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Research| Volume 103, ISSUE 9, P8048-8061, September 2020

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Milk production, methane emissions, nitrogen, and energy balance of cows fed diets based on different forage systems

Open AccessPublished:July 01, 2020DOI:https://doi.org/10.3168/jds.2019-18134

      ABSTRACT

      Eight lactating Italian Friesian cows were housed in individual respiration chambers in a repeated Latin square design to determine their dry matter intake (DMI) and their milk and methane production, as well as to collect the total feces and urine to determine the N and energy balances. Four diets, based on the following forages (% of dry matter, DM), were tested: corn silage (CS, 49.3), alfalfa silage (AS, 26.8), wheat silage (WS, 20.0), and a typical hay-based Parmigiano Reggiano cheese production diet (PR, 25.3 of both alfalfa and Italian ryegrass hay). The greatest DMI was observed for cows fed PR (23.4 vs. 20.7 kg/d, the average of the other 3 diets). The DM digestibility was lower for PR (64.5 vs. 71.7%, the average of the other diets). The highest ash-free neutral detergent fiber digestibility values were obtained for CS (50.7%) and AS (47.4%). In the present study, no differences in milk production were observed between diets, although PR showed a higher milk yield trend. The highest milk urea N concentration (mg/dL) was found for the cows fed the WS diet (13.8), and the lowest was observed for the cows fed AS (9.24). The highest milk urea N concentration for the cows fed WS was also correlated with the highest urinary N excretion (g/d), which was found for the cows fed that same diet (189 vs. 147 on average for the other diets). The protein digestibility was higher for the cows fed the CS and WS diets (on average 68.5%) than for the cows fed AS and PR (on average 57.0%); dietary soybean inclusion was higher for CS and WS than for AS and PR. The rumen fermentation pattern was affected by the diet; the cows fed the PR diet showed a higher rumen pH and decreased propionate production than those fed CS, due to the lower nonfiber carbohydrate content and higher ash-free neutral detergent fiber content of the PR diet than the CS diet. Feeding cows with PR diet increased the acetate:propionate ratio in comparison with the CS diet (3.30 vs. 2.44 for PR and CS, respectively). Cows fed the PR diet produced a greater daily amount of methane and had a greater methane energy loss (% of digestible energy intake) than those fed the CS diet (413 vs. 378 g/d and 8.67 vs. 7.70%), but no differences were observed when methane was expressed as grams per kilogram of DMI or grams per kilogram of milk. The PR diet resulted in a smaller net energy for lactation content than the CS diet (1.36 vs. 1.70 Mcal/kg of DM for the PR and CS diets, respectively). Overall, our research suggests that a satisfactory milk production can be attained by including different high-quality forages in balanced diets without any negative effect on milk production or on the methane emissions per kilogram of milk.

      Key words

      INTRODUCTION

      The concentrations of greenhouse gases (GHG) in the atmosphere have increased over the years and the rate of increase over the past century is unprecedented (
      • Prentice I.C.
      • Farquhar G.D.
      • Fasham M.J.R.
      • Goulden M.L.
      • Heimann M.
      • Jaramillo V.J.
      • Kheshgi H.S.
      • LeQuéré C.
      • Scholes R.J.
      • Wallace Douglas W.R.
      The carbon cycle and atmospheric carbon dioxide in Climate Change 200.
      ). The livestock sector is responsible for about 14.5% of human-induced GHG emissions, with enteric methane being the single largest source (
      • McAllister T.A.
      • Newbold C.J.
      Redirecting rumen fermentation to reduce methanogenesis.
      ;
      • Gerber P.J.
      • Steinfeld H.
      • Henderson B.
      • Mottet A.
      • Opio C.
      • Dijkman J.
      • Falcucci A.
      • Tempio G.
      Tackling Climate Change Through Livestock—A Global Assessment of Emissions and Mitigation Opportunities.
      ). The type and amount of forage used in ruminant diets have direct effects on enteric methane production, and mitigation strategies can be achieved by altering the rumen fermentation pattern (
      • Benchaar C.
      • Pomar C.
      • Chiquette J.
      Evaluation of dietary strategies to reduce methane production in ruminants: A modelling approach.
      ). Forage maturity at harvest and the forage preservation method can be used to manipulate methane production in ruminants. The inclusion of early cut forages in a diet reduces methane production and improves OM and NDF digestibility compared with more mature forages (
      • Brask M.
      • Lund P.
      • Hellwing A.L.F.
      • Poulsen M.
      • Weisbjerg M.R.
      Enteric methane production, digestibility and rumen fermentation in dairy cows fed different forages with and without rapeseed fat supplementation.
      ). Furthermore, methane production was found to be lower for alfalfa used as silage rather than as hay (
      • Benchaar C.
      • Pomar C.
      • Chiquette J.
      Evaluation of dietary strategies to reduce methane production in ruminants: A modelling approach.
      ). The production of methane could also be depressed by the use of legumes (e.g., alfalfa) instead of grass, due to the difference in their chemical composition (
      • Benchaar C.
      • Pomar C.
      • Chiquette J.
      Evaluation of dietary strategies to reduce methane production in ruminants: A modelling approach.
      ). The on-farm production of forages and feeds may contribute to the mitigation of emissions from the dairy sector. For example, the dietary inclusion of high protein forages (e.g., alfalfa) can lead to a reduction of soybean meal (SBM) in the diet, a feed that involves a great environmental impact, mostly related to the change in land use. The intensity of agricultural practices may have a direct effect on emissions linked to feed production (
      • Tabacco E.
      • Comino L.
      • Borreani G.
      Production efficiency, costs and environmental impacts of conventional and dynamic forage systems for dairy farms in Italy.
      ;
      • Zucali M.
      • Bacenetti J.
      • Tamburini A.
      • Nonini L.
      • Sandrucci A.
      • Bava L.
      Environmental impact assessment of different cropping systems of home-grown feed for milk production.
      ), whereas the carbon sequestration potential of forage systems can indirectly mitigate the GHG emissions of the livestock sector (
      • Soussana J.F.
      • Tallec T.
      • Blanfort V.
      Mitigating the greenhouse gas balance of ruminant production systems through carbon sequestration in grasslands.
      ). Farming systems, based on permanent meadows or multiannual rotational grass and legume forages, might represent a significant GHG mitigation strategy, as they increase soil C sequestration in the OM of the soil (
      • Stanley P.L.
      • Rowntree J.E.
      • Beede D.K.
      • DeLonge M.S.
      • Hamm M.W.
      Impacts of soil carbon sequestration on life cycle greenhouse gas emissions in Midwestern USA beef finishing systems.
      ). On the other hand, annual crops, which require several external inputs and soil management practices for their growth (e.g., agrochemicals, synthetic nitrogen fertilizers, frequent plowing), reduce the soil C sequestration potential and may increase the consumption of direct and indirect energy (
      • Soussana J.F.
      • Tallec T.
      • Blanfort V.
      Mitigating the greenhouse gas balance of ruminant production systems through carbon sequestration in grasslands.
      ).
      Water availability and the destination of milk for protected designation of origin (PDO) cheeses are the main drivers of the organization of forage systems in the Po Plain (
      • Mantovi P.
      • Dal Prà A.
      • Pacchioli M.T.
      • Ligabue M.
      Forage production and use in the dairy farming systems of Northern Italy.
      ). The availability of water and the high soil fertility in the north of the Po River have historically favored the cultivation of corn (whole-plant silage and dry grain), which is recognized to produce a high DM yield per hectare and to be more suitable for an easy conservation by ensiling (
      • Borreani G.
      • Coppa M.
      • Revello-Chion A.
      • Comino L.
      • Giaccone D.
      • Ferlay A.
      • Tabacco E.
      Effect of different feeding strategies in intensive dairy farming systems on milk fatty acid profiles, and implications on feeding costs in Italy.
      ;
      • Gislon G.
      • Ferrero F.
      • Bava L.
      • Borreani G.
      • Dal Prà A.
      • Pacchioli M.T.
      • Sandrucci A.
      • Zucali M.
      • Tabacco E.
      Forage systems and sustainability of milk production: Feed efficiency, environmental impacts and soil carbon stocks.
      ) than permanent meadows and legume forage crops. An emerging forage system called dynamic forage system (
      • Tabacco E.
      • Comino L.
      • Borreani G.
      Production efficiency, costs and environmental impacts of conventional and dynamic forage systems for dairy farms in Italy.
      ) is replacing the conventional system based on monocropped corn silage by reintroducing the use of legume forages and producing whole-ear silage, thereby producing high-quality forages and increasing farm protein self-sufficiency. South of the Po River, an area that is characterized by water scarcity, corn cultivation is less productive and has been replaced by winter cereals and multiannual legume crops, such as alfalfa, which are conserved as hay in the Parmigiano Reggiano PDO production area. Overall, the main target of dairy systems should be focused on feeding better quality diets to increase feed efficiency and lower the environmental impacts per kilogram of milk, namely methane emissions and N excretion, the sources of ammonia and nitrous oxide releases.
      As different forage systems can have different environmental effects and implications on soil C sequestration, on the inclusion of SBM in the diet and on fiber digestibility and enteric methane production, the aim of the present study has been to provide data on the performance of animals fed diets characterized by different forages produced on commercial farms in Northern Italy. The hypothesis of this experiment is that including different high-quality forages in balanced diets with low soybean meal levels can lead to milk production similar to that achievable with conventional diets based on corn silage, without increasing the methane emission and N excretion per unit of product.

