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Repeatability of methane emissions from sheep
Animal Feed Science and Technology 166–167 (2011) 210–218
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Repeatability of methane emissions from sheep ˜ ∗ , J.C. McEwan, K.G. Dodds, E.A. Cárdenas 1 , R.S. Hegarty 2 , C.S. Pinares-Patino J.P. Koolaard, H. Clark AgResearch Limited, Grasslands Research Centre, Tennent Drive, Private Bag 11008, Palmerston North, New Zealand
a r t i c l e
Keywords: Methane emission Sheep Repeatability Heritability Animal variation Grass
i n f o
a b s t r a c t Breeding of ruminant livestock for low CH4 emission is an attractive means of mitigating enteric CH4 emissions. However success requires that the mechanism responsible for among animal variation in emissions is repeatable and heritable and has a negligible negative impact on production and functional traits. This study was designed to estimate repeatability and heritability of the CH4 emission trait in sheep, and to determine whether the ranking of sheep based on their CH4 emissions is maintained over a range of contrasting diets. A flock of 105 ewe lambs (10 months old) of a progeny testing program were screened for their CH4 yields (i.e., emissions/unit feed dry matter (DM) intake) when a molasses containing grass silage was fed at restricted intake (1.3 × maintenance metabolisable energy requirements). Methane emissions were measured on a single day in respiration chambers over 4 measurement periods with 13–15 d between consecutive measurements (i.e., screening phase). Mean CH4 yield of lambs was 18.4 ± 0.38 g/kg DM intake during the screening phase, and estimates of repeatability and heritability for CH4 yield were 0.16 and 0.30, respectively. Methane yield in the screening phase was 7.9% higher for the high versus low ranked sheep (19.2 ± 0.18 versus 17.8 ± 0.26 g/kg DM intake). The 10 lowest (low rank) and the 10 highest (high rank) CH4 yielding sheep were selected and retained for further study. Two repeated measurements of CH4 yield were conducted, the first measurement while sheep were fed fresh cut perennial ryegrass pasture (grass), the second with the same sheep fed a 400:600 concentrate:forage (wheat grain:lucerne hay; fresh basis) pelleted diet (pellet). Repeated measurements revealed that rankings were maintained among diets, but that there was a CH4 rank × diet interaction for CH4 yield. When fed the grass diet, the high ranked sheep had 13% higher CH4 yield than the low ranked sheep, but when fed the pelleted diet, the high ranked sheep had 36% higher CH4 yield than the low ranked sheep. Emissions of hydrogen were only measurable when sheep were fed the pelleted diet. This study is the first to report that ranking of sheep for CH4 emissions is consistent among diets, although the magnitude of difference among the rankings was affected by diet, suggesting that among animal variation in CH4 emission could be exploited to breed animals for low CH4 emission.
Abbreviations: BRR, birth/rearing rank; CPT, Central Progeny Test program; DM, dry matter; DMD, DM digestibility; FOR, fractional outflow rate; GHG, greenhouse gas; LW, liveweight; TMR, total mixed ration. ∗ Corresponding author. Tel.: +64 6 3518049; fax: +64 6 3518003. ˜ E-mail address: [email protected] (C.S. Pinares-Patino). 1 Present address: Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional de Colombia, Carrera 45 No. 26-85, Edificio Uriel Garcia, Bogotá D.C., Colombia. 2 Present address: Industry & Investment NSW, Armidale, New South Wales, Australia.
0377-8401/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2011.04.068
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This paper is part of the special issue entitled: Greenhouse Gases in Animal Agriculture – Finding a Balance between Food and Emissions, Guest Edited by T.A. McAllister, Section Guest Editors; K.A. Beauchemin, X. Hao, S. McGinn and Editor for Animal Feed Science and Technology, P.H. Robinson. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Methane is a potent greenhouse gas (GHG) whose global warming potential is 25 times that of CO2 on a weight basis (Solomon et al., 2007). Globally, ruminant livestock are the single most important source of anthropogenic CH4 emissions (∼33% of total manmade CH4 ; Stern and Kaufmann, 1996; Eckard et al., 2010), totalling 2.2 Gt of CO2 equivalents (CO2 -e; Gill et al., 2010). Pastoral ruminant farming is an important component of the economies of most developing countries and some developed countries, where ruminant CH4 emissions, mostly of enteric origin, are a measureable share of national GHG emissions (de Klein et al., 2008; van Beek et al., 2010). In 2007, on a CO2 -e basis, agriculture in New Zealand generated 48% of national GHG emissions, with enteric CH4 emissions accounting ˜ et al., 2009). As global demand for for 75% of this, representing 90% of the national CH4 emissions (Pinares-Patino livestock products is rapidly increasing due to population growth and a shift in human food consumption patterns ˜ et al., (van Beek et al., 2010), livestock enteric CH4 emissions will continue to rise without mitigation (Pinares-Patino 2009). Currently, there are few technologies to mitigate CH4 emissions from ruminants (Eckard et al., 2010). For example, mitigations based on feed additives and supplements have shown some potential to reduce emissions, but these approaches may be unsuitable and impractical for grazing systems where animals are not handled on a daily basis, and supplementary feeding and use of additives may undermine the price competitiveness of the industry. Thus, research into development of cost effective sustainable CH4 mitigations suitable for pastoral farming systems is needed to ensure that these systems remain competitive while meeting consumers expectations of environmental sustainability. A potential cost effective sustainable mitigation for reducing enteric CH4 emissions is to exploit natural among animal variation in CH4 emissions by selecting and breeding ruminants for low CH4 emission. Methane emission measurements conducted in New Zealand and elsewhere have the common feature of large among-animal variation in CH4 emissions (Blaxter and Clapperton, 1965; Lassey et al., 1997; Hammond et al., 2009). For example, our database of CH4 emissions from sheep (n = 196) measured in respiration chambers indicated a 50% difference in CH4 yield (i.e., emissions/unit feed dry matter (DM) intake) between the lowest and highest CH4 yielding individuals when pasture forage was fed (Hammond et al., 2009). This suggests that if the trait responsible for CH4 emission is heritable, it would be possible to breed for lower CH4 emissions, provided that there are no unwanted associations with production and functional traits. This may offer a cost effective mitigation of enteric CH4 emissions with potential widespread application and, most importantly, with benefits accruing across generations of animal selection. Although it is known that CH4 is produced by methanogens, the physiological and behavioural mechanisms responsible ˜ et al. (2003) reported that fractional outflow rate (FOR) of feed for among animal variation are unknown. Pinares-Patino particles from the rumen was responsible for a large proportion (0.57) of the among sheep variation in CH4 emission, and consistent among animal differences in FOR has been reported from studies in sheep and cattle (e.g., Ørskov et al., 1988). In contrast, our research aimed at demonstrating repeatability of the CH4 emission trait using the SF6 tracer technique has ˜ 2000) and cattle (Pinares-Patino ˜ et al., 2007) were had mixed results. For example, when grazing sheep (Pinares-Patino, selected for extreme CH4 yields (emissions/unit ofeed intake), differences in emissions at grazing consistently disappeared in subsequent indoor measurements using fresh and conserved forages. Aware of the higher variability associated with the ˜ et al., 2008), we became convinced that SF6 tracer technique compared to the respiration chamber method (Pinares-Patino continuing to pursue identification of contrasting emission animals using the tracer technique would not be fruitful. Respiration chambers yield the most accurate measures of total enteric CH4 emissions from ruminants. An 8 chamber sheep respiration facility was built at AgResearch Grasslands in 2007 and, using this facility, we conducted repeated measurements on 12 sheep and achieved, for the first time, identification of individuals with consistently contrasting CH4 ˜ and Clark, 2009). emissions (i.e., 3 low and 3 high emitters, with a 12% difference in CH4 yield between them; Pinares-Patino Repeatability is a measure of the extent to which variation in a particular trait is a result of genetic and permanent environmental effects, whereas heritability is the relative importance of genetic effects in determining variation in phenotypic expression (Holmes et al., 2002). This study estimated repeatability and heritability of CH4 emissions on a large number of sheep using respiration chambers. Subsequently, extreme CH4 emission sheep were re-measured on two contrasting diets in order to assess whether consistency of CH4 emission ranking is affected by diet.
