Excreta emissions in progeny of low and high enteric methane yield selection line sheep fed pasture of different qualities

Animal Feed Science and Technology 257 (2019) 114289

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Excreta emissions in progeny of low and high enteric methane yield selection line sheep fed pasture of different qualities

T

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A. Jonkera, , S. MacLeana, C. Woyimo Wojua,d,e, M. Garcia Rendon Calzadaa,f, W. Yua,g,h, G. Molanoa, S. Hickeyb, C.S. Pinares-Patiñoa,i, J.C. McEwanc, P.H. Janssena, E. Sandovala, S. Lewisa, S. Rowec a

Grasslands Research Centre, AgResearch Ltd., Private Bag 11008, Palmerston North, New Zealand Ruakura Research Centre, AgResearch Ltd., Private Bag 3115, Hamilton, New Zealand c Invermay Agricultural Centre, AgResearch Ltd., Private Bag 50034, Mosgiel, New Zealand d Ethiopian Biotechnology Institute, Addis Ababa, Ethiopia e Mizan-Tepi University, College of Agriculture and Natural Resources, Department of Animal Science, Mizan, Ethiopia f Universidad Nacional Autónoma de México, Facultad de Medicina Veterinaria y Zootecnia, Mexico City, Mexico g Animal Nutrition Group, Wageningen University and Research, P.O. Box 338, 6700 AN, Wageningen, The Netherlands h Department of Animal Science, Animal Nutrition and Physiology, Aarhus University, 8830 Tjele, Denmark i International coordinator ‘The New Zealand Peru Dairy Support Project’, MINAGRI, Jr. Yauyos 258, Lima, Peru b

A R T IC LE I N F O

ABS TRA CT

Keywords: Greenhouse gas Nitrous oxide Urine Animal variation Breeding value Repeatability

Selection of sheep with low enteric methane (CH4) emissions is a greenhouse gas (GHG) mitigation option suitable for pastoral systems. However, the effect of breeding sheep with low enteric CH4 emissions on excreta output and associated CH4 and nitrous oxide (N2O) emissions and therefore total GHG emissions are not known. The objective of the current experiments were to determine excreta output, and estimate associated GHG emissions, from progeny of low and high enteric CH4 per unit of dry matter intake (DMI) selection line sheep (CH4/DMI). The animals were fed two qualities of cut perennial ryegrass-based pasture (very mature vs. vegetative, 12 animals per CH4/DMI line) in Exp. 1 and cut pasture in two repeated seasons (autumn and winter; 15 animals per CH4/DMI line × 2 seasons) in Exp. 2. Total faecal and urine output was determined on individual animals, followed by enteric CH4 emission measurements in respiration chambers. GHG emissions from urine (N2O) and faeces (CH4 and N2O) were estimated based on New Zealand Agricultural GHG Inventory methodology. There was no interaction between CH4/ DMI selection line and diet quality in Exp. 1 or seasons in Exp.2. Total daily faecal output of DM, organic matter (OM) and neutral detergent fibre (NDF; all g/d) and associated calculated faecal CH4 emissions were greater for low compared to high CH4/DMI sheep in Exp. 1 (P < 0.05), while being similar between CH4/DMI selection lines in Exp. 2. Nitrogen (N) excretion and N partitioning into urine, faeces and body retention, and calculated excreta N emissions, were mostly similar between CH4/DMI selection line sheep in both experiments. Except, faecal N output (g/d and per unit of N intake) and associated calculated direct faecal N2O-N emissions (g/ d) were greater in low compared to high CH4/DMI sheep in Exp. 1 (P < 0.05). Enteric CH4 emissions were numerically 8% less (P = 0.15) in Exp.1 and 10% less (P = 0.004) in Exp. 2 and total animal level GHG emissions (CH4 and N2O) were numerically 7% less (P = 0.21) in Exp. 1

Abbreviations: ADF, acid detergent fibre; aNDF, neutral detergent fibre; LW, live-weight; CH4, methane; CO2, carbon dioxide; CPT, central progeny test; DM, dry matter; DMI, dry matter intake; GHG, greenhouse gas; MfE, ministry for the environment; N, nitrogen; N2O, nitrous oxide; NH3, ammonia; NO3, nitrate; NZ, New Zealand; OM, organic matter ⁎ Corresponding author at: Tennent Drive, 11 Dairy Farm Road, Palmerston North 4442, New Zealand. E-mail address: [email protected] (A. Jonker). https://doi.org/10.1016/j.anifeedsci.2019.114289 Received 1 May 2019; Received in revised form 15 August 2019; Accepted 5 September 2019 0377-8401/ © 2019 Elsevier B.V. All rights reserved.

