Download BOARD-INVITED REVIEW: Opportunities and challenges in using

Published November 24, 2014
BOARD-INVITED REVIEW: Opportunities and challenges
in using exogenous enzymes to improve ruminant production
S. J. Meale,*† K. A. Beauchemin,† A. N. Hristov,‡ A. V. Chaves,* and T. A. McAllister†1
*Faculty of Veterinary Science, The University of Sydney, NSW 2006, Australia;
†Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB T1J 4B1, Canada;
and ‡Department of Animal Science, Pennsylvania State University, University Park 16802
ABSTRACT: The ability of ruminants to convert plant
biomass unsuitable for human consumption into meat
and milk is of great societal and agricultural importance. However, the efficiency of this process is largely
dependent on the digestibility of plant cell walls. Supplementing ruminant diets with exogenous enzymes has
the potential to improve plant cell wall digestibility and
thus the efficiency of feed utilization. Understanding
the complexity of the rumen microbial ecosystem and
the nature of its interactions with plant cell walls is the
key to using exogenous enzymes to improve feed utilization in ruminants. The variability currently observed
in production responses can be attributed to the array
of enzyme formulations available, their variable activities, the level of supplementation, mode of delivery, and
the diet to which they are applied as well as the productivity level of the host. Although progress on enzyme
technologies for ruminants has been made, considerable
research is still required if successful formulations are to
be developed. Advances in DNA and RNA sequencing
and bioinformatic analysis have provided novel insight
into the structure and function of rumen microbial populations. Knowledge of the rumen microbial ecosystem
and its associated carbohydrases could enhance the
likelihood of achieving positive responses to enzyme
supplementation. The ability to sequence microbial
genomes represents a valuable source of information in
terms of the physiology and function of both culturable
and unculturable rumen microbial species. The advent
of metagenomic, metatranscriptomic, and proteomic
techniques will further enhance our understanding of the
enzymatic machinery involved in cell wall degradation
and provide a holistic view of the microbial community
and the complexities of plant cell wall digestion. These
technologies should provide new insight into the identification of exogenous enzymes that act synergistically
with the rumen microbial populations that ultimately
dictate the efficiency of feed digestion.
Key words: carbohydrases, cattle, exogenous enzymes, rumen, plant cell wall
© 2014 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2014.92:427–442
doi:10.2527/jas2013-6869
INTRODUCTION
The efficiency by which ruminants obtain energy
from structural plant polysaccharides and in turn produce high quality meat and milk protein is increasingly
important if the demands of an expanding human population are to be met. Due to both economic considerations
and maintenance of rumen health, forage will almost always be a component of the diet of ruminants (Krause
et al., 2003). However, digestibility of forage cell walls
1Corresponding
author: [email protected]
Received July 5, 2013.
Accepted November 22, 2013.
427
ultimately limits nutrient availability as conditions for
fiber digestion are often suboptimal in the rumen.
Exogenous enzymes are increasingly considered
as a cost-effective means of improving feed efficiency
(Krause et al., 2003), yet production responses to exogenous enzymes are still highly variable. Several reviews
(Krause et al., 2003; Beauchemin et al., 2004; Beauchemin and Holtshausen, 2011; Tricarico et al., 2008) have
been published on the use of exogenous enzymes in ruminants; however, this paper aims to provide an updated
perspective on current research and new techniques being used to use these products as a means of improving
feed efficiency. Most research has centered on the use of
fibrolytic enzymes to increase fiber digestion and thus
digestible energy intake, but responses have been vari-
428
Meale et al.
able. Increases in milk (Gado et al., 2009; Klingerman
et al., 2009), ADG (Beauchemin et al., 1999; McAllister
et al., 1999), and DM and fiber digestion in situ, in vitro
(Yang et al., 1999; Hristov et al., 2008), and in vivo (Rode
et al., 1999; Beauchemin et al., 2000) have been reported.
However, in many cases, the efficiency of growth or milk
production in ruminants has not been improved (ZoBell et
al., 2000; Eun et al., 2009; Arriola et al., 2011).
Characterization of rumen microbial populations using “-omics” technologies provides insight into the functionality of rumen microbiota (Morgavi et al., 2012) and
the construction of gene catalogues through metagenomics and genomic sequencing expands our understanding
of the interactions between community members and the
carbohydrases they produce. These knowledge advancements are providing new insight into the formulation of
exogenous enzymes that act synergistically with the carbohydrases of rumen microbial communities in a manner
that enhances the efficiency of plant cell wall digestion.
SOURCES OF ENZYMES
Commercial enzyme products used in the livestock
industry are of fungal (Aspergillus oryzae and Trichoderma reesei) and bacterial (Bacillus subtilis, Lactobacillus
acidophilus, Lactobacillus plantarum, and Enterococcus faecium spp.) origin (Muirhead, 1996; McAllister et
al., 2001). Beginning with a seed culture and growth media, enzymes for the feed industry are produced through
microbial fermentation and although the source organisms only constitute a very limited group, the types and
activity of enzymes produced can be diverse depending
on the strain selected, the substrate they are grown on,
and the culture conditions used (Considine and Coughlan, 1989; Gashe, 1992; Lee et al., 1998).
A vast array of enzymes is required to degrade the
complex arrangements of structural carbohydrates in
plant cell walls (Morgavi et al., 2012). Although commercial enzyme preparations are commonly referred to
as cellulases or xylanases, secondary enzyme activities
such as amylases, proteases, esterases, or pectinases are
invariably present as these preparations seldom consist
of a single pure enzyme (McAllister et al., 2001). This
diversity is advantageous, as it facilitates targeting of a
range of substrates using a single product, yet it complicates the identification of the specific enzymes responsible for any positive responses observed in feed digestion. Degradation of cellulose and hemicellulose alone
requires a number of glycosidic hydrolases (Chesson
and Forsberg, 1997; Krause et al., 2003) and differences
in the relative proportions and activity of individual enzymes affects the overall efficacy of cell wall degradation (McAllister et al., 2001). A common approach is to
use an enzyme that may not be suited to a specific feed
but instead to formulate enzyme mixtures that are suitable for a range of feed types (Beauchemin et al., 2003).
However, this approach of adding enzymes to diets without consideration for specific substrates has contributed
to the highly variable results observed when enzymes
are used in ruminants, an outcome that has undoubtedly
discouraged and delayed the adoption of the technology.