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2. Materials and methods 2.1. Experimental, feeds and feeding The study was conducted at the Ulyatt-Reid Large Animal Facility of AgResearch Grasslands Research Centre in Palmerston North (New Zealand) and the experimental protocols followed were approved by the Animal Ethics Committee of AgResearch Limited. Between July and September 2008, a screening process hereafter named ‘screening’ phase, involving a highly recorded flock (n = 105) of ewe lambs (10 ± 0.28 months old) belonging to the Central Progeny Test program (CPT) of Meat and Wool New Zealand (McLean et al., 2006) was conducted. Lambs were fed a molassed perennial ryegrass silage which was commercially prepared in vacuum sealed quantities of 25 kg (fresh weight) by Great Hage in Reporoa (New Zealand) hereafter named ‘silage’. The screening process was to identify individuals with contrasting CH4 yields (g CH4 /kg DM intake). Based on this screening phase, the 10 lowest and the 10 highest CH4 yielding sheep were selected and retained for further study. Two subsequent repeated periods of CH4 measurement were conducted on the selected sheep between May and July 2009. During the first measurement period, the sheep were fed fresh perennial ryegrass (Lolium perenne) pasture forage hereafter named grass, and during the second measurement period they were fed a mixed forage:concentrate pelleted diet hereafter named pellet. Throughout the study the sheep were fed 1.3 times their individual maintenance metabolisable energy requirement (CSIRO, 2007) and daily feed allowances were fed in equal portions twice daily at 08:30 and 16:00 h. Two samples (∼500 g) of feed were collected daily and feed refusals were collected twice daily. These samples were oven dried at 65 ◦ C for 72 h for determination of DM and to calculate DM intake. 2.2. The screening phase (‘screening’) The screening phase included four 1 d measurements (i.e., M1, M2, M3, M4) of CH4 emissions over a period of 3 months. The 105 ewe lambs (liveweight (LW) 41.9 ± 4.70 kg) were sourced from three farms being: Poukawa (Hawkes Bay, North Island), Lincoln (Canterbury, South Island) and Woodlands (Invercargill, South Island). The lambs were the progeny of 15 sires of 7 breeds. The sheep were transported to AgResearch Grasslands in Palmerston North and fed the silage diet described previously. The sheep were randomly subdivided into 13 groups of 8 and the groups were staggered in time to match availability of the 8 respiration chambers. The sheep were acclimatised to the feed for a minimum of 3 weeks, during which time they were kept in pens where they were group fed. They were then moved to metabolic crates for 5 d and fed individually. Following acclimatisation, the sheep were transferred to respiration chambers for a 1 d CH4 emission measurement (M1). The 1 d CH4 emission measurements from each sheep were repeated thrice (i.e., M2, M3, M4) with the same feeding level of 1.3× maintenance metabolisable energy requirements maintained among measurements. The interval between measurements was 13–15 d. Prior to measurements M2, M3 and M4, sheep were re-acclimatised to the metabolic crates for 5 d. Individual feed intakes were measured during the acclimatisation (in metabolic crates) and measurement periods. The LW was measured at the end of the acclimatisation period as well as at the end of each CH4 measurement. Data from M1 were discarded because most of the sheep failed to achieve feed intakes above 80% of offer (i.e., we considered they were not acclimatised to the experimental conditions). Sheep were ranked using a model based on their CH4 yields from M2, M3 and M4, and the 10 lowest and the 10 highest emission sheep were selected and retained for further study. 2.3. The repeated measurement on the grass diet (‘grass’) At the end of the screening phase the selected high rank and low rank CH4 yielding sheep were released to graze at AgResearch Grasslands. Then, in May 2009, the sheep were brought indoors and acclimatised to fresh perennial ryegrass forage over 10 d before a single 2 d CH4 measurement period. The sheep were subdivided into 3 groups of 8, 8 and 4 and measurements were staggered in time. The groups contained equal numbers of low and high CH4 emission ranking sheep. During the first 5 d of acclimatisation, sheep were group fed in pens, whereas during the remaining 5 d of acclimatisation they were individually fed in metabolic crates. The perennial ryegrass forage was cut daily in the mornings (∼10:00 h) and delivered ∼12:00 h. At delivery, samples of feed were collected for DM analysis (oven drying at 65 ◦ C for 72 h) and the afternoon and the following morning meals were weighed and stored (4 ◦ C) until feeding. Feed intake was measured during the last 3 d when sheep were in the metabolic crates, as well as during the CH4 measurement period. Refusals were collected twice daily before feeding and oven dried, and this information was used in calculation of DM intake. 2.4. The repeated measurement on the pelleted diet (‘pellet’) Measurement of CH4 emissions from sheep fed a pelleted diet were in June 2009 immediately following measurements for the grass diet. The three working groups of sheep were maintained, and again the measurements were staggered in time. The pelleted diet consisted of 400:600 concentrate:forage (wheat grain:lucene hay; fresh basis) and was fed as a pellet. Sheep were acclimatised to the pelleted diet over 21 d and, to ensure fibrous material was available for chewing, a small amount of
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Table 1 Chemical compositiona (g/kg dry matter) of the diets fed to the sheep during the screening phase (silage) and repeated measurements (grass and pellet).
Dry matter Crude protein Neutral detergent fibre Acid detergent fibre Soluble sugars Starch DMD
Silage
Grass
Pellet
388 12.7 62.6 38.7 5.0 nd 54.7
170 21.5 40.1 26.5 11.1 nd 75.0
851 15.6 26.9 15.0 4.2 23.3 74.5
nd = not determined. a Chemical composition and dry matter digestibility (DMD) of grass silage (silage) and fresh grass (grass) were estimated by near infrared analysis, whereas the corresponding values for the pelleted diet (pellet) are determined using chemical analysis and in vitro DMD.
