Patent Publication Number: US-11391710-B2

Title: Prediction method for methane (CH4) emission of a dairy cow

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a U.S. National Phase Application of International Patent Application No. PCT/EP2018/073327, filed Aug. 30, 2018, which claims the benefit of priority to European Patent Application No. 17189083.3, filed Sep. 1, 2017, the entire contents of which are hereby incorporated by reference herein. 
     FIELD OF THE INVENTION 
     The present invention relates to a method for predicting the methane (CH 4 ) emission of a dairy cow comprising: determining the total amount of milk of a dairy cow per one day (a); determining the energy corrected milk value (ECM) of the milk of the dairy cow of the same day as in a) (b); determining the percentage amount of saturated fatty acids (SFAs) of the total milk fat of the dairy cow of the same day as in a) and the percentage amount of stearic acid (C18:0) of the total milk fat of the dairy cow of the same day as in a) (c); and calculating the daily amount of methane emitted by the dairy cow based on the ECM as determined according to (b), the percentage amount of SFAs of the total milk fat as determined according to (c) and the percentage amount of C18:0 of the total milk fat as determined according to (c). 
     BACKGROUND 
     Livestock contributes about 18% to global anthropogenic greenhouse gas emission (Hristov et al. 2013), wherein cattle represent a main portion of livestock. Methane mitigation opportunities by feeding and genetic selection have been reported to range from 2.5 to 19% in cattle (Knapp et al. 2014; Pickering et al. 2015). Although breeding low-methane emitting cows seems to be possible through genetic selection, this requires determination of methane emission in large numbers of individuals using respiration chambers or the SF6 method. Large scale measurements in respiration chambers, although the gold standard for methane quantification, are not feasible (Hill et al. 2016) for farm animals. Biochemical relationships between rumen fermentation, CH 4  production, and milk fatty acid composition suggest the usefulness of milk fatty acid profiles to predict methane emission (liter/day) (Knapp et al. 2014; van Lingen et al. 2014). There are several reports on the relationship between milk fatty acids and methane emission as well as on equations predicting methane yield defined as liter of CH 4  per kg dry matter intake and methane intensity defined as liter of CH 4  per kg energy corrected milk (Castro-Montoya et al. 2016; Dijkstra et al. 2011; van Lingen et al. 2014). 
     Quantification of fatty acids is usually done by gas chromatography, the standard procedure to measure the concentration of a large number of fatty acids with high accuracy and precision. However, gas chromatography is unsuitable for routine milk fatty acids determination, but infrared spectroscopy reliably predicted major milk fatty acids and groups of fatty acids (De Marchi et al. 2014; Soyeurt et al. 2011). Infrared spectra from milk are inexpensively recordable on a large scale but have only a moderate power for predicting minor single milk fatty acids (Van Gastelen and Dijkstra 2016; De Marchi et al., 2014). 
     Although earlier studies reported prediction models based on global infrared milk spectra (Dehareng et al. 2012; Vanlierde et al. 2015), there is so far no published methane prediction equation based on fatty acid composition derived from infrared spectroscopy. 
     Dry matter intake is the main determinant for CH 4  emission (Hristov et al. 2013; Knapp et al. 2014), but cow-individual feed intake information is not available on farms. The object of the present invention was therefore the provision of a method for predicting the methane emission of a dairy cow which does not require knowledge of the daily dry matter intake. The method should be usable for dairy cows, of which feed intake on an individual basis is not known. 
     SUMMARY 
     According to the present invention, it was found that this object can be solved by a method for predicting the methane (CH 4 ) emission of a dairy cow comprising:
     a) determining the total amount of milk (M) of a dairy cow per one day;   b) determining the energy corrected milk value (ECM) of the milk of the dairy cow of the same day as in a);   c) determining the percentage amount of saturated fatty acids (SFAs) of the total milk fat of the dairy cow of the same day as in a) and the percentage amount of stearic acid (C18:0) of the total milk fat of the dairy cow of the same day as in a);   d) calculating the daily amount of methane emitted by the dairy cow based on the ECM as determined according to (b), the percentage amount of SFAs of the total milk fat as determined according to (c) and the percentage amount of C18:0 of the total milk fat as determined according to (c).   

    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  illustrates measured CH 4  emission (x axis) and predicted CH 4  emission (y axis) derived from the multiple regression equation for individual experimental diets are shown (grass silage without linseed, corn silage without linseed, grass silage with linseed and corn silage with linseed) considering both periods (filled circle=period A; open circle=period B). The graphical representation highlights the effectiveness of the deduced prediction model. A Pearson correlation coefficient (r) and the probability (p) are given. 
     
