Animal Feed Science and Technology 282 (2021) 115120

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Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci

A meta-analysis of faecal output and nutrient composition, and

potential methane emission from manure of dairy cows
Pekka Huhtanen a, *, Sophie J. Krizsan b, Mohammad Ramin b
a
Production Systems, Natural Resources Institute Finland (LUKE), 31600 Jokioinen, Finland
b
Department of Agricultural Research for Northern Sweden, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden

A R T I C L E I N F O A B S T R A C T

Keywords: A meta-analysis was conducted to predict potential methane emission from manure in dairy cows.
Manure methane Biogas production from manure offers considerable environmental benefits and renewable energy
Fermentable substrates is produced. The developed models predicted faecal concentrations of potentially digestible
Digestible fiber
neutral detergent fibre, neutral detergent solubles and crude protein with adjusted root mean
square errors of 15.4, 12.9 and 5.0 g/kg dry matter, respectively. Average model predicted po­
tential methane production (adjusted root mean square error) of 1040 (53.9) g/d per cow, 51.9
(2.5) g/kg dry matter intake and 203 (3.2) g/kg faecal organic matter (volatile solids). Potential
methane production from dairy cow manure can be predicted from diet and animal character­
istics. Potential methane production is positively related to diet digestibility and daily feed intake.
Determination of indigestible neutral detergent fibre concentration by a long ruminal in situ
incubation is a useful tool in predictions of potential methane production in dairy cow manure.

1. Introduction

Ruminants contribute to greenhouse gas (GHG) emissions by producing methane (CH4) in enteric fermentation with amounts
depending on intake and diet composition. In addition, CH4 is also produced from manure. Manure contribution to GHG emissions
depends on many factors, such as the chemical composition of manure, storage conditions and temperature (Hassanat and Benchaar,
2019). Sweden and some other Nordic countries use the IPCC Tier 2 methodology in estimating CH4 emissions from manure (IPCC
Intergovernmental Panel on Climate Change, 2006). Methane emission factor (kg of CH4/cow per year) of manure depends mainly on
the amount of organic matter (OM) excreted from the animal (IPCC Intergovernmental Panel on Climate Change, 2006). Organic
matter is a term used in animal science that corresponds to volatile solids (VS) used in biogas research. The diet of dairy cows plays a
significant role on the amount of OM excreted in the faeces (Massé et al., 2008; Hassanat and Benchaar, 2019). In attempting to reduce
CH4 emissions from ruminants, the main focus has been in enteric fermentation (Grainger and Beauchemin, 2011) with less attention

Abbreviations: ADF, acid detergent fibre; AIA, acid insoluble ash; AICc, Akaike’s information criterion with correction; BW, body weight; CH4,
methane; CP, crude protein; CPD, crude protein digested; DM, dry matter; DMI, dry matter intake; FA, fatty acids; GHG, greenhouse gas; iNDF,
indigestible neutral detergent fibre; MFOM, metabolic faecal organic matter; MRT, mean retention time; NDF, neutral detergent fibre; NDFD, neutral
detergent fibre digestibility; NDS, neutral detergent solubles; OM, organic matter; OMD, organic matter digestibility; pdNDF, potentially digestible
neutral detergent fibre; pdOM, potentially digestible organic matter; PMP, potential methane production; PMY, potential methane yield; RMSE, root
mean square errors; TUDCP, truly undigested crude protein; VS, volatile solids.
* Corresponding author.
E-mail address: pekka.huhtanen@luke.fi (P. Huhtanen).

https://doi.org/10.1016/j.anifeedsci.2021.115120
Received 25 May 2021; Received in revised form 12 October 2021; Accepted 14 October 2021
Available online 18 October 2021
0377-8401/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
P. Huhtanen et al. Animal Feed Science and Technology 282 (2021) 115120

