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

Influence of main dietary chemical constituents on the in vitro gas and methane production in... Background: Modification of chemical composition of diets fed to dairy cows might be a good strategy to reduce methane (CH ) production in the rumen. Notable reductions of CH production compared to conventional high- 4 4 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 CH production. Results: 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 CH 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 CH 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 CH production per g of TDMd. Conclusions: It was concluded that, under commercial conditions, changes in dietary composition would produce small or negligible alterations of CH production per unit of TDMd, but greater differences in GP and CH production 4 4 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. Keywords: Dairy cows, Dietary manipulation, Gas production, In vitro techniques, Methane production Background type and content of dietary carbohydrates [2] and lipids Mitigation of methane (CH ) production from rumen [3]. In practice, notable reductions of CH production 4 4 fermentation represents an important target for animal compared to conventional high-roughages rations were nutritionists, as also this gas is responsible for global more frequently observed for very concentrated diets [4] warming. Thus, the manipulation of dietary nutrient or when fat supplements [3] were used. In these cases composition is often proposed as a strategy that farmers the reduction in the gas emission was mainly a conse- may exploit to reduce the proportion of energy lost by quence of an overall impairment of rumen function with a animals as eructated gases (CH ) and to improve feed reduction of fiber digestibility [5]. Thus, these strategies and energy efficiency [1]. There is evidence that the do not always comply with the feeding standards used in amount of CH produced in the rumen is influenced by intensive dairy farms and they are usually not applied owing to the risks of negative health and economic conse- * Correspondence: mirko.cattani@unipd.it quences [6]. Compared to carbohydrates and lipids, minor Department of Comparative Biomedicine and Food Science (BCA), effects on rumen gas production and methanogenesis are University of Padova, Viale dell’Università 16, 35020 Legnaro (PD), Italy usually attributed to the crude protein (CP). In this regard, Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Maccarana et al. Journal of Animal Science and Biotechnology (2016) 7:54 Page 2 of 8 [7] observed that CH production related to CP fermenta- flaxseed (Linoies, Cortal Extrasoy, Cittadella, Italy), present tion was lower compared to that due to carbohydrate fer- in the reference diet, and increasing the content of corn, mentation. To date, changes in the dietary CP content barley and soybean meal. The high EE diet (61 g/kg DM) have been mainly addressed to reduce feeding costs and N was achieved by including whole soybean seeds, extruded excretion [8, 9]. However, dietary strategies to reduce N soybean (Soyfull, Cortal Extrasoy, Cittadella, Italy) and ex- excretion could also have an impact because CH produc- truded flaxseed. All diets were prepared at the laboratory of tion may decline when dietary CP is replaced by rumen the University of Padova. For preparation, about 1 kg of bypass nutrients, including starch, escaping rumen fer- each diet was ground to 1 mm using a hammer mill (Pull- mentation [10]. Despite this, little information is currently erisette 19, Fritsch GmbH, Laborgeratebau, Germany). available on the effects on CH production due to changes For each diet, 23 samples were randomly collected, 20 of in dietary CP content. which were used for the incubations (5 per each of the 4 This in vitro study was aimed at evaluating the extent of incubations), whereas the remaining were used for chem- alterations of true dry matter degradability (TDMd), total ical analysis. gas (GP) and CH productions caused by changes in the Diets were analyzed in triplicate for dry matter (DM: # proportions of the main feed ingredients and of the diet- 934.01; [13]), nitrogen (# 976.05; [13]), EE (# 920.29; [13]), ary constituents (structural and non-structural carbohy- and ash (# 942.05; [13]). Neutral detergent fibre (NDF), in- drates, CP and lipids) in TMR samples representative of clusive of residual ash, was determined with α-amylase rations commonly used in intensive farms in North Italy. using the Ankom Fibre Analyzer (Ankom Technology, NY, USA). Acid detergent fibre (ADF), inclusive of residual Methods ash, and sulphuric acid lignin (lignin ) were determined (sa) Chemical composition of the diets sequentially after NDF determination [14]. Starch content Diets used in this experiment were defined after an was determined after hydrolysis to glucose [13] by liquid analysis of a database containing information about chromatography [15]. Gross energy content of diets (MJ/ ingredient and chemical composition of the rations used kg DM) was determined in duplicate by a bomb calorim- by 90 farms considered to be representative of the dairy eter method [16]. farm system in North Italy [11, 12]. A corn silage-diet, containing 361, 158, and 33 g/kg of Incubation NDF, CP, and lipids, respectively, was used as a reference The 7 diets were incubated in 4 repeated incubation (Table 1). Six other diets were formulated to produce runs, conducted in 4 successive wk. Two incubations variations in the proportion of some feed ingredients, were stopped at 24 h, whereas the other two were and hence in content of a given chemical constituent in stopped after 48 h. The incubation times of 24 and 48 h favor or at the expense of starchy feeds, with respect to were chosen as they are, respectively, the reference times the reference diet. Thus, changes in the dietary content for measuring in vitro GP [17] and in vitro degradability of NDF, CP and lipids, were always obtained at the of NDF [18]. In each of the four incubation runs, we expense or in favor of starch. Two diets with a low tested 7 diets × 5 replications, plus 5 blanks (bottles con- (325 g/kg DM) or a high (435 g/kg DM) content of NDF taining only the buffered rumen fluid; 5 blanks/run), for were formulated by replacing, accordingly, roughages a total of 160 bottles incubated. A commercial GP RF (corn silage, alfalfa hay, and ryegrass hay) with corn and apparatus (Ankom Gas Production System, Ankom barley grains in the form of meal. The diet with the high Technology®, NY, USA) was used, consisting of 40 NDF content did not contain corn silage, taking into bottles equipped with pressure sensors (pressure range: consideration dairy farms that are not allowed to use this from - 69 to 3,447 kPa; resolution: 0.27 kPa; accuracy: ± feed as they produce milk to be processed as Italian 0.1 % of measured value) and wireless connected to a Protected Designation of Origin (PDO) Parmigiano- computer. Each bottle (317 mL) was filled with 1.000 ± Reggiano cheese. Other two diets, with a low (115 g/kg 0.0010 g of diet, 100 mL of a buffer solution, and 50 mL DM) or a high CP content (194 g/kg DM), were formu- of rumen fluid (headspace volume = 167 mL), keeping lated by replacing, accordingly, soybean meal with cereal the headspace of bottles flushed with CO . grains (corn and barley meal). It must be underlined that The buffer solution was prepared according to [17], the upper level of CP tested in this study corresponded to heated in a water bath at 39 ± 0.4 °C and purged con- the maximum value found in the considered database of tinuously with CO for 30 min, to maintain anaerobic 90 farms. This value is high if compared with ranges in CP conditions. Rumen fluid was collected by an esophageal contents (118–186 g/kg DM) reported for rations used in probe, as described by [19], 2 h before morning feeding dairy farms of North-Italy [12]. Two diets with different from 3 dry Holstein-Friesian cows housed at the experi- ether extract (EE) content were also formulated. A low EE mental farm of the University of Padova (Italy) and fed diet (26 g/kg DM) was achieved by excluding the extruded hay ad libitum and 2.5 kg/d of concentrates (0.5 kg of Maccarana et al. Journal of Animal Science and Biotechnology (2016) 7:54 Page 3 of 8 Table 1 Feed ingredients, chemical composition and gross energy content of seven diets Reference Low NDF High NDF Low CP High CP Low Lipid High Lipid Ingredients, g/kg DM Corn silage 351 430 – 375 281 351 351 Alfalfa hay 89 23 134 66 156 89 89 Ryegrass hay 47 – 231 43 52 47 56 Meadow hay 47 – 227 47 52 47 60 Corn grain 205 228 152 258 147 218 160 Barley grain 119 171 92 160 90 122 100 Soybean meal, (sol. extr., 44) 113 119 126 27 188 126 18 Whole soybean seeds, cracked –– – – – – 