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Methane production and digestion of different physical forms of rapeseed as fat supplements in dairy cows

Open ArchivePublished:February 18, 2013DOI:https://doi.org/10.3168/jds.2011-5239

      Abstract

      The purpose of this experiment was to study the effect of the physical form of rapeseed fat on methane (CH4) mitigation properties, feed digestion, and rumen fermentation. Four lactating ruminal-, duodenal-, and ileal-cannulated Danish Holstein dairy cows (143 d in milk, milk yield of 34.3 kg) were submitted to a 4 × 4 Latin square design with 4 rations: 1 control with rapeseed meal (low-fat, CON) and 3 fat-supplemented rations with either rapeseed cake (RSC), whole cracked rapeseed (WCR), or rapeseed oil (RSO). Dietary fat concentrations were 3.5 in CON, 5.5 in RSC, 6.2 in WCR, and 6.5% in RSO. The amount of fat-free rapeseed was kept constant for all rations. The forage consisted of corn silage and grass silage and the forage to concentrate ratio was 50:50 on a dry matter basis. Diurnal samples of duodenal and ileal digesta and feces were compiled. The methane production was measured for 4 d in open-circuit respiration chambers. Additional fat reduced the CH4 production per kilogram of dry matter intake and as a proportion of the gross energy intake by 11 and 14%, respectively. Neither the total tract nor the rumen digestibility of organic matter (OM) or neutral detergent fiber were significantly affected by the treatment. Relating the CH4 production to the total-tract digested OM showed a tendency to decrease CH4 per kilogram of digested OM for fat-supplemented rations versus CON. The acetate to propionate ratio was not affected for RSC and WCR but was increased for RSO compared with CON. The rumen ammonia concentration was not affected by the ration. The milk and energy-corrected milk yields were unaffected by the fat supplementation. In conclusion, rapeseed is an appropriate fat source to reduce the enteric CH4 production without affecting neutral detergent fiber digestion or milk production. The physical form of fat did not influence the CH4-reducing effect of rapeseed fat. However, differences in the volatile fatty acid pattern indicate that different mechanisms may be involved.

      Key words

      Introduction

      Globally, agriculture accounts for 47% of total anthropogenic methane (CH4) emissions, with enteric fermentation contributing 32% of the total non-CO2 emissions from agriculture in 2005 (
      • Smith P.
      • Martino D.
      • Cai Z.
      • Gwary D.
      • Janzen H.
      • Kumar P.
      • McCarl S.
      • Ogle S.
      • O’Mara F.
      • Rice C.
      • Scholes B.
      • Sirotenko O.
      Agriculture.
      ). The CH4 production per animal varies depending on the feed composition, feed quality, and production level from 2 to 12% of the gross energy (GE) intake (
      • Johnson K.A.
      • Johnson D.E.
      Methane emissions from cattle.
      ) under extreme circumstances, but values between 3 and 7% (
      • 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 more realistic in intensive dairy production.
      Numerous studies have discussed nutritional possibilities to reduce the enteric CH4 production (
      • Boadi D.
      • Benchaar C.
      • Chiquette J.
      • Masse D.
      Mitigation strategies to reduce enteric methane emissions from dairy cows: Update review.
      ;
      • Beauchemin K.A.
      • Kreuzer M.
      • O’Mara F.
      • McAllister T.A.
      Nutritional management for enteric methane abatement: A review.
      ), and fat supplementation is among the most promising tools to depress CH4 production from ruminants (
      • 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.
      ). Furthermore, fat is fed to dairy cows to increase the energy density of the ration or to alter the product quality (
      • Beauchemin K.A.
      • McGinn S.M.
      • Petit H.V.
      Methane abatement strategies for cattle: Lipid supplementation of diets.
      ). Several oils and oil seeds have been tested for their potential to reduce the CH4 production, and effects of chain length and saturation have been reported. The degree of saturation is important, as the negative effect on bacterial growth increases with the degree of unsaturation, inhibiting both fibrolytic bacteria and methanogens (
      • Giger-Reverdin S.
      • Morand-Fehr P.
      • Tran G.
      Literature survey on the influence of dietary fat composition on methane production in dairy cattle.
      ). With reduced fiber digestibility and a shift in fermentation pattern, less hydrogen arises and, thus, less CH4 (
      • Boadi D.
      • Benchaar C.
      • Chiquette J.
      • Masse D.
      Mitigation strategies to reduce enteric methane emissions from dairy cows: Update review.
      ). Additionally, fat often replaces carbohydrates in the ration, thereby directly reducing rumen fermentation. Reduced fiber digestibility is associated with reduced DMI and milk production; therefore it has to be considered whether the overall reduction of CH4 production from the animal due to the addition of fat is accompanied by a reduction per kilogram of product or per kilogram of feed digested.
      Feeding whole seeds or cake, a by-product from the plant oil production, as well as pure oil, are tools to increase the dietary fat concentration, but the difference in physical form might influence the effect of fat in the rumen.
      • Czerkawski J.W.
      • Blaxter K.L.
      • Wainman F.W.
      Effect of linseed oil and of linseed oil fatty acids incorporated in diet on metabolism of sheep.
      showed that the effect of FA on CH4 production was stronger when the same amount was infused to the rumen once daily, compared with continuous infusion. Similarly,
      • Machmüller A.
      • Ossowski D.A.
      • Kreuzer M.
      Comparative evaluation of the effects of coconut oil, oilseeds and crystalline fat on methane release, digestion and energy balance in lambs.
      concluded that this could be of importance to achieve momentarily high fat concentrations in the rumen rather than a constant presence at a lower level. Oil in seeds is stored intracellularly, and the fat release depends on the digestion and breakdown of the cell wall, which leads to a slower release compared with feeding oil directly (
      • Steele W.
      • Noble R.C.
      • Moore J.H.
      Effects of 2 methods of incorporating soybean oil into diet on milk yield and composition in cow.
      ). This indicates that pure oil may increase the rumen FA concentration faster and reduce the CH4 production more effectively compared with seeds or cake (
      • 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.
      ).
      Oilseed rape (Brassica napus) is widely grown in many countries. The by-products, rapeseed meal and cake, remaining after oil extraction are common feed components in dairy cow rations. Rapeseed meal has a low crude fat concentration (about 4%) compared with rapeseed cake (10–20%) and whole seeds (approximately 50%). The aim of the current experiment was to study the effect of rapeseed fat and the physical form in which it was fed on enteric CH4 production, rumen fermentation, and digestion.