been paid on CH4 emissions from manure. A default value of 240 ± 36 L/kg of OM of maximal CH4 emission of manure was reported by
IPCC Intergovernmental Panel on Climate Change (2006) for dairy cows in North America. It should be noted that this value does not
consider the changes in the chemical composition of manure OM. However, it could be expected that diet composition and feeding
level affect manure composition, and consequently CH4 emissions from manure are not constant. It is well known that diet digestibility
decreases with feeding level in ruminants with depression being greater for highly digestible diets (NRC, 2001; Huhtanen et al., 2009).
Therefore, it is possible that some mitigation strategies such as increased concentrate feeding increases faecal output of potentially
digestible organic matter (pdOM). Genetic selection for low CH4 emitting cows is considered a promising strategy, as it is sustainable,
persistent, and cumulative over subsequent generations (Difford et al., 2018). However, low CH4 emitting cows digest the diet less
efficiently (Løvendahl et al., 2018), and therefore reduced enteric emissions can partly be compensated for by increased emissions from
manure.
Biogas production from manure offers considerable environmental benefits and renewable energy is produced. Predicting potential
CH4 yield (PMY) from manure is important for both biogas production and GHG inventories. Amon et al. (2007) used Weende feed
analysis components to predict potential CH4 production (PMP). However, relative regression coefficients for the Weende components
did not agree with the coefficients calculated according to Buswell and Hatfield (1952). This can be because only a small fraction of
biogas substrates (manure, forages) are nutritionally uniform entities (Van Soest, 1994) according to the Weende analysis, i.e. have a
constant true digestibility. Neutral detergent extraction (Van Soest, 1967) divides feed into neutral detergent fibre (NDF) and neutral
detergent solubles (NDS). The NDS fraction, defined either as dry matter (DM) – NDF or organic matter (OM) - NDF, is a uniform entity
with a constant true digestibility close to 100% (Van Soest, 1994; Weisbjerg et al., 2004). The NDF component is possible to divide into
two components: indigestible NDF (iNDF) that cannot be digested by rumen microbes even in extended digestion time and potentially
digestible NDF (pdNDF = NDF - iNDF). The iNDF fractions is by definition a uniform nutritional entity as its true digestibility is zero.
The pdNDF is also a more uniform entity than the total NDF as its digestibility is less variable than the digestibility of total NDF or crude
fibre and N-free extractives (Huhtanen et al., 2006). Indigestible NDF have been used as an internal marker for digestibility de­
terminations (Lippke et al., 1986). The concentration of iNDF determined by 12-d in situ incubation using nylon bags with small pore
size (6–16 µm) predicted in vivo digestibility of forages precisely and uniformly for different forage types (Huhtanen et al., 2006). The
accuracy of manure CH4 emission predictions (e.g. IPCC) could therefore be improved by considering relevant digestible components
in the manure predicted from dietary and animal factors.
The first objective of this study was to develop models predicting faecal output and faecal concentrations of potentially digestible
components that can produce CH4 from manure or be used as a substrate in biogas production. Second objective was to develop
equations predicting potential CH4 emission from manure. Finally, we determined between-cow variation in faecal output of poten­
tially digestible components from two datasets of individual cow observations.

2. Material and methods

Meta-analyses were conducted to evaluate between-animal variation and the effects of dietary and animal factors on faecal
composition and output of components related to PMP from manure, i.e. manure biogas potential. Between-cow variability was
estimated from two datasets from change-over studies with individual cow/period observations. These datasets are described in detail
by Huhtanen et al. (2015) and Gabezas-Garcia et al. (2017). Digestion studies in Gabezas-Garcia et al. (2017) dataset (Data1) was
conducted using rumen cannulated cows. Only studies (28 studies, 428 cow/period observations) in which dietary iNDF concentration
was determined were included in the current analysis. Digestibility was determined by total faecal collection method. In the dataset
(Data 2) of Huhtanen et al. (2015) diet digestibility was determined in change-over production studies (10 studies, 452 cow/period
observations) using acid insoluble ash (AIA, Van Keulen and Young, 1977) or iNDF as internal markers. Data on dietary iNDF con­
centration was not available, but because iNDF concentration was constant within a diet, between-cow variation in faecal output of
total OM and NDF also represent the variability of digestible components of these fractions. Treatment means dataset (Data3; Nou­
siainen et al., 2009) consisted of 501 diets in 94 studies. Diet digestibility in cows was determined by the total faecal collection method
or by using AIA as an internal marker. When forage iNDF concentrations were not determined by in situ incubation, the estimates were
derived from in vitro or in vivo OM digestibility (OMD) using an equation derived from relationship between iNDF concentration OMD
(Huhtanen et al., 2006).

iNDF (g/kg DM) = 0.758 × (832 – OMD (g/kg)

Concentrate iNDF concentrations were based on either determined values or values derived from unpublished MTT datasets. In all
datasets, if the chemical composition of concentrates was not reported, default feed table values were used.
Faecal output of pdNDF was calculated as faecal NDF output – iNDF intake. Faecal NDS output was calculated as a difference
between faecal OM and NDF. This fraction consists mainly endogenous components, undigested rumen microbial matter and microbial
cells produced in the hind-gut fermentation. The Lucas test (regression of the intake of the digestible dietary entity on the intake of the
respective entity; see Van Soest, 1994) was performed for CP and NDS. The negative intercept of regression represents metabolic and
faecal OM output (MFOM) and the slope of regression is true digestibility.
Potential manure CH4 emissions were estimated per cow/d (PMP), and per kg DMI (PMY per kg DMI (PMYDMI) and per kg faecal
OM (PMYOM). Methane yields were estimated using the stoichiometric equations developed by Buswell and Hatfield (1952):

CxHyOz + (x – y/4 – z/2) H2O → (x/2 + y/8 – z/4) CH4 + (x/2 – y/8 + z/4) CO2⋅

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This equation estimates the PMP from different components assuming complete degradability of biomass. The proportion of PMP
achieved from faecal OM components depends on their bioavailability. Indigestible NDF cannot, by definition, be digested in anaerobic
ruminal fermentation. It may therefore be assumed that anaerobic microbes in the biogas systems cannot reach a higher potential NDF
digestibility than rumen microbes. Faecal NDS fraction consists mainly MFOM with a small part of protein that is not truly digested by
ruminants. The amount of truly undigested crude protein (TUDCP) per kg DMI was calculated as: TUDCP (g/kg DM) = (1 – true CPD)
× CP (g/kg DM). True CPD was derived from the Lucas test (see above). Faecal digestible CP was calculated as a difference between
total CP and TUDCP. Faecal fatty acids (FA) output per kg DMI was predicted as a difference between FA intake and digested FA
estimated from a quadratic relationship between dietary FA concentration and digestibility, i.e., true fat digestibility decreases with its
increased dietary concentration. The following equation was derived from our datasets:

Digested FA (g/kg DMI) = − 6⋅0 + 1⋅13 × FA (g/kg DM) – 0.0042 × FA2 (g/kg DM)

Carbohydrate fraction from faecal NDS was estimated as a difference between faecal NDS – faecal CP – Faecal FA.