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and 8% less (P = 0.006) in Exp.2 for progeny of the low compared to the high CH4/DMI line sheep. In conclusion, the magnitude of difference in enteric CH4 (expressed as CO2-equivalent) between low and high CH4/DMI selection line sheep were still present when CH4 from faeces and N2O emissions from urine and faeces were also accounted for. The animal genetic traits were expressed independent of environmental factors, i.e. pasture quality and season.

1. Introduction Enteric methane emissions from ruminants account for two-thirds of total agricultural greenhouse gas (GHG) emissions in New Zealand (MfE, 2018) and globally ruminants are a major source of anthropogenic CH4 emissions (Hristov et al., 2018). Selection of sheep with a low yield of enteric CH4, expressed per unit of dry matter intake (CH4/DMI), has been identified as a feasible mitigation option (Pinares-Patiño et al., 2013; Jonker et al., 2018c) and this option is suitable for implementation in pastoral livestock systems (Jonker et al., 2017, 2018c). Breeding for sheep with low enteric CH4/DMI could be implemented as a mitigation strategy in the short term in New Zealand. However, the effect of this selection on excreta output and associated GHG emissions is not known. In grazing systems, nitrous oxide (N2O), the second largest source of agricultural GHGs, is mainly formed from urinary N excreted onto soil during grazing (MfE, 2018). Furthermore, CH4 and N2O are emitted from dung excreted onto soil. Sheep selected with low CH4/DMI were found to have a shorter retention time of feed in the rumen (Pinares-Patiño et al., 2003; Goopy et al., 2014; Jonker et al., 2018b) which might be associated with improved microbial N capture in the rumen (Dijkstra et al., 2002; Oddy et al., 2018). Sheep differing in CH4/DMI also had differences in rumen microbial community compositions (Kittelmann et al., 2014), different fermentation pathways and end-product ratios (Jonker et al., 2019) and differences in rumen content stratification (Bain et al., 2014; Goopy et al., 2014), and so could result in differing ruminal N dynamics. On the other hand, some previous studies found that sheep selected with low CH4/DMI had increased faecal DM and organic matter (OM) excretion (Goopy et al., 2014; Jonker et al., 2018b), which would lead to increased excreta CH4 emissions. Therefore, the effect of breeding animals for low enteric CH4 emissions on additional sources of GHGs (excreta N2O and CH4) should be determined before recommending to implement breeding sheep for low enteric CH4 emissions to the farming industry. The objectives of the current experiments were to determine excreta output, and calculate associated excreta CH4 and N2O emissions, from progeny of low and high CH4/DMI selection line sheep. The sheep were fed cut perennial ryegrass-based pasture of two qualities in Exp. 1 and cut pasture representative of two seasons in Exp. 2. The hypothesis was that excreta output, and therefore associated excrete GHG emissions, would be similar between progeny of low and high CH4/DMI selection line sheep, independent of pasture quality or season, and therefore total animal level GHG emissions would be less for low CH4/DMI sheep. 2. Materials and methods The two animal experiments conducted adhered to the guidelines of the 1999 New Zealand Animal Welfare Act and AgResearch Code of Ethical Conduct and were approved by the AgResearch Grasslands Animal Ethics Committee (Palmerston North, NZ). 2.1. Methane per unit of dry mater intake selection line animals The ‘CH4/DMI selection’ flock animals were generated by measuring progeny of maternal dual-purpose sires generated by the New Zealand industry CPT program (McLean et al., 2006) for CH4/DMI (Pinares-Patiño et al., 2013), with the 10% most extreme low and high animals (sires and dams) retained for further breeding. The lines were closed in 2012 and all sires and dams used from 2012 onwards were born in the high and low CH4/DMI selection flocks (Jonker et al., 2018c). The animals used in the current experiments were born at AgResearch Woodlands Research Farm (Woodlands near Invercargill, NZ) and transported to AgResearch Aorangi Research Farm (Rongotea near Palmerston North, NZ), before the start of the experiments, where the animals were managed on perennial ryegrass-based pasture. Both experiments were performed at AgResearch Grasslands Research Centre (Palmerston North, NZ). 2.2. Experiment 1 Exp.1 was performed with 12 low and 12 high CH4/DMI selection line flock male progeny (intact rams) (mean ± standard deviation; 40.0 ± 3.0 kg live weight; LW) born in spring (September) 2013. Data of rumen characteristics and CH4 emissions from Exp. 1 have been published elsewhere (Jonker et al., 2018b). The experiment was conducted from 21 March to 23 May 2014 with animals initially housed in group pens for 10 days and then moved into individual crates four days before a further six days of total excreta collection and then another two days of CH4 measurements in respiration chambers (details of measurements are described below). Animals, balanced for CH4/DMI selection line, were randomly allocated to be fed good or poor quality pasture offered at approximately 2% of body weight. The good quality pasture was managed for another trial (Cosgrove et al., 2015; Jonker et al., 2018d) and was harvested from an irrigated plot with three perennial ryegrass cultivars sown as mono-cultures in strips with plants at a vegetative growth stage. Poor quality pasture was harvested from an unimproved pasture consisting mainly of weed grasses at a very mature stage of growth with mainly dead material. Both pastures were cut daily around 1100 h using a drum mower (PZ220; 2

