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Sheep fed forage chicory (Cichorium intybus) or perennial ryegrass (Lolium perenne) have similar methane emissions
Animal Feed Science and Technology 172 (2012) 217–225
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Sheep fed forage chicory (Cichorium intybus) or perennial ryegrass (Lolium perenne) have similar methane emissions ˜ X.Z. Sun ∗ , S.O. Hoskin, G.G. Zhang 1 , G. Molano, S. Muetzel, C.S. Pinares-Patino, H. Clark, D. Pacheco Grasslands Research Centre, AgResearch Limited, Private Bag 11008, Palmerston North 4442, New Zealand
a r t i c l e
i n f o
Article history: Received 24 May 2011 Received in revised form 14 November 2011 Accepted 15 November 2011
Keywords: Forage chicory Perennial ryegrass Methane Sheep Feeding level Rumen outflow rate
a b s t r a c t Forage chicory (Cichorium intybus) has the potential to mitigate methane emissions from ruminants. It was reported that the reduction can be up to 30% compared with perennial ryegrass (Lolium perenne). To accurately evaluate the reduction, fresh chicory and perennial ryegrass in the vegetative state were fed to 24 wethers, 8 of which rumen-fistulated, at 1.3 and 2.2 times maintenance metabolisable energy requirements. Dry matter (DM) intake, whole tract apparent digestibility, rumen fermentation parameters and rumen liquid passage rate were measured in metabolism crates, and methane emissions determined using a calorimetric technique. Chemical analyses showed that chicory contained less DM, organic matter (OM), crude protein, neutral detergent fibre (aNDF), acid detergent fibre (ADF), cellulose and hemicellulose, but more hot water-soluble carbohydrate and pectin, than perennial ryegrass. Methane yield (g/kg DM intake) of wethers fed chicory did not differ from that of those fed perennial ryegrass. Yield was lower at the high versus the low feeding level of ryegrass. Apparent digestibility of DM and OM was higher, and aNDF, ADF, hemicellulose and cellulose was lower, in wethers fed chicory versus perennial ryegrass. In situ DM degradation rate of chicory was higher than that of perennial ryegrass. Rumen liquid passage rate was the same for wethers fed the two forages and higher at the high feeding level. The reduction in methane emissions by feeding vegetative chicory to wethers was limited, but increased feeding level reduces methane yields per unit of DM intake. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Emissions of methane from grazed ruminants accounts for 31% of CO2 equivalents from the New Zealand greenhouse gas emissions inventory (Ministry for the Environment, 2009). In a recent review of effects of nutritional management to abate enteric methane emissions it was concluded that methane emissions from dairy cows and sheep fed some specific forage plants were sometimes lower than emissions from those fed grasses (Beauchemin et al., 2008). Forage chicory (Cichorium intybus), a plant of the dicotyledons family Asteraceae, is a palatable forage with a high feeding value, high dry matter (DM) yield and can be grown in New Zealand (Li and Kemp, 2005). Kusmartono et al. (1997) reported that deer fed chicory had faster rumen outflow rate than their counterparts fed perennial ryegrass, and faster rumen outflow rates have been linked
Abbreviations: ADF, acid detergent fibre; aNDF, neutral detergent fibre; C2/C3, ratio of acetate to propionate; Co-EDTA, cobalt ethylene diaminetetraacetic acid; DM, dry matter; HWSC, hot water soluble carbohydrates; ME, metabolisable energy; OM, organic matter; RFC, readily fermentable carbohydrates; SC, structural carbohydrates; VFA, volatile fatty acids. ∗ Corresponding author. Tel.: +64 6 351 8150; fax: +64 6 351 8032. E-mail addresses: [email protected], [email protected] (X.Z. Sun). 1 Present address: Department of Animal Sciences and Technology, Shandong Agricultural University, Tai’an, Shandong, 271018, PR China. 0377-8401/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2011.11.007
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˜ et al., 2003a; Hegarty, 2004). Rumen outflow rates and passage can also be to reduced methane production (Pinares-Patino altered by feed intake level (Volden, 1999) and methane yield per unit of DM intake declines with increased feed intakes (Blaxter and Clapperton, 1965; Yan et al., 2009). Reports of methane emissions from ruminants fed forage chicory are scarce, but in two studies (Waghorn et al., 2002; Swainson et al., 2008) sheep fed chicory emitted up to 30% less methane than those fed perennial ryegrass-based pasture, although more recent research suggests no difference in methane yield from sheep fed mature chicory or perennial ryegrass (Sun et al., 2011). The primary objective was to compare methane emissions from sheep fed vegetative forage chicory or perennial ryegrass at two levels of intake using open respiration calorimetry. A secondary objective was to measure in vivo and in situ digestion of the forages to determine causes of the expected differences in methane yield (CH4 /DM intake). 2. Materials and methods 2.1. Experimental design The study was conducted during autumn (8 April to 9 May, 2009) at AgResearch Grasslands (Palmerston North, New Zealand) with 24 Romney wether sheep, including 16 intact and 8 with rumen fistulate. The study was a 2×2 factorial design, using two forages (chicory and perennial ryegrass) and two levels of intake (1.2 and 2.2 times maintenance metabolisable energy (ME) requirement). Four intact and two rumen-fistulated sheep were randomly allocated to each treatment level. Sheep were acclimatised to the forage diets for 7 d while grazing and then acclimatised to the treatment levels over 21 d in pens (8 d) and metabolic crates (13 d). The experimental period was 11 d of feed intake, feed digestibility, rumen outflow and rumen fermentation parameters measurement, followed by 2 d of methane emission measurement in respiration chambers. After 21 d of feeding the respective forages at appropriate intakes, a 6 d faeces and urine collection was undertaken in all sheep to determine diet digestibility after which the sheep were transferred to modified metabolism crates and put into ˜ et al., 2008) for 48 h methane measurement. The sheep were released to pasture respiration calorimeters (Pinares-Patino following methane determinations. The procedure for the fistulated animals included measurements of rumen liquid turnover with rumen sampling for measurement of pH, volatile fatty acids (VFA) and ammonia. These measurements commenced when the sheep had been fed chicory or ryegrass for at least 18 d. For the rumen turnover measurement, the sheep were adapted to hourly feeding for 6 d, with forage supplied using automated belt-driven feeders. Following the rumen turnover measurements, the hourly feeding was discontinued for the digestibility and methane measurement periods when the fistulated sheep were fed twice daily. In addition to measurements with sheep, in sacco DM degradation kinetic studies were undertaken with two rumen fistulated non-lactating cattle fed cut ryegrass pasture. These measurements were completed after the sheep experiment, but using the same herbage, which had been stored frozen. Animal ethics approval for the study was granted by the AgResearch Animal Ethics Committee (Approval 11671). 2.2. Forages Forage chicory (cv. Choice) had been established about 8 mo prior to the experiment, and the last grazing was 6 wk prior to the experiment. Chicory fed to the sheep (grazed or harvested) was in the vegetative stage and ∼50 cm in height (from the ground to the top of plants). Perennial ryegrass (cv. Quartet) used in this study was established two years earlier and was in vegetative stage at the time of study (height ∼35 cm). The grass sward was grazed 4 wk prior to the start of this study. The grazed areas of chicory crop and perennial ryegrass pasture used for the 7 d acclimatisation to diets were 500 and 600 m2 . Herbage allowance during this period was set at generous levels. During the indoor feeding period, forages were harvested and stored as described by Sun et al. (2011). 2.3. Animals and feeding The 24 wethers were about 20 mo of age and weighed 50 ± 4.1 kg. Eight were fitted with a rumen cannula (30 mm internal diameter; Beruc Equipment Ltd., Benoni, South Africa) 4 mo prior to the study and two of them were allocated to each treatment group. At the start of the acclimatisation period, all sheep were drenched with a broad spectrum anthelmintic (Scanda; Schering-Plough Animal Health New Zealand Limited, Wellington, New Zealand). Following the 7 d dietary acclimatisation while grazing ryegrass or chicory, the sheep were fed twice daily throughout with the exception that the rumen fistulated ones were fed hourly from feeders placed above metabolism crates in order to enable measurement of rumen turnover. Indoors, feed was delivered to the non-fistulated animals in two equal meals at 09:00 and 16:30 h. Feeding level was set at 1.3 and 2.2 times maintenance ME requirements (Australian Agricultural Council, 1990) and this was maintained throughout the study. At daily arrival of cut forage, meals were weighed and, during this process, two samples of the herbages (∼200 g fresh weight) were collected and dried at 65 ◦ C for 48 h for chemical analysis. Refusals were collected daily prior to morning