MEASUREMENT OF ENZYME ACTIVITY
The activity of enzymes is assayed by measuring the
generation of the product from the biochemical reaction
that the enzyme catalyzes over time and is expressed as
the amount of product produced per unit of time (McAllister et al., 2001; Beauchemin et al., 2003). In the case
of carbohydrases, the production of free sugars is the
most common product measured. Measurement of enzyme activities must be conducted under closely regulated conditions as variations in temperature, pH, ionic
strength, substrate concentration, and substrate type influence enzyme activity (McAllister et al., 2001). Synthetic substrates can also be used to assay enzyme activity by measuring the release of a dye or chromophore
(Higginbotham et al., 1996). However, these conditions
fail to replicate those of the digestive tract, nor are the
substrates representative of intact feeds that are fed to
ruminants (McAllister et al., 2001). Consequently, these
assays may have limited relevance to the potential worth
of an enzyme as a feed additive for ruminants.
Biological assays using mixed ruminal microorganisms incubated with complex substrates has been one
approach to identify enzyme preparations that are more
suitable for use in ruminants. After the addition of enzymes, these in vitro incubations measure the digestion
of ingredients commonly included in ruminant diets (i.e.,
grains, hay, silage, or straw) by recording the production
of gas that arises from the fermentation process and the
digestibility of the feed DM and fiber at a given incubation time. Using this system, several enzyme preparations can be simultaneously screened for their effectiveness with different application methods and rates
(Muirhead, 1996; Iwaasa et al., 1998; Morgavi et al.,
2000b). However, these systems are not very representative of in vivo conditions (Hristov et al., 2012) and do
not account for animal-to-animal variation in the microbial community, making extrapolation of these results to
whole animal scenarios challenging. Additionally, these
systems do not account for the possible impact of exogenous enzymes on biological parameters such as feed
intake, rate of passage, or postruminal digestion of nutrients (McAllister et al., 2001). Even in vitro systems may
require more enzyme than what is typically produced
by most laboratory-based expression systems. To overcome this problem we have developed a micro-in vitro
Exogenous enzymes in ruminant production
system that enables the synergistic interactions of milligram quantities of exogenous enzymes with rumen microbial enzymes to be assessed in a reaction mixture as
small as 250 μL (Badhan, A., Wang, Y., and McAllister,
T.A. [Agriculture and Agri food Canada, AB]; Patton, D.,
Powlowski, J., and Tsang A. [Centre for Structural and
Functional Genomics, Concordia University, QC]). We
used the system to identify carbohydrases from thermophilic and anaerobic fungi that could enhance the liberation of sugars from alfalfa hay and barley straw by
mixed rumen enzymes. Such mini-assays could prove
invaluable for identifying those enzyme activities that
merit production scale up for animal experiments and
the formulation of efficacious enzyme cocktails.
In vitro screening of exogenous enzymes provides
a cost effective, less time consuming means of screening large numbers of products with specific substrates
and can be used to predict possible in vivo responses.
However, the final assessment of the true value of exogenous enzymes for ruminants in terms of improving
feed utilization can only be assessed through the use of
animal production trials.
PRODUCTION RESPONSES
TO EXOGENOUS ENZYMES
Dairy Cattle
A search of the Commonwealth Agricultural Bureaux International (CABI) database ( www.cabdirect.
org) and the Journal of Dairy Science ( www.journalofdairyscience.org) for “enzyme” and “dairy” in the title
returned 328 and 39 publications, respectively. From
these, 28 were selected and are summarized in Table 1.
Criteria for this selection were that the study had to be
published in English in a refereed journal, be with lactating dairy cows, and have a valid experimental design.
“Field” trials where control over cow grouping was not
clearly described or if there was potential for confounding factors were excluded from the analysis. Studies
judged as having major flaws in experimental design or
statistical analysis of the data were also excluded from
Table 1. A large portion of the studies were not included
as they did not involve the use of lactating cows.
Naturally, the main objective of using exogenous
enzymes in dairy cow nutrition is to improve milk production and yield of milk components and as previous
reviews (McAllister et al., 2001; Beauchemin et al., 2004;
Tricarico et al., 2008) have focused on the dairy-specific
effects of exogenous enzymes on ruminal fermentation
and intestinal digestion, this review will focus on production effects. Most products tested in dairy cows are described as cellulases and/or xylanases, with proteases and
429
amylases being investigated in fewer instances. It is practically impossible to compare exogenous enzyme preparations on an equal activity basis, as there is a distinct
lack of standardization in the methodology used to assess
enzyme activities among labs. Even when the same methods are used, it is difficult to standardize enzyme products
because they contain multiple activities and can only be
standardized for 1 or 2 activities at a time. For example,
when 2 products are used to supply the same level of endoglucanase activity, they may supply vastly different
levels of xylanase activity. Another common limitation of
the enzyme literature is the lack of repeatability of the effects and repeated investigations of a common exogenous
enzyme preparation as most are only examined in a single
experiment. Experimental cost is perhaps a major reason
for this lack of repetition, but the possibility that unfavorable results were not published cannot be excluded.
Interestingly, the commercial α-amylase product Amaize
(Alltech Inc., Nicholasville, KY) has been examined in 3
studies discussed in this review (DeFrain et al., 2005; Tricarico et al., 2005; Klingerman et al., 2009). All of these
studies fed a diet containing either alfalfa hay, alfalfa haylage and maize silage, or a mixed legume hay and maize
silage diet and observed no effects on milk yield or composition of milk components. The dose of enzyme and the
portion of the diet to which it was applied varied across
studies raising questions about the enzymes applicability
for use in lactating dairy cows fed current diets.
Few studies have reported positive effects of exogenous enzymes on milk components; for example, Beauchemin et al. (2000) reported a 2% increase in milk true protein with a β-glucanase/xylanase/endoglucanase product.
Similarly, Bowman et al. (2002), Sutton et al. (2003), and
Eun and Beauchemin (2005) reported increased milk fat
or protein. A few instances of increasing milk yield have
also been observed. For example, Yang et al. (1999) found
milk yield was increased by 1.9 kg/d when an exogenous
enzyme (Pro-Mote; Biovance Technologies Inc., Omaha,
NE) composed predominantly of cellulase and xylanase
activities was applied to hay at 2 g of enzyme mixture/
kg. This effect was attributed to a 12% increase in nutrient
digestibility. Despite these results, the application of exogenous enzymes to dairy cow diets has shown extremely
variable results and largely failed to improve production
efficiency. Of the studies compared in this paper, the
majority showed no effect on milk yield (DeFrain et al.,
2005; Hristov et al., 2008; Bernard et al., 2010; Peters et
al., 2010; Ferraretto et al., 2011) or the production of milk
components (Yang et al., 2000; Holtshausen et al., 2009;
Bernard et al., 2010; Arriola et al., 2011).
