Available online at www.sciencedirect.

com

Animal Feed Science and Technology

145 (2008) 409–417

Anise and capsicum as alternatives to monensin to

modify rumen fermentation in beef heifers
fed a high concentrate diet夽
I. Fandiño, S. Calsamiglia ∗ , A. Ferret, M. Blanch
Grup de Recerca en Nutrició, Maneig i Benestar Animal, Departament de Ciència Animal i dels Aliments,
Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain

Accepted 10 April 2007

Abstract

Four Holstein heifers (229 ± 24 kg BW) fitted with ruminal trocars were used in a 4 × 4 Latin
square design experiment to evaluate effects of monensin (MON), anise extract (ANI) and capsicum
(CAP) on rumen microbial fermentation. Heifers were fed a 100:900 forage:concentrate diet (157 g
CP/kg and 223 g NDF/kg on a dry matter (DM) basis). Treatments were: no extract (CTR), 238 mg/d
of MON, 500 mg/d of ANI or 500 mg/d of CAP. Each period consisted of 15 d of adaptation and 9 d of
sampling. From d 16 to 21 of each period, DM intake was measured while, from d 22 to 24, ruminal
contents were sampled 0, 4, 8 and 12 h after feeding to determine ruminal pH, volatile fatty acids (VFA)
concentration, large peptide (LPep), small peptide plus amino acid (SPep + AA), and ammonia N. Sta-
tistical differences were declared if P<0.05. Monensin reduced VFA from 112.6 to 108.8 mM, acetate
proportion from 55.3 to 54.4 mol/100 mol, acetate to propionate ratio from 2.03 to 1.92 mol/100 mol,
branch-chained VFA (BCVFA) from 2.5 to 2.1 mM, NH3 N from 15.3 to 13.4 mg/100 ml, proto-
zoa counts from 5.69 to 5.46 log10 counts/ml, and increased propionate proportion from 25.2 to

Abbreviations: ADFom, acid detergent fibre; ANI, anise extract; BCVFA, branch-chained VFA; CAP, cap-
sicum; CP, crude protein; CTR, control treatment; DM, dry matter; LPep, large peptide; MON, monensin; aNDFom,
neutral detergent fibre; NH3 N, ammonia N; SPep + AA, small peptide plus amino acid; TCA, trichloroacetic acid;
TA, tungstic acid
夽 This paper is part of a special issue entitled “Enzymes, Direct Fed Microbials and Plant Extracts in Ruminant

Nutrition” guest edited by R. J. Wallace, D. Colombatto and P. H. Robinson.

∗ Corresponding author at: Facultat de Veterinària, Edifici V, Universitat Autònoma de Barcelona, 08193 Bel-

laterra, Spain. Tel.: +34 93 581 1495; fax: +34 93 581 1494.
E-mail address: Sergio.Calsamiglia@uab.es (S. Calsamiglia).

0377-8401/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.anifeedsci.2007.04.018
410 I. Fandiño et al. / Animal Feed Science and Technology 145 (2008) 409–417

27.5 mol/100 mol, SPep + AA N from 15.3 to 18.1 mg/100 ml and LPep N from 11.2 to 14.3 mg/100 ml
versus control. Capsicum increased DM intake from 6.57 to 7.42 kg/d and butyrate proportion from
13.0 to 14.1 mol/100 mol, and reduced acetate proportion from 55.3 to 54.0 mol/100 mol versus con-
trol. Anis reduced VFA from 112.6 to 110.8 mM, BCVFA from 2.5 to 2.1 mM and NH3 N from 15.3
to 13.6 mg/100 ml, acetate proportion from 55.3 to 53.5 mol/100 mol, acetate to propionate from 2.03
to 1.90, protozoa counts from 5.69 to 5.56 log10 counts/ml, and increased propionate proportion from
25.2 to 26.9 mol/100 mol and SPep + AA N from 15.3 to 18.0 mg/100 ml versus control. Capsicum may
be useful in stimulating appetite, and ANI and MON had similar effects on rumen fermentation except
for changes in molar proportion of butyrate, which suggests that they have a different mode of action.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Rumen fermentation; Plant extract

