191
Equine α–amylase: does it limit starch digestion in
the small intestine of the horse?
N. Richards1, M. Choct1, G.N. Hinch1 and J.B. Rowe2
1School
of Rural Science and Agriculture, Animal Science, University of New England, Armidale NSW 2351
Sheep Industry CRC, Locked Bag 1, Armidale NSW 2350
[email protected]
2Australian
Summary
The amylolytic enzyme α–amylase is essential for the
digestion of starch in the small intestine of the horse.
While it had been previously documented that the
concentration of this amylolytic enzyme in the equine
small intestine is low, the ability of equine α–amylase
to degrade cereal grain starch is poorly understood. In
an experiment designed to compare the in vitro starch
digesting capacity (activity) of equine α–amylase with
that of bacterial α–amylase it was observed that the
equine α–amylase digested an average of 20% less
starch during the in vitro incubations. This result
suggests that the apparently limited capacity of equine
α–amylase to digest starch along with the low
concentrations of α–amylase reported to be present in
the equine small intestine may limit pre–caecal starch
digestion. A second experiment investigated if the
addition of exogenous glycanase enzymes to cereal grain
diets fed to horses would improve small intestinal starch
digestion. The results showed that the addition of
α–amylase and amyloglucosidase (AMG) to a digestible
starch source significantly elevated postprandial
glycaemic responses suggesting an improvement in pre–
caecal starch digestion. Thus it appears that the
concentration and activity of equine α–amylase may
be limiting small intestinal starch digestion in the horse.
Keywords: horse, amylase, amyloglucosidase, starch
digestion, grain
Introduction
Although horses evolved primarily consuming grass and
grass–like plant species (Budiansky 1998), cereal grains
now form a common component of performance horse
diets. A recent survey of 72 thoroughbred trainers in
rural, provincial and metropolitan areas of New South
Wales, Australia showed that thoroughbred horses in
training are being fed an average of around 7 kg of grain
concentrate/d (Richards 2003). Cereal grains are fed to
these high performance animals to provide starch as a
relatively cheap energy source to help to meet the
increased energy demands placed on them during work.
Ideally the starch from these cereal grains will be
digested by amylolytic enzymes in the small intestine
and absorbed as glucose.
The digestion of starch in the small intestine of
the horse occurs via a three–step process, beginning
with the degradation of starch by the enzyme α–amylase.
This enzyme does not act on α1–6 or any α1–4 links
adjacent to reducing ends, but efficiently breaks down
α1–4 links in the two principle forms of starch, amylose
and amylopectin, into disaccharide (maltose),
trisaccharide (maltotriose) and α–dextrin units. The
second phase of starch digestion involves the hydrolysis
of maltose, maltotriose and α–dextrin units by small
intestinal brush border glycanases, primarily AMG
which successively releases glucose units from the non–
reducing ends, and α–dextrinase which acts on α1–6,
to form free glucose units (Gray 1992). Na+–dependant
active transport and Na +–independent facilitated
diffusion then transport glucose from the lumen of the
small intestine, through intestinal epithelial cells and
into the bloodstream of the animal (Bird et al. 1996;
Huntington 1997; Thorens 1993).
α–Amylase thus plays an essential role in the
digestion of starch in the small intestine of the horse. It
has been recognised that α–amylases from various plant
and animal sources vary in their activity, their capacity
to degrade cereal grain starch, a function possibly related
to the number of sub–sites (an area within an active site
capable of interacting with one glucose molecule) at
each of the enzyme’s active sites and the affinity of each
of these sub–sites for a glucose molecule (Anindyawati
et al. 1998; MacGregor 1988; MacGregor 1993;
MacGregor et al. 2001). While it has been documented
that horses perhaps have low concentrations of
α–amylase in their small intestine (Comline et al. 1969;
Kienzle et al. 1994; Roberts 1974) the capacity of equine
α–amylase to degrade cereal grain starch is not clear.
This paper describes two experiments; the first
investigates the ability of equine α–amylase to degrade
starch from various cereal grain sources in vitro and
the second examines the use of exogenous amylolytic
Recent Advances in Animal Nutrition in Australia, Volume 14 (2003)
192
Richards et al.
enzymes to improve the digestion of starch in the equine
small intestine.
