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Influence of main dietary chemical constituents on the in vitro gas and methane production in diets for dairy cows



Modification of chemical composition of diets fed to dairy cows might be a good strategy to reduce methane (CH4) production in the rumen. Notable reductions of CH4 production compared to conventional high-roughages rations were more frequently observed for very concentrated diets or when fat supplements were used. In these cases, the reduction in the gas emission was mainly a consequence of an overall impairment of rumen function with a reduction of fiber digestibility. These strategies do not always comply with feeding standards used in intensive dairy farms and they are usually not applied owing to the risks of negative health and economic consequences.

Thus, the present study evaluated the effects of seven commercial diets with contents of neutral detergent fiber (NDF), protein and lipids ranging 325 to 435 g/kg DM, 115 to 194 g/kg DM, and 26 to 61 g/kg DM, respectively, on in vitro degradability, gas (GP), and CH4 production.


In this experiment, changes in the dietary content of NDF, crude protein (CP) and lipids were always obtained at the expense or in favor of starch. A decreased of the dietary NDF content increased NDF (NDFd) and true DM (TDMd) degradability, and increased CH4 production per g of incubated DM (P < 0.001), but not that per g of TDMd. An increase of the dietary CP level did not change in vitro NDFd and TDMd, decreased GP per g of incubated DM (P < 0.001), but CH4 production per g of TDMd was not affected. An increased dietary lipid content reduced NDFd, TDMd, and GP per g of incubated DM, but it had no consequence on CH4 production per g of TDMd.


It was concluded that, under commercial conditions, changes in dietary composition would produce small or negligible alterations of CH4 production per unit of TDMd, but greater differences in GP and CH4 production would be expected when these amounts are expressed per unit of DM intake. The use of TDMd as a standardizing parameter is proposed to account for possible difference in DM intake and productivity.


Mitigation of methane (CH4) production from rumen fermentation represents an important target for animal nutritionists, as also this gas is responsible for global warming. Thus, the manipulation of dietary nutrient composition is often proposed as a strategy that farmers may exploit to reduce the proportion of energy lost by animals as eructated gases (CH4) and to improve feed and energy efficiency [1]. There is evidence that the amount of CH4 produced in the rumen is influenced by type and content of dietary carbohydrates [2] and lipids [3]. In practice, notable reductions of CH4 production compared to conventional high-roughages rations were more frequently observed for very concentrated diets [4] or when fat supplements [3] were used. In these cases the reduction in the gas emission was mainly a consequence of an overall impairment of rumen function with a reduction of fiber digestibility [5]. Thus, these strategies do not always comply with the feeding standards used in intensive dairy farms and they are usually not applied owing to the risks of negative health and economic consequences [6]. Compared to carbohydrates and lipids, minor effects on rumen gas production and methanogenesis are usually attributed to the crude protein (CP). In this regard, [7] observed that CH4 production related to CP fermentation was lower compared to that due to carbohydrate fermentation. To date, changes in the dietary CP content have been mainly addressed to reduce feeding costs and N excretion [8, 9]. However, dietary strategies to reduce N excretion could also have an impact because CH4 production may decline when dietary CP is replaced by rumen bypass nutrients, including starch, escaping rumen fermentation [10]. Despite this, little information is currently available on the effects on CH4 production due to changes in dietary CP content.

This in vitro study was aimed at evaluating the extent of alterations of true dry matter degradability (TDMd), total gas (GP) and CH4 productions caused by changes in the proportions of the main feed ingredients and of the dietary constituents (structural and non-structural carbohydrates, CP and lipids) in TMR samples representative of rations commonly used in intensive farms in North Italy.


Chemical composition of the diets

Diets used in this experiment were defined after an analysis of a database containing information about ingredient and chemical composition of the rations used by 90 farms considered to be representative of the dairy farm system in North Italy [11, 12].