1. Introduction

One strategy to improve nutritional efficiency in ruminants is to reduce the loss of energy,
as methane, and N, as ammonia, from the rumen (Tamminga, 1996). Antibiotics added to
diets have been successful in reducing these losses (Schelling, 1984; Dinius et al., 1976;
Van Nevel and Demeyer, 1977) but, due to the risk of transferring residues into meat
and milk, the European Union prohibited its use after 1st January 2006 (Official Journal
of the European Union, 2003). Some plant extracts modify rumen fermentation reducing
methane and ammonia N production in the rumen (Busquet et al., 2005; Calsamiglia et al.,
2005; Cardozo et al., 2006) and are classified as generally recognized as safe for human
consumption by the US FDA (2004).
Previous in vitro studies conducted in our laboratory (Busquet et al., 2005, 2006; Cardozo
et al., 2004) found that use of plant extracts and secondary metabolites can be beneficial as
modifiers of ruminal fermentation, but most research has been conducted with dairy cattle
rumen fluid and diets, and results may not apply to beef cattle fed high concentrate diets
because effects appear to be diet and pH dependent (Castillejos et al., 2005; Cardozo et al.,
2005). Cardozo et al. (2005) examined a wide range of essential oils and active components
in vitro using a feedlot-type environment (i.e., high concentrate diets and low pH), and
indicated that anise, cinnamaldehyde, capsicum and garlic were potentially useful rumen
fermentation modifiers.
The objective was to evaluate effects of anise and capsicum oils on dry matter (DM)
intake and ruminal fermentation in growing heifers fed a high concentrate diet.

2. Materials and methods

2.1. Animal, treatments and housing

Four heifers (average initial BW 229.2 ± 24.1 kg) fitted with 1 cm i.d. plastic ruminal
trocars (Divasa Farmavic SA, Vic, Spain) were used in a 4 × 4 Latin square design exper-
iment. Heifers were individually housed in tie-stalls at the Unitat de Granges i Camps
Experimentals of the Universitat Autònoma de Barcelona (Spain), and randomly assigned
to one of four experimental diets. The basal diet consisted of (DM basis) barley straw
 I. Fandiño et al. / Animal Feed Science and Technology 145 (2008) 409–417 411

(100 g/kg), ground barley grain (279 g/kg), ground corn grain (256 g/kg), soybean meal
(109 g/kg), soybean hulls (67 g/kg), corn gluten feed (57 g/kg), tapioca (57 g/kg), sunflower
meal (72 g/kg) and a mineral and vitamin mixture (3 g/kg; 1 kg DM of which contained
1562k IU vitamin A; 150k IU vitamin D; 2500 mg vitamin E; 3500 mg zinc; 2000 mg iron;
400 mg manganese; 250 mg copper; 50 mg cobalt; 38 mg iodine; 25 mg selenium). The
diet (910 g/kg DM, 157 g/kg CP, 223 g/kg aNDFom and 117 g/kg ADFom; DM basis) was
designed to meet or exceed nutrient recommendations of a 300 kg BW Holstein heifers
with an ADG of 0.89 kg/d (NRC, 1996). Treatments were: no additive (control), anise oil
(500 mg/d), capsicum oil (500 mg/d) or monensin (238 mg/d). Concentrations of anise and
capsicum oils were selected based on previous in vitro studies (Cardozo et al., 2005) and
the expected concentrations in the rumen were based on calculations by Cardozo et al.
(2006), and monensin was used at the recommended level. Capsicum and anise oils were
provided encapsulated by Pancosma SA (Pancosma, Switzerland) to reduce palatability and
handling problems while allowing their availability in the rumen. Each experimental period
consisted of 24 d (15 d adaptation and 9 d for sample collection). Animals had ad libitum
access to concentrate and barley straw offered once daily at 0900 h. The research proto-
col was approved by The Campus Laboratory Animal Care Committee of the Universitat
Autònoma de Barcelona (Spain).