      Materials and methods

      Animals and Rations

      The experiment complied with the guidelines of the Danish Ministry of Justice with respect to animal experimentation and care of animals under study. Four lactating Danish Holstein dairy cows (1 primiparous and 3 multiparous) were assigned to 1 of 4 rations over 4 periods according to a balanced Latin square design; each period consisted of 4 wk. One cow was omitted from the last period due to disease.
      The cows were 143 DIM (SD = 74 d), had a milk yield of 34.3 kg (SD = 8.6 kg), and a BW of 592 kg (SD = 81 kg) at the beginning of the experiment. All animals were fitted with a ruminal cannula (#1C, Bar Diamond Inc., Parma, ID), a duodenal cannula (open T-piece placed 60 cm caudal to pylorus), and an ileal cannula (open T-piece placed 20 cm cranial to the cecum). The cows were housed in a tie stall with rubber mats and sawdust as bedding and had free access to water. They were milked and fed twice daily at 0500 and 1700 h. Total mixed rations were prepared once a day and fed to the cows on an ad libitum basis after milking. The feed intake was recorded on a daily basis. The animals were weighed at the start of the experiment as well as just before and after the respiration chamber measurements (the last week of each period).
      The rations were a control ration (CON) and 3 high-fat rations with fat supplemented as either rapeseed cake (RSC), whole cracked rapeseed (WCR), or rapeseed oil (RSO), respectively. The amount of fat-free rapeseed was equal for all rations, as the basic rapeseed meal content in the CON was reduced according to the fat-free rapeseed which was supplemented with either cake or seed in the treatment rations. Rapeseed cake, whole rapeseed, and rapeseed oil were obtained from Danraps (DLG Food Oil, Dronninglund, Denmark). The rapeseed used in this study was double-00 rape, equivalent to what is known as canola in North America.
      The chemical composition of ingredients is shown in Table 1. All rations were fed as TMR with a forage to concentrate ratio of 50:50 (Table 2). The forage consisted of 54% corn silage and 46% prewilted perennial ryegrass silage (on DM basis). The corn silage was stored in a bunker silo and the grass silage in bales.
      Table 1Chemical composition (g/kg of DM unless otherwise noted) of feedstuffs.
      ItemBarleyBeet pulpRS
      RS=rapeseed.
      meal
      RS cakeWhole RSRS oilGrass silageCorn silage
      DM, g/kg of fresh matter878903904968940424295
      OM980958922937960896966
      CP11510238130019216399
      Crude fat30.04.854.71734791,00030.830.0
      NDF161408260201150420392
      INDF
      INDF=indigestible NDF.
      36.428.296.790.053.656.892.1
      Gross energy, MJ/kg of DM18.217.519.622.328.739.617.818.7
      NEL, MJ/kg of DM9.037.908.689.9914.3522.36.557.00
      OMD,
      OMD=in vitro OM digestibility.
      %
      75.272.7
      Fatty acids, g/kg of DM
       C16:06.301.993.538.4419.043.72.933.73
       C18:04.200.060.742.466.3515.50.303.60
       C18:12.980.8220.280.32185370.854.28
       C18:210.81.1911.733.480.41912.729.10
       C18:30.960.233.4613.738.291.410.42.39
       Total FA
      Sum of fatty acids includes, beside those shown, C16:1, C20:0, C20:1, C20:2, and C22:0.
      22.44.6046.215138593018.621.0
      1 RS = rapeseed.
      2 INDF = indigestible NDF.
      3 OMD = in vitro OM digestibility.
      4 Sum of fatty acids includes, beside those shown, C16:1, C20:0, C20:1, C20:2, and C22:0.
      Table 2Ration ingredients and chemical composition (g/kg of DM unless otherwise noted).
      ItemRation
      CON=control, RSC=rapeseed cake, WCR=whole cracked rapeseed, RSO=rapeseed oil.
      CONRSCWCRRSO
      Barley143137137138
      Beet pulp dried143137137138
      Rapeseed meal, 4% fat19062149184
      Rapeseed cake, 17% fat015600
      Rapeseed, cracked00690
      Rapeseed oil00033
      Corn silage238232232232
      Grass silage286275275275
      DM, g/kg of fresh matter479500492494
      OM934937939939
      CP169171168171
      Crude fat35556265
      Fatty acids
      Calculated values based on analysis of ingredients.
      26435053
      NDF332328326322
      Gross energy, MJ/kg of DM18.418.919.119.1
      NEL, MJ/kg of DM7.67.88.08.1
      1 CON = control, RSC = rapeseed cake, WCR = whole cracked rapeseed, RSO = rapeseed oil.
      2 Calculated values based on analysis of ingredients.

      Measurements

      The milk production and composition were measured once a week during morning and evening milkings. Weekly samples of the feed ingredients were stored (−20°C) and pooled during the whole experiment. Samples of TMR and refusals were taken daily in connection with the afternoon feeding, stored (−20°C), and pooled for each period from d 15 to 20.
      Chromic oxide was used as a flow marker, and 10 g was administrated to the rumen via the ruminal cannula during each of the 2 daily feedings, except when the cows were in the respiration chambers.
      Samples of duodenal chyme (600 mL), ileal chyme (300 mL), and feces (350 mL) were taken from d 15 to 19 at 1000, 1800 (d 15), 0200, 1200, 2000 (d 16), 0400, 1400, 2200 (d 17), 0600, 1600, 2400 (d 18), and 0800 h (d 19; 12 samples, representing every second hour of the day). Samples from the duodenum and ileum were taken in tube-formed plastic bags which were mounted to the cannulas with plastic knees. Duodenal, ileal, and fecal samples were added to the frozen pooled sample from previous samples at each sampling time. At the end of the period, representative subsamples from thawed material were taken and freeze-dried for chemical analysis. At the 12 sampling times, rumen liquid was sampled from the ventral ruminal sac with a collection tube (#RT, Bar Diamond Inc.). The rumen liquid pH was measured immediately, and two 8-mL samples were taken and frozen (−20°C) immediately for VFA and ammonia (NH3) analysis at each sampling time.