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feeding, weighed and dried for determination of DM concentration as for the herbages offeral. Drinking water was available ad libitum. 2.4. Determination of methane emissions ˜ et al. Methane emissions were measured using the 8 chamber sheep respiration facility described by Pinares-Patino (2011). Sheep were placed in the chambers in a balanced way (i.e., at all times there were 2 sheep from each of the 4 treatments (2 feeding levels×2 forages)) in the respiration chambers. The measuring procedure, data calculation, chamber operation and routine management used the method described by Sun et al. (2011). 2.5. Apparent digestibility Apparent digestibility was calculated from measurements of feed intakes and faecal outputs over a 6 d period. Daily oven-dried (65 ◦ C, 48 h) feed refusals were pooled within sheep and then ground using a Wiley mill to pass a 1 mm screen for chemical analysis. Total faecal outputs were collected from each sheep using faecal collection harnesses. Faeces were collected daily before the morning feeding, weighed, sub-sampled and samples stored frozen at −20 ◦ C. Faecal samples were later pooled within sheep, oven-dried (65 ◦ C, 48 h) and ground in a Wiley mill to pass a 1 mm screen for later chemical analysis. 2.6. Rumen liquid passage rate A single dose of cobalt ethylene diaminetetraacetic acid (Co-EDTA) was used as a fluid phase marker to measure rumen liquid outflow rate. Forages were placed on overhead feeders at 09:00 and 21:00 h and feed delivery was hourly. Following a 5 d period of hourly feeding, rumen contents were sampled for determination of background Co concentrations. Then, a solution of Co-EDTA solution (50 ml containing 0.5 g of Co-EDTA or 68 mg of Co) was dosed into the ventral rumen via the rumen cannula. Rumen contents were sampled at 1, 2, 4, 7, 9, 12, 24 h after Co dosing and frozen immediately after collection. Analysis of Co was after samples were thawed and centrifuged (20,000×g for 10 min at 20 ◦ C) to obtain the supernatant. Co concentration was analysed using inductively coupled plasma optical emission spectrometry (model Optima 2000 DV; Perkin Elmer, Inc., Waltham, MA, USA; Varian Techtron, 1979; Boumans, 1980). 2.7. Rumen fermentation parameters Measurements of rumen pH, and VFA and ammonia concentrations were made on the final day of the digestibility period when the rumen-fistulated sheep were fed twice daily. Rumen fluid samples (∼20 ml) were taken via rumen fistula at 09:00, 10:00, 11:00, 12:00, 13:00 and 15:00 h (i.e., 1, 2, 3, 4, 6 h post-feeding). pH was determined immediately (PHM210 standard pH meter; Radiometer Analytical, France) and then the sample was centrifuged (20,000×g for 5 min at 4 ◦ C) and 0.9 ml of supernatant added to 0.1 ml of acid solution containing 20 mg of ortho-phosphoric acid and 20.0 mM 2-ethyl butyric acid as internal standard and subsequently frozen (−20 ◦ C). Samples were thawed, centrifuged (20,000×g for 5 min at 4 ◦ C) and 800 l used for VFA analyses using a Hewlett Packard HP6890 Series GC system with a HP6890 series injector as described by Tavendale et al. (2005). Remaining sample was used for ammonia determination based on the nitroprusside method of Weatherburn (1967). The rumen sample collected at 11:00 h was used for protozoa counting. Rumen contents (50 g) were added to 50 ml of methyl green fixative containing 4 g of formaldehyde, 0.6 g of methyl green and 8 g of NaCl/L, mixed and stored in the dark at 4 ◦ C. Before counting, the fixed rumen contents were diluted (1:1) with 10 mM phosphate buffered saline (pH 7.2) and transferred to a Fuchs-Rosenthal counting chamber (0.6 l) under the bright field of a Leica microscope with 200 times magnification using the procedure of Kouri et al. (2003). Counting was in 3 fields in each chamber with 8 chambers each sample. 2.8. In situ rumen DM degradation kinetics Due to the small size of the cannulae in the fistulated sheep, in situ rumen DM degradation kinetics of chicory and perennial ryegrass were determined in cattle using the method of Sun et al. (2010). The same chicory and perennial ryegrass used in the sheep study was frozen, minced and placed into Dacron bags for incubation in the rumen of two Friesian×Jersey cows fed cut perennial ryegrass pasture at 1.3 times maintenance ME requirements (Australian Agricultural Council, 1990). Cows were fed twice a day at 08:00 and 17:00 h. Forty incubation bags were filled with each forage (∼5 g DM/bag). Then, all bags were placed in the rumen and 4 bags from each forage removed after 2, 4, 6, 8, 10, 11, 13, 24 and 72 h, but the 0 h bags not incubated in the rumen. Incubated and 0 h bags were washed and freeze dried for residual DM analysis.
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2.9. Chemical composition analyses Dry matter of forages offered and refused and faeces was determined by oven-drying at 105 ◦ C for 16 h (Method 930.15; AOAC, 2005 and Method 925.10; AOAC, 1990). Neutral detergent fibre (aNDF) and acid detergent fibre (ADF) were sequentially determined using the Tecator Fibretec System (Robertson and Van Soest, 1981) with cellulose calculated as ADF less lignin (sa) and hemicellulose as aNDF less ADF. Total N was determined by a Leco analyser (AC350, Leco Corporation, St. Joseph, MI, USA) in a total combustion method (Method 968.06; AOAC, 2000). Organic matter (OM) was measured by ashing samples in a furnace at 500 ◦ C for 16 h (Method 942.05; AOAC, 1990). Hot water soluble carbohydrates (HWSC) and pectin were extracted by the method of Blumenkrantz and Asboe Hansen (1973). The ME content of the forages was estimated using near infrared reflectance spectroscopy (FeedTECH, AgResearch, Palmerston North, New Zealand) as described by Sun et al. (2010) and used to determine feed allowance. 2.10. Data calculation and statistical analysis Rumen liquid passage rate was calculated using Eq. (1) (Faichney, 2005) with the NLIN procedure of SAS (2003) as: Ct = C0 e−kt
(1)
where Ct is the concentration of Co in the rumen (mg/L) at time t (h), C0 is the initial concentration of Co (mg/L) at 0 h and k is the rumen liquid passage rate (/h). Rumen liquid pool size was calculated using Eq. (2) by Faichney (2005) as: V=
D C0
(2)
where V is rumen liquid pool size (L), D is the amount of Co added to the rumen (mg) and C0 is Co concentration in the marker (mg/L) at 0 h. In situ rumen DM degradation kinetics parameters were calculated using Eq. (3) by Ørskov and McDonald (1979) as: P = A + Be−kt
(3)
where P is DM loss (mg/g) at time t (h), A is the soluble fraction (mg/g) which was calculated from DM disappearance at 0 h before estimation of other parameters, B is the potentially degradable fraction (mg/g) and k is the degradation rate (/h) of the B fraction. The indigestible fraction C was calculated as 1 − (A + B). The in situ rumen DM degradation kinetics were estimated using the NLIN procedure (SAS, 2003). Comparison between chicory and perennial ryegrass for chemical composition and in situ rumen DM degradation kinetics used one way ANOVA (SAS, 2003). All other variables were analysed using factorial ANOVA with forage species and feeding level as experimental factors and least significant differences were used to test differences between treatments at P=0.05. 3. Results 3.1. Chemical composition of forages Both forages were in a leafy vegetative state throughout the experiment, and the composition of chicory and ryegrass differed substantially (Table 1). The DM concentrations averaged 119 g/kg for chicory and 165 g/kg for ryegrass. The OM concentration of chicory was lower than that of ryegrass. Chicory contained more water-soluble carbohydrate and pectin, but Table 1 Chemical composition of forage chicory and perennial ryegrass. Chemical constituent (g/kg DM except as noted)
Chicory (n = 3)
Perennial ryegrass (n = 3)
SEM
P
Dry matter (g/kg)a Organic matter Crude protein Hot water-soluble carbohydrates Pectin Readily fermentable carbohydratesb aNDF ADF Hemicellulose Cellulose RFC:SCc
119 804 114 153 75.2 228 239 188 51 106 1.48
165 873 197 114 10.4 124 423 218 204 191 0.31
9.0 3.7 4.7 3.3 4.0 1.9 5.1 4.2 3.0 9.3 0.089
<0.001 <0.001 <0.001 0.002 <0.001 <0.001 <0.001 0.003 <0.001 0.003 <0.001
DM: dry matter; aNDF: neutral detergent fibre assayed with a heat stable amylase and expressed inclusive of residual ash; ADF: acid detergent fibre expressed inclusive of residual ash. a n = 18. b Hot water-soluble carbohydrates plus pectin. c Ratio of readily fermentable carbohydrates: structural carbohydrates (hemicellulose + cellulose).
