Methane emission by goats consuming diets with different levels of condensed tannins from lespedeza

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Animal Feed Science and Technology 144 (2008) 212–227

Methane emission by goats consuming diets with different levels of condensed tannins from lespedeza G. Animut a , R. Puchala a , A.L. Goetsch a,∗ , A.K. Patra a , T. Sahlu a , V.H. Varel b , J. Wells b a

E (Kika) de la Garza American Institute for Goat Research, P.O. Box 730, Langston University, Langston, OK 73050, USA b US Meat Animal Research Center, P.O. Box 166, Clay Center, NE 68933, USA

Received 18 June 2007; received in revised form 16 October 2007; accepted 23 October 2007

Abstract Twenty-four yearling Boer × Spanish wethers (7/8 Boer; initial body weight (BW) of 34.1 ± 1.02 kg) were used to determine effects on methane (CH4 ) emission of dietary levels of a condensed tannin (CT)-containing forage, Kobe lespedeza (Lespedeza striata; K), and a forage very low in CT, sorghum-sudangrass (Sorghum bicolor; G). Treatments were dietary K levels (dry matter (DM) basis) of 1.00, 0.67, 0.33, and 0 (100, 67, 33, and 0 K, respectively). Forages were harvested daily and fed at approximately 1.3 times maintenance metabolizable energy requirement. The experiment lasted 21 days, with most measures on the last 8 days. The CT concentration was 0.3 and 151 g/kg DM in G and K, respectively. DM intake was similar among treatments (i.e., 682, 675, 654, and 648 g/day; S.E. = 30.0) and gross energy (GE) digestibility increased linearly (P<0.05) with decreasing K (0.472, 0.522, 0.606, and 0.666 for 100, 67, 33, and 0 K, respectively). CH4 emission changed quadratically (P<0.05) with decreasing K (10.9, 13.8, 17.6, and 26.2 l/day; 32, 42, 57, and 88 kJ/MJ GE; 69, 81, 94, and 133 kJ/MJ digestible energy for 100, 67, 33, and 0 K, respectively). In vitro CH4 emission by Abbreviations: ADF, acid detergent fiber; aNDF, neutral detergent fiber with residual ash; BW, body weight; CP, crude protein; CT, condensed tannins; DE, digestible energy; DM, dry matter; EE, energy expenditure; G, sorghumsudangrass; GE, gross energy; IVTDMD, in vitro true DM digestibility; K, Kobe lespedeza; ME, metabolizable energy; OM, organic matter; PEG, polyethylene glycol; RE, recovered energy; VFA, volatile fatty acids. ∗ Corresponding author. Tel.: +1 405 466 6164; fax: +1 405 466 6180. E-mail address: [email protected] (A.L. Goetsch). 0377-8401/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2007.10.014

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incubation of ruminal fluid for 3 weeks with a medium for methanogenic bacteria and other conditions promoting activity by methanogens also was affected quadratically (P<0.05) by K level (7.0, 8.1, 9.2, and 16.1 ml for 100, 67, 33, and 0 K, respectively). The total bacterial count of ruminal samples was similar among K levels, but the number of total protozoa increased linearly (P<0.05) as K declined (8.3, 11.8, 15.6, and 27.1 × 105 ml−1 for 100, 67, 33, and 0 K, respectively). The CT-containing forage K decreased CH4 emission by goats regardless of its feeding level, although the effect per unit of K increased with decreasing K. Forage type (i.e., legume versus grass) may have contributed to the effect of K on CH4 emission, but most of the change appeared attributable to CT, which appeared to directly impact activity of methanogenic bacteria, although alterations of protozoal activity could have been involved. These findings suggest that relatively low dietary levels of CT could be employed to lessen CH4 emission without a marked detrimental effect on other conditions such as total tract protein digestion. © 2007 Elsevier B.V. All rights reserved. Keywords: Goats; Methane; Condensed tannins

1. Introduction ‘Greenhouse’ gasses such as methane (CH4 ), carbon dioxide (CO2 ), and nitrous oxide have the capacity to raise the earth’s temperature through absorption of long wave radiation. After CO2 , which is emitted mainly from combustion of fossil fuels, CH4 is the greenhouse gas of greatest importance to global warming. Approximately 700 g/kg of methane production arises from an anthropogenic sources, of which agriculture accounts for about two-thirds with enteric fermentation being responsible for one-third of methane from agriculture (Moss et al., 2000). About 80 million Mt of CH4 is produced annually by livestock through enteric fermentation, of which about 730 g/kg is attributable to cattle (Johnson and Johnson, 1995). Assuming 9 kg of ruminal CH4 emission per sheep annually (Mbanzamihigo et al., 2002) and the 1057 million sheep and 677 million goats in the world in 1996 (Morand-Fehr and Boyazoglu, 1999), sheep and goats account for about 200 g/kg of CH4 emission from enteric fermentation. CH4 from enteric fermentation by ruminants is not only an important greenhouse gas associated with environmental problems, but it also represents a loss of feed energy (20–150 kJ/MJ intake; Johnson and Johnson, 1995; Singh et al., 2005). Therefore, developing feeding strategies to minimize CH4 emission is desirable in long-term mitigation of emission of greenhouse gasses into the atmosphere and for short-term economic benefits. There have been reports that condensed tannins (CT) lower CH4 emission by ruminants (Carulla et al., 2005; Puchala et al., 2005). Supplementation with 29 g Acacia mearnsii CT/kg dietary dry matter (DM) reduced CH4 emission by 130 kJ/MJ in sheep fed a mixture of Lolium perenne and Trifolium pratense or M. sativa (Carulla et al., 2005). Puchala et al. (2005) noted CH4 emission relative to DM intake by goats fed Lespedeza cuneata (177 g CT/kg DM) 0.55 of that by goats consuming a mixture of grasses (Digitaria ischaemum and Festuca arundinacea; 5 g CT/kg DM). Hess et al. (2003) reported that when one-third of a grass-based diet was replaced by the tropical legume Calliandra calothyrsus (270 g CT/kg DM), in vitro CH4 emission was decreased by one-half.

