Available via license: CC BY-NC-ND 4.0
Content may be subject to copyright.
Biotechnology Reports 41 (2024) e00824
Available online 17 December 2023
2215-017X/© 2023 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Research Article
Inclusion of exogenous enzymes in feedlot cattle diets: Impacts on
physiology, rumen fermentation, digestibility and fatty acid prole in
rumen and meat
Alexandre L. Simon
a
,
*
, Priscila M. Copetti
b
, Rafael V.P. Lago
a
, Maksuel G. Vitt
a
,
Aline L. Nascimento
a
, Luiz Eduardo Lobo e Silva
c
, Roger Wagner
c
, Bruna Klein
a
,
Camila Soares Martins
d
, Gilberto V. Kozloski
d
, Aleksandro S. Da Silva
a
a
Departamento de Zootecnia, Universidade do Estado de Santa Catarina, Chapec´
o, Brazil
b
Programa de P´
os-graduaç˜
ao em Bioquímica Toxicol´
ogica, Universidade Federal de Santa Maria (UFSM), Santa Maria, Brazil
c
Departamento de Ciˆ
encias de Alimentos, UFSM, Santa Maria, Brazil
d
Departamento de Zootecnia, UFSM, Santa Maria, Brazil
ARTICLE INFO
Keywords:
Amylase
Protease
Cellulase
Xylanase
Beta glucanase
ABSTRACT
The objective of this study was to evaluate if the inclusion of a blend composed of exogenous enzymes (amylase,
protease, cellulase, xylanase and beta glucanase) in the individual and combined form in the feedlot steers diet
has benets on the physiology, rumen fermentation, digestibility and fatty acid prole in rumen and meat. The
experiment used 24 animals, divided into 4 treatments, described as: T1-CON, T2-BLEND (0.5 g mixture of
enzyme), T3-AMIL (0.5 g alpha-amylase), T4-BLEND+AMIL (0.5 g enzyme blend+0.5 g amylase). The con-
centration of mineral matter was higher in the meat of cattle of T4-BLEND+AMIL. A higher proportion of
monounsaturated fatty acids was observed in the T3-AMIL group when compared to the others. The percentage
of polyunsaturated fatty acids was higher in the T2-BLEND and T4-BLEND+AMIL compared to the T1-CON. The
combination of exogenous enzymes in the diet positively modulate nutritional biomarkers, in addition to benets
in the lipid and oxidative prole meat.
1. Introduction
The demand for beef increases every year, which is why the need to
intensify beef production systems has arisen. However, there is a huge
challenge to this intensication since the 90 s, which is to produce in a
protable and sustainable way [1]. Still in the 1980s, there was the
perception that a reduction in the production cycle, an increase in
carcass yield, greater production per area, freeing up pasture areas for
other categories and a faster return on investment are some of the ad-
vantages of the conned system [2]. However, the high cost of the diet
provided to the animals was a limitation of this production system, due
to the high added value of the ingredients, mainly cereals; nonetheless,
the animal connement system has grown worldwide.
Aiming to maximize the diet, the use of enzymatic additives is a good
alternative to increase the performance of the animals through the
mutual action of the enzymes provided and produced by the ruminal
microbiota [3], which aims to increase digestion and seeks greater use of
feed, and consequently greater weight gain. In order to maximize the use
of the diet, the process of protein degradation in the rumen undergoes
the action of the protease enzyme, involved in the breakdown of mol-
ecules to generate free amino acids through hydrolysis caused by the
action of water and the action of enzymes [4]. In this context, in addition
to the new formation of protein chains, the remaining amino acids serve
as a substrate for microorganisms that will be sources of microbial
protein, which is characterized as the main metabolizable source in
ruminants, which leads to the understanding that the multiplication of
microorganisms promotes improvements both in performance and in the
health of these animals. The use of brolytic enzymes such as cellulase
and xylanase has shown good results in the digestibility of dry matter
and ber, since the forage digestion process is often considered incom-
plete [5], which justies the use of exogenous brolytic enzymes to act
in conjunction with enzymes produced by microorganisms in the
ruminal environment, thus enhancing ber degradation [6–8].
Starchy grains are the main source of energy in conned ruminant
* Corresponding author.
Contents lists available at ScienceDirect
Biotechnology Reports
journal homepage: www.elsevier.com/locate/btre
https://doi.org/10.1016/j.btre.2023.e00824
Received 24 September 2023; Received in revised form 9 December 2023; Accepted 16 December 2023
Biotechnology Reports 41 (2024) e00824
2
diets, mainly corn [9]. With the processing it is possible to change the
physical and chemical characteristics of the starch [10]. Knowing this,
research with the inclusion of alpha amylase enzymes aims to optimize
the digestion of starch, which can pass intact through the rumen. Due to
the consistency of the endosperm, hard or farinaceous and the protein
matrix that covers the starch, because it works as a physical and
chemical barrier that prevents the hydrolysis of the starch molecule [11,
12]. However, this is extremely necessary in the digestion process to
generate a maltose molecule that is equivalent to two glucose molecules,
necessary to increase feed efciency.
There are studies on the addition of exogenous enzymes in the diet of
production animals, but it is considered insufcient when the experi-
mental condition is in enzymatic association in order to evaluate di-
gestibility, rumen environment and their effects on performance [13,
14]. We also agree that further studies are needed in order to seek to
improve production in connement, where a detail can reect on
protability and consequently sustainability. Therefore, our hypothesis
is that the combination of protease and amylase as an additive in the
bovine diet will improve the digestibility. For this reason, this study was
to evaluate if the inclusion of a blend composed of exogenous enzymes
(amylase, protease, cellulase, xylanase and beta glucanase) in the indi-
vidual and combined form in the feedlot steers diet has benets on the
physiology, rumen fermentation, digestibility and fatty acid prole in
rumen and meat.
2. Material and methods
2.1. Facilities and animals
The study was carried out in the ruminant sector at the experimental
farm of the Universidade do Estado de Santa Catarina (FECEO/UDESC)
in the municipality of Guatambú (Latitude: 27◦8
′
5
′
’ South, Longitude:
52◦47
′
15
′
’ West), located in the western region of Santa Catarina. The
animals were housed in appropriate connement, allocated in individ-
ual pens measuring 1.5 ×7.0 m with a concrete oor, equipped with
automatic drinkers and a freely accessible feeder. The animals’ feeding
area had cover, which allowed the animals to shelter from the weather
conditions. The shed had north-south solar orientation, which allowed
contact of the animals with sunlight. Twenty-four castrated male Hol-
stein steers with an average weight of 336±4.68 kg and an average age
of 12 months were used as an experimental model.
2.2. Test products
Tecmax Pro-Ruminantes® (Toledo – PR, Brazil) is a fermented
product of Bacillus subtilis, algaroba bran, inactivated and dehydrated
sugar cane yeast, fermentation product of Aspergillus Niger; which pre-
sents the following guarantee levels in its technical sheet: Protease
(min.) 7500 U/g, cellulase (min.) 2700 U/g, xylanase (min.) 1200 U/g,
beta glucanase (min.) 300 U/g. The alpha-amylase (corn starch and
amylase) have guaranteed levels of amylase (min.) 1000 U/g. Therefore,
the exogenous enzymes used were amylase and an enzyme mixture
containing mainly protease in addition to cellulase, xylanase and beta
glucanase; as well as they combined.
2.3. Experimental design, diets and feeding
The experiment was a randomized controlled design to standardize
the initial body weight, with the animals divided into 4 treatments with
6 repetitions each: T1-CON (traditional connement diet, T2-BLEND
(0.5 g mixture of protease enzymes, cellulase, xylanase and beta glu-
canase per kg of DM diet), T3-AMIL (0.5 g alpha-amylase per kg of DM
diet), T4-BLEND+AMIL (0.5 g enzyme blend +0.5 g amylase per kg of
DM).
Previously, the animals went through a period of adaptation to the
diet (15 days), where the protocol of gradual inclusion of concentrate
was used (40:60, 60:40, 70:30–concentrate ratio: roughage) in the pe-
riods 1–5, 5–10, 10–15 days of experiment, respectively. This gradual
adaptation of the animals occurred because previously the animals were
on winter pasture (oats and ryegrass), receiving protein in the feeder
once a day. The diets were calculated according to the nutritional re-
quirements of the animal category for an estimated average daily gain
(ADG) of 1.5 kg of body weight (BR-CORTE, 2016). The daily diet was
divided into two similar meals (08 AM and 16 PM), being supplied as a
Total Mixed Ration (TMR: concentrate +silage). Enzymes were added to
the TMR in the animal feeder, a top-dressed mixture.