    
    
     DETAILED DESCRIPTION 
     In a preferred embodiment, the calculation of the daily amount of methane emitted by the dairy cow according to (d) is based on regression equation 1:
 
CH 4  [liter/day]=−1363.7+9.58×ECM [kg/day]+18.5×SFAs [%]+32.4×C18:0[%]   —equation 1—
 
In contrast to cow-individual intake information, individual records of energy corrected milk, which is highly related to dry matter intake (Hristov et al. 2013), are readily available. It was found that CH 4  emission could be predicted from milk fatty acids determined by infrared spectroscopy and that the integration of energy corrected milk improves the prediction power.
 
     The method of the present invention, preferably based on regression equation 1, can be used irrespective of the diet type and applies to ad libitum feeding, meaning that the dairy cow, who&#39;s milk is used for the method, has been under an ad libitum feeding regime. 
     The total amount of milk of a dairy cow per one day (M) is determined by means known in the art. For example, the milk obtained by milking can be collected and the weight thereof can be determined, preferably by measuring the total volume of the milk of the dairy cow per day and calculating, preferably under consideration of the milk&#39;s density, its total weight. M is preferably expressed in kg per day. It goes without saying that all determination steps (a), (b) and (c) and also the calculation step (d) are done based on milk or a milk sample respectively, which have been obtained by milking. Since milking is the common central step in milk industry, milk and milk samples respectively are available per se. Thus, the method is typically an ex vivo method carried out on existing milk of a dairy cow and does not require any physical interaction with said dairy cow. 
     In the context of the present invention the term “percentage” always refers to weight percentage and “%” represents “weight-%”. Determining the percentage amount of saturated fatty acids in the total milk fat of the dairy cow of the same day as in a) and the percentage amount of stearic acid of the total milk fat of the dairy cow of the same day as in a) can be done by means and methods known in the art. Preferably, infrared spectroscopy is applied. Determining the percentage amount of saturated fatty acids (SFAs) of the total milk fat of the dairy cow of the same day as in a) and the percentage amount of stearic acid (C18:0) of the total milk fat of the dairy cow of the same day as in a) by infrared spectroscopy means that an infrared spectrum of the milk of the dairy cow is recorded and the respective values such as percentage amount of saturated fatty acids and separately the percentage amount of stearic acid, each based on the total milk fat, are determined from the spectrum by means and methods known to the skilled person. For example, the determination can be done using a MilcoScan milk analysator, for example a MilcoScan FT6000 (Foss, Hilleroed, Denmark), which is based on FTIR technology and works with the mid-Infrared region of the spectrum from 3-10 μm corresponding to 1000-5000 cm −1 . The percentage amount of saturated fatty acids of the total milk fat according to (c) is at least the sum of the percentage amounts of capric acid (C10:0), myristic acid (C14:0), palmitic acid (C16:0) and stearic acid (C18:0), preferably the sum of at least the percentage amounts of butanoic acid (C4:0), hexanoic acid (C6:0), octanoic acid (C8:0), capric acid (C10:0), lauric acid (C12:0), iso-lauric acid (C12:0-iso), anteiso-lauric acid (C12:0-anteiso), tridecanoic acid (C13:0), myristic acid (C14:0), iso-myristic acid (C14:0-iso), anteiso-myristic acid (C14:0-anteiso), pentadecanoic acid (C15:0), iso-pentadecanoic acid (C15:0-iso), palmitic acid (C16:0), iso-palmitic acid (C16:0-iso), anteiso-palmitic acid (C16:0-anteiso), heptadecanoic acid (C17:0), iso-heptadecanoic acid (C17:0-iso), anteiso-heptadecanoic acid (C17:0-anteiso), stearic acid (C18:0), nonadecanoic acid (C19:0), eicosanoic acid (C20:0) and docosanoic acid (C22:0) of the total milk fat. The percentage amount of stearic acid (C18:0) of the total milk fat, is quantified individually in addition. The expression “total milk fat” means all fat which is detectable in milk as done, for example, in milk testing laboratories. Preferably, the total milk fat is determined by infrared spectroscopy according to DIN ISO 9622 (2017-04), preferably at wave lengths in the range of from 25-2.5 μm. Alternatively, total milk fat can be determined by chemical methods, for example, Röse-Gottlieb- (RG-), Schmidt-Bondzynski-Ratzlaff- (SBR-) or Weibull-Berntrop- (WB-) method. 
     The energy corrected milk value (ECM) of the milk of the dairy cow of the same day as in a) is also determined by a method known in the art, i.e. the ECM according to (b) is determined according to regression equation 2 (Spiekers et al. 2004):
 
ECM [kg/day]=((1.05+0.38× F  [%]+0.21× P  [%])/3.28)× M  [kg/day]   —equation 2—
 
wherein:
     “M” means the total amount of milk of a dairy cow per one day as determined in (a);   “F” means the percentage amount of total milk fat contained in the milk of the dairy cow per one day as determined in (a); and   “P” means the percentage amount of protein contained in the milk of a dairy cow per one day as determined in (a).   