68 Extruded soybean seeds –– – – – – 68 Extruded flaxseed seeds 29 29 38 24 34 – 29 Chemical composition, g/kg DM Crude protein (CP) 158 152 161 115 194 161 158 Starch 273 273 100 332 176 265 233 NDF 361 325 435 358 357 359 360 Hemicellulose 169 171 189 172 158 171 169 ADF 192 154 246 186 199 188 191 Cellulose 163 134 203 160 167 163 163 Lignin 29 20 43 26 32 25 28 (sa) NFC 395 443 302 446 357 402 367 Ether extract 33 34 38 34 34 26 61 Ash 53 46 64 47 58 52 54 Gross energy, MJ/kg DM 16.8 16.9 17.3 16.8 17.3 16.5 16.7 NFC Not Fiber Carbohydrates computed as 100 - NDF - CP - EE - Ash Measured by a bomb calorimeter method [16] dry sugar beet pulp, 1 kg of corn grain, and 1 kg of sun- bottle and stored at −20 °C with 1 mL of metaphos- flower meal). During the collection of rumen fluid, cows phoric acid (25 %, w/v) to be later analyzed for ammonia were handled according to the Italian law on animal care N and volatile fatty acids (VFA). The content of ammo- (Legislative Decree No. 26 of March 14, 2014). Rumen nia N was measured using the FIAstar™ 5000 Analyzer fluid was poured into thermal flasks preheated to 39 ± (FOSS Analytical, Hilleroed, Denmark). The VFA profile 0.5 °C, immediately transferred to the laboratory, strained was analyzed by GC with flame ionization detection through 3 layers of cheesecloth, to eliminate feed particles, (7820A GC system, Agilent Technologies, Milan, Italy) and mixed with buffer solution in a 1 to 2 ratio [17]. Oper- using a 30-m stainless steel column (J&W DB-FFAP, ations were conducted under anaerobic conditions, by Agilent Technologies, Milan, Italy) and H as carrier gas flushing with CO , and required less than 30 min to be (flow rate: 30 mL/min; isothermal oven temperature: completed. Bottles were placed in a ventilated oven at 39 150 °C). Fermentation fluids were filtered into weighed ± 0.4 °C and automatically vented at a fixed pressure (6.8 crucibles (30 mL, Robu Glasfilter-Geräte GMBH®, Hattert, kPa), to avoid overpressure conditions and alterations of Germany) and analyzed for residual NDF using a gas and CH measures [20]. In vitro GP was monitored in Fibretech Analyzer (VELP® Scientifica, Milan, Italy). continuous, using a setting of the GP system that allows to At the end of each incubation (24 or 48 h), gas was record pressure values every minute. Other in vitro collected with a 10-mL gas-tight syringe (Artsana S.p.A., parameters (rumen degradability, VFA and N-NH concen- Como, Italy) from the bottle headspace (HS). At each trations, CH production) were measured only at the end of sampling, the syringe was flushed in order to collect a incubation (at 24 or 48 h), to avoid opening of the oven dur- homogeneous sample, which was immediately trans- ing the incubation, with alteration of fermentation process. ferred into a 9-mL vacuette (Greiner Bio-One GmbH, At the end of incubations (24 or 48 h), two aliquots Kremsmunster, Austria). From each vacuette, an aliquot (5 mL) of fermentation fluid were collected from each (10 μL) of gas was sampled with a gas-tight syringe Maccarana et al. Journal of Animal Science and Biotechnology (2016) 7:54 Page 4 of 8 (1701 N, Hamilton, Bonaduz, Switzerland) and immedi- Results ately analyzed for CH concentration by GC with flame Changes of the feed ingredients proportions and of diet- ionization detection (7820A GC system, Agilent Tech- ary contents of chemical constituents had influence on nologies, Milan, Italy) using a 15-m carbon layer column the various parameters of in vitro fermentation (Table 2). (GS-CarbonPLOT, Agilent Technologies, Milan, Italy) As expected, pH values measured at the end of fermen- and H as carrier gas (flow rate: 1.6 mL/min; isothermal tation were not influenced by the dietary changes. The oven temperature: 40 °C). An 11-point calibration curve ammonia N content increased with increasing dietary was generated from eleven gas mixtures containing 2, 4, CP content (P = 0.014). No influence of the diets was 8, 16, 24, 32, 60, 100, 140, 180, and 240 mL of CH /L observed on the VFA production, but the proportion of (99.5 % pure, SAPIO s.r.l., Monza, Italy), respectively, acetate or butyrate decreased (P <0.001) or increased and known volumes of air. Mixtures were prepared (P = 0.004), respectively, with a decrease of NDF con- using the same graduated gas-tight syringe (1701 N, tent, whereas the proportion of propionate decreased Hamilton). The calibration regression had R > 0.99. (P = 0.048) with increasing dietary CP. Thus, the ratio between acetate and propionate decreased with de- Computations creasing level of NDF (P = 0.001) and of CP (P < 0.001), The NDF degradability (NDFd) and the true DM de- and the corresponding increasing level of starch. gradability (TDMd) were calculated according to [18]. Increasing proportions of dietary CP increased the pro- Recently, [20], using vented bottles connected to tight portion of other VFA (P < 0.001) found in the rumen bags for gas collection, calculated CH production (mL) fluid. Changes of dietary EE content had no conse- as: [CH concentration in HS] × [HS volume] + [CH quence on the various rumen fluid parameters. The 4 4 concentration in the gas bag × GP]. To evaluate the prolongation of the incubation time from 24 to 48 h in- possibility of avoiding the use of bags, to save space and creased the VFA production (P = 0.029), but it did not increase the number of replicates, amount of CH lost influence pH and ammonia N values. with gas venting was computed using the unpublished The NDFd, TDMd, and the GP expressed per unit of in- data of a previous study, where 4 forages and 3 concen- cubated DM or per unit of TDMd (Table 3) increased trates were incubated in 42 bottles (6 bottles/feed) for 6, with a decrease of NDF content (P < 0.001 for all). When 24, or 48 h using the same GP equipment and the same the NDF content decreased, the CH production increased operative conditions of the present experiment. It was per unit of incubated DM (P < 0.001) and per g of digested found that total CH production is predictable, with ac- NDF (P = 0.002), but not per unit of TDMd. The increased ceptable precision and accuracy, as: − 0.0064 × [CH con- dietary CP content, with the corresponding decrease of centration in HS × (HS volume + GP)] + 0.9835 × [CH starch, had no influence on NDFd or TDMd, but GP was concentration in HS × (HS volume + GP)]. This equation lowered. No influence was observed on the production of had a residual standard deviation of only 0.1770 mL, and CH , except when this was expressed as a proportion of R = 0.9993. Thus, the present experiment was conducted GP (mL CH /100 mL GP; P < 0.001). An increased inclu- without the use of tight bags for gas collection. The CH sion of extruded oilseeds in the diet reduced both NDFd production was computed using the above described (P = 0.003) and TDMd (P = 0.028), and the measured GP equation and it was expressed as mL/g DM incubated, expressed per g of incubated DM (P = 0.017), but no influ- mL/g of digested NDF (dNDF), mL/g TDMd, or mL/ ences were observed on the CH yield. A prolonged dur- 100 mL GP. In vitro GP and CH were also predicted from ationofthe incubation,from24to48h,increased NDFd VFA production, according to [21]. (P = 0.009), TDMd (P =0.007), and CH production, both per unit of incubated DM (P = 0.014) and per unit of Statistical analysis TDMd (P = 0.027). The correlation between measured and The mean of the 5 replications for each diet in each incu- predictedvaluesshowedR to be 0.78 and 0.74, respect- bation run was computed. These 28 means were analyzed ively, for gas and CH measures (data not shown), and the using PROC MIXED of SAS [22] using a model consider- relationship obtained by regressing measured values of CH ing the diet (D; 6 df), the incubation time (IT, 1 df), and (mL/g DM; y) against those predicted (mL/g DM; x) was the interaction diet × incubation time as fixed factors, the the following: measured CH =0.95×predicted CH – 2.6. 