2.4. Sample and data collection
2.4.1. Growth performance
Zootechnical performance was evaluated as complementary infor-
mation for this research. Steers were weighed individually at 9 times
(days 1, 15, 30, 45, 60, 75, 90, 105 and 120 of the experiment). All
weighings were performed in the morning, with the animals fasting,
with the aid of a digital electronic scale. With the body weight data, it
was possible to calculate weight gain (WG) (WG =initial BW – nal BW)
and average daily gain (ADG) [(nal BW–initial BW) / number of days)].
This body weight information was used to individually calculate the
animals’ diet for the subsequent 15 days; thus, each animal received the
volume of feed proportional to its body weight. Feed leftovers were
weighed and recorded daily during the morning; important information
to calculate feed efciency.
2.4.2. Sample collection
Blood collections were performed on days 1, 15, 60 and 120, through
the caudal vein, with the aid of needles and vacuolated tubes, without
anticoagulants to obtain serum for biochemical analysis and levels of
oxidants and antioxidants. Vacuolated tubes with anticoagulants
(EDTA) were also used for hematological analyses. The tubes were kept
refrigerated at 10 ◦C, in an isothermal box until arrival at the laboratory.
For serum separation, tubes were centrifuged without anticoagulant
(7500 RPM for 10 min). The serum was transferred to microtubes,
identied and stored at -20 ◦C until laboratory analysis. Using an
esophageal probe with a vacuum system, the collection of ruminal uid
from bovines was carried out. This material had pH analyzed instantly
and the rest of the material was frozen for analysis of volatile fatty acids.
On day 121 of the experiment, the animals were slaughtered in a
specialized slaughterhouse for cattle. The slaughters followed current
legislation, and were under veterinary inspection. A fragment of liver
and longissimus dorsi muscle were collected for analysis of fatty acid
proles, stored at −20 ◦C until analysis.
2.5. Laboratory analysis
2.5.1. Chemical composition of diet and feces
Feed and feces samples were dried in a forced-air oven at 56 ◦C for at
least 72 h and ground in a 1 mm sieve for analysis. Total DM contents in
feed and feces samples were determined by oven drying at 110 ◦C for 24
h. Ash was determined by combustion at 600 ◦C for 3 h, and organic
matter was determined by mass difference. Total nitrogen was assayed
using the Kjeldahl method (Method 984.13; AOAC 1997). The neutral
detergent ber (NDF) analyses included ash and were based on the
procedures described by Mertens (2002). The chemical composition of
feeds is shown in Table 1.
2.5.2. Apparent digestibility coeciente
Indigestible neutral detergent ber (iNDF) was used as an internal
marker to calculate apparent feed digestibility (Cochran et al., 1986;
Huhtanen et al., 1994). The feed and feces samples were weighed in bags
with 16-µm porosity and incubated in the rumen of cattle for 288 h.
Then they were washed with tap water, treated with neutral detergent in
an autoclave (SENGER et al., 2008), and dried in a forced-air ventilation
A.L. Simon et al.
Biotechnology Reports 41 (2024) e00824
3
oven at 55 ◦C. Digestibility was calculated as 1 – (iNDF in feed (% of
DM)/iNDF in feces (% of DM).
2.5.3. Hematology
Hemoglobin concentration, total erythrocyte and leukocyte count
and percentage of hematocrit were performed immediately upon arrival
at the laboratory, with the aid of the Sysmex electronic device (model
KX-21 N). The leukocyte differential followed the technique described
by [15], using blood smear and light microscope identication.
2.5.4. Serum biochemistry
Levels of total proteins, glucose, albumin, cholesterol and urea were
made using the Bio-2000 (BioPlus®) semi-automatic analyzer and
commercial kits Analisa®, and their respective methodologies. The
levels of globulins were obtained with mathematical calculation (total
proteins - albumin).
2.5.5. Tissue oxidative status
Liver and meat fragments were homogenized (1v/9v) in saline so-
lution, centrifuged for 10 min at 5600 g [16]. Then, the supernatant was
collected, stored in microtubes under freezing (-20 ◦C) until analysis.
The levels of non-enzymatic antioxidants in meat and liver (protein
thiols) were evaluated, following the methodology described by [17]
and the results were expressed in nmol SH/mg protein. Glutathione
S-transferase (GST) activity in the homogenate was measured based on
the method described by researchers [18,19] and the result was
expressed as µmol CDNB/min/mg protein. Superoxide dismutase (SOD)
activity was performed according to the methodology described by [20],
with the results expressed U SOD/mg protein. The catalase activity
(CAT) in the homogenized meat and liver followed the technique
described by [21], and the data were expressed U CAT/mg protein.
Serum lipid peroxidation was measured as the amount of thio-
barbituric acid reactive substances (TBARS) according to researchers
[22,23]. The reaction was read in a spectrophotometer at 535 nm. The
result will be expressed in nmoles of malondialdehyde/ml of homoge-
nate. The determination of reactive oxygen species (ROS) was based on
the technique described by researchers [24–26] and the results were
expressed in U DCFH/mg protein.
2.5.6. Determination of short chain fatty acids in ruminal liquid
The rumen uid samples were thawed to 5 ◦C and agitated manually
in order to homogenize them. 1 mL aliquots of the supernatant from
rumen uid samples were collected in polypropylene microtubes (2 mL)
and then centrifuged for 5 min (12,300 ×g). Then 250
μ
L of the su-
pernatant was removed and transferred to a new microtube containing
250
μ
L of formic acid. The mixture was manually shaken and centrifuged
for 3 min. After centrifugation, 250
μ
L of the supernatant of the mixture
was collected into an injection vial. 500
μ
L of 3-octanol solution (665
μ
g
mL
−1
in methanol) was added, used as an internal standard, and ho-
mogenized. The samples were injected into a gas chromatograph
equipped with a ame ionization detector (GC-FID; Varian Star 3400,
Palo Alto, USA) and an autosampler (Varian 8200CX, Palo Alto, USA). 1
μ
L of the extract was injected in split mode at 1:10. The carrier gas used
was hydrogen at a constant pressure of 20 psi. The analytes (acetic,
propionic, butyric, valeric, and isovaleric acids) were separated by a CP
WAX 52CB capillary column (60 m x 0.25 mm; 0.25
μ
m stationary phase
thickness). The initial column temperature was set at 80 ◦C for 1 min and
increasing to 120 ◦C at a rate of 8 ◦C min
–1
, than up to 230 ◦C by 20 ◦C
min
–1
, where it remained for 1 min. Injector and detector temperatures
were set at 250 ◦C. The validation of the method comprised the
following parameters: selectivity, linearity, linear range, repeatability,
precision, limit of detection (LOD) and limit of quantication (LOQ) for
acetic, propionic, butyric, valeric and isovaleric acids. Analytical pa-
rameters are shown in Table Supplementary 1. Linearity was evaluated
by calculating a regression equation using the least squares method.
LOD and LOQ values were achieved by sequential dilutions up to signal-
to-noise ratios of 3:1 and 6:1, respectively. Precision was assessed by
analyzing the repeatability of six replicate samples. Accuracy was
determined by recovering known amounts of standard substances added
to a diluted sample. The results were expressed in mmol L
−1
of each
SCFA in rumen uid.
2.5.7. Prole of fatty acids in meat and feed
The extraction was performed by the Bligh and Dyer method [27]
with some modications. 1.5 g of bovine muscle samples, 0.5 mL of
water, 5 mL of methanol and 2.5 mL of chloroform were added to a 15
mL polypropylene tube and mechanical stirring was performed for 60
min. Subsequently, 2.5 mL of chloroform and 1.5 % Na2SO4 solution
were added to promote a biphasic system [28]. This mixture was shaken
for 2 min, and then centrifuged for 15 min at 2000 rpm. Lipids obtained
from the chloroform phase were subjected to fatty acid analysis.
Methylation was performed by a transesterication method pro-
posed by [29]. We added to the extracted lipids 1 mL of 0.4 M koh
methanolic solution in a test tube and vortexed for 1 min. The samples
were kept in a water bath for 10 min at the boiling point. Subsequently,
they were cooled to room temperature and 3 mL of 1 M H
2
SO
4
solution
was added and vortexed and kept in a water bath for 10 min. After
cooling, 2 mL of hexane was added and centrifuged at 2000 rpm for 10
min. Finally, hexane with fatty acid methyl esters (FAME) was subjected
to chromatography analysis.