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Table 2 Methane emissions from sheep fed fresh forage chicory or perennial ryegrass at 1.3 and 2.2× maintenance metabolisable energy requirement. Chicory
DM intake (g/d) CH4 (g/d) CH4 (g/kg DM intake) CH4 (g/kg digestible DM intake) CH4 (g/kg digestible OM intake)
Perennial ryegrass
P
1.3×
2.2×
1.3×
2.2×
SEM
Forage
Feeding level
Forage×Feeding level
666 15.1 22.6 29.5 27.2
1114 23.8 21.4 28.1 25.8
685 17.5 25.6 34.6 33.1
1053 22.7 21.5 28.5 27.5
29.8 0.74 0.59 2.44 2.30
0.626 0.521 0.090 0.030 0.004
<0.001 <0.001 0.008 0.006 0.006
0.362 0.112 0.107 0.058 0.066
DM: dry matter; OM: organic matter. n = 16.
less aNDF than perennial ryegrass, whereas crude protein (CP) concentration of chicory was only 0.6 times that of perennial ryegrass. The ratio of readily fermentable carbohydrates (RFC) to structural carbohydrates (i.e., cellulose and hemicellulose) in chicory forage was 4.8 times higher than of perennial ryegrass.
3.2. Methane yield The DM intakes of sheep fed chicory during methane measurements were 666 and 1114 g/d, and those fed ryegrass ate 685 and 1053 g/d, at the low and high feeding levels while OM intakes were 548, 916, 604 and 929 g/d for the respective groups (Table 2). There were no differences between forages in methane yield (i.e., g CH4 /kg DM intake), which averaged 24.1 and 21.4 for chicory and for ryegrass, respectively. Methane emissions expressed as g/kg digestible OM intake were lower (P=0.004) when wethers were fed chicory (26.3) compared to ryegrass (30.3). The high feeding level increased methane production (g/d) (P<0.001), but reduced methane yield (g/kg DM intake, g/kg digestible DM intake, and g/kg digestible OM intake) (P<0.01).
3.3. Apparent digestibility During the period of digestibility measurements, DM intakes were similar for both chicory and perennial ryegrass at each feeding level (Table 3). There were no interactions between forages and feeding level and no effect of feeding level on nutrient apparent digestibility, but chicory had a 2.3% higher DM digestibility (P=0.046) and a 6.8% higher OM digestibility (P<0.001) than did perennial ryegrass. In contrast, apparent digestibilities of CP (P<0.001) and aNDF (P<0.001) were lower for chicory than for perennial ryegrass.
3.4. Rumen liquid passage rate Rumen liquid volume of sheep fed chicory (Table 4) was smaller (P=0.009) than that of those fed perennial ryegrass (2.8 versus 3.9 L). Rumen liquid passage rate was faster (P=0.020) at the higher, than at the lower, feeding level (0.187 versus 0.128/h), but was similar for sheep fed chicory or perennial ryegrass. Total water intake from drinking and forage water was higher (P=0.011) for sheep fed at 2.2× versus 1.3× ME. Rumen fluid outflow (kg/d) was lower (P=0.009) from sheep fed chicory versus ryegrass, and outflow was increased more at 2.2× versus 1.3× in sheep fed ryegrass versus chicory.
Table 3 Dry matter intake and apparent digestibility of fresh forage chicory and perennial ryegrass fed to sheep at 1.3 and 2.2× maintenance metabolisable energy requirement. Chicory 1.3× Dry matter intake (g/d) Apparent digestibility (g/kg) Dry matter Organic matter Crude protein aNDF ADF
2.2×
Perennial ryegrass
P
1.3×
2.2×
SEM
Forage
Feeding level
Forage×Feeding level
700
1116
675
1080
25.7
0.356
<0.001
0.887
765 833 568 607 656
761 829 568 588 635
741 775 731 760 664
753 782 730 777 719
5.4 5.0 8.4 11.6 11.2
0.046 <0.001 <0.001 <0.001 0.072
0.722 0.809 0.981 0.988 0.492
0.318 0.507 0.739 0.331 0.746
DM: dry matter; aNDF: neutral detergent fibre assayed with a heat stable amylase and expressed inclusive of residual ash; ADF: acid detergent fibre expressed inclusive of residual ash. n = 24.
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Table 4 Rumen liquid volume and liquid passage rate in sheep fed fresh chicory or perennial ryegrass at 1.3 and 2.2× maintenance metabolisable energy requirement. Chicory 1.3× Rumen liquid volume (L) Rumen liquid passage rate (/h)a Water intake (kg/d) Drink Feed Total Rumen outflow (kg/d)b Net water balance (kg/d)c
2.71 0.133 0.3 4.9 5.2 8.8 b 3.6
2.2× 2.93 0.186 0.1 8.8 8.9 13.5 a 4.6
Perennial ryegrass
P
1.3×
SEM
2.2×
3.48 0.122
4.32 0.188
1.9 3.9 5.8 10.3 b 7.2
Forage
0.160 0.0112
1.8 8.0 9.8 19.9 a 10.1
0.60 0.05 0.60 0.59 4.62
Feeding level
Forage×Feeding level
0.009 0.794
0.079 0.020
0.242 0.715
0.129 <0.001 0.447 0.009 0.015
0.844 <0.001 0.011 <0.001 0.081
0.942 0.269 0.865 0.041 0.417
Means with different letters (a, b) with a forage type differ (P<0.05). n = 8. a Expressed as a proportion of total rumen liquid volume. b Rumen liquid volume×liquid passage rate. c Calculated rumen outflow – total drunk and in feed. Table 5 In situ ruminal degradation parameters of forage chicory and perennial ryegrass dry matter.a
Soluble fraction A (g/kg DM)b Potentially degradable fraction B (g/kg DM) Indigestible fraction C (g/kg DM) Dry matter degradation rate k (/h)
Chicory
Perennial ryegrass
SEM
P
0.553 0.434 0.013 0.144
0.580 0.372 0.048 0.103
0.0074 0.0043 0.0043 0.0069
0.040 <0.001 0.001 0.006
DM: dry matter. a In situ ruminal incubations used 4 replicates in the rumens of two cows fed perennial ryegrass. b Soluble fraction A calculated from DM disappearance at the 0 h incubation.
3.5. The in situ degradation of forages in cattle The in situ DM degradation parameters measured in cows showed that chicory has less proportions of soluble and indigestible fractions, but the proportion of potentially degradable fraction 17% more and DM degradation rate 40% faster than perennial ryegrass (P<0.01; Table 5). 3.6. Rumen fermentation parameters and protozoa populations There were no effects of forage type, feeding level and their interaction on rumem fluid pH, total VFA concentration, proportions of acetate, propionate and n-butyrate, and ratio of acetate to propionate (Table 6). Although the proportion of minor VFA including iso-butyrate, iso-valerate and n-valerate was statistically significantly lower with chicory than with ryegrass (P<0.001), the difference was only 1.0 mol/100 mol. Mean ammonia concentrations in the rumen fluid of wethers were lower (P=0.001) when fed chicory compared with perennial ryegrass, but feeding level had no effect (P=0.299). There was no interaction between forage type and feeding level for protozoa counts in the rumen fluid (P=0.532). Sheep fed chicory tended to have higher protozoa counts in their rumen (2.9 ± 0.4×105 /ml) than those fed perennial ryegrass (1.9 ± 0.2×105 /ml) (P=0.102). There was no effect of feeding level for rumen protozoa counts (P=0.955). Table 6 pH, the concentrations of ammonia and total volatile fatty acids (VFA), the molar proportions of individual VFA and protozoa counts in the rumen fluid of wethers fed fresh forage chicory or perennial ryegrass at 1.3 and 2.2× maintenance metabolisable energy requirement (mean of 6 sampling times at 09:00, 10:00, 11:00, 12:00, 13:00, 15:00 h, samples for protozoa counting collected only at 11:00 h). Chicory
pH Ammonia (mM) Total VFA (mM) VFA (mol/100 mol) Acetate Propionate n-butyrate Minor VFA Acetate/propionate Protozoa counts (×105 /ml)
Perennial ryegrass
P
1.3×
2.2×
1.3×
2.2×
SEM
6.44 9.8 95.2
6.30 5.6 105.9
6.35 28.3 83.8
6.18 27.1 92.2
0.178 3.11 10.52
64.5 20.4 12.4 1.9 3.21 2.65
66.5 18.6 12.8 1.5 3.75 3.06
66.9 18.3 10.9 2.7 3.67 2.02
66.5 19.8 10.1 2.6 3.38 1.68
1.52 1.28 0.56 0.17 0.359 0.652
Minor VFA includes iso-butyrate, iso-valerate and n-valerate. n = 8.