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Khun Farm Machinery UK, Telford, UK) at approximately 7 cm above ground level. Cut pasture was subdivided into two equal portions for each treatment and stored in a chiller (at 4 °C) until feeding at approximately 1530 h and 0830 h the next morning. Representative samples were taken from each daily pasture cut and triplicate subsamples (˜200 g) were dried at 105 °C for 24 h to determine the dry matter (DM) content. During the measurement periods, another subsample was dried at 65 °C for 48 h and ground to pass through a 1 mm screen for wet-chemical analysis (see below). 2.3. Experiment 2 Exp. 2 was performed with 15 low and 15 high CH4/DMI selection line flock male progeny (intact rams) born in spring 2016 with measurements repeated in two seasons. The two measurement periods were conducted from 6 March to 6 April (autumn; LW = 40.8 ± 4.7 kg) and from 6 June to 6 July (winter; LW = 48.2 ± 4.9 kg), 2017, respectively. The 30 animals were randomly selected from a cohort of 72 animals, balanced for CH4/DMI selection line and represented by progeny of all sires within CH4/DMI line. Animals were initially housed in group pens for at least 10 days and then moved into individual crates at two days before gas emission measurement in respiration chambers. Then the animals were moved back into group pens before being moved into metabolism crates three days before another seven days of total excreta collection (details of measurements are described below). Pasture was harvested daily around 1100 h, as described above, from a paddock at Aorangi Research Farm. Pasture in autumn consisted of approximately 28% grass (mainly perennial ryegrass), 45% clover (white and red clover), 21% dead matter and 6% weeds (e.g. thistles, dock, butter cup etc.). Pasture in winter consisted of 58% grass, 9% clover, 21% dead matter and 12% weeds. Feeding, storage and sampling of cut pasture was the same as described for Exp. 1. During the measurement periods, a subsample of cut pasture was stored in the freezer at −20 °C until after the experiment was completed. Then these samples were freeze-dried and ground to pass through a 1 mm screen for wet-chemical analysis (see below). Animals were returned to graze pasture at Aorangi farm between the two measurement seasons. During this period between the autumn and winter measurements, animals suffered from parasites resistant to the antiparasitic drench (Arrest Hi Mineral, Merial New Zealand Ltd., Auckland, New Zealand) that was used on-farm. The resistance was identified after some animal died and others got sick. With advice of a veterinarian, all animals were injected with Amphoprim, a broad-spectrum Sulphonamide antibiotic (Virbac NZ, Hamilton, NZ) and drenched with a new antiparasitic drench (Matrix Hi Mineral, Merial New Zealand Ltd.). Two low and one high CH4/DMI selection line sheep died or were euthanized during this period. The final analysis is therefore based on 27 animals (13 low and 14 high CH4/DMI line sheep). 2.4. Excreta collection For excreta collection, sheep were moved into metabolism crates at four and three days before excreta collection started in Exp. 1 and 2, respectively. Harnesses were fitted to each sheep during this time for attachment of faecal collection bags. The metabolism crates had a mesh floor with a funnel tray underneath for collection of urine into a plastic bucket with 100 mL of 6 M sulfuric acid added each day to minimize urinary NH3-N volatilization. Feed refusals, faeces and urine were collected for six consecutive days in Exp. 1 and seven days in both seasons of Exp. 2. The collection of these samples were performed before afternoon feeding of pasture from the daily pasture cut. Refusals were dried daily at 65 °C for 48 h to determine DM and the daily residual pooled over the 6–7 days. Faeces and urine were weighed daily and aliquots (10% for faeces and 1% for urine) per sheep pooled and stored in the freezer at −20 °C and the rest discarded. Faeces were freeze-dried at the end of each N balance measurement periods and ground through a 1 mm screen for wet-chemical analysis (see below). 2.5. Enteric methane measurements Methane emissions in both experiments were determined individually on two consecutive days in 24 open-circuit respiration chambers. The design of this facility is described in detail by Pinares-Patiño et al. (2012) and animal management and operation by Jonker et al. (2018d). For both seasons in Exp. 2 the 72 sheep were randomly allocated to three cohorts of 24 sheep, with the 30 sheep to be used for excreta collection allocated to the first two cohorts. The cohorts were moved consecutively for 48 h periods into respiration chambers. Doors of all chambers were opened twice daily in both experiments for approximately 15 min (0800 h and Table 1 Chemical composition of poor and good quality pasture fed in Exp. 1 and pasture fed in autumn and winter of Exp. 2. Pasture