For FAME determination, a TRACE 1310 gas chromatograph model
equipped with a ame ionization detector (Thermo Scientic) was used.
One microliter of samples was injected into a split/splitless injector
operated in the 1:20 split ratio mode at 250 ◦C. Hydrogen was used as
carrier gas at a constant ow rate of 1.5 mL/min. Separation of FAMEs
was performed using an RT 2560 chromatography column (100 m ×
0.25 mm ×0.20
μ
m thick lm, Restek, USA). The initial oven temper-
ature was programmed at 130 ◦C for 5 min and increased to 180 ◦C at a
rate of 8 ◦C/min. Then, increasing to 210 ◦C, at a rate of 4 ◦C/min, and
nally to 250 ◦C, increasing 20 ◦C/min, and maintained for 7 min in
isotherm. The detector temperature was held constant at 250 ◦C. FAME
compounds were identied by comparing the experimental retention
time with the authentic standard (FAME Mix-37, Sigma Aldrich, St.
Louis, MO). The results were presented as a percentage of each FA
identied in the lipid fraction, considering the equivalent factor of PPI
chain size for FID and ester conversion factor for the respective acid,
according to Visentainer [30,31]. Results of the fatty acid prole of feeds
were presented in Supplementary Material 1.
2.5.8. Statistical analysis
All data were analyzed using the SAS MIXED procedure (SAS Inst.
Inc., Cary, NC, USA; version 9.4), with Satterthwaite approximation to
determine denominator degrees of freedom for the xed effects test.
Growth performance data (exception for body weight -BW), carcass
traits were tested for xed treatment effect using animal (treatment) as
random effect. BW, blood count, serum biochemistry, and ruminal
Table 1
Ingredients and chemical composition of the diet.
Feed Dry matter (kg/animal/day)
Corn silage 2.29
Concentrate
1
7.04
Total 9.33
Chemical composition% Corn silage Concentrate
Dry matter 35.33 89.07
Crude protein 5.97 17.70
NDF 43.02 31.24
ADF 21.76 5.93
Ethereal extract 1.16 4.10
Ashes 4.56 6.59
Starch 22.6 37.35
A.L. Simon et al.
Biotechnology Reports 41 (2024) e00824
4
variables were analyzed as repeated measures and tested for xed effects
of treatment, day, and treatment ×day, using animal (treatment) as the
random effect. The d 1 results were included as an independent covar-
iate. Also, for these variables, to generate the average per treatment, the
d 1 results were removed from the dataset, but were kept as a covariate.
The rst-order autoregressive covariance structure was selected ac-
cording to the lowest Akaike information criterion. Means were sepa-
rated using the PDIFF method (Tukey test) and all results were reported
as LSMEANS followed by SEM. Signicance was dened when P ≤0.05
and trend when P >0.05 and ≤0.10.
3. Results
3.1. Performance of steers supplemented with exogenous enzymes
The zootechnical results are shown in Fig. 1 and Supplementary
material 3. The body weight gain tended to be greater with the treat-
ment in the T4-BLEND+AMIL in the period between days 15 and 120, (P
=0.08–Fig. 1), when compared to the T3-AMIL treatment, as well as
veried, T4-BLEND+AMIL had greater body weight gain throughout the
entire experimental period, from days 1 to 120 (P =0.05–Fig. 1) when
compared to T3-AMIL. Mean daily gain tended to be higher on treatment
at T4, on days 16 to 120 (P =0.09), when compared to T3-AMIL. Total
dry matter intake, feed conversion and feed efciency did not differ
between treatments.
3.1.1. Blood count of steers
The results of the hemograms are shown in Table 2. Higher eryth-
rocyte counts showed a trend of interaction between treatment x day,
being higher (P =0.08) in the T3-AMIL animals compared to the other
treatments on day 15, on day 120 the treatment T4-BLEND+AMIL
presented higher counts when compared to T2-BLEND. The percentage
of hematocrit showed a trend towards treatment effect, that is, lower (P
=0.10) in T3-AMIL at 15 days. There was also an interaction of days
versus treatment for hematocrit, with T2-BLEND being lower in the
blood of cattle (P =0.05) in the 120 days of the experiment. Hemoglobin
levels showed a trend of interaction between days and treatment (P =
0.08) on days 15 and 120 of the experiment, following a similar behavior
for erythrocyte count and hematocrit. The numbers of leukocytes,
lymphocytes, monocytes, granulocytes, distribution of red cells and
number of platelets did not differ between treatments.
3.1.2. Serum biochemistry
The biochemistry results are shown in Table 3. There was a tendency
for treatment to inuence glucose levels, the T1-CON group was superior
to T2-BLEND and T3-AMIL, while T4-BLEND+AMIL was superior to T2-
BLEND and equal to T3-AMIL (P =0.005). Albumin, globulin, total
protein, urea and cholesterol levels did not differ between treatments.
3.1.3. Apparent digestibility coeciente
Apparent digestibility coefcients are presented in Table 4. There
was a treatment effect for digestibility of ether extract (EE) and starch.
The T3-AMIL and T4-BLEND+AMIL treatments showed higher EE di-
gestibility compared to the others (P =0.01), which did not differ from
each other. Starch digestibility was higher in groups T3-AMIL and T4-
BLEND+AMIL compared to T1-CON; T2-BLEND did not differ (P =
0.05). The crude protein digestibility coefcient tended to be higher in
groups T2-BLEND and T3-AMIL compared to T1-CON (P =0.07). The
coefcients of dry matter, organic matter, insoluble ber in neutral
detergent, insoluble ber in acid detergent did not differ between
treatments.
3.1.4. Prole of short-chain fatty acids in ruminal uid
The results of the prole of short-chain fatty acids in the rumen are
shown in Table 5. The T4-BLEND+AMIL group had a higher concen-
tration of acetic acid than the other groups that were equal to each other
(P =0.05). T3-AMIL and T4-BLEND+AMIL had a higher concentration
of propionic acid (P =0.01) than T1-CON, with T2-BLEND being similar
for all groups. Butyric acid concentration had a strong tendency to be
higher in T4-BLEND+AMIL (P =0.06) compared to T1-CON. There was
no treatment effect for isovaleric and valeric acid concentration between
groups.
Total short-chain fatty acids (SCFA) in the ruminal uid was higher
in T4-BLEND+AMIL animals, referring to the interaction between
treatment and day (day 60) compared to the other groups. Interaction
treatment x day was also veried on day 120, with higher SCFA in all
groups that consumed the enzymes compared to T1-CON. Treatment
effect was veried in the SCFA concentration, higher in all treatments
(T1, T2 and T3) compared to control (T1).
3.1.5. Meat composition and carcass weight/yield
The results of the centesimal composition of meat and carcass are
Fig. 1. Mean and standard error (SEM) of body weight gain and feed efciency
of steers (n =6 per group) fed with exogenous enzymes during the nishing
period in a connement system. P ≤0.05 (different) and P ≥0.05 to ≤0.1
(trend) were illustrated by different letters on the bar (a, b, c), which reports
each group.
A.L. Simon et al.
Biotechnology Reports 41 (2024) e00824
5
shown in Table 6. There was no effect of treatment on weight and
carcass yield. There was inuence of the treatment on the percentage of
fat in the meat (P =0.007) in animals from T3-AMIL, higher levels when
compared to T2-BLEND and T4-BLEND+AMIL. The concentration of
mineral matter was higher in the meat of cattle that consumed the
enzymatic combination T4-BLEND+AMIL when compared to T1-CON
and T2-BLEND. As for the content of dry matter and protein there was
no difference between treatments.
3.1.6. Meat fatty acid prole
The results of the fatty acid prole in meat are shown in Table 7. We
veried the inuence of treatments for the presence of saturated,
monounsaturated and polyunsaturated fatty acids in different treat-
ments. We highlight the lower amount of saturated fatty acids in the
meat of cattle that consumed the mixture of enzymes T4-BLEND+AMIL
compared to T1-CON and T3-AMIL. A higher proportion of mono-
unsaturated fatty acids was observed in the T3-AMIL group when
compared to the others. The percentage of polyunsaturated fatty acids
was higher in the T2-BLEND and T4-BLEND+AMIL groups when
compared to the T1-CON control, and lower in the meat of the T3-AMIL
cattle compared to the other groups.