grass hay (∼20 g/d) of unknown chemical composition was provided at each meal. The last 5 d of the acclimatisation period was in metabolic crates. The CH4 emission measurements were over two consecutive days. Feed intake was measured during the last 3 d while sheep were in metabolic crates, as well as during the CH4 measurement period. 2.5. Gas analysis ˜ et al. (in this issue) but, in brief, they had an The respiratory chambers were previously described by Pinares-Patino aluminium frame with clear walls and a net volume of 1.8 m3 (i.e., 1.8 m long, 0.85 m wide, 1.2 m high). Each chamber was fitted with a fresh air inlet at the front and an air outlet at the back and air flow was ducted via flexible (38 mm OD) polyurethane hoses. Wet air flow was measured using diaphragm gas meters (AL425, American Meter Company, Nebraska City, NE, USA). Air flow was adjusted to dry standard temperature and pressure conditions. For purposes of air circulation, the 8 chambers constituted two sets of 4 chambers each and, within set, all 4 outlets from the chambers and individual gas meters were connected to a common air pumping system composed of 2 air pumps (UNI-JET 40, ESAM, Parma, Italy) assembled in parallel, which provided air circulation in the range 250–265 L/min. Continuous streams (2 L/min) of exhaust gases from each chamber and the ambient were selectively and sequentially sampled using a multiport switching system (S.W. & W.S. Burrage, Ashford, Kent, UK). A custom designed adaptive switching algorithm was implemented to optimise the sampling frequency by ensuring that the standard deviation (SD) of three successive measurements was below an acceptable threshold (<1 ppm). This process resulted in sampling periods for each chamber that varied from 35 to 60 s and ensured that the sample gas concentrations measurements from the gas analyser were stable. Methane concentrations on dried samples were measured using a Servomex 4900 gas analyser (Servomex Group Ltd., East Sussex, UK). Based on previous experience at our institute (Takenaka et al., 2008) we expected some degree of H2 emissions from mixed diets, and thus the gas analyser was fitted with an electrochemical H2 detector (7HYT Citicel, City Technology Ltd., Portsmouth, Hampshire, UK). The detection ranges for CH4 and H2 were 0–200 and 0–50 ppm with accuracies of 0.5 and 5 ppm, respectively. Gas recovery tests of the respiration chamber system were immediately prior to our study by mass flow metering of CH4 and H2 (independently). These tests revealed recovery rates of 98.2 ± 0.60 and 100.5 ± 4.01 for CH4 and H2 , respectively. 2.6. Feed chemical analyses Chemical compositions of silage and grass samples were determined by near infrared analysis, whereas pellet samples were analysed by chemical analysis. Contents of crude protein, neutral detergent fibre, acid detergent fibre and soluble ˜ et al. (2007), whereas total starch contents sugars in the pelleted diet were analysed using procedures of Pinares-Patino and in vitro DM digestibility (DMD) were determined according to AOAC method 996.11 (1990) and Roughan and Holland (1977), respectively. Table 1 shows the chemical composition and the DMD of the diets fed at each measurement. 2.7. Calculations and statistical analysis The CH4 emission data from the screening process and repeated measurements on grass and pellet diets were expressed as CH4 yield/unit feed intake (g/kg DM intake). Emission data of CH4 and H2 for the repeated measurements were also expressed as gross emissions (g/d) and, in terms of energy excretion using a heat of combustion of 891 and 286 kJ/mol of CH4 and H2 , respectively (Anonymous, 2004). Methane yield data for the sheep screening phase (i.e., M2, M3, M4) were analysed by residual maximum likelihood (REML) using a repeated measures model with each measurement constituting an observation and individual animals as a random effect (PROC MIXED; SAS, 2004). The model considered farm source (n = 3), measurement group (n = 13), measurement time (n = 3 as M1 was omitted) and birth/rearing rank (BRR, singles, multiples, multiple born but single reared) as fixed factors, whereas animal nested within farm source and sire (n = 15) were random factors. The LW at the time of measurement and day of birth were fixed covariates. Sire breed (a fixed factor) was excluded from the model as there were few (i.e., 1–3)
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CH 4 yield (g/kg DM intake)
30
n= 105 25 M2
20
M3 M4
15
10
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Fig. 1. Data for CH4 yield during the screening phase. The measurements M2, M3, M4 were conducted sequentially, but separated by at least 13–15 d. These data were used for the estimations of heritability and repeatability of CH4 yield and also for the ranking of sheep. Dashed line indicates mean CH4 yield (18.4 g/kg DM intake).
CH4 yield (g/kg DM intake)
22 21 20 19 18 17 16 15
Sheep
Fig. 2. Methane yields (error bars indicate standard deviations) for the 10 lowest (low = ) and 10 highest (high = ) ranked sheep during the screening phase. Dashed line indicates mean CH4 yield (18.4 g/kg dry matter intake [DM intake]) for the overall flock (n = 105).
sires/breed. The LW and day of birth were dropped from the model due to lack of significance (i.e., P>0.05). This analysis was used to obtain heritability and repeatability estimates. In order to select the extremes, an additional model was fitted. It was found that BRR and sire had effects but, because BRR is a permanent attribute and our interest was to incorporate the genetic differences due to sires into the individual animal estimates, both BRR and sire were dropped from the model. There were no farm source and measurement time effects, but these factors were retained in this model because they are important management factors. There was an effect of group of measurement and so it was retained in the model. Individual sheep were ranked on the basis of their predicted effects on CH4 yield. To evaluate consistency of CH4 emission rankings, data for repeated measurements on grass and pellet were analysed with a mixed effects model using REML (GenStat; Payne et al., 2009), with ‘working groups’ and ‘chamber’ as random effects and rank of CH4 emission (low and high), diet (grass and pellet) and CH4 rank × diet as fixed effects. In our study, diet and period effects were confounded and so the assumption was made that effects were due to diet only. One high CH4 emission sheep, which could not acclimatise to the pelleted diet, was removed. A similar statistical analysis was completed on the H2 emission data. A least significant difference was used for means separation at P<0.05. 3. Results Fig. 1 shows CH4 yields from individual sheep at each of the measurements except M1 in the screening phase. Mean CH4 yield was 18.4 ± 0.38 g/kg DM intake. The estimate of repeatability was 0.16 ± 0.10, whereas the heritability estimate was 0.30 ± 0.26. The 10 lowest and 10 highest CH4 emission sheep selected from the screening phase had CH4 yields of 17.8 ± 0.26 and 19.2 ± 0.18 g/kg DM intake, respectively (Fig. 2). The LW for the selected low and high CH4 emission sheep did not differ at this stage (42.5 ± 5.39 and 42.4 ± 4.62 kg, respectively). Repeated measurements of sheep while fed the grass and pelleted diets showed that there were rank and diet effects on CH4 emissions (Table 2). Low CH4 emission ranked sheep consistently had lower (P<0.01) CH4 yields than their high CH4 emission counterparts, and emissions from the pelleted diet were lower (P<0.001) than from the grass diet. However, there was a rank × diet interaction (P=0.01) for CH4 emission. For the grass diet, the high CH4 emission ranked sheep had 13% higher CH4 yield than their low emission counterparts, whereas for the pelleted diet the value was 36%. However, there
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Table 2 Methane emission rank of sheep (R, low and high) as affected by diet (D, grass and pellet) on CH4 and hydrogen (H2 ) emissions. Low rank
CH4 (g/d) CH4 (g/kg DM intake) CH4 energy (kJ/d) H2 (g/d) H2 (g/kg DM intake) H2 energy (kJ/d) H2 energy (% of gaseous emissions)
High rank
SEM
Grass
Pellet
Grass
Pellet
22.7a 22.1a 1263a 0.01a 0.01a 1.7a 0.13a
18.6b 17.8b 1034b 0.15b 0.14b 18.9b 1.90b
26.4a 24.9a 1470a 0.02a 0.02a 2.2a 0.16a
26.1a 24.2a 1453a 0.19b 0.18b 23.5b 1.70b
1.69 1.21 93.9 1.102 1.078 1.27 1.169
P Rank
Diet
R×D
<0.01 <0.01 <0.01 0.43 0.49 0.72 0.81
0.02 <0.001 0.02 <0.001 <0.001 <0.001 <0.001
0.06 0.01 0.06 0.59 0.59 0.90 0.21
a,b Values within a row with different superscript letters differ P<0.05. DM, dry matter.