2.1. Statistical analysis

Multiple regression models were developed from treatment means data by using the mixed model procedure of SAS (Littell et al.,
1996). The model was Y = B0 + B1X1ij + b0 + b1X1ij + B2X2ij + . + BnXnij + eij, where Yij = the expected value for the dependent
variable X in study i, B0, = overall intercept, B1, B2, … Bn are fixed regression coefficients of Y on independent variables X1, X2,…Xnij; b0
and b1 are the random effects of study i on the intercept and regression coefficient of Y on X1 in study i; and eij = the residual error. Only
one random independent variable was used to avoid overparameterized models and to improve convergence (St-Pierre, 2001). The
models were evaluated based on Akaike’s information criterion with correction (AICc) and residual variance. Covariance structure was
specified using the TYPE = UN option (unstructured covariance) in the RANDOM statement with SUBJECT = study. Root mean square
errors (RMSE) were adjusted for random study effects as described by St-Pierre (2001).
To evaluate between-cow variation in faecal output of dietary components the variance components were estimated using the
PROC MIXED procedure of SAS (version 9.3; SAS Institute Inc., Cary, NC) with experiment (Exp), diet within experiment [Diet(Exp)],
period within experiment [Period(Exp)], and cow within experiment [Cow(Exp)] as random factors. Covariance structure was
specified using the TYPE = VC option in the RANDOM statement. The coefficient of variation for each factor were calculated as the
square root of the variance estimate divided by the respective mean value of each factor, respectively.

Table 1
Descriptive data from treatment means dataset.
N Mean SD Min Max

Intake, kg dry matter (DM) per day

Forage 501 11.6 1.80 4.6 17.4
Concentrate 501 8.3 2.41 0.0 18.4
Total 501 19.9 2.64 10.9 26.0
Proportion of concentrate, g/kg 501 412 95.8 0 800
Diet composition, g/kg DM
Organic matter (OM) 501 923 11.8 850 956
Crude protein (CP) 501 165 20.8 111 246
Neutral detergent fiber (NDF) 501 413 49.4 219 570
Indigestible NDF 501 73 20.8 12 148
Potential digestible NDF (pdNDF) 501 341 47.0 188 528
Ether extract 501 43 7.4 22 74
Starch 501 141 54.4 0 259
Production data
Milk yield, kg/d 501 28.3 4.8 13.0 45.8
Energy corrected milk, kg/d 501 29.3 4.7 12.8 42.1
Milk fat, g/kg 501 43.5 4.1 32.1 55.0
Milk protein, g/kg 501 32.9 2.0 24.0 39.0
Body weight, kg 501 591 46.1 417 732
Digestibility, g/kg
OM 501 725 36.5 621 816
CP 477 685 42.9 542 782
NDF 501 622 72.5 427 830
pdNDF 501 754 64.8 564 946
Faecal output, kg/d
OM 501 5.07 1.074 2.36 8.11
CP 477 1.02 0.194 0.49 1.65
NDF 501 3.11 0.849 1.04 5.68
pdNDF 501 1.66 0.532 0.33 3.16
Neutral detergent solubles (NDS) 501 1.95 0.438 0.80 3.89

Note: SD is standard deviation.

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3. Results

Descriptive data on feed intake, diet composition, milk production, diet digestibility and faecal output are described in Table 1 for
treatment means dataset and in Table 2 for two individual cow datasets. As expected, large variation was displayed in both intake and
diet composition, and consequently in milk production, diet digestibility and faecal output. The diets represent typical Nordic dairy
cow diets.
The variance component of cow(exp) was significant (at least P < 0.01) for all faecal components (Table 3), but quantitatively the
variability was small. Diet(Exp) variability was on average 3-fold compared with cow(exp) variability for faecal OM output and 6-fold
for faecal NDF output, respectively. Between-cow variability was usually greater for NDF, and especially for pdNDF in Data1 than for
NDS. The mean concentration of CP in faecal NDS was 517 g/kg assuming NDF as CP free.
The models predicting faecal concentrations of components that potentially can produce CH4 from manure are presented in Table 4.
Root mean square errors adjusted for random study effects were 15.4, 12.9 and 5.0 g/kg DM for faecal concentrations of pdNDF, NDS
and CP, respectively. Faecal pdNDF concentration was positively (P < 0.001) associated with feeding level and the proportion of
concentrates and negatively (P < 0.001) to dietary concentrations of iNDF and CP. The model fit was slightly better when the quadratic
effect of the proportion of concentrate was used in the model. Faecal concentrations of NDS and CP were negatively (P < 0.001) related
to dietary concentration of iNDF and positively (P < 0.001) to that of CP. Increased feeding level had a small positive (P = 0.05) effect
on faecal CP concentration. Dietary iNDF concentration was closely associated with faecal OM composition (Fig. 1). Potentially
fermentable components (NDS and pdNDF) were negatively (P < 0.001) related to dietary iNDF concentration, whereas dietary and
faecal iNDF concentrations were positively (P < 0.001) related to each other.
Models predicting faecal output of potentially digestible components are shown in Table 5. With one variable models, the intake of
pdNDF was the most important factors influencing faecal pdNDF output. Faecal output of pdNDF increased (at least P < 0.01) with
increased proportion of concentrate in the diet (quadratic effect), dietary starch concentration and feeding level, whereas it decreased
(P < 0.001) with increased dietary CP concentration. Faecal output of NDS (MFOM) was mainly related to DMI. When concentrate
intake was expressed as kg DM/d instead of CProp × CProp, the regression coefficient of concentrate DMI was 29 ( ± 8.6) g/kg DM on
faecal pdNDF output. Faecal NDS output was mainly related to DMI, and it increased (at least P < 0.01) with dietary concentrations of
CP and starch. However, quantitatively the effects of CP and starch were small. Faecal CP output was mainly related to DMI being
slightly, although significantly, increased with dietary CP concentration. Adjusted RMSE values were 122, 101 and 40 g/d for faecal
output of pdNDF, NDS and CP, respectively.
Neutral detergent solubles and CP met the criterion of uniform nutritional entity, i.e. the negative intercept representing MFOM