      Chemical Analysis

      Ash was determined by combustion at 525°C for 6 h. Nitrogen was determined by the Dumas principle (
      • Hansen B.
      Determination of nitrogen as elementary-N, an alternative to Kjeldahl.
      ), and CP was calculated as N × 6.25. Crude fat was analyzed as Soxhlet extraction with petroleum ether (Soxtec 2050, Foss Analytical, Hillerød, Denmark) after hydrolyzing with HCl (
      • Stoldt W.
      Vorschlag zur Vereinheitlichung der Fettbestimmung in Lebensmitteln.
      ). The NDF content was analyzed by neutral detergent extraction according to
      • Mertens D.R.
      Gravimetric determination of amylase-treated neutral detergent fiber in feeds with refluxing in beakers or crucibles: Collaborative study.
      with a Fibertec M6 System (Foss Analytical) using heat-stable amylase and corrected for ash. The indigestible NDF in freeze-dried ground (1.5mm) feed samples was determined as residual NDF after 288 h (12 d) of Dacron bag incubation in the rumen of 3 heifers fed a standard ration (
      • Åkerlind M.
      • Weisbjerg M.R.
      • Eriksson T.
      • Tøgersen R.
      • Udén P.
      • Ólafsson B.L.
      • Harstad O.M.
      • Volden H.
      Feed analyses and digestion methods.
      ). The GE was determined by adiabatic bomb calorimeter (Parr 6300 Oxygen Bomb Calorimeter, Parr Instrument Company, Moline, IL).
      The concentrations of VFA were analyzed according to the method described by
      • Canibe N.
      • Højbjerg O.
      • Badsberg J.H.
      • Jensen B.B.
      Effect of feeding fermented liquid feed and fermented grain on gastrointestinal ecology and growth performance in piglets.
      using a Hewlett Packard gas chromatograph (model 6890; Agilent Technologies Inc., Wilmington, DE) equipped with a flame ionization detector and a 30-m SGE BP1 column (Scientific Instrument Services, NJ). Fatty acids in feed were analyzed by GC after an acidic Bligh and Dyer extraction with hydrochloric acid-water-chloroform and methanol, and subsequent methylation as described by
      • Jensen S.K.
      Improved Bligh & Dyer extraction procedure.
      . For determination of NH3, the rumen fluid was made alkaline with KOH, and NH3 was determined by titration after distillation.
      Chromic oxide was determined by colorimetry after oxidation to chromate (
      • Schürch A.F.
      • Lloyd L.E.
      • Crampton E.W.
      The use of chromic oxide as an index for determining the digestibility of a diet.
      ). The OM digestibility was determined in vitro for grass and corn silage as described by
      • Tilley J.M.A.
      • Terry R.A.
      A two-stage technique for in vitro digestion of forage crops.
      . Milk concentrations of fat, protein, and lactose were analyzed by a Milkoscan Msc4000 infrared analyzer (Foss Analytical).

      Methane Measurements

      During the fourth week of each period, the CH4 production was measured for 2 × 48 h in four 17-m3 open-circuit respiration chambers (
      • Hellwing A.L.F.
      • Lund P.
      • Weisbjerg M.R.
      • Brask M.
      • Hvelplund T.
      Technical note: Test of a low-cost and animal-friendly system for measuring methane emissions from dairy cows.
      ). The animals were housed individually. The chambers were covered with transparent polycarbonate and placed in a square so that the cows faced each other. The chambers were located in the barn where the cows were usually housed to minimize changes in the environment and the daily routines during the CH4 measurements were identical to the period outside the chambers. The mean ambient temperature in the chambers was 21.1°C, ranging from 15.6 to 29.5°C.
      The cows changed chambers diagonally after the first 48 h to balance out any differences in background levels of CH4 and CO2. Cow and chamber were confounded over periods and, therefore, every ration was tested in every chamber.
      The chambers were opened twice daily, at 0500 and 1700 h, for about 20 min during milking and subsequent feeding. Methane was measured as the accumulated amount (L) over 24 h and is reported under standard conditions (0°C, 101.325 kPa). The measurements during the openings of the chambers for milking and feeding were deleted (about 60 min/d). The CH4 production during this period was assumed to correspond to the mean of the rest of the day.
      The air flow was measured by a HFM-200 flow meter with a laminar flow element from Teledyne Hastings Instruments (Hampton, VA). The background (inlet air), as well as the chamber outlet air concentration of CH4, was measured every 12 1/2 min with an infrared analyzer. All instruments were from Columbus Instruments (Columbus, OH). The air flow was adjusted individually for every animal depending on the BW and milk yield to obtain a CO2 concentration in the chamber below, but close to 9,000 ppm. The instruments were calibrated every second day with zero gas (nitrogen) and a span gas with nitrogen and 20.55% O2, 5,000 ppm CO2, and 800 ppm CH4 (Yara Praxair AS, Oslo, Norway). The temperature, humidity, and CO2 concentration of the chamber air were monitored with independent sensors (Veng System A/S, Roslev, Denmark) for alarm purposes.