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However, effects of different levels of a specific CT-containing forage on CH4 emission have not been investigated. Such knowledge would be important in the design of future strategies to decrease CH4 emission. For instance, a low level of CT administration might have minimal effects on overall animal digestion but the same impact on CH4 emission as higher levels, thus minimizing or averting possible negative effects of CT on animal performance. The present study was, therefore, conducted to compare effects of dietary levels of a CTcontaining forage (Kobe lespedeza; Lespedeza Striata) and grass (Sorghum-sudangrass; Sorghum bicolor) on digestibility, N and energy balances, energy expenditure, CH4 emission, and characteristics of the ruminal microflora of meat goats. 2. Materials and methods 2.1. Animals and treatments This experiment was conducted at the E (Kika) de la Garza American Institute for Goat Research of Langston University, Langston, OK, USA, and was approved by the Langston University Animal Care Committee. Twenty-four yearling Boer × Spanish (7/8 Boer) goat wethers born in the spring of 2004 with an initial body weight (BW) of 34.1 ± 1.02 kg were used. Treatments entailed feeding different levels of a forage with a negligible level of CT, sorghum-sudangrass (S. bicolor; G), and one with a relatively high level, Kobe lespedeza (L. striata; K). Levels of K on a DM basis were 1.00, 0.67, 0.33, and 0 (100, 67, 33, and 0, respectively). Goats were assigned to six groups by ranking according to BW in ascending order. Goats were then randomly assigned within these groups to the four treatments. Thereafter, a small number of treatment assignments were changed to achieve more similar mean BW and variation in BW among the treatments. First growth forages were harvested daily using a small self-propelled Troy-Bilt sickle bar mower (Garden Way Incorporated, Troy, NY, USA) at an approximate height of 6 cm. Although forage stands were generally mono-species, other plant species when present were hand-removed after harvest. Forages were fed in equal portions at 8:00 and 15:00 h. For goats fed both forages, one-half of the daily allowance of each was hand-mixed and fed together. Feeding was at 1.3 times the assumed metabolizable energy (ME) requirement for maintenance of goats incurring normal pen or stall activity (452 kJ ME/kg BW0.75 ; Luo et al., 2004; Sahlu et al., 2004). To calculate the amount of fresh forage for feeding, DM content was determined on forage samples collected immediately before the experiment began. These forage samples were also analyzed for in vitro true DM digestibility (IVTDMD), used to derive digestible energy (DE) assuming a gross energy (GE) concentration of 18.447 MJ/kg DM. The ME content of the forages was then calculated as 0.82 of DE (AFRC, 1998) and used to determine the amount of forage fed to the animals. DM and IVTDMD content of the forages were determined as noted later in Section 2.3. All animals had free access to water and trace mineralized salt (Big 6® Mineral Salt, American Stockman, Overland Park, KS, USA; 2400 ppm Mn, 2400 ppm Fe, 260–380 ppm Cu, 320 ppm Zn, 70 ppm I, and 40 ppm Co).

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2.2. Measurements Prior to the experiment with housing in 1.2 m × 2 m pens, wethers grazed a pasture primarily of Jose tall wheatgrass (Agropyron elongatum) for 3 weeks with free access to water and trace mineralized salt blocks (Big 6® Mineral Salt). The experiment was 21 days in length and occurred in October 2005. The indirect, open-circuit respiration calorimetry system used in this study had four head boxes (Sable Systems International, Henderson, NV, USA) allowing gas exchange measurement on four animals at the same time. Thus, six groups of four goats each, with one assigned randomly from each treatment, were formed. These groups began the experiment sequentially, 3 days apart. Upon being moved to pens, all goats were fed G for the first 3 days to become accustomed to that diet. On day 4, while still being fed only G, goats were moved to the calorimetry system. The CH4 and CO2 emission and oxygen (O2 ) consumption were measured on day 4 and also on days 5 and 6. However, on days 5 and 6 the treatment diets were fed, which were the first and second days, respectively, of consuming those diets. O2 concentration was analyzed using a fuel cell FC-1B oxygen analyzer (Sable Systems, Henderson, NV, USA), and CH4 and CO2 concentrations were measured with infrared analyzers (CA-1B for CO2 and MA-1 for CH4 ; Sable Systems). Prior to gas exchange measurements for each goat group, analyzers were calibrated with gases of known concentrations. Ethanol burn tests were performed to assure complete recovery of O2 and CO2 produced with the same flow rates as used during measurements. Measures on days 4–6 assessed the rapidity, or speed, of potential effects of feeding a CT-containing forage on CH4 emission. That is, these results would indicate whether partial or full effects occur quickly, such as in the first and/or second day of consumption, or if a longer period of adaptation to a CT-containing forage is necessary to realize full effects. Heat production or energy expenditure (EE) was determined according to the Brouwer (1965) equation. After these measures, goats were housed in 1.2 m × 2 m pens and feeding of the treatment diets continued for the remainder of the experiment. There was a period of 7 days after day 6 before subsequent measurements. The length of the experiment was minimized to avoid large changes in forage composition. However, these goats had been used in a companion experiment approximately 3 weeks in length immediately before the 3 weeks of grazing preceding this study. Hence, they were well adapted to experimental cages and consumption of comparable fresh forage. Following the total 9-day period of adaptation to treatment diets (i.e., days 5 and 6 followed by 7 days), there was an 8-day period for total feces and urine collections and sampling. On the last 2 days of this 8-day sampling period, CH4 and CO2 emission and O2 consumption were again measured. It was deemed preferable to average gas exchange values on days 20 and 21 to minimize variability. However, for days 4–6, since change with time was of interest, daily averages were computed from these data. CH4 emission per kg DM intake on day 4–6 was based on intake on those individual days, whereas intake, feces, and urine data over the 8-day digestibility period were used for gas exchange measures on days 20–21. Feed was sampled each day and weekly composite samples of feedstuffs were formed. Feed refusals were weighed daily and sampled during the 8-day digestibility period. Feces was collected in wire-screen baskets placed under the floor of the metabolism crates in order to keep feces and urine separate, and urine was collected with a funnel sloping or draining into plastic buckets containing 10 ml of 0.1 (v/v) of sulfuric acid. Aliquots of feces

