within a row, means without a common superscript differ (P<0.05).
      4.82
      Within a row, means without a common superscript differ (P<0.05).
      0.202<0.001
      cis-13 22:10.77
      Within a row, means without a common superscript differ (P<0.05).
      0.74
      Within a row, means without a common superscript differ (P<0.05).
      0.77
      Within a row, means without a common superscript differ (P<0.05).
      47.2
      Within a row, means without a common superscript differ (P<0.05).
      1.81<0.001
       22:2n-60.00
      Within a row, means without a common superscript differ (P<0.05).
      0.00
      Within a row, means without a common superscript differ (P<0.05).
      0.00
      Within a row, means without a common superscript differ (P<0.05).
      1.43
      Within a row, means without a common superscript differ (P<0.05).
      0.053<0.001
       24:02.26
      Within a row, means without a common superscript differ (P<0.05).
      2.21
      Within a row, means without a common superscript differ (P<0.05).
      2.30
      Within a row, means without a common superscript differ (P<0.05).
      3.42
      Within a row, means without a common superscript differ (P<0.05).
      0.152<0.001
       26:01.781.731.791.610.0880.14
       28:01.311.271.321.140.0650.06
       30:00.880.860.890.770.0440.06
       Other16.6
      Within a row, means without a common superscript differ (P<0.05).
      16.1
      Within a row, means without a common superscript differ (P<0.05).
      16.7
      Within a row, means without a common superscript differ (P<0.05).
      33.6
      Within a row, means without a common superscript differ (P<0.05).
      1.38<0.001
       Σ SFA93.9
      Within a row, means without a common superscript differ (P<0.05).
      91.5
      Within a row, means without a common superscript differ (P<0.05).
      95.1
      Within a row, means without a common superscript differ (P<0.05).
      168
      Within a row, means without a common superscript differ (P<0.05).
      7.29<0.001
       Σ MUFA102
      Within a row, means without a common superscript differ (P<0.05).
      99.3
      Within a row, means without a common superscript differ (P<0.05).
      103
      Within a row, means without a common superscript differ (P<0.05).
      387
      Within a row, means without a common superscript differ (P<0.05).
      15.2<0.001
       Σ PUFA200
      Within a row, means without a common superscript differ (P<0.05).
      195
      Within a row, means without a common superscript differ (P<0.05).
      203
      Within a row, means without a common superscript differ (P<0.05).
      653
      Within a row, means without a common superscript differ (P<0.05).
      26.0<0.001
       Total403
      Within a row, means without a common superscript differ (P<0.05).
      392
      Within a row, means without a common superscript differ (P<0.05).
      408
      Within a row, means without a common superscript differ (P<0.05).
      1,213
      Within a row, means without a common superscript differ (P<0.05).
      48.7<0.001
      a,b Within a row, means without a common superscript differ (P < 0.05).
      1 Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5 g/d of 1 of 2 strains of live yeasts A or B or 60 g/kg DM of camelina oil (CO).
      2 Calculated as NDF – indigestible NDF.

      Rumen Fermentation and Microbial Populations

      Relative to the control and treatment A, treatment CO tended (P = 0.06) to decrease rumen pH, but treatments had no effect (P > 0.05) on ruminal ammonia-N and total VFA concentrations (Table 5). Treatments had no influence (P > 0.05) on the molar proportions of major VFA or on the molar acetate-to-propionate ratio. However, treatment CO lowered (P < 0.001) the molar proportion of isobutyrate and tended (P = 0.06) to decrease molar proportions of valerate and caproate, and treatment B decreased (P < 0.001) the molar proportion of isobutyrate compared with the control (Table 5). Abundance of F. succinogenes, R. flavefaciens, total bacteria, methanogenic archaea, protozoa, and fungi were not affected (P > 0.05) by experimental treatments (Table 5).
      Table 5Effect of dietary supplements of 2 live yeast strains or camelina oil on rumen fermentation characteristics and rumen microbial populations of lactating cows
      ItemTreatment
      Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5g/d of 1 of 2 strains of live yeasts A or B or 60g/kg DM of camelina oil (CO).
      SEMP-value
      ControlABCO
      pH6.656.656.526.400.0650.06
      Ammonia-N (mmol/L)6.436.426.285.530.6210.31
      Total VFA (mmol/L)98.697.21031003.600.67
      Molar proportion (mmol/mol)
       Acetate6726706686754.20.67
       Propionate1891921921984.00.22
       Butyrate86.988.689.480.73.450.21
       Isobutyrate10.0
      Within a row, means without a common superscript differ (P<0.05).
      9.70
      Within a row, means without a common superscript differ (P<0.05).
      9.16
      Within a row, means without a common superscript differ (P<0.05).
      7.73
      Within a row, means without a common superscript differ (P<0.05).
      0.225<0.001
       Valerate15.915.015.714.20.410.06
       Isovalerate17.017.116.614.50.910.17
       Caproate9.598.949.257.920.3920.06
      Molar ratio
      Acetate:propionate3.583.523.473.410.0810.45
      Microbial numbers (log gene copies/µg of DNA)
       Total bacteria10.0810.1310.1610.130.0320.44
       Methanogens7.277.357.307.300.0340.26
       Protozoa7.637.597.607.420.0830.23
       Fungi6.106.075.986.020.0560.43
      Fibrobacter succinogenes7.647.587.777.610.0710.31
      Ruminococcus flavefaciens6.506.346.476.750.1000.12
      a–c Within a row, means without a common superscript differ (P < 0.05).
      1 Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5 g/d of 1 of 2 strains of live yeasts A or B or 60 g/kg DM of camelina oil (CO).

      Nutrient Utilization

      Apparent whole-tract nutrient digestibility, other than that of starch and GE, were unaffected (P > 0.05) by treatments (Table 6). Digestibility of starch (P = 0.05) and GE tended (P = 0.08) to be lower for treatment CO compared with the control and treatment B. Relative to the control, treatment CO decreased (P < 0.05) N intake, urinary N excretion, and milk N secretion, and tended (P = 0.07) to lower fecal N output. Compared with treatment B, treatment CO increased (P < 0.05) the efficiency of N utilization. However, treatments had no effect (P > 0.05) on milk energy secretion or energy excreted in feces, whereas treatment CO tended (P = 0.06) to lower energy losses as CH4 and in urine.
      Table 6Effect of dietary supplements of 2 live yeast strains or camelina oil on apparent total-tract digestibility of nutrients and energy and nitrogen metabolism of lactating cows
      ItemTreatment
      Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5g/d of 1 of 2 strains of live yeasts A or B 60g/kg of DM of camelina oil (CO).
      SEMP-value
      ControlABCO
      Apparent digestibility (g/kg, unless otherwise stated)
       OM7247167246938.50.13
       CP71171071268810.50.31
       NDF58557058954513.00.18
       Potentially digestible NDF
      Calculated as NDF – indigestible NDF.
      69168069365511.40.17
       ADF59558260055713.90.22
       Starch982
      Within a row, means without a common superscript differ (P<0.05).
      981
      Within a row, means without a common superscript differ (P<0.05).
      983
      Within a row, means without a common superscript differ (P<0.05).
      977
      Within a row, means without a common superscript differ (P<0.05).
      1.10.05
       Gross energy (KJ/MJ)7097007096758.30.08
      Energy (MJ/d)
       Intake35534635933317.70.36
       Feces1041041051075.20.70
       Urine12.811.612.210.90.770.06
       Methane21.219.320.215.11.380.06
       Milk83.379.978.874.77.460.19
       Digestible energy
      Digestible energy=energy intake – fecal energy.
      25224225422513.50.20
       ME
      Metabolizable energy=digestible energy – urinary energy – methane energy.
      21821122219912.80.43
      Nitrogen (g/d)
       Intake494
      Within a row, means without a common superscript differ (P<0.05).
      481
      Within a row, means without a common superscript differ (P<0.05).
      500
      Within a row, means without a common superscript differ (P<0.05).
      405
      Within a row, means without a common superscript differ (P<0.05).
      22.90.006
       Feces1431391441265.70.07
       Urine184
      Within a row, means without a common superscript differ (P<0.05).
      179
      Within a row, means without a common superscript differ (P<0.05).
      181
      Within a row, means without a common superscript differ (P<0.05).
      153
      Within a row, means without a common superscript differ (P<0.05).
      11.50.01
       Milk138
      Within a row, means without a common superscript differ (P<0.05).
      132
      Within a row, means without a common superscript differ (P<0.05).
      129
      Within a row, means without a common superscript differ (P<0.05).
      120
      Within a row, means without a common superscript differ (P<0.05).
      10.00.04
       Retained N
      Retained N=N intake – fecal N – urinary N – milk N.
      32.133.549.09.5710.620.14
       Milk N/intake N0.274
      Within a row, means without a common superscript differ (P<0.05).
      0.270
      Within a row, means without a common superscript differ (P<0.05).
      0.254
      Within a row, means without a common superscript differ (P<0.05).
      0.290
      Within a row, means without a common superscript differ (P<0.05).
      0.02030.04
      a,b Within a row, means without a common superscript differ (P < 0.05).
      1 Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5 g/d of 1 of 2 strains of live yeasts A or B 60 g/kg of DM of camelina oil (CO).
      2 Calculated as NDF – indigestible NDF.
      3 Digestible energy = energy intake – fecal energy.
      4 Metabolizable energy = digestible energy – urinary energy – methane energy.
      5 Retained N = N intake – fecal N – urinary N – milk N.