      MATERIALS AND METHODS

      The study was conducted at the Università degli Studi di Milano “Cascina Baciocca” Research Center at Cornaredo (Milan, Italy). All of the animal procedures were conducted with the approval of the University of Milan Ethics Committee for Animal Use and Care and in accordance with the guidelines of the Italian law on animal welfare for experimental animals (

      Italian Ministry of Health. 2014. Protezione degli animali utilizzati a fini sperimentali o ad altri fini scientifici. D.Lgs 26/2014. Rome, Italy.

      ) under authorization 980/2017.

      Cows, Experimental Design, and Methane Determination

      Eight multiparous lactating Italian Friesian cows were used in a replicated 4×4 Latin square design. Each experimental period lasted 28 d: 23 d of diet adaptation and 5 d of sample collection. At the start of the trial, the cows averaged 127 DIM (SD: ±19.6) with an average BW of 608 kg and a milk yield of 38.7 kg/d (SD: ±3.61).
      The cows were fed the experimental TMR ad libitum twice daily. The animals had free access to drinking water. Orts were recorded daily, and the feeding rate was adjusted to obtain at least 5% of the supplied amount as orts (on an as-fed basis). During the adaptation periods, the animals were housed in individual tiestalls, which were equipped with rubber mattresses and bedded with straw. Each cow was weighed at the beginning and at the end of each experimental period. The cows spent the last 7 d of each experimental period in respiration chambers: the first 2 d to adapt to the chambers and the last 5 d for the sample collection. Four individual open-circuit respiration chambers were used to enable the measurement of CH4, CO2 emissions, and O2 consumption. The chambers measured 3.6 m (length) × 2.4 m (width) × 2.3 m (height), and each contained a small pre-chamber for the personnel entrance, and wide glass walls to allow the cows to see each other and outside. Each respiration chamber was equipped with a feeder and contained a 2.5 m × 1.5 m stanchion that allowed the animal to stand or lie down. The air temperature in the chambers was maintained at 18 ± 1°C and a low negative pressure was maintained inside the chambers to prevent CH4 losses produced by the cows. The air flow through the chambers was measured using a diaphragm flow-meter (PH 20/335 G 25, 40 m3/h, Sacofgas, Città di Castello, Perugia, Italy). The air flux was on average maintained at 35 ± 1 m3/h. The daily O2 consumption and the CO2 and CH4 production were determined by measuring the volume of air circulating in the system in 24 h (and by referring to the standard temperature and pressure conditions) and multiplying this volume by the difference between the relative concentrations of the gases measured continuously in the ingoing and the outgoing air. The CH4 and CO2 concentrations were measured using an URAS 4 analyzer (Hartmann and Braun AG, Frankfurt am Main, Germany). The oxygen concentration was measured using a Magnos 6G analyzer (Hartmann and Braun AG). The gas concentrations were measured every 575 s, considering 105 s of air change and 10 s of O2, CO2, and CH4 determination for each chamber and the external air, for a total of 150 observations/d for each gas and each cow. Corrections were applied to account for the entrance of personnel.
      The total heat production was determined using the Brouwer equation (
      • Brouwer E.
      Report of sub-committee on constants and factors.
      ): heat production (kcal/d) = 3.866O2 + 1.200CO2 − 1.431N − 0.518CH4, where gas volumes (L/d) are expressed at standard conditions and N (g/d) is the urinary N. The ME requirement necessary for maintenance was assumed to be 115 kcal/metabolic BW (
      • Van Es A.J.H.
      Feed evaluation for ruminants. I. The systems in use from May 1977-onwards in The Netherlands.
      ).
      Urine and feces were collected separately daily as follows: cows were fitted with Foley urinary catheters (model 1855H24, C. R. Bard Inc., Covington, GA) and urine was collected in plastic bins containing sulfuric acid (20% vol/vol) to maintain the pH below 2.5 and to prevent ammonia losses. Feces left the chamber through openings in the floor at the back of the stanchion and were collected in tanks located underneath the floor of the chambers, as reported by
      • Colombini S.
      • Galassi G.
      • Crovetto G.M.
      • Rapetti L.
      Milk production, nitrogen balance, and fiber digestibility prediction of corn, whole plant grain sorghum, and forage sorghum silages in the dairy cow.
      . The feces and urine were weighed daily, sampled (2% of the total weight), and pooled per cow during each collection period.
      The cows were milked twice daily (0730 and 1830 h), and the milk production was recorded at each milking. Milk samples from individual cows were taken at each milking during the sample collection period and 2-bromo-2-nitropropan-1,3-diol was added to the milk as a preservative. Feces, TMR, and ort samples were dried in a ventilation oven at 55°C until constant weight. After drying, the samples were ground to 1 mm using a Fritsch mill (Pulverisette 19, Fritsch GmbH, Idar-Oberstein, Germany). A fresh feces subsample was used for N analysis. The N balance was determined by considering the N volatilized in the chamber, which was measured from the N concentration of the water condensed by the air conditioning system. This process involved collecting the total volume of condensed water in plastic canisters containing 20% sulfuric acid (vol/vol), which were placed inside of the chambers. The water volume was weighed daily, sampled to obtain a composite sample, and stored at −20°C for the subsequent ammonia N (N-NH3) analysis.
      Ruminal fluid was collected from cows after they left the chambers at the end of each experimental period. Ruminal liquid was taken 5 h after the morning feeding and samples were taken using an esophageal probe. To avoid saliva contamination, the first collected rumen sample (with the possible presence of saliva) was discarded. Approximately 0.6 L of rumen fluid was strained through 4 layers of cheesecloth. The pH was measured immediately after sampling, and 1 aliquot was stored at −20°C for subsequent VFA analysis.

      Diets

      The experimental treatments were based on the typical forage systems that have been identified as the most representative of the Po Plain (Northern Italy;
      • Gislon G.
      • Ferrero F.
      • Bava L.
      • Borreani G.
      • Dal Prà A.
      • Pacchioli M.T.
      • Sandrucci A.
      • Zucali M.
      • Tabacco E.
      Forage systems and sustainability of milk production: Feed efficiency, environmental impacts and soil carbon stocks.
      ). The 4 dietary treatments were as follows: (1) a corn silage-based system (CS), which was considered representative of the most widespread intensive forage system in the Po Plain; (2) a forage system based on double-cropped corn (harvested as whole-ear silage), alfalfa (harvested as silage at an early growth stage), and Italian ryegrass (harvested as silage at an early growth stage; AS); (3) a forage system based on double-cropped corn (harvested as whole-ear silage) and winter cereal (harvested as silage, wheat in the present experiment; WS); and (4) a representative forage system of Parmigiano Reggiano cheese production (PDO), based on dried forages from alfalfa and permanent meadows (PR). The diets were formulated using the CNCPS model (version 6.5, Cornell University, Ithaca, NY) to provide a similar MP and energy concentration. Wrapped bales of TMR, prepared on 3 different commercial farms, using fodders produced directly on each farm, were used for the 3 silage-based diets. The TMR bales were made using an MP 2,000 compactor (Orkel, Fannrem, Norway). The PR diet was provided, as small TMR bales, by a feed compounder of the Parmigiano Reggiano area. The chemical composition of the forages and the VFA, lactic acid, and alcohol contents of the silages are reported in Table 1, Table 2, respectively.
      Table 1Chemical composition of the main forages included in the 4 experimental diets
      Experimental diets: CS = corn silage; AS = alfalfa silage; WS = wheat silage; PR = Parmigiano Reggiano.
      ItemChemical composition
      EE = ether extract; aNDFom = NDF assayed with a heat-stable amylase and expressed exclusive of residual ash; ADFom = ADF expressed exclusive of residual ash; ADL = lignin content determined by solubilization of cellulose with sulfuric acid; NFC = 100 − (ash + CP + EE + aNDFom).
      (% of DM unless noted)
      DM (%)AshCPEEaNDFomADFomADLNFC
      CS
       Corn silage38.34.307.293.1941.024.32.9244.2
       Italian ryegrass hay89.07.0013.33.0060.036.05.3516.7
      AS
       Alfalfa silage44.616.221.44.4040.029.36.8516.0
       Italian ryegrass silage44.511.78.633.3155.035.35.0821.4
      WS
       Alfalfa hay (mixed hay)87.010.013.03.0056.538.26.2717.5
       Wheat silage26.97.509.843.3462.737.85.3916.6
       Alfalfa silage53.09.0521.54.4040.031.56.8225.1
      PR
       Alfalfa hay90.010.018.02.5058.734.27.2625.7
       Italian ryegrass hay90.010.57.993.0061.342.06.2417.2
      1 Experimental diets: CS = corn silage; AS = alfalfa silage; WS = wheat silage; PR = Parmigiano Reggiano.
      2 EE = ether extract; aNDFom = NDF assayed with a heat-stable amylase and expressed exclusive of residual ash; ADFom = ADF expressed exclusive of residual ash; ADL = lignin content determined by solubilization of cellulose with sulfuric acid; NFC = 100 − (ash + CP + EE + aNDFom).
      Table 2The pH and fermentative profiles of the silages used in the experimental diets
      Experimental diets: CS = corn silage; AS = alfalfa silage; WS = wheat silage.
      of the experiment
      ItempHContent (g/kg of DM)
      Lactic acidAcetic acidPropionic acidButyric acidEthanol1,2 Propandiol
      CS
       Corn silage3.6064.322.20.00.015.110.2
      AS
       Alfalfa silage4.9733.428.61.710.14.71.6
       Italian ryegrass silage4.2858.327.30.00.03.54.4
       High-moisture corn3.9619.25.90.00.03.20.3
      WS
       Wheat silage4.2649.343.54.34.913.82.7
       Alfalfa silage5.2113.839.90.21.40.00.0
       High-moisture corn3.7635.611.80.00.09.40.7
      1 Experimental diets: CS = corn silage; AS = alfalfa silage; WS = wheat silage.