     “F” and “P” can be determined by methods known in the art. For example, the percentage amount of total milk fat contained in the milk of the dairy cow per one day can also be determined by infrared spectroscopy. Methods and means for determining the percentage amount of total milk fat contained in the milk of the dairy cow are known in the art. For example, the determination can also be done using a MilcoScan milk analysator, for example a MilcoScan FT6000 (Foss, Hilleroed, Denmark), which is based on Fourier Transform InfraRed (FTIR) analysis. It preferably works with the mid-Infrared region of the spectrum from 3-10 μm corresponding to 1000-5000 cm −1 . The percentage amount of protein contained in the milk of a dairy cow, can be determined by, for example, by a MilcoScan FT6000 (Foss, Hilleroed, Denmark), which is based on FTIR technology and works with the mid-Infrared region of the spectrum from 3-10 μm corresponding to 1000-5000 cm −1 . 
     Below, the work done by the inventors in order to arrive at regression equation 1 is outlined in more detail. 
     Materials and Methods 
     1. Animals, Experimental Design and Diets 
     Ethical approval of the study was obtained (permission no. 7221.3-1-014/14). Twenty half-sib German Holstein cows (106±28 days in milk, 29.5±7.7 kg energy corrected milk per day, 580±57 kg bodyweight, mean±standard deviation) in second (15 cows) or third lactation (5 cows) were used. Cows were kept in tie-stalls, with water and total mixed rations for ad libitum intake. Two isoenergetic total mixed rations, in which the major forage component was either corn silage or grass silage, supplemented with or without linseed were used (7 MJ NEL/kg dry matter; Table 1). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Ingredients and chemical composition of experimental total mixed ration 
               
               
                 consisting of basal rations based on grass silage or corn silage with and  
               
               
                 without linseed supplementation (means ± standard deviation; n = 5).  
               
               
                 Dry matter, fresh matter, ash-free neutral detergent fiber, acid detergent  
               
               
                 fiber, and net energy for lactation are abbreviated as DM, FM, aNDF, ADF  
               
               
                 and NE L . The linseed product (Omegalin 60; Spezialfutter Neuruppin 
               
               
                 GmbH und Co. KG, Neuruppin, Germany) contained 60% of extruded  
               
               
                 linseed (Tradilin ®) and 40% of wheat bran with 25% crude fat including  
               
               
                 6% palmitic acid (C16:0), 18.4% oleic acid (C18:1cis9), 18.5% linoleic  
               
               
                 acid (C18:2cis9, cis12) and 55% linolenic acid (C18:3cis9, cis12,  
               
               
                 cis15). Concentrate (MF 2000; Vollkraft Mischfutterwerke GmbH,  
               
               
                 Güstrow, Germany) consisted of 33% extracted soy meal, 20% corn,  
               
               
                 17% wheat gluten, 8% extracted rapeseed meal, 5% sugar beet  
               
               
                 pulp, 2% sodium hydrogen carbonate, 1.3% calcium carbonate, 0.2% 
               
               
                 sodium chloride; “—” not added. 
               
            
           
           
               
               
               
            
               
                 Ingredient 
                 Grass silage 
                 Corn silage 
               
            
           
           
               
               
               
               
               
            
               
                 (g/kg DM) 
                 No linseed 
                 Linseed 
                 No linseed 
                 Linseed 
               
               
                   
               
               
                 Grass silage 
                 343 ± 31.0 
                 380 ± 15.0 
                 122 ± 21.5 
                 142 ± 34.3 
               
               
                 Corn silage 
                 181 ± 33.2 
                 195 ± 37.9 
                 445 ± 46.7 
                 452 ± 44.0 
               
               
                 Straw, barley 
                 52 ± 7.0 
                  61 ± 23.2 
                 40 ± 7.2 
                  34 ± 10.3 
               
               
                 Grass hay 
                  81 ± 12.0 
                  94 ± 22.0 
                 43 ± 3.1 
                 44 ± 3.0 
               
               
                 Corn, ground 
                 —   — 
                 —   — 
                 93 ± 9.2 
                  25 ± 27.2 
               
               
                 Soy extract 
                  25 ± 26.8 
                  40 ± 19.2 
                 114 ± 14.2 
                 87 ± 9.2 
               
               
                 meal 
                   
                   
                   
                   
               
               
                 Barley, ground 
                 100 ± 17.9 
                 —   — 
                 —   — 
                 —   — 
               
               
                 Wheat, ground 
                 101 ± 9.8  
                 —   — 
                 —   — 
                 —   — 
               
               
                 Linseed 
                 —   — 
                 143 ± 11.4 
                 —   — 
                 137 ± 9.9  
               
               
                 product 
                   
                   
                   
                   
               