4 4 run within incubation time (2 df) as a random blocking Predicted values of GP and CH productions were not in- factor and the residual error term e (18 df). As the diet × fluenced either by dietary changes or by incubation time. incubation time interaction was never significant, it was excluded from the model. Contrasts were run to analyze Discussion statistical differences among diets with different contents General considerations of a given chemical constituent, using the Bonferroni ad- The diets were formulated using feed ingredients com- justment to perform multiple comparisons. monly used in the Po valley (North-East of Italy) and Maccarana et al. Journal of Animal Science and Biotechnology (2016) 7:54 Page 5 of 8 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 pH Ammonia N, mg/L VFA, mmoL/L Acetate (Ac), % VFA Propionate (Pr), % VFA Ac:Pr Butyrate, % VFA Other VFA, % VFA Diet Reference 6.87 202 5.08 56.1 23.5 2.40 16.0 4.47 Low NDF 6.88 192 5.37 55.8 23.7 2.36 16.2 4.29 A A B B High NDF 6.90 223 4.44 58.5 23.1 2.53 13.3 4.26 Low CP 6.85 171 5.06 56.0 24.3 2.31 16.1 3.71 A A High CP 6.87 222 4.89 57.2 22.8 2.51 14.6 4.85 Low lipid 6.87 207 4.96 56.4 23.7 2.39 15.5 4.26 High lipid 6.90 197 4.99 56.7 23.5 2.41 15.4 4.36 SEM 0.043 28.5 0.231 0.25 0.64 0.062 0.91 0.158 P-value 0.46 0.008 0.25 <0.001 0.06 0.001 0.002 <0.001 P-value of contrasts Low vs High NDF 0.99 0.44 0.23 <0.001 0.99 0.001 0.004 0.99 Low vs High CP 0.99 0.014 0.99 0.048 0.048 <0.001 0.99 <0.001 Low vs High lipid 0.99 0.99 0.99 0.99 0.99 0.65 0.99 0.99 Incubation time 24 h 6.95 202 4.47 57.2 22.9 2.50 15.8 3.87 48 h 6.80 202 5.47 56.1 24.1 2.33 14.7 4.76 SEM 0.055 38.7 0.123 0.13 0.82 0.077 1.15 0.201 P-value 0.31 0.99 0.029 0.026 0.39 0.26 0.58 0.09 RMSE 0.032 17.5 0.462 0.49 0.60 0.112 0.87 0.147 Values with different superscripts within column are significantly (P < 0.05) higher (A) or lower (B) compared to the reference diet (containing 361, 158, and 33 g/kg DM of NDF, CP, and lipids) Low NDF low NDF diet (325 g/kg DM); High NDF high NDF diet (435 g/kg DM), Low CP low protein diet (CP, 115 g/kg DM), High CP high protein diet (CP, 194 g/kg DM), Low lipid low lipid diet (EE, 26 g/kg DM), High lipid high lipid diet (EE, 61 g/kg DM) composed mainly by cereal grains, corn silage and vari- After 24 h of fermentation, the measured GP of the ous hays. It should be preliminarily considered that in various diets was, on average, 250 mL/g DM, suggesting this study the variation in the content of the various nu- that a cow consuming 20 kg/d DM might produce about trients, namely, NDF, CP and lipid, were always achieved 5,000 L/d of gas. The CH production from fermentation by decreasing or increasing the proportion of dietary of these diets ranged 30.8 to 34.4 mL/g DM, suggesting starch. In the scientific literature, in vitro evaluation of that, for a DM intake of 20 kg/d, a cow might produce gas and CH productions is commonly carried out using 616 to 688 L/d of CH . Sauer et al. (1998) reported 4 4 single feeds, mainly forages, whereas less information is in vivo CH production of lactating cows in the order of available for complete diets [23]. In vivo measurement of 622 L/d, corresponding to 38.9 mL/g DM intake. In the gas and CH production requires expensive equipment study of [25], CH production from dairy cows was 4 4 and it is labor and time consuming. In vitro techniques 29.2 mL/g DM intake, whereas [26] reported for sheep would permit a much more simple determination of the an averaged CH production of 31.0 mL/g DM intake. dietary characteristics which can influence the potential In a continuous culture fermenter, [27] measured an emission of gas and CH from their fermentation in a averaged CH production of 33.0 mL/g DM. The in vitro 4 4 simulated rumen environment [20]. Studies of the CH production of the dairy rations tested by [23] varied relationships between in vitro and in vivo gas and CH from 30.1 to 35.9 mL/g DM, a range consistent with productions are lacking [23]. However, a recent study results obtained in this study. However, comparison suggested that in vitro gas and CH measurements can with data from literature is difficult because huge varia- be indicative of the trend of in vivo CH production ori- tions in gas and CH productions are commonly ob- 4 4 ginating from different combinations of feed ingredients served across experiments, even for diets with similar [24]. This study was aimed at evaluating if changes in composition. This is the consequence of a combination the diet composition might or might not have notable of different biological, as rumen fluid characteristics, influence on gas and CH productions. and technical factors, as fermentation procedures and 4 Maccarana et al. Journal of Animal Science and Biotechnology (2016) 7:54 Page 6 of 8 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 (CH ) production, and predicted values of GP and CH production 4 4 Degradability Actual GP, ml per: Actual CH mL per Predicted GP Predicted CH 4 4 NDFd, g/kg NDF TDMd, g/kg DM g DM g TDMd g DM g dNDF g TDMd 100 mL GP mL/g DM mL/g DM Diet Reference 567 840 274 325 34.2 168 40.8 12.5 244 38.8 A A A Low NDF 612 872 288 331 34.4 174 39.3 11.9 250 39.6 B B B B B B A High NDF 480 767 228 300 30.8 151 39.9 13.5 220 35.9 Low CP 545 835 277 333 33.4 169 39.9 12.0 242 38.0 B A High CP 542 831 255 310 34.0 176 41.0 13.3 234 37.8 Low lipid 583 845 265 315 33.1 159 39.0 12.3 251 37.2 B B B High lipid 531 826 253 304 32.2 169 38.8 12.5 238 37.9 SEM 10.3 4.2 5.6 7.2 0.53 3.4 0.63 0.40 10.9 2.09 P-value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.025 <0.001 0.33 0.70 P-value of contrasts Low vs High NDF <0.001 <0.001 <0.001 <0.001 <0.001 0.002 0.99 <0.001 0.99 0.99 Low vs High CP 0.99 0.99 <0.001 0.009 0.99 0.99 0.99 <0.001 0.99 0.99 Low vs High lipid 0.003 0.028 0.017 0.99 0.99 0.99 0.99 0.99 0.99 0.99 Incubation time 24 h 472 801 250 314 29.6 172 37.0 11.9 214 34.9 48 h 631 860 276 320 36.7 161 42.6 13.2 260 41.5 SEM 10.5 3.5 7.4 8.8 0.59 1.8 0.66 0.54 7.6 1.69 P-value 0.009 0.007 0.14 0.69 0.014 0.046 0.027 0.22 0.06 0.12 RMSE 15.5 7.2 4.0 7.6 0.69 3.2 0.94 0.25 21.9 3.78 Values with different superscripts within column are significantly (P < 0.05) higher (A) or lower (B) compared to the reference diet (containing 361, 158, and 33 g/kg DM of NDF, CP, and lipids) Low NDF low NDF diet (325 g/kg DM), High NDF high NDF diet (435 g/kg DM), Low CP low protein diet (CP, 115 g/kg DM), High CP high protein diet (CP, 194 g/kg DM), Low lipid low lipid diet (EE, 26 g/kg DM), High lipid high lipid diet (EE, 61 g/kg DM) equipment for collection, measurement and analysis On the contrary, the diet with the high level of NDF [28]. In this experiment the observed GP and CH were (435 g/kg DM) was based on different hays and did not regressed against the GP and CH values predicted contain corn silage. This is of interest in Italy as use of from the stoichiometry of the VFA production, to ob- silages for the production of important Protected De- tain an internal control of consistency of the data. The nomination of Origin (PDO) cheeses (i.e., Parmigiano- correlations found (R = 0.78 and 0.74, respectively, for Reggiano) is forbidden by specific feeding regulations. gas and CH measures) are acceptable considering the From the results it emerged that NDF degradability was rather narrow range of variation in GP and CH pro- negatively influenced by the dietary NDF content, par- duction caused by the dietary changes. It was observed tially because of the complete replacement of corn silage that the SEM of predicted GP and CH production with hays. This would be in agreement with previous (21.9 and 3.78 mL/g DM, respectively) was about five observations that the NDF fraction of hay samples col- times greater than the corresponding values of the lected in the same area of the present experiment were measured GP and CH production. In other words, the less degradable than corn silage samples [19]. measure of the VFA production was less precise than In diets similar for ingredient composition to those the GP and CH measures. used in this experiment, [12] observed an increased CH 4 4 production (35.6 to 44.3 mL/g DM) with decreasing Effects on NDF degradability, gas and CH production NDF content. In the present study, a decrease from 435 due to changes in the NDF content to 325 g NDF/kg DM increased CH production from In this experiment, the diet with the low level of NDF 30.8 to 34.4 mL/g DM (+11.6 %). This is consistent with (325 g/kg DM) was based on corn silage and a small an increased true degradability of the feed (+14 %), but amount of alfalfa hay (23 g/kg DM) as forage sources. also of the NDF fibrous fraction (+27 %). This seems to Maccarana et al. Journal of Animal Science and Biotechnology (2016) 7:54 Page 7 of 8 be contradictory with current literature, as a decrease of affect its availability in the rumen, and these factors ap- dietary NDF commonly reduces the NDF degradability pear to be more important than the FA profile [35]. In [12]. However, also [29] evidenced that a decrease of the this regard, [36], using dairy cows housed in respiratory dietary NDF content increased by 43 % in vivo CH chambers, found that, compared to the control, the production expressed per unit of dNDF. Results also evi- average reduction in CH (L/kg DMI) per 10 g/kg of denced that GP increased with a decrease of NDF con- crude fat added was persistent throughout lactation. tent even when expressed per unit of TDMd (+10 %), The same authors observed that the most effective lipid but no influences were observed on the CH production source in reducing methanogenesis was a commercial per unit of TDMd. A different trend emerged