2.2. Sample collection and analyses

The DM and water intake were measured individually from d 16 to 21 of each period
to avoid interference with rumen sampling protocols. Feed refusals were recorded before
feeding. Refused concentrate and barley straw were manually separated with a sieve (0.5 cm
screen), and weighed separately. Feed and refusal samples were collected daily and analyzed
for DM to determine DM intake. Dry matter was determined by oven drying feed at 105 ◦ C
for 24 h (AOAC, 1990; ID 950.01), ash by heating feed at 550 ◦ C for 4 h (AOAC, 1990;
ID 942.05) and OM was determined by difference. The N content was determined using a
Kjeldahl procedure (AOAC, 1990; ID 976.05), ash corrected NDF was determined using
␣-amylase and sodium sulphite (Van Soest et al., 1991), and ADF determined sequentially
(AOAC, 1990; ID 989.03).
From d 22 to 24 of each period, samples of whole ruminal contents were collected 0,
4, 8 and 12 h after the morning feeding. Rumen pH was measured immediately with a
portable pH-meter (model 507, Crison Instruments SA, Alella, Spain). Ruminal fluid was
strained through two layers of cheesecloth and five sub-samples of filtrate were collected
for determination of VFA, large peptides (LPep), small peptides plus AA (SPep + AA),
ammonia N and enumeration of protozoa.
Samples for VFA were prepared as described by Jouany (1982). One milliliter of a
solution comprising 2 g/l of mercuric chloride, 2 g/l 4-methylvaleric acid as an internal
standard, and 20 ml/l of orthophosphoric acid was added to 4 ml of filtered rumen fluid
and frozen. Samples were centrifuged at 15,000 × g for 15 min at 7 ◦ C, and the supernatant
fluid was analyzed by gas chromatography (model 6890; Hewlett-Packard, Palo Alto, CA,
USA) using a polyethylene glycol nitroterephthalic acid-treated capillary column (BP21;
SGE, Europe Ltd., Buckinghamshire, UK) at 275 ◦ C in the injector and at a gas flow rate
of 29.9 ml/min.
412 I. Fandiño et al. / Animal Feed Science and Technology 145 (2008) 409–417

For ammonia N determination, a 4 ml sample of filtered rumen fluid was acidified with
4 ml of 0.2N HCl and frozen. Samples were centrifuged at 15,000 × g for 15 min at 7 ◦ C,
and the supernatant was analyzed by spectrophotometry (Libra S21, Biochrom Analytical
Instruments, Cambridge, UK) for ammonia N (Chaney and Marbach, 1962).
The TCA and TA N were determined as described by Winter et al. (1964). To determine
TA soluble N, 16 ml sample of filtered rumen fluid was added to 4 ml of 100 g/l sodium
tungstate and 4 ml of 1.07N sulphuric acid. After allowing the tubes to stand at 5 ◦ C for 4 h,
they were centrifuged at 9000 × g for 15 min, and supernatant was frozen until analyzed
for TA soluble N by the Kjeldahl procedure (AOAC, 1990; ID 976.05). To determine TCA
soluble N, 4 ml of 500 g/l TCA solution was added to 16 ml of filtered rumen fluid. After 4 h
at 5 ◦ C, tubes were centrifuged at 9000 × g for 15 min, and the supernatant was frozen until
analyzed for TCA soluble N by the Kjeldahl procedure. Results were used to calculate the
following (mg/100 ml): (1) LPep = TCA soluble N-TA soluble N, and (2) SPep + AA = TA
soluble N – ammonia N.
For enumeration of protozoa, 8 ml of filtered rumen fluid were mixed with 2 ml of
methyl green:formaldehyde (380 g/l) solution. Entodiniomorphs and holotrichs were iden-
tified (Dehority, 1993) and counted as described by Veira et al. (1983) using a Neubauer
Improved Bright-Line counting chamber (Hauser Scientific Partnership, Horsham, PA,
USA). Protozoa counts were transformed to base 10 logarithms before statistical analysis.

2.3. Statistical analyses

Results were analyzed using the PROC MIXED procedure for repeated measures (Littel
et al., 1998) using SAS (version 8.2; SAS Inst., Inc., Cary, NC, USA). The model accounted
for effects of treatments and days for DM intake, or effects of treatments, days and hours for
ruminal pH, the concentration and proportion of VFA, LPep N, SPep + AA N, ammonia N,
and protozoa counts, and the interaction of treatment by days or treatment by hours. Period
was considered as a random effect. Each variable was subjected to three covariance struc-
tures: compound symmetric, autoregressive order one, and unstructured covariance. The
covariance structure that yielded the largest Schwarz’s Bayesian criterion was considered to
be the most desirable analysis, and least square means for treatments are reported. For com-
parisons among treatments at each hour, and between 0 and 4, 8 and 12 h within treatments,
the Bonferroni comparison test was used, and differences were declared if P<0.05. Differ-
ences between treatments and control were declared at P<0.05 using Dunnett’s multiple
comparison test and least squares means for treatments are reported.