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and urine (150 g/kg) and approximately 40 g orts were sampled daily and used to form composite samples for each goat. All samples were stored at −20 ◦ C until analyses. Immediately after gas measurements concluded at the very end of day 21, ruminal fluid was sampled via stomach tube and blood via jugular veinpuncture. Approximately 50 ml of ruminal digesta (i.e., one sample per goat) was collected about 17 h after the last meal at 15:00 h of day 21. A subsample was taken in a sterilized oxygen-free container within 1 h of sampling for ruminal bacterial counts. The pH of the remaining ruminal fluid was measured immediately, followed by placement of 1 ml into a tube containing 4 ml of a solution of methyl green, formalin, and saline (0.06 g methyl green, 0.85 g sodium chloride, 10 ml of 0.7 (v/v) formaldehyde solution, and 90 ml deionized water) for protozoa enumeration (Kamra et al., 1991), 5 ml into a tube with 1 ml of 0.25 (w/v) metaphosphoric acid for volatile fatty acid (VFA) analysis, and 3 ml into a tube with 2 ml of 3 M hydrochloric acid (HCl) for ammonia analysis. Samples for VFA and ammonia were frozen at −20 ◦ C until analysis. After harvesting via centrifugation at 3500 × g for 15 min at 4 ◦ C, plasma samples (one per goat) were also stored at −20 ◦ C. Goats were weighed initially, at the beginning and end of the digestibility period, and after calorimetry measurements. 2.3. Laboratory analyses Samples of forages, refusals, and feces were ground to pass a 1 mm screen after drying in a forced air oven at 55 ◦ C for 48 h. Samples were analyzed for DM (ID 967.03), ash (ID 942.05), and Kjeldahl N (ID 976.06) of AOAC (2006). Samples were also analyzed for GE using a bomb calorimeter (Parr 6300; Parr Instrument Co. Inc., Moline, IL, USA) and neutral detergent fiber (aNDF; Van Soest et al., 1991) with the addition of a heat stable alpha amylase enzyme and sodium sulfite. In addition, forage samples were analyzed for acid detergent fiber (ADF (ID 973.18); AOAC, 2006). Both aNDF and ADF were determined using an ANKOM200 Fiber Analyzer (filter bag technique; ANKOM Technology Corp., Fairport, NY, USA) and were expressed inclusive of residual ash. IVTDMD of forage samples was determined by placing 0.25 g samples in ANKOM F57 filter bags and incubation in a DaisyII incubator (ANKOM Technology Corp.). Bags were heat sealed and incubated in buffered rumen fluid using a previously described method (Wilman and Adesogan, 2000), with aNDF as the end-point measure. Ruminal fluid for IVTDMD was collected from four Boer crossbred wether goats grazing native grass pasture, consisting primarily of Cynodon dactylon, Sorghastrum nutans, Andropogon scoparium, and Andropogon gerardi, and supplemented with approximately 7.5 g/kg BW (DM basis) of a pelleted concentrate containing 203 g/kg crude protein (CP) on a DM basis. The ingredient composition (DM basis) of the supplemental concentrate was 150 g/kg cottonseed hulls, 200 g/kg dehydrated alfalfa meal, 134 g/kg wheat middlings, 250 g/kg ground maize grain, 188.5 g/kg soybean meal, 50 g/kg pelleting agent, 4 g/kg dicalcium phosphate, 11 g/kg calcium carbonate, 5 g/kg salt, 5 g/kg ammonium chloride, and 2.5 g/kg trace mineralized salt and vitamin premix. Urine samples were analyzed for DM (lyophilization), and N and GE concentrations in lyophilized urine samples were determined as described above. Subsamples of G and K were also lyophilized for the analysis of CT with the butanol–HCl colorimetric procedure of Terrill et al. (1992) using CT extracted (Sephadex LH-20, Sigma Chemical Co., St. Louis, MO, USA) from Sericea lespedeza (L. cuneata) as the standard (Jackson et al., 1996). Ruminal fluid was