These results are related to individual changes in fatty acids, which
when added together changed the fatty acid prole of the meat. We
found that C8:0 levels were higher (P =0.010) in T4-BLEND+AMIL
Table 2
Mean and standard error (SEM) of blood count of steers supplemented with amylase and blend enzymes.
Items Treatments
1
SEM P – values
T1 (n ¼6) Control T2 (n ¼6) Blend T3 (n ¼6) Amylase T4 (n ¼6) BlendþAmylase Treat Treat £Day
Erythrocytes (x10
6
µL) 0.31 0.08
d 1 7.92 7.03 6.73 7.69 0.44
d 15 8.21
a
7.58
a
6.47
b
8.03
a
0.42
d 60 6.82 6.69 6.64 7.17 0.44
d 120 7.09
ab
6.52
b
7.29
ab
7.89
a
0.42
Average
2
7.40 6.93 6.80 7.70 0.34
Hematocrit (%) 0.10 0.05
d 1 32.3 30.1 30.1 31.9 1.44
d 15 32.9
a
32.0
ab
29.3
b
32.6
a
1.41
d 60 29.9 29.8 31.5 30.6 1.62
d 120 32.0
bc
28.8
c
34.1
ab
36.2
a
1.41
Average
2
31.6
ab
30.2
b
31.7
ab
33.1
a
0.66
Hemoglobin (g/dL) 0.26 0.08
d 1 11.0 10.4 10.2 11.0 0.55
d 15 11.3
a
10.9
ab
9.75
b
11.7
a
0.55
d 60 10.7 10.8 11.0 11.3 0.63
d 120 11.5
ab
10.4
b
12.3
a
12.9
a
0.55
Average
2
11.2 10.6 11.0 12.0 0.47
Leukocytes (x10
3
µL) 0.93 0.88
d 1 3.84 3.79 3.32 2.87 0.71
d 15 4.79 4.57 4.71 4.39 0.71
d 60 5.51 6.30 6.50 6.22 0.81
d 120 4.56 5.41 5.74 6.21 0.71
Average
2
5.09 5.55 5.61 5.39 0.58
Lymphocytes (x10
3
µL) 0.49 0.88
d 1 2.63 2.69 2.43 2.39 0.49
d 15 3.50 3.14 3.53 2.61 0.49
d 60 3.65 3.55 4.38 3.98 0.56
d 120 3.25 3.25 4.11 3.74 0.49
Average
2
3.50 3.36 3.98 3.39 0.30
Monocytes (x10
3
µL)
d 1 0.28 0.27 0.28 0.24 0.09 0.19 0.29
d 15 0.31 0.27 0.32 0.28 0.10
d 60 0.85 1.21 0.95 0.81 0.12
d 120 0.63 0.88 0.74 0.77 0.10
Average
2
0.60 0.79 0.67 0.62 0.07
Granulocytes (x10
3
µL) 0.12 0.59
d 1 1.15 0.95 0.69 0.89 0.20
d 15 1.30 1.30 0.87 1.09 0.21
d 60 1.33 1.69 1.16 0.99 0.24
d 120 0.99 1.42 0.87 1.29 0.21
Average
2
1.20 1.47 0.97 1.13 0.18
RDW-CV 0.88 0.50
d 1 21.2 22.3 21.8 21.3 1.30
d 15 21.2 23.6 22.4 21.6 1.39
d 60 26.2 24.7 25.9 25.3 1.67
d 120 22.9 21.0 23.2 22.2 1.39
Average
2
23.4 23.1 23.8 23.0 1.00
Platelets (x10
3
µL) 0.89 0.16
d 1 395 345 329 372 55.7
d 15 380 289 305 423 62.0
d 60 235 119 145 161 70.7
d 120 130 283 223 229 62.0
Average
2
248 230 224 271 45.9
1
Treatments were: T1 Control- Control treatment, T2 blend– Treatment with 0.5 g of blend per kg of DM in the diet, T3 Amylase- Treatment with 0.5 g of amylase per
kg of DM in the diet, T4 blend +Amylase- Treatment with 0.5 g enzyme blend+0 0.5 g amylase per kg of DM in the diet.
2
The d 1 results were removed from the data set to generate the average per treatment in the statistical analysis.
a,b,c
Note: P ≤0.05 (different) and P ≥0.05 to ≤0.1 (trend) were illustrated by different letters (a,b,c) on the same line.
A.L. Simon et al.
Biotechnology Reports 41 (2024) e00824
6
when compared to T3-AMIL, the same was repeated for C14:0 (P =
0.010), but T4-BLEND+AMIL showed lower concentration than T3-
AMIL, for C16:0 (P =0.071) the T4-BLEND+AMIL treatment and T2-
BLEND showed a lower concentration than the control treatment, for
C18:0 (P =0.100) the T2-BLEND and T3-AMIL were superior when
compared to T4-BLEND+AMIL, for C:20 (P =0.023) all treatments had a
higher concentration than the control, for C22:0 (P =0.050) T4-
BLEND+AMIL was superior when compared to all other treatments, for
C24:0 (P =0.050) T2-BLEND was superior when compared to T3-AMIL.
The concentration of C18:1n9c (P =0.001) had a treatment effect,
with higher concentrations in T2-BREND when compared to control, T1-
CON and T4-BLEND+AMIL; for C20:1n9 (P =0.060) all treatments
showed lower concentration compared to control (T1-CON).
The concentration of C18:3n3 (P =0.001) in meat was higher in T4-
BLEND+AMIL when compared to T3-AMIL, T2-BLEND, and T1-CON; for
the levels of C20:3n6 (P =0.043) we found that T4-BLEND+AMIL had a
higher concentration when compared to T3-AMIL, but did not differ
from the other treatments; the levels of C20:4n6 (0.001) in T4-
BLEND+AMIL were higher when compared to T3-AMIL, T2-BLEND, and
T1-CON.
3.1.7. Oxidative status in liver and meat
The results of oxidative status in liver and meat are shown in Table 8.
In the liver, in general, we observed a lower concentration of TBARS in
Table 3
Mean and standard error (SEM) of serum biochemistry of steers supplemented with amylase and blend enzymes.
Items Treatments
1
SEM P – values
T1 (n ¼6) Control T2 (n ¼6) Blend T3 (n ¼6) Amylase T4 (n ¼6) BlendþAmylase Treat Treat £Day
Albumin (g/dL) 0.88 0.45
d 1 6.36 5.86 6.07 5.58 0.29
d 15 2.50 2.63 2.64 2.78 0.29
d 60 2.76 2.83 2.52 3.40 0.29
d 120 3.05 3.01 2.89 2.90 0.29
Average
2
2.89 2.79 2.72 2.91 0.14
Globulin (g/dL) 0.48 0.20
d 1 3.30 2.91 3.51 3.21 0.79
d 15 6.02 5.81 5.99 5.71 0.79
d 60 9.12 11.8 12.3 9.99 0.79
d 120 4.15 4.99 3.9 5.14 0.79
Average
2
6.45 7.43 7.48 6.94 0.53
Total protein (g/dL) 0.73 0.36
d 1 9.68 8.74 9.61 8.78 0.83
d 15 8.53 8.41 8.65 8.48 0.83
d 60 11.9 14.6 14.8 13.4 0.83
d 120 7.21 7.98 6.8 8.03 0.83
Average
2
9.38 10.2 10.3 9.81 0.57
Glucose (mg/dL) 0.06 0.86
d 1 97.7 98.6 97.0 93.9 6.37
d 15 84.8 77.1 82.4 87.7 6.37
d 60 105 86.6 89.4 106 6.37
d 120 96.4 72.9 85.4 84.9 6.37
Average
2
95.5
a
78.9
c
85.7
bc
92.6
ab
3.24
Urea (mg/dL) 0.98 0.18
d 1 23.6 20.8 22.6 21.6 1.87
d 15 26.2 23.3 22.9 23.3 1.87
d 60 26.6 25.3 25.6 28.1 1.87
d 120 27.1 31.7 32.1 28.4 1.87
Average
2
27.1 26.3 27.1 26.4 1.51
Cholesterol (mg/dL) 0.75 0.51
d 1 108 97.6 86.8 97.2 11.7
d 15 103 87.1 90.4 117 11.7
d 60 98.6 106 97.1 103 11.7
d 120 116 113 138 125 11.7
Average
2
109 102 105 115 9.40
1
Treatments were: T Control- Control treatment, T2 blend– Treatment with 0.5 g of blend per kg of DM in the diet, T3 Amylase- Treatment with 0.5 g of amylase per
kg of DM in the diet, T4 blend +Amylase- Treatment with 0.5 g enzyme blend +0 0.5 g amylase per kg of DM in the diet.