The small intestine of each horse was cut into the
duodenal, jejunal and ileal sections. Each was weighed
individually when full of digesta; the contents were then
emptied from each section into labelled plastic
containers, the mucosa was lightly scraped to sample
the brush border, and each section was reweighed empty.
All small intestinal digesta was immediately placed on
ice. On return to the laboratory all digesta samples were
centrifuged at 8000 g and the supernatant and pellet
were stored separately at –18°C prior to analysis.
Experiment 1: assessing the
activity of equine α–amylase
Jejunal supernatant collected from a horse following
slaughter was used during this study as a source of
equine α–amylase in an in vitro assay that enabled
assessment of how much starch was degraded by this
enzyme during 15– and 60–minute incubations at
physiological temperature. The in vitro starch digestion
assay of Bird et al. (1999) was used to provide an
estimation of the ability of α–amylase from a bacterial
source to degrade starch. It was hypothesised that the
equine and bacterial α–amylases would have the same
activity and, due to the more extensive range of digestive
enzymes assumed to be present in the equine jejunal
supernatant, incubation in the supernatant would result
in a more extensive degradation of cereal grain starch
in vitro than during the standard enzyme digestion assay
of Bird et al. (1999) which employed only the amylolytic
enzymes α–amylase and amyloglucosidase (AMG).
Measurement and analytical procedure
α–Amylase concentration in the digesta supernatant was
measured using the Megazyme Ceralpha Method
(Megazyme International Ireland Ltd, Ireland) using the
Amylase HR reagent (specific for α–amylase) and non–
reducing–end blocked p–nitrophenyl maltoheptaoside
(BPNPG7) as substrate. AMG concentration in the small
intestinal digesta supernatant was determined using the
Megazyme assay for amyloglucosidase using
p–nitrophenyl–β–maltoside (4 mM) and β–glucosidase
(25 U/ml) as substrate.
The method of Bird et al. (1999), which
utilises 300 U of α–amylase, derived from Bacillus
licheniformis and 20 U of AMG derived from
Aspergillus niger, per grain sample, with both 15 and
60 minute incubation periods was used to assess the
starch digesting capacity of bacterial α–amylase. Two
in vitro starch digestion assays, using a modification
of the method of Bird et al. (1999) which involved
using the jejunal supernatant from horse A to supply
α–amylase, were then carried out.
These assays involved the incubation of 100 mg
of cereal grain in 5.3 ml of equine jejunal fluid (300 U
α–amylase and 5.6 U AMG) and 14.4 U of AMG,
derived from Aspergillus niger (Megazyme). Incubation
periods of 15 and 60 minutes were used. Tubes with no
grain sample were run as blanks for both assays.
Twelve grain feeds commonly included in equine
diets were examined during this study. These feeds were
two varieties of corn, one variety of each of rice, barley,
triticale and oats in unprocessed form, and five of these
grains in processed forms. The grains processed were
Sample collection
Two horses (one thoroughbred gelding aged 9 years and
one mare, breeding unknown, aged >20) that were being
sent to slaughter by their owners who gave permission
to collect gastrointestinal tract samples from the horses
at the abattoir. The horses had been fed a cereal grain
diet for 7 days prior to slaughter, with each horse
consuming 2.8 kg of oats and 2 kg of extruded rice in
two meals/d. Lucerne hay was fed ad libitum as the
only roughage component of the diet and was the only
feed available to the animals for the 18 h prior to
slaughter. On the morning of slaughter, horses were
transported to a commercial pet food abattoir and were
slaughtered using a captive bolt gun and the
gastrointestinal tract was immediately removed. Cotton
ties were promptly placed at the junctions of the stomach
with duodenum, duodenum with jejunum, jejunum with
ileum, and the ileum with caecum to segregate these
sections of the gut.
Table 1 The α–amylase concentration (U/mL) and the total units of α–amylase in the digesta
supernatant collected from the duodenum, jejunum and ileum of horses A and B. Total
units were calculated as U/mL multiplied by the total supernatant volume for each
segment of the small intestine.
a–Amylase concentration
Horse A
U/mL
Duodenum
Horse B
Total units (U)
U/mL
Total units (U)
166
1238
8
1450
Jejunum
57
47870
28
24630
Ileum
80
1400
22
1210
Equine α–amylase and starch digestion in the horse
193
The percent of starch digested during incubations
in jejunal fluid with equine α–amylase was typically
lower than that digested during the standard in vitro
assay for both the 15 and 60 minute incubation times
(Table 3).
corn 1 extruded, corn 2 micronised, white rice extruded,
barley expanded, triticale expanded, and triticale steam
rolled; there was no processed oats. All grains were
finely ground in a mill prior to analysis.