when CH vegetal rumen protected fat fortified with hydroxy- production was referred to the total GP. In this case, the methionine-analog-isobutyrate (−5.5 % of CH ), decrease of NDF lowered the CH proportion by about followed by vegetal rumen protected fat (−2.3 %), and 12 % (13.5 to 11.9 mL CH /100 mL GP, for the high and by whole cracked rapeseed (−0.8 %). In the experiment the low content of NDF, respectively). Results suggest of [35], only crushed canola seeds lowered CH produc- that a decrease of dietary NDF content, achieved from a tion per unit of digestible DM intake (−15 %), whereas complete replacement of hays with corn silage and crushed flaxseeds and crushed sunflower seeds did not cereal grains, might increase feed digestibility without reduce CH production compared to the control diet (a changing GP and CH produced per unit of digested diet supplemented with a commercial source of calcium material. salts of long chain fatty acids). In the current experi- ment, fat addition reduced feed degradability, particu- larly that of the NDF fraction. No influences were Effects on gas and CH production due to changes in the found on total VFA production and on the proportion CP content of acetate, propionate and butyrate, whereas GP and In this experiment an increased proportion of CP, in CH productions decreased by 8 and 6 %, respectively, replacement of starch, caused a reduction of GP. The compared to the reference diet. However, differences negative influence of dietary CP on GP has been ob- were greatly reduced when GP and CH were expressed served by others in the past. Such an effect was at- per unit of TDMd suggesting that, under the commer- tributed to the buffer capacity of CP, that reduces the cial conditions evaluated in this study, small reductions indirect CO released from the buffered rumen fluid, of CH might be achieved. and to the stoichiometry of protein fermentation, that differs from that of carbohydrates [30, 31]. In this Conclusions study, increase of the CP content was associated to a Changes of the ingredient and chemical composition of decrease of dietary starch. As a consequence, some diets were analyzed to evaluate benefits in the amount of VFA as iso-butyric and iso-valeric acids were in- CH produced, for the north eastern Italian dairy pro- creased, being end-products of protein degradation duction chain. It was found that a replacement of hays [32], whereas the production of propionate decreased with corn silage and cereals might increase GP and CH and the ratio between acetate and propionate in- per unit of DM intake. An increase of the dietary CP creased. Changes in the dietary CP proportion had no content would reduce GP with no influences in the effect on CH production when expressed both per amount of CH produced, whereas a moderate addition unit of incubated DM and per unit of TDMd. Thus, of cracked soybean seeds, and extruded flaxseed had as CP depresses GP, an increased proportion of CP few, or any, influence on the in vitro GP and CH produc- increases the CH concentration on total GP. tions. In general, none of the various strategies tested in the present work was able to reduce the amount of CH Effects on gas and CH production due to changes in the produced, especially if this production is expressed per lipid content unit of digestible DM intake. More research is needed to In this experiment changes in the dietary fat content of evaluate the effectiveness of strategies to reduce the CH the diets were achieved by changing the proportions of emissions, and relationships between in vitro and in vivo extruded flaxseeds, extruded soybean and whole soybean gas and CH productions need to be developed. seeds. The threshold of 60 g fat/kg DM was considered Abbreviations to be the upper limit to avoid a possible impairment of ADF, Acid detergent fibre expressed inclusive of residual ash; CP, Crude feed digestibility [6, 33]. protein; DM, Dry matter; EE, Ether extract; GP, Gas production; NDF, Neutral The effect of dietary lipids on CH production is detergent fibre assayed with a heat stable amylase and expressed inclusive of residual ash; NDFd, NDF degradability; lignin(sa), Lignin determined by dependent on the source, FA profile, level of inclusion, solubilization of cellulose with sulphuric acid; NRC, National Research and diet composition [34]. The level of supplementa- Council; RMSE, Root mean square error; SEM, Standard error of the mean; tion and the physical form of the lipid supplement TDMd, True DM degradability; VFA, Volatile fatty acids Maccarana et al. Journal of Animal Science and Biotechnology (2016) 7:54 Page 8 of 8 Acknowledgments 16. ISO 9831. Animal feeding stuffs, animal products, and faeces or urine – This work was financed by the project “ARCHAEA - Feeding strategies to Determination of gross calorific value – Bomb calorimeter method. Geneva: reduce methane emissions from dairy cows”– Veneto Region Rural ISO 9831; 1988. Development Programme (RDP) 2007–2013. 17. Menke KH, Steingass H. Estimation of the energetic feed value obtained from chemical analysis and gas production using rumen fluid. Anim Res Dev. 1988;28:7–55. Authors’ contributions 18. Goering HK, Van Soest PJ. Forage fiber analysis (apparatus, reagents, All authors conceived of this experiment, due to experience gained over the procedures, and some applications). Agricultural hand-book no. 379. last years in the use of in vitro gas production technique for the evaluation Washington, DC: USDA; 1970. of ruminant feeds. LM and MC gave substantial contributions to analytical 19. Tagliapietra F, Cattani M, Hindrichsen IK, Hansen HH, Colombini S, Bailoni L, procedures, statistical analysis, and writing of the manuscript. FT, LB, and SS et al. True dry matter digestibility of feeds evaluated in situ with different contributed in revising critically the manuscript. All authors read and bags and in vitro using rumen fluid collected from intact donor cows. Anim approved the final manuscript. Prod Sci. 2012;52(5):338–346. 20. Cattani M, Tagliapietra F, Maccarana L, Hansen HH, Bailoni L, Schiavon S. Competing interests Technical note: In vitro total gas and methane production measurements The authors declare that they have no competing interests. from closed or vented rumen batch culture systems. J Dairy Sci. 2014;97(3): 1736–741. Author details 21. Blümmel M, Aiple KP, Steingass H, Becker K. A note on the stoichiometrical Department of Comparative Biomedicine and Food Science (BCA), relationship of short chain fatty acid production and gas formation in vitro University of Padova, Viale dell’Università 16, 35020 Legnaro (PD), Italy. in feedstuffs of widely differing quality. J Anim Physiol Anim Nutr. 1999; Department of Agronomy, Food, Natural resources, Animals and 81(3):157–67. Environment (DAFNAE), University of Padova, Viale dell’Università 16, 35020 22. Institute SAS. SAS User’s Guide: Basics. Cary: SAS Inst. Inc.; 2007. Legnaro (PD), Italy. 23. Getachew G, Robinson PH, Depeters EJ, Taylor SJ, Gisi DD, Higginbotham GE, Riordan TJ. Methane production from commercial dairy rations Received: 14 January 2016 Accepted: 17 August 2016 estimated using an in vitro gas technique. Anim Feed Sci Technol. 2005; 123–124(1):391–402. 24. Hatew B, Cone JW, Pellikaan WF, Podesta SC, Bannink A, Hendriks WA, et al. 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Methane production by mixed ruminal 4. Johnson KA, Johnson DE. Methane emissions from cattle. J Anim Sci. 1995; cultures incubated in dual-flow fermenters. J Dairy Sci. 2004;87(1):112–21. 73(8):2483–92. 28. Maccarana L, Cattani M, Mantovani R, Tagliapietra F, Schiavon S, Bailoni L. 5. Patra AK. Enteric methane mitigation technologies for ruminant livestock: a Assessment of factors influencing in vitro gas and methane production by synthesis of current research and future directions. Environ Monit Assess. meta-analysis. Milan: Proceedings of the 21st ASPA Congress; 2015. p. 22. 2012;184(4):1929–952. 29. Pirondini M, Colombini S, Mele M, Malagutti L, Rapetti L, Galassi G, et al. 6. Kumar S, Choudhury PK, Carro MD, Griffith GW, Dagar SS, Puniya M, et al. Effects of dietary starch concentration and fish oil supplementation on milk New aspects and strategies for methane mitigation fromruminants. 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The Analysis of Dietary Fiber in Food. New York: Marcel Dekker Inc; 1981. p. 123–58. 15. Bouchard J, Chornet E, Overend RP. High-performance liquid chromatographic monitoring carbohydrate fractions in partially hydrolyzed corn starch. J Agric Food Chem. 1988;36(6):1188–192. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Animal Science and Biotechnology Springer Journals

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

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Copyright © 2016 by The Author(s).