      Calculations and Statistical Analysis

      The net energy content in feeds was calculated in Scandinavian Feed Units (SFU;
      • Weisbjerg M.R.
      • Hvelplund T.
      Estimation of net energy content (FU) in feeds for cattle.
      ) and presented in megajoules of NEL by using a fixed conversion factor of 7.89 MJ of NEL/SFU, as described by
      • Hvelplund T.
      • Weisbjerg M.R.
      • Lund P.
      Energy and protein evaluation systems used for dairy cows in Denmark.
      . Content of SFU is calculated based on content of ash, CP, crude fiber, crude fat, and in vitro OM digestibility.
      The apparent rumen digestibility was calculated as the feed intake minus the duodenal flow divided by feed intake. Apparent small intestine digestibility was calculated as the duodenal flow minus ileal flow divided by duodenal flow and, accordingly, apparent large intestine digestibility as ileal flow minus fecal flow divided by ileal flow.. Apparent total-tract digestibility was calculated as the feed intake minus fecal flow divided by feed intake.
      Average ECM yield for each cow per period was calculated according to
      • Sjaunja L.O.
      • Baevre L.
      • Junkkarinen L.
      • Pedersen J.
      • Setala J.
      A Nordic proposal for an energy corrected Milk (ECM) formula.
      as follows: ECM = milk yield × (383 × fat % + 242 × protein % + 783.2)/3,140. For samples with repeated measures (VFA, NH3, and pH), an average for each cow-period was calculated before the statistical analysis.
      The data was evaluated with the MIXED procedure (SAS 9.2 version, SAS Institute Inc., Cary, NC) with treatment and period as fixed effects and cow as random effect.
      The results are reported as LSM and SEM for each treatment. The SEM was different for RSO because one cow receiving RSO was omitted. Therefore, the RSO SEM is reported and a factor for the SEM of the other treatments is noted under each table. Apart from the mean treatment effect, the significance of orthogonal contrasts were calculated for CON versus fat supplement, RSC and WCR versus RSO, and RSC versus WCR. P-values < 0.05 were regarded as significant and P < 0.1 as a tendency.

      Results

      Milk Production

      Average milk production and ECM were not significantly affected by the treatment (Table 3). The milk fat content was numerically lower in fat supplemented rations compared with CON. Milk protein content was numerically higher on RSC and WCR than on RSO (P = 0.71). Daily milk fat production in g/d was 1,187, 1,170, 1,164, and 968 for CON, RSC, WCR, and RSO, respectively. Daily milk protein production was 915, 1,030, 928, and 849 g/d for CON, RSC, WCR, and RSO. The yield of milk solids per day was not affected by the ration.
      Table 3Milk production and composition.
      ItemRations
      CON=control, RSC=rapeseed cake, WCR=whole cracked rapeseed, RSO=rapeseed oil.
      SEM
      SEM for RSO; the SEM for the other treatments is the presented SEM×0.93.
      P-value
      Fixed effect of treatment.
      Contrast (P-value)
      Linear orthogonal contrasts of treatment.
      CONRSCWCRRSOCON vs. FatRSC and WCR vs. RSORSC vs. WCR
      Milk, kg/d27.431.228.126.34.610.230.520.160.18
      Milk fat, g/kg40.439.141.237.94.390.900.780.620.64
      Milk protein, g/kg33.533.132.932.41.130.710.350.510.83
      Milk lactose, g/kg46.446.846.146.41.280.260.800.990.07
      ECM, kg/d27.230.225.128.44.370.120.610.040.29
      1 CON = control, RSC = rapeseed cake, WCR = whole cracked rapeseed, RSO = rapeseed oil.
      2 SEM for RSO; the SEM for the other treatments is the presented SEM × 0.93.
      3 Fixed effect of treatment.
      4 Linear orthogonal contrasts of treatment.

      Feed Intake

      The DMI was not affected by the treatments. The intake was numerically greater for cows consuming CON and RSC than WCR and RSO. The higher GE content in the 3 fat-supplemented rations compensated for the numerically lower DMI, and the GE intake was similar between CON and fat-supplemented rations. As planned, fat intake increased in the fat-supplemented rations, and NDF and CP intakes were not affected, as shown in Table 4. Adding rapeseed to the ration increased the intake of total FA from 471 g/d in CON to 804, 835, and 892 g/d for RSC, WCR, and RSO, respectively. Compared with CON, adding fat almost doubled the absolute intake of C18:0 (P = 0.001) and tripled the intake of C18:1 (P < 0.003).
      Table 4Intake and apparent digestibility of nutrients.
      Ration
      CON=control, RSC=rapeseed cake, WCR=whole cracked rapeseed, RSO=rapeseed oil.
      Contrast (P-value)
      Linear orthogonal contrasts of treatment.
      ItemCONRSCWCRRSOSEM
      SEM for RSO; the SEM for the other treatments is the presented SEM×0.86.
      P-value
      Fixed effect of treatment.
      CON vs. FatRSC and WCR vs. RSORSC vs. WCR
      DM
       Intake, kg/d18.318.917.915.82.070.540.620.220.58
      OM
       Intake, kg/d17.117.716.814.91.960.560.640.230.61
       Duodenal flow, kg/d9.849.8110.39.191.300.850.940.480.67
       Rumen digestibility, %42.544.638.140.62.340.230.570.790.06
       Total-tract digestibility, %73.274.372.071.91.360.410.710.410.16
      NDF
       Intake, kg/d6.106.205.835.180.740.590.540.270.60
       Duodenal flow, kg/d2.352.162.171.930.320.430.180.310.96
       Rumen digestibility, %61.765.562.763.92.570.220.140.930.13
       Hindgut digestibility, %1.559.641.281.836.390.610.660.630.29
       Total-tract digestibility, %61.763.360.461.12.370.600.980.720.23
      Crude fat
       Intake, kg/d0.651.031.111.000.100.010.0030.480.38
       Duodenal flow, kg/d0.831.181.351.270.120.010.0040.980.15
       Rumen digestibility, %−27.5−15.7−22.5−26.46.210.460.370.360.39
       Small intestine digestibility, %75.075.075.069.93.180.330.440.100.99
       Total-tract digestibility, %65.467.865.158.04.290.250.590.070.50
       FA intake, g/d47180483589289.10.0090.0020.880.30
      CP
       Intake, kg/d3.093.233.002.700.350.530.680.230.48
       Duodenal flow, kg/d3.663.463.613.260.450.780.510.500.69
       Ruminal digestibility, %−19.0−6.88−21.3−16.35.160.210.440.730.06
       Total-tract digestibility, %65.166.964.564.51.630.520.850.520.22
      1 CON = control, RSC = rapeseed cake, WCR = whole cracked rapeseed, RSO = rapeseed oil.
      2 SEM for RSO; the SEM for the other treatments is the presented SEM × 0.86.
      3 Fixed effect of treatment.
      4 Linear orthogonal contrasts of treatment.