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analyzed for ammonia N (Broderick and Kang, 1980) and VFA (Lu et al., 1990). Plasma was analyzed for urea N concentration colorimetrically using a Technicon Autoanalyzer II System (Technicon Instruments, Tarrytown, NY, USA). For ruminal microbial analysis, serial 10-fold dilutions of ruminal fluid were prepared for each sample using the anaerobic dilution solution of Bryant and Burkey (1953). The dilution range used was from 10−8 to 10−10 for counts of total viable bacteria and 10−7 to 10−9 for cellulolytic bacteria. Total viable counts were determined in roll tubes using the complete medium of Leedle and Hespell (1980). The cellulolytic medium used was described by Halliwell and Bryant (1963). Cellulolytic bacterial counts were determined by the most probable number method (Morvan et al., 1994). All tubes were incubated at 39 ◦ C for 2 weeks. Fifty millilitres of culture media for methanogens (Morvan et al., 1994) was dispensed into serum bottles and inoculated with 1 ml of 10−4 diluted ruminal fluid and incubated for 3 weeks for estimation of CH4 gas. Methanogenic cultures were pressurized to 202 kPa with 800 g/kg H2 and 200 g/kg CO2 . Total viable counts were determined by direct count. CH4 produced in serum bottles was analyzed using a infrared analyzer (MA-1, Sable Systems). The gas mixture from the 150 ml bottles used for incubation of methanogens was transferred into 250 ml glass syringe and injected at a rate of 400 ml/min into the infrared analyzer through a 5 cm × 1.5 cm column filled with granules of calcium sulfate as a desiccant (WA Hammond Drierite Company, Xenia, OH, USA). Ciliate protozoa were enumerated microscopically using a 0.1 mm deep Neubauer hemocytometer counting chamber (Hausser Scientific, Horsham, PA, USA) after fixing with methyl green formalin saline solution. 2.4. Calculations and statistical analysis DE was calculated as the difference between GE and fecal energy, energy lost as CH4 was total CH4 emitted in l/day × 39.5388 kJ/l (Brouwer, 1965), ME was the difference between DE and the sum of energy in urine and CH4 , and recovered energy (RE) was the difference between ME and EE. Data were analyzed using mixed model procedures of SAS (Version 8.2, 2001, SAS Inst. Inc., Cary, NC, USA). Calorimetry measurement data on days 4–6 were analyzed with the repeated measure of day according to the mixed model procedure of Littell et al. (1998). The model was Yijk = μ + Ti + Rij + Dk + (TD)ik + eijk, where Yijk is the dependent variable, μ the overall mean, Ti the fixed effect of treatment i, Rij the random effect of animal j in Ti, Dk the fixed effect of day k, (TD)ik the fixed interaction effect of Ti × Dk, and eijk is the random error. Main effect dietary treatment means were presented when the treatment × day interaction (i.e., (TD)ik) was not statistically significant; treatment × day means were reported when the interaction was significant. Other data (not involving the repeated measure of day) were analyzed with the model: Yij = μ + Ti + eij, where Yij is the dependent variable, μ the overall mean, Ti the fixed treatment effect, and eij is the residual. Polynomial contrasts were used to determine linear and quadratic components of the dietary treatment response. When the treatment × day interaction for calorimetry measures on days 4–6 was statistically significant, polynomial contrasts were used to determine linear and quadratic components of the dietary treatment response on the three individuals days (i.e., days 4–6). Differences within treatments among days were evaluated by least significant difference.

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3. Results 3.1. Forage composition K was higher in CP than G (Table 1). As expected, G had a negligible level of CT, whereas the level in K was appreciable. The level of CT in K in the present study was similar to that previously found in Sericea lespedeza (Merkel et al., 2003; Puchala et al., 2005). The GE content was higher for K, which appeared primarily related to the difference in ash content. The aNDF level and difference between aNDF and ADF were smaller for K versus G. Makkar et al. (1995b) addressed influences of CT on fiber determinations including estimates of in vivo digestion. Hence, means of in vivo digestibility of aNDF have been omitted, and relatively low IVTDMD of K may also involve analytical effects of CT. 3.2. Gas exchange measures on days 4–6 DM intake was similar among treatments on days 4–6 and not influenced by day (Table 2). The respiratory quotient was near 1, but increased linearly (P<0.05) with decreasing level of K. There were interactions (P<0.05) between day and treatment in EE in MJ/day and all expressions of CH4 emission. CH4 emission was similar among treatments on day 4 when all goats were fed G, whereas CH4 emission increased quadratically or linearly (P<0.05), depending on the method of expression, on day 5 and quadratically on day 6 as the level of K decreased. CH4 emission was numerically similar between days 5 and 6 for each treatment. CH4 emission for the 0 K treatment was similar among days 4–6. Conversely, for 100 and 67 K CH4 emission on day 4 was higher (P<0.05) than on days 5 and 6. For treatment 33 K, differences among days in CH4 emission varied depending on the method of expression. CH4 emission in l/day was higher on day 4 compared with other days, in l/kg DM intake was similar among days, and in l/MJ GE intake was higher (P<0.05) on day 4 versus day 6. Table 1 Nutritivea value of forages offered to Boer wether goats Kobe lespedezab DM (g/kg) N (g/kg DM) Ash (g/kg DM) aNDF (g/kg DM) ADF (g/kg DM) IVTDMD (coefficient)c Gross energy (MJ/kg DM) Condensed tannins (g/kg DM)d

367 22.2 53 478 458 0.680 19.8 151.1

± ± ± ± ± ± ± ±

18.1 1.59 1.90 6.76 6.08 0.0084 0.11 2.80

Sorghum-sudangrassb 160 16.8 102 678 391 0.854 17.9 0.3

± ± ± ± ± ± ± ±

12.9 0.36 9.47 11.48 9.33 0.0062 0.16 0.03

a DM: dry matter; aNDF: neutral detergent fiber with residual ash; ADF: acid detergent fiber with residual ash; IVTDMD: in vitro true DM digestibility. b Values are mean ± S.E.M.; number of weekly composite samples = 4. c Filter bag technique, with aNDF as the end-point measure. d Analyzed using extracted condensed tannins from Sericea lespedeza as the standard.

