Directory UMM :Data Elmu:jurnal:A:Animal Feed Science and Technology:Vol84.Issue3-4.May2000:
Animal Feed Science and Technology
84 (2000) 243±256
Yield and quality characteristics of bahiagrass
(Paspalum notatum) exposed to ground-level ozone
R.B. Muntiferinga,*, D.D. Crosbya, M.C. Powella,
A.H. Chappelkab
a
Department of Animal and Dairy Sciences, Auburn University, Auburn, AL 36849, USA
b
School of Forestry and Wildlife Sciences, Auburn University, Auburn, AL 36849, USA
Received 30 September 1999; received in revised form 20 January 2000; accepted 10 February 2000
Abstract
Early and late season-planted bahiagrass (Paspalum notatum Flugge, cultivar `Pensacola') were
grown in open-top chambers (OTC) to which added air had been carbon-®ltered (CF),
representative of that found at pristine air quality sites; non-®ltered (NF), characteristic of ambient
air in Auburn, AL and representative of that found in rural agricultural areas; or enriched with
ozone (O3) to twice-ambient O3 concentration (2X), representative of that found in the vicinity of
large metropolitan areas. Primary-growth and regrowth forages from each planting were harvested
periodically throughout the experiment from each of six OTC (two OTC/air treatment). Mean
daytime (09:00±21:00 h) O3 concentrations over the entire 24-week experiment (7 May±23 October
1997) were 22, 45 and 91 Zl lÿ1, respectively, for CF, NF and 2X treatments. Mean daytime
ambient O3 concentrations peaked in mid-May and again in late August±late September at 50±
60 Zl lÿ1, and highest individual ambient O3 concentrations were recorded in late June, late July,
late August and mid-September at 90 Zl lÿ1. Dry matter (DM) yield was greater for CF than for
NF primary-growth forage, and concentrations of neutral detergent ®ber (NDF) were higher in 2X
than in NF primary-growth and regrowth forages from the early-season planting. Concentration of
acid detergent ®ber (ADF) tended to be higher in 2X than in NF primary-growth forage and was
higher in 2X than in NF regrowth forage, whereas acid detergent lignin (ADL) concentration was
higher in 2X than in NF primary-growth forage and tended to be higher in 2X than in NF regrowth
forage from the early-season planting. Crude protein (CP) concentrations were lower in CF than in
NF regrowth forage from the early-season planting and in CF than in NF primary-growth forage
from the initial harvest of the late-season planting. No differences were observed among treatments
in DM yield or concentrations of cell wall constituents in primary-growth or regrowth forages from
the late-season planting, although concentrations of CP, NDF and ADF tended to be higher in 2X
*
Corresponding author. Tel.: 1-334-844-1533; fax: 1-334-844-1519.
E-mail address: rmuntife@acesag.auburn.edu (R.B. Muntifering)
0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 0 0 ) 0 0 1 2 4 - 3
244
R.B. Muntifering et al. / Animal Feed Science and Technology 84 (2000) 243±256
than in NF regrowth forage. No differences were observed among treatments in concentrations of
total phenolics in primary-growth or regrowth forages from either planting, although concentrations
of total phenolics tended to be higher in CF than in NF primary-growth forage from the late-season
planting. Particularly in the case of early-planted bahiagrass, alterations in DM yield and quality of
primary-growth and vegetative regrowth forages were of suf®cient magnitude to have nutritional
and possibly economic implications to their utilization for ruminant animal production under
existing and projected global climate scenarios. # 2000 Elsevier Science B.V. All rights reserved.