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Life Sciences; Agriculture; Biotechnology; Food Science; Animal Genetics and Genomics; Animal Physiology
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Abstract

Background: Modification of chemical composition of diets fed to dairy cows might be a good strategy to reduce methane (CH ) production in the rumen. Notable reductions of CH production compared to conventional high- 4 4 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 CH production. Results: 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 CH 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 CH 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 CH production per g of TDMd. Conclusions: It was concluded that, under commercial conditions, changes in dietary composition would produce small or negligible alterations of CH production per unit of TDMd, but greater differences in GP and CH production 4 4 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. Keywords: Dairy cows, Dietary manipulation, Gas production, In vitro techniques, Methane production Background type and content of dietary carbohydrates [2] and lipids Mitigation of methane (CH ) production from rumen [3]. In practice, notable reductions of CH production 4 4 fermentation represents an important target for animal compared to conventional high-roughages rations were nutritionists, as also this gas is responsible for global more frequently observed for very concentrated diets [4] warming. Thus, the manipulation of dietary nutrient or when fat supplements [3] were used. In these cases composition is often proposed as a strategy that farmers the reduction in the gas emission was mainly a conse- may exploit to reduce the proportion of energy lost by quence of an overall impairment of rumen function with a animals as eructated gases (CH ) and to improve feed reduction of fiber digestibility [5]. Thus, these strategies and energy efficiency [1]. There is evidence that the do not always comply with the feeding standards used in amount of CH produced in the rumen is influenced by intensive dairy farms and they are usually not applied owing to the risks of negative health and economic conse- * Correspondence: mirko.cattani@unipd.it quences [6]. Compared to carbohydrates and lipids, minor Department of Comparative Biomedicine and Food Science (BCA), effects on rumen gas production and methanogenesis are University of Padova, Viale dell’Università 16, 35020 Legnaro (PD), Italy usually attributed to the crude protein (CP). In this regard, Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Maccarana et al. Journal of Animal Science and Biotechnology (2016) 7:54 Page 2 of 8 [7] observed that CH production related to CP fermenta- flaxseed (Linoies, Cortal Extrasoy, Cittadella, Italy), present tion was lower compared to that due to carbohydrate fer- in the reference diet, and increasing the content of corn, mentation. To date, changes in the dietary CP content barley and soybean meal. The high EE diet (61 g/kg DM) have been mainly addressed to reduce feeding costs and N was achieved by including whole soybean seeds, extruded excretion [8, 9]. However, dietary strategies to reduce N soybean (Soyfull, Cortal Extrasoy, Cittadella, Italy) and ex- excretion could also have an impact because CH produc- truded flaxseed. All diets were prepared at the laboratory of tion may decline when dietary CP is replaced by rumen the University of Padova. For preparation, about 1 kg of bypass nutrients, including starch, escaping rumen fer- each diet was ground to 1 mm using a hammer mill (Pull- mentation [10]. Despite this, little information is currently erisette 19, Fritsch GmbH, Laborgeratebau, Germany). available on the effects on CH production due to changes For each diet, 23 samples were randomly collected, 20 of in dietary CP content. which were used for the incubations (5 per each of the 4 This in vitro study was aimed at evaluating the extent of incubations), whereas the remaining were used for chem- alterations of true dry matter degradability (TDMd), total ical analysis. gas (GP) and CH productions caused by changes in the Diets were analyzed in triplicate for dry matter (DM: # proportions of the main feed ingredients and of the diet- 934.01; [13]), nitrogen (# 976.05; [13]), EE (# 920.29; [13]), ary constituents (structural and non-structural carbohy- and ash (# 942.05; [13]). Neutral detergent fibre (NDF), in- drates, CP and lipids) in TMR samples representative of clusive of residual ash, was determined with α-amylase rations commonly used in intensive farms in North Italy. using the Ankom Fibre Analyzer (Ankom Technology, NY, USA). Acid detergent fibre (ADF), inclusive of residual Methods ash, and sulphuric acid lignin (lignin ) were determined (sa) Chemical composition of the diets sequentially after NDF determination [14]. Starch content Diets used in this experiment were defined after an was determined after hydrolysis to glucose [13] by liquid analysis of a database containing information about chromatography [15]. Gross energy content of diets (MJ/ ingredient and chemical composition of the rations used kg DM) was determined in duplicate by a bomb calorim- by 90 farms considered to be representative of the dairy eter method [16]. farm system in North Italy [11, 12]. A corn silage-diet, containing 361, 158, and 33 g/kg of Incubation NDF, CP, and lipids, respectively, was used as a reference The 7 diets were incubated in 4 repeated incubation (Table 1). Six other diets were formulated to produce runs, conducted in 4 successive wk. Two incubations variations in the proportion of some feed ingredients, were stopped at 24 h, whereas the other two were and hence in content of a given chemical constituent in stopped after 48 h. The incubation times of 24 and 48 h favor or at the expense of starchy feeds, with respect to were chosen as they are, respectively, the reference times the reference diet. Thus, changes in the dietary content for measuring in vitro GP [17] and in vitro degradability of NDF, CP and lipids, were always obtained at the of NDF [18]. In each of the four incubation runs, we expense or in favor of starch. Two diets with a low tested 7 diets × 5 replications, plus 5 blanks (bottles con- (325 g/kg DM) or a high (435 g/kg DM) content of NDF taining only the buffered rumen fluid; 5 blanks/run), for were formulated by replacing, accordingly, roughages a total of 160 bottles incubated. A commercial GP RF (corn silage, alfalfa hay, and ryegrass hay) with corn and apparatus (Ankom Gas Production System, Ankom barley grains in the form of meal. The diet with the high Technology®, NY, USA) was used, consisting of 40 NDF content did not contain corn silage, taking into bottles equipped with pressure sensors (pressure range: consideration dairy farms that are not allowed to use this from - 69 to 3,447 kPa; resolution: 0.27 kPa; accuracy: ± feed as they produce milk to be processed as Italian 0.1 % of measured value) and wireless connected to a Protected Designation of Origin (PDO) Parmigiano- computer. Each bottle (317 mL) was filled with 1.000 ± Reggiano cheese. Other two diets, with a low (115 g/kg 0.0010 g of diet, 100 mL of a buffer solution, and 50 mL DM) or a high CP content (194 g/kg DM), were formu- of rumen fluid (headspace volume = 167 mL), keeping lated by replacing, accordingly, soybean meal with cereal the headspace of bottles flushed with CO . grains (corn and barley meal). It must be underlined that The buffer solution was prepared according to [17], the upper level of CP tested in this study corresponded to heated in a water bath at 39 ± 0.4 °C and purged con- the maximum value found in the considered database of tinuously with CO for 30 min, to maintain anaerobic 90 farms. This value is high if compared with ranges in CP conditions. Rumen fluid was collected by an esophageal contents (118–186 g/kg DM) reported for rations used in probe, as described by [19], 2 h before morning feeding dairy farms of North-Italy [12]. Two diets with different from 3 dry Holstein-Friesian cows housed at the experi- ether extract (EE) content were also formulated. A low EE mental farm of the University of Padova (Italy) and fed diet (26 g/kg DM) was achieved by excluding the extruded hay ad libitum and 2.5 kg/d of concentrates (0.5 kg of Maccarana et al. Journal of Animal Science and Biotechnology (2016) 7:54 Page 3 of 8 Table 1 Feed ingredients, chemical composition and gross energy