were no effects of rank or rank × diet for H2 emission, but emissions of H2 differed (P<0.001) with diet. For the grass diet, H2 emissions were not measurable, whereas for the pelleted diet emissions represented up to 1.9% of enteric gaseous energy emissions (i.e., CH4 + H2 energy; Table 2). Emissions of H2 did not differ with sheep rank, which is contrary to what would be expected since CH4 formation requires H2 . Fig. 3 shows typical within day profiles of emissions of CH4 and H2 for a sheep ranked low and a sheep ranked high for CH4 emission when fed grass or pelleted diets. Overall, for the pelleted diet, there was not a clear pattern of emission profile differences between low and high CH4 emission rankings, but immediately after feeding the peaks of emission of H2 for high emission sheep were lower and declined more slowly than for their low CH4 emission ranking counterparts. In addition, for the pelleted diet, 4 sheep ranked low for CH4 emission and 2 sheep ranked high had negligible H2 emissions (<0.6 g/d). 4. Discussion This study, using measurements of CH4 emission and feed intake, provides the first estimates of heritability (0.30 ± 0.26) and repeatability (0.16 ± 0.10) of the CH4 yield trait for sheep. However, as the repeatability value indicates the upper limit of heritability and because the SE are large for both estimates, further investigations involving larger numbers of sheep from diverse genetic backgrounds are needed. Robinson et al. (2010) used short-term (i.e., 1 h) measurements of CH4 emissions from 708 sheep of diverse genotypes and reported a heritability estimate of 0.13 for the CH4 emission trait adjusted for LW. The main aim of the screening phase was to identify sheep with contrasting CH4 emissions in order to advance research into factors responsible for among-animal differences in CH4 emissions. The repeated measurements of CH4 emission on contrasting diets (i.e., grass versus pellet) revealed that the sheep maintained their initial rankings over subsequent measurements, but that the magnitude of the differences between the rankings differed with diet. The potential role of animal breed on CH4 emission is largely unknown. Münger and Kreuzer (2008) measured CH4 emissions in respiration chambers from 10 Holstein, 10 Simmental and 10 Jersey dairy cows over weeks 8–41 of lactation and reported that there were no differences in CH4 yield between and within the breeds, and that individual cows did not maintain consistent CH4 yields among measurement periods. Robertson and Waghorn (2002) measured CH4 yields using the SF6 tracer technique from dairy genotypes selected on a pasture based system typical of New Zealand or on a total mixed ration (TMR) typical of diets fed in the northern hemisphere, and reported that CH4 yields increased throughout lactation with a clear genotype × diet interaction. In early lactation, and on both diets, the northern hemisphere cows had lower CH4 yields than their New Zealand counterparts, whereas within genotypes there were no differences between diets at this stage of lactation. However in late lactation CH4 yields of cows on pasture diets were much higher than for those fed TMR, with no differences among genotypes. Genotype × environment interactions for production and functional traits of livestock are widely reported in the literature (e.g., Hammami et al., 2009). The studies of Robertson and Waghorn (2002) and Münger and Kreuzer (2008) involved random individuals within breeds, whereas in the current study individuals were pre-selected to evaluate consistency of CH4 emission rankings, which may help explain the observed scaling effect (Hammami et al., 2009) of diet (i.e., the difference in CH4 yield between the emission rankings widened from the grass to the pellet diet). The rank × diet interaction in our study may indicate that low CH4 emission genotypes may perform as such both on low forage and high forage systems. ˜ et al., 2003; Among-animal differences in CH4 yield has been attributed to rumen FOR and volume (Pinares-Patino Hegarty, 2004), with low CH4 yielding animals having smaller rumen volumes and faster FOR than their high CH4 yielding counterparts. Structural carbohydrates are fermented at slower rates than non-structural carbohydrates such as starch and sugars to yield more CH4 /unit substrate fermented (Czerkawski, 1969; Holter and Young, 1992; Moe and Tyrrell, 1979). Thus, slower rumen FOR and higher rumen volumes increase rate of CH4 emission, most probably by allowing increased digestion of structural carbohydrates and providing a better environment for growth of methanogens. Studies with sheep (Lambourne, 1957; Faichney, 1993) and cattle (Campling et al., 1961; Ørskov et al., 1988) reported that rankings of animals on the basis of rumen FOR were maintained among diets and feeding levels, and Smuts et al. (1995) reported that rumen retention time of digesta is a repeatable physiological trait (repeatability, 0.45). Whether FOR and/or feeding levels were implicated in differences in CH4 yield in our study between low and high CH4 yielding sheep is not known.
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Fig. 3. Within day profiles of emission of methane (CH4 ) (solid lines) and hydrogen (H2 ) (broken lines) from a sheep ranked low (ID 2019) or high (ID 2060) for CH4 emission when fed a grass diet (top panel) or a grain-containing pelleted diet (bottom panel). Emissions were measured over two consecutive days. [Arrows indicate feeding times.]
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The lower CH4 yield of sheep fed the pelleted diet containing wheat grain than when fed the grass diet was expected, as it is well established that increasing levels of concentrate in the diet leads to a reduction in CH4 yield. This is associated with lower ruminal pH and higher rates of ruminal fermentation favouring a shift of fermentation from acetate to propionate (Boadi et al., 2004; Martin et al., 2010). However, a major factor contributing to decreased CH4 yield with the pelleted diet compared to that of the grass was probably the physical form of diet. Despite the pellet containing 600 g lucerne hay/kg, it is likely that the 10 mm diameter pellet could accommodate only a small proportion of particles >10 mm and, therefore, it favoured a rapid rate of passage and a lower CH4 yield (Thomson, 1972; Hironaka et al., 1996). This study confirmed our previous observations of emissions of H2 from diets containing grain (Takenaka et al., 2008). The literature (e.g., Czerkawski, 1972; Benchaar et al., 2001) states that, because of highly effective interspecies H2 transfer in the rumen, the H2 balance resulting from difference between H2 production and utilisation processes is fully utilised by methanogens for production of CH4 . Our current data suggest that, with forage diets, emissions of H2 are negligible but, on diets containing concentrates, this does not always hold. In our study, sheep were fed at restricted levels with feed delivered in two equal meals. Contrary to what occurred for the grass diet, sheep fed the pelleted diet ate their feed allowance within 30 min of feeding and the highest peaks of H2 emissions occurred soon after feeding (Fig. 3). Thus, it can be hypothesised that diet composition, diet form and feeding rate may all have caused the sudden increase in rate of fermentation and H2 production, which was not coupled to the rate of methanogenic activity. Nevertheless when the pelleted diet was fed, the share of H2 emissions on total gaseous energy emissions was <2%. 5. Conclusions The repeatability (0.16 ± 0.10) and heritability (0.30 ± 0.26) estimates for CH4 emissions from sheep are preliminary and need to be substantiated in further investigations involving larger numbers of sheep from diverse genetic backgrounds. Nevertheless, this is the first study to report the persistence of individual CH4 emission rankings for sheep. That the CH4 yield difference between sheep rankings was much higher for a pelleted diet than for a grass diet suggests that the trait or mechanism responsible for such a difference may be potentially exploited to breed low CH4 sheep for forage and intensive feeding systems. Hydrogen emissions from sheep fed grain containing pellets indicates that full conversion of excess H2 into CH4 does not always occur, especially with high concentrate diets containing rapidly fermenting carbohydrates. Conflict of interest None Acknowledgements This study was funded by the New Zealand Pastoral Greenhouse Gas Research Consortium (PGGRC) and M. Aspin, of PGGRC, was instrumental in facilitating this study. We acknowledge the access to animals and lambing and performance data from the Meat & Wool New Zealand Central Progeny Test and the staff involved: N. Jopson, N. McLean, K. Knowler, C. Logan and P. Muir. AgResearch Grasslands engineering and technical staff, P. Lovejoy, J. West, C. Hunt, G. Molano, S. MacLean and E. Sandoval, are all thanked for their commitments to the sheep screening process. E. Cárdenas (from Colombia) received a fellowship from the Livestock Emissions Abatement Research Network (LEARN) of the New Zealand Ministry of Agriculture and Forestry. References AOAC, 1990. Official Methods of Analysis, 15th ed. Assoc. Off. Anal. Chem., Arlington, VA, USA. Benchaar, C., Pomar, C., Chiquette, J., 2001. 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Repeatability of methane emissions from sheep ˜ ∗ , J.C. McEwan, K.G. Dodds, E.A. Cárdenas 1 , R.S. Hegarty 2 , C.S. Pinares-Patino J.P. Koolaard, H. Clark AgResearch Limited, Grasslands Research Centre, Tennent Drive, Private Bag 11008, Palmerston North, New Zealand