3. Results

Capsicum increased (P<0.05) concentrate and tended to increase (P<0.10) total DM

intake, with no effects of anise and monensin (Table 1). Because there were no interactions
between treatment and time after feeding for any of the ruminal fermentation metabo-
lites measured, only averages over time are shown in Tables 2 and 3. Monensin increased
(P<0.05) ruminal pH, the molar proportion of propionate, the concentration of LPep and
the concentration of SPep + AA, and reduced (P<0.05) the total VFA concentration, the
 I. Fandiño et al. / Animal Feed Science and Technology 145 (2008) 409–417 413

Table 1
Effect of monensin, capsicum and anise on dry matter intake
Control Monensin Capsicum Anise S.E.M.
DM intake (kg/d) 7.5 7.6 8.3+ 7.7 0.43
Concentrate intake 6.6 6.8 7.4* 6.8 0.32
Barley straw intake 0.9 0.9 0.9 0.9 0.08
* Within a row, means are different from control (P<0.05).
+ Within a row, means are different from control (P<0.10).

Table 2
Effect of monensin, capsicum and anise on average rumen pH, volatile fatty acids concentration
Control Monensin Capsicum Anise S.E.M.
pH 5.77 5.91* 5.78 5.85 0.06
Total VFA (mM) 112.6 108.8* 112.0 110.8* 1.58
BCVFAa (mM) 2.47 2.11* 2.56 2.10* 0.10
Individual VFA (mol/100 mol)
Acetate 55.3 54.5* 54.0* 53.5* 0.45
Propionate 25.2 27.5* 25.6 26.9* 0.30
Butyrate 13.0 12.2* 14.1* 13.5 0.28
C2:C3b 2.03 1.92* 1.98 1.90* 0.04?
* Within a row, means are different from control (P<0.05).
a BCVFA = branched-chain VFA, includes isobutyrate and isovalerate.
b Acetate:propionate.

molar proportion of acetate, butyrate and the BCVFA concentration, the concentration of
ammonia N, and the acetate to propionate ratio, versus the control (Tables 2 and 3). Cap-
sicum reduced (P<0.05) the molar proportion of acetate, and increased the molar proportion
of butyrate, versus the control (Table 2). Anise increased (P<0.05) the molar proportion
of propionate, and the concentration of SPep + AA, and reduced (P<0.05) the total VFA
concentration, the molar proportion of acetate and the BCVFA concentration, the concen-
tration of ammonia N and the acetate to propionate ratio, versus the control (Tables 2 and 3).
There was no time of sampling × treatment interaction for protozoa counts, and average
treatment effects are shown in Table 4. Monensin and anise reduced (P<0.05) the con-
centration of total protozoa, as well as entodiniomorphs and holotrichs, versus the control
(Table 4).

Table 3
Effect of monensin, capsicum and anise on average nitrogen fraction concentrations in the rumen
Control Monensin Capsicum Anise S.E.M.
LPep (mg/1)a 112 143* 132 125 11.7
SPep + AA (mg/l)b 153 181* 168 180* 10.9
Ammonia-N (mg/1) 153 134* 157 136* 14.7
* Within a row, means are different from control (P<0.05).
a LPep = large peptide.
b SPep + AA = small peptide plus amino acid.
414 I. Fandiño et al. / Animal Feed Science and Technology 145 (2008) 409–417

Table 4
Effect of monensin, capsicum and anise on average protozoa populations in the rumen
Control Monensin Capsicum Anise S.E.M.
Protozoa, log10 (counts/ml)
Entodiniomorphs 5.65 5.43* 5.63 5.51* 0.07
Holotrichs 4.56 4.38* 4.55 4.47* 0.12
Total 5.69 5.46* 5.67 5.56* 0.09
* Within a row, means are different from control (P<0.05).