content of seven diets Reference Low NDF High NDF Low CP High CP Low Lipid High Lipid Ingredients, g/kg DM Corn silage 351 430 – 375 281 351 351 Alfalfa hay 89 23 134 66 156 89 89 Ryegrass hay 47 – 231 43 52 47 56 Meadow hay 47 – 227 47 52 47 60 Corn grain 205 228 152 258 147 218 160 Barley grain 119 171 92 160 90 122 100 Soybean meal, (sol. extr., 44) 113 119 126 27 188 126 18 Whole soybean seeds, cracked –– – – – – 68 Extruded soybean seeds –– – – – – 68 Extruded flaxseed seeds 29 29 38 24 34 – 29 Chemical composition, g/kg DM Crude protein (CP) 158 152 161 115 194 161 158 Starch 273 273 100 332 176 265 233 NDF 361 325 435 358 357 359 360 Hemicellulose 169 171 189 172 158 171 169 ADF 192 154 246 186 199 188 191 Cellulose 163 134 203 160 167 163 163 Lignin 29 20 43 26 32 25 28 (sa) NFC 395 443 302 446 357 402 367 Ether extract 33 34 38 34 34 26 61 Ash 53 46 64 47 58 52 54 Gross energy, MJ/kg DM 16.8 16.9 17.3 16.8 17.3 16.5 16.7 NFC Not Fiber Carbohydrates computed as 100 - NDF - CP - EE - Ash Measured by a bomb calorimeter method [16] dry sugar beet pulp, 1 kg of corn grain, and 1 kg of sun- bottle and stored at −20 °C with 1 mL of metaphos- flower meal). During the collection of rumen fluid, cows phoric acid (25 %, w/v) to be later analyzed for ammonia were handled according to the Italian law on animal care N and volatile fatty acids (VFA). The content of ammo- (Legislative Decree No. 26 of March 14, 2014). Rumen nia N was measured using the FIAstar™ 5000 Analyzer fluid was poured into thermal flasks preheated to 39 ± (FOSS Analytical, Hilleroed, Denmark). The VFA profile 0.5 °C, immediately transferred to the laboratory, strained was analyzed by GC with flame ionization detection through 3 layers of cheesecloth, to eliminate feed particles, (7820A GC system, Agilent Technologies, Milan, Italy) and mixed with buffer solution in a 1 to 2 ratio [17]. Oper- using a 30-m stainless steel column (J&W DB-FFAP, ations were conducted under anaerobic conditions, by Agilent Technologies, Milan, Italy) and H as carrier gas flushing with CO , and required less than 30 min to be (flow rate: 30 mL/min; isothermal oven temperature: completed. Bottles were placed in a ventilated oven at 39 150 °C). Fermentation fluids were filtered into weighed ± 0.4 °C and automatically vented at a fixed pressure (6.8 crucibles (30 mL, Robu Glasfilter-Geräte GMBH®, Hattert, kPa), to avoid overpressure conditions and alterations of Germany) and analyzed for residual NDF using a gas and CH measures [20]. In vitro GP was monitored in Fibretech Analyzer (VELP® Scientifica, Milan, Italy). continuous, using a setting of the GP system that allows to At the end of each incubation (24 or 48 h), gas was record pressure values every minute. Other in vitro collected with a 10-mL gas-tight syringe (Artsana S.p.A., parameters (rumen degradability, VFA and N-NH concen- Como, Italy) from the bottle headspace (HS). At each trations, CH production) were measured only at the end of sampling, the syringe was flushed in order to collect a incubation (at 24 or 48 h), to avoid opening of the oven dur- homogeneous sample, which was immediately trans- ing the incubation, with alteration of fermentation process. ferred into a 9-mL vacuette (Greiner Bio-One GmbH, At the end of incubations (24 or 48 h), two aliquots Kremsmunster, Austria). From each vacuette, an aliquot (5 mL) of fermentation fluid were collected from each (10 μL) of gas was sampled with a gas-tight syringe Maccarana et al. Journal of Animal Science and Biotechnology (2016) 7:54 Page 4 of 8 (1701 N, Hamilton, Bonaduz, Switzerland) and immedi- Results ately analyzed for CH concentration by GC with flame Changes of the feed ingredients proportions and of diet- ionization detection (7820A GC system, Agilent Tech- ary contents of chemical constituents had influence on nologies, Milan, Italy) using a 15-m carbon layer column the various parameters of in vitro fermentation (Table 2). (GS-CarbonPLOT, Agilent Technologies, Milan, Italy) As expected, pH values measured at the end of fermen- and H as carrier gas (flow rate: 1.6 mL/min; isothermal tation were not influenced by the dietary changes. The oven temperature: 40 °C). An 11-point calibration curve ammonia N content increased with increasing dietary was generated from eleven gas mixtures containing 2, 4, CP content (P = 0.014). No influence of the diets was 8, 16, 24, 32, 60, 100, 140, 180, and 240 mL of CH /L observed on the VFA production, but the proportion of (99.5 % pure, SAPIO s.r.l., Monza, Italy), respectively, acetate or butyrate decreased (P <0.001) or increased and known volumes of air. Mixtures were prepared (P = 0.004), respectively, with a decrease of NDF con- using the same graduated gas-tight syringe (1701 N, tent, whereas the proportion of propionate decreased Hamilton). The calibration regression had R > 0.99. (P = 0.048) with increasing dietary CP. Thus, the ratio between acetate and propionate decreased with de- Computations creasing level of NDF (P = 0.001) and of CP (P < 0.001), The NDF degradability (NDFd) and the true DM de- and the corresponding increasing level of starch. gradability (TDMd) were calculated according to [18]. Increasing proportions of dietary CP increased the pro- Recently, [20], using vented bottles connected to tight portion of other VFA (P < 0.001) found in the rumen bags for gas collection, calculated CH production (mL) fluid. Changes of dietary EE content had no conse- as: [CH concentration in HS] × [HS volume] + [CH quence on the various rumen fluid parameters. The 4 4 concentration in the gas bag × GP]. To evaluate the prolongation of the incubation time from 24 to 48 h in- possibility of avoiding the use of bags, to save space and creased the VFA production (P = 0.029), but it did not increase the number of replicates, amount of CH lost influence pH and ammonia N values. with gas venting was computed using the unpublished The NDFd, TDMd, and the GP expressed per unit of in- data of a previous study, where 4 forages and 3 concen- cubated DM or per unit of TDMd (Table 3) increased trates were incubated in 42 bottles (6 bottles/feed) for 6, with a decrease of NDF content (P < 0.001 for all). When 24, or 48 h using the same GP equipment and the same the NDF content decreased, the CH production increased operative conditions of the present experiment. It was per unit of incubated DM (P < 0.001) and per g of digested found that total CH production is predictable, with ac- NDF (P = 0.002), but not per unit of TDMd. The increased ceptable precision and accuracy, as: − 0.0064 × [CH con- dietary CP content, with the corresponding decrease of centration in HS × (HS volume + GP)] + 0.9835 × [CH starch, had no influence on NDFd or TDMd, but GP was concentration in HS × (HS volume + GP)]. This equation lowered. No influence was observed on the production of had a residual standard deviation of only 0.1770 mL, and CH , except when this was expressed as a proportion of R = 0.9993. Thus, the present experiment was conducted GP (mL CH /100 mL GP; P < 0.001). An increased inclu- without the use of tight bags for gas collection. The CH sion of extruded oilseeds in the diet reduced both NDFd production was computed using the above described (P = 0.003) and TDMd (P = 0.028), and the measured GP equation and it was expressed as mL/g DM incubated, expressed per g of incubated DM (P = 0.017), but no influ- mL/g of digested NDF (dNDF), mL/g TDMd, or mL/ ences were observed on the CH yield. A prolonged dur- 100 mL GP. In vitro GP and CH were also predicted from ationofthe incubation,from24to48h,increased NDFd VFA production, according to [21]. (P = 0.009), TDMd (P =0.007), and CH production, both per unit of incubated DM (P = 0.014) and per unit of Statistical analysis TDMd (P = 0.027). The correlation between measured and The mean of the 5 replications for each diet in each incu- predictedvaluesshowedR to be 0.78 and 0.74, respect- bation run was computed. These 28 means were analyzed ively, for gas and CH measures (data not shown), and the using PROC MIXED of SAS [22] using a model consider- relationship obtained by regressing measured values of CH ing the diet (D; 6 df), the incubation time (IT, 1 df), and (mL/g DM; y) against those predicted (mL/g DM; x) was the interaction diet × incubation time as fixed factors, the the following: measured CH =0.95×predicted CH – 2.6. 