A recent study by Holtshausen et al. (2011) screened
5 doses of a fibrolytic enzyme additive (AB Vista, Marlborough, UK) and further assessed its efficacy in situ
before the enzyme additive was fed to lactating Holstein
Cellulose and xylanase
0.7 to 1.5 L/t forage
DM
Cellulase and xylanase 0.5 to 1 g/kg TMR DM
β-glucanase, xylanase, 1.22 to 3.67 L/t TMR
and endocellulase
Cellulase, hemicellulase, 2 to 10 L/t fresh forage
and xylanase
Xylanase
50 mg/kg TMR DM
FinnFeeds Int.7
Natugrain 33-L11
Vicini et al.,
200314
DeFrain et al.,
200516
Eun and
Beauchemin, 2005
Tricarico et al.,
Latin square (20)
2005
Elwakeel et al.,
Split plot (24)
2007
Knowlton et al.,
Completely randomized
2007
(24)
Reddish and Kung,
Switchover (24)
2007
Hristov et al., 2008
Latin square (4)
continued
204 g/t TMR
10 L/t fresh forage
Xylanase and cellulose
Cellulase
Cellulase and xylanase
Promote N.E.T.15
Loveland Industries Inc.,
Greeley, CO
FinnFeeds Int.7
Cellulase and phytase
Cellulose and xylanase
Amylase and xylanase
Alltech Inc.17
Alltech, Inc.17
10 g/cow per d
10 g/cow per d
297 g/t TMR DM
240 to 720 dextrinizing
units/kg TMR DM
β-glucanase and xylanase
15 g/cow per d
Amylase
1.25 mg/kg TMR DM
Protease
Cattle-Ase-P20
Saf Agri, Milwaukee, WI
Amaize17
47 to 69%
0.1% TMR DM
40%
50%
37%
37%
55%
34 to 60%
43 to 57%
57%
1.25 to 2 L/t TMR DM
2 kg/t TMR DM
45%
45 to 61%
55%
50 to 65%
38%
50%
45%
55%
55%
39%
Xylanase and
endoglucanase
Xylanase and
endoglucanase
Amylase
1 g/cow per d
1.25 L/t forage DM
Cellulase and xylanase
Biovance Technol.
Inc.5
Bovizyme 40117
FinnFeeds Int.7
Biovance Technologies,
Inc.5
Completely randomized FinnFeeds Int.14 and Biovance
(257 and 122)
Technologies Inc.5
Completely randomized
Amaize17
block (24)
Latin square (8)
Protex 6L18
Beauchemin et al.,
Latin square (6)
2000
Kung et al., 200014 Completely randomized
(30)
Yang et al., 20009 Completely randomized
block (43)
Zheng et al., 2000 Completely randomized
block (48)
Latin square (8)
Bowman et al.,
20029
Knowlton et al.,
Switchover (12)
2002
Kung et al., 2002 Completely randomized
(30)
Sutton et al., 20038
Latin square (4)
Pro-Mote5
1.3 kg/t TMR6 DM
Completely randomized
(20)
Schingoethe et al., Completely randomized
1999
block (50)
Yang et al., 19999
Latin square (4)
Xylanase and cellulase
34%
Pro-Mote5
Forage level
in basal diet
Application
level
Rode et al., 1999
Declared primary
activities
209 g/t3
Product/manufacturer
Completely randomized Digest M, Loveland Industries Amylase and protease
(36)
Inc., Greeley, CO
Experimental design
(number of cows)
Chen et al., 1995
Source1
30
40
37 to 39
43 to 44
29 to 30
41 to 48
32 to 33 and 28
to 29
38
34 to 36
36 to 39
31 to 43
29 to 30
33 to 37
33 to 35 and 36
to 39
35 to 37
30 to 31
24 to 26
25 to 28
36 to 40
34 to 37
-
-
-
-
NR
↓
-
-
-
-
-
-
-
-
-
↑
-
-
-
-
Milk
production, kg/d DMI
↑MMP
-
-
↓MF
-
-
-
-
-
-8
↓
-
-
-
-
-
-
-
-
-
-
-
↑DM, OM, and CP
-22
-21
NR
NR
↑All
↑MF; and ↓MMP19
-
NR
NR
↓DM, and OM
NR
-
↑All4
NR
-
-
↑MMP
-
-
↑MF and MMP
-
↑DM
NR
↑DM, and ↓NDF13
↑OM, and NDF
↑DM, OM, NDF, ADF,
and CP
NR
↑CP4
Milk components Total tract digestibility
↑ (both - or ↓MF and MMP
experiments)
↑
-
-
↑10
-8
-
-
Effects2
Milk yield
Table 1. Summary of exogenous polysaccharide-degrading enzyme effects on production traits and total tract apparent digestibility of nutrients in lactating dairy cows
430
Meale et al.
Experimental design
(number of cows)
Product/manufacturer
Roxazyme G2 Liquid23
Declared primary
activities
Application
level
Forage level
in basal diet
chronological order.
32 to 36
49.5 to 52.1
50%
-
26 to 27
52 to 67%
-
40 to 42
14Two
Feed Technologies, Greeley, CO.
Biology Laboratory of the Ain Shams University, Cairo, Egypt. Questionable data; see discussion.
Nutrition, Minneapolis, MN.
yield and digestibilities increased only by low level of an experimental amylase enzyme.
26Cargill Animal
25Milk
24Molecular
Nutritional Products Ltd., Basel, Switzerland.
experiment with sheep.
for increased NDF and ADF digestibility.
22Digestion
21Trends
23DSM
International, Rochester, NY.
low-forage (34%) diet.
20Animal
19Only
diets were fed from 21 d before expected calving through 21 d in milk.
Inc., Nicholasville, KY.
18Genencor
17Alltech
16Experimental
International, St. Louis, MO.
the high enzyme application level.
experiments.
13Only
15Agribrands
the low enzyme application level.