      Digestibility

      Duodenal, ileal, and fecal flows were unaffected by the treatment, except for an increased fat flow in the fat-supplemented rations (data not shown). Neither total-tract nor ruminal apparent digestibility was affected by the treatment for any measured nutrient (Table 4). The flow of crude fat to the duodenum was higher than intake by an average of 210 g/d, or 22.7%; no difference was observed between rations in ruminal fat digestibility (P = 0.46). The NDF digestibility in the hind gut was close to zero for all rations except RSC.

      VFA, Ammonia, and Rumen pH

      Fat supplementation did not affect VFA concentrations in the rumen (P = 0.24). Bound-fat supplements (rapeseed cake, whole rapeseed) resulted in higher propionic acid proportion (P = 0.005), and tended to reduce the acetic acid proportion (P = 0.07) compared with free supplement (oil). Consequently, the acetate to propionate ratio was 2.95 for RSO and 2.76, 2.73, and 2.74 for CON, RSC, and WCR, respectively, resulting in a significant difference (P = 0.01) between the 2 bound-fat supplements and RSO (Table 5).
      Table 5Rumen VFA, ammonia, and pH (mean of 12 diurnal samples).
      ItemRation
      CON=control, RSC=rapeseed cake, WCR=whole cracked rapeseed, RSO=rapeseed oil.
      SEM
      SEM for RSO; the SEM for the other treatments is the presented SEM×0.91.
      P-value
      Fixed effect of treatment.
      Contrast (P-value)
      Linear orthogonal contrasts of treatment.
      CONRSCWCRRSOCON vs. FatRSC and WCR vs. RSORSC vs. WCR
      Total VFA, mmol/L10910510398.63.520.240.100.200.63
      VFA, % of total VFA
       Acetic acid (A)60.559.860.161.00.670.220.680.070.50
       Butyric acid15.115.715.415.80.520.210.080.400.34
       Isobutyric acid0.740.780.720.840.060.320.390.170.29
       Propionic acid (P)22.122.122.120.80.930.020.100.0050.91
       Valeric acid1.641.671.701.620.060.440.510.220.58
       A:P ratio2.762.732.742.950.150.050.340.010.89
      NH3-N, mg/100 g14.818.814.516.11.920.190.330.780.06
      Average pH6.326.286.266.280.050.730.310.750.73
      1 CON = control, RSC = rapeseed cake, WCR = whole cracked rapeseed, RSO = rapeseed oil.
      2 SEM for RSO; the SEM for the other treatments is the presented SEM × 0.91.
      3 Fixed effect of treatment.
      4 Linear orthogonal contrasts of treatment.
      The ammonia concentration in the rumen fluid was not influenced by the ration (P = 0.19), but a tendency for a higher concentration in RSC than in WCR (P = 0.06) was noted. The rumen pH was within the normal physiological range: an average of 6.29 for the 16 observations ranging from 5.60 to 6.91.

      Methane Production

      Adding rapeseed fat to the ration reduced the enteric CH4 production significantly in liters per day and also related to the feed intake and ECM yield (Table 6). The average reduction (L of CH4 per kg of DMI per percent fat added) was 4.6, 4.8, and 3.8% for RSC, WCR, and RSO, respectively. A tendency was observed for a reduction in CH4 per kilogram of OM digested in the whole digestive tract (P = 0.08) when fat was added, but the ration had no effect on CH4 per kilogram of totally digested carbohydrate or NDF and per kilogram of ruminally digested OM and NDF.
      Table 6Methane production.
      Ration
      CON=control, RSC=rapeseed cake, WCR=whole cracked rapeseed, RSO=rapeseed oil.
      Contrasts (P-value)
      Linear orthogonal contrasts of treatment.
      CH4productionCONRSCWCRRSOSEM
      SEM for RSO; the SEM for the other treatments is the presented SEM×0.88.
      P-value
      Fixed effect of treatment.
      CON vs. FatRSC and WCR vs. OilRSC vs. WCR
      L/d56953147846251.00.040.020.180.10
      L/kg of ECM20.419.016.916.72.030.0080.0030.110.02
      L/kg of DMI
      DMI during CH4 measurements.
      29.626.925.826.41.780.070.020.990.37
      % of GE
      GE=gross energy.
      intake
      6.325.605.315.400.030.002<0.0010.040.02
      L/kg of total digested OM46.140.339.843.03.100.220.080.360.87
      L/kg of total digested NDF15413513714610.30.200.180.390.85
      L/kg of total digested CHO
      CHO=carbohydrate calculated as OM minus fat minus CP.
      57.251.451.154.93.680.330.160.340.92
      L/kg of rumen digested OM79.869.979.179.37.490.540.570.550.26
      L/kg of rumen digested NDF15413213214012.30.450.160.600.99
      1 CON = control, RSC = rapeseed cake, WCR = whole cracked rapeseed, RSO = rapeseed oil.
      2 SEM for RSO; the SEM for the other treatments is the presented SEM × 0.88.
      3 Fixed effect of treatment.
      4 Linear orthogonal contrasts of treatment.
      5 DMI during CH4 measurements.
      6 GE = gross energy.
      7 CHO = carbohydrate calculated as OM minus fat minus CP.