4. Discussion

4.1. Dry matter intake

Capsicum enhanced consumption of concentrate by 12.1% which is consistent with

previous research that reported that capsicum increased DM intake by 9% (Cardozo et al.,
2006). It is unlikely that the increase in the DM intake was due to improved palatability
since the capsicum oil was encapsulated. There is no other research on effects of capsicum
on DM intake in ruminants but capsaicin, the active component of capsicum oil, increased
DM and water intake in rats (Zafra et al., 2003) and may have stimulated appetite in humans
(Calixto et al., 2000). Increased DM intake can be desirable at some times, such as in during
adaptation periods where stressors of fasting and dehydration associated with transportation
can markedly affect DM intake (Brown et al., 2006). In contrast, neither monensin nor anise
resulted in changes in DM intake, which is consistent with previous reports. For example,
monensin maintained or reduced DM intake (Schelling, 1984), and effects of anise on DM
intake in ruminants are limited and contradictory. While no effects were reported in our
study, and that of Nombekela et al. (1994) in dairy cows, Cardozo et al. (2006) reported a
trend to higher concentrate intake when anise was supplied at 2 g/d to beef heifers fed high
concentrate diets.

4.2. Rumen fermentation

VFA are end products of rumen microbial fermentation and represent the main supply
of metabolizable energy for ruminants (Van Soest, 1982). The reduction in the ruminal
concentration of total VFA due to feeding monensin agrees with previous reports (Wallace
et al., 1980; Fuller and Johnson, 1981; Richardson et al., 1976), but suggests that digestion
of nutrients in the rumen decreased. Fuller and Johnson (1981) and Chalupa et al. (1980)
observed that ionophores decreased DM and OM digestion in vitro, probably due to a
decrease in fibre degradation, and this was attributed to the higher sensitivity of cellulolytic
strains to the antibiotic. In contrast, in vivo research suggests that monensin increased DM
digestibility (Dinius et al., 1976; Poss et al., 1979), probably due to adaptation of cellulolytic
bacteria resistant to monensin, such as Fibrobacter succinogenes (Chen and Wolin, 1979),
that may replace other bacterial populations to avoid the decrease in fibre degradation
(Schelling, 1984).
Anise also reduced the concentration of total VFA, although this reduction was less
than that of monensin. These results agree with observations of Cardozo et al. (2005), who
 I. Fandiño et al. / Animal Feed Science and Technology 145 (2008) 409–417 415

reported that high doses of anise, evaluated in vitro, reduced total VFA concentrations. In
contrast, anise did not affect ruminal pH, probably due to the small decrease in total VFA.
The reduction in the acetate to propionate ratio has been reported previously for monensin
(Wallace et al., 1980; Richardson et al., 1976) and anise in vitro (Cardozo et al., 2005) and
in vivo (Cardozo et al., 2006).
Anise resulted in effects similar to monensin, except for the changes in molar proportion
of butyrate, that decreased in monensin but was unaffected by anise versus the control.
The decrease in butyrate production with monensin is due to inhibition of the gram-positive
bacteria Butyrivibrio fibrisolvens, a major butyrate producer in the rumen (Bryant and Small,
1956). The difference in the butyrate concentration suggests that monensin and anise may
have different modes of action on rumen microbial metabolism.
BCVFA are derived from deamination of AA, and changes in these metabolites are
frequently parallel (Hino and Russell, 1985). Monensin reduced BCVFA and ammonia-N
concentrations, and increased the concentration of the LPep and SPep + AA, which suggests
that monensin reduced deamination and may have inhibited proteolytic enzymes (Van Nevel
and Demeyer, 1977). Anise also reduced BCVFA and ammonia-N concentrations, and
increased concentrations of SPep + AA N, supporting the hypothesis that anise inhibits
deamination. These results are consistent with changes observed under similar in vivo
conditions (Cardozo et al., 2006).

4.3. Protozoa population

Rumen ciliate protozoa play diverse roles in rumen metabolism and, in their absence,
the numbers of bacteria and starch degradation increase, and ammonia N concentration
decreases (Veira, 1986; Van Nevel and Demeyer, 1988). Monensin reduced the concentra-
tion of total, entodiniomorph and holotrich populations, consistent with their antiprotozoal
effects (Richardson et al., 1976; Poss et al., 1979; Hino and Russell, 1986). There is
very limited information on effects of plant extracts and secondary compounds on rumi-
nal protozoa counts. Newbold et al. (2004) found no effects of a blend of essential oils
on protozoal numbers. However, in the present experiment, anise reduced concentrations
of total, entodiniomorphs and holotrichs, supporting the hypothesis of its antiprotozoal
effect. Although the mechanism of action of anise is not well understood, Cardozo et al.
(2006) proposed that their lipophilic nature may allow them to cross the cell membrane of
protozoa.