Keywords: Ozone; Air pollution; Climate change; Bahiagrass; Forage quality
1. Introduction
Tropospheric (i.e. ground-level) ozone (O3) is formed near the Earth's surface from the
photo-oxidation of hydrocarbons and oxides of nitrogen (NOx) from automobiles,
factories, power plants and other sources of high-temperature combustion of fossil fuels
(National Research Council, 1991). It is considered the most important phytotoxic
gaseous pollutant in the eastern USA (US Environmental Protection Agency, 1996), and it
has become ubiquitous in this and other northern mid-latitude regions which collectively
dominate global industrial and agricultural productivity; viz. Europe, eastern China and
Japan (Chameides et al., 1994). Once thought to be con®ned to large metropolitan areas,
air pollutants such as ground-level O3 are now known to be transported long distances to
rural areas such that many of the world's most productive agricultural regions are
currently exposed to harmful concentrations of O3 (Chameides et al., 1994). Computer
models predict that tropospheric O3 concentrations will continue to increase globally, on
average, between 0.3 and 1.0% per year for the next 50 years (Thompson, 1992). Global
simulation of atmospheric reactive nitrogen compounds, conducted recently by
Chameides et al. (1994), estimates that exposure to phytotoxic concentrations of O3
pollution (50 Zl lÿ1 for highly sensitive crops such as winter wheat to 70 Zl lÿ1 for
less sensitive crops such as rice) could triple by the year 2025 if rising anthropogenic NOx
emissions are not abated. According to their analysis, by 2025 as much as 30 to 75% of
the world's cereals may be grown in regions with O3 concentrations exceeding the 50±
70 Zl lÿ1 threshold.
Ozone can directly injure plant tissue and disrupt normal patterns of resource
acquisition and allocation such that chronic exposure over a growing season ultimately
reduces crop yield. Generally, root biomass in graminoids is reduced more than shoot
biomass (Lechowicz, 1987), and this change in priority of biomass allocation to different
plant organs leads to reduced seed size and decreased seed and grain production in O3sensitive species (Miller, 1988). Long-term research programs such as the U.S. National
Crop Loss Assessment Program (Heck et al., 1988) and the European Open-top Chamber
Program (Commission of the European Communities, 1993) have been directed largely
toward O3 stress in cereal crops, because of their economic importance as sources of food
for human consumption. Forage crops have historically received much less attention in
spite of their importance for animal production, and most of the experimental studies
involving forage crops have been restricted to cool-season (C3 photosynthetic pathway)
grasses and legumes (Fuhrer, 1997). Also, there are few experimental studies involving
R.B. Muntifering et al. / Animal Feed Science and Technology 84 (2000) 243±256
245
interactions between O3 and forage management; e.g. mechanical harvesting, grazing,
etc. (Davison and Barnes, 1998).
Available data suggest that forage yield decreases with increasing O3, but very few
studies have investigated changes in quality characteristics in response to O3 stress.
Fuhrer et al. (1994) reported marginal changes in calcium, crude protein and crude ®ber
concentrations of a mixed fescue±clover pasture fumigated with O3. Flagler and
Youngner (1985) reported that O3 reduced crude fat, crude ®ber and total nonstructural
carbohydrates, but increased calcium and crude protein concentrations at the expense of
yield in tall fescue. Ozone reduced shoot total nonstructural carbohydrate and increased
mineral concentrations of ladino clover (Blum et al., 1982), and it decreased digestibility
of mixed fescue±clover regrowth forage (Blum et al., 1983). Also, O3 exposure has been
shown to increase the activities of select phenylpropanoid and ¯avonoid pathway
enzymes, which results in foliar accumulation of phenolic compounds associated with
accelerated senescence and death of plant tissue (Runeckles and Krupa, 1994). Recent
biochemical and molecular biological evidence suggests that such defense reactions are
similar to those induced by other abiotic and certain biotic stressors (KangasjaÈrvi et al.,
1994). Whether this general defensive response to stress occurs in O3-exposed forage
with resultant implications to nutritional quality for livestock has not been investigated.
Phenolics are also effective protractors of decomposition and nitri®cation by soil-borne
micro-organisms, and it is conceivable that O3 impacts on these constituents could have
subsequent effects on nutrient cycling in grassland ecosystems. Kim et al. (1998) have
reported increased lignin content and reduced rate of litter decomposition in an O3exposed blackberry±broomsedge mixture.