Forage
Feeding level
Forage×Feeding level
0.612 0.001 0.137
0.431 0.299 0.205
0.937 0.415 0.640
0.472 0.731 0.019 <0.001 0.606 0.102
0.616 0.902 0.732 0.272 0.946 0.955
0.457 0.270 0.357 0.437 0.289 0.532
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4. Discussion 4.1. Methane emissions from sheep fed chicory This study, using forages in vegetative stage, showed no differences in methane yield from sheep as g/kg DM intake between chicory and perennial ryegrass. This is consistent with our previous study in which mature chicory and ryegrass were fed to sheep (Sun et al., 2011), but contrasts with findings of Waghorn et al. (2002) and Swainson et al. (2008), who reported up to 30% lower methane yields from chicory than from perennial ryegrass dominated pasture. The difference between the findings of our study and Waghorn et al. (2002) and Swainson et al. (2008) may be due to differences in experimental situations and the techniques. For example, we used the respiration calorimetry technique, whereas Waghorn et al. (2002) and Swainson et al. (2008) used the SF6 tracer technique. Furthermore, there were some factors, including animal type and age, botanical and chemical compositions of diets, season and feeding level that differed between these two previous studies and our study. Consequently, these differences in experimental conditions make it difficult to directly compare the results. Our previous study (Sun et al., 2011) was conducted under similar conditions to the present one. For example, animal species, sex, age and liveweight were similar, and the feeding level in the previous study was similar to the lower level (1.3× ME maintenance) in the present study. Furthermore, in both experiments methane emission and feed intake were measured using the same protocol. However, the major difference between these two studies was the phenology of the forages used (i.e., vegetative state in the present study versus pre-flowering or flowering state in the previous study). With forages in a reproductive stage, methane yields were 22.8 and 23.8 g/kg DM intake for chicory and ryegrass, respectively (Sun et al., 2011); whereas for forages in a vegetative stage (present study) the corresponding methane yields were 22.0 and 23.6 g/kg DM intake. Thus, it seems that methane yield is not influenced by forage species or maturity. The latter is consistent with lack of difference in methane production when these forages were incubated in vitro using both vegetative and reproductive materials (Sun et al., 2011). 4.2. Feeding level and methane emissions That methane yields as g/kg DM intake decreased with an increase in feeding level is in agreement with other studies of Blaxter and Clapperton (1965), Yan et al. (2009) and Muetzel et al. (2009). Yan et al. (2009) summarised methane emission data from 108 growing-to-finishing beef steers measured using respiration chambers and found methane yield (g CH4 /kg DM intake) was negatively correlated to increasing feeding level. Muetzel et al. (2009) fed non-lactating and lactating ewes at 1.0–2.5 and 1.8–3.6 times maintenance ME requirements, respectively, and reported that feeding level explained 53% of the total variation in methane yield. Our results with wethers fed at 1.3 and 2.2× maintenance ME requirements are similar where, at the high feeding level, the wethers emitted less methane (g/kg DM intake) than those fed at the low level. 4.3. Feed digestion and methane emissions Compared with perennial ryegrass, chicory contains a large amount of readily fermentable carbohydrates, and much lower level of structural carbohydrates, which are rich in pectin (Sun et al., 2006), whereas perennial ryegrass contains little pectin (Carpita, 1996). Despite large differences in chemical composition, these forages did not differ in methane yield (g/kg DM intake). Hammond et al. (2009) analysed over 3000 methane emission records from sheep fed fresh perennial ryegrass and found that chemical composition explained less than 2% of the total variation in methane yields estimated using the SF6 tracer technique, but 20% when respiration chambers were used. Thus, excluding the secondary metabolites, it seems that conventional chemical composition of chicory and ryegrass has little effect on methane yield. That DMD and OMD were higher for chicory than perennial ryegrass is in agreement with Barry (1998) and Waghorn et al. (2002). An effect of apparent feed digestibility on methane emission did not occur in our study, confirming reports ˜ et al., 2003b) and sheep (Molano and Clark, 2008). from previous studies with cattle (Pinares-Patino Although methane yield (g/kg DM intake) was higher at the low than high feeding level, feeding level had no effects on rumen fluid pH and VFA. This suggested rumen fermentation was not a reason for the difference in methane yield between two levels of feeding. There was no difference in rumen protozoal counts between the two levels of feeding, but there was a tendency for protozoa counts to increase in the rumen of sheep fed chicory than those fed ryegrass. Although protozoa numbers are generally considered to be positively related to methane emissions, a number of reports did not find such a relationship (e.g., Morgavi et al., 2010). Our data are not supportive of a protozoal association with methane emissions. Digesta retention time, either total mean retention time or ruminal retention time, has a central role in the digestion ˜ et al. (2003a) and Hegarty (2004) found that retention time of digesta in the rumen is related to process. Pinares-Patino methane emissions (MJ CH4 /MJ gross energy intake). Ushida et al. (1997) proposed that a faster rumen fluid fractional outflow rate may have a washing out effect on methanogens, resulting in lower methanogen numbers and a consequent reduction in methane production, although methanogen activity has recently been suggested to be more important than numbers (Morgavi et al., 2010). In our study, fluid outflow rate of sheep chicory was almost the same as that of those fed
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perennial ryegrass. However, fluid outflow rate was higher for the high feeding level, suggesting that rumen fluid outflow rate was the mechanism for the effect of feeding level on methane yield. 5. Conclusions Despite differences in chemical composition, feed digestibility and rumen fermentation parameters between chicory and perennial ryegrass forages, no differences occurred in methane yield expressed per unit DM intake. These findings contrast with previous studies which reported reduced emissions with chicory feeding. However differences in methodology and animal characteristics between the current study and previous work could be responsible for the different outcomes. Nevertheless, our results confirm previous reports that increased feeding level of forages leads to decreases in methane yield, per unit DM intake. Acknowledgements We thank Dr. Chris Grainger and Professors Garry Waghorn and Tom Barry for suggestions either in experimental design or in results interpretation and for assistance with the manuscript; Dr’s John Koolaard and Dongwen Luo for advice in statistical analysis; Sarah Maclean, Edgar Sandoval, Natalie Butcher and Grant Taylor for assistance with animal feeding and sampling. This study was funded by the New Zealand Pastoral Greenhouse Gas Research Consortium (PGgRc). Xuezhao Sun thanks the New Zealand Foundation for Research, Science and Technology (FRST) for a postdoctoral fellowship. Guiguo Zhang thanks the New Zealand government for a Livestock Emissions & Abatement Research Network (MAF-LEARN) fellowship. References AOAC, 1990. Official Method of Analysis, 15th ed. Association of Official Analytical Chemists, Washington, DC, USA. AOAC, 2000. Official Method of Analysis, vol. I., 17th ed. Association of Official Analytical Chemists, Inc, MD, USA. AOAC, 2005. Official Methods of Analysis of AOAC International, 18th ed. AOAC International, Gaithersburg, MD, USA. Australian Agricultural Council, 1990. Feeding Standards for Australian livestock – Ruminants. Ruminants Subcommittee, Australian Agricultural Council. CSIRO Publications, Sydney, Australia. Barry, T.N., 1998. The feeding value of chicory (Cichorium intybus) for ruminant livestock. J. Agric. Sci. 131, 251–257. Beauchemin, K.A., Kreuzer, M., O’Mara, F., McAllister, T.A., 2008. Nutritional management for enteric methane abatement: a review. Aust. J. Exp. Agric. 48, 21–27. Blaxter, K.L., Clapperton, J.L., 1965. Prediction of the amount of methane produced by ruminants. Brit. J. Nutr. 19, 511–522. Blumenkrantz, N., Asboe Hansen, G., 1973. New method for quantitative determination of uronic acids. Anal. Biochem. 