      Chemical Analyses

      The feed ingredients, TMR, orts, and feces were analyzed for chemical composition. The DM was determined by oven drying at 55°C until constant weight. Analytical DM was determined by drying in a ventilated oven at 100°C overnight (
      • AOAC International
      Official Methods of Analysis.
      ; method 945.15). The ash content was determined by incineration at 550°C overnight in a muffle furnace (
      • AOAC International
      Official Methods of Analysis.
      ; method 942.05). The CP (N × 6.25) was determined according to the Dumas method, using MAX N exceed (Elementar Analysensystem GmbH, Langenselbold, Germany). The concentration of fiber was determined as described by
      • Mertens D.R.
      Gravimetric determination of amylase-treated neutral detergent fiber in feeds using refluxing in beakers or crucibles: Collaborative study.
      , with the inclusion of heat-stable α-amylase and sodium sulfite, and expressed exclusive of residual insoluble ash (aNDFom). Acid detergent fiber (ADFom) and ADL, determined according to the method of
      • Van Soest P.J.
      • Robertson J.B.
      • Lewis B.A.
      Methods of dietary fiber, neutral detergent fiber and non-polysaccharides in relation to animal nutrition.
      , were expressed exclusive of residual insoluble ash; lignin was determined by solubilization of cellulose with sulfuric acid. The NDF and ADF procedures were adapted for use in an Ankom200 fiber analyzer (Ankom Technology Corp., Fairport, NY). The ether extract was determined according to
      • AOAC International
      Official Methods of Analysis.
      method 920.29. The gross energy of the TMR, orts, feces, urine, and milk was determined using an adiabatic calorimeter (IKA 6000; IKA Werke GmbH and Co. KG, Staufen, Germany). The concentration of N in the acidified urine, in the condensed water collected in the chamber, in the fresh feces, and in composite milk samples was determined according to the Dumas method, using MAX N exceed.
      The milk fat and lactose concentrations were determined using a Fourier transform infrared analyzer (MilkoScan FT6000; Foss Analytical A/S, Hillerod, Denmark). The MUN concentration was determined using a differential pH technique (method 14637;
      • ISO (International Organization for Standardization)
      Milk: Determination of urea content. Enzymatic method using difference in pH (Reference method).
      ). The ECM (3.5% fat and 3.2% protein) was calculated according to
      • Tyrrell H.F.
      • Reid J.T.
      Prediction of the energy value of cow's milk.
      .
      Rumen samples were analyzed for VFA using an Agilent 3000A micro GC gas chromatograph (Agilent Technologies, Santa Clara, CA) according to
      • Pirondini M.
      • Malagutti L.
      • Colombini S.
      • Amodeo P.
      • Crovetto G.M.
      Methane yield from dry and lactating cows diets in the Po Plain (Italy) using an in vitro gas production technique.
      .
      A silage sample was divided into 2 subsamples. The first subsample was extracted for pH determination using a Stomacher blender (Seward Ltd., Worthing, UK) for 4 min in distilled water at a 9:1 water-to-sample material (fresh weight) ratio. The second subsample was extracted using a Stomacher blender for 4 min in 0.05 M sulfuric acid (H2SO4) at a 5:1 acid-to-sample material (fresh weight) ratio. A 40-mL aliquot of silage acid extract was filtered with a 0.20-μm syringe filter and used to quantify the fermentation products. The lactic and monocarboxylic acids (acetic, propionic, and butyric acids) were determined by means of HPLC in the acid extract (
      • Canale A.
      • Valente M.E.
      • Ciotti A.
      Determination of volatile carboxylic acids (C1–C5) and lactic acid in aqueous acid extracts of silage by high performance liquid chromatography.
      ). Ethanol and 1,2-propanediol were determined by means of HPLC coupled to a refractive index detector in an Aminex HPX-87H column (Bio-Rad Laboratories, Richmond, CA).

      Statistical Analysis

      Statistical analysis was performed using the Mixed procedure of SAS, version 9.2 (
      • SAS Institute
      User's Guide: Statistics. Release 8.01.
      ). The data were analyzed with the following model:
      Yij(k)m = μ + Sm + Cim + Pjm + T(k) + eijm,


      where Yij(k)m represents the dependent variable, calculated as the mean of the daily measurements during each sampling period ij(k)m; μ is the overall mean; Sm represents the fixed effect of square meter, with m = 1, 2; Cim represents the random effect of cow i within square meter, with i = 1, ..., 4; Pjm represents the fixed effect of period j, with j = 1, ..., 4 within square meter; T(k) represents the fixed effect of treatment k, with k = 1, ..., 4; and eijm represents the residual error. Estimates of the least squares means are reported. Significance was declared at P ≤ 0.05 and trends at P ≤ 0.10 for all of the statistical analyses.

      RESULTS

      Forage and Diet Composition

      As previously described, the TMR bales were prepared on different farms with different forages, explaining the differences in terms of chemical composition within the same forage category. Unexpectedly, the ash content was very high in the alfalfa silage of the AS diet (16.2% on DM). Alfalfa was the forage with the highest CP content (% of DM), with higher values for silages (21.5, on average) than for hays (15.5, on average).
      The forages were characterized by a wide variability of the fiber concentrations. The corn and alfalfa silages had the lowest aNDFom concentrations, but they were characterized by a different ADL content, which was lower for corn silage (2.92% of DM) than for alfalfa silages (6.82% of DM, on average). The Italian ryegrass, alfalfa hays, and wheat silage were characterized by the highest aNDFom concentrations. Corn silage, as expected, had the highest NFC content (44.2% of DM).
      The pH and fermentative profiles of the silages included in the diets are reported in Table 2. The corn silage had the highest lactic acid content (64.3 g/kg of DM), followed by the Italian ryegrass silage (58.3), and wheat silage (49.3). Moderate concentrations of butyric acid were detected in the alfalfa silage of both the AS and WS diets (10.1 and 4.9 g/kg of DM, respectively).
      The ingredients and chemical composition of the 4 experimental diets are shown in Table 3. The diets were formulated to allow the maximum inclusion of forages. Corn grain meal was present in a higher proportion in the PR diet (22.8% of diet DM) than in the other treatments (12.1%, on average) because no forages with a high starch content or high-moisture ear corn were used. High-moisture ear corn was used in the AS and WS diets. Soybean meal inclusion was higher for CS and WS than for AS and PR. The aNDFom concentration (% of DM) was higher for the PR diet (36.6) and lower for the AS diet (27.1), with intermediate values for CS and WS (32.9%, on average).
      Table 3Composition and chemical analysis of the 4 experimental diets
      ItemDiet
      Experimental diets: CS = corn silage; AS = alfalfa silage; WS = wheat silage; PR = Parmigiano Reggiano.
      CSASWSPR
      Composition (% of DM)
       Corn silage49.3000
       Alfalfa silage026.810.40
       Italian ryegrass silage019.100
       Italian ryegrass hay17.30025.3
       Alfalfa hay0010.625.3
       Wheat silage0020.00
       High-moisture ear corn028.629.10
       Corn grain12.111.412.722.8
       Solvent soybean meal, 48% CP15.78.109.0
       Solvent soybean meal, 44% CP0012.70
       Corn gluten feed, dry0004.4
       Corn grain, flaked0008.6
       Sugarcane3.03.51.92.2
       Mineral and vitamin supplement
      Mineral and vitamin supplements composition for the 3 silage-based diets (CS, AS, WS) and PR diet, respectively: 37.4 and 19.4% calcium carbonate, 24.0 and 12.9% sodium bicarbonate, 14.2 and 9.8% sodium chloride, 12.2 and 11.3% magnesium oxide, 8.2 and 4.3% dicalcium phosphate, 5.0 and 5.2 microminerals and vitamins, 0 and 37.1% wheat bran. Provided (per kg): 870 and 918 mg of Fe, 1,558 and 641 mg of Zn, 691 and 160 mg of Cu, 1,105 and 822 mg of Mn, 26 and 19 mg of I, 14 and 12 mg of Se, 400 and 224 kIU of vitamin A, 60 and 36.4 kIU of vitamin D, 1,000 and 1,400 IU of vitamin E.
      2.52.52.52.4
       Rumen-protected methionine0.030.030.030.03
      Chemical analysis
      EE = ether extract; aNDFom = NDF assayed with a heat-stable amylase and expressed exclusive of residual ash; ADFom = ADF expressed exclusive of residual ash; NFC = 100 − (ash + CP + EE + aNDFom).
      (% of DM unless noted)
       DM (%)53.054.651.289.6
       OM92.489.691.992.1
       Ash7.5110.38.128.00
       CP15.015.315.714.3
       EE2.342.872.832.52
       aNDFom32.827.133.736.6
       ADFom22.022.723.827.7
       NFC41.244.338.638.5
       Predicted ME content (Mcal/kg of DM)2.632.432.552.50
      1 Experimental diets: CS = corn silage; AS = alfalfa silage; WS = wheat silage; PR = Parmigiano Reggiano.
      2 Mineral and vitamin supplements composition for the 3 silage-based diets (CS, AS, WS) and PR diet, respectively: 37.4 and 19.4% calcium carbonate, 24.0 and 12.9% sodium bicarbonate, 14.2 and 9.8% sodium chloride, 12.2 and 11.3% magnesium oxide, 8.2 and 4.3% dicalcium phosphate, 5.0 and 5.2 microminerals and vitamins, 0 and 37.1% wheat bran. Provided (per kg): 870 and 918 mg of Fe, 1,558 and 641 mg of Zn, 691 and 160 mg of Cu, 1,105 and 822 mg of Mn, 26 and 19 mg of I, 14 and 12 mg of Se, 400 and 224 kIU of vitamin A, 60 and 36.4 kIU of vitamin D, 1,000 and 1,400 IU of vitamin E.
      3 EE = ether extract; aNDFom = NDF assayed with a heat-stable amylase and expressed exclusive of residual ash; ADFom = ADF expressed exclusive of residual ash; NFC = 100 − (ash + CP + EE + aNDFom).