Results
Discussion
Table 1 shows the concentrations of α–amylase in
the three segments of the equine small intestine of
both horses. Horse A had higher concentrations of
α–amylase/mL in all three segments of the small
intestine than horse B, but owing to a greater digesta
pool size in the gastrointestinal tract of horse B the total
number of α–amylase units in the duodenum and ileum
did not differ between horses.
AMG concentrations in the small intestine of
horses A and B are shown in Table 2 and again horse A
tended to have higher concentrations of AMG/mL in
all sections of the small intestine. However, horse B
had a greater number of total units of AMG than horse
A in the duodenum and ileum due to a greater digesta
pool size.
The α–amylase concentrations measured in these two
horses, if calculated on a units/g wet mucosa basis, are
comparable to those reported by Kienzle et al. (1994).
Also in support of earlier observations (Kienzle et al.
1994; Roberts 1974), the concentration of enzymes
appears to be variable between horses, Horse A
having a higher concentration than B of α–amylase on
a units/mL basis in all three segments of the small
intestine.
Despite previous reports from Kienzle et al. (1993)
and Roberts et al. (1974) indicating that small intestinal
brush border glycanase concentrations in the equine
small intestine are high and comparable to other
monogastrics, the AMG concentration measured in the
Table 2 The amyloglucosidase (AMG) concentration (U/mL) and the total units of AMG in the
digesta supernatant collected from the duodenum, jejunum and ileum of horses A and
B. Total units were calculated as U/mL multiplied by the total supernatant volume for
each segment of the small intestine.
AMG concentration
Horse A
Horse B
U/mL
Total units (U)
U/mL
Total units (U)
1.68
13
0.94
170
Jejunum
1.00
840
0.60
528
Ileum
1.71
30
0.70
39
Duodenum
Table 3 The % starch digested from the specified grains during the standard assay (Bird et al. 1999), employing bacterial
derived enzymes with 15 and 60 minute incubation periods and the % starch digested during the jejunal fluid assays
employing equine α–amylase with 15 and 60 minute incubation periods.
In vitro starch digestion (% starch digested in specified time)
Standard assay
(15 min)
Jejunal fluid
(15 min)
Standard assay
(60 min)
Jejunal fluid
(60 min)
Unprocessed corn 2
12.3
5.8
24.4
15.2
Unprocessed corn 1
15.8
8.2
30.6
21.6
Unprocessed white rice
18.7
10.8
32.8
29.5
Cracked barley
25.7
12.7
50.3
30.7
Micronised corn
34.0
13.2
49.4
28.8
Expanded barley
39.5
19.2
62.1
39.5
Oats
40.6
27.2
69.5
61.6
Cracked triticale
41.9
16.7
73.8
37.1
Expanded triticale
50.1
21.3
80.7
45.0
Steam rolled triticale
60.4
25.1
82.3
52.5
Extruded corn
73.9
40.4
84.0
63.4
Extruded rice
80.2
44.9
86.2
58.0
194
Richards et al.
small intestine of these horses appears to be low.
However, brush border glycanases, such as AMG, are
attached to the intestinal brush border by a short terminal
hydrophobic section of their protein chain (Gray 1992)
and thus, even though the epithelium was scraped, it is
likely that a majority of AMG remained attached to
the small intestinal brush border. The pattern of
distribution of AMG throughout the small intestine was,
however, consistent with the pattern of distribution of
α–glucosidases reported by Roberts et al. (1974).
It was hypothesised that the bacterial and equine
α–amylases would have the same activity and so, due
to the greater array of protease and glycanase enzymes
assumed to be present in equine jejunal fluid, the in
vitro starch digestion within jejunal fluid would be
higher than that observed in the standard in vitro starch
digestion assay employing only Bacillus licheniformis
α–amylase and Aspergillus niger AMG. However, an
average of around 20% less starch was digested in
jejunal fluid in comparison to that digested during the
standard starch digestion assay (Table 3), suggesting
that perhaps the activity of the equine α–amylase is
inferior to that of the B. licheniformis α–amylase. Such
variation between amylases in ability to degrade starch
has been previously reported by Anindyawati et al.