      Ruminal CH4 and CO2 Production

      Treatments A and B had no effect (P > 0.05) on ruminal CH4 production, whereas treatment CO decreased (P < 0.05) ruminal CH4 output (Table 7). However, treatments had no effect (P > 0.05) on ruminal CH4 production intensity (g/kg of OM or NDF digested in total digestive tract or g/kg of milk) or the proportion of GE lost as CH4. Compared with the control, treatment CO decreased (P < 0.05) ruminal CO2 production. Ruminal CO2 output per kilogram of milk tended (P = 0.09) to be lower for treatment CO compared with the control and treatment B.
      Table 7Effect of dietary supplements of 2 live yeast strains or camelina oil on ruminal gas production of lactating cows
      ItemTreatment
      Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5g/d of 1 of 2 strains of live yeasts A or B or 60g/kg DM of camelina oil (CO).
      SEMP-value
      ControlABCO
      Ruminal methane
       g/d407
      Within a row, means without a common superscript differ (P<0.05).
      365
      Within a row, means without a common superscript differ (P<0.05).
      372
      Within a row, means without a common superscript differ (P<0.05).
      287
      Within a row, means without a common superscript differ (P<0.05).
      22.30.03
       g/kg of DMI21.419.719.417.61.270.30
       g/kg of OMD
      OM apparently digested in total digestive tract.
      32.730.429.627.92.120.51
       g/kg of NDFD
      NDF apparently digested in total digestive tract.
      57.853.352.549.73.600.51
       g/kg of milk15.613.815.013.11.330.27
       % of GEI
      Gross energy intake.
      6.315.825.734.860.3680.14
      Ruminal carbon dioxide
       g/d3,493
      Within a row, means without a common superscript differ (P<0.05).
      3,006
      Within a row, means without a common superscript differ (P<0.05).
      3,172
      Within a row, means without a common superscript differ (P<0.05).
      2,295
      Within a row, means without a common superscript differ (P<0.05).
      226.40.02
       g/kg of DMI18416316613810.50.11
       g/kg of OMD28125125222016.80.18
       g/kg of NDFD49744044739228.50.18
       g/kg of milk13311412710410.40.09
      a,b Within a row, means without a common superscript differ (P < 0.05).
      1 Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5 g/d of 1 of 2 strains of live yeasts A or B or 60 g/kg DM of camelina oil (CO).
      2 OM apparently digested in total digestive tract.
      3 NDF apparently digested in total digestive tract.
      4 Gross energy intake.

      Milk Production and Composition

      Compared with treatment A and the control, treatment CO decreased (P < 0.05) the yields of milk and milk protein (Table 8). Furthermore, treatment CO decreased (P < 0.05) yields of ECM and milk lactose compared with the control and treatments A and B, and lowered (P < 0.05) milk fat yield relative to the control and treatment B. Treatments had no effect (P > 0.05) on milk fat and lactose concentrations, whereas milk protein content tended (P = 0.08) to be lower for treatment A and CO.
      Table 8Effect of dietary supplements of 2 live yeast strains or camelina oil on milk yield and milk composition of lactating cows
      ItemTreatment
      Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5g/d of 1 of 2 strains of live yeasts A or B or 60g/kg DM of camelina oil (CO).
      SEMP-value
      ControlABCO
      Yield
       Milk (kg/d)27.0
      Within a row, means without a common superscript differ (P<0.05).
      26.5
      Within a row, means without a common superscript differ (P<0.05).
      25.6
      Within a row, means without a common superscript differ (P<0.05).
      22.4
      Within a row, means without a common superscript differ (P<0.05).
      2.300.05
       ECM
      Energy-corrected milk=milk (kg/d) × [38.3 × fat (g/kg) + 24.2 × protein (g/kg) + 16.54 × lactose (g/kg) + 20.7]/3,140 (Sjaunja et al. 1990).
      (kg/d)
      26.4
      Within a row, means without a common superscript differ (P<0.05).
      25.5
      Within a row, means without a common superscript differ (P<0.05).
      25.7
      Within a row, means without a common superscript differ (P<0.05).
      22.1
      Within a row, means without a common superscript differ (P<0.05).
      2.180.04
       Fat (g/d)1,070
      Within a row, means without a common superscript differ (P<0.05).
      1,020
      Within a row, means without a common superscript differ (P<0.05).
      1,062
      Within a row, means without a common superscript differ (P<0.05).
      920
      Within a row, means without a common superscript differ (P<0.05).
      96.40.05
       Protein (g/d)859
      Within a row, means without a common superscript differ (P<0.05).
      827
      Within a row, means without a common superscript differ (P<0.05).
      824
      Within a row, means without a common superscript differ (P<0.05).
      696
      Within a row, means without a common superscript differ (P<0.05).
      57.50.01
       Lactose (g/d)1,240
      Within a row, means without a common superscript differ (P<0.05).
      1,228
      Within a row, means without a common superscript differ (P<0.05).
      1,183
      Within a row, means without a common superscript differ (P<0.05).
      1,017
      Within a row, means without a common superscript differ (P<0.05).
      115.70.03
      Composition (g/kg)
       Fat39.938.341.640.81.310.24
       Protein32.431.232.431.31.080.08
       Lactose45.846.346.244.61.110.45
      a,b Within a row, means without a common superscript differ (P < 0.05).
      1 Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5 g/d of 1 of 2 strains of live yeasts A or B or 60 g/kg DM of camelina oil (CO).
      2 Energy-corrected milk = milk (kg/d) × [38.3 × fat (g/kg) + 24.2 × protein (g/kg) + 16.54 × lactose (g/kg) + 20.7]/3,140 (
      • Sjaunja L.O.
      • Baevre L.
      • Junkkarinen L.
      • Pedersen J.
      • Setälä J.
      ).

      Milk FA Composition

      Administration of live yeasts into the rumen had little influence on milk FA composition (Table 9). In contrast, treatment CO resulted in substantial changes in milk FA composition that were characterized by decreases (P < 0.05) in 6:0, 8:0, 10:0, 12:0, 14:0, 16:0, and total SFA and increases (P < 0.05) in 18:0, total 18:1, 18:2, CLA, 18:3n-3, total 20- and 22-carbon, MUFA, PUFA, and trans FA concentrations (Table 9).
      Table 9Effect of dietary supplements of 2 live yeast strains or camelina oil on milk FA composition of lactating cows
      FA