As very little is known about O3 effects on yield and quality of warm-season (C4
photosynthetic pathway) forages commonly used for pasture and hay production, the
effect of exposing bahiagrass, planted twice in the growing season, to three ground-level
O3 scenarios on forage DM yield and concentrations of select chemical constituents
in¯uencing its nutritive quality for ruminant animals was studied.
2. Materials and methods
2.1. O3 exposure system
The O3 exposure system comprised six large (4.8-m height4.5-m diameter) open-top
chambers (OTC), each consisting of an aluminum frame surrounded by clear plastic and
perforated at the bottom to allow introduction and circulation of air by large fans (Heagle
et al., 1989). The chambers were assigned randomly to three air treatments, resulting in
two OTC/air treatment: carbon-®ltered (CF), non-®ltered (NF) or enriched with O3 to
twice-ambient O3 concentration (2X). Carbon-®ltered chambers were calibrated to
remove approximately 50% of O3 from ambient air, resulting in a range of O3 concentrations (20±45 Zl lÿ1) currently found in relatively unpolluted environments around the
world (Lefohn et al., 1990). Ambient air in the Auburn, AL area is representative of that
found in rural agricultural and forested regions of the southeastern USA, with typical
summer daytime O3 concentrations of 50 Zl lÿ1 and occasional episodes above 100 Zl lÿ1.
246
R.B. Muntifering et al. / Animal Feed Science and Technology 84 (2000) 243±256
The 2X treatment approximated O3 concentrations representative of those in the vicinity
of major urban centers and other areas which are frequently violative of the current
National Ambient Air Quality Standard for O3 of 120 Zl lÿ1 for 1 h (US Environmental
Protection Agency, 1996). Ozone for the 2X treatment was generated by passing pure O2
through a high-intensity electrical discharge source and was added to the chambers daily
between 09:00 and 21:00 h. Ozone concentrations in all OTC were monitored
continuously using a US EPA-approved monitor with a rapid response time (20 s).
Monitoring was time-shared such that the monitoring port for each OTC was read twice
per hour. Monitoring instrumentation was calibrated according to US EPA quality
assurance guidelines, and both the monitoring system and calibrator were audited by the
Alabama Department of Environmental Management prior to initiation of treatments.
2.2. Forage establishment, management and sampling
Bahiagrass (Paspalum notatum Flugge, cultivar `Pensacola') was planted in large (15-l
volume, 30.5-cm top diameter) pots and placed into OTC on 7 May 1997 (early-planted)
and again on 10 July 1997 (late-planted). Pots were ®lled to capacity with a Norfolk
sandy loam soil, and seeds were planted to a depth of 0.6 cm at 50 seeds per pot. Each
OTC was delineated into quadrants, and eight pots of each planting were assigned
randomly to quadrants with the added restriction that no quadrant could be represented
more than once in an air treatment replicate. Pots were arranged in an isosceles triangular
con®guration which was right-angled with respect to the center of the OTC. Six weeks
postseeding, each pot of early-planted bahiagrass was topdressed with 4 g each of a
controlled-release fertilizer (14:14:14 of N:P2O5:K2O) and a conventional pelleted
fertilizer (29:3:4 of N:P2O5:K2O). Late-planted bahiagrass was topdressed with 4 g of
each fertilizer source at the time of planting. Pots were watered as necessary to maintain
moisture at near ®eld capacity. Precipitation was allowed to fall into chambers through
the open tops, and fans were turned off at night from 22:01 to 06:59 h to permit natural
dew formation within the OTC.