A corn silage-diet, containing 361, 158, and 33 g/kg of NDF, CP, and lipids, respectively, was used as a reference (Table 1). Six other diets were formulated to produce variations in the proportion of some feed ingredients, and hence in content of a given chemical constituent in favor or at the expense of starchy feeds, with respect to the reference diet. Thus, changes in the dietary content of NDF, CP and lipids, were always obtained at the expense or in favor of starch. Two diets with a low (325 g/kg DM) or a high (435 g/kg DM) content of NDF were formulated by replacing, accordingly, roughages (corn silage, alfalfa hay, and ryegrass hay) with corn and barley grains in the form of meal. The diet with the high NDF content did not contain corn silage, taking into consideration dairy farms that are not allowed to use this feed as they produce milk to be processed as Italian Protected Designation of Origin (PDO) Parmigiano-Reggiano cheese. Other two diets, with a low (115 g/kg DM) or a high CP content (194 g/kg DM), were formulated by replacing, accordingly, soybean meal with cereal grains (corn and barley meal). It must be underlined that the upper level of CP tested in this study corresponded to the maximum value found in the considered database of 90 farms. This value is high if compared with ranges in CP contents (118–186 g/kg DM) reported for rations used in dairy farms of North-Italy [12]. Two diets with different ether extract (EE) content were also formulated. A low EE diet (26 g/kg DM) was achieved by excluding the extruded flaxseed (Linoies, Cortal Extrasoy, Cittadella, Italy), present in the reference diet, and increasing the content of corn, barley and soybean meal. The high EE diet (61 g/kg DM) was achieved by including whole soybean seeds, extruded soybean (Soyfull, Cortal Extrasoy, Cittadella, Italy) and extruded flaxseed. All diets were prepared at the laboratory of the University of Padova. For preparation, about 1 kg of each diet was ground to 1 mm using a hammer mill (Pullerisette 19, Fritsch GmbH, Laborgeratebau, Germany). For each diet, 23 samples were randomly collected, 20 of which were used for the incubations (5 per each of the 4 incubations), whereas the remaining were used for chemical analysis.

Table 1 Feed ingredients, chemical composition and gross energy content of seven diets

Diets were analyzed in triplicate for dry matter (DM: # 934.01; [13]), nitrogen (# 976.05; [13]), EE (# 920.29; [13]), and ash (# 942.05; [13]). Neutral detergent fibre (NDF), inclusive of residual ash, was determined with α-amylase using the Ankom220 Fibre Analyzer (Ankom Technology, NY, USA). Acid detergent fibre (ADF), inclusive of residual ash, and sulphuric acid lignin (lignin(sa)) were determined sequentially after NDF determination [14]. Starch content was determined after hydrolysis to glucose [13] by liquid chromatography [15]. Gross energy content of diets (MJ/kg DM) was determined in duplicate by a bomb calorimeter method [16].


The 7 diets were incubated in 4 repeated incubation runs, conducted in 4 successive wk. Two incubations were stopped at 24 h, whereas the other two were stopped after 48 h. The incubation times of 24 and 48 h were chosen as they are, respectively, the reference times for measuring in vitro GP [17] and in vitro degradability of NDF [18]. In each of the four incubation runs, we tested 7 diets × 5 replications, plus 5 blanks (bottles containing only the buffered rumen fluid; 5 blanks/run), for a total of 160 bottles incubated. A commercial GP apparatus (AnkomRF Gas Production System, Ankom Technology®, NY, USA) was used, consisting of 40 bottles equipped with pressure sensors (pressure range: from - 69 to 3,447 kPa; resolution: 0.27 kPa; accuracy: ± 0.1 % of measured value) and wireless connected to a computer. Each bottle (317 mL) was filled with 1.000 ± 0.0010 g of diet, 100 mL of a buffer solution, and 50 mL of rumen fluid (headspace volume = 167 mL), keeping the headspace of bottles flushed with CO2.

The buffer solution was prepared according to [17], heated in a water bath at 39 ± 0.4 °C and purged continuously with CO2 for 30 min, to maintain anaerobic conditions. Rumen fluid was collected by an esophageal probe, as described by [19], 2 h before morning feeding from 3 dry Holstein-Friesian cows housed at the experimental farm of the University of Padova (Italy) and fed hay ad libitum and 2.5 kg/d of concentrates (0.5 kg of dry sugar beet pulp, 1 kg of corn grain, and 1 kg of sunflower meal). During the collection of rumen fluid, cows were handled according to the Italian law on animal care (Legislative Decree No. 26 of March 14, 2014). Rumen fluid was poured into thermal flasks preheated to 39 ± 0.5 °C, immediately transferred to the laboratory, strained through 3 layers of cheesecloth, to eliminate feed particles, and mixed with buffer solution in a 1 to 2 ratio [17]. Operations were conducted under anaerobic conditions, by flushing with CO2, and required less than 30 min to be completed. Bottles were placed in a ventilated oven at 39 ± 0.4 °C and automatically vented at a fixed pressure (6.8 kPa), to avoid overpressure conditions and alterations of gas and CH4 measures [20]. In vitro GP was monitored in continuous, using a setting of the GP system that allows to record pressure values every minute. Other in vitro parameters (rumen degradability, VFA and N-NH3 concentrations, CH4 production) were measured only at the end of incubation (at 24 or 48 h), to avoid opening of the oven during the incubation, with alteration of fermentation process.