Poor Good Autumn Winter

DM g/kg Experiment 1 380 173 Experiment 2 130 124

ash g/kg DM

CP

fat

NDF

ADF

lignin

115 140

103 219

26 39

631 515

315 256

34 21

108 116

212 219

27 31

443 442

283 267

50 39

DM, dry matter; CP, crude protein; aNDF, neutral detergent fibre; ADF, acid detergent fibre. 3

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1530 h) for feed-refusal collection, excreta removal and feeding. Emissions for these periods were extrapolated by taking the average of the last 12 values before opening the door. 2.6. Laboratory analysis The ground samples of feed were analysed by the Nutrition Laboratory of Massey University (Palmerston North, NZ) according to procedures of the Association of Official Analytical Chemists (AOAC 1990) for dry matter, ash (AOAC #942.05), crude fat (AOAC #991.36), nitrogen (AOAC #968.06), neutral detergent fibre (aNDF) assayed with heat stable α-amylase, acid detergent fibre (ADF) (both expressed inclusive of residual ash) and lignin after extraction of ADF residue in sulphuric acid (Robertson and Van Soest, 1981) (Table 1). Faeces and refusals were analysed for DM, ash, N and aNDF as described above and urine was analysed for N as described above. 2.7. Calculations Body retained-N was calculated as: N intake – faecal-N output – urinary-N output. Excreta GHG emissions were estimated according to equations used for the national GHG inventory of New Zealand (MfE, 2018). This involves multiplying excreta output (faecal DM, urine N and faecal N [g/d]) with emission factors relevant to pastoral grazing systems. Excreta CH4 emissions were calculated as faecal DM output (kg/d) × 0.69 g/kg faecal DM. Direct N2O-N emissions (g/d) were calculated from urine N (kg/d) and faecal N (kg/d) multiplied by the respective emission factor of 10.0 and 2.5 g N2O-N/kg N output. Indirect N2O-N emissions from NH3-N emissions and NO3-N leached were calculated as excreta (faecal + urine) N (kg/d) × 100 g NH3-N/kg excreta-N × N2O-N from NH3 (0.01 g N2O-N/g NH3-N) and as excreta-N (kg/d) × 70.0 g NO3-N/kg excreta-N × N2O-N from NO3 (0.0075 g N2O-N/g NO3-N), respectively. To express emissions in CO2-equivalent, the 100 year global warming potential values of 25 kg and 298 kg CO2equivalent/kg CH4 and N2O, respectively, were used. 2.8. Statistical analysis The two experiments were analysed separately. Data from Exp. 1 were analysed using a standard least square model (SAS, 2012) with CH4/DMI selection line (CH4 low, CH4 high), feed quality (good, poor) and their interaction fitted as fixed effects. Data from Exp. 2 were analysed in a mixed model using ASREML (Gilmour et al., 2009) with CH4/DMI selection line (CH4 low, CH4 high), measurement season (autumn and winter) and their interaction fitted as fixed effects and animal as random effect to Table 2 Mean intake, faecal output and apparent total tract digestibility of dry matter (DM), organic matter (OM) and neutral detergent fibre (NDF) and estimated manure CH4 emissions (M_CH4; g/d) in progeny of low and high CH4 yield, per unit of dry matter intake (CH4/DMI), selection line sheep (CH4 low and CH4 high) fed poor or good quality pasture in Exp. 1 and in two seasons in Exp. 2. Intake (kg/d) DM CH4 selection line CH4 high CH4 low SED P-value Pasture quality Poor Good SED P-value CH4 selection line CH4 high CH4 low SED P-value Season Autumn Winter SED P-value Repeatability1 SE

Faecal output (g/d) OM

Experiment 1 0.83 0.72 0.77 0.68 0.024 0.020 0.117 0.116

Total tract digestibility (g/kg)

NDF

DM

OM

NDF

M_CH4

DM

OM

NDF

0.47 0.44 0.014 0.117

278 303 7.6 0.037

221 242 6.6 0.047

151 167 4.6 0.034

0.19 0.21 0.005 0.037

642 615 10.5 0.096

674 648 10.4 0.112

665 633 10.6 0.059

0.72 0.63 0.89 0.77 0.024 0.020 < 0.001 0.001 Experiment 2 1.09 0.97 1.15 1.02 0.024 0.021 0.026 0.026