a r t i c l e
Keywords: Methane emission Sheep Repeatability Heritability Animal variation Grass
i n f o
a b s t r a c t Breeding of ruminant livestock for low CH4 emission is an attractive means of mitigating enteric CH4 emissions. However success requires that the mechanism responsible for among animal variation in emissions is repeatable and heritable and has a negligible negative impact on production and functional traits. This study was designed to estimate repeatability and heritability of the CH4 emission trait in sheep, and to determine whether the ranking of sheep based on their CH4 emissions is maintained over a range of contrasting diets. A flock of 105 ewe lambs (10 months old) of a progeny testing program were screened for their CH4 yields (i.e., emissions/unit feed dry matter (DM) intake) when a molasses containing grass silage was fed at restricted intake (1.3 × maintenance metabolisable energy requirements). Methane emissions were measured on a single day in respiration chambers over 4 measurement periods with 13–15 d between consecutive measurements (i.e., screening phase). Mean CH4 yield of lambs was 18.4 ± 0.38 g/kg DM intake during the screening phase, and estimates of repeatability and heritability for CH4 yield were 0.16 and 0.30, respectively. Methane yield in the screening phase was 7.9% higher for the high versus low ranked sheep (19.2 ± 0.18 versus 17.8 ± 0.26 g/kg DM intake). The 10 lowest (low rank) and the 10 highest (high rank) CH4 yielding sheep were selected and retained for further study. Two repeated measurements of CH4 yield were conducted, the first measurement while sheep were fed fresh cut perennial ryegrass pasture (grass), the second with the same sheep fed a 400:600 concentrate:forage (wheat grain:lucerne hay; fresh basis) pelleted diet (pellet). Repeated measurements revealed that rankings were maintained among diets, but that there was a CH4 rank × diet interaction for CH4 yield. When fed the grass diet, the high ranked sheep had 13% higher CH4 yield than the low ranked sheep, but when fed the pelleted diet, the high ranked sheep had 36% higher CH4 yield than the low ranked sheep. Emissions of hydrogen were only measurable when sheep were fed the pelleted diet. This study is the first to report that ranking of sheep for CH4 emissions is consistent among diets, although the magnitude of difference among the rankings was affected by diet, suggesting that among animal variation in CH4 emission could be exploited to breed animals for low CH4 emission.
Abbreviations: BRR, birth/rearing rank; CPT, Central Progeny Test program; DM, dry matter; DMD, DM digestibility; FOR, fractional outflow rate; GHG, greenhouse gas; LW, liveweight; TMR, total mixed ration. ∗ Corresponding author. Tel.: +64 6 3518049; fax: +64 6 3518003. ˜ E-mail address: [email protected] (C.S. Pinares-Patino). 1 Present address: Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional de Colombia, Carrera 45 No. 26-85, Edificio Uriel Garcia, Bogotá D.C., Colombia. 2 Present address: Industry & Investment NSW, Armidale, New South Wales, Australia.
0377-8401/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2011.04.068
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This paper is part of the special issue entitled: Greenhouse Gases in Animal Agriculture – Finding a Balance between Food and Emissions, Guest Edited by T.A. McAllister, Section Guest Editors; K.A. Beauchemin, X. Hao, S. McGinn and Editor for Animal Feed Science and Technology, P.H. Robinson. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Methane is a potent greenhouse gas (GHG) whose global warming potential is 25 times that of CO2 on a weight basis (Solomon et al., 2007). Globally, ruminant livestock are the single most important source of anthropogenic CH4 emissions (∼33% of total manmade CH4 ; Stern and Kaufmann, 1996; Eckard et al., 2010), totalling 2.2 Gt of CO2 equivalents (CO2 -e; Gill et al., 2010). Pastoral ruminant farming is an important component of the economies of most developing countries and some developed countries, where ruminant CH4 emissions, mostly of enteric origin, are a measureable share of national GHG emissions (de Klein et al., 2008; van Beek et al., 2010). In 2007, on a CO2 -e basis, agriculture in New Zealand generated 48% of national GHG emissions, with enteric CH4 emissions accounting ˜ et al., 2009). As global demand for for 75% of this, representing 90% of the national CH4 emissions (Pinares-Patino livestock products is rapidly increasing due to population growth and a shift in human food consumption patterns ˜ et al., (van Beek et al., 2010), livestock enteric CH4 emissions will continue to rise without mitigation (Pinares-Patino 2009). Currently, there are few technologies to mitigate CH4 emissions from ruminants (Eckard et al., 2010). For example, mitigations based on feed additives and supplements have shown some potential to reduce emissions, but these approaches may be unsuitable and impractical for grazing systems where animals are not handled on a daily basis, and supplementary feeding and use of additives may undermine the price competitiveness of the industry. Thus, research into development of cost effective sustainable CH4 mitigations suitable for pastoral farming systems is needed to ensure that these systems remain competitive while meeting consumers expectations of environmental sustainability. A potential cost effective sustainable mitigation for reducing enteric CH4 emissions is to exploit natural among animal variation in CH4 emissions by selecting and breeding ruminants for low CH4 emission. Methane emission measurements conducted in New Zealand and elsewhere have the common feature of large among-animal variation in CH4 emissions (Blaxter and Clapperton, 1965; Lassey et al., 1997; Hammond et al., 2009). For example, our database of CH4 emissions from sheep (n = 196) measured in respiration chambers indicated a 50% difference in CH4 yield (i.e., emissions/unit feed dry matter (DM) intake) between the lowest and highest CH4 yielding individuals when pasture forage was fed (Hammond et al., 2009). This suggests that if the trait responsible for CH4 emission is heritable, it would be possible to breed for lower CH4 emissions, provided that there are no unwanted associations with production and functional traits. This may offer a cost effective mitigation of enteric CH4 emissions with potential widespread application and, most importantly, with benefits accruing across generations of animal selection. Although it is known that CH4 is produced by methanogens, the physiological and behavioural mechanisms responsible ˜ et al. (2003) reported that fractional outflow rate (FOR) of feed for among animal variation are unknown. Pinares-Patino particles from the rumen was responsible for a large proportion (0.57) of the among sheep variation in CH4 emission, and consistent among animal differences in FOR has been reported from studies in sheep and cattle (e.g., Ørskov et al., 1988). In contrast, our research aimed at demonstrating repeatability of the CH4 emission trait using the SF6 tracer technique has ˜ 2000) and cattle (Pinares-Patino ˜ et al., 2007) were had mixed results. For example, when grazing sheep (Pinares-Patino, selected for extreme CH4 yields (emissions/unit ofeed intake), differences in emissions at grazing consistently disappeared in subsequent indoor measurements using fresh and conserved forages. Aware of the higher variability associated with the ˜ et al., 2008), we became convinced that SF6 tracer technique compared to the respiration chamber method (Pinares-Patino continuing to pursue identification of contrasting emission animals using the tracer technique would not be fruitful. Respiration chambers yield the most accurate measures of total enteric CH4 emissions from ruminants. An 8 chamber sheep respiration facility was built at AgResearch Grasslands in 2007 and, using this facility, we conducted repeated measurements on 12 sheep and achieved, for the first time, identification of individuals with consistently contrasting CH4 ˜ and Clark, 2009). emissions (i.e., 3 low and 3 high emitters, with a 12% difference in CH4 yield between them; Pinares-Patino Repeatability is a measure of the extent to which variation in a particular trait is a result of genetic and permanent environmental effects, whereas heritability is the relative importance of genetic effects in determining variation in phenotypic expression (Holmes et al., 2002). This study estimated repeatability and heritability of CH4 emissions on a large number of sheep using respiration chambers. Subsequently, extreme CH4 emission sheep were re-measured on two contrasting diets in order to assess whether consistency of CH4 emission ranking is affected by diet.