Primary-growth forage from early-planted bahiagrass was harvested from pots
randomly selected at 12 (four pots), 18 (two pots) and 24 (two pots) weeks postseeding
at approximately early-vegetative, late-vegetative and early-bloom stages of maturity,
respectively. Vegetative regrowth forage from the ®rst (i.e. 12-week) primary-growth
cutting was harvested three times at 4-week intervals. Primary growth forage from lateplanted bahiagrass was harvested from pots randomly selected at 9 (four pots) and 15
(four pots) weeks postseeding at approximately early-vegetative and early-bloom stages
of maturity, respectively. Vegetative regrowth forage from the ®rst (i.e. 9-week) primarygrowth cutting was harvested twice at 3-week intervals. Forages were cut to leave a 2.5cm aboveground stubble, composited on an OTC basis, dried to constant weight at 508C
and ground in a Wiley mill to pass a 1-mm screen.
2.3. Chemical analyses
Forage samples were analyzed for dry matter (DM) and crude protein (CP) according
to Association of Of®cial Analytical Chemists (1995) procedures. Forage concentrations
R.B. Muntifering et al. / Animal Feed Science and Technology 84 (2000) 243±256
247
of neutral detergent ®ber (NDF) and acid detergent ®ber (ADF) were determined
sequentially, and of acid detergent lignin (ADL) separately according to procedures of
Goering and Van Soest (1970). The prussian blue assay (Price and Butler, 1977) as
modi®ed by Graham (1992) was used to estimate forage concentration of total phenolics
by reference to a gallic acid standard.
2.4. Statistical analyses
Data were analyzed using general linear model procedures of the Statistical Analysis
System Institute Inc. (1989) for a completely randomized split-plot design. Error term (a)
for testing main effects of O3 treatment (main plots) was OTC within treatments, and
residual mean squares was the error term (b) for testing harvest period (subplots) effects
and the harvest periodO3 treatment interaction. Dunnett's procedure was used to
independently compare CF and 2X with NF means in order to evaluate biological
response to decreased and increased O3 concentrations, respectively, compared with
ambient conditions. In recognition of the low statistical power characteristic of ®eld
studies which employ limited numbers of OTC, treatment differences were considered
signi®cant when p
84 (2000) 243±256
Yield and quality characteristics of bahiagrass
(Paspalum notatum) exposed to ground-level ozone
R.B. Muntiferinga,*, D.D. Crosbya, M.C. Powella,
A.H. Chappelkab
a
Department of Animal and Dairy Sciences, Auburn University, Auburn, AL 36849, USA
b
School of Forestry and Wildlife Sciences, Auburn University, Auburn, AL 36849, USA
Received 30 September 1999; received in revised form 20 January 2000; accepted 10 February 2000
Abstract
Early and late season-planted bahiagrass (Paspalum notatum Flugge, cultivar `Pensacola') were
grown in open-top chambers (OTC) to which added air had been carbon-®ltered (CF),
representative of that found at pristine air quality sites; non-®ltered (NF), characteristic of ambient
air in Auburn, AL and representative of that found in rural agricultural areas; or enriched with
ozone (O3) to twice-ambient O3 concentration (2X), representative of that found in the vicinity of
large metropolitan areas. Primary-growth and regrowth forages from each planting were harvested
periodically throughout the experiment from each of six OTC (two OTC/air treatment). Mean
daytime (09:00±21:00 h) O3 concentrations over the entire 24-week experiment (7 May±23 October
1997) were 22, 45 and 91 Zl lÿ1, respectively, for CF, NF and 2X treatments. Mean daytime
ambient O3 concentrations peaked in mid-May and again in late August±late September at 50±
60 Zl lÿ1, and highest individual ambient O3 concentrations were recorded in late June, late July,
late August and mid-September at 90 Zl lÿ1. Dry matter (DM) yield was greater for CF than for
NF primary-growth forage, and concentrations of neutral detergent ®ber (NDF) were higher in 2X
than in NF primary-growth and regrowth forages from the early-season planting. Concentration of
acid detergent ®ber (ADF) tended to be higher in 2X than in NF primary-growth forage and was
higher in 2X than in NF regrowth forage, whereas acid detergent lignin (ADL) concentration was
higher in 2X than in NF primary-growth forage and tended to be higher in 2X than in NF regrowth
forage from the early-season planting. Crude protein (CP) concentrations were lower in CF than in
NF regrowth forage from the early-season planting and in CF than in NF primary-growth forage
from the initial harvest of the late-season planting. No differences were observed among treatments
in DM yield or concentrations of cell wall constituents in primary-growth or regrowth forages from
the late-season planting, although concentrations of CP, NDF and ADF tended to be higher in 2X
*
Corresponding author. Tel.: 1-334-844-1533; fax: 1-334-844-1519.