               
                 Concentrate 
                 109 ± 20   
                  79 ± 13.0 
                 129 ± 7.3  
                  65 ± 32.6 
               
               
                 Mineral/vita- 
                  9 ± 0.5 
                 10 ± 0.6 
                 10 ± 0.6 
                 10 ± 0.6 
               
               
                 min mix 
                   
                   
                   
                   
               
               
                 Calcium 
                 —   — 
                 —   — 
                 4.1 ± 0.3  
                  4 ± 0.3 
               
               
                 carbonate 
                   
                   
                   
                   
               
               
                 DM 
                 468 ± 33.0 
                 452 ± 450  
                 446 ± 32.0 
                 439 ± 33.0 
               
               
                 (g/kg of FM) 
                   
                   
                   
                   
               
            
           
           
               
            
               
                 Nutrients (g/kg of DM) 
               
            
           
           
               
               
               
               
               
            
               
                 Crude ash 
                 69.9 ± 2.8   
                 75.8 ± 2.5   
                 66.1 ± 3.0   
                 65.7 ± 4.2   
               
               
                 Crude protein 
                 161 ± 9.0  
                 173 ± 14.4 
                 169 ± 11.7 
                 163 ± 11.1 
               
               
                 Crude fiber 
                 166 ± 7.9  
                 186 ± 7.1  
                 155 ± 3.3  
                 161 ± 4.5  
               
               
                 Crude fat 
                 28.1 ± 1.7   
                 58.3 ± 7.8   
                 29.8 ± 2.4   
                 56.4 ± 6.5   
               
               
                 Sugar 
                 51.3 ± 15.7  
                 50.1 ± 16.2  
                 31.4 ± 7.9   
                 30.3 ± 11.1  
               
               
                 Starch 
                 218 ± 12.0 
                 110 ± 18.7 
                 261 ± 25.4 
                 216 ± 34.5 
               
               
                 aNDF 
                 371 ± 18.1 
                 410 ± 12.9 
                 330 ± 10.9 
                 352 ± 9.2  
               
               
                 ADF 
                 198 ± 9.8  
                 224 ± 7.9  
                 186 ± 5.4  
                 194 ± 5.0  
               
               
                 NE L  (MJ/kg of 
                 6.9 ± 0.2  
                 6.9 ± 0.1  
                 7.0 ± 0.1  
                 7.1 ± 0.1  
               
               
                 DM) 
               
               
                   
               
            
           
         
       
     
     Randomly selected 10 cows were fed on corn silage based diets for 5 weeks without and for another 5 weeks with linseed supplementation in randomized order (Periods A and B: experimental weeks 1 to 5, and 7 to 11, respectively; step-wise change of rations in experimental weeks 0 and 6). The other 10 cows were allocated randomly to grass silage diets without or with linseed supplementation for 5 weeks each. The study was conducted in 5 experimental runs each with 4 cows. All four diets were fed simultaneously in each run. Cows were fed twice daily at 0730 and 1730 hours; dry matter intake was recorded daily. Three cows feeding on the grass silage based diet supplemented with linseed had to be removed because of illness or feed refusal. The nutrient contents of the diets were in line with recommendations of the German Society of Nutritional Physiology (GfE 2001). 
     2. Determination of Feed and Milk Composition and Methane Production 
     Analyses of feed nutrient composition (Table 1) were performed by a certified feed laboratory. During the respiration measurements aliquots of one evening and morning milking were pooled. Milk fat and fatty acid composition was analysed using infrared spectroscopy (MilcoScan FT6000; Foss, Hilleroed, Denmark) by a commercial laboratory (Comité du Lait, Battice, Belgium). Saturated and unsaturated fatty acids, as well as isomers of C18:1cis and trans and ω-3 fatty acids were quantified as groups (Table 2). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Composition of groups of milk fatty acids analyzed  
               
               
                 using infrared spectroscopy. 
               
            
           
           
               
               
            
               
                 Group 
                 Fatty acids 
               
               
                   
               
               
                 Saturated fatty acids 
                 C4:0 
               
               
                   
                 C6:0 
               
               
                   
                 C8:0 
               
               
                   
                 C10:0 
               
               
                   
                 C12:0 
               
               
                   
                 C12:0-iso 
               
               
                   
                 C12:0-anteiso 
               
               
                   
                 C13:0-iso 
               
               
                   
                 C14:0 
               
               
                   
                 C14:0-iso 
               
               
                   
                 C14:0-anteiso 
               
               
                   
                 C14:0 
               
               
                   
                 C15:0 
               
               
                   
                 C15:0-iso 
               
               
                   
                 C16:0 
               
               
                   
                 C16:0-iso 
               
               
                   
                 C16:0-anteiso 
               
               
                   
                 C17:0 
               
               
                   
                 C17:0-iso 
               
               
                   
                 C17:0-anteiso 
               
               
                   