12Only
Corporation, Ludwigshafen, Germany.
one of the application methods.
11BASF
10For
International, Marlborough, Wiltshire, UK.
increase of 3.5% fat corrected milk.
studies investigated enzyme application method.
8Reported
9These
Inc., Omaha, NE.
= total mixed ration.
7FinnFeeds
6TMR
5Biovance Technologies
with grain processing.
to the grain portion of the diet.
4Interaction
-
-
↓
↑
44 to 47
38
↑
13 to 16
52%
-
28 to 29
Milk
production, kg/d DMI
= increase; ↓ = decrease; - = no statistically significant effect; NR = not reported; MF = milk fat percentage; MMP = milk protein percentage; All = all nutrients studied.
3Applied
2↑
1In
Holtshausen et al., Completely randomized AB Vista, Marlborough, UK
Xylanase and
0.5 to 1.0 mL/kg DM
2011
(60)
endoglucanase
Arriola et al., 2011 Completely randomized Dyadic International Inc., Xylanase, exoglucanase, 3.4 mg/g TMR DM
block (66)
Jupiter, FL
and endoglucanase
Ferraretto et al.,
Completely randomized Ronozyme RumiStar CT23
Amylase
300 kilo novo units/kg
2011
(45)
TMR DM
Xylanase and
2.15 and 4.30 mL/kg Pasture and 6.7 kg/d
endoglucanase
concentrate
grain supplement
Gado et al., 200924 Completely randomized
ZADO24
Protease, amylase, and
40 g/cow per d
70%
(20)
cellulase
Klingerman et al.,
Latin square (28)
Amaize17 and an experimental
Amylase
0.4 g/kg TMR DM and
50%
2009
preparation23
0.88 to 4.4 mL/kg
Bernard et al.,
Completely randomized
Promote N.E.T.-L26
Cellulase
4 g/cow per d
50 to 54%
2010
(44)
Peters et al., 2010
Switchover (6)
Roxazyme G223
Cellulase and xylanase 6.2 mL/kg TMR DM
50%
Miller et al., 2008 Completely randomized
block (72)
Source1
Table 1. (continued)
-
-
-
-
↓MMP
-
-
-
-
-
↑25
-
-
-
NR
↑All
NR
-
↑DM, OM, NDF, and
ADF
↑DM, OM, CP, and
NDF25
NR
NR
Milk components Total tract digestibility
↑
-
Effects2
Milk yield
Exogenous enzymes in ruminant production
431
432
Meale et al.
dairy cows. The enzyme product improved fat corrected
milk (FCM) production efficiency in a dose dependent
manner up to 11.3%. Similarly, Arriola et al. (2011)
screened varying amounts of a fibrolytic enzyme product in situ before conducting a feeding trial. Milk production efficiency was increased in cows fed this enzyme product with a low-concentrate diet as compared
with those fed either an untreated low-concentrate diet
or a high-concentrate diet (treated or untreated). Therefore, it is evident that careful attention needs to be paid
to the type and dose of enzymes being applied to dairy
cattle diets. The use of in vitro screening can help to
elucidate appropriate doses and predict production responses when combined with specific substrates. However, it remains difficult to assess the consistency of animal responses to individual enzyme products, as most
products are experimental and can vary in activity over
time. In many instances the same product formulation is
often used in only a limited number of studies of widely
differing experimental conditions.
Beef Cattle
Although the first reports of exogenous enzymes improving beef cattle BW gains were over 50 yr ago (Burroughs et al., 1960), adoption of enzyme technology has
been slow as the cost of enzymes outweighs that of other
additives, such as ionophores, antibiotics, and implants
(Beauchemin et al., 2006). We used a similar criterion as
described above for dairy production studies to identify
beef production studies (CABI and the Journal of Animal Science [ www.journalofanimalscience.org]) with a
search for “enzyme” and “beef cattle” in the title returning 383 and 70 titles, respectively. From these, 11 studies
were selected and summarized in Table 2. Experimental
criteria for selection were the same as for dairy cattle,
with studies having to be conducted with growing, backgrounding, or finishing beef cattle. Although responses to
exogenous enzymes are expected to be greater in beef cattle fed roughage-based diets as compared with high-grain
diets, many exogenous enzyme formulations have shown
promising effects in cattle fed high-grain finishing diets,
at least when included in barley-based diets (Beauchemin
and Holtshausen, 2011). However, these responses are still
variable depending on the dosage of the enzyme applied,
the time of application in relation to feeding, and the portion of the diet to which they are applied. Application of
a mixture of xylanase and cellulase products (Xylanase B
[Biovance Technologies Inc., Omaha, NE] and Spezyme
CP [Genencor, Rochester, NY]) increased ADG of steers
fed alfalfa hay or timothy hay by 30 and 36%, respectively,
but had no effect when applied to barley silage (Beauchemin et al., 1995). These positive responses were attributed
to an increase in digestible DM intake; however, it was
noted that forage type influenced the optimal dose required to elicit these responses (0.25 to 1.0 L/t DM for alfalfa hay versus 4 L/t DM for timothy hay), demonstrating
the importance of interactions between dosage, enzyme,
and substrate. A subsequent study with steers assessed the
same enzyme formulation in high-concentrate diets (95%
DM) containing either barley or corn grain (Beauchemin
et al., 1997). Application to a barley grain diet improved
feed efficiency by 11%, yet performance was unaffected
when added to corn. Supplementing a similar exogenous
enzyme mixture (FinnFeeds Int. Ltd., Marlborough, UK)
increased ADG of steers by 10% when applied to both
the grain and forage portions of the diet (McAllister et al.,
1999) and resulted in a 28% increase in ADF digestibility
(Krause et al., 1998). These studies indicate the application of a xylanase and cellulase enzyme formulation is
promising in terms of increasing ADG when applied to
either barley grain or forage diets; however, the use of this
enzyme formulation is not recommended in diets based
on corn grain or barley silage due to its apparent lack of
effectiveness with these feeds. Conversely, a study by
Tricarico et al. (2007) reported an A. oryzae extract containing α-amylase activity quadratically increased ADG
when included in either a cracked corn or high-moisture
corn and corn silage diet but had no effect when included
with alfalfa hay, cotton seed hulls, or steam-flaked corn.