At the end of incubations (24 or 48 h), two aliquots (5 mL) of fermentation fluid were collected from each bottle and stored at −20 °C with 1 mL of metaphosphoric acid (25 %, w/v) to be later analyzed for ammonia N and volatile fatty acids (VFA). The content of ammonia N was measured using the FIAstar™ 5000 Analyzer (FOSS Analytical, Hilleroed, Denmark). The VFA profile was analyzed by GC with flame ionization detection (7820A GC system, Agilent Technologies, Milan, Italy) using a 30-m stainless steel column (J&W DB-FFAP, Agilent Technologies, Milan, Italy) and H2 as carrier gas (flow rate: 30 mL/min; isothermal oven temperature: 150 °C). Fermentation fluids were filtered into weighed crucibles (30 mL, Robu Glasfilter-Geräte GMBH®, Hattert, Germany) and analyzed for residual NDF using a Fibretech Analyzer (VELP® Scientifica, Milan, Italy).

At the end of each incubation (24 or 48 h), gas was collected with a 10-mL gas-tight syringe (Artsana S.p.A., Como, Italy) from the bottle headspace (HS). At each sampling, the syringe was flushed in order to collect a homogeneous sample, which was immediately transferred into a 9-mL vacuette (Greiner Bio-One GmbH, Kremsmunster, Austria). From each vacuette, an aliquot (10 μL) of gas was sampled with a gas-tight syringe (1701 N, Hamilton, Bonaduz, Switzerland) and immediately analyzed for CH4 concentration by GC with flame ionization detection (7820A GC system, Agilent Technologies, Milan, Italy) using a 15-m carbon layer column (GS-CarbonPLOT, Agilent Technologies, Milan, Italy) and H2 as carrier gas (flow rate: 1.6 mL/min; isothermal oven temperature: 40 °C). An 11-point calibration curve was generated from eleven gas mixtures containing 2, 4, 8, 16, 24, 32, 60, 100, 140, 180, and 240 mL of CH4/L (99.5 % pure, SAPIO s.r.l., Monza, Italy), respectively, and known volumes of air. Mixtures were prepared using the same graduated gas-tight syringe (1701 N, Hamilton). The calibration regression had R2 > 0.99.


The NDF degradability (NDFd) and the true DM degradability (TDMd) were calculated according to [18].

Recently, [20], using vented bottles connected to tight bags for gas collection, calculated CH4 production (mL) as: [CH4 concentration in HS] × [HS volume] + [CH4 concentration in the gas bag × GP]. To evaluate the possibility of avoiding the use of bags, to save space and increase the number of replicates, amount of CH4 lost with gas venting was computed using the unpublished data of a previous study, where 4 forages and 3 concentrates were incubated in 42 bottles (6 bottles/feed) for 6, 24, or 48 h using the same GP equipment and the same operative conditions of the present experiment. It was found that total CH4 production is predictable, with acceptable precision and accuracy, as: − 0.0064 × [CH4 concentration in HS × (HS volume + GP)]2 + 0.9835 × [CH4 concentration in HS × (HS volume + GP)]. This equation had a residual standard deviation of only 0.1770 mL, and R2 = 0.9993. Thus, the present experiment was conducted without the use of tight bags for gas collection. The CH4 production was computed using the above described equation and it was expressed as mL/g DM incubated, mL/g of digested NDF (dNDF), mL/g TDMd, or mL/100 mL GP. In vitro GP and CH4 were also predicted from VFA production, according to [21].