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2. Materials and methods 2.1. Experimental, feeds and feeding The study was conducted at the Ulyatt-Reid Large Animal Facility of AgResearch Grasslands Research Centre in Palmerston North (New Zealand) and the experimental protocols followed were approved by the Animal Ethics Committee of AgResearch Limited. Between July and September 2008, a screening process hereafter named ‘screening’ phase, involving a highly recorded flock (n = 105) of ewe lambs (10 ± 0.28 months old) belonging to the Central Progeny Test program (CPT) of Meat and Wool New Zealand (McLean et al., 2006) was conducted. Lambs were fed a molassed perennial ryegrass silage which was commercially prepared in vacuum sealed quantities of 25 kg (fresh weight) by Great Hage in Reporoa (New Zealand) hereafter named ‘silage’. The screening process was to identify individuals with contrasting CH4 yields (g CH4 /kg DM intake). Based on this screening phase, the 10 lowest and the 10 highest CH4 yielding sheep were selected and retained for further study. Two subsequent repeated periods of CH4 measurement were conducted on the selected sheep between May and July 2009. During the first measurement period, the sheep were fed fresh perennial ryegrass (Lolium perenne) pasture forage hereafter named grass, and during the second measurement period they were fed a mixed forage:concentrate pelleted diet hereafter named pellet. Throughout the study the sheep were fed 1.3 times their individual maintenance metabolisable energy requirement (CSIRO, 2007) and daily feed allowances were fed in equal portions twice daily at 08:30 and 16:00 h. Two samples (∼500 g) of feed were collected daily and feed refusals were collected twice daily. These samples were oven dried at 65 ◦ C for 72 h for determination of DM and to calculate DM intake. 2.2. The screening phase (‘screening’) The screening phase included four 1 d measurements (i.e., M1, M2, M3, M4) of CH4 emissions over a period of 3 months. The 105 ewe lambs (liveweight (LW) 41.9 ± 4.70 kg) were sourced from three farms being: Poukawa (Hawkes Bay, North Island), Lincoln (Canterbury, South Island) and Woodlands (Invercargill, South Island). The lambs were the progeny of 15 sires of 7 breeds. The sheep were transported to AgResearch Grasslands in Palmerston North and fed the silage diet described previously. The sheep were randomly subdivided into 13 groups of 8 and the groups were staggered in time to match availability of the 8 respiration chambers. The sheep were acclimatised to the feed for a minimum of 3 weeks, during which time they were kept in pens where they were group fed. They were then moved to metabolic crates for 5 d and fed individually. Following acclimatisation, the sheep were transferred to respiration chambers for a 1 d CH4 emission measurement (M1). The 1 d CH4 emission measurements from each sheep were repeated thrice (i.e., M2, M3, M4) with the same feeding level of 1.3× maintenance metabolisable energy requirements maintained among measurements. The interval between measurements was 13–15 d. Prior to measurements M2, M3 and M4, sheep were re-acclimatised to the metabolic crates for 5 d. Individual feed intakes were measured during the acclimatisation (in metabolic crates) and measurement periods. The LW was measured at the end of the acclimatisation period as well as at the end of each CH4 measurement. Data from M1 were discarded because most of the sheep failed to achieve feed intakes above 80% of offer (i.e., we considered they were not acclimatised to the experimental conditions). Sheep were ranked using a model based on their CH4 yields from M2, M3 and M4, and the 10 lowest and the 10 highest emission sheep were selected and retained for further study. 2.3. The repeated measurement on the grass diet (‘grass’) At the end of the screening phase the selected high rank and low rank CH4 yielding sheep were released to graze at AgResearch Grasslands. Then, in May 2009, the sheep were brought indoors and acclimatised to fresh perennial ryegrass forage over 10 d before a single 2 d CH4 measurement period. The sheep were subdivided into 3 groups of 8, 8 and 4 and measurements were staggered in time. The groups contained equal numbers of low and high CH4 emission ranking sheep. During the first 5 d of acclimatisation, sheep were group fed in pens, whereas during the remaining 5 d of acclimatisation they were individually fed in metabolic crates. The perennial ryegrass forage was cut daily in the mornings (∼10:00 h) and delivered ∼12:00 h. At delivery, samples of feed were collected for DM analysis (oven drying at 65 ◦ C for 72 h) and the afternoon and the following morning meals were weighed and stored (4 ◦ C) until feeding. Feed intake was measured during the last 3 d when sheep were in the metabolic crates, as well as during the CH4 measurement period. Refusals were collected twice daily before feeding and oven dried, and this information was used in calculation of DM intake. 2.4. The repeated measurement on the pelleted diet (‘pellet’) Measurement of CH4 emissions from sheep fed a pelleted diet were in June 2009 immediately following measurements for the grass diet. The three working groups of sheep were maintained, and again the measurements were staggered in time. The pelleted diet consisted of 400:600 concentrate:forage (wheat grain:lucene hay; fresh basis) and was fed as a pellet. Sheep were acclimatised to the pelleted diet over 21 d and, to ensure fibrous material was available for chewing, a small amount of
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Table 1 Chemical compositiona (g/kg dry matter) of the diets fed to the sheep during the screening phase (silage) and repeated measurements (grass and pellet).
Dry matter Crude protein Neutral detergent fibre Acid detergent fibre Soluble sugars Starch DMD
Silage
Grass
Pellet
388 12.7 62.6 38.7 5.0 nd 54.7
170 21.5 40.1 26.5 11.1 nd 75.0
851 15.6 26.9 15.0 4.2 23.3 74.5
nd = not determined. a Chemical composition and dry matter digestibility (DMD) of grass silage (silage) and fresh grass (grass) were estimated by near infrared analysis, whereas the corresponding values for the pelleted diet (pellet) are determined using chemical analysis and in vitro DMD.