      Intake and Digestibility

      The apparent total-tract digestibility of the nutrients and DMI are reported in Table 4. The DMI (kg/d) was higher (P = 0.008) for cows fed the PR diet (23.4) than for the other diets (20.7, on average). The lowest DM digestibility was observed for the PR diet (64.5%) and the highest for the CS diet (73.3%), and AS and WS diets showed intermediate values (70.9%, on average; P < 0.001). The CS and AS diets had the highest values of OM digestibility (75.1 and 74.7%, respectively), WS had intermediate an intermediate value (72.0%), and the PR diet had the lowest value (67.1%; P < 0.001). Significant differences (P < 0.001) between treatments were observed for CP digestibility, with the highest values attained by the CS and WS diets (69.0 and 67.9%, respectively) and the lowest by the AS and PR diets (58.4 and 55.6%, respectively). The aNDFom digestibility was higher for the CS and AS diets (50.7 and 47.4%, respectively); the aNDFom digestibility of the PR diet (38.5%) was similar to that of the WS diet (40.5%; P < 0.001).
      Table 4Intake and total-tract apparent digestibility of the nutrients of lactating cows fed diets based on different forages
      ItemDiet
      Experimental diets: CS = corn silage; AS = alfalfa silage; WS = wheat silage; PR = Parmigiano Reggiano.
      SEMP-value
      CSASWSPR
      DMI (kg/d)20.3
      Means in the same row with different superscript letters differ (P < 0.05).
      20.9
      Means in the same row with different superscript letters differ (P < 0.05).
      20.9
      Means in the same row with different superscript letters differ (P < 0.05).
      23.4
      Means in the same row with different superscript letters differ (P < 0.05).
      0.700.008
      Digestibility
      aNDFom = NDF assayed with a heat-stable amylase and expressed exclusive of residual ash; ADFom = ADF expressed exclusive of residual ash.
      (%)
       DM73.3
      Means in the same row with different superscript letters differ (P < 0.05).
      71.4
      Means in the same row with different superscript letters differ (P < 0.05).
      70.3
      Means in the same row with different superscript letters differ (P < 0.05).
      64.5
      Means in the same row with different superscript letters differ (P < 0.05).
      0.90<0.001
       OM75.1
      Means in the same row with different superscript letters differ (P < 0.05).
      74.7
      Means in the same row with different superscript letters differ (P < 0.05).
      72.0
      Means in the same row with different superscript letters differ (P < 0.05).
      67.1
      Means in the same row with different superscript letters differ (P < 0.05).
      0.91<0.001
       CP69.0
      Means in the same row with different superscript letters differ (P < 0.05).
      58.4
      Means in the same row with different superscript letters differ (P < 0.05).
      67.9
      Means in the same row with different superscript letters differ (P < 0.05).
      55.6
      Means in the same row with different superscript letters differ (P < 0.05).
      1.86<0.001
       aNDFom50.7
      Means in the same row with different superscript letters differ (P < 0.05).
      47.4
      Means in the same row with different superscript letters differ (P < 0.05).
      40.5
      Means in the same row with different superscript letters differ (P < 0.05).
      38.5
      Means in the same row with different superscript letters differ (P < 0.05).
      2.23<0.001
       ADFom36.829.229.231.82.760.114
      a–c Means in the same row with different superscript letters differ (P < 0.05).
      1 Experimental diets: CS = corn silage; AS = alfalfa silage; WS = wheat silage; PR = Parmigiano Reggiano.
      2 aNDFom = NDF assayed with a heat-stable amylase and expressed exclusive of residual ash; ADFom = ADF expressed exclusive of residual ash.

      Milk Production and Composition

      The milk production and composition data are presented in Table 5. Production (milk and ECM, kg/d) was not affected by the diet, nor were the milk composition and yield in terms of fat, CP, and lactose. However, there was a tendency (P = 0.057) for cows fed the PR diet to show a higher milk production than those fed the other diets. We also found a slightly higher tendency (P = 0.075, P = 0.052, and P = 0.051) in the lactose concentration, CP, and lactose yield for cows fed the PR diet than those fed other diets. Feed efficiency (milk/DMI and ECM/DMI) was not statistically affected by the diet. Significant differences (P < 0.001) between diets were observed for the MUN concentration (mg/dL), which was the highest for WS (13.8 mg/dL) and the lowest for AS (9.24 mg/dL; P < 0.05).
      Table 5Milk production and milk composition of lactating cows fed diets based on different forages
      ItemDiet
      Experimental diets: CS = corn silage; AS = alfalfa silage; WS = wheat silage; PR = Parmigiano Reggiano.
      SEMP-value
      CSASWSPR
      Production (kg/d)
       Milk27.027.328.229.30.920.057
       ECM
      ECM (3.5% fat and 3.2% protein) according to Tyrrell and Reid (1965).
      30.531.433.132.71.000.122
      Composition (%)
       Fat4.384.604.714.260.180.172
       CP3.583.523.563.530.090.349
       Lactose5.025.035.065.090.030.075
      Yield (kg/d)
       Fat1.161.241.311.240.050.174
       CP0.960.951.001.030.040.052
       Lactose1.361.371.431.490.040.051
      LS
      LS = linear score, logarithmic transformation of SCC.
      2.092.672.033.030.640.496
      MUN (mg/dL)11.8
      Means in the same row with different superscript letters differ (P < 0.05).
      9.24
      Means in the same row with different superscript letters differ (P < 0.05).
      13.8
      Means in the same row with different superscript letters differ (P < 0.05).
      11.5
      Means in the same row with different superscript letters differ (P < 0.05).
      0.18<0.001
      Acetone (mmol/L)0.0160.0200.0190.0040.010.053
      BHB (mmol/L)0.030.050.030.030.010.093
      Feed efficiency
       Milk/DMI1.331.311.351.250.0260.163
       ECM/DMI1.501.511.581.400.0390.119
      a–c Means in the same row with different superscript letters differ (P < 0.05).
      1 Experimental diets: CS = corn silage; AS = alfalfa silage; WS = wheat silage; PR = Parmigiano Reggiano.
      2 ECM (3.5% fat and 3.2% protein) according to
      • Tyrrell H.F.
      • Reid J.T.
      Prediction of the energy value of cow's milk.
      .
      3 LS = linear score, logarithmic transformation of SCC.