54, 484–489. Boumans, P., 1980. Line Coincidence for ICPAES Spectroscopy, vols. 1 and 2. Pergamon Press, Oxford, England. Carpita, N.C., 1996. Structure and biogenesis of the cell walls of grasses. Annu. Rev. Physiol. Plant Mol. Biol. 47, 445–476. Faichney, G.J., 2005. Digesta flow. In: Dijkstra, J., Forbes, J.M., France, J. (Eds.), Quantitative Aspects of Ruminant Digestion and Metabolism. , 2nd ed. CAB International Wallingford, Oxfordshire, UK, pp. 49–86. ˜ C.S., Burke, J.L., Hoskin, S.O., 2009. The variation in methane emissions from sheep and cattle is Hammond, K.J., Muetzel, S., Waghorn, G.C., Pinares-Patino, not explained by the chemical composition of ryegrass. Proc. N. Z. Soc. Anim. Prod. 69, 174–178. Hegarty, R.S., 2004. Genotype differences and their impact on digestive tract function of ruminants: a review. Aust. J. Exp. Agric. 44, 459–467. Kouri, T., Gyory, A., Rowan, R.M., Fernandes, B., DeMatteo, T., Dotson, M., Koontz, A., Michaels, A., Laippala, P., 2003. ISLH recommended reference procedure for the enumeration of particles in urine. Lab. Hematol. 9, 58–63. Kusmartono, Shimada, A., Barry, T.N., 1997. Rumen digestion and rumen outflow rate in deer fed fresh chicory (Cichorium intybus) or perennial ryegrass (Lolium perenne). J. Agric. Sci. 128, 87–94. Li, G., Kemp, P.D., 2005. Forage Chicory (Cichorium intybus L.): a review of its agronomy and animal production. Adv. Agron. 88, 187–222. Ministry for the Environment, N.Z., 2009. New Zealand’s Greenhouse Gas Inventory 1990–2007, Wellington, New Zealand. Molano, G., Clark, H., 2008. The effect of level of intake and forage quality on methane production by sheep. Aust. J. Exp. Agric. 48, 219–222. Morgavi, D.P., Forano, E., Martin, C., Newbold, C.J., 2010. Microbial ecosystem and methanogenesis in ruminants. Animal 4, 1024–1036. Muetzel, S., Knight, T.W., Hoskin, S.O., Molano, G., Maclean, S., Silva-Villacorta, D., Clark, H., 2009. Level of intake and physiological state influences methane emissions from sheep fed fresh pasture. In: Chilliard, Y., Glasser, F., Faulconnier, Y., Bocquier, F., Veissie, I., Doreau, M. (Eds.), Ruminant physiology: digestion, metabolism, and effects of nutrition on reproduction and welfare. Proc. of the XIth Int. Symp. on Ruminant Physiology. Wageningen Academic Publishers, Clermont-Ferrand, France, pp. 90–91. Ørskov, E.R., McDonald, I., 1979. The estimation of protein degradability in the rumen from incubation measurements weighed according to rate of passage. J. Agric. Sci. 92, 499–503. ˜ C.S., Baumont, R., Martin, C., 2003b. Methane emissions by Charolais cows grazing a monospecific pasture of timothy at four stages of Pinares-Patino, maturity. Can. J. Anim. Sci. 83, 769–777. ˜ C.S., Lassey, K.R., Martin, R.J., Molano, G., Fernandez, M., MacLean, S., Sandoval, E., Luo, D., Clark, H., 2011. Assessment of the sulphur Pinares-Patino, hexafluoride (SF6 ) tracer technique using respiration chambers for estimation of methane emissions from sheep. Anim. Feed Sci. Technol. 166–167, 201–209. ˜ C.S., Lovejoy, P., Hunt, C., Martin, R., Molano, G., Waghorn, G.C., Clark, H., 2008. A versatile sheep respiration chamber system for measurement Pinares-Patino, of methane emission. Proc. N. Z. Soc. Anim. Prod. 68, 29–30. ˜ C.S., Ulyatt, M.J., Lassey, K.R., Barry, T.N., Holmes, C.W., 2003a. Rumen function and digestion parameters associated with differences between Pinares-Patino, sheep in methane emissions when fed chaffed lucerne hay. J. Agric. Sci. 140, 205–214. Robertson, J.B., Van Soest, P.J., 1981. The detergent system of analysis and its application to human foods. In: James, W.P.T., Theander, O. (Eds.), The Analysis of Dietary Fiber in Food. Marcel Dekker, New York, NY, USA, pp. 123–158. SAS, 2003. Statistical Analysis System. User’s Guide: Statistics, SAS Institute, Cary, NC, USA. Sun, X.Z., Andrew, I.G., Joblin, K.N., Harris, P.J., McDonald, A., Hoskin, S.O., 2006. Polysaccharide compositions of leaf cell walls of forage chicory (Cichorium intybus L.). Plant Sci. 170, 18–27. Sun, X.Z., Hoskin, S.O., Muetzel, S., Molano, G., Clark, H., 2011. Effects of chicory (Cichorium intybus) and perennial ryegrass (Lolium perenne) on methane emissions in vitro and from sheep. Anim. Feed Sci. Technol. 166–167, 391–397. Sun, X.Z., Waghorn, G.C., Clark, H., 2010. Cultivar and age of regrowth effects on physical, chemical and in sacco degradation kinetics of vegetative perennial ryegrass (Lolium perenne L.). Anim. Feed Sci. Technol. 155, 172–185.
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Sheep fed forage chicory (Cichorium intybus) or perennial ryegrass (Lolium perenne) have similar methane emissions ˜ X.Z. Sun ∗ , S.O. Hoskin, G.G. Zhang 1 , G. Molano, S. Muetzel, C.S. Pinares-Patino, H. Clark, D. Pacheco Grasslands Research Centre, AgResearch Limited, Private Bag 11008, Palmerston North 4442, New Zealand
a r t i c l e
i n f o
Article history: Received 24 May 2011 Received in revised form 14 November 2011 Accepted 15 November 2011
Keywords: Forage chicory Perennial ryegrass Methane Sheep Feeding level Rumen outflow rate
a b s t r a c t Forage chicory (Cichorium intybus) has the potential to mitigate methane emissions from ruminants. It was reported that the reduction can be up to 30% compared with perennial ryegrass (Lolium perenne). To accurately evaluate the reduction, fresh chicory and perennial ryegrass in the vegetative state were fed to 24 wethers, 8 of which rumen-fistulated, at 1.3 and 2.2 times maintenance metabolisable energy requirements. Dry matter (DM) intake, whole tract apparent digestibility, rumen fermentation parameters and rumen liquid passage rate were measured in metabolism crates, and methane emissions determined using a calorimetric technique. Chemical analyses showed that chicory contained less DM, organic matter (OM), crude protein, neutral detergent fibre (aNDF), acid detergent fibre (ADF), cellulose and hemicellulose, but more hot water-soluble carbohydrate and pectin, than perennial ryegrass. Methane yield (g/kg DM intake) of wethers fed chicory did not differ from that of those fed perennial ryegrass. Yield was lower at the high versus the low feeding level of ryegrass. Apparent digestibility of DM and OM was higher, and aNDF, ADF, hemicellulose and cellulose was lower, in wethers fed chicory versus perennial ryegrass. In situ DM degradation rate of chicory was higher than that of perennial ryegrass. Rumen liquid passage rate was the same for wethers fed the two forages and higher at the high feeding level. The reduction in methane emissions by feeding vegetative chicory to wethers was limited, but increased feeding level reduces methane yields per unit of DM intake. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Emissions of methane from grazed ruminants accounts for 31% of CO2 equivalents from the New Zealand greenhouse gas emissions inventory (Ministry for the Environment, 2009). In a recent review of effects of nutritional management to abate enteric methane emissions it was concluded that methane emissions from dairy cows and sheep fed some specific forage plants were sometimes lower than emissions from those fed grasses (Beauchemin et al., 2008). Forage chicory (Cichorium intybus), a plant of the dicotyledons family Asteraceae, is a palatable forage with a high feeding value, high dry matter (DM) yield and can be grown in New Zealand (Li and Kemp, 2005). Kusmartono et al. (1997) reported that deer fed chicory had faster rumen outflow rate than their counterparts fed perennial ryegrass, and faster rumen outflow rates have been linked
Abbreviations: ADF, acid detergent fibre; aNDF, neutral detergent fibre; C2/C3, ratio of acetate to propionate; Co-EDTA, cobalt ethylene diaminetetraacetic acid; DM, dry matter; HWSC, hot water soluble carbohydrates; ME, metabolisable energy; OM, organic matter; RFC, readily fermentable carbohydrates; SC, structural carbohydrates; VFA, volatile fatty acids. ∗ Corresponding author. Tel.: +64 6 351 8150; fax: +64 6 351 8032. E-mail addresses: [email protected], [email protected] (X.Z. Sun). 1 Present address: Department of Animal Sciences and Technology, Shandong Agricultural University, Tai’an, Shandong, 271018, PR China. 0377-8401/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2011.11.007