Table 2
Descriptive data from two individual cow datasets.
Dataset1 Dataset2

N Mean SD Min Max N Mean SD Min Max

Intake, kg dry matter (DM) per day

Forage 428 11.1 2.47 2.5 18.8 452 12.7 2.05 7.4 18.6
Concentrate 428 7.7 2.41 0.0 15.5 452 8.5 2.32 2.5 13.1
Total 428 18.8 2.89 7.5 25.8 452 21.3 3.21 11.4 29.3
Proportion of concentrate, g/kg 428 406 109.2 0 797 452 396 77.7 190 591
Diet composition, g/kg DM
Organic matter (OM) 428 923 11.7 885 967 452 912 16.9 864 935
Crude protein (CP) 428 157 22.1 121 239 452 171 26.2 115 240
Neutral detergent fibre (NDF) 428 402 50.7 252 534 452 408 55.8 255 546
Indigestible NDF 428 71 16.4 37 123
Potential digestible NDF (pdNDF) 428 331 50.9 187 478
Ether extract 428 44 16.2 14 106
Starch 412 147 59.8 0 400
Production data
Milk yield, kg/d 426 26.1 6.5 5.0 41.8 452 30.7 6.75 10.5 47.4
Energy corrected milk, kg/d 426 26.2 5.9 5.2 40.1 452 32.6 6.36 13.2 52.3
Milk fat, g/kg 426 41.0 6.2 16.5 55.9 452 45.5 7.00 31.4 66.6
Milk protein, g/kg 426 33.3 4.0 21.3 58.0 452 33.0 3.01 22.9 42.3
Body weight, kg 426 616 61.3 459 808 426 595 69.8 453 806
Digestibility, g/kg
OM 428 739 33.7 660 837 452 712 32 607 827
CP 428 682 50.6 520 876 448 675 42 525 772
NDF 428 640 70.7 413 825 450 613 66 404 790
pdNDF 428 780 69.2 526 997
Faecal output, kg/d
OM 428 4.56 1.015 1.35 7.06 452 5.59 1.098 2.85 8.34
CP 428 0.93 0.205 0.28 1.62 448 1.18 0.295 0.50 2.07
NDF 428 2.71 0.704 0.81 4.92 450 3.34 0.836 1.61 5.61
pdNDF 428 1.37 0.492 0.01 2.84
Neutral detergent solubles (NDS) 428 1.86 0.452 0.55 3.43 450 2.25 0.529 1.10 3.74

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Table 3
Between cow variation in faecal output (g/kg dry matter intake) of different dietary components.
Dataset1 Dataset2

N Mean Cow variance Between-cow CV, % N Mean Cow variance Between-cow CV, %

Organic matter 428 239 74 3.6 452 266 69 3.1

NDF 428 142 32 4.0 450 159 41 4.0
Potential digestible NDF 428 69.0 35 8.6
NDS 428 97.7 16 4.1 450 106 9.2 2.9
Crude protein (CP) 428 49.0 3.4 3.8 448 55.5 3.7 3.5
CP/NDS, g/kg 428 508 115 2.1 448 526 107 2.0

CV = coefficient of variation, NDF = neutral detergent fibre; NDS = neutral detergent solubles.

Table 4
Diet factors influencing faecal composition (g/kg dry matter, DM).
Unit Estimate SE P-value

Faecal pdNDF g/kg DM

Intercept 427 31.0 < 0.001
iNDF g/kg NDF -0.62 0.096 < 0.001
DMI/BW g/kg 2.51 0.741 < 0.001
Crude protein g/kg DM -0.83 0.082 < 0.001
CProp × CProp Fraction of 1 95 21.8 < 0.001
Faecal neutral detergent solubles g/kg DM
Intercept 341 19.0 < 0.001
iNDF g/kg DM -1.09 0.170 < 0.001
Crude protein g/kg DM 0.43 0.071 < 0.001
Faecal CP g/kg DM
Intercept 113 9.9 < 0.001
iNDF g/kg DM -0.56 0.072 < 0.001
CP g/kg DM 0.53 0.030 < 0.001
DMI/BW g/kg 0.43 0.221 0.05

SE = standard error, pdNDF = potential digestible neutral detergent fibre, iNDF = indigestible neutral detergent fibre, DMI = dry matter intake, BW
= body weight, CProp is concentrate proportion.