4 4 run within incubation time (2 df) as a random blocking Predicted values of GP and CH productions were not in- factor and the residual error term e (18 df). As the diet × fluenced either by dietary changes or by incubation time. incubation time interaction was never significant, it was excluded from the model. Contrasts were run to analyze Discussion statistical differences among diets with different contents General considerations of a given chemical constituent, using the Bonferroni ad- The diets were formulated using feed ingredients com- justment to perform multiple comparisons. monly used in the Po valley (North-East of Italy) and Maccarana et al. Journal of Animal Science and Biotechnology (2016) 7:54 Page 5 of 8 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 pH Ammonia N, mg/L VFA, mmoL/L Acetate (Ac), % VFA Propionate (Pr), % VFA Ac:Pr Butyrate, % VFA Other VFA, % VFA Diet Reference 6.87 202 5.08 56.1 23.5 2.40 16.0 4.47 Low NDF 6.88 192 5.37 55.8 23.7 2.36 16.2 4.29 A A B B High NDF 6.90 223 4.44 58.5 23.1 2.53 13.3 4.26 Low CP 6.85 171 5.06 56.0 24.3 2.31 16.1 3.71 A A High CP 6.87 222 4.89 57.2 22.8 2.51 14.6 4.85 Low lipid 6.87 207 4.96 56.4 23.7 2.39 15.5 4.26 High lipid 6.90 197 4.99 56.7 23.5 2.41 15.4 4.36 SEM 0.043 28.5 0.231 0.25 0.64 0.062 0.91 0.158 P-value 0.46 0.008 0.25 <0.001 0.06 0.001 0.002 <0.001 P-value of contrasts Low vs High NDF 0.99 0.44 0.23 <0.001 0.99 0.001 0.004 0.99 Low vs High CP 0.99 0.014 0.99 0.048 0.048 <0.001 0.99 <0.001 Low vs High lipid 0.99 0.99 0.99 0.99 0.99 0.65 0.99 0.99 Incubation time 24 h 6.95 202 4.47 57.2 22.9 2.50 15.8 3.87 48 h 6.80 202 5.47 56.1 24.1 2.33 14.7 4.76 SEM 0.055 38.7 0.123 0.13 0.82 0.077 1.15 0.201 P-value 0.31 0.99 0.029 0.026 0.39 0.26 0.58 0.09 RMSE 0.032 17.5 0.462 0.49 0.60 0.112 0.87 0.147 Values with different superscripts within column are significantly (P < 0.05) higher (A) or lower (B) compared to the reference diet (containing 361, 158, and 33 g/kg DM of NDF, CP, and lipids) Low NDF low NDF diet (325 g/kg DM); High NDF high NDF diet (435 g/kg DM), Low CP low protein diet (CP, 115 g/kg DM), High CP high protein diet (CP, 194 g/kg DM), Low lipid low lipid diet (EE, 26 g/kg DM), High lipid high lipid diet (EE, 61 g/kg DM) composed mainly by cereal grains, corn silage and vari- After 24 h of fermentation, the measured GP of the ous hays. It should be preliminarily considered that in various diets was, on average, 250 mL/g DM, suggesting this study the variation in the content of the various nu- that a cow consuming 20 kg/d DM might produce about trients, namely, NDF, CP and lipid, were always achieved 5,000 L/d of gas. The CH production from fermentation by decreasing or increasing the proportion of dietary of these diets ranged 30.8 to 34.4 mL/g DM, suggesting starch. In the scientific literature, in vitro evaluation of that, for a DM intake of 20 kg/d, a cow might produce gas and CH productions is commonly carried out using 616 to 688 L/d of CH . Sauer et al. (1998) reported 4 4 single feeds, mainly forages, whereas less information is in vivo CH production of lactating cows in the order of available for complete diets [23]. In vivo measurement of 622 L/d, corresponding to 38.9 mL/g DM intake. In the gas and CH production requires expensive equipment study of [25], CH production from dairy cows was 4 4 and it is labor and time consuming. In vitro techniques 29.2 mL/g DM intake, whereas [26] reported for sheep would permit a much more simple determination of the an averaged CH production of 31.0 mL/g DM intake. dietary characteristics which can influence the potential In a continuous culture fermenter, [27] measured an emission of gas and CH from their fermentation in a averaged CH production of 33.0 mL/g DM. The in vitro 4 4 simulated rumen environment [20]. Studies of the CH production of the dairy rations tested by [23] varied relationships between in vitro and in vivo gas and CH from 30.1 to 35.9 mL/g DM, a range consistent with productions are lacking [23]. However, a recent study results obtained in this study. However, comparison suggested that in vitro gas and CH measurements can with data from literature is difficult because huge varia- be indicative of the trend of in vivo CH production ori- tions in gas and CH productions are commonly ob- 4 4 ginating from different combinations of feed ingredients served across experiments, even for diets with similar [24]. This study was aimed at evaluating if changes in composition. This is the consequence of a combination the diet composition might or might not have notable of different biological, as rumen fluid characteristics, influence on gas and CH productions. and technical factors, as fermentation procedures and 4 Maccarana et al. Journal of Animal Science and Biotechnology (2016) 7:54 Page 6 of 8 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 (CH ) production, and predicted values of GP and CH production 4 4 Degradability Actual GP, ml per: Actual CH mL per Predicted GP Predicted CH 4 4 NDFd, g/kg NDF TDMd, g/kg DM g DM g TDMd g DM g dNDF g TDMd 100 mL GP mL/g DM mL/g DM Diet Reference 567 840 274 325 34.2 168 40.8 12.5 244 38.8 A A A Low NDF 612 872 288 331 34.4 174 39.3 11.9 250 39.6 B B B B B B A High NDF 480 767 228 300 30.8 151 39.9 13.5 220 35.9 Low CP 545 835 277 333 33.4 169 39.9 12.0 242 38.0 B A High CP 542 831 255 310 34.0 176 41.0 13.3 234 37.8 Low lipid 583 845 265 315 33.1 159 39.0 12.3 251 37.2 B B B High lipid 531 826 253 304 32.2 169 38.8 12.5 238 37.9 SEM 10.3 4.2 5.6 7.2 0.53 3.4 0.63 0.40 10.9 2.09 P-value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.025 <0.001 0.33 0.70 P-value of contrasts Low vs High NDF <0.001 <0.001 <0.001 <0.001 <0.001 0.002 0.99 <0.001 0.99 0.99 Low vs High CP 0.99 0.99 <0.001 0.009 0.99 0.99 0.99 <0.001 0.99 0.99 Low vs High lipid 0.003 0.028 0.017 0.99 0.99 0.99 0.99 0.99 0.99 0.99 Incubation time 24 h 472 801 250 314 29.6 172 37.0 11.9 214 34.9 48 h 631 860 276 320 36.7 161 42.6 13.2 260 41.5 SEM 10.5 3.5 7.4 8.8 0.59 1.8 0.66 0.54 7.6 1.69 P-value 0.009 0.007 0.14 0.69 0.014 0.046 0.027 0.22 0.06 0.12 RMSE 15.5 7.2 4.0 7.6 0.69 3.2 0.94 0.25 21.9 3.78 Values with different superscripts within column are significantly (P < 0.05) higher (A) or lower (B) compared to the reference diet (containing 361, 158, and 33 g/kg DM of NDF, CP, and lipids) Low NDF low NDF diet (325 g/kg DM), High NDF high NDF diet (435 g/kg DM), Low CP low protein diet (CP, 115 g/kg DM), High CP high protein diet (CP, 194 g/kg DM), Low lipid low lipid diet (EE, 26 g/kg DM), High lipid high lipid diet (EE, 61 g/kg DM) equipment for collection, measurement and analysis On the contrary, the diet with the high level of NDF [28]. In this experiment the observed GP and CH were (435 g/kg DM) was based on different hays and did not regressed against the GP and CH values predicted contain corn silage. This is of interest in Italy as use of from the stoichiometry of the VFA production, to ob- silages for the production of important Protected De- tain an internal control of consistency of the data. The nomination of Origin (PDO) cheeses (i.e., Parmigiano- correlations found (R = 0.78 and 0.74, respectively, for Reggiano) is forbidden by specific feeding regulations. gas and CH measures) are acceptable considering the From the results it emerged that NDF degradability was rather narrow range of variation in GP and CH pro- negatively influenced by the dietary NDF content, par- duction caused by the dietary changes. It was observed tially because of the complete replacement of corn silage that the SEM of predicted GP and CH production with hays. This would be in agreement with previous (21.9 and 3.78 mL/g DM, respectively) was about five observations that the NDF fraction of hay samples col- times greater than the corresponding values of the lected in the same area of the present experiment were measured GP and CH production. In other words, the less degradable than corn silage samples [19]. measure of the VFA production was less precise than In diets similar for ingredient composition to those the GP and CH measures. used in this experiment, [12] observed an increased CH 4 4 production (35.6 to 44.3 mL/g DM) with decreasing Effects on NDF degradability, gas and CH production NDF content. In the present study, a decrease from 435 due to changes in the NDF content to 325 g NDF/kg DM increased CH production from In this experiment, the diet with the low level of NDF 30.8 to 34.4 mL/g DM (+11.6 %). This is consistent with (325 g/kg DM) was based on corn silage and a small an increased true degradability of