2
The d 1 results were removed from the data set to generate the average per treatment in the statistical analysis.
a,b,c
Note: P ≤0.05 (different) and P ≥0.05 to ≤0.1 (trend) were illustrated by different letters (a,b,c) on the same line.
Table 4
Mean and standard error (SEM) of apparent digestibility coeciente (ADC) of cattle fed exogenous enzymes.
Variables (%) T1 (n ¼6) Control T2 (n ¼6) Blend T3 (n ¼6) Amylase T4 (n ¼6) BlendþAmylase SEM P- trat
Dry Matter 61.7 65.4 67.6 66.4 2.41 0.39
Organic Matter 65.4 68.8 71.2 70.3 2.93 0.26
Crude protein 54.2
b
58.1
a
61.3
a
57.1
ab
1.08 0.07
NDF 59.6 62.9 61.5 64.5 2.58 0.46
ADF 46.1 46.6 51.0 49.5 2.36 0.37
Ether extract 36.6
b
42.8
b
53.0
a
55.2
a
2.79 0.01
Starch 73.0
b
76.9
ab
81.3
a
81.8
a
2.01 0.05
1
Treatments were: T1 Control- Control treatment, T2 blend– Treatment with 0.5 g of blend per kg of DM in the diet, T3 Amylase- Treatment with 0.5 g of amylase per kg
of DM in the diet, T4 blend +Amylase- Treatment with 0.5 g enzyme blend+0 0.5 g amylase per kg of DM in the diet.
a,b,c
Note: P ≤0.05 (different) and P ≥0.05 to ≤0.1 (trend) were illustrated by different letters (a,b,c) on the same line.
A.L. Simon et al.
Biotechnology Reports 41 (2024) e00824
7
T2-BLEND animals (P =0.050), when compared to T1-CON. The lowest
production of ROS (P =0.001) was veried in T3-CON, when compared
to T2-BLEND and control, on the other hand SOD activity (P =0.015)
did not differ between T2-BLEND, T3-AMIL and T4-BLEND+AMIL, but it
was higher in the T1-CON control. We also veried the highest CAT
activity (P =0.052) in the liver of the T4-BLEND+AMIL animals when
compared to the other treatments. As for meat, we found that animals
from T4-BLEND+AMIL (P =0.094) tended towards a higher proportion
of ROS than T3-AMIL and T1-CON, in contrast to meat from steers from
T4-BLEND+AMIL (P =0.001) showed higher GST activity when
compared to T3-AMIL and control. PSH levels (P =0.050) were higher in
T3-AMIL when compared to T4-BLEND+AMIL and T2-BLEND; while the
highest SOD activity (P =0.045) was higher in T4-BLEND+AMIL and
T2-BLEND when compared to control T1-CON.
4. Discussion
Addition of exogenous enzymes amylase, protease, cellulase, xyla-
nase and beta glucanase positively affected weight gain, but without
affecting the nal weight. Similar results were veried by Rodríguez-
Carías et al. [32] who used a commercial product based on exogenous
enzymes beta-glucanase, xylanase, pectinase, mannanase, xylogluca-
nase, laminarase, β-glucosidase, β-xylosidase,
α
-L-arabinofuranosidase,
amylase and protease in lambs, and found an effect on animal perfor-
mance. These authors explain that this greater weight gain occurs due to
the improvement in nutrient digestibility, since the product did not
affect DM intake [32]. It is believed that when using proteolytic enzymes
they act by removing protein structures from the cell wall of the forage,
allowing faster access for ruminal microorganisms [33], which leads us
to justify the gains in performance when using the enzymes in
combination.
Researchers found that the inclusion of amylase promotes rapid
release of starch oligosaccharides that are used by both amylolytic and
non-amylolytic bacteria [34], for this reason starch is fermented quickly.
This was observed in our study, in both groups of steers that consumed
amylase in the diet, the starch digestibility coefcient was higher; as
well as increasing the concentration of total volatile fatty acids in the
rumen; but this was not enough when the exogenous enzyme was added
to the diet to enhance weight gain. The reasons for this are not known
and complementary results do not help explain the mechanisms
Table 5
Mean and standard error (SEM) of total short-chain fatty acids (SCFA) in ruminal uid and prole of volatile fatty acids in the rumen of cattle that consumed exogenous
enzymes.
Variables T1 (n ¼6) Control T2 (n ¼6) Blend T3 (n ¼6) Amylase T4 (n ¼6) BlendþAmylase SEM P- trat P- trat x day
Acetic acid (mmol/L 0.05 0.05
d60 64.7
ab
51.5
b
58.2
ab
69.0
a
1.98
d120 68.4
b
77.2
a
75.1
ab
77.9
a
1.57
Average 66.5
b
64.3
b
66.6
b
73.4
a
1.45
Propionic acid (mmol/L) 0.01 0.01
d60 15.6
bc
13.7
c
17.0
ab
19.7
a
0.74
d120 18.0
b
26.9
a
29.4
a
28.8
a
0.81
Average 16.8
b
20.3
ab
23.2
a
24.2
a
0.75
Butiric acid (mmol/L) 0.06 0.04
d60 10.9 10.81 10.6 12.6 0.65
d120 13.3
b
15.5
ab
15.6
ab
17.1
a
0.66
Average 12.1
b
13.1
ab
13.1
ab
14.9
a
0.62
Isovaleric acid (mmol/L) 0.59 0.23
d60 1.82 1.60 1.49 1.70 0.11
d120 1.72 2.23 1.74 2.05 0.09
Average 1.77 1.91 1.61 1.87 0.1
Valeric acid (mmol/L) 0.44 0.62
d60 1.77 1.51 1.59 1.85 0.21
d120 2.01 2.50 2.35 2.61 0.36
Average 1.89 2.00 1.97 2.23 0.25
SCFA (mmol/L) 0.01 0.01
d60 94.7
b
79.1
c
88.8
b
104.8
a
2.04
d120 103.4
b
124.3
a
124.2
a
128.4
a
1.96
Average 99.0
b
101.7
b
106.5
b
116.6
a
2.01
1
Treatments were: T1 Control- Control treatment, T2 blend– Treatment with 0.5 g of blend per kg of DM in the diet, T3 Amylase- Treatment with 0.5 g of amylase per kg
of DM in the diet, T4 blend +Amylase- Treatment with 0.5 g enzyme blend+0 0.5 g amylase per kg of DM in the diet.
2
The d 1 results were removed from the data set to generate the average per treatment in the statistical analysis.
a,b,c
Note: P ≤0.05 (different) and P ≥0.05 to ≤0.1
(trend) were illustrated by different letters (a,b,c) on the same line.
Table 6
Mean and standard error (SEM) of meat and carcass composition of steers supplemented with amylase and protease enzymes.
Items Treatments
1
SEM P-value
T1 (n ¼6) Control T2 (n ¼6) Blend T3 (n ¼6) Amylase T4 (n ¼6) BlendþAmylase Trat
Carcass variables
Carcass weight (kg) 251 244 239 246 9.79 0.86
Carcass yield (%) 50.5 49.2 49.5 48.9 0.71 0.43
Meat variables
Dry matter (%) 31.8 30.6 32.3 33.1 1.13 0.48
Protein (%) 25.1 25.7 23.8 25.7 0.78 0.30
Fat (%) 2.79
b
2.05
b
3.84
a
1.97
b
0.38 0.007
Ash (%) 3.91
bc
2.85
c
4.66
ab
5.43
a
0.40 0.001
1
Treatments were: T1 Control- Control treatment, T2 blend– Treatment with 0.5 g of blend per kg of DM in the diet, T3 Amylase- Treatment with 0.5 g of amylase per
kg of DM in the diet, T4 blend +Amylase- Treatment with 0.5 g enzyme blend+0 0.5 g amylase per kg of DM in the diet.
a,b,c
Note: P ≤0.05 (different) and P ≥0.05 to ≤0.1 (trend) were illustrated by different letters (a,b,c) on the same line.
A.L. Simon et al.
Biotechnology Reports 41 (2024) e00824
8
involved.