      Discussion

      Intake and Milk Production

      The rations were calculated to have identical crude fat content. However, the fat content in the rapeseed cake was not as high as planned, which resulted in a lower fat content in RSC.
      We hypothesized that fat supplementation at the present levels would only reduce CH4 production with out affecting feed energy intake and milk yield. Negative effects of fat supplementation on DMI have been reported in some (
      • Harvatine K.J.
      • Allen M.S.
      Effects of fatty acid supplements on ruminal and total tract nutrient digestion in lactating dairy cows.
      ;
      • 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.
      ) but not all previous studies (
      • Johnson K.A.
      • Kincaid R.L.
      • Westberg H.H.
      • Gaskins C.T.
      • Lamb B.K.
      • Cronrath J.D.
      The effect of oilseeds in diets of lactating cows on milk production and methane emissions.
      ;
      • Moate P.J.
      • Williams S.R.O.
      • Grainger C.
      • Hannah M.C.
      • Ponnampalam E.N.
      • Eckard R.J.
      Influence of cold-pressed canola, brewers grains and hominy meal as dietary supplements suitable for reducing enteric methane emissions from lactating dairy cows.
      ). The effect of fat on DMI and GE intake depends on the level of supplementation. When cows are fed a low level of supplementation, DMI remains unaffected and GE intake increases, due to a higher energy density. When a moderate amount of fat is supplemented, DMI is reduced but GE intake is unchanged, whereas a further increase of the fat concentration in the ration can reduce the DMI to an extent where the GE intake is also reduced (
      • Grainger C.
      • Beauchemin K.A.
      Can enteric methane emissions from ruminants be lowered without lowering their production?.
      ). Neither DMI nor GE intake was affected significantly in the present study, indicating that the fat concentration in the rations (5.5–6.5% of DM) was within the nutritionally acceptable range. The rapeseed oil supplementation numerically reduced the DMI by 2.2 kg compared with CON, but the cows responded differently to the addition of oil, which is illustrated by the higher standard deviation between cows for DMI on RSO (5.3 kg) compared with the other rations (3.1 kg). This was also supported by heterogeneous variance for DMI, as a likelihood ratio test showed. Dry matter intake depression can be expected when the dietary fat concentration exceeds 6 to 7% (
      • Beauchemin K.A.
      • McGinn S.M.
      • Petit H.V.
      Methane abatement strategies for cattle: Lipid supplementation of diets.
      ). A fat concentration of 6.5% in RSO in the present study may therefore be at a borderline where it affects some, but not necessarily all, animals. This was illustrated by the pronounced depressing effect of oil supplementation on DMI for the 2 cows with the highest feed intake level (per kg of BW) compared with the 2 cows with the lowest feed intake level (values not shown).

      NDF Digestibility

      Ruminal NDF digestibility was 63% on average, which is in accordance with previous studies with similar ration compositions (
      • Lund P.
      • Weisbjerg M.R.
      • Hvelplund T.
      • Knudsen K.E.B.
      Determination of digestibility of different forages in dairy cows using indigestible NDF as marker.
      ). Rapeseed fat includes mainly monounsaturated FA and was presented at moderate concentrations in the present study; therefore, it did not affect NDF digestibility. The same effect has been observed with comparable additions of rapeseed (
      • Chelikani P.K.
      • Bell J.A.
      • Kennelly J.J.
      Effects of feeding or abomasal infusion of canola oil in Holstein cows 1. Nutrient digestion and milk composition.
      ;
      • Beauchemin K.A.
      • McGinn S.M.
      • Benchaar C.
      • Holtshausen L.
      Crushed sunflower, flax, or canola seeds in lactating dairy cow diets: Effects on methane production, rumen fermentation, and milk production.
      ).
      Few studies have compared how different physical forms of the same fat affect digestion. Even though effects of the physical form and processing of whole seeds were reported for linseed (
      • 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.
      ), this might not be valid for rapeseed.
      • Pallister S.M.
      • Smithard R.R.
      The digestion, by sheep, of diets containing different physical forms of rapeseed.
      found no effect of whole or extruded rapeseed or rapeseed oil on fiber digestion, using a comparable fat content in the control ration and slightly higher levels in the fat-supplemented rations compared with the present experiment.
      • Ferlay A.
      • Legay F.
      • Bauchart D.
      • Poncet C.
      • Doreau M.
      Effect of supply of raw or extruded rapeseed on digestion in dairy cows.
      hypothesized that extrusion ruptures cell membranes and increases the availability of fat. However, in an experiment with feeding raw and extruded rapeseed to dairy cows, no effect on digestion was noted (
      • Ferlay A.
      • Legay F.
      • Bauchart D.
      • Poncet C.
      • Doreau M.
      Effect of supply of raw or extruded rapeseed on digestion in dairy cows.
      ). This might be due to the high fat content in rapeseed compared with other oilseeds (
      • Ferlay A.
      • Legay F.
      • Bauchart D.
      • Poncet C.
      • Doreau M.
      Effect of supply of raw or extruded rapeseed on digestion in dairy cows.
      ).
      Total-tract NDF digestibility was lower than the ruminal NDF digestibility for all fat-supplemented rations. That problem has been observed earlier in connection with flow markers (
      • Faichney G.J.
      Assessment of chromic oxide as an indigestible marker for digestion studies in sheep.
      ;
      • Lund P.
      • Weisbjerg M.R.
      • Hvelplund T.
      • Knudsen K.E.B.
      Determination of digestibility of different forages in dairy cows using indigestible NDF as marker.
      ) and different types of duodenal cannulas (
      • Stensig T.
      • Robinson P.H.
      Digestion and passage kinetics of forage fiber in dairy cows as affected by fiber-free concentrate in the diet.
      ). The hindgut digestibility of NDF was higher for RSC compared with the other rations, but still within the physiological range (
      • Huhtanen P.
      • Ahvenjärvi S.
      • Weisbjerg M.R.
      • Nørgaard P.
      Digestion and passage of fibre in ruminants.
      ).