      (g/100 g of FA)
      Treatment
      Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5g/d of 1 of 2 strains of live yeasts A or B or 60g/kg DM of camelina oil (CO).
      SEMP-value
      ControlABCO
      4:03.103.183.113.180.2250.98
      6:01.90
      Within a row, means without a common superscript differ (P<0.05).
      1.93
      Within a row, means without a common superscript differ (P<0.05).
      1.88
      Within a row, means without a common superscript differ (P<0.05).
      1.56
      Within a row, means without a common superscript differ (P<0.05).
      0.1160.03
      8:01.12
      Within a row, means without a common superscript differ (P<0.05).
      1.11
      Within a row, means without a common superscript differ (P<0.05).
      1.11
      Within a row, means without a common superscript differ (P<0.05).
      0.79
      Within a row, means without a common superscript differ (P<0.05).
      0.0650.001
      10:02.66
      Within a row, means without a common superscript differ (P<0.05).
      2.62
      Within a row, means without a common superscript differ (P<0.05).
      2.60
      Within a row, means without a common superscript differ (P<0.05).
      1.55
      Within a row, means without a common superscript differ (P<0.05).
      0.132<0.001
      cis-9 10:10.296
      Within a row, means without a common superscript differ (P<0.05).
      0.276
      Within a row, means without a common superscript differ (P<0.05).
      0.303
      Within a row, means without a common superscript differ (P<0.05).
      0.193
      Within a row, means without a common superscript differ (P<0.05).
      0.0131<0.001
      12:03.24
      Within a row, means without a common superscript differ (P<0.05).
      3.13
      Within a row, means without a common superscript differ (P<0.05).
      3.17
      Within a row, means without a common superscript differ (P<0.05).
      1.84
      Within a row, means without a common superscript differ (P<0.05).
      0.126<0.001
      cis-9 12:10.082
      Within a row, means without a common superscript differ (P<0.05).
      0.075
      Within a row, means without a common superscript differ (P<0.05).
      0.083
      Within a row, means without a common superscript differ (P<0.05).
      0.043
      Within a row, means without a common superscript differ (P<0.05).
      0.0041<0.001
      trans-9 12:10.081
      Within a row, means without a common superscript differ (P<0.05).
      0.073
      Within a row, means without a common superscript differ (P<0.05).
      0.082
      Within a row, means without a common superscript differ (P<0.05).
      0.046
      Within a row, means without a common superscript differ (P<0.05).
      0.0042<0.001
      14:012.1
      Within a row, means without a common superscript differ (P<0.05).
      11.8
      Within a row, means without a common superscript differ (P<0.05).
      11.9
      Within a row, means without a common superscript differ (P<0.05).
      8.10
      Within a row, means without a common superscript differ (P<0.05).
      0.32<0.001
      cis-9 14:11.12
      Within a row, means without a common superscript differ (P<0.05).
      1.02
      Within a row, means without a common superscript differ (P<0.05).
      1.15
      Within a row, means without a common superscript differ (P<0.05).
      0.77
      Within a row, means without a common superscript differ (P<0.05).
      0.073<0.001
      trans-9 14:10.013
      Within a row, means without a common superscript differ (P<0.05).
      0.011
      Within a row, means without a common superscript differ (P<0.05).
      0.013
      Within a row, means without a common superscript differ (P<0.05).
      0.009
      Within a row, means without a common superscript differ (P<0.05).
      0.00070.01
      Σ 152.36
      Within a row, means without a common superscript differ (P<0.05).
      2.31
      Within a row, means without a common superscript differ (P<0.05).
      2.22
      Within a row, means without a common superscript differ (P<0.05).
      1.55
      Within a row, means without a common superscript differ (P<0.05).
      0.1260.004
      16:034.4
      Within a row, means without a common superscript differ (P<0.05).
      35.8
      Within a row, means without a common superscript differ (P<0.05).
      34.6
      Within a row, means without a common superscript differ (P<0.05).
      21.3
      Within a row, means without a common superscript differ (P<0.05).
      1.32<0.001
      Σ cis 16:12.20
      Within a row, means without a common superscript differ (P<0.05).
      2.09
      Within a row, means without a common superscript differ (P<0.05).
      2.23
      Within a row, means without a common superscript differ (P<0.05).
      1.64
      Within a row, means without a common superscript differ (P<0.05).
      0.1530.02
      Σ trans 16:10.230
      Within a row, means without a common superscript differ (P<0.05).
      0.227
      Within a row, means without a common superscript differ (P<0.05).
      0.236
      Within a row, means without a common superscript differ (P<0.05).
      0.456
      Within a row, means without a common superscript differ (P<0.05).
      0.0148<0.001
      Σ 16:12.432.322.462.100.1520.14
      Σ 171.18
      Within a row, means without a common superscript differ (P<0.05).
      1.16
      Within a row, means without a common superscript differ (P<0.05).
      1.16
      Within a row, means without a common superscript differ (P<0.05).
      0.86
      Within a row, means without a common superscript differ (P<0.05).
      0.031<0.001
      18:08.78
      Within a row, means without a common superscript differ (P<0.05).
      9.41
      Within a row, means without a common superscript differ (P<0.05).
      8.33
      Within a row, means without a common superscript differ (P<0.05).
      12.9
      Within a row, means without a common superscript differ (P<0.05).
      0.7780.003
      10-oxo-18:00.465
      Within a row, means without a common superscript differ (P<0.05).
      0.457
      Within a row, means without a common superscript differ (P<0.05).
      0.456
      Within a row, means without a common superscript differ (P<0.05).
      0.247
      Within a row, means without a common superscript differ (P<0.05).
      0.0430<0.001
      13-oxo-18:00.0220.0230.0230.0170.00190.12
      Σ cis 18:117.5
      Within a row, means without a common superscript differ (P<0.05).
      16.4
      Within a row, means without a common superscript differ (P<0.05).
      18.4
      Within a row, means without a common superscript differ (P<0.05).
      25.3
      Within a row, means without a common superscript differ (P<0.05).
      1.540.02
      Σ trans 18:12.31
      Within a row, means without a common superscript differ (P<0.05).
      2.23
      Within a row, means without a common superscript differ (P<0.05).
      2.28
      Within a row, means without a common superscript differ (P<0.05).
      6.71
      Within a row, means without a common superscript differ (P<0.05).
      0.280<0.001
      Σ 18:119.8
      Within a row, means without a common superscript differ (P<0.05).
      18.6
      Within a row, means without a common superscript differ (P<0.05).
      20.6
      Within a row, means without a common superscript differ (P<0.05).
      32.0
      Within a row, means without a common superscript differ (P<0.05).
      1.340.001
      Σ 18:2
      Sum of nonmethylene interrupted 18:2 isomers.
      1.71
      Within a row, means without a common superscript differ (P<0.05).
      1.63
      Within a row, means without a common superscript differ (P<0.05).
      1.69
      Within a row, means without a common superscript differ (P<0.05).
      2.65
      Within a row, means without a common superscript differ (P<0.05).
      0.095<0.001
      Σ CLA0.38
      Within a row, means without a common superscript differ (P<0.05).
      0.36
      Within a row, means without a common superscript differ (P<0.05).
      0.39
      Within a row, means without a common superscript differ (P<0.05).
      0.95
      Within a row, means without a common superscript differ (P<0.05).
      0.047<0.001
      18:3n-30.454
      Within a row, means without a common superscript differ (P<0.05).
      0.437
      Within a row, means without a common superscript differ (P<0.05).
      0.437
      Within a row, means without a common superscript differ (P<0.05).
      0.489
      Within a row, means without a common superscript differ (P<0.05).
      0.02240.007
      18:3n-60.015
      Within a row, means without a common superscript differ (P<0.05).
      0.016
      Within a row, means without a common superscript differ (P<0.05).
      0.016
      Within a row, means without a common superscript differ (P<0.05).
      0.007
      Within a row, means without a common superscript differ (P<0.05).
      0.00120.005
      cis-9,trans-11,cis-15 18:30.036
      Within a row, means without a common superscript differ (P<0.05).
      0.031
      Within a row, means without a common superscript differ (P<0.05).
      0.032
      Within a row, means without a common superscript differ (P<0.05).
      0.056
      Within a row, means without a common superscript differ (P<0.05).
      0.00500.02
      20:00.178
      Within a row, means without a common superscript differ (P<0.05).
      0.169
      Within a row, means without a common superscript differ (P<0.05).
      0.146
      Within a row, means without a common superscript differ (P<0.05).
      1.69
      Within a row, means without a common superscript differ (P<0.05).
      0.056<0.001
      Σ cis 20:10.247
      Within a row, means without a common superscript differ (P<0.05).
      0.211
      Within a row, means without a common superscript differ (P<0.05).
      0.218
      Within a row, means without a common superscript differ (P<0.05).
      2.48
      Within a row, means without a common superscript differ (P<0.05).
      0.089<0.001
      Σ trans 20:10.040
      Within a row, means without a common superscript differ (P<0.05).
      0.031
      Within a row, means without a common superscript differ (P<0.05).