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˜ et al., 2003a; Hegarty, 2004). Rumen outflow rates and passage can also be to reduced methane production (Pinares-Patino altered by feed intake level (Volden, 1999) and methane yield per unit of DM intake declines with increased feed intakes (Blaxter and Clapperton, 1965; Yan et al., 2009). Reports of methane emissions from ruminants fed forage chicory are scarce, but in two studies (Waghorn et al., 2002; Swainson et al., 2008) sheep fed chicory emitted up to 30% less methane than those fed perennial ryegrass-based pasture, although more recent research suggests no difference in methane yield from sheep fed mature chicory or perennial ryegrass (Sun et al., 2011). The primary objective was to compare methane emissions from sheep fed vegetative forage chicory or perennial ryegrass at two levels of intake using open respiration calorimetry. A secondary objective was to measure in vivo and in situ digestion of the forages to determine causes of the expected differences in methane yield (CH4 /DM intake). 2. Materials and methods 2.1. Experimental design The study was conducted during autumn (8 April to 9 May, 2009) at AgResearch Grasslands (Palmerston North, New Zealand) with 24 Romney wether sheep, including 16 intact and 8 with rumen fistulate. The study was a 2×2 factorial design, using two forages (chicory and perennial ryegrass) and two levels of intake (1.2 and 2.2 times maintenance metabolisable energy (ME) requirement). Four intact and two rumen-fistulated sheep were randomly allocated to each treatment level. Sheep were acclimatised to the forage diets for 7 d while grazing and then acclimatised to the treatment levels over 21 d in pens (8 d) and metabolic crates (13 d). The experimental period was 11 d of feed intake, feed digestibility, rumen outflow and rumen fermentation parameters measurement, followed by 2 d of methane emission measurement in respiration chambers. After 21 d of feeding the respective forages at appropriate intakes, a 6 d faeces and urine collection was undertaken in all sheep to determine diet digestibility after which the sheep were transferred to modified metabolism crates and put into ˜ et al., 2008) for 48 h methane measurement. The sheep were released to pasture respiration calorimeters (Pinares-Patino following methane determinations. The procedure for the fistulated animals included measurements of rumen liquid turnover with rumen sampling for measurement of pH, volatile fatty acids (VFA) and ammonia. These measurements commenced when the sheep had been fed chicory or ryegrass for at least 18 d. For the rumen turnover measurement, the sheep were adapted to hourly feeding for 6 d, with forage supplied using automated belt-driven feeders. Following the rumen turnover measurements, the hourly feeding was discontinued for the digestibility and methane measurement periods when the fistulated sheep were fed twice daily. In addition to measurements with sheep, in sacco DM degradation kinetic studies were undertaken with two rumen fistulated non-lactating cattle fed cut ryegrass pasture. These measurements were completed after the sheep experiment, but using the same herbage, which had been stored frozen. Animal ethics approval for the study was granted by the AgResearch Animal Ethics Committee (Approval 11671). 2.2. Forages Forage chicory (cv. Choice) had been established about 8 mo prior to the experiment, and the last grazing was 6 wk prior to the experiment. Chicory fed to the sheep (grazed or harvested) was in the vegetative stage and ∼50 cm in height (from the ground to the top of plants). Perennial ryegrass (cv. Quartet) used in this study was established two years earlier and was in vegetative stage at the time of study (height ∼35 cm). The grass sward was grazed 4 wk prior to the start of this study. The grazed areas of chicory crop and perennial ryegrass pasture used for the 7 d acclimatisation to diets were 500 and 600 m2 . Herbage allowance during this period was set at generous levels. During the indoor feeding period, forages were harvested and stored as described by Sun et al. (2011). 2.3. Animals and feeding The 24 wethers were about 20 mo of age and weighed 50 ± 4.1 kg. Eight were fitted with a rumen cannula (30 mm internal diameter; Beruc Equipment Ltd., Benoni, South Africa) 4 mo prior to the study and two of them were allocated to each treatment group. At the start of the acclimatisation period, all sheep were drenched with a broad spectrum anthelmintic (Scanda; Schering-Plough Animal Health New Zealand Limited, Wellington, New Zealand). Following the 7 d dietary acclimatisation while grazing ryegrass or chicory, the sheep were fed twice daily throughout with the exception that the rumen fistulated ones were fed hourly from feeders placed above metabolism crates in order to enable measurement of rumen turnover. Indoors, feed was delivered to the non-fistulated animals in two equal meals at 09:00 and 16:30 h. Feeding level was set at 1.3 and 2.2 times maintenance ME requirements (Australian Agricultural Council, 1990) and this was maintained throughout the study. At daily arrival of cut forage, meals were weighed and, during this process, two samples of the herbages (∼200 g fresh weight) were collected and dried at 65 ◦ C for 48 h for chemical analysis. Refusals were collected daily prior to morning
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feeding, weighed and dried for determination of DM concentration as for the herbages offeral. Drinking water was available ad libitum. 2.4. Determination of methane emissions ˜ et al. Methane emissions were measured using the 8 chamber sheep respiration facility described by Pinares-Patino (2011). Sheep were placed in the chambers in a balanced way (i.e., at all times there were 2 sheep from each of the 4 treatments (2 feeding levels×2 forages)) in the respiration chambers. The measuring procedure, data calculation, chamber operation and routine management used the method described by Sun et al. (2011). 2.5. Apparent digestibility Apparent digestibility was calculated from measurements of feed intakes and faecal outputs over a 6 d period. Daily oven-dried (65 ◦ C, 48 h) feed refusals were pooled within sheep and then ground using a Wiley mill to pass a 1 mm screen for chemical analysis. Total faecal outputs were collected from each sheep using faecal collection harnesses. Faeces were collected daily before the morning feeding, weighed, sub-sampled and samples stored frozen at −20 ◦ C. Faecal samples were later pooled within sheep, oven-dried (65 ◦ C, 48 h) and ground in a Wiley mill to pass a 1 mm screen for later chemical analysis. 2.6. Rumen liquid passage rate A single dose of cobalt ethylene diaminetetraacetic acid (Co-EDTA) was used as a fluid phase marker to measure rumen liquid outflow rate. Forages were placed on overhead feeders at 09:00 and 21:00 h and feed delivery was hourly. Following a 5 d period of hourly feeding, rumen contents were sampled for determination of background Co concentrations. Then, a solution of Co-EDTA solution (50 ml containing 0.5 g of Co-EDTA or 68 mg of Co) was dosed into the ventral rumen via the rumen cannula. Rumen contents were sampled at 1, 2, 4, 7, 9, 12, 24 h after Co dosing and frozen immediately after collection. Analysis of Co was after samples were thawed and centrifuged (20,000×g for 10 min at 20 ◦ C) to obtain the supernatant. Co concentration was analysed using inductively coupled plasma optical emission spectrometry (model Optima 2000 DV; Perkin Elmer, Inc., Waltham, MA, USA; Varian Techtron, 1979; Boumans, 1980). 2.7. Rumen fermentation parameters Measurements of rumen pH, and VFA and ammonia concentrations were made on the final day of the digestibility period when the rumen-fistulated sheep were fed twice daily. Rumen fluid samples (∼20 ml) were taken via rumen fistula at 09:00, 10:00, 11:00, 12:00, 13:00 and 15:00 h (i.e., 1, 2, 3, 4, 6 h post-feeding). pH was determined immediately (PHM210 standard pH meter; Radiometer Analytical, France) and then the sample was centrifuged (20,000×g for 5 min at 4 ◦ C) and 0.9 ml of supernatant added to 0.1 ml of acid solution containing 20 mg of ortho-phosphoric acid and 20.0 mM 2-ethyl butyric acid as internal standard and subsequently frozen (−20 ◦ C). Samples were thawed, centrifuged (20,000×g for 5 min at 4 ◦ C) and 800 l used for VFA analyses using a Hewlett Packard HP6890 Series GC system with a HP6890 series injector as described by Tavendale et al. (2005). Remaining sample was used for ammonia determination based on the nitroprusside method of Weatherburn (1967). The rumen sample collected at 11:00 h was used for protozoa counting. Rumen contents (50 g) were added to 50 ml of methyl green fixative containing 4 g of formaldehyde, 0.6 g of methyl green and 8 g of NaCl/L, mixed and stored in the dark at 4 ◦ C. Before counting, the fixed rumen contents were diluted (1:1) with 10 mM phosphate buffered saline (pH 7.2) and transferred to a Fuchs-Rosenthal counting chamber (0.6 l) under the bright field of a Leica microscope with 200 times magnification using the procedure of Kouri et al. (2003). Counting was in 3 fields in each chamber with 8 chambers each sample. 2.8. In situ rumen DM degradation kinetics Due to the small size of the cannulae in the fistulated sheep, in situ rumen DM degradation kinetics of chicory and perennial ryegrass were determined in cattle using the method of Sun et al. (2010). The same chicory and perennial ryegrass used in the sheep study was frozen, minced and placed into Dacron bags for incubation in the rumen of two Friesian×Jersey cows fed cut perennial ryegrass pasture at 1.3 times maintenance ME requirements (Australian Agricultural Council, 1990). Cows were fed twice a day at 08:00 and 17:00 h. Forty incubation bags were filled with each forage (∼5 g DM/bag). Then, all bags were placed in the rumen and 4 bags from each forage removed after 2, 4, 6, 8, 10, 11, 13, 24 and 72 h, but the 0 h bags not incubated in the rumen. Incubated and 0 h bags were washed and freeze dried for residual DM analysis.