Fig. 1. The effects of dietary indigestible neutral detergent fiber (iNDF) concentration (g/kg dry matter, DM) on faecal composition (g/kg organic
matter, OM). Faecal neutral digestible solubles, NDS (g/kg OM) = 493 ± 18.3 – 1.39 ± 0.232 × Diet iNDF (Adj. RMSE = 14.6). Faecal potential
digestible NDF, pdNDF (g/kg OM) = 432 ± 21.7–1.57 ± 0.278 × Diet iNDF (Adj. RMSE = 18.5). Faecal iNDF (g/kg OM) = 78 ± 6.9 –
2.89 ± 0.107 × Diet iNDF (Adj. RMSE = 9.4).

output per kg DM intake and the slope representing true digestibility:

Digestibly NDS = − 76±8⋅4 + 0.96±0⋅017 × NDS (Adj⋅RMSE = 4⋅8)

Digestible CP = − 33±2⋅3 + 0.89±0⋅014 × NDS (Adj⋅RMSE = 1⋅9)

All are expressed as g/kg DM. Faecal output of the components can be calculated as difference between dietary concentrations and
respective digested concentration.
Models predicting potential CH4 production (PMP) are in Table 6. The average PMP (mean ± SD) was 1040 ± 22.4 g/d per cow,

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Table 5
Models predicting faecal output (g/d) of different components.
Unit Estimate SE P-value

Faecal output of pdNDF g/d

Intercept -289 261 0.27
pdNDF intake kg/d 227 35.6 < 0.001
Crude Protein g/kg DM -4.57 0.79 < 0.001
Starch g/kg DM 1.96 0.39 < 0.001
CProp × CProp Fraction of 1 691 225.7 0.002
DMI/BW g/kg 23 8.0 0.005
Faecal output of neutral detergent solubles g/d
Intercept -158 143 0.27
DM intake kg/d 90.2 7.15 < 0.001
Crude protein g/kg DM 1.55 0.498 0.002
Starch g/kg DM 0.54 0.185 0.004
Faecal output of CP g/d
Intercept -611 67.3 < 0.001
DM intake kg/d 61.1 2.91 < 0.001
CP g/kg DM 2.39 0.204 < 0.001
Indigestible NDF g/kg DM 0.43 0.237 0.07

SE = standard error, pdNDF = potential digestible neutral detergent fibre, DMI = dry matter intake, BW = body weight, CProp is concentrate
proportion.

Table 6
Potential methane (CH4) production from faeces.
Unit Estimate SE P-value

Max CH4/d, g
Intercept 210 75 0.006
Dry matter intake kg/d 59.2 3.3 < 0.001
Crude protein g/kg DM -2.69 0.268 < 0.001
Fat g/kg DM 4.01 1.095 < 0.001
CH4, g/kg DMI
Intercept 61.7 3.95 < 0.001
DMI kg/d 0.48 0.140 < 0.001
Crude protein g/kg DM -0.15 0.017 < 0.001
Fat g/kg DM 0.19 0.051 < 0.001
CH4, g/kg faecal OM
Intercept 286 5.5 < 0.001
iNDF g/kg DM -0.83 0.036 < 0.001
Crude protein g/kg DM -0.25 0.017 < 0.001
Fat g/kg DM 0.33 0.067 < 0.001
DMI/BW g/kg BW 0.40 0.127 0.002

SE = standard error, DMI = dry matter intake, OM = organic matter, iNDF = indigestible neutral detergent fibre, BW = body weight.

51.9 ± 7.3 g/kg DMI and 203 ± 17.0 g/kg faecal OM respectively. Adjusted RMSE were 53.9, 2.5 and 3.2 for the respective three
models.
Daily PMP was positively (P < 0.001) related to DMI, and it increased (P < 0.001) with dietary FA concentration and decreased
(P < 0.001) with that of CP. The effect of iNDF was not significant. Although the effects of CP and FA were highly significant, adjusted
RMSE decreased only from 57.7 to 53.9 when these variables were included in the model with DMI. When expressed per daily DMI,
PMP increased (P < 0.001) with DMI and dietary FA concentration and decreased (P < 0.001) with increased CP concentration. When
expressed per kg faecal OM PMYOM, dietary concentrations of iNDF and CP decreased (P < 0.001) and dietary FA concentration and
feeding level expressed as DMI per kg BW had positive (P < 0.001) effects on CH4 per kg faecal OM.