      0.031
      Within a row, means without a common superscript differ (P<0.05).
      0.585
      Within a row, means without a common superscript differ (P<0.05).
      0.0293<0.001
      Σ 20:10.287
      Within a row, means without a common superscript differ (P<0.05).
      0.242
      Within a row, means without a common superscript differ (P<0.05).
      0.250
      Within a row, means without a common superscript differ (P<0.05).
      3.07
      Within a row, means without a common superscript differ (P<0.05).
      0.112<0.001
      20:2n-60.024
      Within a row, means without a common superscript differ (P<0.05).
      0.023
      Within a row, means without a common superscript differ (P<0.05).
      0.022
      Within a row, means without a common superscript differ (P<0.05).
      0.063
      Within a row, means without a common superscript differ (P<0.05).
      0.0044<0.001
      20:3n-30.008
      Within a row, means without a common superscript differ (P<0.05).
      0.008
      Within a row, means without a common superscript differ (P<0.05).
      0.007
      Within a row, means without a common superscript differ (P<0.05).
      0.046
      Within a row, means without a common superscript differ (P<0.05).
      0.0033<0.001
      20:3n-60.047
      Within a row, means without a common superscript differ (P<0.05).
      0.049
      Within a row, means without a common superscript differ (P<0.05).
      0.047
      Within a row, means without a common superscript differ (P<0.05).
      0.033
      Within a row, means without a common superscript differ (P<0.05).
      0.00680.01
      20:4n-30.034
      Within a row, means without a common superscript differ (P<0.05).
      0.035
      Within a row, means without a common superscript differ (P<0.05).
      0.036
      Within a row, means without a common superscript differ (P<0.05).
      0.026
      Within a row, means without a common superscript differ (P<0.05).
      0.00340.03
      20:4n-60.066
      Within a row, means without a common superscript differ (P<0.05).
      0.065
      Within a row, means without a common superscript differ (P<0.05).
      0.059
      Within a row, means without a common superscript differ (P<0.05).
      0.046
      Within a row, means without a common superscript differ (P<0.05).
      0.00680.04
      20:5n-30.049
      Within a row, means without a common superscript differ (P<0.05).
      0.052
      Within a row, means without a common superscript differ (P<0.05).
      0.048
      Within a row, means without a common superscript differ (P<0.05).
      0.032
      Within a row, means without a common superscript differ (P<0.05).
      0.00380.01
      22:00.054
      Within a row, means without a common superscript differ (P<0.05).
      0.057
      Within a row, means without a common superscript differ (P<0.05).
      0.050
      Within a row, means without a common superscript differ (P<0.05).
      0.157
      Within a row, means without a common superscript differ (P<0.05).
      0.0078<0.001
      cis-9 22:10.011
      Within a row, means without a common superscript differ (P<0.05).
      0.011
      Within a row, means without a common superscript differ (P<0.05).
      0.011
      Within a row, means without a common superscript differ (P<0.05).
      0.054
      Within a row, means without a common superscript differ (P<0.05).
      0.0038<0.001
      cis-13 22:10.014
      Within a row, means without a common superscript differ (P<0.05).
      0.012
      Within a row, means without a common superscript differ (P<0.05).
      0.012
      Within a row, means without a common superscript differ (P<0.05).
      0.232
      Within a row, means without a common superscript differ (P<0.05).
      0.0139<0.001
      Σ 22:10.025
      Within a row, means without a common superscript differ (P<0.05).
      0.023
      Within a row, means without a common superscript differ (P<0.05).
      0.023
      Within a row, means without a common superscript differ (P<0.05).
      0.286
      Within a row, means without a common superscript differ (P<0.05).
      0.0155<0.001
      22:4n-60.0180.0180.0200.0130.00330.33
      22:5n-30.060
      Within a row, means without a common superscript differ (P<0.05).
      0.055
      Within a row, means without a common superscript differ (P<0.05).
      0.055
      Within a row, means without a common superscript differ (P<0.05).
      0.038
      Within a row, means without a common superscript differ (P<0.05).
      0.00640.001
      Σ 220.157
      Within a row, means without a common superscript differ (P<0.05).
      0.152
      Within a row, means without a common superscript differ (P<0.05).
      0.147
      Within a row, means without a common superscript differ (P<0.05).
      0.494
      Within a row, means without a common superscript differ (P<0.05).
      0.0248<0.001
      24:00.039
      Within a row, means without a common superscript differ (P<0.05).
      0.037
      Within a row, means without a common superscript differ (P<0.05).
      0.035
      Within a row, means without a common superscript differ (P<0.05).
      0.031
      Within a row, means without a common superscript differ (P<0.05).
      0.00380.002
      cis-15 24:10.010
      Within a row, means without a common superscript differ (P<0.05).
      0.010
      Within a row, means without a common superscript differ (P<0.05).
      0.010
      Within a row, means without a common superscript differ (P<0.05).
      0.028
      Within a row, means without a common superscript differ (P<0.05).
      0.0017<0.001
      26:00.030
      Within a row, means without a common superscript differ (P<0.05).
      0.029
      Within a row, means without a common superscript differ (P<0.05).
      0.029
      Within a row, means without a common superscript differ (P<0.05).
      0.013
      Within a row, means without a common superscript differ (P<0.05).
      0.0025<0.001
      Other
      Sum of 8-oxo-16:0, 10-oxo-16:0, 16:2n-4, 9-oxo-18:0, 15-oxo-18:0, cis-9,trans-11,trans-15 18:3, 18:4n-3, 22:2n-6, 22:3n-3, 22:6n-3, and 28:0.
      0.161
      Within a row, means without a common superscript differ (P<0.05).
      0.153
      Within a row, means without a common superscript differ (P<0.05).
      0.155
      Within a row, means without a common superscript differ (P<0.05).
      0.234
      Within a row, means without a common superscript differ (P<0.05).
      0.0088<0.001
      Summary
       Σ trans FA3.37
      Within a row, means without a common superscript differ (P<0.05).
      3.21
      Within a row, means without a common superscript differ (P<0.05).
      3.30
      Within a row, means without a common superscript differ (P<0.05).
      11.8
      Within a row, means without a common superscript differ (P<0.05).
      0.412<0.001
       Σ SFA72.1
      Within a row, means without a common superscript differ (P<0.05).
      73.7
      Within a row, means without a common superscript differ (P<0.05).
      71.3
      Within a row, means without a common superscript differ (P<0.05).
      56.0
      Within a row, means without a common superscript differ (P<0.05).
      1.50<0.001
       Σ MUFA24.7
      Within a row, means without a common superscript differ (P<0.05).
      23.3
      Within a row, means without a common superscript differ (P<0.05).
      25.6
      Within a row, means without a common superscript differ (P<0.05).
      39.1
      Within a row, means without a common superscript differ (P<0.05).
      1.47<0.001
       Σ PUFA2.89
      Within a row, means without a common superscript differ (P<0.05).
      2.78
      Within a row, means without a common superscript differ (P<0.05).
      2.86
      Within a row, means without a common superscript differ (P<0.05).
      4.46
      Within a row, means without a common superscript differ (P<0.05).
      0.164<0.001
      Total FA (g/100 g of fat)94.694.394.394.30.130.47
      a–c Within a row, means without a common superscript differ (P < 0.05).
      1 Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5 g/d of 1 of 2 strains of live yeasts A or B or 60 g/kg DM of camelina oil (CO).
      2 Sum of nonmethylene interrupted 18:2 isomers.
      3 Sum of 8-oxo-16:0, 10-oxo-16:0, 16:2n-4, 9-oxo-18:0, 15-oxo-18:0, cis-9,trans-11,trans-15 18:3, 18:4n-3, 22:2n-6, 22:3n-3, 22:6n-3, and 28:0.
      Relative to the control, treatment A lowered (P < 0.05) milk cis-10 16:1 concentration and treatment CO increased (P < 0.05) cis-10 16:1, cis-12 16:1 and trans-9 to -13 16:1 content and decreased (P < 0.05) cis-9 16:1 and cis-13 16:1 concentrations (Supplemental Table S1; http://dx.doi.org/10.3168/jds.2014-7976). Treatments A and B had no effect (P > 0.05) on the distribution of milk fat 18:1 isomers, whereas treatment CO increased (P < 0.05) cis-9, -12, -15, and -16 18:1 and trans (Δ4–16) 18:1 concentrations. Furthermore, treatment CO enriched (P < 0.05) milk fat cis (Δ9–15) and trans (Δ9–13) 20:1 concentrations.
      Treatments A and B had no influence (P > 0.05) on milk fat 18:2 or CLA isomer concentrations (Supplemental Table S2; http://dx.doi.org/10.3168/jds.2014-7976). In contrast, treatment CO increased (P < 0.05) the abundance of nonmethylene interrupted 18:2 isomers, including cis-12,cis-15 18:2, cis-9,trans-13 18:2, and trans-11,cis-15 18:2, but decreased (P < 0.05) the concentration of 18:2n-6. Relative to other treatments, treatment CO increased (P < 0.01) milk fat cis-9,trans-11 CLA, cis,trans CLA (12,14), trans,cis CLA (7,9; 9,11; 10,12; 11,13; and 12,14), and trans,trans CLA (11,13 and 12,14) concentrations.
      Treatments A and B had no influence (P > 0.05) on the concentrations of the majority of odd- and branched-chain FA (OBCFA) in milk fat, with the exception of a decrease (P < 0.001) in 11-cyclohexyl 11:0 concentration for treatment B compared with the control (Supplemental Table S3; http://dx.doi.org/10.3168/jds.2014-7976). Compared with other treatments, treatment CO lowered (P < 0.05) the abundance of most OBCFA in milk fat and increased (P < 0.001) 21:0 and cis-12 21:1 concentrations.