0.45 0.45 0.014 0.934

380 200 9.8 < 0.001

316 148 8.5 < 0.001

229 89 5.9 < 0.001

0.26 0.14 0.007 < 0.001

519 739 13.5 < 0.001

547 775 13.4 < 0.001

538 760 15.6 < 0.001

0.48 0.51 0.011 0.026

389 401 9.5 0.244

316 324 8.1 0.400

164 166 6.3 0791

0.27 0.28 0.007 0.244

652 642 8.2 0.253

681 675 7.8 0.437

670 666 12.3 0.830

1.12 1.11 0.017 0.545 0.309 0.181

0.50 0.50 0.008 0.459 0.310 0.181

409 381 6.8 < 0.001 0.273 0.187

343 298 4.8 < 0.001 0.267 0.189

178 151 3.7 < 0.001 0.340 0.179

0.28 0.26 0.005 < 0.001 0.273 0.187

636 659 5.8 < 0.001 0.291 0.185

658 699 5.5 < 0.001 0.294 0.185

642 694 8.1 < 0.001 0.362 0.176

1.00 0.98 0.015 0.239 0.310 0.181

SED, standard error of the difference; SE, standard error. Interactions between CH4 selection line and pasture quality in Exp. 1 and CH4 selection line and period in Exp. 2 were not significant and are therefore not shown. 1 The repeatability between the repeat measures on the same animal in the two seasons was estimated from the residual variance (Vr) and animal variance (Va) as: repeatability = Va / (Va + Vr). 4

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enable estimation of repeatability between the two seasons. The repeatability between the repeat measures on the same animal in the two seasons in Exp. 2 was estimated from the residual variance (Vr) and animal variance (Va), generated using the ASRML model, as: repeatability = Va / (Va + Vr) as in Oddy et al. (2018). It is well known that differences in DMI will affect excreta output and enteric CH4 emissions. Therefore, measured DMI was fitted as a covariate in the statistical model for both experiments, except in the analysis of intake variables. Significance was declared at P < 0.05, trends at P < 0.10 and repeatability was classified as low (< 0.35), moderate (0.35 – 0.75) and high (> 0.75). Interactions between CH4/DM selection line and pasture quality in Exp. 1 and CH4/DMI selection line and period in Exp. 2 were not significant and are therefore not shown. 3. Results 3.1. Intake, faecal output and total tract digestibility Intakes of DM, OM and NDF were greater for the low CH4/DMI selection line sheep than the high CH4/DMI selection line sheep in Exp. 2 (P < 0.05), while intakes were similar between seasons in Exp. 2 and between the two CH4/DMI selection lines in Exp. 1 (Table 2). Intakes of DM and OM were greater for sheep fed good quality pasture than those fed poor quality pasture in Exp. 1 (P < 0.05), with intake of NDF being similar between sheep fed good and poor quality pasture. Total daily faecal output of DM, OM and NDF (g/d) and calculated excreta CH4 emissions were greater for low compared to high CH4/DMI sheep in Exp. 1 (P < 0.05), for good vs. poor quality pasture in Exp. 1 (P < 0.001) and for autumn compared to winter in Exp. 2 (P < 0.001), and were similar between the two CH4/DMI selection lines in Exp. 2. Total tract apparent digestibility of DM, OM and NDF were similar between the CH4/DMI selection line sheep in both experiments, but digestibility of DM and NDF tended (P < 0.10) to be less for low than for high CH4/DMI sheep in Exp. 1. Not surprisingly, good quality pasture had greater total tract digestibility of all components (DM, OM, NDF) than poor quality pasture in Exp. 1 (P < 0.01). In Exp. 2, pasture in winter had greater apparent total tract digestibility of all components (DM, OM, NDF) than pasture in autumn (P < 0.01). 3.2. Nitrogen balance and partitioning Nitrogen excretion and partitioning in urine, faeces and retained in the body, as well as associated calculated excreta N emissions, were mostly similar between sheep of the two CH4/DMI selection lines in both experiments. However, faecal N output (g/d and per unit of N intake) and associated calculated direct faecal N2O-N emissions (g/d) were greater in low compared to high CH4/DMI sheep Table 3 Nitrogen intake (NI; g/d), balance, partitioning and emissions in progeny of low and high CH4 yield, per unit of dry matter intake (CH4/DMI), selection line sheep (CH4 low and CH4 high) fed poor or good quality pasture in Exp. 1 and in two seasons in Exp. 2. N balance (g/d) NI CH4 selection line CH4 high CH4 low SED P-value Pasture quality Poor Good SED P-value CH4 selection line CH4 high CH4 low SED P-value Season Autumn Winter SED P-value Repeatability1 SE

UN

Experiment 1 21.9 14.0 20.6 14.2 0.58 0.45 0.144 0.723

N partitioning (g/kg NI)

N emissions (g/d)