4. Discussion

The dataset represents typical Nordic dairy cow diets with rather wide ranges in intake and diet composition. The dataset did not
include low digestibility diets and only a few high fat diets. Digestibility was determined by total faecal collection for individual cows
in Data1 and using either AIA or iNDF as internal markers for individual cows in Data2. For the two individual cow datasets, residual
SD of faecal OM output per kg DM intake was similar (4.9% of observed mean) indicating that the marker method did not increase
random variation. In the treatment means dataset diet digestibility was determined using total faecal collection (n = 176) or using AIA
as an internal marker (n = 325). The relationship between OMD estimates based on total faecal collection and AIA in 5 studies
including 21 diets was good, as indicated by a high (0.82) R2 value (Nousiainen et al., 2009).
Potential CH4 production from organic matter is possible to estimate based on its contents of C, H and O (Buswell and Hatfield,
1952). Validation of their model with independent data showed a poor prediction performance and large bias (Herrmann and Rath,

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2012). This is not surprising since different materials used in biogas systems contain variable proportions of lignified cell walls that are
not degraded in anaerobic conditions. Keymer and Schilcher (1999) suggested that the difference between theoretical and actual CH4
yield can be explained by the digestibility of crops. Digestible carbohydrates, protein and fat can be calculated from chemical com­
ponents using digestibility coefficients derived from feed tables. Ohl and Hartung (2010); cited by Herrmann and Rath, 2012) reported
that this method underestimated PMY and concluded that this method was inappropriate for biogas systems. Weissbach et al. (2010)
suggested an approach assuming that PMY can be derived from fermentable OM that is a difference between OM and OM unavailable
for anaerobic digestion derived by sheep digestion trials, and the unavailable OM can be estimated from forage quality parameters.
Weissbach et al. (2010) suggested models based on forage crude fibre or acid detergent fibre (ADF) concentrations to predict
fermentable OM. However, these variables have not been good predictors of forage digestibility (Van Soest, 1994), especially of
different forage types (Huhtanen et al., 2006).
Underestimation of CH4 yield is most likely related to the fact that tabulated digestibility coefficients are based on apparent
nutrient digestibility, whereas the fermentation times are much longer in the biogas systems compared with the rumen. The longer
retention time in the biogas digesters allows a more complete digestion of pdNDF, whereas within shorter retention times in the rumen
pdNDF remains partly undigested. In sheep fed different forages at maintenance the digestibility of pdNDF was 85% (Huhtanen et al.,
2006) and 75% in dairy cows fed diets based mainly on grass silage and concentrates (Nousiainen et al., 2009). Apparent digestibility
coefficients presented in feed tables ignores MFOM output that is typically about 100 g/kg DMI (Weisbjerg et al., 2004; Nousiainen
et al., 2009). When MFOM was expressed as DM – NDF instead of OM – NDF the amount was about 140 g/kg DMI (Van Soest, 1994).
This fraction consists of unabsorbed endogenous secretions, sloughed epithelia, undigested microbial cells from rumen fermentation
and microbial cells produced in the hindgut (Van Soest, 1994). It can be calculated that faecal OM from a dairy cow diet containing
400 g NDF/kg DM (pdNDF 320) contains about 180 g potentially digestible OM (0.25 × 320 + 100) per kg DMI.
It is unlikely that biogas digesters can digest NDF that is not digested by rumen microbes in long-term (10 – 12 d) in vitro (Raf­
frenato et al., 2018) or in situ (Huhtanen et al., 1994) incubations. If the potential extent of digestion after long incubation time is not
different in the rumen and biogas digesters, it is reasonable to assume that PMY is related to the concentration of potentially digestible
OM (pdOM) in the substrate. Lignin is often used as a proxy of non-fermentable material. In the study of Triolo et al. (2011) lignin
concentration of the substrate (manure and energy crops) predicted biochemical PMY well (R2 = 0.88, RMSE 9.8% of observed mean).
Biodegradability defined as a ratio of observed CH4 production to potential maximal CH4 production at zero lignin concentration was
0.93. This is close to potential maximum of 1.00 considering that some fermentable substrates are incorporated to microbial cells.
Appuhamy et al. (2018) defined biodegradable OM as OM – Lignin. However, this does not consider that lignin inhibits digestion of
other cell wall components. Van Soest et al. (2005) suggested a factor of 2.4 × Lignin to describe iNDF of forages presuming this factor
to be universal among forage types. Later studies (Huhtanen et al., 2006; Raffrenato et al., 2018) showed that the ratio iNDF/lignin is
not constant among forage types. The ratio was not constant even within a forage type with higher ratios in forages with lower lignin
concentration (Raffrenato et al., 2018). Forage iNDF concentration predicted OM digestibility of different forage types in sheep more
precisely and uniformly than lignin concentration (Huhtanen et al., 2006). In this dataset dietary iNDF concentration predicted faecal
concentration of pdOM [(NDS + pdNDF) / DM] better than lignin (R2 = 0.80 vs. 0.37; RMSE 6.0% and 10.6% of observed mean).
Therefore, it could be expected that iNDF concentration can improve predictions of CH4 potential of different substrates compared
with lignin concentration. Faecal recoveries of iNDF were close to 100% in dairy cows (Ahvenjärvi et al., 2001) suggesting that dietary
iNDF concentration can be a useful tool in predicting faecal CH4 potential.