      Discussion

      Intake and Nutrient Digestion

      Treatments A and B had no effect on DMI, but given the relatively small numbers of cows used in our experiment, these findings require confirmation in a larger-scale experiment. However, an extensive evaluation of data from multiple studies concluded that a single live strain of Saccharomyces cerevisiae had no effect on intake of lactating cows (
      • De Ondarza M.B.
      • Sniffen C.J.
      • Dussert L.
      • Chevaux E.
      • Sullivan J.
      • Walker N.
      Case study: Multiple-study analysis of the effect of live yeast on milk yield, milk component content and yield, and feed efficiency.
      ). Nevertheless, some indications were found that the influence of live yeasts on fiber digestion are dependent on the inherent digestibility of dietary forages, with the largest improvements reported in cows fed low-quality forages (
      • Guedes C.M.
      • Goncalves D.
      • Rodrigues M.A.M.
      • Dias-da-Silva A.
      Effects of a Saccharomyces cerevisiae yeast on ruminal fermentation and fibre degradation of maize silages in cows.
      ). Furthermore, strains of live yeasts have been shown to be effective in the prevention of ruminal acidosis (
      • Fonty G.
      • Chaucheyras-Durand F.
      Effects and modes of action of live yeasts in the rumen.
      ), a condition not prevalent in this experiment as indicated from measurements of rumen pH.
      At relatively high levels of supplementation (≥50 g of oil/kg of DM) plant oils and oilseeds typically lower DMI, a response often attributed to various mechanisms including the adverse effect of unsaturated FA on ruminal microbial communities, lower OM and NDF digestion in the rumen, a tendency to shift the site of nutrient digestion from the rumen to the intestines, and elevate plasma gut peptide concentrations (
      • Allen M.S.
      Effects of diet on short-term regulation of feed intake by lactating dairy cows.
      ;
      • Lock A.L.
      • Shingfield K.J.
      Optimizing milk composition.
      ). Compared with the control diet (fat content 24.3 g/kg of DM), treatment CO containing 85.9 g of fat/kg of DM resulted in a 12.1% decrease in DMI. An earlier study also reported that camelina lipid supplements might lower intake in lactating cows. Supplementing maize silage-based diets with camelina seeds (29 g/kg of DM) or camelina meal (95 g/kg of DM), both providing 10.3 g of additional oil/kg of DM, equivalent to 39 and 35% of total dietary lipid content was associated with decreases in DMI of −1.9 and −5.7%, respectively (
      • Hurtaud C.
      • Peyraud J.L.
      Effects of feeding Camelina (seeds or meal) on milk fatty acid composition and butter spreadability.
      ). In cows fed red clover silage-based diets, supplements of camelina oil (14.9 g/kg of DM) or camelina expeller (106 g/kg of DM), providing an additional 17.6 g of FA/kg of DM, were found to have no effect on DMI (
      • Halmemies-Beauchet-Filleau A.
      • Kokkonen T.
      • Lampi A.M.
      • Toivonen V.
      • Shingfield K.J.
      • Vanhatalo A.
      Effect of plant oils and camelina expeller on milk fatty acid composition in lactating cows fed diets based on red clover silage.
      ). However, the effects of plant oils on DMI are known to vary depending on the composition and lipid content of the basal diet and the source and inclusion rate of lipid supplements (
      • Chilliard Y.
      Dietary fat and adipose tissue metabolism in ruminants, pigs, and rodents: A review.
      ;
      • Benchaar C.
      • Romero-Pérez G.A.
      • Chouinard P.Y.
      • Hassanat F.
      • Eugene M.
      • Petit H.V.
      • Côrtes C.
      Supplementation of increasing amounts of linseed oil to dairy cows fed total mixed rations: Effects on digestion, ruminal fermentation characteristics, protozoal populations, and milk fatty acid composition.
      ;
      • Rabiee A.R.
      • Breinhild K.
      • Scott W.
      • Golder H.M.
      • Block E.
      • Lean I.J.
      Effect of fat additions to diets of dairy cattle on milk production and components: A meta-analysis and meta-regression.
      ).
      Decreases in DMI on the treatment CO were accompanied by numerical decreases in total-tract OM, NDF, pdNDF, starch, or GE digestibility coefficients (−4.3, −6.8, −5.2, −0.5, and −4.8%, respectively), differences that may well be determined significant if the current study was repeated with more animals. Plant oils and oilseeds have variable effects on diet digestibility in lactating cows depending on the amount and source of lipid added and composition of the basal diet, with decreases often being attributed to the inhibitory adverse effect of unsaturated FA on the growth of microbial populations in the rumen (
      • Palmquist D.L.
      • Jenkins T.C.
      Fat in lactation rations.
      ;
      • Jenkins T.C.
      Lipid metabolism in the rumen.
      ). In cows fed alfalfa and barley silage, including 88 g of rapeseed oil/kg of diet, DM in the ration had no influence on ruminal or total-tract OM and NDF digestion (
      • Chelikani P.K.
      • Bell J.A.
      • Kennelly J.J.
      Effect of feeding or abomasal infusion of canola oil in Holstein cows 1. Nutrient digestion and milk composition.
      ). Sunflower oil offered up to 50 g/kg of DM was shown to progressively decrease ruminal pdNDF and total NDF digestibility in cows fed grass silage-based diets (
      • Shingfield K.J.
      • Ahvenjärvi S.
      • Toivonen V.
      • Vanhatalo A.
      • Huhtanen P.
      • Griinari J.M.
      Effect of incremental levels of sunflower-seed oil in the diet on ruminal lipid metabolism in lactating cows.
      ). When fed as part of a TMR based on grass hay, linseed oil (40 g/kg of diet DM) had no negative effects on nutrient digestion (
      • Benchaar C.
      • Romero-Pérez G.A.
      • Chouinard P.Y.
      • Hassanat F.
      • Eugene M.
      • Petit H.V.
      • Côrtes C.
      Supplementation of increasing amounts of linseed oil to dairy cows fed total mixed rations: Effects on digestion, ruminal fermentation characteristics, protozoal populations, and milk fatty acid composition.
      ), whereas linseed oil or whole or processed linseeds supplying 57 g of lipid/kg of diet DM lowered total-tract OM and NDF digestibility in cows fed maize silage-based diets (
      • Martin C.
      • Rouel J.
      • Jouany J.P.
      • Doreau M.
      • Chilliard Y.
      Methane output and diet digestibility in response to feeding dairy cows crude linseed, extruded linseed, or linseed oil.
      ). In cows fed a diet based on grass and maize silage, supplemental lipid from linseed oil (400 g/d) and linseed hulls (1.8 kg/d containing 29.4 g of fat/kg of DM) had more adverse effects on intake and total-tract fiber digestibility when administered in the rumen than the abomasum (
      • Kazama R.
      • Côrtes C.
      • da Silva D.
      • Gagnon N.
      • Benchaar C.
      • Zeoula L.M.
      • Santos G.T.D.
      • Petit H.V.
      Abomasal or ruminal administration of flax oil and hulls on milk production, digestibility, and milk fatty acid profile of dairy cows.
      ). Direct comparisons are limited, but at low levels of inclusion (10 g/kg of DM), no differences in total-tract nutrient digestion have been reported for camelina oil compared with rapeseed or sunflower oil in cows fed red clover silage (
      • Halmemies-Beauchet-Filleau A.
      • Kokkonen T.
      • Lampi A.M.
      • Toivonen V.
      • Shingfield K.J.
      • Vanhatalo A.
      Effect of plant oils and camelina expeller on milk fatty acid composition in lactating cows fed diets based on red clover silage.
      ).
      Regulation of the intake of lactating cows involves both physical and metabolic feedback mechanisms (
      • Allen M.S.
      Effects of diet on short-term regulation of feed intake by lactating dairy cows.
      ). No measurements of rumen pool sizes and digestion kinetics were made to explore physical constraints on intake. Nevertheless, the numerical decreases in total-tract digestibility do not appear to fully explain the decrease in DMI on the treatment CO, suggesting that metabolic factors may also have contributed to the decrease in intake in response to camelina oil. Decreases in DMI to high amounts of lipid supplements have often been explained as a consequence of alterations in ruminoreticular motility, the release of gut hormones, including cholecystokinin-octapeptide, and oxidation of fat in the liver (
      • Chilliard Y.
      Dietary fat and adipose tissue metabolism in ruminants, pigs, and rodents: A review.
      ;
      • Allen M.S.
      Effects of diet on short-term regulation of feed intake by lactating dairy cows.
      ). Abomasal infusion of unsaturated FA from soybean (178–534 g/d;
      • Litherland N.B.
      • Thire S.
      • Beaulieu A.D.
      • Reynolds C.K.
      • Benson J.A.
      • Drackley J.K.
      Dry matter is decreased more by abomasal infusion of unsaturated free fatty acids than by unsaturated triglycerides.
      ) or linseed oil (250 and 500 g/d;
      • Côrtes C.
      • Kazama R.
      • da Silva-Kazama D.
      • Benchaar C.
      • Zeoula L.M.
      • Santos G.T.D.
      • Petit H.V.
      Digestion, milk production and milk fatty acid profile of dairy cows fed flax hulls and infused with flax oil in the abomasum.
      ) have been shown to lower DMI without altering diet digestibility. These studies also demonstrated that free FA induce more pronounced decreases in DMI compared with esterified FA (
      • Litherland N.B.
      • Thire S.
      • Beaulieu A.D.
      • Reynolds C.K.
      • Benson J.A.
      • Drackley J.K.
      Dry matter is decreased more by abomasal infusion of unsaturated free fatty acids than by unsaturated triglycerides.
      ), and that the decreases in intake to infusions of linseed oil were greater in cows fed diets containing 24.7 than 73.2 g of fat/kg of DM (
      • Côrtes C.
      • Kazama R.
      • da Silva-Kazama D.
      • Benchaar C.
      • Zeoula L.M.
      • Santos G.T.D.
      • Petit H.V.
      Digestion, milk production and milk fatty acid profile of dairy cows fed flax hulls and infused with flax oil in the abomasum.
      ). Even though increases in FA at the small intestine have been shown to stimulate enterocyte secretion of glucagon-like peptide-1 and cholecystokinin-octapeptide (
      • Litherland N.B.
      • Thire S.
      • Beaulieu A.D.
      • Reynolds C.K.
      • Benson J.A.
      • Drackley J.K.
      Dry matter is decreased more by abomasal infusion of unsaturated free fatty acids than by unsaturated triglycerides.
      ), the mechanisms involved in the regulation of intake in cows fed lipid supplements merit further investigation.
      Across all diets, the efficiency of N utilization, defined as milk N/N intake, averaged 0.272, which is consistent with a mean of 0.277 for cows fed grass silage-based diets containing on average 165 g of CP/kg of DM (
      • Huhtanen P.
      • Hristov A.N.
      A meta-analysis of the effects of protein concentration and degradability on milk protein yield and milk N efficiency in dairy cows.
      ). Camelina oil improved N utilization compared with treatment B, which can, for the most part, be explained by a lower N intake. It is well established that dietary N intake is a major determinant of the efficiency of N utilization for milk production in lactating cows (
      • Huhtanen P.
      • Hristov A.N.
      A meta-analysis of the effects of protein concentration and degradability on milk protein yield and milk N efficiency in dairy cows.
      ), whereas partitioning into urine increases exponentially when N intake exceeds 400 g/d (
      • Castillo A.R.
      • Kebreab E.
      • Beever D.E.
      • France J.
      A review of efficiency of nitrogen utilisation in lactating dairy cows and its relationship with environmental pollution.
      ).