FN

RN

UN/NI

FN/NI

RN/NI

UN/FN

N2O-N

NH3-N

NO3-N

6.7 7.7 0.24 0.013

0.5 −0.7 0.49 0.113

667 680 27.9 0.757

401 449 15.0 0.046

−68 −129 37.1 0.281

2.11 1.91 0.111 0.218

0.19 0.20 0.005 0.391

2.07 2.19 0.050 0.109

1.45 1.54 0.035 0.109

11.3 7.8 31.2 20.4 0.76 0.58 < 0.001 < 0.001 Experiment 2 38.0 23.3 39.1 23.6 0.83 0.87 0.028 0.622

8.2 6.3 0.309 0.0027

−2.7 2.5 0.63 0.0002

612 735 35.9 0.062

629 221 19.3 < 0.001

−241 44 47.7 0.003

0.86 3.15 0.136 < 0.001

0.12 0.26 0.007 < 0.001

1.59 2.67 0.063 < 0.001

1.11 1.87 0.045 < 0.001

11.6 11.9 0.29 0.463

3.6 3.0 0.96 0.488

603 617 23.1 0.521

302 308 7.5 0.485

95 76 25.3 0.430

2.00 2.01 0.086 0.828

0.32 0.32 0.010 0.573

3.49 3.55 0.095 0.502

2.44 2.49 0.067 0.500

37.6 39.5 0.60 0.085 0.307 0.181

11.6 11.9 0.20 0.087 0.325 0.184

4.4 2.2 0.68 0.003 0.287 0.186

579 641 15.9 0.001 0.312 0.184

305 304 5.0 0.818 0.339 0.182

116 55 18.1 0.002 0.276 0.187

1.90 2.12 0.054 < 0.001 0.402 0.173

0.30 0.34 0.007 < 0.001 0.2958 0.185

3.34 3.70 0.068 < 0.001 0.2803 0.1863

2.34 2.59 0.048 < 0.001 0.2803 0.1863

21.8 25.1 0.60 < 0.001 0.307 0.184

SED, standard error of the difference; SE, standard error. Interactions between CH4 selection line and pasture quality in Exp. 1 and CH4 selection line and period in Exp. 2 were not significant and are therefore not shown. NI, nitrogen intake; FN, faecal nitrogen; UN, urinary nitrogen; RN; calculated retained nitrogen (NI – FN – UN). 1 The repeatability between the repeat measures on the same animal in the two seasons was estimated from the residual variance (Vr) and animal variance (Va) as: repeatability = Va / (Va + Vr). 5

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in Exp. 1 (P < 0.05) (Table 3). Calculated NH3-N, NO3-N and total N2O-N emissions were similar between the two CH4/DMI selection lines in both experiments. Expressed as g/d, faecal N excretion was greater and urinary N excretion and retained N were less for sheep fed poor quality pasture compared to those fed good quality pasture in Exp. 1 (P < 0.001), while urine N excretion was less and body retained N was greater in autumn than in the winter of Exp. 2 (P < 0.01). As a proportion of N intake, faecal N was greater and body retained N was less (P < 0.01), and urine N tended to be less (P = 0.06), for sheep fed poor than those fed good quality pasture in Exp. 1 and urine N was less and body retained N greater in autumn than in winter in Exp. 2. 3.3. Total animal level CO2 equivalent gas emissions Enteric CH4 emissions were numerically 8% less (P = 0.15) in Exp.1 and 10% less (P = 0.004) in Exp. 2 for progeny of the low compared to the high CH4/DMI line sheep (Table 4). Excreta emissions were similar between the two CH4/DMI selection lines in both experiments, except that faecal CH4 and N2O were greater (P < 0.04) in progeny of the low compared to progeny of the high CH4/ DMI line sheep in Exp. 1. As a result, total animal level GHG emissions were numerically 7% less (P = 0.21) in Exp. 1 and 8% less (P = 0.006) in Exp. 2 for progeny of the low compared to the high CH4/DMI line sheep. Enteric CH4 emissions tended to be greater (P = 0.07) and excreta and total GHG emissions were greater (P < 0.003) for sheep eating good quality pasture compared to those eating poor quality pasture in Exp. 1. In Exp. 2, all sources of GHG emissions were less in autumn than in winter (P < 0.002; P = 0.096 for faecal N2O), except excreta CH4 emissions, which were greater in autumn (P < 0.001). 4. Discussion The main finding of the current study was that total excreta GHG emissions, estimated from measured faecal and urinary excretion, were similar for progeny of low and high CH4/DMI selection line sheep. Furthermore, there was no interaction between CH4/ DMI selection line and diet quality (Exp. 1) or seasons (Exp.2), suggesting that the GHG genetic traits are expressed independent of environmental factors like diet and season. 4.1. Nitrogen excretion and emissions Importantly, urinary N excretion (g/d and % of N intake), the main source for N emissions (N2O, NH3 and NO3) in pastoral Table 4 Enteric methane and estimated excreta greenhouse gas (GHG) emissions (all expressed as g/d CO2 equivalent) from progeny of low and high CH4 yield, per unit of dry matter intake (CH4/DMI), selection line sheep (CH4 low and CH4 high) fed poor or good quality pasture in Exp. 1 and in two seasons in Exp. 2. Enteric CH4 CH4 selection line CH4 high CH4 low SED P-value Pasture quality Poor Good SED P-value CH4 selection line CH4 high CH4 low SED P-value Season Autumn Winter SED P-value Repeatability1 SE