      Ruminal Fermentation Characteristics

      The ruminal fermentation characteristics are reported in Table 6. The mean ruminal pH value was affected by the diet. The ruminal pH was significantly lower (P = 0.020) for cows fed the CS diet (6.23) than those fed the PR diet (6.60), with AS (6.47) and WS (6.43) showing intermediate values.
      Table 6Ruminal pH, total VFA, and VFA molar proportion of the ruminal fluid of lactating dairy cows fed diets based on different forages
      ItemDiet
      Experimental diets: CS = corn silage; AS = alfalfa silage; WS = wheat silage; PR = Parmigiano Reggiano.
      SEMP-value
      CSASWSPR
      pH6.23
      Means in the same row with different superscript letters differ (P < 0.05).
      6.47
      Means in the same row with different superscript letters differ (P < 0.05).
      6.43
      Means in the same row with different superscript letters differ (P < 0.05).
      6.60
      Means in the same row with different superscript letters differ (P < 0.05).
      0.080.020
      Total VFA (mmol/L)13110910410010.80.096
      VFA (mol/100 mol)
       Acetate58.9
      Means in the same row with different superscript letters differ (P < 0.05).
      57.5
      Means in the same row with different superscript letters differ (P < 0.05).
      60.0
      Means in the same row with different superscript letters differ (P < 0.05).
      62.7
      Means in the same row with different superscript letters differ (P < 0.05).
      1.160.021
       Propionate24.621.821.519.11.330.056
       Butyrate12.6
      Means in the same row with different superscript letters differ (P < 0.05).
      16.4
      Means in the same row with different superscript letters differ (P < 0.05).
      14.1
      Means in the same row with different superscript letters differ (P < 0.05).
      15.1
      Means in the same row with different superscript letters differ (P < 0.05).
      0.560.002
       Isobutyric acid0.75
      Means in the same row with different superscript letters differ (P < 0.05).
      0.88
      Means in the same row with different superscript letters differ (P < 0.05).
      0.94
      Means in the same row with different superscript letters differ (P < 0.05).
      0.68
      Means in the same row with different superscript letters differ (P < 0.05).
      0.050.011
      n-Valeric acid1.541.901.641.480.140.117
       Isovaleric acid1.64
      Means in the same row with different superscript letters differ (P < 0.05).
      1.54
      Means in the same row with different superscript letters differ (P < 0.05).
      1.82
      Means in the same row with different superscript letters differ (P < 0.05).
      0.79
      Means in the same row with different superscript letters differ (P < 0.05).
      0.16<0.001
      Acetate:propionate ratio2.44
      Means in the same row with different superscript letters differ (P < 0.05).
      2.71
      Means in the same row with different superscript letters differ (P < 0.05).
      2.83
      Means in the same row with different superscript letters differ (P < 0.05).
      3.30
      Means in the same row with different superscript letters differ (P < 0.05).
      0.190.032
      a–c Means in the same row with different superscript letters differ (P < 0.05).
      1 Experimental diets: CS = corn silage; AS = alfalfa silage; WS = wheat silage; PR = Parmigiano Reggiano.
      The total VFA concentration (mmol/L) was not affected by the treatment; however, there was a tendency (P = 0.096) for the CS diet to show the highest total VFA concentration (131).
      The proportions of VFA were affected by the treatment. The acetate percentage was the smallest (P = 0.021) for AS (57.5%) and the highest for PR (62.7%), with WS and CS showing intermediate values; the butyrate content was lower in CS (12.6%) than in AS and PR (16.4 and 15.1, respectively), and was intermediate for WS (14.1; P = 0.002); isobutyric acid was lower for PR and higher for AS and WS, and was intermediate for CS. Isovaleric acid was lower in PR than in the other diets. Feeding cows with the PR diet increased the acetate:propionate ratio in comparison with CS (3.30 vs. 2.44 for PR and CS, respectively; P < 0.05).

      Enteric Methane Production

      The dietary effects related to rumen methanogenesis are reported in Table 7. Methane production (g/d) was higher (P = 0.047) for the cows fed the PR diet (413) than those fed the CS diet (378). The dietary treatment did not affect methane emissions in terms of enteric emissions related to intake or milk production. On average, the cows showed a methane production of 18.6 g/kg of DMI and 14.5 g/kg of milk. Differences were detected for the methane emission as percent of digestible energy (DE) intake, which was the highest for PR (8.67), intermediate for AS and WS (8.15 and 8.17, respectively), and the lowest for CS (7.70, P = 0.018).
      Table 7Methane production of lactating dairy cows fed diets based on different forages
      Composition of methaneDiet
      Experimental diets: CS = corn silage; AS = alfalfa silage; WS = wheat silage; PR = Parmigiano Reggiano.
      SEMP-value
      CSASWSPR
      g/d378
      Means in the same row with different superscript letters differ (P < 0.05).
      396
      Means in the same row with different superscript letters differ (P < 0.05).
      396
      Means in the same row with different superscript letters differ (P < 0.05).
      413
      Means in the same row with different superscript letters differ (P < 0.05).
      10.40.047
      g/kg of DMI18.619.019.017.80.590.547
      g/kg of OM digested26.828.328.728.90.740.126
      % GE intake
      GE = gross energy.
      5.675.925.785.590.190.543
      % DE intake
      DE = digestible energy.
      7.70
      Means in the same row with different superscript letters differ (P < 0.05).
      8.15
      Means in the same row with different superscript letters differ (P < 0.05).
      8.17
      Means in the same row with different superscript letters differ (P < 0.05).
      8.67
      Means in the same row with different superscript letters differ (P < 0.05).
      0.210.018
      g/kg of milk14.414.814.414.20.560.582
      g/kg of ECM
      Milk yields, ECM, milk fat, and milk protein were measured over the collection period.
      12.512.712.112.70.370.389
      g/kg of milk fat
      Milk yields, ECM, milk fat, and milk protein were measured over the collection period.
      3263233053359.690.187
      g/kg of milk protein
      Milk yields, ECM, milk fat, and milk protein were measured over the collection period.
      40042140440320.80.505
      a,b Means in the same row with different superscript letters differ (P < 0.05).
      1 Experimental diets: CS = corn silage; AS = alfalfa silage; WS = wheat silage; PR = Parmigiano Reggiano.
      2 GE = gross energy.
      3 DE = digestible energy.
      4 Milk yields, ECM, milk fat, and milk protein were measured over the collection period.

      Nitrogen Balance

      The results concerning the N balance are presented in Table 8. Nitrogen intake was significantly lower (P = 0.012) for the CS diet (490 g/d) than for the others (on average 540 g/d). Fecal excretion (DM, kg/d) was lower for CS, but not different from AS (P < 0.001). Urinary excretion (kg/d) was higher for AS than for CS (25.0 and 22.8, respectively; P = 0.046).
      Table 8Nitrogen balance of lactating dairy cows fed diets based on different forages
      ItemDiet
      Experimental diets: CS = corn silage; AS = alfalfa silage; WS = wheat silage; PR = Parmigiano Reggiano.
      SEMP-value
      CSASWSPR
      N intake (g/d)490
      Means in the same row with different superscript letters differ (P < 0.05).
      533
      Means in the same row with different superscript letters differ (P < 0.05).
      542
      Means in the same row with different superscript letters differ (P < 0.05).
      546
      Means in the same row with different superscript letters differ (P < 0.05).
      17.40.012
      Fecal excretion
       DM (kg/d)5.44
      Means in the same row with different superscript letters differ (P < 0.05).
      6.00
      Means in the same row with different superscript letters differ (P < 0.05).
      6.24
      Means in the same row with different superscript letters differ (P < 0.05).
      8.23
      Means in the same row with different superscript letters differ (P < 0.05).
      0.361<0.001
       Total N (g/d)152
      Means in the same row with different superscript letters differ (P < 0.05).
      223
      Means in the same row with different superscript letters differ (P < 0.05).
      177
      Means in the same row with different superscript letters differ (P < 0.05).
      241
      Means in the same row with different superscript letters differ (P < 0.05).
      12.1<0.001
       Total N (% of N intake)31.0
      Means in the same row with different superscript letters differ (P < 0.05).
      41.6
      Means in the same row with different superscript letters differ (P < 0.05).
      32.1
      Means in the same row with different superscript letters differ (P < 0.05).
      44.4
      Means in the same row with different superscript letters differ (P < 0.05).
      1.86<0.001
      Urinary excretion
       Urine (kg/d)22.8
      Means in the same row with different superscript letters differ (P < 0.05).
      25.0
      Means in the same row with different superscript letters differ (P < 0.05).
      23.4
      Means in the same row with different superscript letters differ (P < 0.05).
      24.7
      Means in the same row with different superscript letters differ (P < 0.05).
      0.8130.046
       Total N (g/d)152
      Means in the same row with different superscript letters differ (P < 0.05).
      133
      Means in the same row with different superscript letters differ (P < 0.05).
      189
      Means in the same row with different superscript letters differ (P < 0.05).
      156
      Means in the same row with different superscript letters differ (P < 0.05).
      9.43<0.001
       Total N (% of N intake)31.0
      Means in the same row with different superscript letters differ (P < 0.05).
      24.9
      Means in the same row with different superscript letters differ (P < 0.05).
      35.6
      Means in the same row with different superscript letters differ (P < 0.05).
      29.2
      Means in the same row with different superscript letters differ (P < 0.05).
      2.360.006
      Manure excretion
       Total N (g/d)304
      Means in the same row with different superscript letters differ (P < 0.05).
      355
      Means in the same row with different superscript letters differ (P < 0.05).
      369
      Means in the same row with different superscript letters differ (P < 0.05).
      397
      Means in the same row with different superscript letters differ (P < 0.05).
      7.91<0.001
       Total N (% of N intake)62.0
      Means in the same row with different superscript letters differ (P < 0.05).
      66.6
      Means in the same row with different superscript letters differ (P < 0.05).
      67.7
      Means in the same row with different superscript letters differ (P < 0.05).
      73.7
      Means in the same row with different superscript letters differ (P < 0.05).
      1.830.006
      Milk excretion
       Total N (g/d)1501491561625.550.052
       Total N (% of N intake)30.7
      Means in the same row with different superscript letters differ (P < 0.05).
      28.0
      Means in the same row with different superscript letters differ (P < 0.05).
      28.9
      Means in the same row with different superscript letters differ (P < 0.05).
      29.6
      Means in the same row with different superscript letters differ (P < 0.05).
      1.040.026
      N balance
       N retained (g/d)36.6
      Means in the same row with different superscript letters differ (P < 0.05).
      28.6
      Means in the same row with different superscript letters differ (P < 0.05).
      17.7
      Means in the same row with different superscript letters differ (P < 0.05).
      −13.8
      Means in the same row with different superscript letters differ (P < 0.05).
      13.20.023
       N retained (% of N intake)7.23
      Means in the same row with different superscript letters differ (P < 0.05).
      5.46
      Means in the same row with different superscript letters differ (P < 0.05).
      3.17
      Means in the same row with different superscript letters differ (P < 0.05).
      −3.32
      Means in the same row with different superscript letters differ (P < 0.05).
      2.470.018
      a–c Means in the same row with different superscript letters differ (P < 0.05).
      1 Experimental diets: CS = corn silage; AS = alfalfa silage; WS = wheat silage; PR = Parmigiano Reggiano.
      Fecal N excretion (g/d) was higher (P < 0.001) for both the PR and AS diets (241 and 223, respectively), intermediate for WS (177), and lower for CS (152). Cows fed the WS treatment produced the largest amount of urinary N excretion (189 g/d, P < 0.001). Urinary N excretion, as a percentage of N intake, was lower for AS (24.9) than for CS (31.0) and WS (35.6, P = 0.06). The lowest manure N excretion value (g/d) was observed (P < 0.001) for the cows fed the CS diet (304); the PR diet had the largest value (397), but was not statistically different from WS (369). Dietary N utilization for milk protein synthesis (milk N excretion, % of N intake) differed between the CS (30.7) and AS (28.0) diets (P = 0.026). The N balance (N retained, % of N intake) for the cows fed the PR diet (−3.32) was lower than for the CS or AS diets (P = 0.018), but was not different from the WS diet (3.17).