Similarly, DiLorenzo et al. (2011) observed no effects of
supplementing an amylase enzyme formulation (600 kilo
novo units/kg of dietary DM; RumiStar; DSM Nutritional
Products, Inc., Kaiseraugst, Switzerland) to either a driedrolled corn or steam-flaked corn diet, indicating the need
for further investigation of the various enzyme types and
their applicability for feeds commonly supplied to beef
cattle. A lack of response to amylases may reflect the fact
that starch digestion is generally not limited in the rumen,
provided that the grains are adequately processed (McAllister and Cheng, 1996).
Recently, the addition of an experimental exogenous
proteolytic enzyme (Danisco-Agtech, Waukesha, WI)
during the growing phase increased DMI of steers by
14.8%, but an increase in ruminal passage rate reduced
NDF digestibility (4.1%) and as a result this increase in
DMI was not reflected in improvements in BW gain or
feed efficiency nor were any effects observed when this
same enzyme was added to a finishing diet (Vera et al.,
2012). ZoBell et al. (2000) observed no effects on ADG or
feed efficiency when applying the same enzyme product
to either a barley-based growing (65:35 forage to concentrate ratio; DM basis) or finishing diet (20:80 forage to
concentrate ratio; DM basis) as McAllister et al. (1999),
who observed an increase in DMI when this enzyme was
applied (0.5 L/t DM) to barley silage as well as increase
an in ADG when it was applied at (3.5 L/t total mixed ration [TMR]) to a finishing TMR. Comparatively, Balci et
433
Exogenous enzymes in ruminant production
al. (2007) applied Promote N.E.T. (60 g/d; Agribands Int.,
St. Louis, MO) with cellulase and xylanase activities to a
corn and barley diet and observed increases in ADG and
feed conversion efficiency. Eun et al. (2009) supplemented both growing and finishing diets with a commercial
enzyme product (Fibrozome; Alltech Inc.) and observed
no effect on growth performance, despite minor improvements in carcass characteristics. Lewis et al. (1996) applied Grasszyme (FinnFeeds Int. Ltd., Marlborough, UK)
to a grass hay and barley diet (70:30) and measured the
impact of application time before feeding and the portion
of the diet to which the enzyme was applied. No effects
on DMI were observed; however, digestibility of DM,
NDF, and ADF increased when the enzyme was added
to the forage either 24 h before or at the time of feeding.
Similarly, Krueger et al. (2008) applied an enzyme mixture (Biocellulase A20; Loders Croklaan, Channahon, IL)
to Bermudagrass hay at 3 different stages, immediately
after cutting, at bailing, or at feeding and although enzyme treatment at cutting increased DMI, no effect was
observed on final live weight, ADG, or G:F, regardless of
the time of application. Evidently, most studies have observed inconsistent responses after enzyme supplementation in beef cattle despite the commonality of a barleybased diet, hampering the adoption of this technology on
an industry-wide scale. Similarly, the studies that exhibited increases in ADG used different application methods whereby the enzyme was applied to the TMR in the
study by McAllister et al. (1999) in the study of Balci et
al. (2007), it was only applied to the concentrate portion
of the diet, and as these studies were the only reports of
positive effects on production performance, it is difficult
to discern at this point which method, if either, would best
ensure the efficacy of exogenous enzymes in beef cattle.
Small Ruminants
Generally, the application of exogenous enzymes
to the diets of small ruminants has had little impact on
production performance. Miller et al. (2008) fed a barley-based diet treated with a commercial exogenous enzyme (Roxazyme G2 Liquid; DSM Nutritional Products
Pty Ltd, Basel, Switzerland) to Dorset-cross ewe lambs
and observed no effects on DMI, ADG, feed conversion,
or wool growth. Similarly, no effects were observed on
milk yield, milk composition, or DMI when Promote
(Agribrands Int., St. Louis, MO) was applied to the diets of lactating Manchega and Lacaune ewes (Flores et
al., 2008). Additionally, Rojo et al. (2005) fed exogenous
amylases from Bacillus licheniformis and Aspergillus
niger (up 2.90 g enzyme/kg DM sorghum; ENMAX,
Mexico City, Mexico) and observed no effects on production performance in Suffolk lambs. As such, the majority of exogenous enzyme studies found in sheep and
goats have focused on investigating the impact of exogenous enzymes on diet digestibility. In a study by Reddish and Kung (2007) lambs were fed a commercial diet
supplemented with an enzyme mixture (4 g/lamb daily;
Alltech Inc.); however, no effect on apparent digestibility of DM, ADF, NDF, or N was observed. A study by
Avellaneda et al. (2009) fed Suffolk lambs Guinea grass
in conjunction with fibrolytic enzymes (3 g/lamb daily;
Fibrozyme; Alltech Inc.) and also reported no effects on
DMI, ruminal fermentation, or ruminal or total tract digestion. Giraldo et al. (2008) delivered exogenous fibrolytic enzymes (12 g/lamb daily; Fibrozyme; Alltech Inc.)
directly into the rumen of fistulated Merino sheep fed a
grass–hay concentrate diet (70:30; DM basis) without affecting diet digestibility. By supplementing the enzyme
directly into the rumen the prefeeding feed–enzyme
interaction was negated, yet the enzymes were able to
stimulate fibrolytic activity and the growth of cellulolytic bacteria. Conversely, Bala et al. (2009) applied 2 levels of exogenous enzymes, described as a cellulase and
a xylanase (4,000 and 12,500 or 8,000 and 18,750 IU/
kg, respectively), to the diets of lactating Beetle-sannen
crossbred goats. The greatest amounts of supplementation decreased DMI (g/kg FCM yield) and increased the
digestibility of DM, OM, CP, NDF, ADF, and total carbohydrates and improved FCM yield (kg/d) in the last quarter of lactation. These studies suggest that the application
of existing exogenous enzymes to lamb diets has limited
impact on diet digestibility or growth performance. Additional studies are required to determine if the response
to exogenous enzymes in lactating goats is a reflection of
their high metabolic demand for milk production.