Statistical analysis

The mean of the 5 replications for each diet in each incubation run was computed. These 28 means were analyzed using PROC MIXED of SAS [22] using a model considering the diet (D; 6 df), the incubation time (IT, 1 df), and the interaction diet × incubation time as fixed factors, the run within incubation time (2 df) as a random blocking factor and the residual error term e (18 df). As the diet × incubation time interaction was never significant, it was excluded from the model. Contrasts were run to analyze statistical differences among diets with different contents of a given chemical constituent, using the Bonferroni adjustment to perform multiple comparisons.


Changes of the feed ingredients proportions and of dietary contents of chemical constituents had influence on the various parameters of in vitro fermentation (Table 2). As expected, pH values measured at the end of fermentation were not influenced by the dietary changes. The ammonia N content increased with increasing dietary CP content (P = 0.014). No influence of the diets was observed on the VFA production, but the proportion of acetate or butyrate decreased (P < 0.001) or increased (P = 0.004), respectively, with a decrease of NDF content, whereas the proportion of propionate decreased (P = 0.048) with increasing dietary CP. Thus, the ratio between acetate and propionate decreased with decreasing level of NDF (P = 0.001) and of CP (P < 0.001), and the corresponding increasing level of starch. Increasing proportions of dietary CP increased the proportion of other VFA (P < 0.001) found in the rumen fluid. Changes of dietary EE content had no consequence on the various rumen fluid parameters. The prolongation of the incubation time from 24 to 48 h increased the VFA production (P = 0.029), but it did not influence pH and ammonia N values.

Table 2 Effect of diets and incubation time on pH, ammonia N concentration, volatile fatty acid production (VFA) and molar proportions of acetate, propionate, butyrate and other VFA

The NDFd, TDMd, and the GP expressed per unit of incubated DM or per unit of TDMd (Table 3) increased with a decrease of NDF content (P < 0.001 for all). When the NDF content decreased, the CH4 production increased per unit of incubated DM (P < 0.001) and per g of digested NDF (P = 0.002), but not per unit of TDMd. The increased dietary CP content, with the corresponding decrease of starch, had no influence on NDFd or TDMd, but GP was lowered. No influence was observed on the production of CH4, except when this was expressed as a proportion of GP (mL CH4/100 mL GP; P < 0.001). An increased inclusion of extruded oilseeds in the diet reduced both NDFd (P = 0.003) and TDMd (P = 0.028), and the measured GP expressed per g of incubated DM (P = 0.017), but no influences were observed on the CH4 yield. A prolonged duration of the incubation, from 24 to 48 h, increased NDFd (P = 0.009), TDMd (P = 0.007), and CH4 production, both per unit of incubated DM (P = 0.014) and per unit of TDMd (P = 0.027). The correlation between measured and predicted values showed R2 to be 0.78 and 0.74, respectively, for gas and CH4 measures (data not shown), and the relationship obtained by regressing measured values of CH4 (mL/g DM; y) against those predicted (mL/g DM; x) was the following: measured CH4 = 0.95 × predicted CH4 – 2.6. Predicted values of GP and CH4 productions were not influenced either by dietary changes or by incubation time.

Table 3 Effects of diets and incubation time on in vitro degradability of NDF (NDFd) and of true DM (TDMd), gas production (GP) and methane (CH4) production, and predicted values of GP and CH4 production


General considerations

The diets were formulated using feed ingredients commonly used in the Po valley (North-East of Italy) and composed mainly by cereal grains, corn silage and various hays. It should be preliminarily considered that in this study the variation in the content of the various nutrients, namely, NDF, CP and lipid, were always achieved by decreasing or increasing the proportion of dietary starch. In the scientific literature, in vitro evaluation of gas and CH4 productions is commonly carried out using single feeds, mainly forages, whereas less information is available for complete diets [23]. In vivo measurement of gas and CH4 production requires expensive equipment and it is labor and time consuming. In vitro techniques would permit a much more simple determination of the dietary characteristics which can influence the potential emission of gas and CH4 from their fermentation in a simulated rumen environment [20]. Studies of the relationships between in vitro and in vivo gas and CH4 productions are lacking [23]. However, a recent study suggested that in vitro gas and CH4 measurements can be indicative of the trend of in vivo CH4 production originating from different combinations of feed ingredients [24]. This study was aimed at evaluating if changes in the diet composition might or might not have notable influence on gas and CH4 productions.