      Rumen Fermentation

      Treatments A and B had no significant influence on ruminal fermentation, which is in agreement with the findings of an earlier experiment in growing cattle (
      • McGinn S.M.
      • Beauchemin K.A.
      • Coates T.
      • Colombatto D.
      Methane emissions from beef cattle: effects of monensin, sunflower oil, enzymes, yeast, and fumaric acid.
      ). In dry cows, 1 strain of Saccharomyces cerevisiae was shown to increase propionate and decrease acetate whereas another had no influence on ruminal molar VFA proportions, highlighting the variability in responses to yeast supplements in vivo (
      • Chung Y.-H.
      • Walker N.D.
      • McGinn S.M.
      • Beauchemin K.A.
      Differing effects of 2 active dried yeast (Saccharomyces cerevisiae) strains on ruminal acidosis and methane production in nonlactating dairy cows.
      ).
      Camelina oil had limited influence on rumen fermentation, other than a tendency to lower rumen pH and decrease the molar proportion of isobutyrate. In cows fed maize silage, camelina seeds or meal have been shown to lower molar proportions of acetate and increase those of propionate and valerate in rumen VFA (
      • Hurtaud C.
      • Peyraud J.L.
      Effects of feeding Camelina (seeds or meal) on milk fatty acid composition and butter spreadability.
      ). Differences between experiments may relate to the form in which camelina lipid is included in the diet and the composition of other feed ingredients. Dietary plant oil supplements may influence molar VFA proportions depending on the composition of the basal diet, amount of added lipid, and the amount of lipid in the basal diet (
      • Chelikani P.K.
      • Bell J.A.
      • Kennelly J.J.
      Effect of feeding or abomasal infusion of canola oil in Holstein cows 1. Nutrient digestion and milk composition.
      ;
      • Shingfield K.J.
      • Ahvenjärvi S.
      • Toivonen V.
      • Vanhatalo A.
      • Huhtanen P.
      • Griinari J.M.
      Effect of incremental levels of sunflower-seed oil in the diet on ruminal lipid metabolism in lactating cows.
      ;
      • Benchaar C.
      • Romero-Pérez G.A.
      • Chouinard P.Y.
      • Hassanat F.
      • Eugene M.
      • Petit H.V.
      • Côrtes C.
      Supplementation of increasing amounts of linseed oil to dairy cows fed total mixed rations: Effects on digestion, ruminal fermentation characteristics, protozoal populations, and milk fatty acid composition.
      ). A recent meta-analysis of 106 treatment means derived from experiments in cattle fed grass silage-based diets indicated that differences in silage lactate concentrations have a greater influence on rumen fermentation patterns compared with dietary fat content (
      • Huhtanen P.
      • Jaakkola S.
      • Nousiainen J.
      An overview of silage research in Finland: From ensiling innovation to advances in dairy cow feeding.
      ). Plant oils and oilseeds typically have adverse effects on rumen fermentation when included in rations to provide 60 to 70 g of fat/kg of diet DM (
      • Jenkins T.C.
      Lipid metabolism in the rumen.
      ;
      • Grainger C.
      • Beauchemin K.A.
      Can enteric methane emissions from ruminants be lowered without lowering their production?.
      ), but little evidence exists from this or earlier studies (
      • Shingfield K.J.
      • Ahvenjärvi S.
      • Toivonen V.
      • Vanhatalo A.
      • Huhtanen P.
      • Griinari J.M.
      Effect of incremental levels of sunflower-seed oil in the diet on ruminal lipid metabolism in lactating cows.
      ) to suggest that high amounts of plant oils (≥50 g/kg of DM) result in substantial changes in rumen pH or molar VFA proportions in cows fed high-forage diets based on silage prepared from mixed timothy and meadow fescue.