MODES OF ACTION
Preconsumption Effects
Application of exogenous enzymes before consumption appears to be the most effective when they
are applied in a liquid form to dry as opposed to wet
forage as there appears to be components in silage that
can inhibit exogenous enzymes (Morgavi et al., 2000a;
Nsereko et al., 2000; Wallace et al., 2001). Even the
low moisture content in dry feeds, such as hay and
grain, appears sufficient to enable hydrolysis of carbohydrates from complex polymers (Morgavi et al.,
2000b). This release of sugars arises, at least partially,
from the solubilization of NDF and ADF (Morgavi
et al., 2000b; Morrison and Miron, 2000; Devillard
et al., 2004) encouraging rapid microbial growth and
reducing the lag time required for microbial colonization (Beauchemin et al., 2004). The type of exogenous
enzyme and substrate determines the degree of sugar
release, yet this represents only a minute portion of the
Promote N.E.T.14
Completely randomized (16)
chronological order.
Danisco-Agtech,
Waukesha, WI
RumiStar19
Inc., Omaha, NE.
continued
Nutrition, Minneapolis, MN.
= total mixed ration.
14Cargill Animal
13TMR
experiment with sheep.
11Only
12Digestion
the greatest amount of enzyme application (5.0 L/t) in the backgrounding study.
in the finishing stage.
10Only
International, Marlborough, Wiltshire, UK.
on forage and application rate (increases seen at alfalfa level 1, 2, and 3 and at timothy hay level 5).
9Finnfeeds
on forage and application rate (increases seen at alfalfa level 3).
8Dependant
= filter paper units of cellulase.
Rochester, NY.
Protease
7Dependant
6FPU
5Genencor,
4Biovance Technologies
= feed conversion rate.
= increase; ↓ = decrease; - = no statistically significant effect; NR = not reported.
3FCR
2↑
1In
Completely randomized block
(32)
Completely randomized (48)
Completely randomized block
Amaize15
(120, 96, and 56)
Completely randomized (50) Biocellulase A20, Loders
Croklaan, Channahon, IL
Completely randomized (60)
Fibrozyme15
Xylanase
and cellulase
Endoglucanase, exoglucanase,
xylanase, and amylase
Amylase
Xylanase
and cellulase
Amylase
FinnFeeds Int.9
FinnFeeds Int.9
Pro-Mote4
Completely randomized block
(1,200)
Completely randomized (98 and
66)
Completely randomized (32)
Completely randomized block
(56)
Latin square
Latin square (5)
Lewis et al.,
1996
Beauchemin et al.,
1997
Krause et al.,
1998
Beauchemin et al.,
1999
McAllister et al.,
1999
ZoBell et al.,
2000
Balci et al.,
2007
Tricarico et al.,
2007
Krueger et al.,
2008
Eun et al.,
2009
DiLorenzo et al.,
2011
Vera et al.,
2012
Xylanase
and cellulase
Xylanase
and cellulase
Xylanase
and cellulase
Xylanase
and cellulase
Xylanase
and cellulase
Xylanase
and cellulase
Xylanase and endoglucanase
Xylanase B4 and
Spezyme CP5
Grasszyme, FinnFeeds
Int.9
Xylanase B4 and
Spezyme CP5
Pro-Mote4
Completely randomized (72)
Declared primary
activities
Product/
manufacturer
Experimental
design (number of cows)
Beauchemin et al.,
1995
Source1
400 kilo novo units/
kg DM
0.52 g/kg DM TMR
1 to 2 g/kg TMR DM
580 to 1,160 DU16/
kg DM
16.5 g/t
15,880 and 5,580 IU/
kg TMR13 DM
60 g/d
1.25 to 5.0 L/t DM
1.4 L/t DM
40 to 316 FPU6/kg
DM
1.65 mL/kg forage
DM
4.0 L/t concentrate
DM
1.5 g/kg DM
Application
level
25 to 63.4%
5.1%
Ad libitum access
to hay
20 to 58%
Ad libitum wheat
straw
20 to 65%
70 to 82.5%
-
↑20
-
-
↑18
-
↑17
↑
-
↑11
↑
NR
-
NR
↑8
Effects2
ADG
-
NR
-
↑10
-
-
5%
7.8%
-
-
↑7
DMI
4.90%
70%
91 to 96.7%
Forage level
in basal diet
-
-
-
-
-
↑
-
-
-
NR
-
NR
-
FCR3
↓NDF,11 ↑DM, N,
NDF, and ADF11
-
NR
↑DM, NDF, and CP18
(in vitro)
NR
NR
-12
NR
↑ADF
NR
↑DM, NDF, and ADF
NR
Total tract
digestibility
Table 2. Summary of exogenous polysaccharide-degrading enzyme effects on production traits and total tract apparent digestibility of nutrients in beef cattle
434
Meale et al.
Exogenous enzymes in ruminant production
total carbohydrate present in
the diet. As such, it is difficult to attribute production
responses solely to the generation of soluble carbohydrates before consumption.
Fibrolytic enzymes that
bind to the feed appear to
be more active, possibly because of increased resistance
to proteolytic inactivation
in the rumen (Fontes et al.,
1995). Similarly, maximizing the proportion of the diet
to which the enzyme is added is considered to increase
the chances that the enzymes
will remain active in the rumen. Bowman et al. (2002)
reported that enzymes were
more effective when added
to rolled grain, which comprised 45% of the diet, compared with if they were added to a finely ground premix,
which comprised 0.2% of
the diet (DM basis).
in growing phase.
20Only
on time of enzyme application before feeding.
Nutritional Products, Inc., Kaiseraugst, Switzerland.
19DSM
increase observed in experiment 2 only.
18Dependent
17Quadratic
Inc., Nicholasville, KY.
= dextrinizing unit.
16DU
15Alltech
Table 2. (continued)
Ruminal Effects
Exogenous
enzymes
have been shown to be more
stable in the rumen environment than originally proposed (Hristov et al., 1998b;
Morgavi et al., 2000b). For
example, Morgavi et al.
(2001) found 4 commercial
enzymes remained stable
when incubated in ruminal
fluid, pepsin, or pancreatin.
Enzyme stability in the rumen is considered to be a
result of glycosylation and is
usually enhanced by adding
exogenous enzymes to feed
before consumption (Fontes
et al., 1995). However, nonglycosylated enzymes may
also resist ruminal proteolysis, but their persistence in
the rumen may depend on the
microbial source from which
they were derived (Fontes et
435
al., 1995). Variation in enzyme stability may contribute
to the inconsistent production responses observed when
enzymes are included in ruminant diets.