E-mail address: rmuntife@acesag.auburn.edu (R.B. Muntifering)
0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 0 0 ) 0 0 1 2 4 - 3
244
R.B. Muntifering et al. / Animal Feed Science and Technology 84 (2000) 243±256
than in NF regrowth forage. No differences were observed among treatments in concentrations of
total phenolics in primary-growth or regrowth forages from either planting, although concentrations
of total phenolics tended to be higher in CF than in NF primary-growth forage from the late-season
planting. Particularly in the case of early-planted bahiagrass, alterations in DM yield and quality of
primary-growth and vegetative regrowth forages were of suf®cient magnitude to have nutritional
and possibly economic implications to their utilization for ruminant animal production under
existing and projected global climate scenarios. # 2000 Elsevier Science B.V. All rights reserved.
Keywords: Ozone; Air pollution; Climate change; Bahiagrass; Forage quality
1. Introduction
Tropospheric (i.e. ground-level) ozone (O3) is formed near the Earth's surface from the
photo-oxidation of hydrocarbons and oxides of nitrogen (NOx) from automobiles,
factories, power plants and other sources of high-temperature combustion of fossil fuels
(National Research Council, 1991). It is considered the most important phytotoxic
gaseous pollutant in the eastern USA (US Environmental Protection Agency, 1996), and it
has become ubiquitous in this and other northern mid-latitude regions which collectively
dominate global industrial and agricultural productivity; viz. Europe, eastern China and
Japan (Chameides et al., 1994). Once thought to be con®ned to large metropolitan areas,
air pollutants such as ground-level O3 are now known to be transported long distances to
rural areas such that many of the world's most productive agricultural regions are
currently exposed to harmful concentrations of O3 (Chameides et al., 1994). Computer
models predict that tropospheric O3 concentrations will continue to increase globally, on
average, between 0.3 and 1.0% per year for the next 50 years (Thompson, 1992). Global
simulation of atmospheric reactive nitrogen compounds, conducted recently by
Chameides et al. (1994), estimates that exposure to phytotoxic concentrations of O3
pollution (50 Zl lÿ1 for highly sensitive crops such as winter wheat to 70 Zl lÿ1 for
less sensitive crops such as rice) could triple by the year 2025 if rising anthropogenic NOx
emissions are not abated. According to their analysis, by 2025 as much as 30 to 75% of
the world's cereals may be grown in regions with O3 concentrations exceeding the 50±
70 Zl lÿ1 threshold.
Ozone can directly injure plant tissue and disrupt normal patterns of resource
acquisition and allocation such that chronic exposure over a growing season ultimately
reduces crop yield. Generally, root biomass in graminoids is reduced more than shoot
biomass (Lechowicz, 1987), and this change in priority of biomass allocation to different
plant organs leads to reduced seed size and decreased seed and grain production in O3sensitive species (Miller, 1988). Long-term research programs such as the U.S. National
Crop Loss Assessment Program (Heck et al., 1988) and the European Open-top Chamber
Program (Commission of the European Communities, 1993) have been directed largely
toward O3 stress in cereal crops, because of their economic importance as sources of food
for human consumption. Forage crops have historically received much less attention in
spite of their importance for animal production, and most of the experimental studies
involving forage crops have been restricted to cool-season (C3 photosynthetic pathway)
grasses and legumes (Fuhrer, 1997). Also, there are few experimental studies involving
R.B. Muntifering et al. / Animal Feed Science and Technology 84 (2000) 243±256
245
interactions between O3 and forage management; e.g. mechanical harvesting, grazing,
etc. (Davison and Barnes, 1998).