      Ruminal Gas Production and Microbial Ecology

      Administration of live yeast strains was associated with numerical decreases in ruminal CO2 and CH4 production compared with the control, but because of the limited number of observations and inherent sensitivity of the SF6 tracer technique it did not establish these differences as significant. Further evaluation of live yeast strains A and B using a larger number of cows would be required to confirm possible efficacy. In growing cattle, probiotic yeasts have been shown to result in variable and often nonsignificant differences in ruminal CH4 production of between −3 and 6% (
      • McGinn S.M.
      • Beauchemin K.A.
      • Coates T.
      • Colombatto D.
      Methane emissions from beef cattle: effects of monensin, sunflower oil, enzymes, yeast, and fumaric acid.
      ). Consistent with minimal changes in rumen fermentation patterns and gas production, treatments A and B had no effect on the abundance of microbial groups or targeted ruminal cellulolytic bacterial species. Although it has been suggested that change-over designs may not be the most appropriate for assessing the effects of live yeast effects on rumen function due to possible treatment carry-over effects on the rumen microbiota (
      • Chaucheyras-Durand F.
      • Chevaux E.
      • Martin C.
      • Forano E.
      Use of yeast probiotics in ruminants: Effects and mechanisms of action on rumen pH, fiber degradation, and microbiota according to the diet.
      ), a 14-d washout between treatments was used in the present study to minimize these effects.
      Dietary supplements of camelina oil resulted in a 4.9% decrease in CH4 production per 10 g/kg of diet DM increase in diet oil content, a response similar to that of 4.8% reported for cows fed diets containing 58 g of linseed oil/kg of diet DM (
      • Martin C.
      • Rouel J.
      • Jouany J.P.
      • Doreau M.
      • Chilliard Y.
      Methane output and diet digestibility in response to feeding dairy cows crude linseed, extruded linseed, or linseed oil.
      ). The decrease in both ruminal CH4 and CO2 production on treatment CO (−29.5 and −34.3%, respectively) can be explained, at least to some extent, by the lower DMI on treatment CO (−12.1%). Supplementing the diet of growing cattle with 46 g of rapeseed oil/kg of diet DM was also reported to lower absolute CH4 emissions (
      • Beauchemin K.A.
      • McGinn S.M.
      Methane emissions from beef cattle: Effects of fumaric acid, essential oil, and canola oil.
      ). However, energy losses of CH4 as a percentage of digestible energy intake did not differ among treatments, indicating that the changes in ruminal CH4 formation to rapeseed oil reported by
      • Beauchemin K.A.
      • McGinn S.M.
      Methane emissions from beef cattle: Effects of fumaric acid, essential oil, and canola oil.
      ) could be explained by a lower DMI.
      Both 18:2n-6 and 18:3n-3 contained in camelina oil exert toxic effects on ciliate protozoa and bacteriostatic effects on cellulolytic bacteria (
      • Doreau M.
      • Ferlay A.
      Effect of dietary lipids on nitrogen metabolism in the rumen: A review.
      ;
      • Jenkins T.C.
      • Wallace R.J.
      • Moateand P.J.
      • Mosley E.E.
      Board-invited review: Recent advances in biohydrogenation of unsaturated fatty acids within the rumen microbial ecosystem.
      ). Both protozoal and bacterial populations contribute to fiber digestion and H2 production in the rumen, indicating that PUFA may potentially inhibit ruminal CH4 production (
      • Martin C.
      • Morgavi D.P.
      • Doreau M.
      Methane mitigation in ruminants: From microbe to the farm scale.
      ). Based on the analysis of samples collected prefeeding, camelina oil had no effect on the abundance of protozoa in the rumen, or on the populations of fiber-degrading bacteria F. succinogenes and R. flavefaciens. There are no reports on the effects of camelina oil on rumen microbial populations. However, the FA composition of camelina oil is more similar to linseed oil than other plant oils typically fed to ruminants (
      • Halmemies-Beauchet-Filleau A.
      • Kokkonen T.
      • Lampi A.M.
      • Toivonen V.
      • Shingfield K.J.
      • Vanhatalo A.
      Effect of plant oils and camelina expeller on milk fatty acid composition in lactating cows fed diets based on red clover silage.
      ), and therefore the effects of linseed and camelina oils on rumen microbial ecology could be expected to be rather similar.
      Dietary linseed oil supplements were reported to have no effect on rumen microbial populations in samples collected from sheep before feeding, but decreased protozoal numbers with no influence on fibrolytic bacteria in samples collected 3 h postfeeding (
      • Mosoni P.
      • Rochette Y.
      • Graviou D.
      • Martin C.
      • Forano E.
      • Morgavi D.P.
      ). In contrast, linseed oil was demonstrated to have no effect on the total number or the genera distribution of protozoa in cattle collected 2 h postfeeding (
      • Benchaar C.
      • Romero-Pérez G.A.
      • Chouinard P.Y.
      • Hassanat F.
      • Eugene M.
      • Petit H.V.
      • Côrtes C.
      Supplementation of increasing amounts of linseed oil to dairy cows fed total mixed rations: Effects on digestion, ruminal fermentation characteristics, protozoal populations, and milk fatty acid composition.
      ). Consistent with earlier findings (
      • Mosoni P.
      • Rochette Y.
      • Graviou D.
      • Martin C.
      • Forano E.
      • Morgavi D.P.
      ), the lack of treatment effects on rumen protozoal numbers in our study may also be related to the collection of ruminal digesta immediately before morning feeding. An in vitro study reported that crushed linseeds and sunflower seeds decrease CH4 production without changes in protozoal numbers, suggesting that other mechanisms than an inhibition of protozoa may also be involved, including changes in molar VFA proportions or changes in the diversity or activity of archaea (
      • Popova M.
      • Martin C.
      • Eugène M.
      • Mialon M.M.
      • Doreau M.
      • Morgavi D.P.
      Effect of fibre- and starch-rich finishing diets on methanogenic Archaea diversity and activity in the rumen of feedlot bulls.
      ). Even though treatment CO decreased daily CH4 production, the abundance of ruminal methanogens was not affected, suggesting that rather than influencing numbers, camelina oil may modulate the composition and functioning of this microbial population (
      • Popova M.
      • Martin C.
      • Eugène M.
      • Mialon M.M.
      • Doreau M.
      • Morgavi D.P.
      Effect of fibre- and starch-rich finishing diets on methanogenic Archaea diversity and activity in the rumen of feedlot bulls.
      ).

      Milk Production

      Treatments A and B had no influence on milk yield, composition, or energy secretion in milk, consistent with no changes in intake or digestibility compared with the control. However, the number of cows used to assess the effects of live yeasts on animal performance in our study was limited, with the corollary that a larger experiment with more animals would be required to establish fully the effects of treatments A and B on intake and milk production. A recent meta-analysis based on data from 14 experiments including measurements of more than 1,600 cows fed a range of diets indicated that live yeasts can be expected to improve FCM yield and stimulate an increase in milk protein and fat secretion (
      • De Ondarza M.B.
      • Sniffen C.J.
      • Dussert L.
      • Chevaux E.
      • Sullivan J.
      • Walker N.
      Case study: Multiple-study analysis of the effect of live yeast on milk yield, milk component content and yield, and feed efficiency.
      ).
      Compared with the control, treatment CO lowered the yields of milk, milk fat, protein, and lactose. In cows fed diets based on red clover silage, camelina oil (14.9 g/kg of DM) or expeller (106 g/kg of DM) had no adverse effects on milk yield and composition (
      • Halmemies-Beauchet-Filleau A.
      • Kokkonen T.
      • Lampi A.M.
      • Toivonen V.
      • Shingfield K.J.
      • Vanhatalo A.
      Effect of plant oils and camelina expeller on milk fatty acid composition in lactating cows fed diets based on red clover silage.
      ). However, camelina meal (95 g/kg of DM), but not camelina seeds (29 g/kg of DM), was shown to decrease milk protein yield in cows fed maize silage-based diets (
      • Hurtaud C.
      • Peyraud J.L.
      Effects of feeding Camelina (seeds or meal) on milk fatty acid composition and butter spreadability.
      ). It is generally accepted that energy intake is the major nutritional factor influencing milk protein synthesis, but the relation between increases in ME and milk protein secretion only holds true for dietary protein and carbohydrate components (
      • Lock A.L.
      • Shingfield K.J.
      Optimizing milk composition.
      ). Depending on inclusion rate, dietary lipid supplements including animal fats and plant oils typically result in a 1- to 4-g/kg decrease in milk protein concentration (
      • Wu Z.
      • Huber J.T.
      Relationship between dietary-fat supplementation and milk protein-concentration in lactating cows—A review.
      ). Often the decrease has been attributed to increases in milk yield rather than lowered milk protein output, but there is evidence to indicate that the decrease in milk protein percentage is not simply due to dilution, but reflects true physiological responses to fat supplements (
      • Wu Z.
      • Huber J.T.
      Relationship between dietary-fat supplementation and milk protein-concentration in lactating cows—A review.
      ). Compared with the control, treatment CO lowered milk fat output that was related to the overall decrease in milk yield, rather than a decrease in milk fat content.