      Energy Balance

      The results concerning the energy balance are presented in Table 9. The energy intake (Mcal/d) was greater (P = 0.020) for the cows fed PR (98.3) than for those fed CS and AS (88.1 and 88.3, respectively). Digestible energy (% of gross energy intake, GEI) was significantly lower (P < 0.001) for PR (64.7) than for the other diets; CS and AS showed the highest values (73.6 and 72.6, respectively) and WS was intermediate (70.9). The PR diet resulted in a greater (P = 0.047) methane energy loss (5.45 Mcal/d) than the CS diet (5.00 Mcal/d), but there were no differences when methane production was expressed as a percentage of the GEI. The ME (as a % of the GEI) was lower for the PR diet (56.7%, P < 0.001) than for the other diets. As far as the NEL (Mcal/DM) energy content is concerned, the PR treatment was characterized by the lowest value (1.36, P < 0.001) and CS by the highest (1.70), but the latter was not significantly different from AS (1.57); WS (1.53) was different from both PR and CS.
      Table 9Energy balance of lactating dairy cows fed diets based on different forages
      ItemDiet
      Experimental diets: CS = corn silage; AS = alfalfa silage; WS = wheat silage; PR = Parmigiano Reggiano.
      SEMP-value
      CSASWSPR
      GEI
      GEI = gross energy intake.
      (Mcal/d)
      88.1
      Means in the same row with different superscript letters differ (P < 0.05).
      88.3
      Means in the same row with different superscript letters differ (P < 0.05).
      90.7
      Means in the same row with different superscript letters differ (P < 0.05).
      98.3
      Means in the same row with different superscript letters differ (P < 0.05).
      2.960.020
      Fecal energy (Mcal/d)23.4
      Means in the same row with different superscript letters differ (P < 0.05).
      24.3
      Means in the same row with different superscript letters differ (P < 0.05).
      26.3
      Means in the same row with different superscript letters differ (P < 0.05).
      34.8
      Means in the same row with different superscript letters differ (P < 0.05).
      1.62<0.001
       Digestible energy (Mcal/d)64.764.064.163.51.600.665
      Urinary energy (Mcal/d)2.492.462.442.240.140.622
      Methane energy (Mcal/d)5.00
      Means in the same row with different superscript letters differ (P < 0.05).
      5.21
      Means in the same row with different superscript letters differ (P < 0.05).
      5.21
      Means in the same row with different superscript letters differ (P < 0.05).
      5.45
      Means in the same row with different superscript letters differ (P < 0.05).
      0.140.047
       ME (Mcal/d)57.356.356.455.81.650.569
      Heat production (Mcal/d)29.9
      Means in the same row with different superscript letters differ (P < 0.05).
      30.6
      Means in the same row with different superscript letters differ (P < 0.05).
      31.2
      Means in the same row with different superscript letters differ (P < 0.05).
      30.8
      Means in the same row with different superscript letters differ (P < 0.05).
      0.720.019
      Milk energy (Mcal/d)21.422.123.423.00.700.111
       Retained energy (Mcal/d)5.88
      Means in the same row with different superscript letters differ (P < 0.05).
      3.59
      Means in the same row with different superscript letters differ (P < 0.05).
      1.75
      Means in the same row with different superscript letters differ (P < 0.05).
      1.89
      Means in the same row with different superscript letters differ (P < 0.05).
      1.270.031
      Fecal energy (% of GEI)26.4
      Means in the same row with different superscript letters differ (P < 0.05).
      27.4
      Means in the same row with different superscript letters differ (P < 0.05).
      29.1
      Means in the same row with different superscript letters differ (P < 0.05).
      35.3
      Means in the same row with different superscript letters differ (P < 0.05).
      0.98<0.001
       Digestible energy (% of GEI)73.6
      Means in the same row with different superscript letters differ (P < 0.05).
      72.6
      Means in the same row with different superscript letters differ (P < 0.05).
      70.9
      Means in the same row with different superscript letters differ (P < 0.05).
      64.7
      Means in the same row with different superscript letters differ (P < 0.05).
      0.98<0.001
      Urinary energy (% of GEI)2.842.782.712.320.190.328
      Methane energy (% of GEI)5.675.925.785.590.190.543
       ME (% of GEI)65.1
      Means in the same row with different superscript letters differ (P < 0.05).
      63.9
      Means in the same row with different superscript letters differ (P < 0.05).
      62.2
      Means in the same row with different superscript letters differ (P < 0.05).
      56.7
      Means in the same row with different superscript letters differ (P < 0.05).
      0.86<0.001
      Heat production (% of GEI)34.234.834.731.60.760.058
      Milk energy (% of GEI)24.425.125.723.50.680.274
       Retained energy (% of GEI)6.51
      Means in the same row with different superscript letters differ (P < 0.05).
      3.98
      Means in the same row with different superscript letters differ (P < 0.05).
      1.49
      Means in the same row with different superscript letters differ (P < 0.05).
      1.51
      Means in the same row with different superscript letters differ (P < 0.05).
      1.480.029
      NEL (Mcal/kg of DM)1.70
      Means in the same row with different superscript letters differ (P < 0.05).
      1.57
      Means in the same row with different superscript letters differ (P < 0.05).
      1.53
      Means in the same row with different superscript letters differ (P < 0.05).
      1.36
      Means in the same row with different superscript letters differ (P < 0.05).
      0.06<0.001
      kl
      kl = milk energy/(ME − 110 kcal/BW0.75), where milk energy and ME are expressed as kcal/BW0.75; ME for maintenance was assumed to be 110 kcal/BW0.75 (Van Es, 1978).
      0.63
      Means in the same row with different superscript letters differ (P < 0.05).
      0.60
      Means in the same row with different superscript letters differ (P < 0.05).
      0.58
      Means in the same row with different superscript letters differ (P < 0.05).
      0.59
      Means in the same row with different superscript letters differ (P < 0.05).
      0.020.037
      a–c Means in the same row with different superscript letters differ (P < 0.05).
      1 Experimental diets: CS = corn silage; AS = alfalfa silage; WS = wheat silage; PR = Parmigiano Reggiano.
      2 GEI = gross energy intake.
      3 kl = milk energy/(ME − 110 kcal/BW0.75), where milk energy and ME are expressed as kcal/BW0.75; ME for maintenance was assumed to be 110 kcal/BW0.75 (
      • Van Es A.J.H.
      Feed evaluation for ruminants. I. The systems in use from May 1977-onwards in The Netherlands.
      ).