Available data suggest that forage yield decreases with increasing O3, but very few
studies have investigated changes in quality characteristics in response to O3 stress.
Fuhrer et al. (1994) reported marginal changes in calcium, crude protein and crude ®ber
concentrations of a mixed fescue±clover pasture fumigated with O3. Flagler and
Youngner (1985) reported that O3 reduced crude fat, crude ®ber and total nonstructural
carbohydrates, but increased calcium and crude protein concentrations at the expense of
yield in tall fescue. Ozone reduced shoot total nonstructural carbohydrate and increased
mineral concentrations of ladino clover (Blum et al., 1982), and it decreased digestibility
of mixed fescue±clover regrowth forage (Blum et al., 1983). Also, O3 exposure has been
shown to increase the activities of select phenylpropanoid and ¯avonoid pathway
enzymes, which results in foliar accumulation of phenolic compounds associated with
accelerated senescence and death of plant tissue (Runeckles and Krupa, 1994). Recent
biochemical and molecular biological evidence suggests that such defense reactions are
similar to those induced by other abiotic and certain biotic stressors (KangasjaÈrvi et al.,
1994). Whether this general defensive response to stress occurs in O3-exposed forage
with resultant implications to nutritional quality for livestock has not been investigated.
Phenolics are also effective protractors of decomposition and nitri®cation by soil-borne
micro-organisms, and it is conceivable that O3 impacts on these constituents could have
subsequent effects on nutrient cycling in grassland ecosystems. Kim et al. (1998) have
reported increased lignin content and reduced rate of litter decomposition in an O3exposed blackberry±broomsedge mixture.
As very little is known about O3 effects on yield and quality of warm-season (C4
photosynthetic pathway) forages commonly used for pasture and hay production, the
effect of exposing bahiagrass, planted twice in the growing season, to three ground-level
O3 scenarios on forage DM yield and concentrations of select chemical constituents
in¯uencing its nutritive quality for ruminant animals was studied.
2. Materials and methods
2.1. O3 exposure system
The O3 exposure system comprised six large (4.8-m height4.5-m diameter) open-top
chambers (OTC), each consisting of an aluminum frame surrounded by clear plastic and
perforated at the bottom to allow introduction and circulation of air by large fans (Heagle
et al., 1989). The chambers were assigned randomly to three air treatments, resulting in
two OTC/air treatment: carbon-®ltered (CF), non-®ltered (NF) or enriched with O3 to
twice-ambient O3 concentration (2X). Carbon-®ltered chambers were calibrated to
remove approximately 50% of O3 from ambient air, resulting in a range of O3 concentrations (20±45 Zl lÿ1) currently found in relatively unpolluted environments around the
world (Lefohn et al., 1990). Ambient air in the Auburn, AL area is representative of that
found in rural agricultural and forested regions of the southeastern USA, with typical
summer daytime O3 concentrations of 50 Zl lÿ1 and occasional episodes above 100 Zl lÿ1.
246
R.B. Muntifering et al. / Animal Feed Science and Technology 84 (2000) 243±256
The 2X treatment approximated O3 concentrations representative of those in the vicinity
of major urban centers and other areas which are frequently violative of the current
National Ambient Air Quality Standard for O3 of 120 Zl lÿ1 for 1 h (US Environmental
Protection Agency, 1996). Ozone for the 2X treatment was generated by passing pure O2
through a high-intensity electrical discharge source and was added to the chambers daily
between 09:00 and 21:00 h. Ozone concentrations in all OTC were monitored
continuously using a US EPA-approved monitor with a rapid response time (20 s).