the feed (+14 %), but amount of alfalfa hay (23 g/kg DM) as forage sources. also of the NDF fibrous fraction (+27 %). This seems to Maccarana et al. Journal of Animal Science and Biotechnology (2016) 7:54 Page 7 of 8 be contradictory with current literature, as a decrease of affect its availability in the rumen, and these factors ap- dietary NDF commonly reduces the NDF degradability pear to be more important than the FA profile [35]. In [12]. However, also [29] evidenced that a decrease of the this regard, [36], using dairy cows housed in respiratory dietary NDF content increased by 43 % in vivo CH chambers, found that, compared to the control, the production expressed per unit of dNDF. Results also evi- average reduction in CH (L/kg DMI) per 10 g/kg of denced that GP increased with a decrease of NDF con- crude fat added was persistent throughout lactation. tent even when expressed per unit of TDMd (+10 %), The same authors observed that the most effective lipid but no influences were observed on the CH production source in reducing methanogenesis was a commercial per unit of TDMd. A different trend emerged when CH vegetal rumen protected fat fortified with hydroxy- production was referred to the total GP. In this case, the methionine-analog-isobutyrate (−5.5 % of CH ), decrease of NDF lowered the CH proportion by about followed by vegetal rumen protected fat (−2.3 %), and 12 % (13.5 to 11.9 mL CH /100 mL GP, for the high and by whole cracked rapeseed (−0.8 %). In the experiment the low content of NDF, respectively). Results suggest of [35], only crushed canola seeds lowered CH produc- that a decrease of dietary NDF content, achieved from a tion per unit of digestible DM intake (−15 %), whereas complete replacement of hays with corn silage and crushed flaxseeds and crushed sunflower seeds did not cereal grains, might increase feed digestibility without reduce CH production compared to the control diet (a changing GP and CH produced per unit of digested diet supplemented with a commercial source of calcium material. salts of long chain fatty acids). In the current experi- ment, fat addition reduced feed degradability, particu- larly that of the NDF fraction. No influences were Effects on gas and CH production due to changes in the found on total VFA production and on the proportion CP content of acetate, propionate and butyrate, whereas GP and In this experiment an increased proportion of CP, in CH productions decreased by 8 and 6 %, respectively, replacement of starch, caused a reduction of GP. The compared to the reference diet. However, differences negative influence of dietary CP on GP has been ob- were greatly reduced when GP and CH were expressed served by others in the past. Such an effect was at- per unit of TDMd suggesting that, under the commer- tributed to the buffer capacity of CP, that reduces the cial conditions evaluated in this study, small reductions indirect CO released from the buffered rumen fluid, of CH might be achieved. and to the stoichiometry of protein fermentation, that differs from that of carbohydrates [30, 31]. In this Conclusions study, increase of the CP content was associated to a Changes of the ingredient and chemical composition of decrease of dietary starch. As a consequence, some diets were analyzed to evaluate benefits in the amount of VFA as iso-butyric and iso-valeric acids were in- CH produced, for the north eastern Italian dairy pro- creased, being end-products of protein degradation duction chain. It was found that a replacement of hays [32], whereas the production of propionate decreased with corn silage and cereals might increase GP and CH and the ratio between acetate and propionate in- per unit of DM intake. An increase of the dietary CP creased. Changes in the dietary CP proportion had no content would reduce GP with no influences in the effect on CH production when expressed both per amount of CH produced, whereas a moderate addition unit of incubated DM and per unit of TDMd. Thus, of cracked soybean seeds, and extruded flaxseed had as CP depresses GP, an increased proportion of CP few, or any, influence on the in vitro GP and CH produc- increases the CH concentration on total GP. tions. In general, none of the various strategies tested in the present work was able to reduce the amount of CH Effects on gas and CH production due to changes in the produced, especially if this production is expressed per lipid content unit of digestible DM intake. More research is needed to In this experiment changes in the dietary fat content of evaluate the effectiveness of strategies to reduce the CH the diets were achieved by changing the proportions of emissions, and relationships between in vitro and in vivo extruded flaxseeds, extruded soybean and whole soybean gas and CH productions need to be developed. seeds. The threshold of 60 g fat/kg DM was considered Abbreviations to be the upper limit to avoid a possible impairment of ADF, Acid detergent fibre expressed inclusive of residual ash; CP, Crude feed digestibility [6, 33]. protein; DM, Dry matter; EE, Ether extract; GP, Gas production; NDF, Neutral The effect of dietary lipids on CH production is detergent fibre assayed with a heat stable amylase and expressed inclusive of residual ash; NDFd, NDF degradability; lignin(sa), Lignin determined by dependent on the source, FA profile, level of inclusion, solubilization of cellulose with sulphuric acid; NRC, National Research and diet composition [34]. The level of supplementa- Council; RMSE, Root mean square error; SEM, Standard error of the mean; tion and the physical form of the lipid supplement TDMd, True DM degradability; VFA, Volatile fatty acids Maccarana et al. Journal of Animal Science and Biotechnology (2016) 7:54 Page 8 of 8 Acknowledgments 16. ISO 9831. Animal feeding stuffs, animal products, and faeces or urine – This work was financed by the project “ARCHAEA - Feeding strategies to Determination of gross calorific value – Bomb calorimeter method. Geneva: reduce methane emissions from dairy cows”– Veneto Region Rural ISO 9831; 1988. Development Programme (RDP) 2007–2013. 17. Menke KH, Steingass H. Estimation of the energetic feed value obtained from chemical analysis and gas production using rumen fluid. Anim Res Dev. 1988;28:7–55. Authors’ contributions 18. Goering HK, Van Soest PJ. Forage fiber analysis (apparatus, reagents, All authors conceived of this experiment, due to experience gained over the procedures, and some applications). Agricultural hand-book no. 379. last years in the use of in vitro gas production technique for the evaluation Washington, DC: USDA; 1970. of ruminant feeds. LM and MC gave substantial contributions to analytical 19. Tagliapietra F, Cattani M, Hindrichsen IK, Hansen HH, Colombini S, Bailoni L, procedures, statistical analysis, and writing of the manuscript. FT, LB, and SS et al. True dry matter digestibility of feeds evaluated in situ with different contributed in revising critically the manuscript. All authors read and bags and in vitro using rumen fluid collected from intact donor cows. Anim approved the final manuscript. Prod Sci. 2012;52(5):338–346. 20. Cattani M, Tagliapietra F, Maccarana L, Hansen HH, Bailoni L, Schiavon S. Competing interests Technical note: In vitro total gas and methane production measurements The authors declare that they have no competing interests. from closed or vented rumen batch culture systems. J Dairy Sci. 2014;97(3): 1736–741. Author details 21. Blümmel M, Aiple KP, Steingass H, Becker K. A note on the stoichiometrical Department of Comparative Biomedicine and Food Science (BCA), relationship of short chain fatty acid production and gas formation in vitro University of Padova, Viale dell’Università 16, 35020 Legnaro (PD), Italy. in feedstuffs of widely differing quality. J Anim Physiol Anim Nutr. 1999; Department of Agronomy, Food, Natural resources, Animals and 81(3):157–67. Environment (DAFNAE), University of Padova, Viale dell’Università 16, 35020 22. Institute SAS. SAS User’s Guide: Basics. Cary: SAS Inst. Inc.; 2007. Legnaro (PD), Italy. 23. Getachew G, Robinson PH, Depeters EJ, Taylor SJ, Gisi DD, Higginbotham GE, Riordan TJ. Methane production from commercial dairy rations Received: 14 January 2016 Accepted: 17 August 2016 estimated using an in vitro gas technique. Anim Feed Sci Technol. 2005; 123–124(1):391–402. 24. Hatew B, Cone JW, Pellikaan WF, Podesta SC, Bannink A, Hendriks WA, et al. 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