Supplementing ruminant diets with exogenous enzymes increases the rate but seldom the extent of feed
digestion. This suggests that positive responses to present exogenous enzymes are not a result of these preparations solubilizing substrates that would not be normally digested if retained in the rumen for a sufficient
period of time. However, an increase in total enzymatic
activity in the rumen can increase ruminal hydrolytic
capacity, which can enhance the digestibility of the
complete diet rather than just being limited to the specific components targeted by the enzyme (Beauchemin
et al., 2004). As such, digestibility of both nonfibrous
and fibrous fractions can increase, explaining why fibrolytic enzymes can also be effective in increasing the
digestibility of nonfiber fractions in high grain diets
(Beauchemin et al., 1999).
Given that exogenous enzymes represent only a
fraction of enzyme activity in the rumen combined
with the inherent capacity of the ruminal microbiota
to digest fiber, it is difficult to attribute an increase in
fiber degradation by exogenous enzymes to direct hydrolysis alone (McAllister et al., 2001). A synergistic
relationship between exogenous enzymes and rumen
microbiota and an increase in bacterial attachment are
other likely modes of action of exogenous enzymes in
the rumen. Synergism acts to increase the effects of
both indigenous ruminal microbes and exogenous enzymes so that the combined response exceeds the additive effects of each individual component (Morgavi
et al., 2000a). Identification and supplementation with
exogenous enzymes not produced by ruminal microbes
could be theorized to further heighten this synergistic
response. Current enzyme preparations do not appear to
introduce novel enzyme activity into the rumen as they
typically increase only the rate and not the extent of
cell wall digestion (Wallace et al., 2001; Krause et al.,
2003; Jalilvand et al., 2008). Exogenous enzymes can
also stimulate the attachment of ruminal microbes to
plant fiber (Morgavi et al., 2000a), yet the mechanism
by which this occurs is unknown. Reduced amounts of
exogenous enzymes have been shown to enhance attachment of ruminal bacteria to fiber resulting in a disruption in the hydrogen bonds within the cellulose matrix (White et al., 1993). However, increased amounts
of exogenous enzymes can also compete with the ruminal microbial population for cellulose binding sites on
feed (Morgavi et al., 2000b), potentially explaining the
lack of or even negative responses observed with the
increased amounts of exogenous enzyme supplementation in vivo. For exogenous enzymes to be effective, it
is important that they complement and not replace the
436
Meale et al.
existing natural enzyme activities produced by ruminal
microbes.
Enzymes have also been associated with a reduction in digesta viscosity in poultry (Choct, 2006);
if a similar reduction was observed in ruminants, an
increase in passage rate through the rumen would be
expected, reducing gut fill and subsequently increasing intake. From a production perspective, increasing
intake is beneficial. However, if the enzymes are in the
fluid phase and there is a rapid flow of digesta through
the rumen, they may not be afforded sufficient time to
degrade the fibrous portion of the diet before they are
largely inactivated by exposure to the low pH and pepsin in the abomasum (Hristov et al., 1998b).
Postruminal Effects
Hristov et al. (1996, 1998a) were the first to report
that approximately 30% of xylanases can escape ruminal fermentation and are active in intestinal digesta of
ruminants. These findings confirmed previous reports in
vitro (Fontes et al., 1995) and studies with pigs (Chesson, 1993; Inborr et al., 1994). Depending on application level, other enzymes may also bypass the rumen
and increase polysaccharide-degrading activities in
intestinal digesta (Chesson, 1994). Glycosylation confers proteolytic stability to exogenous enzymes (Gorbacheva and Rodionova, 1977), but as demonstrated
by Fontes et al. (1995), nonglycosylated enzymes may
also resist proteolysis. Hristov et al. (1998a) identified
the abomasum as a major barrier to active exogenous
enzymes entering the intestine. A follow-up study by
Morgavi et al. (2001) confirmed that some exogenous
enzymes survive ruminal fermentation and the abomasal environment and may exert activity for a period of
time in the small intestine. In general, xylanases are
more stable in the rumen and abomasum than cellulases and consequently xylanase activity in the small
intestine that is attributable to exogenous enzymes is
usually greater than cellulase activity.
The few studies designed to investigate ruminal stability and bypass of exogenous enzymes have
clearly shown that some exogenous enzymes are remarkably resistant to microbial proteases, bypass the
abomasums, and remain active in the small intestine and have even been shown to linearly increase
polysaccharide-degrading activities in feces (Hristov
et al., 2000). However, the practical implication of
these effects remains unclear. In theory, exogenous
enzymes could improve animal performance by not
only enhancing ruminal carbohydrate degradability
but also by reducing the viscosity of digesta and improving postruminal nutrient absorption. Presently,
although postruminal effects can be documented, they
are thought to account for a minor component of any
positive responses observed with existing enzyme
preparations with improvements primarily arising
from positive alterations in rumen function.
NEW APPROACHES
TO FORMULATING EXOGENOUS ENZYMES
Role of Metagenomics in Enzyme Discovery
Before the development and application of the
“-omics” disciplines, culturing isolates in a laboratory
was the only method of identifying rumen microbial
species. Such cultivation-based techniques applied
to the rumen identified approximately 60 to 70 genera and 300 to 400 species of bacteria, protozoa, and
fungi (Krause et al., 2007). However, this represents
less than 10% of the total microbial species that inhabit this environment (Edwards et al., 2004; Krause
et al., 2007), raising the question as to whether the
numerically dominant or functionally significant members of the rumen remained unidentified. The diversity and complexity of the rumen ecosystem possess
a major challenge in terms of determining the functionality and prevalence of the various carbohydrases
produced by ruminal microorganisms over a range of
dietary conditions (Morgavi et al., 2012). Although the
genomes of 3 of the most highly fibrolytic culturable
bacterial species (Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) have
been sequenced (Krause et al., 2003), the enzymatic
strategies used by each of these species to hydrolyze
cellulose are still not well understood. Consequently,
culture-independent approaches such as metagenomics,
metatranscriptomics, and proteomics have been used
to further characterize the carbohydrases produced in
the rumen without necessarily attributing their presence to any 1 specific microorganism. With the rapid
advancements that have occurred in these sequence- or
function-based technologies, an integrated and holistic
picture of the metabolic potential and activity of this
complex microbial ecosystem is being generated.
Although sequenced-based metagenomics may
use various techniques and approaches, the main aim
remains to develop a catalogue of all genes present
in the rumen (Brulc et al., 2009). A recent study by
Hess et al. (2011) assembled 15 microbial genomes
that were previously unculturable and identified more
than 27,000 putative genes coding for carbohydrases.