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2.9. Chemical composition analyses Dry matter of forages offered and refused and faeces was determined by oven-drying at 105 ◦ C for 16 h (Method 930.15; AOAC, 2005 and Method 925.10; AOAC, 1990). Neutral detergent fibre (aNDF) and acid detergent fibre (ADF) were sequentially determined using the Tecator Fibretec System (Robertson and Van Soest, 1981) with cellulose calculated as ADF less lignin (sa) and hemicellulose as aNDF less ADF. Total N was determined by a Leco analyser (AC350, Leco Corporation, St. Joseph, MI, USA) in a total combustion method (Method 968.06; AOAC, 2000). Organic matter (OM) was measured by ashing samples in a furnace at 500 ◦ C for 16 h (Method 942.05; AOAC, 1990). Hot water soluble carbohydrates (HWSC) and pectin were extracted by the method of Blumenkrantz and Asboe Hansen (1973). The ME content of the forages was estimated using near infrared reflectance spectroscopy (FeedTECH, AgResearch, Palmerston North, New Zealand) as described by Sun et al. (2010) and used to determine feed allowance. 2.10. Data calculation and statistical analysis Rumen liquid passage rate was calculated using Eq. (1) (Faichney, 2005) with the NLIN procedure of SAS (2003) as: Ct = C0 e−kt
(1)
where Ct is the concentration of Co in the rumen (mg/L) at time t (h), C0 is the initial concentration of Co (mg/L) at 0 h and k is the rumen liquid passage rate (/h). Rumen liquid pool size was calculated using Eq. (2) by Faichney (2005) as: V=
D C0
(2)
where V is rumen liquid pool size (L), D is the amount of Co added to the rumen (mg) and C0 is Co concentration in the marker (mg/L) at 0 h. In situ rumen DM degradation kinetics parameters were calculated using Eq. (3) by Ørskov and McDonald (1979) as: P = A + Be−kt
(3)
where P is DM loss (mg/g) at time t (h), A is the soluble fraction (mg/g) which was calculated from DM disappearance at 0 h before estimation of other parameters, B is the potentially degradable fraction (mg/g) and k is the degradation rate (/h) of the B fraction. The indigestible fraction C was calculated as 1 − (A + B). The in situ rumen DM degradation kinetics were estimated using the NLIN procedure (SAS, 2003). Comparison between chicory and perennial ryegrass for chemical composition and in situ rumen DM degradation kinetics used one way ANOVA (SAS, 2003). All other variables were analysed using factorial ANOVA with forage species and feeding level as experimental factors and least significant differences were used to test differences between treatments at P=0.05. 3. Results 3.1. Chemical composition of forages Both forages were in a leafy vegetative state throughout the experiment, and the composition of chicory and ryegrass differed substantially (Table 1). The DM concentrations averaged 119 g/kg for chicory and 165 g/kg for ryegrass. The OM concentration of chicory was lower than that of ryegrass. Chicory contained more water-soluble carbohydrate and pectin, but Table 1 Chemical composition of forage chicory and perennial ryegrass. Chemical constituent (g/kg DM except as noted)
Chicory (n = 3)
Perennial ryegrass (n = 3)
SEM
P
Dry matter (g/kg)a Organic matter Crude protein Hot water-soluble carbohydrates Pectin Readily fermentable carbohydratesb aNDF ADF Hemicellulose Cellulose RFC:SCc
119 804 114 153 75.2 228 239 188 51 106 1.48
165 873 197 114 10.4 124 423 218 204 191 0.31
9.0 3.7 4.7 3.3 4.0 1.9 5.1 4.2 3.0 9.3 0.089
<0.001 <0.001 <0.001 0.002 <0.001 <0.001 <0.001 0.003 <0.001 0.003 <0.001
DM: dry matter; aNDF: neutral detergent fibre assayed with a heat stable amylase and expressed inclusive of residual ash; ADF: acid detergent fibre expressed inclusive of residual ash. a n = 18. b Hot water-soluble carbohydrates plus pectin. c Ratio of readily fermentable carbohydrates: structural carbohydrates (hemicellulose + cellulose).
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Table 2 Methane emissions from sheep fed fresh forage chicory or perennial ryegrass at 1.3 and 2.2× maintenance metabolisable energy requirement. Chicory
DM intake (g/d) CH4 (g/d) CH4 (g/kg DM intake) CH4 (g/kg digestible DM intake) CH4 (g/kg digestible OM intake)
Perennial ryegrass
P
1.3×
2.2×
1.3×
2.2×
SEM
Forage
Feeding level
Forage×Feeding level
666 15.1 22.6 29.5 27.2
1114 23.8 21.4 28.1 25.8
685 17.5 25.6 34.6 33.1
1053 22.7 21.5 28.5 27.5
29.8 0.74 0.59 2.44 2.30
0.626 0.521 0.090 0.030 0.004
<0.001 <0.001 0.008 0.006 0.006
0.362 0.112 0.107 0.058 0.066
DM: dry matter; OM: organic matter. n = 16.
less aNDF than perennial ryegrass, whereas crude protein (CP) concentration of chicory was only 0.6 times that of perennial ryegrass. The ratio of readily fermentable carbohydrates (RFC) to structural carbohydrates (i.e., cellulose and hemicellulose) in chicory forage was 4.8 times higher than of perennial ryegrass.
3.2. Methane yield The DM intakes of sheep fed chicory during methane measurements were 666 and 1114 g/d, and those fed ryegrass ate 685 and 1053 g/d, at the low and high feeding levels while OM intakes were 548, 916, 604 and 929 g/d for the respective groups (Table 2). There were no differences between forages in methane yield (i.e., g CH4 /kg DM intake), which averaged 24.1 and 21.4 for chicory and for ryegrass, respectively. Methane emissions expressed as g/kg digestible OM intake were lower (P=0.004) when wethers were fed chicory (26.3) compared to ryegrass (30.3). The high feeding level increased methane production (g/d) (P<0.001), but reduced methane yield (g/kg DM intake, g/kg digestible DM intake, and g/kg digestible OM intake) (P<0.01).
3.3. Apparent digestibility During the period of digestibility measurements, DM intakes were similar for both chicory and perennial ryegrass at each feeding level (Table 3). There were no interactions between forages and feeding level and no effect of feeding level on nutrient apparent digestibility, but chicory had a 2.3% higher DM digestibility (P=0.046) and a 6.8% higher OM digestibility (P<0.001) than did perennial ryegrass. In contrast, apparent digestibilities of CP (P<0.001) and aNDF (P<0.001) were lower for chicory than for perennial ryegrass.
3.4. Rumen liquid passage rate Rumen liquid volume of sheep fed chicory (Table 4) was smaller (P=0.009) than that of those fed perennial ryegrass (2.8 versus 3.9 L). Rumen liquid passage rate was faster (P=0.020) at the higher, than at the lower, feeding level (0.187 versus 0.128/h), but was similar for sheep fed chicory or perennial ryegrass. Total water intake from drinking and forage water was higher (P=0.011) for sheep fed at 2.2× versus 1.3× ME. Rumen fluid outflow (kg/d) was lower (P=0.009) from sheep fed chicory versus ryegrass, and outflow was increased more at 2.2× versus 1.3× in sheep fed ryegrass versus chicory.
Table 3 Dry matter intake and apparent digestibility of fresh forage chicory and perennial ryegrass fed to sheep at 1.3 and 2.2× maintenance metabolisable energy requirement. Chicory 1.3× Dry matter intake (g/d) Apparent digestibility (g/kg) Dry matter Organic matter Crude protein aNDF ADF
2.2×
Perennial ryegrass
P
1.3×
2.2×
SEM
Forage
Feeding level
Forage×Feeding level
700
1116
675
1080
25.7
0.356
<0.001
0.887
765 833 568 607 656
761 829 568 588 635
741 775 731 760 664
753 782 730 777 719
5.4 5.0 8.4 11.6 11.2
0.046 <0.001 <0.001 <0.001 0.072
0.722 0.809 0.981 0.988 0.492
0.318 0.507 0.739 0.331 0.746
DM: dry matter; aNDF: neutral detergent fibre assayed with a heat stable amylase and expressed inclusive of residual ash; ADF: acid detergent fibre expressed inclusive of residual ash. n = 24.
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Table 4 Rumen liquid volume and liquid passage rate in sheep fed fresh chicory or perennial ryegrass at 1.3 and 2.2× maintenance metabolisable energy requirement. Chicory 1.3× Rumen liquid volume (L) Rumen liquid passage rate (/h)a Water intake (kg/d) Drink Feed Total Rumen outflow (kg/d)b Net water balance (kg/d)c
2.71 0.133 0.3 4.9 5.2 8.8 b 3.6
2.2× 2.93 0.186 0.1 8.8 8.9 13.5 a 4.6
Perennial ryegrass
P
1.3×
SEM
2.2×
3.48 0.122
4.32 0.188
1.9 3.9 5.8 10.3 b 7.2
Forage
0.160 0.0112
1.8 8.0 9.8 19.9 a 10.1
0.60 0.05 0.60 0.59 4.62
Feeding level
Forage×Feeding level
0.009 0.794
0.079 0.020
0.242 0.715
0.129 <0.001 0.447 0.009 0.015
0.844 <0.001 0.011 <0.001 0.081
0.942 0.269 0.865 0.041 0.417
Means with different letters (a, b) with a forage type differ (P<0.05). n = 8. a Expressed as a proportion of total rumen liquid volume. b Rumen liquid volume×liquid passage rate. c Calculated rumen outflow – total drunk and in feed. Table 5 In situ ruminal degradation parameters of forage chicory and perennial ryegrass dry matter.a
Soluble fraction A (g/kg DM)b Potentially degradable fraction B (g/kg DM) Indigestible fraction C (g/kg DM) Dry matter degradation rate k (/h)
Chicory
Perennial ryegrass
SEM
P
0.553 0.434 0.013 0.144
0.580 0.372 0.048 0.103
0.0074 0.0043 0.0043 0.0069
0.040 <0.001 0.001 0.006
DM: dry matter. a In situ ruminal incubations used 4 replicates in the rumens of two cows fed perennial ryegrass. b Soluble fraction A calculated from DM disappearance at the 0 h incubation.