4.1. Manure composition

Faecal concentrations of pdNDF and NDS were negatively related to dietary iNDF concentration (Table 4; Fig. 1). This could be
expected since the other components are partly digested leading on accretion of indigestible component in faeces. Positive effect of
feeding level expressed as g DMI/kg BW on faecal pdNDF concentration is related to the well-known negative effect of increased
feeding level on diet digestibility (NRC, 2001; Yan et al., 2002; Huhtanen et al., 2009). Depression of NDF digestibility (NDFD) with
increased feeding level accounted for most of the depression in OMD with increased intake in cows fed grass silage-based diets
(Huhtanen et al., 2009). The decrease in digestibility with increased intake was closely associated with the decrease in ruminal digesta
retention time in sheep and cows fed different diets (Colucci et al., 1989, 1990). Negative effect of dietary CP concentration on faecal
pdNDF concentration is related to the positive effects of protein supplementation on diet digestibility (Oldham, 1984; Nousiainen
et al., 2009). The effects was mainly derived from improved NDFD (Nousiainen et al., 2009). The quadratic increase of faecal pdNDF
concentration with increased proportion of concentrates in the diet is related to well-known effects of rapidly fermentable carbohy­
drates on ruminal cell wall digestion.
Faecal concentrations of NDS and CP were mainly affected by dilution to dietary iNDF concentration. The positive effect of dietary
CP concentration on faecal CP and NDS concentrations indicates that a small proportion of CP is indigestible. Indigestible CP is often
defined as N bound to ADF (Sniffen et al., 1992; NRC, 2001).

4.2. Manure output

Total output of pdOM in manure depends both on DMI and diet composition. Faecal output of pdNDF was closely related to the
intake of this fraction with regression coefficient (227) indicating that 77% of pdNDF intake was digested by dairy cows. The extent of
pdNDF digestion depends both on intrinsic feed characteristics and extrinsic animal factors that affect digestion rate of pdNDF.
Intrinsic maximal digestion rate is reached in optimal rumen conditions when e.g. pH or nutrient supply do not limit digestion rate and

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P. Huhtanen et al. Animal Feed Science and Technology 282 (2021) 115120

is influenced by plant species and maturity (Mertens, 1993). Extrinsic factors (diet composition, feeding level and their interactions)
determine to what extent the potential of intrinsic feed characteristics is reached. The positive regression coefficient of concentrate
proportion (quadratic effect) is often related to reduced rumen pH caused by rapid fermentation of concentrates. The positive effect of
dietary starch concentration on faecal pdNDF output can be related to the negative influence of starchy concentrates on ruminal cell
wall digestion compared with fibrous concentrate supplements. In addition, lower pdNDF digestibility of whole crop silages can in­
crease faecal output of pdNDF compared with other forages. Brask et al. (2013a) reported a lower pdNDF digestibility of diets based on
maize silage compared with diets based on grass silage. The difference between the silages could be related to the slower intrinsic
digestion rate and smaller particle size (shorter rumen retention time) of maize silage, and the adverse effect of starch in maize silage
on the digestion rate of pdNDF. The negative effect of dietary CP concentration on faecal pdNDF output is related to stimulation of
ruminal cell wall digestion. Appuhamy et al. (2018) reported a negative effect of dietary CP concentration on faecal output of OM and
biodegradable OM defined as OM – Lignin. Shorter digesta retention time with increased intake explains the effect of feeding level on
faecal pdNDF output. Dietary fat concentration did not affect faecal pdNDF output, probably because the current dataset did not have
many diets with high fat content. According to a recent meta-analysis by Weld and Armentano (2017), only supplementation of fat as
medium-chain fatty acids decreased NDF digestibility in dairy cows. However, high sunflower oil supplementation (50 g/kg DMI)
decreased pdNDF digestibility in dairy cows with high (650 g/kg on DM basis) but not with low (350 g/kg) proportion of concentrate
in grass silage-based diets (Bayat et al., 2017). Similarly, linseed oil supplementation decreased NDF digestibility in cows fed maize
silage-based diets, but not in those fed red clover-based diets (Benchaar et al., 2015).
Faecal NDS output was closely related to DMI with minor positive effects of dietary CP and starch concentrations. Positive effect of
CP is related to indigestible CP. Quantitatively the effect of CP was small, at DMI of 20 kg/d a difference of 10 g/kg DM in dietary CP
concentration makes less than 1% difference in faecal NDS output. In the current dataset the faecal NDS increased only marginally with
increased dietary starch concentration reflecting high digestibility of barley and oats starch. However, the effect of starch source
should be considered since it can influence on faecal NDS output as the digestibility of starch is lower for maize compared with barley,
oats and wheat (Mills et al., 1999; Moharrery et al., 2014).