Functional metagenomics, in this instance, aims to use
these catalogues or libraries to identify and isolate specific hydrolytic enzymes involved in structural plant
digestion in the rumen (Zhao et al., 2010). Ferrer et
Exogenous enzymes in ruminant production
al. (2005) identified 9 endogulcanases, 12 esterases,
and 1 cyclodextrinases within a metagenomic library
derived from the rumen of a dairy cow. The success
of this approach in identifying novel enzymes and
metabolic pathways, however, is still dependent on the
availability of appropriate bioassays and the development of innovative strategies to screen for enzyme activities of interest. Current interest is focusing on the
degradation of cellulose and hemicellulose by enzyme
members of the glycoside hydrolase family 5 (GH5;
Morgavi et al., 2012). This family of glycosyl hydrolyases represents the most abundant cellulases from both
cultured (Krause et al., 2003) and uncultured ruminal
bacteria (Ferrer et al., 2005). As such, it is not surprising that even though Duan et al. (2009) identified and
characterized novel cellulases from the rumen, they
were identified as members of the GH5. Recently a
metatranscriptomic approach was used by our laboratory (Qi et al., 2011) to examine carbohydrases associated with fungi and protozoa in the rumen of musk
oxen, identifying a greater percentage of cellulases per
gigabase of sequence than Hess et al. (2011). The application of this technology can be broadened to study
carbohydrase complexes in the rumen of a variety of
ruminant species over a range of ecological niches. In
fact, New Zealand researchers are presently leading an
international research group with the objective of collecting samples from ruminants across the globe. This
global survey could provide new insight into the carbohydrases that are key to plant cell wall digestion in a
variety of species. Identification of carbohydrases that
are lacking within ruminal environment could provide
the knowledge needed to specifically formulate exogenous enzymes that can fill these deficiencies.
A Focus on Rate-Limiting Enzymes
Lignin is the most recalcitrant of the 3 main heterogeneous polymers in lignocellulose (Himmel et
al., 2007; Sanchez, 2009). The formation of ferulatepolysaccharide-lignin complexes that cross-link cell
wall polymers is a major factor limiting the rate and
extent of enzymatic dissolution of cell walls as it interferes with hydrolysis by preventing the binding of
xylanases to their target substrate (Hatfield et al., 1999;
Buanafina et al., 2008). However, the cellulolytic capacity of the rumen is not considered to limit cellulose digestion; rather, it is the surface area available for
enzyme attachment that limits lignocellulose digestion
in the rumen (Weimer et al., 1990). True degradation
of lignin is an oxidative process that is primarily performed by aerobic fungi. As the rumen is anaerobic,
lignin is not truly degraded, but rather its solubilization is a key step in increasing the amount of cellulose
437
and hemicellulose available for microbial fermentation. Chemical pretreatment with either acidic (e.g.,
sulfuric acid; Himmel et al., 2007) or alkali solutions
(e.g., ammonium hydroxide, sodium hydroxide) aims
to increase the susceptibility of cellulose to enzymatic
hydrolysis by breaking the ester bonds that link lignin with the plant cell wall (Buanafina et al., 2008).
In biorefineries, cellulosic feedstuffs are subjected to
pretreatment strategies including thermochemical pretreatment, where dilute sulfuric acid is applied at 140
to 200°C, rendering the cellulose in cell walls more
accessible to saccharifying enzymes. Alternatively, the
application of alkalines through processes such as ammonia fiber expansion renders cell walls considerably
more susceptible to enzyme hydrolysis (Himmel et al.,
2007). Such pretreatments can reduce the recalcitrance
of cellulose to enzymatic hydrolysis; however, they are
costly, require heat or pressurization, and can reduce
the availability of fermentable carbohydrate (Wyman,
2007; Weimer et al., 2009).
Carbohydrases have advantages over the use of
alkali treatment as chemical treatments can be detrimental to the digestibility of other plant components
and denature plant-associated enzymes (Mathew and
Abraham, 2004). Ferulic acid esterases, produced by A.
oryzae, perform a function similar to that of alkali deesterification of lignin–hemicellulose linkages in plant
cell walls (Tenkanen et al., 1991). Ferulic acid esterases act synergistically with xylanases and pectinases
to facilitate access of hydrolyases to the backbone of
cell wall polymers. However, the source and type of
ferulic acid esterase can influence the amount of ferulic acid released and thus the accessibility of cellulose
(Faulds et al., 2003, 2006).
The success of this technology is reliant on its
ability to encompass a vast array of dietary and production scenarios. Therefore, it becomes essential to
examine variations in ruminal microbial communities across vastly different production environments.
The use of the metagenomic and metatranscriptomic
procedures described above to identify high activity
esterases that could be used to complement existing
enzyme products could be one approach to improving
the efficacy of these preparations (Fig. 1.). Similarly,
the development and refinement of biomass pretreatment strategies that can be used in conjunction with
exogenous enzymes to enhance hydrolysis of cellulose in the rumen could further improve the efficacy of enzyme preparations. Effective use of these
technologies is considered the most promising way
to identify novel enzyme cocktails and thus advance
enzyme technology by minimizing the variability currently seen in production responses of ruminants.
438
Meale et al.
Figure 1. Stages in the development of novel enzymatic pretreatments to enhance ruminal digestion of structural plant polysaccharides.
Conclusion
The use of exogenous enzymes in ruminant diets
is still an emerging technology. Although progress has
been made, previous strategies have been largely unsuccessful at improving ruminant production or produced
highly variable results due to our lack of understanding
of the carbohydrase complex and microbial interactions
within the rumen ecosystem. Enzymes have been added
to diets with the aim of increasing cell wall digestibility and have shown some improvements in milk yield
and feed conversion efficiency, yet the true mechanistic
effects responsible for these positive outcomes remain
elusive. New approaches such as metagenomics, meta-
Exogenous enzymes in ruminant production
transcriptomics, and proteomics are providing novel insights into the structure, interactions, and function of the
rumen microbial community to a degree that was previously impossible with lab-based culture techniques.
Such approaches have the potential to allow for formulation of exogenous enzymes based on optimal properties for specific ruminal conditions. Ideally, responses
to these additives will need to be broad based across a
range of diet types as development of a specific exogenous enzyme formulation and dose rate for each diet
type and feeding level would introduce a degree of complexity in on-farm application that would likely discourage adoption by producers.
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