grass hay (∼20 g/d) of unknown chemical composition was provided at each meal. The last 5 d of the acclimatisation period was in metabolic crates. The CH4 emission measurements were over two consecutive days. Feed intake was measured during the last 3 d while sheep were in metabolic crates, as well as during the CH4 measurement period. 2.5. Gas analysis ˜ et al. (in this issue) but, in brief, they had an The respiratory chambers were previously described by Pinares-Patino aluminium frame with clear walls and a net volume of 1.8 m3 (i.e., 1.8 m long, 0.85 m wide, 1.2 m high). Each chamber was fitted with a fresh air inlet at the front and an air outlet at the back and air flow was ducted via flexible (38 mm OD) polyurethane hoses. Wet air flow was measured using diaphragm gas meters (AL425, American Meter Company, Nebraska City, NE, USA). Air flow was adjusted to dry standard temperature and pressure conditions. For purposes of air circulation, the 8 chambers constituted two sets of 4 chambers each and, within set, all 4 outlets from the chambers and individual gas meters were connected to a common air pumping system composed of 2 air pumps (UNI-JET 40, ESAM, Parma, Italy) assembled in parallel, which provided air circulation in the range 250–265 L/min. Continuous streams (2 L/min) of exhaust gases from each chamber and the ambient were selectively and sequentially sampled using a multiport switching system (S.W. & W.S. Burrage, Ashford, Kent, UK). A custom designed adaptive switching algorithm was implemented to optimise the sampling frequency by ensuring that the standard deviation (SD) of three successive measurements was below an acceptable threshold (<1 ppm). This process resulted in sampling periods for each chamber that varied from 35 to 60 s and ensured that the sample gas concentrations measurements from the gas analyser were stable. Methane concentrations on dried samples were measured using a Servomex 4900 gas analyser (Servomex Group Ltd., East Sussex, UK). Based on previous experience at our institute (Takenaka et al., 2008) we expected some degree of H2 emissions from mixed diets, and thus the gas analyser was fitted with an electrochemical H2 detector (7HYT Citicel, City Technology Ltd., Portsmouth, Hampshire, UK). The detection ranges for CH4 and H2 were 0–200 and 0–50 ppm with accuracies of 0.5 and 5 ppm, respectively. Gas recovery tests of the respiration chamber system were immediately prior to our study by mass flow metering of CH4 and H2 (independently). These tests revealed recovery rates of 98.2 ± 0.60 and 100.5 ± 4.01 for CH4 and H2 , respectively. 2.6. Feed chemical analyses Chemical compositions of silage and grass samples were determined by near infrared analysis, whereas pellet samples were analysed by chemical analysis. Contents of crude protein, neutral detergent fibre, acid detergent fibre and soluble ˜ et al. (2007), whereas total starch contents sugars in the pelleted diet were analysed using procedures of Pinares-Patino and in vitro DM digestibility (DMD) were determined according to AOAC method 996.11 (1990) and Roughan and Holland (1977), respectively. Table 1 shows the chemical composition and the DMD of the diets fed at each measurement. 2.7. Calculations and statistical analysis The CH4 emission data from the screening process and repeated measurements on grass and pellet diets were expressed as CH4 yield/unit feed intake (g/kg DM intake). Emission data of CH4 and H2 for the repeated measurements were also expressed as gross emissions (g/d) and, in terms of energy excretion using a heat of combustion of 891 and 286 kJ/mol of CH4 and H2 , respectively (Anonymous, 2004). Methane yield data for the sheep screening phase (i.e., M2, M3, M4) were analysed by residual maximum likelihood (REML) using a repeated measures model with each measurement constituting an observation and individual animals as a random effect (PROC MIXED; SAS, 2004). The model considered farm source (n = 3), measurement group (n = 13), measurement time (n = 3 as M1 was omitted) and birth/rearing rank (BRR, singles, multiples, multiple born but single reared) as fixed factors, whereas animal nested within farm source and sire (n = 15) were random factors. The LW at the time of measurement and day of birth were fixed covariates. Sire breed (a fixed factor) was excluded from the model as there were few (i.e., 1–3)
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Fig. 1. Data for CH4 yield during the screening phase. The measurements M2, M3, M4 were conducted sequentially, but separated by at least 13–15 d. These data were used for the estimations of heritability and repeatability of CH4 yield and also for the ranking of sheep. Dashed line indicates mean CH4 yield (18.4 g/kg DM intake).
CH4 yield (g/kg DM intake)
22 21 20 19 18 17 16 15
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Fig. 2. Methane yields (error bars indicate standard deviations) for the 10 lowest (low = ) and 10 highest (high = ) ranked sheep during the screening phase. Dashed line indicates mean CH4 yield (18.4 g/kg dry matter intake [DM intake]) for the overall flock (n = 105).
sires/breed. The LW and day of birth were dropped from the model due to lack of significance (i.e., P>0.05). This analysis was used to obtain heritability and repeatability estimates. In order to select the extremes, an additional model was fitted. It was found that BRR and sire had effects but, because BRR is a permanent attribute and our interest was to incorporate the genetic differences due to sires into the individual animal estimates, both BRR and sire were dropped from the model. There were no farm source and measurement time effects, but these factors were retained in this model because they are important management factors. There was an effect of group of measurement and so it was retained in the model. Individual sheep were ranked on the basis of their predicted effects on CH4 yield. To evaluate consistency of CH4 emission rankings, data for repeated measurements on grass and pellet were analysed with a mixed effects model using REML (GenStat; Payne et al., 2009), with ‘working groups’ and ‘chamber’ as random effects and rank of CH4 emission (low and high), diet (grass and pellet) and CH4 rank × diet as fixed effects. In our study, diet and period effects were confounded and so the assumption was made that effects were due to diet only. One high CH4 emission sheep, which could not acclimatise to the pelleted diet, was removed. A similar statistical analysis was completed on the H2 emission data. A least significant difference was used for means separation at P<0.05. 3. Results Fig. 1 shows CH4 yields from individual sheep at each of the measurements except M1 in the screening phase. Mean CH4 yield was 18.4 ± 0.38 g/kg DM intake. The estimate of repeatability was 0.16 ± 0.10, whereas the heritability estimate was 0.30 ± 0.26. The 10 lowest and 10 highest CH4 emission sheep selected from the screening phase had CH4 yields of 17.8 ± 0.26 and 19.2 ± 0.18 g/kg DM intake, respectively (Fig. 2). The LW for the selected low and high CH4 emission sheep did not differ at this stage (42.5 ± 5.39 and 42.4 ± 4.62 kg, respectively). Repeated measurements of sheep while fed the grass and pelleted diets showed that there were rank and diet effects on CH4 emissions (Table 2). Low CH4 emission ranked sheep consistently had lower (P<0.01) CH4 yields than their high CH4 emission counterparts, and emissions from the pelleted diet were lower (P<0.001) than from the grass diet. However, there was a rank × diet interaction (P=0.01) for CH4 emission. For the grass diet, the high CH4 emission ranked sheep had 13% higher CH4 yield than their low emission counterparts, whereas for the pelleted diet the value was 36%. However, there
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Table 2 Methane emission rank of sheep (R, low and high) as affected by diet (D, grass and pellet) on CH4 and hydrogen (H2 ) emissions. Low rank
CH4 (g/d) CH4 (g/kg DM intake) CH4 energy (kJ/d) H2 (g/d) H2 (g/kg DM intake) H2 energy (kJ/d) H2 energy (% of gaseous emissions)
High rank
SEM
Grass
Pellet
Grass
Pellet
22.7a 22.1a 1263a 0.01a 0.01a 1.7a 0.13a
18.6b 17.8b 1034b 0.15b 0.14b 18.9b 1.90b
26.4a 24.9a 1470a 0.02a 0.02a 2.2a 0.16a
26.1a 24.2a 1453a 0.19b 0.18b 23.5b 1.70b
1.69 1.21 93.9 1.102 1.078 1.27 1.169
P Rank
Diet
R×D
<0.01 <0.01 <0.01 0.43 0.49 0.72 0.81
0.02 <0.001 0.02 <0.001 <0.001 <0.001 <0.001
0.06 0.01 0.06 0.59 0.59 0.90 0.21
a,b Values within a row with different superscript letters differ P<0.05. DM, dry matter.