                 C18:0 
               
               
                   
                 C19:0 
               
               
                   
                 C20:0 
               
               
                   
                 C22:0 
               
               
                 Unsaturated fatty acids 
                 C10:1 
               
               
                   
                 C12:1cis of summed isomers 
               
               
                   
                 C14:1cis of summed isomers 
               
               
                   
                 C16:1cis of summed isomers 
               
               
                   
                 C16:1trans of summed isomers 
               
               
                   
                 C18:1cis9 
               
               
                   
                 C18:1cis11 
               
               
                   
                 C18:1cis12 
               
               
                   
                 C18:1trans6 + 7 + 8 + 9 + 10 + 11 isomers 
               
               
                   
                 C18:1trans12 + 13 + 14 isomers 
               
               
                   
                 C18:1cis13 + cis14 + trans16 isomers 
               
               
                   
                 C20:1cis9 
               
               
                   
                 C20:1cis11 
               
               
                   
                 C18:2cis9, trans11 
               
               
                   
                 C18:3cis9, cis12, cis15 
               
               
                   
                 C20:3 ω-6 
               
               
                   
                 C20:4 ω-6 
               
               
                   
                 C20:5 ω-3 
               
               
                   
                 C22:5 ω-3 
               
               
                 Monounsaturated fatty acids 
                 % of unsaturated fatty acids 
               
               
                 Polyunsaturated fatty acids 
                 % of unsaturated fatty acids 
               
               
                 C18:1cis 
                 Total sum of isomers 
               
               
                 C18:1trans 
                 Total sum of isomers (6 to 14) 
               
               
                 Omega-3 fatty acids 
                 C18:3cis9, cis12, cis15 
               
               
                   
                 C20:5 ω-3 
               
               
                   
                 C22:5 ω-3 
               
               
                   
               
            
           
         
       
     
     Mono- and polyunsaturated fatty acids were expressed as percentage of unsaturated fatty acids in total milk fat. Palmitic acid (C16:0) and stearic acid (C18:0) were quantified individually each as percentage in total milk fat. 
     Individual methane emission was quantified in experimental weeks 5 and 11 for two subsequent 24-hours periods (0700 to 0700 hours; i.e. from 07.00 to 07.00 hours next day) using respiration chambers, after cows have been placed in the chambers for &gt;15 h with their respective rations (Demo et al. 2009). The CH 4  recovery in the chambers was 99.8%. In the chambers temperature and relative humidity were 15° C. and 60%, respectively, and light was on from 0600 to 1900 hours. Feed intake was recorded continuously and summarized over 24 hours. Residual feed was removed from the trough before cows were offered fresh feed at 0730 and 1730 hours. Cows were milked at 0630 and 1630 hours. 
     3. Calculations and Statistical Analyses 
     3.1 Analysis of Variance 
     Data were analysed using SAS (SAS/STAT 9.3; SAS Institute Inc. 2011, Cary, N.C.). The dependent variables CH 4  emission, dry matter intake and energy corrected milk averaged per 24 hours were analyzed with repeated measures analysis of variance using PROC MIXED. The model contained the fixed effects of basal ration (corn silage or grass silage based), linseed supplementation (with and without), the interaction effect between basal ration and linseed supplementation as well as effects of experimental runs (1-5), periods (A or B) and the order (linseed in period A or B first). The covariance structure was Compound Symmetry. Effects were considered significant at P≤0.05. There were no interaction effects between basal ration and linseed supplementation and data are presented for basal ration and linseed supplementation categories (Table 3). 
                     TABLE 3                  Dry matter intake (DMI), energy corrected milk (ECM), methane emission(CH 4 )       per day, per energy corrected milk (CH 4 /ECM) and per dry matter intake       (CH 4 /DMI) and milk fatty acid composition of lactating cows housed in respiration       chambers receiving rations based on grass silage (GS) or corn silage (CS)       with (LS) and without (L0) linseed supplementation. Unsaturated fatty       acids are abbreviated as UFA. There was no significant interaction       between basal ration and linseed supplementation (P &gt; 0.14).                                 Linseed           Basal ration   supplementation                                                     GS   CS   SE   P, ≤   L0   LS   SE   P, ≤                                                             DMI (kg/day)   15.4   18.3   0.83   0.024   17.4   16.3   0.66   0.061       ECM (kg/day)   22.3   28.7   1.19   0.002   25.8   25.2   0.92   0.334       Milk fat (g/l)   46.0   40.5   1.67   0.032   44.1   42.4   1.36   0.206       Methane       production       CH 4  (l/day)   480   544   25.11   0.089   547   477   20.24   0.002       CH 4 /ECM (l/kg)   22.3   19.0   1.08   0.050   21.7   19.5   0.85   0.006       CH 4 /DMI (l/kg)   32.3   29.6   1.73   0.289   31.9   30.0   1.27   0.011       Fatty acid (% of       total milk fat)       Saturated   65.7   66.3   1.07   0.684   72.1   59.8   0.84   0.001       Unsaturated   34.4   33.7   1.07   0.684   27.9   40.2   0.84   0.001       Mono- (% UFA)   87.2   86.6   0.23   0.060   87.2   86.7   0.20   0.053       Poly- (% UFA)   12.8   13.4   0.23   0.060   12.8   13.4   0.20   0.053       C16:0   27.8   27.8   0.89   0.988   32.2   23.3   0.75   0.001       C18:0   12.6   12.8   0.27   0.500   10.5   15.0   0.24   0.001       C18:1cis   22.9   22.5   0.76   0.735   18.7   26.7   0.60   0.001       C18:1trans   3.83   3.90   0.18   0.788   2.58   5.15   0.15   0.001       ω -3   0.80   0.82   0.02   0.556   0.70   0.92   0.02   0.001                    
3.2 Regression and Correlations
 