3.5. The in situ degradation of forages in cattle The in situ DM degradation parameters measured in cows showed that chicory has less proportions of soluble and indigestible fractions, but the proportion of potentially degradable fraction 17% more and DM degradation rate 40% faster than perennial ryegrass (P<0.01; Table 5). 3.6. Rumen fermentation parameters and protozoa populations There were no effects of forage type, feeding level and their interaction on rumem fluid pH, total VFA concentration, proportions of acetate, propionate and n-butyrate, and ratio of acetate to propionate (Table 6). Although the proportion of minor VFA including iso-butyrate, iso-valerate and n-valerate was statistically significantly lower with chicory than with ryegrass (P<0.001), the difference was only 1.0 mol/100 mol. Mean ammonia concentrations in the rumen fluid of wethers were lower (P=0.001) when fed chicory compared with perennial ryegrass, but feeding level had no effect (P=0.299). There was no interaction between forage type and feeding level for protozoa counts in the rumen fluid (P=0.532). Sheep fed chicory tended to have higher protozoa counts in their rumen (2.9 ± 0.4×105 /ml) than those fed perennial ryegrass (1.9 ± 0.2×105 /ml) (P=0.102). There was no effect of feeding level for rumen protozoa counts (P=0.955). Table 6 pH, the concentrations of ammonia and total volatile fatty acids (VFA), the molar proportions of individual VFA and protozoa counts in the rumen fluid of wethers fed fresh forage chicory or perennial ryegrass at 1.3 and 2.2× maintenance metabolisable energy requirement (mean of 6 sampling times at 09:00, 10:00, 11:00, 12:00, 13:00, 15:00 h, samples for protozoa counting collected only at 11:00 h). Chicory
pH Ammonia (mM) Total VFA (mM) VFA (mol/100 mol) Acetate Propionate n-butyrate Minor VFA Acetate/propionate Protozoa counts (×105 /ml)
Perennial ryegrass
P
1.3×
2.2×
1.3×
2.2×
SEM
6.44 9.8 95.2
6.30 5.6 105.9
6.35 28.3 83.8
6.18 27.1 92.2
0.178 3.11 10.52
64.5 20.4 12.4 1.9 3.21 2.65
66.5 18.6 12.8 1.5 3.75 3.06
66.9 18.3 10.9 2.7 3.67 2.02
66.5 19.8 10.1 2.6 3.38 1.68
1.52 1.28 0.56 0.17 0.359 0.652
Minor VFA includes iso-butyrate, iso-valerate and n-valerate. n = 8.
Forage
Feeding level
Forage×Feeding level
0.612 0.001 0.137
0.431 0.299 0.205
0.937 0.415 0.640
0.472 0.731 0.019 <0.001 0.606 0.102
0.616 0.902 0.732 0.272 0.946 0.955
0.457 0.270 0.357 0.437 0.289 0.532
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4. Discussion 4.1. Methane emissions from sheep fed chicory This study, using forages in vegetative stage, showed no differences in methane yield from sheep as g/kg DM intake between chicory and perennial ryegrass. This is consistent with our previous study in which mature chicory and ryegrass were fed to sheep (Sun et al., 2011), but contrasts with findings of Waghorn et al. (2002) and Swainson et al. (2008), who reported up to 30% lower methane yields from chicory than from perennial ryegrass dominated pasture. The difference between the findings of our study and Waghorn et al. (2002) and Swainson et al. (2008) may be due to differences in experimental situations and the techniques. For example, we used the respiration calorimetry technique, whereas Waghorn et al. (2002) and Swainson et al. (2008) used the SF6 tracer technique. Furthermore, there were some factors, including animal type and age, botanical and chemical compositions of diets, season and feeding level that differed between these two previous studies and our study. Consequently, these differences in experimental conditions make it difficult to directly compare the results. Our previous study (Sun et al., 2011) was conducted under similar conditions to the present one. For example, animal species, sex, age and liveweight were similar, and the feeding level in the previous study was similar to the lower level (1.3× ME maintenance) in the present study. Furthermore, in both experiments methane emission and feed intake were measured using the same protocol. However, the major difference between these two studies was the phenology of the forages used (i.e., vegetative state in the present study versus pre-flowering or flowering state in the previous study). With forages in a reproductive stage, methane yields were 22.8 and 23.8 g/kg DM intake for chicory and ryegrass, respectively (Sun et al., 2011); whereas for forages in a vegetative stage (present study) the corresponding methane yields were 22.0 and 23.6 g/kg DM intake. Thus, it seems that methane yield is not influenced by forage species or maturity. The latter is consistent with lack of difference in methane production when these forages were incubated in vitro using both vegetative and reproductive materials (Sun et al., 2011). 4.2. Feeding level and methane emissions That methane yields as g/kg DM intake decreased with an increase in feeding level is in agreement with other studies of Blaxter and Clapperton (1965), Yan et al. (2009) and Muetzel et al. (2009). Yan et al. (2009) summarised methane emission data from 108 growing-to-finishing beef steers measured using respiration chambers and found methane yield (g CH4 /kg DM intake) was negatively correlated to increasing feeding level. Muetzel et al. (2009) fed non-lactating and lactating ewes at 1.0–2.5 and 1.8–3.6 times maintenance ME requirements, respectively, and reported that feeding level explained 53% of the total variation in methane yield. Our results with wethers fed at 1.3 and 2.2× maintenance ME requirements are similar where, at the high feeding level, the wethers emitted less methane (g/kg DM intake) than those fed at the low level. 4.3. Feed digestion and methane emissions Compared with perennial ryegrass, chicory contains a large amount of readily fermentable carbohydrates, and much lower level of structural carbohydrates, which are rich in pectin (Sun et al., 2006), whereas perennial ryegrass contains little pectin (Carpita, 1996). Despite large differences in chemical composition, these forages did not differ in methane yield (g/kg DM intake). Hammond et al. (2009) analysed over 3000 methane emission records from sheep fed fresh perennial ryegrass and found that chemical composition explained less than 2% of the total variation in methane yields estimated using the SF6 tracer technique, but 20% when respiration chambers were used. Thus, excluding the secondary metabolites, it seems that conventional chemical composition of chicory and ryegrass has little effect on methane yield. That DMD and OMD were higher for chicory than perennial ryegrass is in agreement with Barry (1998) and Waghorn et al. (2002). An effect of apparent feed digestibility on methane emission did not occur in our study, confirming reports ˜ et al., 2003b) and sheep (Molano and Clark, 2008). from previous studies with cattle (Pinares-Patino Although methane yield (g/kg DM intake) was higher at the low than high feeding level, feeding level had no effects on rumen fluid pH and VFA. This suggested rumen fermentation was not a reason for the difference in methane yield between two levels of feeding. There was no difference in rumen protozoal counts between the two levels of feeding, but there was a tendency for protozoa counts to increase in the rumen of sheep fed chicory than those fed ryegrass. Although protozoa numbers are generally considered to be positively related to methane emissions, a number of reports did not find such a relationship (e.g., Morgavi et al., 2010). Our data are not supportive of a protozoal association with methane emissions. Digesta retention time, either total mean retention time or ruminal retention time, has a central role in the digestion ˜ et al. (2003a) and Hegarty (2004) found that retention time of digesta in the rumen is related to process. Pinares-Patino methane emissions (MJ CH4 /MJ gross energy intake). Ushida et al. (1997) proposed that a faster rumen fluid fractional outflow rate may have a washing out effect on methanogens, resulting in lower methanogen numbers and a consequent reduction in methane production, although methanogen activity has recently been suggested to be more important than numbers (Morgavi et al., 2010). In our study, fluid outflow rate of sheep chicory was almost the same as that of those fed
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perennial ryegrass. However, fluid outflow rate was higher for the high feeding level, suggesting that rumen fluid outflow rate was the mechanism for the effect of feeding level on methane yield. 5. Conclusions Despite differences in chemical composition, feed digestibility and rumen fermentation parameters between chicory and perennial ryegrass forages, no differences occurred in methane yield expressed per unit DM intake. These findings contrast with previous studies which reported reduced emissions with chicory feeding. However differences in methodology and animal characteristics between the current study and previous work could be responsible for the different outcomes. Nevertheless, our results confirm previous reports that increased feeding level of forages leads to decreases in methane yield, per unit DM intake. 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