      DISCUSSION

      The present study tested 4 diets as silages and hays, which included the most widespread forages grown in the Po plain area: corn silage, wheat silage, alfalfa, and Italian ryegrass, under the hypothesis that the presence of different high-quality forages in balanced diets can lead to a similar milk production without increasing methane emissions and N excretion per unit of product. The forages used in the trial were all produced locally on commercial farms and are representative of the most widespread forage systems in Northern Italy (
      • Gislon G.
      • Ferrero F.
      • Bava L.
      • Borreani G.
      • Dal Prà A.
      • Pacchioli M.T.
      • Sandrucci A.
      • Zucali M.
      • Tabacco E.
      Forage systems and sustainability of milk production: Feed efficiency, environmental impacts and soil carbon stocks.
      ). The diets were formulated to meet ME and MP requirements, but some important differences in DMI, digestibility, rumen fermentative pattern, NEL content, N excretion (g/d), and methane emissions (% DE intake) were observed. However, no differences were detected for the emissions per unit of product.
      All of the diets were formulated to allow the maximum inclusion of forage. For example, the inclusion level of corn silage in the CS diet was 49.3% DM, which is a much higher value than the values (29.6 and 29.0) reported by
      • Gislon G.
      • Bava L.
      • Colombini S.
      • Zucali M.
      • Crovetto G.M.
      • Sandrucci A.
      Looking for high-production and sustainable diets for lactating cows: A survey in Italy.
      and by
      • Pirondini M.
      • Malagutti L.
      • Colombini S.
      • Amodeo P.
      • Crovetto G.M.
      Methane yield from dry and lactating cows diets in the Po Plain (Italy) using an in vitro gas production technique.
      for commercial dairy farms in the same area. The use of alfalfa silage is not very common in this area; that is, only 21.0% of the farms use it (
      • Gislon G.
      • Bava L.
      • Colombini S.
      • Zucali M.
      • Crovetto G.M.
      • Sandrucci A.
      Looking for high-production and sustainable diets for lactating cows: A survey in Italy.
      ), and at a lower inclusion level than that used in the AS diet of the present study, which was ∼27% on a DM basis. This suggested an opportunity to re-introduce legume forages into the cropping systems in this area and to increase farm protein self-sufficiency by producing high-quality forages. The inclusion level of whole cereal silage, on a DM basis of the WS diet (20%), fell between the lower level tested by
      • Benchaar C.
      • Hassanat F.
      • Gervais R.
      • Chouinard P.Y.
      • Petit H.V.
      • Massé D.I.
      Methane production, digestion, ruminal fermentation, nitrogen balance, and milk production of cows fed corn silage-or barley silage-based diets.
      for barley silage (27.2%) and the level (wheat silage, 10% of diet DM) tested by
      • Harper M.T.
      • Oh J.
      • Giallongo F.
      • Roth G.W.
      • Hristov A.N.
      Inclusion of wheat and triticale silage in the diet of lactating dairy cows.
      ; to the best of our knowledge, no studies have been conducted on the use of winter cereal silage in the diets of lactating cow in the study area. The PR diet was formulated according to the indications reported in the disciplinary codes for Parmigiano Reggiano cheese production, and for this reason it included a significant amount of hay in the TMR (50.6% of total DM).

      DMI and Digestibility

      The cows fed the PR diet had a significantly higher DMI; this agrees with the results of
      • Brown L.D.
      • Hillman D.
      • Lassiter C.A.
      • Huffman C.F.
      Grass silage vs. hay for lactating dairy cows.
      , who reported an increase in DMI as the level of hay in the diet increased. The PR diet showed a lower digestibility for all of the main constituents. The lower digestibility values of the PR diet are mainly due to the higher fiber content of the diet and to the high ADL of the used alfalfa hay.
      • Broderick G.A.
      Performance of lactating dairy cows fed either alfalfa silage or alfalfa hay as the sole forage.
      showed higher values of apparent nutrient digestibility, together with lower DMI, for alfalfa silage when used as a replacement for alfalfa hay, which we also observed in the present study. This is also consistent with the studies of
      • Colucci P.E.
      • Chase L.E.
      • Van Soest P.J.
      Feed intake, apparent diet digestibility, and rate of particulate passage in dairy cattle.
      and of
      • de Souza R.A.
      • Tempelman R.J.
      • Allen M.S.
      • Weiss W.P.
      • Bernard J.K.
      • VandeHaar M.J.
      Predicting nutrient digestibility in high-producing dairy cows.
      , who reported that the digestibility of a diet is reduced as DMI increases.
      Alfalfa, Italian ryegrass, and corn silages are the 3 most common forages fed to dairy cows in silage-based diets in Northern Italy, although the use of winter cereal silage is also increasing. The best results for aNDFom digestibility were obtained for the CS and AS diets. Therefore, the use of large amounts of alfalfa silage together with ryegrass silage may provide a valuable possibility to increase the proportion of feed ingredients produced on a farm and reduce the inclusion of soybean meal in the TMR. Interestingly, these forages are mainly used as hay in Northern Italy at a lower inclusion level than that used in the present study (
      • Gislon G.
      • Bava L.
      • Colombini S.
      • Zucali M.
      • Crovetto G.M.
      • Sandrucci A.
      Looking for high-production and sustainable diets for lactating cows: A survey in Italy.
      ).
      The diet based on wheat silage was characterized by a lower aNDFom digestibility than CS and AS, unlike what
      • Harper M.T.
      • Oh J.
      • Giallongo F.
      • Roth G.W.
      • Hristov A.N.
      Inclusion of wheat and triticale silage in the diet of lactating dairy cows.
      observed, but in agreement with the results of
      • Benchaar C.
      • Hassanat F.
      • Gervais R.
      • Chouinard P.Y.
      • Petit H.V.
      • Massé D.I.
      Methane production, digestion, ruminal fermentation, nitrogen balance, and milk production of cows fed corn silage-or barley silage-based diets.
      for barley silage as a replacement of corn silage. This difference can be explained by considering several factors, such as different inclusion levels or maturity at harvest;
      • Arieli A.
      • Adin G.
      Effect of wheat silage maturity on digestion and milk yield in dairy cows.
      , for example, showed that the in vivo NDF digestibility of cows fed wheat silage diets was 5 percentage points higher for cows fed a silage harvested at an earlier maturity stage than for a silage harvested at a later maturity.
      One negative effect that has emerged in this study, related to the high proportion of alfalfa in the AS and PR diets, is a lower protein digestibility than the CS and WS diets, which were characterized by a higher amount of soybean meal. Therefore, as far as this aspect is concerned, soybean meal may be more favorable than alfalfa.

      Milk Production and Composition

      As far as milk production is concerned, the results of the present study did not point out any significant differences between diets, even though the cows offered the PR diet had a significantly greater DMI, which resulted in a tendency for a higher milk yield. Overall, feed efficiency (ECM/DMI) was only numerically lower for the PR diet.
      • Broderick G.A.
      Performance of lactating dairy cows fed either alfalfa silage or alfalfa hay as the sole forage.
      also reported a lower dairy efficiency for cows fed alfalfa as hay instead of silage. We found that the milk fat concentration was slightly lower for the cows fed the PR diet than the cows fed the other diets, probably because of a dilution effect due to the higher milk production for the PR diet. Under the experimental conditions of the current study, the smallest MUN was observed for the AS diet. The smaller MUN content of the cows fed the AS diet than those fed the CS diet agrees with other studies (
      • Broderick G.A.
      Alfalfa silage or hay versus corn silage as the sole forage for lactating dairy cows.
      ;
      • Benchaar C.
      • Petit H.V.
      • Berthiaume R.
      • Ouellet D.R.
      • Chiquette J.
      • Chouinard P.Y.
      Effects of essential oils on digestion, ruminal fermentation, rumen microbial populations, milk production, and milk composition in dairy cows fed alfalfa silage or corn silage.
      ) which reported a smaller MUN concentration when cows were fed alfalfa rather than corn silage as the sole source of forage. The cows fed the WS diet had the highest MUN content, a result that agrees with the findings of
      • Harper M.T.
      • Oh J.
      • Giallongo F.
      • Roth G.W.
      • Hristov A.N.
      Inclusion of wheat and triticale silage in the diet of lactating dairy cows.
      , who found a greater MUN for cows fed wheat silage than for cows fed corn silage. However, the CS diet in
      • Harper M.T.
      • Oh J.
      • Giallongo F.
      • Roth G.W.
      • Hristov A.N.
      Inclusion of wheat and triticale silage in the diet of lactating dairy cows.
      was characterized by a lower CP content than their wheat diet, like in our experiment; the WS diet had a slightly higher CP concentration than the others. Moreover, in the present study, the WS diet resulted in a lower OM digestibility than the CS and AS diets, and it can therefore be speculated that there was less energy available for microbial protein synthesis at a rumen level, with a consequent decrease in the capturing of N (as AA or NH3) by rumen microbes. In the present experiment, the WS diet had a relatively high inclusion of alfalfa silage, with a consequent probable high release of ammonia in the rumen. This high release was also correlated with the higher urinary N excretion (g/d) of the cows fed the WS diet.