Excreta CH4

Urine N2O

Faeces N2O

N2O from NH3

N2O from NO3

Total excreta N2O

Total excreta GHG

Total GHG

65.4 66.6 2.13 0.723

7.9 9.0 0.28 0.013

9.7 10.3 0.23 0.109

5.1 5.4 0.12 0.109

88.1 91.3 2.45 0.3907

92.9 96.5 2.49 0.337

499.8 465.3 23.63 0.165

Experiment 407.0 368.8 23.07 0.119

1 4.8 5.2 0.13 0.037

353.1 422.7 34.34 0.061 Experiment 605.5 528.0 23.44 0.002

6.6 3.5 0.17 < 0.001 2 6.7 6.9 0.16 0.244

36.3 95.7 2.74 < 0.001

9.6 7.4 0.36 0.003

7.5 12.5 0.30 < 0.001

3.9 6.6 0.16 < 0.001

57.2 122.1 3.16 < 0.001

63.8 125.6 3.20 < 0.001

416.9 548.3 35.18 0.002

109.0 110.7 4.06 0.622

13.6 13.9 0.34 0.463

16.3 16.6 0.45 0.502

8.6 8.7 0.23 0.502

147.5 149.9 4.78 0.573

154.2 156.8 4.81 0.549

759.7 685.0 24.25 0.004

7.1 6.6 0.12 < 0.001 0.273 0.187

102.2 117.5 2.81 < 0.001 0.307 0.184

13.6 14.0 0.23 0.096 0.325 0.184

15.6 17.3 0.32 < 0.001 0.280 0.186

8.2 9.1 0.17 < 0.001 0.280 0.186

139.6 157.9 3.35 < 0.001 0.296 0.185

146.6 164.5 3.38 < 0.001 0.294 0.185

682.8 761.8 19.41 < 0.001 0.164 0.197

536.2 597.4 18.08 0.002 0.202 0.195

SED, standard error of the difference; SE, standard error. Interactions between CH4 selection line and pasture quality in Exp. 1 and CH4 selection line and period in Exp. 2 were not significant and are therefore not shown. 1 The repeatability between the repeat measures on the same animal in the two seasons was estimated from the residual variance (Vr) and animal variance (Va) as: repeatability = Va / (Va + Vr). 6