(1998) who found that of the three forms of α–amylase
produced by the fungi Aspergillus awamori KT–11 only
two had the ability to degrade raw corn starch, and that
of these two one was more effective than the other.
Equine α–amylase appeared to have greater substrate
specificity for oat starch (Table 3), with almost the same
percentage of starch digested by both α–amylases
during the 60–minute incubation period, which may
possibly explain the superior pre–caecal digestibility
and thus ‘safety’ of oats for horses.
Implications
With a seemingly limited ability of equine α–amylase
to degrade cereal grain starch in vitro and previous
observations that horses have sub–optimal
concentrations of α–amylase in their small intestine
(Comline 1969; Kienzle 1994; Roberts 1974), it is likely
that starch digestion will be limited by the activity and
concentration of α–amylase in the equine small
intestine. Starch that escapes digestion in the small
intestine will be fermented in the horse’s enlarged
hindgut by amylolytic bacteria, in a less energy efficient
process (Black 1971). This hindgut fermentation of
starch commonly causes an accumulation of volatile
fatty acids and lactic acid in the caecum and colon of
equines. Such acid accumulation can lead to hindgut
acidosis (Garner et al. 1977), a metabolic condition
commonly associated with reduced fibre fermentation
(de Fombelle et al. 1999), behavioural changes (Johnson
et al. 1998; Willard et al. 1977) and the crippling and
potentially fatal disease laminitis (Pollitt 2001). Thus
it is desirable from an animal health and feed use
efficiency perspective that cereal grain starch is
completely digested in the small intestine. It is possible
that the addition of exogenous amylolytic enzymes to
cereal grain diets may be used to improve starch
digestion in the small intestine of horses and this was
the focus of experiment 2.
Experiment 2: the use of
exogenous amylolytic enzymes
to improve starch digestion
The hypothesis for experiment 2 was that the addition
of α–amylase or a combination of α–amylase and AMG
to diets containing digestible starch would improve the
digestion of starch in the equine small intestine. It was
also hypothesised that α–amylase is the primary limiting
factor to starch digestion in the equine small intestine
and that the addition of AMG alone would have a non–
significant effect on pre–caecal starch digestion. The
design of the experiment was based on the assumption
that an improvement in small intestinal starch digestion
would be reflected in glycaemic responses that are
elevated above those observed for the control diet.
Methods
Three treatment diets and a control diet were used in
the experiment. Steam–rolled triticale containing per
kg dry matter 662 g starch, 120 g non–starch
polysaccharide, 120 g crude protein, 32 g crude fat,
25 g crude fibre, and 17.0 MJ gross energy, was used as
the control grain to which exogenous enzymes were
added. Steam–rolled triticale had a high in vitro starch
digestibility (82% of starch digested in 1 h) when
incubated at 39°C in the presence of excess
thermostable α–amylase and AMG using the method
of Bird et al. (1999). The control diet consisted of
1.12 kg of steam–rolled triticale meal which
corresponded to 670 g of starch; meal sizes were not
varied according to body weight.
Treatment 1 was 1.12 kg steam–rolled triticale and
3 mL of heat stable α–amylase derived from Bacillus
licheniformis (450 Kilo Novo α–amylase units,
Termamyl® Classic, Novozymes A/S. DK–2880,
Bagsvaerd); treatment 2 was 1.12 kg steam–rolled
triticale and 1 mL AMG, derived from Aspergillus niger
(300 amyloglucosidase units, AMG 300L, Novozymes
A/S. DK–2880, Bagsvaerd); treatment 3 was 1.12 kg
steam–rolled triticale, 1 mL AMG (AMG 300L) and
3 mL of heat stable α–amylase (Termamyl® Classic).
All enzymes were diluted to 25 mL with distilled water
and sprayed over the steam–rolled triticale meal using
a hand held spray nozzle to thoroughly cover the grain.
Enzymes were added to the grain meal 10 minutes prior
to feeding and horses were fed twice daily at 0700 h
and 1730 h.
Twelve horses (eight standardbred horses,
4 geldings and 4 mares, and four thoroughbred
horse geldings) aged 4 to 9 years and weighing
432–512 kg were used in the study. The horses were
Equine α–amylase and starch digestion in the horse
195
that a deficiency of α–amylase is the major limiting
factor to starch digestion in the equine small intestine
and is in agreement with Kienzle et al. (1993) and
Roberts et al. (1974) who suggest that horses possess
naturally high levels of brush border glycanases.