5. Conclusions

Monensin resulted in expected changes in rumen fermentation. Capsicum increased

DM intake, although it also reduced the acetate, and increased the butyrate, proportions
of total ruminal VFA. Anise reduced the acetate proportion, and increased the propionate
proportion while not affecting the butyrate proportion, suggesting a different mechanism of
action on rumen microbial fermentation versus monensin. Results indicate that capsicum
and anise may be useful rumen fermentation modifiers in beef production systems, but
animal performance data is required to confirm their benefits.
416 I. Fandiño et al. / Animal Feed Science and Technology 145 (2008) 409–417

Acknowledgments

The authors thank Pancosma SA and Karizoo for their financial support and technical
assistance. This project was partially funded by the Project CDTI 04-0005 of the Spanish
Government.

References

AOAC, 1990. Official Methods of Analysis, 13th ed. Association of Official Analytical Chemists, Washington,
DC, USA.
Brown, M.S., Ponce, C.H., Pulikanti, R., 2006. Adaptation of beef cattle to high-concentrate diets: performance
and ruminal metabolism. J. Anim. Sci. 84 (E. Suppl.), E25–E33.
Bryant, M.P., Small, N., 1956. The anaerobic monotrichous butyric acid-producing curved rod-shaped bacteria in
the rumen. J. Bacteriol. 72, 16–21.
Busquet, M., Calsamiglia, S., Ferret, A., Kamel, C., 2005. Screening for effects of plant extracts and active
compounds of plants on dairy cattle rumen microbial fermentation in a continuous culture system. Anim. Feed
Sci. Technol. 124, 597–613.
Busquet, M., Calsamiglia, S., Ferret, A., Kamel, C., 2006. Plant extracts affect in vitro rumen microbial fermen-
tation. J. Dairy Sci. 89, 761–771.
Calixto, J.B., Beirith, A., Ferreira, J., Santos, A.R.S., Filho, V.C., Yunes, R.A., 2000. Naturally occurring antinoci-
ceptive substances from plants: a review. Phytother. Res. 14, 401–418.
Calsamiglia, S., Castillejos, L., Busquet, M., 2005. Alternatives to antimicrobial growth promoters in cattle. In:
Garnsworthy, P.C., Wiseman, J. (Eds.), Recent Advances in Animal Nutrition. Nottingham University Press,
Nottingham, UK.
Cardozo, P.W., Calsamiglia, S., Ferret, A., Kamel, C., 2004. Effects of natural plant extracts on protein degradation
and fermentation profiles in continuous culture. J. Anim. Sci. 82, 3230–3236.
Cardozo, P.W., Calsamiglia, S., Ferret, A., Kamel, C., 2005. Screening for the effects of natural plant extracts at
different pH on in vitro rumen microbial fermentation of a high-concentrate diet for beef cattle. J. Anim. Sci.
83, 2572–2579.
Cardozo, P.W., Calsamiglia, S., Ferret, A., Kamel, C., 2006. Effects of alfalfa extract, anise, capsicum, and a
mixture of cinnamaldehyde and eugenol on ruminal fermentation and protein degradation in beef heifers fed
a high-concentrate diet. J. Anim. Sci. 84, 2801–2808.
Castillejos, L., Calsamiglia, S., Ferret, A., Losa, R., 2005. Effects of a specific blend of essential oil compounds
and the type of diet on rumen microbial fermentation and nutrient flow from a continuous culture system.
Anim. Feed Sci. Technol. 119, 29–41.
Chalupa, W., Corbett, W., Brethour, J.R., 1980. Effects of monensin and amicloral on rumen fermentation. J.
Anim. Sci. 51, 170–179.
Chaney, A.L., Marbach, E.P., 1962. Modified reagents for determination of urea and ammonia. Clin. Chem. 8,
130–132.
Chen, M., Wolin, M.J., 1979. Effect of monensin and lasalocid – sodium on the growth of methanogenic and
rumen saccharolytic bacteria. Appl. Environ. Microbiol. 38, 72–77.
Dehority, B.A., 1993. Laboratory Manual for Classification of Rumen Ciliate Protozoa. CRC Press, Boca Raton,
FL, USA.
Dinius, D.A., Simpson, M.E., Marsh, P.B., 1976. Effect of monensin fed with forage on digestion and the ruminal
ecosystem of steers. J. Anim. Sci. 42, 229–234.
FDA, 2004. Food and Drug Administration of the US, 21 CFR 184. http://www.cfsan.fda.gov/(lrd/FCF184.html
Accessed August. 17, 2004.
Fuller, J.R., Johnson, D.E., 1981. Monensin and lasalocid effects on fermentation in vitro. J. Anim. Sci. 53,
1574–1580.
Hino, T., Russell, J.B., 1985. Effect of reducing-equivalent disposal and NADH/NAD on deamination of amino
acids by intact rumen microorganisms and their cell extracts. Appl. Environ. Microbiol. 50, 1368–1374.
 I. Fandiño et al. / Animal Feed Science and Technology 145 (2008) 409–417 417