Feed efciency, feed conversion and dry matter intake showed no
treatment effect; which is a consequence of the greater digestibility of
the EE and starch nutrients, which may be related to the greater weight
gain of T4-BLEND+AMIL cattle. Rose et al. (2010) found similar results
when testing a product based on protease and amylase in the diet of
Guzerat cattle in connement, in which there was also no inuence on
the same productive parameters mentioned above. Andreazzi et al. [35]
when testing amylase in dairy cows, found that there was no change in
dry matter intake, therefore, in milk production, it generated an increase
with diets of at least 30 % starch. In the 90 s, researchers already said
that the use of exogenous enzymes could be more efcient in diets with
low moisture content, less than 30 %, than in diets with high moisture
content [36], as when corn silage is present, which presents approxi-
mately 70 %, which explains the results obtained in our study, since the
silage had a high moisture content.
With the blood count results, we noticed a high percentage of he-
matocrit, with interaction of treatment ×day for T4-BLEND+AMIL,
which was really not expected and we have no explanation of the
mechanisms involved. According to Santos [37], normal blood hemat-
ocrit levels are 30 to 35 %, in this work we noticed that there was a
positive effect when using the enzymes in a combined form
T4-BLEND+AMIL and a negative effect at times when using only
amylase (T3-AMIL), as the percentage was less than 30, indicating mild
anemia, which may have contributed to the body weight not differing.
There is a tendency for treatment to affect glucose levels, with
Table 7
Mean and standard error (SEM) of prole of fatty acids in the meat of steers supplemented with amylase and blend enzymes.
Fatty acid (g/kg) Treatments SEM P-value
T1 (n ¼6) Control T2 (n ¼6) Blend T3 (n ¼6) Amylase T4 (n ¼6) BlendþAmylase
C8:0 (Caprylic) 0.908
ab
1.605
a
0.579
b
1.744
a
0.121 0.010
C12:0 (Lauric) 0.472 0.650 0.532 0.521 0.048 0.654
C14:0 (Myristic) 20.12
ab
19.18
ab
23.598
a
17.861
b
0.823 0.010
C14:1 (Myristoleic) 5.232 4.776 5.616 4.497 0.337 0.102
C15:0 (Pentadecanoic) 2.279 2.401 2.498 2.204 0.083 0.856
C16:0 (Palmitic) 314.8
a
300.0
b
310.0
ab
300.9
b
2.397 0.071
C16:1 (Palmitoleic) 33.59 34.29 36.56 33.02 0.969 0.258
C17:0 (Heptadecanoic) 6.437 6.309 7.475 6.261 0.240 0.182
C18:0 (Stearic) 148.1
a
150.9
a
148.6
a
144.1
b
1.966 0.100
C18:1n9c (Oleic) 384.0
b
380.0
b
406.0
a
378.9
b
4.689 0.001
C18:2n6c (Linoleic) 53.33
b
67.96
a
37.33
c
73.74
a
4.202 0.001
C20:0 (Arachidic) 0.645
b
1.173
a
0.923
a
0.982
a
0.061 0.023
C18:3n6 (?-Linolenic) 0.558 0.626 0.420 0.589 0.047 0.362
C20:1n9 (cis-11-Eicosenoic) 2.382
a
1.000
b
1.304
b
1.347
b
0.054 0.060
C18:3n3 (a-Linolenic) 1.772
b
1.548
b
1.337
b
3.201
a
0.245 0.001
C21:0 (Henicosanoic) 2.014 1.864 2.534 1.867 0.105 0.201
C20:2 (cis-11,14-Eicosadienoic) 0.799 0.832 0.592 0.632 0.027 0.120
C22:0 (Behenic) 0.362
b
0.369
b
0.195
c
0.457
a
0.048 0.050
C20:3n6 (cis-8,11,14-Eicosatrienoic) 4.371
a
4.567
a
2.770
b
4.531
a
0.258 0.043
C22:1n9 (Erucic) 0.283 0.310 0.204 0.384 0.038 0.258
C20:4n6 (Arachidonic) 14.92
cd
16.21
bc
9.575
d
19.28
a
1.217 0.001
C22:2 (cis-13,16-Docosadienoic) 0.187 0.072 0.109 0.185 0.011 0.350
C24:0 (Lignoceric) 0.399
bc
0.551
a
0.266
c
0.461
ab
0.032 0.050
C20:5n3 (cis-5,8,11,14,17-Eicosapentaenoic) 0.692 0.680 0.374 0.792 0.051 0.135
C24:1n9 (Nervonic) 0.220 0.148 0.116 0.162 0.016 0.186
C22:6n3 (cis-4,7,10,13,16,19-Docosahexaenoic) 1.035 0.792 0.361 1.187 0.163 0.632
AGS 496.5
a
485.0
ab
497.2
a
477.4
b
2.053 0.038
MUFA 425.7
b
420.5
b
449.8
a
418.3
b
1.970 0.001
PUFA 77.67
b
93.29
a
52.87
c
104.14
a
2.381 0.001
1
Treatments were: T1 Control- Control treatment, T2 blend– Treatment with 0.5 g of blend per kg of DM in the diet, T3 Amylase- Treatment with 0.5 g of amylase per kg
of DM in the diet, T4 blend +Amylase- Treatment with 0.5 g enzyme blend+0 0.5 g amylase per kg of DM in the diet.
a,b,c
Note: P ≤0.05 (different) and P ≥0.05 to ≤0.1 (trend) were illustrated by different letters (a,b,c) on the same line.
Table 8
Mean and standard error (SEM) of oxidative status in the liver and meat of steers supplemented with amylase and blend enzymes.
Tissue Treatments TBARS ROS GST PSH SOD CAT
Liver(n =6 per
group)
T1 Control 1.436
a
4.623
a
1082 579.7 3.976
a
2.044
ab
T2 Blend 1.183
b
3.235
b
1188 464.0 1.281
b
2.454
ab
T3 Amylase 1.273
ab
2.206
c
1119 566.0 2.061
b
2.375
ab
T4 Blend +Amylase 1.270
ab
2.434
c
1087 488.1 1.729
b
2.880
a
SEM 0.025 0.067 4.142 8.369 0.042 0.013
p-value 0.050 0.001 0.657 0.241 0.015 0.052
Meat(n =6 per
group)
T1 Control 2.704 1.119
ab
2832
b
757.2
c
9.080
b
1.505
T2 Blend 2.702 1.498
a
3202
ab
803.6
b
13.04
a
1.662
T3 Amylase 2.442 0.699
b
2858
b
853.6
a
12.60
ab
1.185
T4 Blend +Amylase 3.110 1.350
a
3478
a
808.0
b
14.38
a
1.876
SEM 0.169 0.056 6.521 5.978 1.006 0.240
p-value 0.147 0.094 0.001 0.050 0.045 0.109
Note: ROS (U DCFH/mg protein); TBARS (nmol MDA/mL), GST (µmolCDNB/min/mg protein), PSH (nmol SH/mg protein), SOD (U SOD/mg protein), CAT (U CAT/mg
of protein).
1
Treatments were: T1 Control- Control treatment, T2 blend– Treatment with 0.5 g of blend per kg of DM in the diet, T3 Amylase- Treatment with 0.5 g of amylase per kg
of DM in the diet, T4 blend +Amylase- Treatment with 0.5 g enzyme blend+0 0.5 g amylase per kg of DM in the diet.
a,b,c
Note: P ≤0.05 (different) and P ≥0.05 to ≤0.1 (trend) were illustrated by different letters (a,b,c) on the same column.
A.L. Simon et al.
Biotechnology Reports 41 (2024) e00824
9
animals in the T2-BLEND and T3-AMIL groups having lower serum
concentrations; however, we expected that the animals that consumed
the enzyme diet would have a higher concentration of glucose in the
blood. This increase in glucose concentration in amylase treatments may
have resulted from increased ruminal starch fermentation and propio-
nate uptake for gluconeogenesis [38]. According to Denardin Silva [39],
starch is composed of amylose and amylopectin, formed by glucose
joined by a-1,4 and a-1,6 bonds, part of which is broken down into
glucose by hydrolysis and another part by the enzymatic action of
amylase. As mentioned by Vigne et al. [14], when using amylase-based
enzyme complexes in feedlot steers, there was an improvement in starch
digestion. We also observed that the addition of
α
-amylase improved the
digestibility of starch and ether extract, which demonstrates the action
of this enzyme in the degradation of these compounds. As a result of the
greater utilization of starch, a higher concentration of short-chain fatty
acids was observed, especially propionic acid, the main precursor of
glucose in ruminants. However, the concentration of glucose in the
blood tended to be lower when the animals consumed the enzymes
individually; which may be related to the difculty of measuring this
enzyme in ruminants, since the animal is rarely fasting; as well as the
rate of passage of feed through the gastrointestinal tract differ between
animals.