However, the addition of AMG to a diet also being
supplemented with exogenous α–amylase increased the
glycaemic response above that observed for the diet
being supplemented with exogenous α–amylase only.
This result indicates that endogenous AMG may not be
sufficient to cope with the excess maltose, maltotriose
and α–dextrin units produced through the addition of
α–amylase to the diet, suggesting that a combination of
α–amylase and AMG may be necessary to effectively
enhance small intestinal starch digestion.
divided into four groups of three; group A was allocated
to the control diet, group B to the α–amylase diet, group
C to the AMG diet, and group D to the α–amylase +
AMG diet. Horses were allowed one and a half days to
acclimatize to the stables and daily routine and from
the evening feed of the second day were fed the
experimental diets. Blood sampling to measure the
glycaemic response took place on the morning of the
fifth and eighth days; samples via jugular catheter were
taken before the start of eating, every quarter hour for
one hour following commencement of eating, and then
every half hour for the next four hours. Plasma glucose
concentrations were determined using the Dimension®
clinical chemistry system on a DADE XL clinical auto–
analyser (Dade Behring Inc, Newark, DE 19714, USA).
The GLU Flex reagent cartridge (Cat No DF39A) was
used as the in vitro reagent.
Conclusions
Results and discussion
It appears that low concentrations of α–amylase in the
equine small intestine and a limited ability of this equine
α–amylase to degrade cereal grain starch may limit
starch digestion in the equine small intestine. Dietary
supplementation with amylolytic enzymes may be used
to overcome these limitations and improve starch
digestion in the equine small intestine.
Four major benefits may be expected following
the dietary supplementation of cereal grain diets with
amylolytic enzymes. These are:
The addition of a combination of α–amylase and AMG
to the steam–rolled triticale diet significantly (P≤0.008)
elevated peak, average and peak minus basal plasma
glucose concentrations above those initiated by the
control diet, while the addition of α–amylase alone
significantly (P≤0.008) increased average glucose
concentrations above those observed for horses
consuming the control diet (Table 4). Thus, in
support of the hypothesis, these findings indicate that
dietary supplementation with amylolytic enzymes
improves small intestinal starch digestion and are
in accordance with Meyer et al. (1993) who observed a
12% improvement in the pre–caecal digestion of
cracked corn following the addition of powdered
α–amylase to the diet.
The addition of AMG to the control diet appeared
to make no improvement to the digestion of starch in
the small intestine, with similar glycaemic responses
observed for horses given the control and AMG diets
(Table 4). Thus in support of the hypothesis it appears
that endogenous concentrations of AMG present in the
equine small intestine are adequate to break down the
products of endogenous α–amylase digestion to
glucose. This observation further supports the theory
1 the feeding efficiency of cereal grains to horses
will be improved as starch will be digested and
absorbed as glucose in the small intestine in a
more energy efficient process than the
fermentation of starch in the equine hindgut;
2 the incidence of hindgut starch fermentation and
hindgut acidosis will be reduced;
3 the occurrence of diseases associated with hindgut
acidosis, such as laminitis, will be reduced;
4 the incidence of adverse behaviours, often
associated with acid accumulation in the equine
hindgut, may be reduced.
Table 4 Mean peak glucose concentration, average glucose concentration, peak minus basal glucose concentration, time to
peak glucose and slope to peak glucose for the control, α–amylase, amyloglucosidase (AMG) and α–amylase + AMG
diets.
a–Amylase
Control
AMG+ a–Amylase
AMG
Mean
SE
Mean
SE
Mean
Peak glucose (mmol/L)
8.8 a
0.19
10.0ab
0.56
8.8a
Average glucose (mmol/L)
6.9 a
0.08
7.9 b
0.41
7.0a
Peak–basal glucose (mmol/L)
3.3b
0.24
4.6 ab
0.54
3.6ab
SE
Mean
SE
0.30
10.8 b
0.22
0.19
8.4 b
0.11
0.31
5.5 a
0.23
Time to peak glucose (hours)
1.6
0.20
1.9
0.20
1.6
0.15
2.2
0.11
Slope to peak glucose (mmol/L/h)
2.2
0.16
2.5
0.33
2.4
0.12
2.8
0.16
Values in same row with different superscripts are significantly different (P≤0.008)
196
Richards et al.
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