      Fat Digestibility

      The fat digestibility in the rumen was negative for all rations.
      • Schmidely P.
      • Glasser F.
      • Doreau M.
      • Sauvant D.
      Digestion of fatty acids in ruminants: A meta-analysis of flows and variation factors. 1. Total fatty acids.
      reported, in a meta-analysis, a slightly positive FA balance from feed to duodenum (duodenal flow – intake). However, they found a net disappearance of FA with higher intake (50–120 g of FA/kg of DMI). In the present study, RSO only slightly exceeded the 50 g of FA/kg of DM, and the other rations had a lower FA content.
      • Scollan N.D.
      • Dhanoa M.S.
      • Choi N.J.
      • Maeng W.J.
      • Enser M.
      • Wood J.D.
      Biohydrogenation and digetion of long chain fatty acids in steers fed on different sources of lipid.
      reported a positive net FA flow to the duodenum with an increase in rumen FA balance by 21.8% when steers receive rations with 60 g/kg of DM fat, which fits well with the present experiment where crude fat increased by 22.7% from feed to duodenum. As observed earlier (
      • Doreau M.
      • Chilliard Y.
      Digestion and metabolism of dietary fat in farm animals.
      ), a net FA synthesis takes place in the rumen at low to moderate fat contents.
      The digestibility of fat in the small intestine is highly variable;
      • Doreau M.
      • Chilliard Y.
      Digestion and metabolism of dietary fat in farm animals.
      reported variations from 55 to 92% in a literature survey. The digestibility of fat in the small intestine depends on the chain length and degree of unsaturation (
      • Weisbjerg M.R.
      • Hvelplund T.
      • Børsting C.F.
      Digestibility of fatty-acids in the gastrointestinal tract of dairy cows fed with tallow or saturated fats rich in stearic acid or palmitic acid.
      ;
      • Doreau M.
      • Chilliard Y.
      Digestion and metabolism of dietary fat in farm animals.
      ). The present digestibility for fat with mainly C18 FA was between 70 and 75%, which is in agreement with earlier reported values (
      • Schmidely P.
      • Glasser F.
      • Doreau M.
      • Sauvant D.
      Digestion of fatty acids in ruminants: A meta-analysis of flows and variation factors. 1. Total fatty acids.
      ). With increasing fat consumption, the intestinal digestibility decreases because the absorptive capacity is limited, but absorption of FA can be higher than 1 kg/d (
      • Doreau M.
      • Chilliard Y.
      Digestion and metabolism of dietary fat in farm animals.
      ). As the highest FA intake in the present experiment was 892 g/d, fat absorption was not affected by treatment.

      VFA and NH3

      Molar concentration of VFA was in agreement with earlier studies with dairy cows receiving rapeseed fat supplementation (
      • Chelikani P.K.
      • Bell J.A.
      • Kennelly J.J.
      Effects of feeding or abomasal infusion of canola oil in Holstein cows 1. Nutrient digestion and milk composition.
      ;
      • Beauchemin K.A.
      • McGinn S.M.
      • Benchaar C.
      • Holtshausen L.
      Crushed sunflower, flax, or canola seeds in lactating dairy cow diets: Effects on methane production, rumen fermentation, and milk production.
      ). The higher acetate to propionate ratio for RSO compared with CON is surprising, because fat addition is believed to inhibit NDF but not starch fermentation, and therefore the propionic acid proportion was expected to increase as observed by
      • Harvatine K.J.
      • Allen M.S.
      Effects of fatty acid supplements on ruminal and total tract nutrient digestion in lactating dairy cows.
      . According to fermentation stoichiometry, a shift in fermentation pattern toward acetate, as observed for RSO, should result in enhanced CH4 production (
      • Boadi D.
      • Benchaar C.
      • Chiquette J.
      • Masse D.
      Mitigation strategies to reduce enteric methane emissions from dairy cows: Update review.
      ). However, cows fed RSO produced less CH4 than on CON. The higher propionate proportion could be due to reduction in total VFA production because less substrate was fermented in the rumen, or because differences were buffered, as the values shown are averages of day and night time measurements.
      The ammonia concentration in the rumen is considered to be the balance between entry sources (degradable feed N, N recycling) and outputs (incorporation into microbes, N absorption, ammonia N outflow;
      • Doreau M.
      • Ferlay A.
      Effect of dietary lipids on nitrogen metabolism in the rumen: A review.
      ). In agreement with previous studies (
      • Oldick B.S.
      • Firkins J.L.
      Effects of degree of fat saturation on fiber digestion and microbial protein synthesis when diets are fed twelve times daily.
      ), fat addition itself did not affect the protein digestion in the rumen.