Rocha et al. [40] concluded that the optimal level of starch in cattle
supplemented with essential oils and amylase is 35 %, thanks to the
possibility that the amylase remains active until the intestine. Further-
more Toseti et al. [41] observed potential for increased microbial pro-
tein with combined supplementation of amylase and essential oils.
Upregulation of several enzymes that promote carbohydrate degrada-
tion in glycolic pathways, gluconeogenesis and oxidative phosphoryla-
tion was observed in cattle supplemented with amylase and essential oils
combined [40]. This justies the increase in starch digestibility and
increase in body weight gain when amylase was given in combination.
Salem et al. [42] observed a 16 % increase in weight gain when
providing exogenous enzymes. Cattle supplemented with an
amylase-based enzymatic complex [43] concluded that starch from corn
caused improvements in subcutaneous fat thickness in feedlot
super-early steers. Another point that we can observe was the concen-
tration of mineral matter, which was higher in the meat of cattle that
consumed the enzymatic combination T4-BLEND+AMIL, but it was not
evaluated which minerals were deposited in the muscle tissue.
Another point that we can observe is the concentration of mineral
matter, which was higher in the meat of cattle that consumed the
enzymatic combination T4-BLEND+AMIL, but it was not evaluated
which minerals were deposited in the muscle tissue. Vigne et al. [44]
used a similar enzyme complex in feedlot steers consuming diets with
high starch content, and also observed greater fat deposition in the
carcass, a consequence of better feed conversion and increased starch
digestibility. As can be seen, the addition of exogenous enzymes can
alter lipid metabolism in meat from cattle conned with high percentage
of concentrate. In addition to greater fat deposition, treatments inu-
enced the fatty acid prole in the meat. We emphasize that the lower
amount of saturated fatty acids in the meat of cattle that consumed the
mixture of T4-BLEND+AMIL enzymes is an excellent result for the
consumer, since saturated fats must be avoided, since in the lipid
metabolism it can increase the levels of LDL (known as bad cholesterol),
which is a low-density lipoprotein, capable of carrying cholesterol par-
ticles from the liver and other places to the arteries, that is, when in
excess in the circulation, it causes accumulation in the vessels that can,
over time, clog or form thrombi [45]. The benets of reducing LDL
cholesterol in humans are observed on a large scale in the literature
[46].
In addition, when isolated or combined enzymes were used in the
bovine diet, we veried a higher percentage of unsaturated fatty acids,
the fat considered ideal for human consumption, since we have the
omegas that increased when consumed in a diet containing only the
analysis, such as acid oleic. Observed effect on the concentration of
polyunsaturated fatty acids (PUFA) in the T4-BLEND+AMIL group;
which is extremely benecial for human health with potential effect on
cancer protection (Chikwabha et al. 2018). We believe that the mech-
anisms that led to changes in the fatty acid prole are indirect, altering
the digestive process, and consequently the lipid metabolism; the
mechanisms involved are unclear and need further research to avoid
speculation.
With changes in the prole of fatty acids in meat, an effect on the
oxidative status of liver and meat was already expected. This is because
it is known that lipids are among the main tissues responsible for pro-
ducing oxidative instability and raising levels of free radicals. Con-
sumption of combined exogenous enzymes and isolated amylase had a
positive effect on redox balance, mainly in meat, but also in liver. We
veried less lipid peroxidation and reactive oxygen species, as well as
greater activity of antioxidant enzymes. In the literature there is no
explanation of how exogenous enzymes modulate the oxidative status,
but it is known that proteases are able to improve the oxidative stability
of feeds with fermented processed items [47].
5. Conclusion
The results allow us to conclude that the combination of digestive
enzymes (amylase, protease, cellulase, xylanase and beta glucanase) in
the bovine diet improves growth performance, in addition to increasing
the amount of unsaturated fatty acids and reducing the amount of
saturated fatty acids in the meat. Consumption of exogenous enzymes
also has a positive effect on oxidative stability in meat.
Ethics committee
Project approved at CEUA/UDESC, protocol number 8717230921.
CRediT authorship contribution statement
Alexandre L. Simon: Writing – original draft, Methodology, Data
curation, Conceptualization. Priscila M. Copetti: Validation, Method-
ology, Formal analysis. Rafael V.P. Lago: Writing – original draft,
Methodology, Investigation, Data curation. Maksuel G. Vitt: Writing –
original draft, Methodology, Investigation, Formal analysis. Aline L.
Nascimento: Writing – original draft, Methodology, Investigation. Luiz
Eduardo Lobo e Silva: Writing – original draft, Methodology. Roger
Wagner: Writing – review & editing, Validation, Supervision, Method-
ology, Formal analysis, Conceptualization. Bruna Klein: Writing – re-
view & editing, Validation, Supervision, Methodology. Camila Soares
Martins: Writing – original draft, Methodology. Gilberto V. Kozloski:
Writing – review & editing, Validation, Supervision, Resources, Meth-
odology, Investigation, Data curation. Aleksandro S. Da Silva: Writing
– review & editing, Supervision, Resources, Project administration,
Methodology, Investigation, Funding acquisition, Formal analysis, Data
curation, Conceptualization.
Declaration of Competing Interest
The authors declare no competing or nancial interests.
Data availability
No data was used for the research described in the article.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.btre.2023.e00824.
A.L. Simon et al.
Biotechnology Reports 41 (2024) e00824
10
References
[1] V.S.P. Wedekin, C.R.F. Bueno, A.M.P. Amaral, Informaç˜
oes Econˆ
omicas,
Informaç˜
oes Econ. 24 (1994) 123–131.
[2] L. Velloso, Terminaç˜
ao de bovinos em connamento. slp, s. ed., 1984.
[3] R.C. Queiroz, A.F. Bergamaschine, J.F.P. Bastoset, et al., Uso de produto `
a base de
enzima e levedura na dieta de bovinos: digestibilidade dos nutrientes e
desempenho em connamento, Rev. Bras. Zootec. 33 (2004) 1548–1556.
[4] Tiago Pereira Guimar˜
aes, Exigˆ
encias Proteicas para bovinos de corte, Multi-Sci. J. 1
(1) (2015) 90–99 (ISSN 2359-6902)v.n.
[5] A. De Souza Martins, et al., Eciˆ
encia de síntese microbiana e atividade enzim´
atica
em bovinos submetidos `
a suplementaç˜
ao com enzimas brolíticas, R. Bras. Zootec.
35 (2006) 45–48.
[6] A. De Souza Martins, et al., Degradabilidade in situ e observaç˜
oes microsc´
opicas de
volumosos em bovinos suplementados com enzimas brolíticas ex´
ogenas1, R. Bras.
Zootec 36 (6) (2007) 1927–1936.
[7] J.E. Edwards, S.A. Huws, E.J. Kim, A.H. Kingston-Smith, Characterization of the
dynamics of initial bacterial colonization of nonconserved forage in the bovine
rumen, FEMS Microbiol. Ecol., Oxford 62 (1) (2007) 323–335.
[8] L.A. Giraldo, M.L. Tejido, M.J. Ranilla, S. Ramos, M.D. Carro, Inuence of direct
feed brolytic enzymes on diet digestibility and ruminal activity in sheep fed a
grass hay-based diet, J. Animal Sci., Champaign 86 (7) (2008) 1617–1620.
[9] A.M. Silvestre, D.D. Millen, The 2019 Brazilian survey on nutritional practices
provided by feedlot cattle consulting nutritionists, Rev. Bras. Zootec. [S.L.] 50
(2021), e20200189.
[10] G. Giuberti, A. Gallo, F. Masoero, L.F. Ferraretto, P.C. Hoffman, R.D. Shaver,
Factors affecting starch utilization in large animal food production system: a
review, Starch-St¨
arke 66 (2014) 72–90.
[11] R.G. Utrilla-Coello, C. Hern´
andez-Jaimes, H. Carrillo-Navas, F. Gonz´
alez,
E. Rodríguez, L.A. Bello-P´
erez, E.J. Vernon-Carter, J. Alvarez-Ramirez, Acid
hydrolysis of native corn starch: Morphology, crystallinity, rheological and thermal
properties, Carbohydr. Polym. 103 (2014) 596–602.