      Ruminal Fermentation Characteristics

      The rumen fermentation pattern was also affected by the different chemical compositions of the diets; PR and CS differed the most. The rumen pH increased in the PR diet, which was characterized by a lower NFC content and higher aNDFom content than the CS diet. The propionate proportion was lower in the PR diet than the CS diet. In agreement with the findings of
      • Broderick G.A.
      Alfalfa silage or hay versus corn silage as the sole forage for lactating dairy cows.
      , we found a higher ruminal pH, a lower propionate ruminal molar proportion, and a higher acetate-to-propionate ratio in the ruminal fluid of the cows fed alfalfa hay than in the ruminal fluid of the cows fed corn silage.
      The rumen pH values were registered 5 h after the morning feeding; the nadir pH appears after this time interval and allows the differences in diets and possible subacidosis issues to be better understood. In this regard, despite the difference between the CS and PR diets, the pH values registered for each diet were adequate to support rumen bacteria growth and fermentation.
      Replacing structural carbohydrates in the diet with nonstructural carbohydrates has resulted in notable modifications of the physical-chemical conditions and microbial populations of the rumen, such as the shift of VFA production from acetate toward propionate, which occurs with the development of starch-fermenting microbes (
      • Martin C.
      • Morgavi D.P.
      • Doreau M.
      Methane mitigation in ruminants: From microbe to the farm scale.
      ). In agreement with this observation, the AS diet in the present study, which showed the smallest aNDFom concentration, resulted in a higher rumen acetate proportion than the PR diet, which was the diet with the highest fiber content.

      Enteric Methane Production and Energy Balance

      The cows fed the PR diet had a greater daily production of methane (g/d) than those fed the CS diet due to the ruminal fermentation profile (i.e., higher pH and higher acetate:propionate ratio) together with the increased DMI.
      • Benchaar C.
      • Pomar C.
      • Chiquette J.
      Evaluation of dietary strategies to reduce methane production in ruminants: A modelling approach.
      found a greater daily methane production for cows fed hay than for cows fed silages. Despite this difference, no significant differences in methane production were observed between diets in terms of grams per kilogram of DMI or grams per kilogram of milk.
      • Harper M.T.
      • Oh J.
      • Giallongo F.
      • Roth G.W.
      • Hristov A.N.
      Inclusion of wheat and triticale silage in the diet of lactating dairy cows.
      did not detect any difference in the methane emissions of cows fed CS or WS diets. On the other hand,
      • Hart K.J.
      • Huntington J.A.
      • Wilkinson R.G.
      • Bartram C.G.
      • Sinclair L.A.
      The influence of grass silage-to-maize silage ratio and concentrate composition on methane emissions, performance and milk composition of dairy cows.
      observed lower methane yields for cows fed a high corn silage ration than those fed a high grass silage ration, but the proportion of NFC (starch) and fiber in the diets in their experiment was more variable than in our study. As far as the overall methane energy losses (% of energy intake) are concerned, the results of the present study agree with those of
      • Pirondini M.
      • Colombini S.
      • Mele M.
      • Malagutti L.
      • Rapetti L.
      • Galassi G.
      • Crovetto G.M.
      Effect of dietary starch concentration and fish oil supplementation on milk yield and composition, diet digestibility, and methane emissions in lactating dairy cows.
      and of
      • Colombini S.
      • Zucali M.
      • Rapetti L.
      • Crovetto G.M.
      • Sandrucci A.
      • Bava L.
      Substitution of corn silage with sorghum silages in lactating cow diets: In vivo methane emission and global warming potential of milk production.
      , with methane produced by rumen fermentation accounting for 5 to 6% of the gross energy ingested by the cows. No difference between diets was found for the methane energy loss as a percentage of GEI, but the methane energy loss as a percentage of DE was lower for the cows fed the CS diet than the cows fed the PR diet. This may partially be related to the observed, previously described rumen fermentation pattern.
      The energy balance results suggest a different utilization of energy, depending on the diet. In agreement with the digestibility results, the hay-based diet (PR) in the present study was characterized by the lowest digestible and ME (% of GEI), which overall resulted in a lower NEL content for PR than for the other diets. The AS diet had a similar NEL content to the CS diet, whereas the WS diet had an intermediate NEL content between the CS and PR diets. The final ranking of the 4 diets suggest that the corn silage, which is rich in starch and with a fairly good NDF digestibility, supplied more NEL than the other forages. Among the latter, the high-quality forages (the AS diet) showed similar values to the CS diet, and the wheat silage and hays provided less digestible fiber, and consequently less NEL. The PR diet, which is a feeding system that is based totally on hays as the forage source, seemed less efficient than those based on silages, thus confirming the lower feeding value of hays than silages.

      Nitrogen Balance

      Any dietary strategy aimed at mitigating the methane emissions of dairy cows should consider the possible effect of N losses in manure, urinary N in particular, to ensure that the reduction in enteric methane emissions is not offset by an increase in nitrous oxide and ammonia emissions (
      • Hassanat F.
      • Gervais R.
      • Benchaar C.
      Methane production, ruminal fermentation characteristics, nutrient digestibility, nitrogen excretion, and milk production of dairy cows fed conventional or brown midrib corn silage.
      ). Nitrous oxide is a powerful GHG, and ammonia, although not a GHG, is environmentally harmful because it favors environmental acidification and the formation of fine particulates, which pollute the air. In the present study, the urine volume was higher for the AS diet than for the CS diet. Other studies (
      • Hristov A.N.
      • Broderick G.A.
      Synthesis of microbial protein in ruminally cannulated cows fed alfalfa silage, alfalfa hay, or corn silage.
      ;
      • Brito A.F.
      • Broderick G.A.
      Effect of varying dietary ratios of alfalfa silage to corn silage on production and nitrogen utilization in lactating dairy cows.
      ) reported greater urine volumes for diets with higher dietary alfalfa silage proportions vs. corn silage. In agreement with the MUN concentration, the cows fed the AS diet showed a lower urinary N excretion (% of N intake) than those fed the CS and WS diets. These results were partially unexpected because a large proportion of CP is converted into NPN during ensiling, thus reducing the efficiency of CP utilization in lactating cows. Feeding carbohydrates that are more extensively fermented in the rumen may improve the utilization of alfalfa NPN through the stimulation of microbial protein synthesis. In this study, a consistent amount of high-moisture ear corn (28.6% of diet DM) was used in the AS diet and probably improved N use in the rumen, as confirmed from the MUN concentration and urinary N excretion (% N intake), which were significantly lower than for the CS and WS diets. Urinary N is largely represented by urea, and is therefore more rapidly nitrified with consequent nitrous oxide emissions (
      • Eckard R.J.
      • Grainger C.
      • De Klein C.A.M.
      Options for the abatement of methane and nitrous oxide from ruminant production: A review.
      ). Thus, urinary N is less desirable, and shifting N excretion from urine to feces may be useful (
      • Brito A.F.
      • Broderick G.A.
      Effects of different protein supplements on milk production and nutrient utilization in lactating dairy cows.
      ). The higher N urinary excretion (total N g/d) of the cows fed the WS diet than that of the other diets agreed with the results of
      • Benchaar C.
      • Hassanat F.
      • Gervais R.
      • Chouinard P.Y.
      • Petit H.V.
      • Massé D.I.
      Methane production, digestion, ruminal fermentation, nitrogen balance, and milk production of cows fed corn silage-or barley silage-based diets.
      , who also observed an increase in N urinary excretion for a larger amount of winter cereal (i.e., barley silage) in the diet. These increased urinary N losses are probably related to a reduced N utilization in the rumen, as confirmed by the higher amount of MUN concentrations than for the other diets.

      CONCLUSIONS

      This study has shown that, despite differences in several variables in the considered diets, no differences concerning methane production (per unit of product or per kilogram of DMI) have been observed between the diets that are commonly used in dairy feeding in Northern Italy. The use of high-quality forages, especially if preserved as silage rather than as hay, is a valuable strategy that can be adopted to increase the feeds produced on a farm, a strategy which is positively related to the development of an environmentally sustainable farming system. The use of a hay-based diet is interesting for the production of PDO cheese and, based on these results, the environmental impact of this feeding system is comparable with that of the other diets. The results of the present study show that corn silage can also have a high nutritive value in terms of fiber digestibility; however, the use of high-moisture ear corn, in combination with high-quality grass and legume silages, is a valuable alternative. Despite the similar milk production, the wheat silage diet showed a lower OM digestibility and a higher urinary N excretion than the AS diet. A long-term study would be useful to evaluate the maintenance of milk production over lactation when winter cereal silage is used as the main forage. Overall, on the basis of these results, an evaluation of the environmental impact of milk production should be performed that considers the entire milk production chain. Particular attention should be paid to the forage production system to identify the best dietary strategy to enhance the environmental sustainability of dairy farms in both agronomic and animal aspects.

      ACKNOWLEDGMENTS

      The study was part of the project Life + Forage4Climate—Forage systems for less GHG emissions and more soil carbon sink in continental and Mediterranean agricultural areas (LIFE15 CCM/IT/000039), funded by the EU Commission (Brussels, Belgium). The authors have not stated any conflicts of interest.

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