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Table 2 Dry matter intake, energy expenditure, and methane emission by Boer wether goats fed sorghum-sudangrass on day 4 and different levels of sorghum-sudangrass and Kobe lespedeza on days 5 and 6 Itema

Day

DM intake (g/day) Respiratory quotient

Mean Mean

EE (MJ/day)

4 5 6

EE (kJ/kg BW0.75 )

Mean

Methane emission l/day

Kobe lespedeza levelb

S.E.

100 K

67 K

33 K

0K

613 0.96

667 0.96

606 1.01

627 1.02

5.72 5.09 5.06

5.53 5.01 5.11

5.52 5.50 5.45

6.04 6.41 6.50

391

398

417

492

Effectc L

Q

30.3 0.014

0.89 0.01

0.60 0.87

0.319

0.53 0.01 0.01

0.29 0.13 0.13

0.01

0.03

14.5

4 5 6

19.1 9.9 10.3

18.3 11.1 10.8

17.3 15.2 15.2

20.4 21.7 22.4

1.09

0.58 0.01 0.01

0.10 0.02 0.01

l/kg DM intake

4 5 6

30.9 15.7 19.4

29.5 17.9 15.3

28.5 26.6 24.4

31.4 34.9 36.8

2.16

0.95 0.01 0.01

0.33 0.17 0.01

l/MJ GE intake

4 5 6

0.118

0.99 0.01 0.01

0.36 0.12 0.01

1.73 0.79 0.96

1.65 0.93 0.79

1.60 1.42 1.30

1.75 1.94 2.04

a

DM: dry matter; EE: energy expenditure; BW: body weight; GE: gross energy. DM offered: 100 K = 1.00 Kobe lespedeza; 67 K = 0.67 Kobe lespedeza plus 0.33 sorghum-sudangrass; 33 K = 0.33 Kobe lespedeza plus 0.67 sorghum-sudangrass; 0 K = 1.00 sorghum-sudangrass. c L and Q: observed significance levels for linear and quadratic effects, respectively. b

3.3. Digestibility and gas exchange on days 20–21 BW and intakes of DM and organic matter (OM) were similar among treatments (Table 3). Feed refusals averaged 121 ± 6.5 g/kg DM offered. Intake of N decreased (P<0.05) as the level of K in the diet decreased. Digestibilities of DM, OM, and N as well as intakes of digestible DM, OM, and N increased linearly (P<0.05) with decreasing dietary K. Fecal N decreased and urinary N increased linearly (P<0.05) with decreasing K, which coupled with change in N intake yielded a linear decrease in N retention (P<0.05) and a difference of 1.45 g/day between 100 and 0 K. Intake of GE decreased linearly (P<0.05) as level of K decreased (Table 4). Energy lost through feces decreased linearly (P<0.05) with decreasing K, which corresponded to a linear increase in DE intake (P<0.05). There was a linear increase (P<0.05) in urinary energy as the level of K decreased. Energy lost as CH4 changed quadratically (P<0.05) as dietary level of K declined, with the value in MJ/day for 100 K 0.42 of that for 0 K. ME intake was similar among treatments. EE was affected quadratically (P<0.05) as K level decreased. RE changed quadratically (P<0.05) as K level in the diet varied (P<0.05). The respiratory quotient on days 20–21 tended (P<0.06) to be affected quadratically as dietary K level decreased (Table 4). CH4 emission (l/day) on days 20 and 21 changed quadratically with decreasing K level (P<0.01). These values are fairly similar to those on

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Table 3 Intake and digestibility of nutrients by Boer wether goats fed different levels of sorghum-sudangrass and Kobe lespedeza during the 8-day digestibility period Itema

Kobe lespedeza levelb 100 K

Body weight (kg) Initial Digestibility phase Nutrient intake (g/day) DM OM N

33 K

0K

Effectc L

Q

34.5 34.1

35.1 34.3

33.4 33.1

33.5 33.1

2.16 1.51

0.63 0.56

0.92 0.94

682 644 14.1

675 630 13.5

654 604 12.3

648 595 11.1

30.0 28.5 0.49

0.38 0.19 0.01

0.97 0.94 0.59

0.01 0.01 0.01

0.95 0.93 0.31

0.01 0.02 0.01

0.99 0.98 0.89

0.01 0.01 0.01

0.43 0.48 0.57

Apparent total tract digestibility DM 0.507 OM 0.517 N 0.404 Digestible nutrient intake (g/day) DM 347 OM 335 N 5.7 Fecal N (g/day) Urinary N (g/day) N retention (g/day)

67 K

S.E.