[12] C. OBERT, C. CHIKWANHA, et al., Nutritional enhancement of sheep meat fatty
acid prole for human health and wellbeing, Food Res. Int., [S.L.] 104 (2018)
25–38.
[13] B.L. ROSA, J.B. Alves, A.C. Bergamaschine, et al., Levels of concentrate and
inclusion of probiotics for cattle Guzera in feedlot, Rev. Bras. de Saude e Prod.
Anim. 11 (2010) 440–451.
[14] G.L.D. Vigne, et al., Digestibilidade do amido e comportamento ingestivo de
novilhos connados sob efeito de doses de complexo enzim´
atico em dietas de alta
densidade energ´
etica, Arquivo Brasileiro de Medicina Veterin´
aria e Zootecnia 71
(2019) 1015–1026, v.
[15] B.F. Feldman, J.G. Zinkl, N.C. Jain, Veterinary Hematology, 1344, Williams &
Wilkins, 2000.
[16] E. Tatsch, G.V. Bochi, R.D.S. Pereira, H. Kober, J.R.D. Oliveira, R.N. Moresco,
Inuence of anticoagulants and storage temperature on blood nitrite levels, J Bras
Patol Med Lab 47 (2011) 147–150.
[17] G.L. Ellman, Tissue sulfhydryl groups, Arch. Biochem. Biophys. (82) (1959) 70–77.
[18] W.H. Habig, M.J. Pabst, W.B. Jakob, Glutathione S-transferases: the rst enzymatic
step in mercapturic acid formation, J. Biol. Chem. 249 (1974) 7130–7139.
[19] A.L.R.BRUNETTO FAVERO F.F., B.F. BISSACOTTI, et al., Phytogenic blend in the
diet of growing Holstein steers: effects on performance, digestibility, rumen
volatile fatty acid prole, and immune and antioxidant responses, Anim. Feed Sci.
Technol. 297 (2023) 115595.
[20] J.M. Mc Cord, I Fridovich, The role of superoxide anion in the autoxidation of
epinephrine and a simple assay for superoxide dismutase, J. Biol. Chem. 247 (10)
(1972) 3170–3175.
[21] D.P. Nelson, L.A. Kiesow, Enthalpy of decomposition of hydrogen peroxide by
catalase at 25◦C (with molar extinction coefcients of H2O2 solutions in the UV),
Anal. Biochem. 49 (2) (1972) 474–478.
[22] A.M. Jentzsch, H. Bachmann, P. Furst, H Biesalski, Improved analysis of
malondialdehyde in human body uids, Free Radical Biol. Medicine 20 (1996)
251–256.
[23] H. Ohkawa, N. Ohishi, K. Yagi, Assay for lipid peroxides in animal tissues by
thiobarbituric acid reaction, Anal. Biochem. 95 (1978) 351–358, 1978.
[24] S.F. Ali, C.P. Lebel, S.C. Bondy, Reactive oxygen species formation as a biomarker
of methylmercury and trimethyltin neurotoxicity, Neurotoxico 113 (1992)
637–648.
[25] B. HALLIWELL, J.M.C. GUTTERIDGE, Free Radicals in Biology and Medicine, 4th
edn., Oxford University Press, New York, 2007.
[26] L.C. Maltez, L.A.L. Barbas, L.F. Nitz, L. Pellegrin, M.H. Okamoto, L.A. Sampaio, J.
M. Monserrat, L. Garcia, Oxidative stress and antioxidant responses in juvenile
Brazilian ounder Paralichthys orbignyanus exposed to sublethal levels of nitrite,
Fish Physiol. Biochem. 44 (2018) 1349–1362.
[27] E.G. Bligh, W.J. Dyer, A rapid method of total lipid extraction and purication, Can
J Biochem Physiol 37 (1959) 911–917.
[28] C.M. Giacomelli, M.S. Marchiori, M. Vedovatto, Silva A.S. DA, Encapsulated pepper
blend in the diet of conned Holstein bullocks: effect on ruminal volatile fatty acid
proles, growth performance, and animal health, Trop. Anim. Health Prod. 55
(2023) 114–221.
[29] L. Hartman, R.C. Lago, Preparaç˜
ao r´
apida de ´
esteres de metila ´
acido graxo partir de
lipídios, Pract. 22 (494) (1973) 475–477.
[30] J.V. Visentainer, M.R.B. Franco. ´
Acidos Graxos em ´
Oleos e Gorduras: Identicaç˜
ao e
Quanticaç˜
ao, 1a ed, Varela:, S˜
ao Paulo, 2006.
[31] Visentainer, Aspectos analíticos da resposta do detector de ionizaç˜
ao em chama
para ´
esteres de ´
acidos graxos em biodiesel e alimentos, Química Nova 35 (2012)
274–279.
[32] A.A. RODR´
IGUEZ-CAR´
IAS, et al., Evaluaci´
on de a-amilasa y proteasa sobre el
consumo y la digestibilidad de nutrientes de las dietas y par´
ametros siol´
ogicos en
ovinos, J.Agric. Univ. Puerto Rico 101 (1) (2017) 63–78, v.n.1 jan.
[33] D. Colombatto, K.A. Beauchemin, A proposed methodology to standardize the
determination of enzymic activities present in enzyme additives used in ruminant
diets, Canadian J. Animal Sci., Sherbrooke 83 (3) (2003) 559–568.
[34] J.M. Tricarico, J.D. Johnston, K.A. Dawson, Dietary supplementation of ruminant
diets with an
α
-amylase from Aspergillus oryzae, Animal Feed Science and
Technology, Amesterd˜
ao 145 (1–4) (2008) 136–150.
[35] A.S.R. Andreazzi, et al., Effect of exogenous amylase on lactation performance of
dairy cows fed a high-starch diet, J. Dairy Sci. 101 (8) (2018) 7199–7207.
[36] K.A. Beauchemin, et al., Effects of brolytic enzymes in corn or barley diets on
performance and carcass characteristics of feedlot cattle, Can. J. Anim. Sci. 77 (4)
(1997) 645–653.
[37] G.T. Santos, Efeito de diferentes volumosos sobre os constituintes sangüíneos de
vacas da raça holandesa, Rev. Bras. Saúde Prod. An 9 (2008) 35–44.
[38] C.K. Reynolds, et al., Net Metabolism of Volatile Fatty Acids, D-β-Hydroxybutyrate,
Nonesteried Fatty Acids, and Blood Gasses by Portal-Drained Viscera and Liver of
Lactating Holstein Cows, J. Dairy Sci. 71 (9) (1988) 2395–2404.
[39] C.C. Denardin, L.P. Da Silva, Estrutura dos grˆ
anulos de amido e sua relaç˜
ao com
propriedades físico-químicas, Ciˆ
encia Rural 39 (3) (2009) 945–954.
[40] L.C. Rocha, et al., Feedlot diets containing different starch levels and additives
change the cecal proteome involved in cattle’s energy metabolism and
inammatory response, Scientic Reports, [S.L.] 12 (2022), 5691.
[41] L.B. Toseti, et al., Effects of a blend of essential oils and exogenous
α
-amylase in
diets containing different roughage sources for nishing beef cattle, Animal Feed
Sci. Technol., [S.L.] 269 (2020), 114643 v.nov.Elsevier BV.
[42] A.Z.M. Salem, et al., Effects of exogenous enzymes on nutrient digestibility,
ruminal fermentation and growth performance in beef steers, Livestock Sci., [S.L.]
154 (1–3) (2013) 69–73.
[43] L.C.V. ´
Itavo, et al., Fontes de amido no concentrado de bovinos superprecoces de
diferentes classes sexuais, Arq. Bras. Med. Vet. Zootec 66 (4) (2014) 1129–1138.
[44] G.L.D. Vigne, et al., Doses of enzyme complex in a high-energy diet on performance
and carcass traits of feedlot steers, Revista Brasileira de Zootecnia 47 (03) (2018) v.
n.
[45] A. Beatriz, et al., Nutriç˜
ao e exercício na prevenç˜
ao e controle das doenças
cardiovasculares, Rev. Bras. Med. Esporte 8 (6) (2002) 244–254.
[46] J.A. Tobert, LDL cholesterol-how low can we go? Endocrinol. Metab. Clin. North.
Am. 51 (3) (2022) 681–690.
[47] J.M. Broncano, et al., Use of proteases to improve oxidative stability of fermented
sausages by increasing low molecular weight compounds with antioxidant activity,
Food Res. Int. 44 (9) (2011) 2655–2659.
A.L. Simon et al.