     Methane emission and CH 4  intensity were estimated using multiple linear regression models with the stepwise explanatory-independent variable selection method for each diet separately. Similarly, methane emission was also estimated by using combined data categorized by basal diets or linseed supplementation, as well as for all available data across 4 diets. For the regression analysis, two data sets with different independent explanatory variables were made available. Explanatory variables of the stepwise variable selection were considered if P≤0.15. The first data set included milk fatty acid concentrations only, whereas the second one additionally included energy corrected milk yield. Agreement between predicted and measured methane emissions was assessed by correlation analysis. 
     Results and Discussion 
     1. Feed Intake, Performance, and Methane Emission 
     Cows fed the corn silage based diets tended to show a greater CH 4  emission as compared to cows fed the grass silage based diets (P=0.089; Table 3). In contrast, CH 4  yield was similar among basal diets, and CH 4  intensity was lower (P=0.050) by about 15% with corn as compared to grass silage based rations. Methane yield of dairy cows fed grass silage and corn silage based diets and/or fat additives reported by others (Benchaar et al. 2015; Martin et al. 2016; Mohammed et al. 2011) were comparable to the present results. The lowering of CH 4  intensity in corn silage based diets was mainly due to higher levels of energy corrected milk (Table 3) irrespective of dry matter intake. Starch fermentation in the rumen favors the production of propionate which occurs at higher levels when corn silage instead of grass silage based diets are fed (Knapp et al. 2014; van Gastelen et al. 2015). This is linked to a decreased rumen pH and a lower production of H 2  which reduces the activity of methanogens (Knapp et al. 2014; van Gastelen et al. 2015). Van Gastelen et al. (2015) observed a reduction in CH 4  yield of 11% when corn silage compared to grass silage was fed. Linseed supplementation decreased (P&lt;0.01) CH 4  yield by 6%, CH 4  intensity by 10% and CH 4  emission by 13%. The methane lowering potential of lipids from linseed is a result of the inhibitory effect on fibrolytic bacteria, a lower number of protozoa and the biohydrogenation of unsaturated fatty acids in the rumen (Maia et al. 2007; Martin et al. 2016). A 10% supplementation of extruded linseed reduced CH 4  yield by 15% in hay based and 8% in corn silage based diets (Martin et al. 2016). Benchaar et al. (2015) examined a 4% linseed oil supplementation to a corn silage diet and a red clover diet, respectively, and observed 20 and 8% reduction of CH 4  yield, respectively. Related to dry matter intake, differences in methane lowering potential in the various studies could be a result of the amount (Martin et al. 2016) and form of linseed supplementation (Martin et al. 2008). 
     2. Milk Fatty Acid Composition 
     A greater total milk fat level was observed with grass silage compared to corn silage based diets (P=0.032). Milk fatty acid groups and individual fatty acids concentrations were unaffected by basal rations with the exception of mono and polyunsaturated fatty acids which tended to be lower and higher with corn silage based rations, respectively (P=0.060; Table 3). Dietary fat determines the concentrations of long chain saturated and unsaturated fatty acids in milk fat (Chilliard et al. 2009; Meignan et al. 2017). Grass and corn silage are rich in C18:3 ω-3 and C18:2 ω-6, respectively, explaining their concentrations and that of their bio-hydrogenation products in milk (van Gastelen et al. 2015). Similar to the present results, replacing grass silage with corn silage did not alter total saturated, mono- and polyunsaturated fatty acids, as well as C18:1cis including cis9 concentrations, but proportions of C6:0 to C14:0 and C18:1trans increased while ω-3 fatty acids decreased. Inconsistent results were reported for C18:0 and C16:0 levels that were similar, increased or decreased (Kliem et al. 2008; van Gastelen et al. 2015). This ambiguity is probably due to different dietary levels of grass silage and corn silage, differences in bio-hydrogenation, and possibly differences in de novo synthesis in the mammary gland (Kliem et al. 2008; van Gastelen et al. 2015). We found, that the higher availability of unsaturated fatty acids in linseed supplemented diets lead to higher concentrations of C18:0, ω-3 milk fatty acids, sums of C18:1cis and trans, and groups of unsaturated fatty acids whereas C16:0 and groups of saturated fatty acid levels were lower (P&lt;0.001; Table 3). Linseed tended to decrease the proportion of monounsaturated fatty acids (P=0.053) whilst increasing the corresponding proportion of polyunsaturated fatty acids (P&lt;0.001). It has been shown that 87% of the C18:3 ω-3 intake was bio-hydrogenated (Meignan et al. 2017). Thus diets containing high amounts of C18:3 ω-3 such as linseed diets lead to higher proportions of bio-hydrogenation intermediates like C18:1trans isomers and their end-product C18:0, which can be converted to oleic acid, as well as unsaturated fatty acids which can be found in milk. In contrast, the observed decrease of saturated fatty acids including C16:0 (P&lt;0.001) may be a result of less organic matter and fiber digestibility in the presence of a higher proportion of polyunsaturated fatty acids in the rumen (Maia et al. 2007; Martin et al. 2016). In addition, fatty acid synthesis is inhibited by long chain fatty acid bio-hydrogenation products (Glasser et al. 2008; van Lingen et al. 2014). Others reported comparable changes in saturated and unsaturated fatty acid, C16:0, C18:0, 18:1cis and trans isomers and ω-3 fatty acids levels in their studies, when extruded linseed was supplemented (Chilliard et al. 2009; Ferlay et al. 2013). Mohammed et al. (2011) observed no difference in C18:1trans milk fatty acid concentrations between a 9.3% linseed supplemented diet and a control diet that included 4% Ca salts of palm oil. Notably, in all studies cited gas chromatography was used to determine milk fatty acid concentrations. Although infrared spectroscopy is considered less specific than gas chromatography and the accuracy of quantification of major fatty acids is better than for minor fatty acids (De Marchi et al. 2014) the present results are largely in line with the literature. High accuracies have been shown for infrared predictions of saturated fatty acids, C18:1trans, C18:1cis9, C18:1cis, groups of saturated, unsaturated and monounsaturated fatty acids, and also for the group of ω-3 fatty acids (De Marchi et al. 2014; Soyeurt et al. 2011). 
     3. Predicting Methane Emission 
     Many studies reporting prediction equations are based on milk fatty acid concentrations analysed by gas chromatography (Castro-Montoya et al. 2016; Rico et al. 2016; van Lingen et al. 2014). To date published prediction models based on global infrared milk spectra (Kandel et al. 2015; Vanlierde et al. 2015) used the model of Dehareng et al. (2012). Dehareng et al. (2012) included methane production data of 11 cows measured by the SF6 method, and reported a predictive power between 0.68 and 0.79 for g of CH 4  per day, and g of CH 4  per kg of milk. In the present study two datasets were used to develop multiple regression equations. The data set 1 comprised nine groups of fatty acids or individual fatty acids measured by infrared spectroscopy, the second data set included additionally energy corrected milk yield (Table 4). 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Summary of multiple regression equations predicting methane emission. Each line summarizes a regression equation specific 
               