8.37 3.16 2.57

0.552 0.565 0.454 373 356 6.1 7.35 4.10 2.04

0.626 0.646 0.567 411 391 7.0

0.673 0.696 0.655 437 414 7.3

5.28 5.74 1.25

3.84 6.17 1.12

0.0158 0.0160 0.0184 22.6 21.5 0.39 0.261 0.358 0.339

a

DM: dry matter; OM: organic matter; N: nitrogen. DM offered: 100 K = 1.00 Kobe lespedeza; 67 K = 0.67 Kobe lespedeza plus 0.33 sorghum-sudangrass; 33 K = 0.33 Kobe lespedeza plus 0.67 sorghum-sudangrass; 0 K = 1.00 sorghum-sudangrass. c L and Q: observed significance levels for linear and quadratic effects, respectively. b

days 5 and 6, with similar quadratic effects of K level in the diet (P<0.05). CH4 emission expressed relative to intakes of DM, digested OM, GE, and DE varied similarly with dietary treatment. 3.4. Ruminal fluid measures and in vitro CH4 emission Ruminal pH decreased linearly (P<0.05) as the dietary K level declined (Table 5). Total VFA concentration was similar among treatments. Molar proportions of acetate, isobutyrate, and isovalerate were not altered by K level. Effects of dietary K level were quadratic for propionate and linear for butyrate and valerate (P<0.05). There was a quadratic change in the acetate to propionate ratio as dietary K level decreased (P<0.05), which involved a value for 33 K considerably different from other treatments. Ruminal ammonia N and plasma urea N concentrations were unaffected by treatment. Total bacteria count was similar among treatments, whereas the total number of ciliate protozoa increased linearly (P<0.01) with decreasing K (Table 5). The number of cellulolytic bacteria were not affected by K level. In vitro CH4 emission changed quadratically as K level decreased (P<0.05); differences among treatments was of similar magnitude as in in vivo CH4 emission.

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Table 4 Energy balance and utilization and methane emission by Boer wether goats fed different levels of sorghumsudangrass and Kobe lespedeza Itema,b

Kobe lespedeza levelc 100 K

Respiratory quotient MJ/day Intake Fecal Digestible Methane Urinary Metabolizable Expenditure Recovered

67 K

S.E. 33 K

0K

Effectd L

Q

1.03

1.05

1.08

1.07

0.008

0.01

0.06

13.51 7.09 6.42 0.43 0.30 5.69 5.08 0.60

12.99 6.20 6.79 0.54 0.48 5.77 4.96 0.81

12.26 4.80 7.45 0.69 0.62 6.14 5.50 0.64

11.80 3.95 7.85 1.03 0.69 6.13 7.01 −0.88

0.578 0.287 0.429 0.032 0.037 0.402 0.223 0.322

0.04 0.01 0.02 0.01 0.01 0.35 0.01 0.01

0.96 0.85 0.98 0.01 0.15 0.92 0.01 0.02

Loss (kJ/MJ intake) Urine Feces

22.4 528

37.0 479

50.6 394

57.9 334

2.21 17.3

0.01 0.01

0.12 0.78

Methane emission l/day l/kg DM intake l/kg OM digested kJ/MJ GE intake kJ/MJ DE intake

10.9 16.2 33.4 32.2 69.0

13.8 20.5 38.9 42.1 81.0

17.6 26.9 45.1 56.8 93.8

26.2 40.4 63.5 87.7 132.5

0.81 1.04 2.34 2.17 5.08

0.01 0.01 0.01 0.01 0.01

0.01 0.01 0.02 0.01 0.02

a Intake, fecal, and urine values are means for days 14–21 and methane and energy expenditure values are means for days 20–21. b DM: dry matter; OM: organic matter; GE: gross energy; DE: digestible energy. c DM offered: 100 K = 1.00 Kobe lespedeza; 67 K = 0.67 Kobe lespedeza plus 0.33 sorghum-sudangrass; 33 K = 0.33 Kobe lespedeza plus 0.67 sorghum-sudangrass; 0 K = 1.00 sorghum-sudangrass. d L and Q: observed significance levels for linear and quadratic effects, respectively.

4. Discussion 4.1. Intake CT from Lotus corniculatus or A. mearnsii at dietary levels less than 50 g/kg DM did not depress forage intake (Barry and McNabb, 1999; Carulla et al., 2005). With higher CT concentrations (e.g., >60 g/kg from Prosopis cineraria shrub foliage), decreases in feed intake have occurred (Bhatta et al., 2002). Nonetheless, in the present study with dietary CT concentrations of 151, 101, 50, and 0 g/kg DM corresponding to CT intake of 103, 68, 33, and 0 g/day for 100, 67, 33, and 0 K, respectively, intakes of DM and OM were unaffected by treatment. Landau et al. (2000) suggested that the negative effect of quebracho CT on feed intake by Holstein heifers was caused by astringency of CT and short-term post-ingestive malaise, which disappeared after 7 days of feeding. A lack of treatment effects on DM intake on days 4–6 in the present experiment, similar to intake later during fecal and urine collections, does not infer great importance of adaptation.