were no effects of rank or rank × diet for H2 emission, but emissions of H2 differed (P<0.001) with diet. For the grass diet, H2 emissions were not measurable, whereas for the pelleted diet emissions represented up to 1.9% of enteric gaseous energy emissions (i.e., CH4 + H2 energy; Table 2). Emissions of H2 did not differ with sheep rank, which is contrary to what would be expected since CH4 formation requires H2 . Fig. 3 shows typical within day profiles of emissions of CH4 and H2 for a sheep ranked low and a sheep ranked high for CH4 emission when fed grass or pelleted diets. Overall, for the pelleted diet, there was not a clear pattern of emission profile differences between low and high CH4 emission rankings, but immediately after feeding the peaks of emission of H2 for high emission sheep were lower and declined more slowly than for their low CH4 emission ranking counterparts. In addition, for the pelleted diet, 4 sheep ranked low for CH4 emission and 2 sheep ranked high had negligible H2 emissions (<0.6 g/d). 4. Discussion This study, using measurements of CH4 emission and feed intake, provides the first estimates of heritability (0.30 ± 0.26) and repeatability (0.16 ± 0.10) of the CH4 yield trait for sheep. However, as the repeatability value indicates the upper limit of heritability and because the SE are large for both estimates, further investigations involving larger numbers of sheep from diverse genetic backgrounds are needed. Robinson et al. (2010) used short-term (i.e., 1 h) measurements of CH4 emissions from 708 sheep of diverse genotypes and reported a heritability estimate of 0.13 for the CH4 emission trait adjusted for LW. The main aim of the screening phase was to identify sheep with contrasting CH4 emissions in order to advance research into factors responsible for among-animal differences in CH4 emissions. The repeated measurements of CH4 emission on contrasting diets (i.e., grass versus pellet) revealed that the sheep maintained their initial rankings over subsequent measurements, but that the magnitude of the differences between the rankings differed with diet. The potential role of animal breed on CH4 emission is largely unknown. Münger and Kreuzer (2008) measured CH4 emissions in respiration chambers from 10 Holstein, 10 Simmental and 10 Jersey dairy cows over weeks 8–41 of lactation and reported that there were no differences in CH4 yield between and within the breeds, and that individual cows did not maintain consistent CH4 yields among measurement periods. Robertson and Waghorn (2002) measured CH4 yields using the SF6 tracer technique from dairy genotypes selected on a pasture based system typical of New Zealand or on a total mixed ration (TMR) typical of diets fed in the northern hemisphere, and reported that CH4 yields increased throughout lactation with a clear genotype × diet interaction. In early lactation, and on both diets, the northern hemisphere cows had lower CH4 yields than their New Zealand counterparts, whereas within genotypes there were no differences between diets at this stage of lactation. However in late lactation CH4 yields of cows on pasture diets were much higher than for those fed TMR, with no differences among genotypes. Genotype × environment interactions for production and functional traits of livestock are widely reported in the literature (e.g., Hammami et al., 2009). The studies of Robertson and Waghorn (2002) and Münger and Kreuzer (2008) involved random individuals within breeds, whereas in the current study individuals were pre-selected to evaluate consistency of CH4 emission rankings, which may help explain the observed scaling effect (Hammami et al., 2009) of diet (i.e., the difference in CH4 yield between the emission rankings widened from the grass to the pellet diet). The rank × diet interaction in our study may indicate that low CH4 emission genotypes may perform as such both on low forage and high forage systems. ˜ et al., 2003; Among-animal differences in CH4 yield has been attributed to rumen FOR and volume (Pinares-Patino Hegarty, 2004), with low CH4 yielding animals having smaller rumen volumes and faster FOR than their high CH4 yielding counterparts. Structural carbohydrates are fermented at slower rates than non-structural carbohydrates such as starch and sugars to yield more CH4 /unit substrate fermented (Czerkawski, 1969; Holter and Young, 1992; Moe and Tyrrell, 1979). Thus, slower rumen FOR and higher rumen volumes increase rate of CH4 emission, most probably by allowing increased digestion of structural carbohydrates and providing a better environment for growth of methanogens. Studies with sheep (Lambourne, 1957; Faichney, 1993) and cattle (Campling et al., 1961; Ørskov et al., 1988) reported that rankings of animals on the basis of rumen FOR were maintained among diets and feeding levels, and Smuts et al. (1995) reported that rumen retention time of digesta is a repeatable physiological trait (repeatability, 0.45). Whether FOR and/or feeding levels were implicated in differences in CH4 yield in our study between low and high CH4 yielding sheep is not known.
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Fig. 3. Within day profiles of emission of methane (CH4 ) (solid lines) and hydrogen (H2 ) (broken lines) from a sheep ranked low (ID 2019) or high (ID 2060) for CH4 emission when fed a grass diet (top panel) or a grain-containing pelleted diet (bottom panel). Emissions were measured over two consecutive days. [Arrows indicate feeding times.]
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The lower CH4 yield of sheep fed the pelleted diet containing wheat grain than when fed the grass diet was expected, as it is well established that increasing levels of concentrate in the diet leads to a reduction in CH4 yield. This is associated with lower ruminal pH and higher rates of ruminal fermentation favouring a shift of fermentation from acetate to propionate (Boadi et al., 2004; Martin et al., 2010). However, a major factor contributing to decreased CH4 yield with the pelleted diet compared to that of the grass was probably the physical form of diet. Despite the pellet containing 600 g lucerne hay/kg, it is likely that the 10 mm diameter pellet could accommodate only a small proportion of particles >10 mm and, therefore, it favoured a rapid rate of passage and a lower CH4 yield (Thomson, 1972; Hironaka et al., 1996). This study confirmed our previous observations of emissions of H2 from diets containing grain (Takenaka et al., 2008). The literature (e.g., Czerkawski, 1972; Benchaar et al., 2001) states that, because of highly effective interspecies H2 transfer in the rumen, the H2 balance resulting from difference between H2 production and utilisation processes is fully utilised by methanogens for production of CH4 . Our current data suggest that, with forage diets, emissions of H2 are negligible but, on diets containing concentrates, this does not always hold. In our study, sheep were fed at restricted levels with feed delivered in two equal meals. Contrary to what occurred for the grass diet, sheep fed the pelleted diet ate their feed allowance within 30 min of feeding and the highest peaks of H2 emissions occurred soon after feeding (Fig. 3). Thus, it can be hypothesised that diet composition, diet form and feeding rate may all have caused the sudden increase in rate of fermentation and H2 production, which was not coupled to the rate of methanogenic activity. Nevertheless when the pelleted diet was fed, the share of H2 emissions on total gaseous energy emissions was <2%. 5. Conclusions The repeatability (0.16 ± 0.10) and heritability (0.30 ± 0.26) estimates for CH4 emissions from sheep are preliminary and need to be substantiated in further investigations involving larger numbers of sheep from diverse genetic backgrounds. Nevertheless, this is the first study to report the persistence of individual CH4 emission rankings for sheep. That the CH4 yield difference between sheep rankings was much higher for a pelleted diet than for a grass diet suggests that the trait or mechanism responsible for such a difference may be potentially exploited to breed low CH4 sheep for forage and intensive feeding systems. Hydrogen emissions from sheep fed grain containing pellets indicates that full conversion of excess H2 into CH4 does not always occur, especially with high concentrate diets containing rapidly fermenting carbohydrates. Conflict of interest None Acknowledgements This study was funded by the New Zealand Pastoral Greenhouse Gas Research Consortium (PGGRC) and M. Aspin, of PGGRC, was instrumental in facilitating this study. We acknowledge the access to animals and lambing and performance data from the Meat & Wool New Zealand Central Progeny Test and the staff involved: N. Jopson, N. McLean, K. Knowler, C. Logan and P. Muir. AgResearch Grasslands engineering and technical staff, P. Lovejoy, J. West, C. Hunt, G. Molano, S. MacLean and E. Sandoval, are all thanked for their commitments to the sheep screening process. E. Cárdenas (from Colombia) received a fellowship from the Livestock Emissions Abatement Research Network (LEARN) of the New Zealand Ministry of Agriculture and Forestry. References AOAC, 1990. Official Methods of Analysis, 15th ed. Assoc. Off. Anal. Chem., Arlington, VA, USA. Benchaar, C., Pomar, C., Chiquette, J., 2001. 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