               
                 to a single or grouped diets based on combinations of basal ration or linseed supplementation. Data set 1 included milk 
               
               
                 fatty acids, whereas data set 2 additionally included energy corrected milk (ECM) as independent variable. Equations 
               
               
                 were developed separately for experimental diets containing grass silage without or with linseed supplementation (GS- 
               
               
                 L0, GS-LS) and corn silage without or with linseed supplementation (CS-L0, CS-LS) as well as for basal rations (GS, CS), 
               
               
                 linseed supplementation (L0, LS) and all experimental diets collectively; P* value of the model; “—” not determined. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 C18:1 
                 C18:1 
                 ω-3 fatty 
                   
                   
               
               
                 CH 4  (l/day) 
                 N 
                 Intercept 
                 ECM 
                 SFA 
                 UFA 
                 MUFA 
                 PUFA 
                 C16:0 
                 C18:0 
                 cis 
                 trans 
                 acids 
                 R 2   
                 P*≤ 
               
               
                   
               
            
           
           
               
            
               
                 Data set 1 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 GS-L0 
                 9 
                 −979.3 
                 Left out 
                 — 
                 — 
                 — 
                 — 
                 46.18 
                 — 
                 — 
                 — 
                 — 
                 0.94 
                 0.001 
               
               
                 GS-LS 
                 7 
                 1002.2 
                 Left out 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 −19.57 
                 — 
                 — 
                 0.70 
                 0.019 
               