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Table 5 Ruminal ammonia N, plasma urea N, ruminal volatile fatty acids, ruminal microbial counts, and in vitro methane emission by Boer wether goats fed different levels of sorghum-sudangrass and Kobe lespedeza Itema

Kobe lespedeza levelb 100 K

Ruminal pH Ruminal fluid VFA Total (mmol/l) mol/100 mol Acetate Propionate Isobutyrate Butyrate Isovalerate Valerate Acetate:propionate ratio

6.72 55.0

67 K 6.69 42.1

SE 33 K 6.60 81.2

0K 6.60 43.2

Effectc L

Q

0.047

0.05

0.76

9.39

0.94

0.20

0.693 0.152 0.019 0.095 0.030 0.011 4.59

0.703 0.164 0.018 0.081 0.026 0.009 4.30

0.676 0.179 0.020 0.083 0.032 0.010 3.80

0.718 0.166 0.016 0.070 0.023 0.008 4.36

0.0116 0.0055 0.0014 0.0050 0.0003 0.0007 0.197

0.37 0.04 0.33 0.01 0.23 0.01 0.19

0.19 0.04 0.31 0.96 0.45 0.86 0.05

Ruminal ammonia N (g/l) Plasma urea N (g/l)

0.126 0.127

0.133 0.113

0.128 0.117

0.120 0.112

0.0111 0.0078

0.63 0.28

0.53 0.58

Microbial count Total bacteria (1010 ml−1 ) Ciliate protozoa (105 ml−1 )

6.6 8.3

7.0 11.8

9.1 15.6

8.6 27.1

3.07 2.03

0.58 0.01

0.87 0.07

Cellulolytic bacteriad In vitro methane production (ml)e

9.5 7.0

9.5 8.1

9.2 9.2

9.2 16.1

0.35 0.73

0.41 0.01

1.00 0.01

a

VFA: volatile fatty acids; N: nitrogen. DM offered: 100 K = 1.00 Kobe lespedeza; 67 K = 0.67 Kobe lespedeza plus 0.33 sorghum-sudangrass; 33 K = 0.33 Kobe lespedeza plus 0.67 sorghum-sudangrass; 0 K = 1.00 sorghum-sudangrass. c L and Q: observed significance levels for linear and quadratic effects, respectively. d Values are most probable number (log/ml). e In vitro methane production by incubating 0.0001 ml of ruminal fluid in a methanogenic microbial medium for 3 weeks. b

4.2. Digestibility, ruminal ammonia, and plasma urea Carulla et al. (2005) decreased total tract apparent digestibility in sheep of OM, CP, NDF, and ADF by adding CT of A. mearnsii to grass or grass/legume diets. Thus, effects of CT on digestion in the present experiment are likely. The magnitude of change in digestion as K level decreased was higher for N than for DM or OM. Expected total tract apparent CP digestibility was 0.69, 0.67, 0.65, and 0.63 for 100, 67, 33, and 0 K, respectively (Moore et al., 2004), with values for diets with K higher than observed. Furthermore, Animut et al. (2008) reported a CP digestibility coefficient of 0.68 for K when fed with polyethylene glycol (PEG), similar to 0.655 for 0 K in the present experiment, and 0.51 without PEG, comparable to 0.404 for 100 K. However, digestibilities of DM and OM were not increased by supplementing goats consuming K with PEG (Animut et al., 2008), which suggests importance to digestion of characteristics of the two forages, such as level and type of lignification. Overall, it would appear that CT affected total tract N digestion in the present experiment, with lesser or no effects on digestibilities of other feed fractions. Regardless

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of causal factors, as indicated by the lack of quadratic effects of dietary forage levels, there were no associative effects in digestibilities between K and G. A. mearnsii CT (25 g/kg DM) supplemented to sheep consuming a mixture of L. perenne and T. pratense or M. sativa reduced ruminal ammonia and plasma urea N concentrations by 0.09 and 0.05, respectively (Carulla et al., 2005). Such changes relate to reduced ruminal CP degradation because of CT binding to protein, normally accompanied by increased CP flow to the small intestine and intestinal amino acid absorption (Min et al., 2003). However, despite increasing total tract apparent N digestibility with decreasing K level, ruminal ammonia and plasma urea levels in the present study were not impacted. Decreasing urinary N excretion as K level in the diet increased, however, suggests decreasing absorption of ammonia from the rumen and urea synthesis in the liver. In a review, Reed (1995) suggested that CT may increase efficiency of N recycling to the rumen by lessening ruminal ammonia concentration to promote urea influx into the rumen. CT may also increase the glycoprotein content of saliva (Reed, 1995) and stimulate higher saliva production (Van Soest, 1994), which could also elevate N recycled to the rumen. Thus, it is possible that dietary K level in the present experiment did impact N recycling, which contributed to the lack of treatment effects on ruminal ammonia and plasma urea concentrations. Furthermore, sampling at 17 h after the last meal, rather than earlier, may have minimized the likelihood of significant K level effects on these measures. 4.3. Methane emission There are reports indicating a decrease in CH4 emission with dietary addition of CT or inclusion of CT-containing forage (Carulla et al., 2005; Puchala et al., 2005), but factors responsible for effects of CT on ruminal CH4 emission are unclear. Negative effects on ruminal fiber digestion, which may relate to a decreased number of cellulolytic bacteria (McSweeney et al., 2001), formation of CT-cellulose complexes that are resistant to enzymatic digestion (Makkar et al., 1995a), and/or impaired substrate adhesion by fibrolytic microbes (Bento et al., 2005a), would reduce H2 availability to lessen methanogenesis (Carulla et al., 2005). In the present experiment, the lack of a linear effect of K level on the acetate to propionate ratio, no change in the number of cellulolytic bacteria, and only a linear change in OM digestibility (rather than also a quadratic effect as in CH4 emission) suggest that factors other than an inhibition of activity of ruminal fibrolytic microorganisms by CT were responsible for changes in CH4 emission due to level of K in the diet. Most plausible conditions responsible for reduced CH4 emission are alterations in the number and/or activity of bacteria other than ones highly fibrolytic, including methanogens (Field et al., 1989) and protozoa (Sch¨onhusen et al., 2003). Such changes may be attributable to characteristics of the two forage types (i.e., legume compared with grass) and/or CT. Studies on effects of dietary inclusion of legumes on enteric CH4 emission have yielded contradictory results. Mbanzamihigo et al. (2002) found no effect of dietary legume proportion (i.e., 0.16, 0.35, 0.41, and 0.51) on CH4 emission of sheep grazing ryegrass–white clover pastures. Carulla et al. (2005) compared haylage from ryegrass alone with a 1:1 mixture of ryegrass and clover or alfalfa haylage and noted higher CH4 emission (kJ/MJ GE) for the ryegrass:clover mixture. Furthermore; when expressed relative to digested OM and NDF, CH4 emission was higher for diets containing legumes. McCaughey et al. (1999)