After 24 h of fermentation, the measured GP of the various diets was, on average, 250 mL/g DM, suggesting that a cow consuming 20 kg/d DM might produce about 5,000 L/d of gas. The CH4 production from fermentation of these diets ranged 30.8 to 34.4 mL/g DM, suggesting that, for a DM intake of 20 kg/d, a cow might produce 616 to 688 L/d of CH4. Sauer et al. (1998) reported in vivo CH4 production of lactating cows in the order of 622 L/d, corresponding to 38.9 mL/g DM intake. In the study of [25], CH4 production from dairy cows was 29.2 mL/g DM intake, whereas [26] reported for sheep an averaged CH4 production of 31.0 mL/g DM intake. In a continuous culture fermenter, [27] measured an averaged CH4 production of 33.0 mL/g DM. The in vitro CH4 production of the dairy rations tested by [23] varied from 30.1 to 35.9 mL/g DM, a range consistent with results obtained in this study. However, comparison with data from literature is difficult because huge variations in gas and CH4 productions are commonly observed across experiments, even for diets with similar composition. This is the consequence of a combination of different biological, as rumen fluid characteristics, and technical factors, as fermentation procedures and equipment for collection, measurement and analysis [28]. In this experiment the observed GP and CH4 were regressed against the GP and CH4 values predicted from the stoichiometry of the VFA production, to obtain an internal control of consistency of the data. The correlations found (R2 = 0.78 and 0.74, respectively, for gas and CH4 measures) are acceptable considering the rather narrow range of variation in GP and CH4 production caused by the dietary changes. It was observed that the SEM of predicted GP and CH4 production (21.9 and 3.78 mL/g DM, respectively) was about five times greater than the corresponding values of the measured GP and CH4 production. In other words, the measure of the VFA production was less precise than the GP and CH4 measures.

Effects on NDF degradability, gas and CH4 production due to changes in the NDF content

In this experiment, the diet with the low level of NDF (325 g/kg DM) was based on corn silage and a small amount of alfalfa hay (23 g/kg DM) as forage sources. On the contrary, the diet with the high level of NDF (435 g/kg DM) was based on different hays and did not contain corn silage. This is of interest in Italy as use of silages for the production of important Protected Denomination of Origin (PDO) cheeses (i.e., Parmigiano-Reggiano) is forbidden by specific feeding regulations. From the results it emerged that NDF degradability was negatively influenced by the dietary NDF content, partially because of the complete replacement of corn silage with hays. This would be in agreement with previous observations that the NDF fraction of hay samples collected in the same area of the present experiment were less degradable than corn silage samples [19].

In diets similar for ingredient composition to those used in this experiment, [12] observed an increased CH4 production (35.6 to 44.3 mL/g DM) with decreasing NDF content. In the present study, a decrease from 435 to 325 g NDF/kg DM increased CH4 production from 30.8 to 34.4 mL/g DM (+11.6 %). This is consistent with an increased true degradability of the feed (+14 %), but also of the NDF fibrous fraction (+27 %). This seems to be contradictory with current literature, as a decrease of dietary NDF commonly reduces the NDF degradability [12]. However, also [29] evidenced that a decrease of the dietary NDF content increased by 43 % in vivo CH4 production expressed per unit of dNDF. Results also evidenced that GP increased with a decrease of NDF content even when expressed per unit of TDMd (+10 %), but no influences were observed on the CH4 production per unit of TDMd. A different trend emerged when CH4 production was referred to the total GP. In this case, the decrease of NDF lowered the CH4 proportion by about 12 % (13.5 to 11.9 mL CH4/100 mL GP, for the high and the low content of NDF, respectively). Results suggest that a decrease of dietary NDF content, achieved from a complete replacement of hays with corn silage and cereal grains, might increase feed digestibility without changing GP and CH4 produced per unit of digested material.

Effects on gas and CH4 production due to changes in the CP content

In this experiment an increased proportion of CP, in replacement of starch, caused a reduction of GP. The negative influence of dietary CP on GP has been observed by others in the past. Such an effect was attributed to the buffer capacity of CP, that reduces the indirect CO2 released from the buffered rumen fluid, and to the stoichiometry of protein fermentation, that differs from that of carbohydrates [30, 31]. In this study, increase of the CP content was associated to a decrease of dietary starch. As a consequence, some VFA as iso-butyric and iso-valeric acids were increased, being end-products of protein degradation [32], whereas the production of propionate decreased and the ratio between acetate and propionate increased. Changes in the dietary CP proportion had no effect on CH4 production when expressed both per unit of incubated DM and per unit of TDMd. Thus, as CP depresses GP, an increased proportion of CP increases the CH4 concentration on total GP.