               
                 CS-L0 
                 10 
                 −702.6 
                 Left out 
                 — 
                 — 
                 — 
                 — 
                 40.36 
                 — 
                 — 
                 — 
                 — 
                 0.68 
                 0.004 
               
               
                 CS-LS 
                 10 
                 1232.7 
                 Left out 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 −27.89 
                 — 
                 — 
                 0.61 
                 0.008 
               
               
                 GS 
                 16 
                 −2216.7 
                 Left out 
                 — 
                 — 
                 — 
                 39.67 
                  6.88 
                 70.81 
                 — 
                 — 
                 — 
                 0.82 
                 0.001 
               
               
                 CS 
                 20 
                 2350.9 
                 Left out 
                 — 
                 — 
                 — 
                 — 
                 −24.80  
                 71.70 
                 −50.77 
                 — 
                 −1093.0 
                 0.75 
                 0.001 
               
               
                 L0 
                 19 
                 −2594.7 
                 Left out 
                 22.54 
                 — 
                 — 
                 — 
                 27.41 
                 61.52 
                 — 
                 — 
                 — 
                 0.86 
                 0.001 
               
               
                 LS 
                 17 
                 1098.9 
                 Left out 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 −23.00 
                 — 
                 — 
                 0.61 
                 0.001 
               
               
                 All diets 
                 36 
                 −2533.7 
                 Left out 
                 33.81 
                 — 
                 — 
                 — 
                 — 
                 54.97 
                 — 
                 30.91 
                 — 
                 0.70 
                 0.001 
               
            
           
           
               
            
               
                 Data set 2 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 GS-L0 
                 9 
                 −979.3 
                 — 
                 — 
                 — 
                 — 
                 — 
                 46.18 
                 — 
                 — 
                 — 
                 — 
                 0.94 
                 0.001 
               
               
                 GS-LS 
                 7 
                 1002.2 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 −19.57 
                 — 
                 — 
                 0.70 
                 0.019 
               
               
                 CS-L0 
                 10 
                 −604.9 
                 18.16 
                 — 
                 — 
                 — 
                 — 
                 20.81 
                 — 
                 — 
                 — 
                 — 
                 0.85 
                 0.001 
               
               
                 CS-LS 
                 10 
                 27.42 
                 16.73 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 0.66 
                 0.004 
               
               
                 GS 
                 16 
                 −1019.9 
                 10.64 
                 — 
                 — 
                 — 
                 — 
                 27.77 
                 38.82 
                 — 
                 — 
                 — 
                 0.82 
                 0.001 
               
               
                 CS 
                 20 
                 284.1 
                 16.82 
                 — 
                 −6.58 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 0.76 
                 0.001 
               
               
                 L0 
                 19 
                 −558.4 
                 10.04 
                 — 
                 — 
                 — 
                 — 
                 24.31 
                 47.88 
                 — 
                 — 
                  −616.6 
                 0.91 
                 0.001 
               
               
                 LS 
                 17 
                 721.3 
                 7.97 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 −16.76 
                 — 
                 — 
                 0.71 
                 0.001 
               
               
                 All diets 
                 36 
                 −1363.7 
                 9.58 
                 18.45 
                 — 
                 — 
                 — 
                 — 
                 32.36 
                 — 
                 — 
                 — 
                 0.79 
                 0.001 
               
               
                   
               
            
           
         
       
     
     Although dry matter intake explains a large part of variation observed in methane emission (Knapp et al. 2014) this information is not available on farms. Thus it cannot be considered as a variable to be implemented in methane prediction equations for on-farm application. This is in contrast to energy corrected milk which is a readily available cow-individual measure reflecting the dry matter intake (Hristov et al. 2013). Consequently, it can be used as a surrogate for dry matter intake. The dry matter intake can explain 52 to 64% of methane emission (Knapp et al. 2014). Using the first data set the goodness of the regression fit for CH 4  emission for individual diets ranged from R 2 =0.61 to 0.94. Measured and predicted methane emission correlated strongly ( FIG. 1 ). Using data of all diets collectively, a coefficient of determination of R 2 =0.70 was obtained and the corresponding correlation coefficient between measured and predicted methane emission was r=0.84 (P&lt;0.001). 
     If energy corrected milk was included (data set 2), the goodness of fit was considerably improved relative to the equations of data set 1 in most cases (Table 4). Correlation between measured and predicted methane emission was r=0.89 (P&lt;0.001) for all diets, and ranged from 0.81 to 0.97 (P&lt;0.019) for individual diets. 
     CONCLUSION 
     This is the first report on multiple regression equations predicting methane emission based on milk fatty acids analyzed by infrared spectroscopy. The prediction of methane emission using milk fatty acid data of all diets collectively showed moderate to high predictive power. The inclusion of energy corrected milk yield as additional independent variables resulted in improvements of predictive power for all diets collectively by 13%. 
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