Hino, T., Russell, J.B., 1986. Relative contributions of ruminal bacteria and protozoa to the degradation of protein
in vitro. J. Anim. Sci. 64, 261–274.
Jouany, J.P., 1982. Volatile fatty acids and alcohol determination in digestive contents, silage juice, bacterial
cultures and anaerobic fermentor contents. Sci. Alim. 2, 131–144.
Littel, R.C., Henry, P.R., Ammerman, C.B., 1998. Statistical analysis of repeated measures data using SAS
procedure. J. Anim. Sci. 76, 1216–1231.
Newbold, C.J., McIntosh, F.M., Williams, P., Losa, R., Wallace, R.J., 2004. Effects of a specific blend of essential
oil compounds on rumen fermentation. Anim. Feed Sci. Technol. 114, 105–112.
Nombekela, S.W., Murphy, M.R., Gonyou, H.W., Marden, J.I., 1994. Dietary preferences in early lactation cows
as affected by primary tastes and some common feed flavors. J. Dairy Sci. 77, 2393–2399.
NRC, 1996. Nutrient Requirements of Beef Cattle, 7th ed. Natl. Acad. Press, Washington, DC, USA.
OJEU, 2003. Regulation (EC) No 1831/2003 of the European Parliament and the Council of 22 September 2003
on Additives for Use in Animal Nutrition. Official Journal of European Union. Page L268/36 in OJEU of
10/18/2003.
Poss, M.I., Hanson, T.L., Klopfenstein, T.J., 1979. Monensin effects on diet digestibility, ruminal protein bypass
and microbial protein synthesis. J. Anim. Sci. 48, 1516–1524.
Richardson, L.F., Raun, A.P., Potter, E.L., Cooley, C.O., 1976. Effect of monensin on rumen fermentation in vitro
and in vivo. J. Anim. Sci. 43, 657–664.
Schelling, G.T., 1984. Monensin mode of action in the rumen. J. Anim. Sci. 58, 1518–1527.
Tamminga, S., 1996. A review on environmental impacts of nutritional strategies in ruminants. J. Anim. Sci. 74,
3112–3124.
Van Nevel, C.J., Demeyer, D.I., 1977. Effect of monensin on rumen metabolism in vitro. Appl. Environ. Microbiol.
34, 251–257.
Van Nevel, C.J., Demeyer, D.I., 1988. In: Hobson, P.N. (Ed.), Manipulation of rumen fermentation. The Rumen
Microbial Ecosystem. Elsevier Applied Science, London, UK, USA, p. 387–444.
Van Soest, P.J., 1982. Nutritional Ecology of the Ruminant. Comstock, Cornell Univ. Press, Ithaca, NY, USA.
Van Soest, P.J., Robertson, J.P., Lewis, B.A., 1991. Methods for dietary fibre, neutral detergent fibre, and nonstarch
polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597.
Veira, D.M., 1986. The role of ciliate protozoa in nutrition of the ruminant. J. Anim. Sci. 63, 1547–1560.
Veira, D.M., Ivan, M., Juli, P.Y., 1983. Rumen ciliate protozoa: effects on digestion in the stomach of sheep. J.
Dairy Sci. 66, 1015–1022.
Wallace, R.J., Cheng, K.-J., Czerkawski, J.W., 1980. Effect of monensin on fermentation characteristics of the
artificial rumen. Appl. Environ. Microbiol. 40, 672–674.
Winter, K.A., Johnson, R.R., Dehority, B.A., 1964. Metabolism of urea nitrogen by mixed cultures of rumen
bacteria grown on cellulose. J. Anim. Sci. 23, 793–797.
Zafra, M.A., Molina, F., Puerto, A., 2003. Effects of perivagal administration of capsaicin on post-surgical food
intake. Auton. Neurosci. 107, 37–44.