      Methane Production

      The CH4 production in absolute numbers (L/d), as well as in relation to intake, was comparable to previous studies (
      • Beauchemin K.A.
      • Kreuzer M.
      • O’Mara F.
      • McAllister T.A.
      Nutritional management for enteric methane abatement: A review.
      ;
      • Moate P.J.
      • Williams S.R.O.
      • Grainger C.
      • Hannah M.C.
      • Ponnampalam E.N.
      • Eckard R.J.
      Influence of cold-pressed canola, brewers grains and hominy meal as dietary supplements suitable for reducing enteric methane emissions from lactating dairy cows.
      ). The reduction in CH4 loss expressed as the proportion of GE intake when rapeseed fat was supplemented was less than found by
      • Beauchemin K.A.
      • McGinn S.M.
      • Benchaar C.
      • Holtshausen L.
      Crushed sunflower, flax, or canola seeds in lactating dairy cow diets: Effects on methane production, rumen fermentation, and milk production.
      with dairy cows (18 vs. 14% in the present study) or
      • Machmüller A.
      • Ossowski D.A.
      • Kreuzer M.
      Comparative evaluation of the effects of coconut oil, oilseeds and crystalline fat on methane release, digestion and energy balance in lambs.
      with lambs (22%). This was most likely due to a higher fat concentration (
      • Beauchemin K.A.
      • McGinn S.M.
      • Benchaar C.
      • Holtshausen L.
      Crushed sunflower, flax, or canola seeds in lactating dairy cow diets: Effects on methane production, rumen fermentation, and milk production.
      ) and a higher forage proportion (
      • Machmüller A.
      • Ossowski D.A.
      • Kreuzer M.
      Comparative evaluation of the effects of coconut oil, oilseeds and crystalline fat on methane release, digestion and energy balance in lambs.
      ) compared with rations in the present study. The reduction in CH4 per kg of DMI compared with CON per percentage fat added was highest for WCR (4.8%) followed by 4.6 for RSC and 3.8% for RSO. The present reductions per kg of DMI were not as high as the 5.6% reduction found in a review by
      • Beauchemin K.A.
      • Kreuzer M.
      • O’Mara F.
      • McAllister T.A.
      Nutritional management for enteric methane abatement: A review.
      , but close to the values presented in the review by
      • Grainger C.
      • Beauchemin K.A.
      Can enteric methane emissions from ruminants be lowered without lowering their production?.
      ; between 4.7 and 5.1% depending on fat level in the ration).
      Earlier studies with rapeseed found contradictory results;
      • Martin C.
      • Pomiès D.
      • Ferlay A.
      • Rochette Y.
      • Martin B.
      • Chilliard Y.
      • Morgavi D.P.
      • Doreau M.
      Methane output and rumen microbiota in dairy cows in response to long-term supplementation with linseed or rapeseed of grass silage- or pasture-based diets.
      added 3% fat as extruded rapeseed to a dairy cow ration without finding any effect on CH4.
      • Beauchemin K.A.
      • McGinn S.M.
      Methane emissions from beef cattle: Effects of fumaric acid, essential oil, and canola oil.
      found a significant reduction in CH4 loss as proportion of GE when feeding a ration with 4.6% rapeseed oil added to heifers, but no effect per kg of DMI. In studies using dairy cows,
      • Moate P.J.
      • Williams S.R.O.
      • Grainger C.
      • Hannah M.C.
      • Ponnampalam E.N.
      • Eckard R.J.
      Influence of cold-pressed canola, brewers grains and hominy meal as dietary supplements suitable for reducing enteric methane emissions from lactating dairy cows.
      found a significant reduction in CH4 per kg of DMI when adding 2.6% fat to the ration as a rapeseed-hominy meal mix, and
      • Beauchemin K.A.
      • McGinn S.M.
      • Benchaar C.
      • Holtshausen L.
      Crushed sunflower, flax, or canola seeds in lactating dairy cow diets: Effects on methane production, rumen fermentation, and milk production.
      found a reduction for 1.7% fat added as crushed rapeseed compared with the control ration.
      • Beauchemin K.A.
      • McGinn S.M.
      Methane emissions from beef cattle: Effects of fumaric acid, essential oil, and canola oil.
      observed a feed intake depression and therefore, no effect on CH4 per kg of DMI was observed when heifers received rapeseed oil. In a later study,
      • Beauchemin K.A.
      • McGinn S.M.
      • Benchaar C.
      • Holtshausen L.
      Crushed sunflower, flax, or canola seeds in lactating dairy cow diets: Effects on methane production, rumen fermentation, and milk production.
      compared the CH4-reducing properties of different oil seeds and emphasized that rapeseed did not depress DMI in contrast to other oil seeds. The present results and those of
      • Moate P.J.
      • Williams S.R.O.
      • Grainger C.
      • Hannah M.C.
      • Ponnampalam E.N.
      • Eckard R.J.
      Influence of cold-pressed canola, brewers grains and hominy meal as dietary supplements suitable for reducing enteric methane emissions from lactating dairy cows.
      confirm that rapeseed fat does not always depress feed intake. The depressed feed intake in heifers found by
      • Beauchemin K.A.
      • McGinn S.M.
      Methane emissions from beef cattle: Effects of fumaric acid, essential oil, and canola oil.
      was probably due to the high fat supplementation level.
      We hypothesized that fat from seeds is released more slowly in the rumen, and therefore might affect both CH4 production and digestion differently than 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.
      found a stronger effect on CH4 production the higher the fat content in the supplement (i.e., extruded linseed being less effective than whole linseed and whole linseed less effective than linseed oil). Conversely,
      • Beauchemin K.A.
      • McGinn S.M.
      • Petit H.V.
      Methane abatement strategies for cattle: Lipid supplementation of diets.
      compared sunflower seeds and sunflower oil and found, in agreement with the present study, no difference in the CH4-depressing properties for different physical forms of the same fat source. The linseed fat in the study by
      • 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.
      was supplemented at a higher level, and linseed FA have a higher degree of unsaturation than rapeseed or sunflower. Furthermore, the different physical forms in the study by
      • 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.
      were confounded with fat contents, as the oil ration included 1.4 and 1.6% per kg of DM more fat than extruded and crude seed rations, respectively.
      The fat concentration in rapeseed cake was lower than planned, which resulted in a lower fat content in RSC than in WCR and RSO. A meta-analysis by
      • Beauchemin K.A.
      • Kreuzer M.
      • O’Mara F.
      • McAllister T.A.
      Nutritional management for enteric methane abatement: A review.
      showed a linear relationship between the percentage of fat added and the reduction in CH4. A rapeseed cake probably would have been more effective in reducing CH4 if it had had a higher fat concentration.
      Fat supplementation depresses CH4 production by lowering the quantity of OM degraded in the rumen, by influencing the microbial activity and ecosystem, and, to a very minor extent, by biohydrogenation of unsaturated FA (
      • Johnson K.A.
      • Johnson D.E.
      Methane emissions from cattle.
      ). Although rumen digestibilities and VFA composition were not altered, the numeric reduction in CH4 per kg of digested carbohydrate indicates that fermentation pathways were affected by fat supplementation in addition to the effect of fat being nonfermentable.
      An optimal rumen microbial ecosystem is a prerequisite for efficient milk production, and it is well known that methanogens and fibrolytic bacteria may be hampered by addition of fat. The methanogen population of the rumen was studied in parallel to the present study by
      • Poulsen M.
      Mitigation of methane emission from Holstein dairy cows: Effects of dietary manipulation on bacterial and methanogen communities.
      ; despite the fact that fat supplementation reduced the CH4 production, the total abundance of the methanogens in the rumen was unaffected by the ration.

      Conclusions

      Supplementation with rapeseed fat up to 6 to 6.5% fat of ration of DM reduced GE loss of enteric CH4 by 14% without compromising the NDF digestibility or milk production. Adding fat by supplementing rapeseed in different forms decreased CH4 production and numerically increased milk and ECM yields in the present study, resulting in less CH4 per kg of product.

      Acknowledgements

      The work was funded by the Danish Ministry of Food, Agriculture and Fisheries, Mælkeafgiftsfonden (Aarhus, Denmark) , and Aarhus University . The authors thank Torkild Jakobsen (Aarhus University, Foulum, Denmark) for skillful assistance during the experiment and Ingolf Nielsen (DLG Food Oil, Dronninglund, Denmark) for providing the rapeseed feeds.