Effects on gas and CH4 production due to changes in the lipid content

In this experiment changes in the dietary fat content of the diets were achieved by changing the proportions of extruded flaxseeds, extruded soybean and whole soybean seeds. The threshold of 60 g fat/kg DM was considered to be the upper limit to avoid a possible impairment of feed digestibility [6, 33].

The effect of dietary lipids on CH4 production is dependent on the source, FA profile, level of inclusion, and diet composition [34]. The level of supplementation and the physical form of the lipid supplement affect its availability in the rumen, and these factors appear to be more important than the FA profile [35]. In this regard, [36], using dairy cows housed in respiratory chambers, found that, compared to the control, the average reduction in CH4 (L/kg DMI) per 10 g/kg of crude fat added was persistent throughout lactation. The same authors observed that the most effective lipid source in reducing methanogenesis was a commercial vegetal rumen protected fat fortified with hydroxy-methionine-analog-isobutyrate (−5.5 % of CH4), followed by vegetal rumen protected fat (−2.3 %), and by whole cracked rapeseed (−0.8 %). In the experiment of [35], only crushed canola seeds lowered CH4 production per unit of digestible DM intake (−15 %), whereas crushed flaxseeds and crushed sunflower seeds did not reduce CH4 production compared to the control diet (a diet supplemented with a commercial source of calcium salts of long chain fatty acids). In the current experiment, fat addition reduced feed degradability, particularly that of the NDF fraction. No influences were found on total VFA production and on the proportion of acetate, propionate and butyrate, whereas GP and CH4 productions decreased by 8 and 6 %, respectively, compared to the reference diet. However, differences were greatly reduced when GP and CH4 were expressed per unit of TDMd suggesting that, under the commercial conditions evaluated in this study, small reductions of CH4 might be achieved.


Changes of the ingredient and chemical composition of diets were analyzed to evaluate benefits in the amount of CH4 produced, for the north eastern Italian dairy production chain. It was found that a replacement of hays with corn silage and cereals might increase GP and CH4 per unit of DM intake. An increase of the dietary CP content would reduce GP with no influences in the amount of CH4 produced, whereas a moderate addition of cracked soybean seeds, and extruded flaxseed had few, or any, influence on the in vitro GP and CH4 productions. In general, none of the various strategies tested in the present work was able to reduce the amount of CH4 produced, especially if this production is expressed per unit of digestible DM intake. More research is needed to evaluate the effectiveness of strategies to reduce the CH4 emissions, and relationships between in vitro and in vivo gas and CH4 productions need to be developed.


ADF, Acid detergent fibre expressed inclusive of residual ash; CP, Crude protein; DM, Dry matter; EE, Ether extract; GP, Gas production; NDF, Neutral detergent fibre assayed with a heat stable amylase and expressed inclusive of residual ash; NDFd, NDF degradability; lignin(sa), Lignin determined by solubilization of cellulose with sulphuric acid; NRC, National Research Council; RMSE, Root mean square error; SEM, Standard error of the mean; TDMd, True DM degradability; VFA, Volatile fatty acids


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This work was financed by the project “ARCHAEA - Feeding strategies to reduce methane emissions from dairy cows” – Veneto Region Rural Development Programme (RDP) 2007–2013.

Authors’ contributions

All authors conceived of this experiment, due to experience gained over the last years in the use of in vitro gas production technique for the evaluation of ruminant feeds. LM and MC gave substantial contributions to analytical procedures, statistical analysis, and writing of the manuscript. FT, LB, and SS contributed in revising critically the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

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Correspondence to Mirko Cattani.

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Maccarana, L., Cattani, M., Tagliapietra, F. et al. Influence of main dietary chemical constituents on the in vitro gas and methane production in diets for dairy cows. J Animal Sci Biotechnol 7, 54 (2016).

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  • Dairy cows
  • Dietary manipulation
  • Gas production
  • In vitro techniques
  • Methane production