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systems (Selbie et al., 2015; MfE, 2018) was similar for progeny of low and high CH4/DMI selection line sheep in both experiments. Therefore, estimated urinary N2O and NH3 emissions and NO3 leaching were similar for progeny of low and high CH4/DMI selection line sheep. Urinary N excretion as a proportion of N intake in the current study was in a similar range as previously reported for sheep fed cut pasture (Zhao et al., 2016b; Jonker et al., 2018a). To our knowledge, the current study was the first study were the N balance of animals selected for low or high CH4/DMI were compared. When different sheep breeds were compared, there was also no difference in N balance and partitioning between breeds in a series of three studies in the UK with forage-based diets (Zhao et al., 2015, 2016a; Zhao et al., 2017). Nitrous oxide emissions (and NH3 emissions and NO3 leaching) from excreta could be reduced if more feed N is excreted in faecal matter as opposed to N excretion via urine (MfE, 2018). Urinary N/faecal N ratio was similar for progeny of CH4/DMI selection line sheep in both experiments of the current study. The N2O emission factor from urinary N might be affected by urinary N composition (Kool et al., 2006; Chadwick et al., 2018) and considerable between animal variation in urinary N composition has been found by others (Bristow et al., 1992). However, others found no effect of urine N constituents other than urea N, the main N component in urine, on N2O emissions (Gardiner et al., 2018). The composition of urinary N in the current study was not determined, nor were N2O emissions from urine deposited onto soil. Nitrogen balance and partitioning parameters and urinary N/faecal N ratio were moderately repeatable (non-significant due to large associated standard errors) across the two seasons in Exp. 2, suggesting that it is possible to select animals with decreased urinary N excretion parameters and consequently decrease direct and indirect N2O emissions. However, decreasing dietary N concentration and increasing feeding level can reduce urinary N excretion to a much larger extent (Zhao et al., 2016b; Jonker et al., 2018a). 4.2. Faecal matter excretion Faecal matter output (DM, OM, NDF and N) was greater for progeny of low CH4/DMI selection line sheep in Exp. 1 of the current study, which was consistent with findings in trials with selected extremes for low and high CH4/DMI (Pinares-Patiño et al., 2011; Goopy et al., 2014). Those low CH4/DMI sheep were found to have shorter retention time of feed particles in the rumen (PinaresPatiño et al., 2011; Goopy et al., 2014; Jonker et al., 2018b), which might reduce the degradation of feed in the rumen, the main site of fibre degradation. Consequently, total tract feed (especially fibre) digestibility might decrease as suggested by mechanistic model simulations of low compared to high CH4/DMI sheep and cows (Huhtanen et al., 2016). The very poor-quality pasture in Exp.1, which had long retention time of particles in the rumen (Jonker et al., 2018b), also tended to result in low enteric CH4 emissions, while increasing faecal matter output compared to sheep fed the good quality pasture. Meta-analysis of data from sheep and cattle respiration chamber/excreta collection trials comparing dietary treatments have also indicated trade-offs between CH4/DMI and faecal matter excretion per unit of DMI (Sauvant et al., 2014; van Lingen et al., 2018). In some other studies there was no clear relationship between CH4/DMI and faecal matter output (Pinares-Patiño et al., 2003; Bond et al., 2018), as was also the case for progeny of the CH4/DMI selection line sheep in Exp. 2. Bond et al. (2018) found negligible correlation between faecal matter output (per unit of intake) and retention time of particles in the rumen (r = 0.02 to 0.04), while retention time correlated moderately with CH4/DMI (r = 0.51). This suggests that the shorter retention time of particles in the rumen can be compensated for by increased degradation of feed in the large intestine to result in no effect on total tract digestibility of feed. Cabezas-Garcia et al. (2017) found, based on meta-analysis of data from dairy cow metabolism trials, a small between animal variation in total tract OM digestibility and therefore argued that this could not account for much of between cow variation in CH4/ DMI. However, faecal output and total tract digestibility of DM, OM and NDF were weakly repeatable (0.23 ± 0.19 to 0.33 ± 0.18), but non-significant, across the two seasons in Exp. 2, which is similar to findings by others who found that total tract digestibility was weakly heritable in sheep and cattle (Lee et al., 2002; Berry et al., 2007). This suggests that it might be possible to select animals with reduced enteric CH4 emissions, but with similar or reduced faecal matter output. This is important because digestibility of the diet is the main determinant of its energy value that can be used by the animal for maintenance and production and therefore affects GHG emissions intensity, i.e. GHG per unit of animal product. To date, there has not been any relationship (genetic correlations) identified between CH4/DMI and animal production traits in CH4/DMI selection line sheep (Pinares-Patiño et al., 2013) suggesting similar nutrient utilization from the diet for sheep of both CH4/DMI selection lines. 5. Conclusions There were only minor differences in excreta output and associated GHG emissions between progeny of the two CH4/DMI sheep selection lines and enteric CH4 made up approximately 80% of total GHG emissions (on a CO2 equivalent basis). Therefore, magnitude of difference in total animal level GHG emissions between the CH4/DMI selection lines was similar to the difference in enteric CH4 (expressed as CO2-equivalent) between them. The animal genetic traits were expressed independent of the environmental factors, being pasture quality and season in the current study. Therefore, breeding for low CH4/DMI in sheep is a viable mitigation option for pastoral systems, which is not offset by excreta GHG emissions. Declaration of Competing Interest The authors declare that there are no conflicts of interest. 7

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Acknowledgements This work was financially supported by the Pastoral Greenhouse Gas Research Consortium (PGgRc; www.pggrc.co.nz) and the New Zealand Agricultural Greenhouse Gas Research Centre (NZAGRC; www.nzagrc.co.nz). Chernet Woyimo Woju was financially supported by the New Zealand Government through the Global Research Alliance Livestock Emissions and Abatement Research Network (LEARN) awards programme (https://livestockemissions.net/). References Bain, W.E., Bezuidenhout, L., Jopson, N.B., Pinares-Patiño, C.S., McEwan, J.C., 2014. Rumen differences between sheep identified as being low or high methane emitters. Proc. Assoc. Advmt. Anim. Breed. Genet 20, 376–378. Berry, D.P., Horan, B., O’Donovan, M., Buckley, F., Kennedy, E., McEvoy, M., Dillon, P., 2007. Genetics of grass dry matter intake, energy balance, and digestibility in grazing Irish dairy cows. J. Dairy Sci. 90, 4835–4845. 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