4.3. Potential CH4 production

The average potential CH4 production was 203 g/kg manure OM (284 L) that is higher than IPCC Intergovernmental Panel on
Climate Change (2006) default value (240). The higher value in the present data is due to the high potential digestibility of OM di­
gestibility (low iNDF concentration) of the diet. This resulted in a high potential digestibility of faecal OM (710 g/kg). Faecal con­
centrations of digestible components (pdNDF and NDS) decreased with increased dietary iNDF concentration, i.e. reduced digestibility
(Fig. 1). Møller et al. (2014) reported a similar average (284 L/kg OM) maximum CH4 potential for 10 faecal samples from two studies
in dairy cows. Total tract OMD was similar (729 vs. 725) in Møller et al. (2014) and in our study suggesting that predicting faecal CH4
potential of faecal OM can be predicted with reasonable accuracy from dietary components and animal factors that are associated with
diet digestibility. In the study of Møller et al. (2014) maximum faecal CH4 was closely related to dietary fat concentration. This is
consistent with the higher maximal CH4 from fat than from carbohydrates in biogas systems and incomplete digestion of fat in cows
(Brask et al., 2013a, 2013b). Møller et al. (2014) reported a negative relationship between enteric CH4 emission and CH4 production
from faeces. Similarly, in the study of Hassanat and Benchaar (2019) linseed oil supplement increased CH4 production from faeces,
whereas enteric CH4 production was reduced by linseed oil supplementation (Benchaar et al., 2015). True digestibility of crude fat
estimated by the Lucas test was only 710 g/kg (R2 = 0.96) in the data used by Møller et al. (2014) and Hassanat and Benchaar (2019)
despite a moderate increase in dietary crude fat concentration (34 vs. 61 g/kg DM). The effects of fat supplementation on observed
faecal or manure PMP were greater in the studies of Møller et al. (2014) and Hassanat and Benchaar (2019) than in our model pre­
dictions. This difference partly can be attributed to the higher predicted values of true fat digestibility in our study and partly to
increased faecal output of NDF with some fat supplements. Improving accuracy of estimation of CH4 emissions from manure requires
the development of equations predicting faecal fat output due to the high CH4 potential of fat.
Feeding maize silage reduced enteric CH4 production but increased CH4 production from faeces compared with red clover-based
diets (Benchaar et al., 2015; Hassanat and Benchaar, 2019). The loss of NDF in the biogas system was almost 2-fold from the manure of
cows fed maize silage-based diets compared with those fed red clover-based diets (Hassanat and Benchaar, 2019). This could be related
to lower rumen pH in cows fed maize silage-based diets (Hassanat and Benchaar, 2019) and the slower intrinsic digestion rate of
pdNDF in maize silage compared with red clover silage according to the Cornell Net Carbohydrate and Protein System feed table
(CNCPS; Tylutki et al., 2008).
Although predicted faecal PMP per unit faecal OM is strongly related to the concentration of pdOM, the effect of dietary iNDF
concentration on potential CH4 per kg DMI or per cow/day was not significant. This is because there is a negative relationship between
faecal OM output and diet digestibility. In the current dataset faecal OM output was negatively (P < 0.001) related to OMD and
positively to (P < 0.001) to dietary iNDF concentration. Therefore, faecal PMP was mainly related to DMI with a negative effect of CP
related to improved digestibility and a positive effect of dietary fat concentration. The same factors explained variation in PMP when
expressed per kg DMI. Increased feeding level decreased enteric CH4 emissions about 10% per multiple of maintenance (Yan et al.,
2000; Ramin and Huhtanen, 2013), but reduced digestibility (increased faecal output of digestible OM) can partly counterbalance this.

4.4. Between-cow variation

Between-cow SD in faecal OM output was 8.5 g/kg DMI that is 1.3% of digestible OM concentration of the diet. This value agrees

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P. Huhtanen et al. Animal Feed Science and Technology 282 (2021) 115120

with between-cow CV of 1.3% in gross energy digestibility in respiration chamber studies (Guinguina et al., 2020). Earlier, Flatt et al.
(1969) reported a CV of 1.9% for the ration of digestible energy to gross energy. Mean retention time (MRT) of feed particles, reciprocal
of passage rate, is probably the main contributor to between-cow variation in digestibility. Ørskov et al. (1988) reported a strong
negative (− 0.80) correlation coefficient between passage rate of Cr-mordanted straw and diet digestibility in 22 cows. Digesta passage
rate from the rumen has been related to between-animal differences in CH4 emission. In studies with sheep, CH4 emissions decreased
with increased fractional outflow rate from the rumen (Pinares-Patiño et al., 2003), and particulate MRT was longer for high emitters
compared with low emitters (Pinares-Patiño and Clark, 2010; Goopy et al., 2014). In several studies (see Løvendahl et al., 2018) low
CH4 emissions were associated with reduced diet digestibility. Therefore, it is possible that low emitters produce more CH4 from
manure. However, it is unlikely that increased CH4 emissions from manure reduced fully compensate for reduced enteric emissions.

5. Conclusions

Faecal PMP can be predicted from diet and animal characteristics. Potential CH4 per kg OM is related to pdOM, i.e., the sum of NDS
and pdNDF in faeces. Knowledge of true fat digestibility will potentially improve model predictions of CH4 per kg VS. Strategies
mitigating enteric emissions are associated with increased manure emissions. However, increased manure CH4 can be utilized in biogas
systems. Although PMP per unit of OM is positively related to diet digestibility, PMP per cow/day is mainly related to feed intake
because higher PMP/kg OM and reduced OM output with improved digestibility will counterbalance each other. It would be useful to
directly compare digestion potential of different substrate in the farm scale biogas systems and in the rumen.

CRediT authorship contribution statement

Pekka Huhtanen: Conceptualization, Data curation, Modelling, Writing – original draft. Mohammad Ramin: Conceptualization,
Writing – review & editing. Sophie Krizsan: Conceptualization, Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to
influence the work reported in this paper.

Acknowledgments

The study was part of the CEDERS project within the FACCE ERAGAS funding scheme as part of the EU Horizon 2020 Research and
Innovation Programme under grant agreement No. 696356. Support by the Swedish Research Council for Environment, Agricultural
Sciences and Spatial Planning (FORMAS; project no. 2017-01750) is also acknowledged. Contributions of colleagues in building the
datasets are also greatly acknowledged.

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