      Milk FA Composition

      Live yeasts do not contain substantial amounts of lipid (
      • Ratledge C.
      • Evans C.T.
      Lipids and their metabolism.
      ), and therefore the possible effects on milk fat composition could be anticipated to involve changes in the relative abundance of OBCFA and specific biohydrogenation intermediates originating from the rumen. The similarities in milk FA composition between the control and treatments A and B would tend to suggest that the live yeast strains tested had no major influence on ruminal lipolysis, biohydrogenation, or microbial lipid synthesis.
      Compared with the control, treatment CO resulted in a substantial decrease in milk fat SFA due to lower 6- to 16-carbon FA concentrations, which represents a typical response to relatively high amounts of dietary plant oil supplements (
      • Chilliard Y.
      • Glasser F.
      • Ferlay A.
      • Bernard L.
      • Rouel J.
      • Doreau M.
      Diet, rumen biohydrogenation and nutritional quality of cow and goat milk fat.
      ;
      • Glasser F.
      • Ferlay A.
      • Chilliard Y.
      Oilseed lipid supplements and fatty acid composition of cow milk: A meta-analysis.
      ;
      • Shingfield K.J.
      • Bonnet M.
      • Scollan N.D.
      Recent developments in altering the fatty acid composition of ruminant-derived foods.
      ). Much of the decrease can be attributed to an increase in the availability of 18-carbon and longer FA, which inhibits acyl-CoA carboxylase activity and decreases the synthesis of 6:0 to 16:0 de novo in the mammary glands (
      • Chilliard Y.
      • Glasser F.
      • Ferlay A.
      • Bernard L.
      • Rouel J.
      • Doreau M.
      Diet, rumen biohydrogenation and nutritional quality of cow and goat milk fat.
      ).
      Treatment CO elevated milk fat cis-MUFA concentrations compared with other treatments, principally due to increases in cis-9 18:1 that can be attributed to both higher intake and ruminal escape of cis-9 18:1, as well as increased availability of 18:0 for desaturation in the mammary glands. Studies in cows indicate that between 44 and 78% of 18:0 extracted from the blood is desaturated in the mammary gland of lactating cows (
      • Shingfield K.J.
      • Bonnet M.
      • Scollan N.D.
      Recent developments in altering the fatty acid composition of ruminant-derived foods.
      ).
      For all treatments, cis-9 16:1 represented the second-most abundant cis-MUFA in milk fat, more than 50% of which is synthesized endogenously via the action of stearoyl CoA desaturase on 16:0 in the mammary gland (
      • Shingfield K.J.
      • Bonnet M.
      • Scollan N.D.
      Recent developments in altering the fatty acid composition of ruminant-derived foods.
      ). Milk fat also contained 14 cis and trans 16:1 isomers that may originate from the isomerization of dietary cis-9 16:1 and trans-3 16:1, or from the oxidation of 18:1 biohydrogenation intermediates in the rumen (
      • Destaillats F.
      • Wolff R.L.
      • Precht D.
      • Molkentin J.
      Study of individual trans- and cis-16:1 isomers in cow, goat and ewe cheese fats by gas-liquid chromatography with emphasis on the trans-Δ3 isomer.
      ).
      Compared with the control, treatment CO increased total trans FA content as a result of elevated trans 16:1, trans 18:1, and trans 18:2 concentrations. Much of the increase in milk trans FA was associated with trans-11 18:1, that represents a common intermediate formed during the penultimate step of PUFA biohydrogenation in the rumen (
      • Harfoot C.G.
      • Hazlewood G.P.
      Lipid metabolism in the rumen.
      ). Enrichment of trans FA in milk by treatment CO can be attributed to the incomplete ruminal biohydrogenation of unsaturated FA (
      • Chilliard Y.
      • Glasser F.
      • Ferlay A.
      • Bernard L.
      • Rouel J.
      • Doreau M.
      Diet, rumen biohydrogenation and nutritional quality of cow and goat milk fat.
      ) in camelina oil. Despite the differences in milk trans 18:1 concentrations, the relative abundance of individual 18:1 isomers did not differ substantially among treatments, indicating that camelina oil did not induce substantial alterations in the major biohydrogenation pathways in the rumen (
      • Jenkins T.C.
      • Wallace R.J.
      • Moateand P.J.
      • Mosley E.E.
      Board-invited review: Recent advances in biohydrogenation of unsaturated fatty acids within the rumen microbial ecosystem.
      ;
      • Shingfield K.J.
      • Bonnet M.
      • Scollan N.D.
      Recent developments in altering the fatty acid composition of ruminant-derived foods.
      ). Compared with an earlier report (
      • Halmemies-Beauchet-Filleau A.
      • Kokkonen T.
      • Lampi A.M.
      • Toivonen V.
      • Shingfield K.J.
      • Vanhatalo A.
      Effect of plant oils and camelina expeller on milk fatty acid composition in lactating cows fed diets based on red clover silage.
      ), the increases in milk trans-11 18:1 concentrations per gram of camelina oil per kilogram of diet DM on the treatment CO were lower than expected, with the implication that unsaturated 18-carbon FA were extensively biohydrogenated to 18:0 in the rumen. Camelina oil also enriched milk fat cis-9,trans-11 CLA concentrations, responses that are in the range that could be expected when plant oils rich in 18:2n-6 and 18:3n-3 are used as supplements for diets of similar composition to the experimental TMR fed in our study (
      • Chilliard Y.
      • Glasser F.
      • Ferlay A.
      • Bernard L.
      • Rouel J.
      • Doreau M.
      Diet, rumen biohydrogenation and nutritional quality of cow and goat milk fat.
      ;
      • Shingfield K.J.
      • Bonnet M.
      • Scollan N.D.
      Recent developments in altering the fatty acid composition of ruminant-derived foods.
      ).
      Even though cows fed treatment CO had a higher intake of 18:2n-6 and 18:3n-3, the enrichment of 18:3n-3 in milk was 7.7% higher compared with the control, whereas 18:2n-6 abundance was decreased, changes that are in line with earlier reports on milk FA composition responses to camelina oil (
      • Halmemies-Beauchet-Filleau A.
      • Kokkonen T.
      • Lampi A.M.
      • Toivonen V.
      • Shingfield K.J.
      • Vanhatalo A.
      Effect of plant oils and camelina expeller on milk fatty acid composition in lactating cows fed diets based on red clover silage.
      ), seeds, or expeller (
      • Hurtaud C.
      • Peyraud J.L.
      Effects of feeding Camelina (seeds or meal) on milk fatty acid composition and butter spreadability.
      ). Lower concentrations of most OBCFA in milk from cows receiving treatment CO are consistent with milk fat composition responses to plant oils in lactating cows (
      • Glasser F.
      • Ferlay A.
      • Chilliard Y.
      Oilseed lipid supplements and fatty acid composition of cow milk: A meta-analysis.
      ). Although the appearance of OBCFA in milk fat originate principally from microbial lipids synthesized de novo in the rumen, changes in the relative concentrations in milk may at least in part be related to a decrease in microbial synthesis or alterations in the relative abundance of specific populations of bacteria and protozoa in the rumen (
      • Vlaeminck B.
      • Fievez V.
      • Cabrita A.R.J.
      • Fonseca A.J.M.
      • Dewhurst R.J.
      Factors affecting odd- and branched-chain fatty acids in milk: A review.
      ).

      Conclusions

      Ruminal administration of live yeast strains had no influence on ruminal CH4 and CO2 production, rumen fermentation, or animal performance in cows fed diets based on grass silage. Supplements of camelina oil decreased ruminal CH4 and CO2 production, responses that were accompanied by lowered intake, yield of milk and milk constituents, and altered milk FA composition without a substantial change in nutrient digestion, rumen fermentation, or ruminal microbial populations. Live yeast strains had no influence on the proportions of the major FA in milk. Dietary supplements of camelina oil decreased 12:0, 14:0, 16:0, and 18:2n-6 and increased cis-9 18:1, trans-11 18:1, cis-9,trans-11 CLA, 18:3n-3, and total trans FA concentrations. The effect of these changes in milk fat composition on human health merits further investigation.

      Acknowledgments

      The authors thank staff in the metabolism unit at Natural Resources Institute (Jokioinen, Finland) for technical support, care of experimental animals, and assistance in sample collection. Competitive funding from the Finnish Ministry of Agriculture (Helsinki) as part of the GreenDairy project is gratefully acknowledged. Plasmids used for protozoa quantification were a generous donation from F. Ossa (Institut Rosell/Lallemand, Montréal, Canada).

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