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reported lower CH4 yield by cattle grazing grass-alfalfa pasture versus cattle grazing grass pasture. Factors responsible for differences among findings are unclear but may include variation in chemical composition of legumes and grasses and associated microbial activity and rate of digesta outflow from the rumen (Pinares-Pati˜no et al., 2003a). Pinares-Pati˜no et al. (2003b) noted that CH4 emission by sheep grazing perennial ryegrass-based pasture, lucerne, and a CT-containing legume L. corniculatus was 0.08, 0.05, and 0.03 of GE intake, respectively, and concluded that CT in such forages are not solely responsible for differences in CH4 emission compared with grass diets. However, Carulla et al. (2005) did not detect an interaction in CH4 emission between replacement of a grass by different legumes and CT supplementation, indicating that effects of forage type (i.e., grass compared with legume) and CT supplementation were independent. CH4 emission relative to GE intake by goats consuming CT from two different legumes or a CT-containing legume plus quebracho CT was 0.44 of that by goats consuming the same diets supplemented with PEG (Animut et al., 2008). If this value of 0.44 is applied to the 0.63 lower CH4 emission for 100 K compared with 0 K, then 0.69 of the total reduction in CH4 emission was attributable to CT and 0.31 was accounted for by differences in dietary characteristics. In vitro CH4 emission indicated that the number or activity of rumen methanogens was impacted by dietary level of K. Whether the reduction in the number and activity of methanogens was due to a direct effect on methanogens or an inhibition of organisms producing H2 is unclear. Since there is no obvious evidence that CT decreased the number or activity of fibrolytic microbes, alterations in protozoal activity may have been important. Ruminal methanogens are often associated with protozoa intracellularly and/or attached to the external cell surface, presumably with interspecies H2 transfer (Krumholz et al., 1983; Finlay et al., 1994). Sch¨onhusen et al. (2003) reported that ruminal CH4 emission increased exponentially when the size of the ruminal protozoal population was increased. Newbold et al. (1995) noted that methanogenic bacteria associated with rumen ciliates accounted for 0.09–0.25 of methanogenesis in ruminal fluid. Thus, changes in ruminal protozoal numbers with CT-containing forage in the current study imply that supply of substrates to methanogens may be partly responsible for reduced CH4 emission. Similar effects of K level on days 5 and 6, compared to days 20 and 21, indicate that the effect of CT and/or forage type on CH4 emission was immediate. Likewise, Bento et al. (2005b), in an in vitro study compared gas production of 15 N-labelled maize shoots incubated alone (control), with mimosa CT, and with mimosa CT plus PEG and noted a decrease in gas production with the inclusion of mimosa CT at all times of incubation (i.e., 2, 4, 6, and 24 h); values when PEG was given were similar to the control levels. The lack of effect of CT-containing forage on cellulolytic bacteria in the present study, with an immediate effect of dietary K level on CH4 emission, suggest direct effect of CT on methanogens, as microbial activity shifts in response to different forage types presumably would necessitate a period of time to be fully realized. In addition to addressing likely contributions of CT and forage type to the difference in CH4 emission between 100 and 0 K diets, it is also important to consider effects of the dietary mixtures of K and G (i.e., 67 and 33 K). The decrease in CH4 energy loss relative to GE was 55.5 kJ/MJ for 100 K compared with 0 K, and if there was a linear effect of K or K CT on CH4

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energy loss (i.e., lack of quadratic change and no associative effects), expected decreases in CH4 were 37.0 and 18.3 kJ/MJ for 67 and 33 K, respectively. Actual depressions in CH4 loss were 45.6 and 30.9 kJ/MJ, which are 0.23 and 0.69 higher than expected for 67 and 33 K, respectively. With an alternative expression (i.e., (observed − expected)/observed), associative effects in methane emission relative to GE were 0.19 and 0.41 for 67 and 33 K, respectively. Hence, the effect of each unit of K or K CT increased as K level decreased.

5. Conclusions The CT-containing forage K decreased CH4 emission by goats regardless of its level and the effect per unit of K or CT increased with decreasing K. It seems that CT were responsible for most of this effect, although influences of forage type (i.e., legume compared with grass) may also have been present. Nonetheless, the impact of K CT on CH4 emission appeared attributable to changes in methanogenic bacterial activity, but which might also involve alterations of protozoal activity. This suggests that relatively low dietary levels of CT could be employed to lessen CH4 emission without marked detrimental effects on other conditions such as total tract N digestion.

Acknowledgments This project was supported by USDA Project Number 2004-38814-15045. The authors thank the farm and laboratory personnel of E (Kika) de la Garza American Institute for Goat Research for assistance in field work and laboratory analysis.

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