Monitoring was time-shared such that the monitoring port for each OTC was read twice
per hour. Monitoring instrumentation was calibrated according to US EPA quality
assurance guidelines, and both the monitoring system and calibrator were audited by the
Alabama Department of Environmental Management prior to initiation of treatments.
2.2. Forage establishment, management and sampling
Bahiagrass (Paspalum notatum Flugge, cultivar `Pensacola') was planted in large (15-l
volume, 30.5-cm top diameter) pots and placed into OTC on 7 May 1997 (early-planted)
and again on 10 July 1997 (late-planted). Pots were ®lled to capacity with a Norfolk
sandy loam soil, and seeds were planted to a depth of 0.6 cm at 50 seeds per pot. Each
OTC was delineated into quadrants, and eight pots of each planting were assigned
randomly to quadrants with the added restriction that no quadrant could be represented
more than once in an air treatment replicate. Pots were arranged in an isosceles triangular
con®guration which was right-angled with respect to the center of the OTC. Six weeks
postseeding, each pot of early-planted bahiagrass was topdressed with 4 g each of a
controlled-release fertilizer (14:14:14 of N:P2O5:K2O) and a conventional pelleted
fertilizer (29:3:4 of N:P2O5:K2O). Late-planted bahiagrass was topdressed with 4 g of
each fertilizer source at the time of planting. Pots were watered as necessary to maintain
moisture at near ®eld capacity. Precipitation was allowed to fall into chambers through
the open tops, and fans were turned off at night from 22:01 to 06:59 h to permit natural
dew formation within the OTC.
Primary-growth forage from early-planted bahiagrass was harvested from pots
randomly selected at 12 (four pots), 18 (two pots) and 24 (two pots) weeks postseeding
at approximately early-vegetative, late-vegetative and early-bloom stages of maturity,
respectively. Vegetative regrowth forage from the ®rst (i.e. 12-week) primary-growth
cutting was harvested three times at 4-week intervals. Primary growth forage from lateplanted bahiagrass was harvested from pots randomly selected at 9 (four pots) and 15
(four pots) weeks postseeding at approximately early-vegetative and early-bloom stages
of maturity, respectively. Vegetative regrowth forage from the ®rst (i.e. 9-week) primarygrowth cutting was harvested twice at 3-week intervals. Forages were cut to leave a 2.5cm aboveground stubble, composited on an OTC basis, dried to constant weight at 508C
and ground in a Wiley mill to pass a 1-mm screen.
2.3. Chemical analyses
Forage samples were analyzed for dry matter (DM) and crude protein (CP) according
to Association of Of®cial Analytical Chemists (1995) procedures. Forage concentrations
R.B. Muntifering et al. / Animal Feed Science and Technology 84 (2000) 243±256
247
of neutral detergent ®ber (NDF) and acid detergent ®ber (ADF) were determined
sequentially, and of acid detergent lignin (ADL) separately according to procedures of
Goering and Van Soest (1970). The prussian blue assay (Price and Butler, 1977) as
modi®ed by Graham (1992) was used to estimate forage concentration of total phenolics
by reference to a gallic acid standard.
2.4. Statistical analyses
Data were analyzed using general linear model procedures of the Statistical Analysis
System Institute Inc. (1989) for a completely randomized split-plot design. Error term (a)
for testing main effects of O3 treatment (main plots) was OTC within treatments, and
residual mean squares was the error term (b) for testing harvest period (subplots) effects
and the harvest periodO3 treatment interaction. Dunnett's procedure was used to
independently compare CF and 2X with NF means in order to evaluate biological
response to decreased and increased O3 concentrations, respectively, compared with
ambient conditions. In recognition of the low statistical power characteristic of ®eld
studies which employ limited numbers of OTC, treatment differences were considered
signi®cant when p