Effects of Arazyme Intake on Growth Performance, Ruminal Fermentation, Nutrient Digestibility, and Blood Parameters in Feedlot Cattle

Simple Summary

Feed additives are commonly used to improve nutrient utilization and performance in beef cattle production systems. Arazyme is an exogenous metalloprotease with high catalytic activity and stability, but its effects in ruminant nutrition are still poorly understood. In this study, arazyme was included in the diet of feedlot cattle to evaluate its effects on growth performance, ruminal fermentation, blood parameters, and nutrient digestibility. The enzyme improved weight gain, feed efficiency, and the digestibility of protein and fiber, while also increasing ruminal production of volatile fatty acids (VFAs) without affecting animal health. These findings suggest that the use of arazyme may be a promising nutritional strategy to improve productivity in feedlot cattle.

Abstract

The use of exogenous enzymes in feedlot cattle nutrition has been investigated as a strategy to increase nutrient utilization efficiency and productive performance. The objective of this study was to evaluate the effects of including arazyme, an exogenous metalloprotease, in the diet of feedlot beef cattle on zootechnical performance, hematological parameters, serum biochemical biomarkers, ruminal fermentation, and apparent nutrient digestibility. Twelve male Holstein cattle, with an average initial body weight of 352.5 ± 20.6 kg, were distributed in a completely randomized design with two treatments: control (no additive) and basal diet with arazyme (0.67 g/kg of dry matter consumed) for 63 days. The diet was formulated with a forage/concentrate ratio of 40:60. Although the sample size was small, the inclusion of arazyme proved effective under these conditions, with animals receiving the enzyme showing greater total weight gain, higher average daily gain, and better feed conversion, without affecting dry matter intake. No significant changes were observed in hematological parameters or in most serum biochemical biomarkers, indicating an absence of adverse effects on the metabolic or hepatic health of the animals. Higher total concentrations of volatile fatty acids in the rumen, particularly acetic, propionic, and butyric acids, as well as a higher ruminal pH on day 63, may be related to the enzyme’s action. Additionally, the apparent digestibility of crude protein and neutral detergent fiber was higher. Therefore, the inclusion of arazyme in the diet of feedlot cattle may be a promising alternative for improving digestive efficiency and intensifying ruminal fermentation, potentially resulting in improved productive performance. Future research with a larger number of replicates and different doses of the additive is necessary to understand its action.

1. Introduction

In response to increasing demand for animal-derived protein, including beef, production systems have undergone significant intensification. Improvements in management and adjustments, particularly in the nutritional component, can make a substantial difference in increasing herd productivity while ensuring animal health, performance and optimization of resource use [1]. Feedlot production is an expanding system in beef cattle production, relying heavily on high-value feed ingredients such as cereal grains, which increases production costs. In this context, the use of feed additives can improve nutrient utilization and positively impact performance, thereby enhancing system efficiency [2].

Among feed additives, the use of exogenous enzymes has emerged as an option to improve digestive efficiency and nutrient utilization, since these enzymes act synergistically with the natural enzymatic activity of the gastrointestinal tract [3]. From an economic perspective, substances capable of improving the utilization of dietary protein are particularly attractive, as protein is one of the most expensive components in animal diets and its inefficient utilization may also lead to environmental issues associated with nitrogen excretion.

Several factors can influence enzyme activity in the ruminal environment. According to Yang et al. [4], changes in ruminal pH may alter the structural conformation of enzymes and reduce their catalytic capacity, especially in more acidic environments resulting from high-concentrate diets. Furthermore, Carrillo-Díaz et al. [5] highlighted that enzyme efficiency depends on their stability under different ruminal conditions and on their ability to interact with the substrates available in the diet. The composition of the microbiota may also interfere with enzymatic activity, since some enzymes can act synergistically with ruminal microorganisms, while others may undergo proteolytic degradation within the rumen [6]. Therefore, enzymes with greater resistance to variations in pH, temperature, and ruminal degradation present greater potential for application in ruminant nutrition. Among these enzymes, arazyme has emerged as a promising protease.

Arazyme is a metalloprotease secreted by Serratia proteamaculans or Aranicola proteolyticus, a Gram-negative bacterium isolated from the digestive tract of spiders [7]. This enzyme is characterized by broad substrate specificity, high catalytic efficiency and remarkable stability across a wide range of pH and temperatures [8]. These properties distinguish arazyme from conventional proteases and support its potential application under the variable physicochemical conditions of the gastrointestinal tract. Experimental studies have demonstrated that arazyme can hydrolyze several protein substrates, including casein, collagen, elastin and hemoglobin, indicating its suitability for improving dietary protein utilization [8,9]. More recently, enzymatic mixtures containing arazyme have been associated with improvements in growth performance, nutrient digestibility, modulation of the intestinal microbiota and reduction of nitrogen residues and harmful gas emissions in monogastric animals [10,11].

In swine and poultry production, the use of enzymes and enzymatic cocktails is already well established, as reported by Sureshkumar et al. [12] in their review. Among the enzymes used, exogenous proteases have been shown to improve animal development, which has been attributed to enhanced protein hydrolysis at the intestinal level, improving nutrient utilization and reducing the excretion of nitrogenous compounds. Several exogenous enzymes with different pH ranges and substrate specificities have demonstrated efficiency in improving the utilization of soybean meal protein in broilers, one of the most widely used protein feedstuffs in animal nutrition, as reported by Zulkifli et al. [13]. These findings highlight the importance of exogenous enzyme use in animal feeding. Considering the benefits associated with exogenous enzymes, arazyme may be a promising alternative due to its ability to adapt to substrates and maintain stability across different pH ranges.

However, experimental studies evaluating the effects of arazyme intake in ruminants are still lacking. Our hypothesis was that intake of arazyme would improve nutrient digestibility, increase ruminal fermentation, and improve growth performance in feedlot cattle without compromising metabolic health. Therefore, the objective of this study was to evaluate whether the inclusion of arazyme in the diet of feedlot finishing cattle could improve the digestibility of dietary nutrients, promote an increase in short-chain fatty acids in the rumen, improve animal weight gain, without impacting dry matter intake, ruminal health, and the metabolic health of the animals.

2. Materials and Methods

2.1. Arazyme

The additive used in this study was obtained from Won Biotech Wcsat Comercial LTDA (Kbovinos^®, Blumenau, SC, Brazil). The product is composed of a metalloprotease produced by the bacterium Aranicola proteolyticus, originally isolated from the intestine of the spider Nephila clavata, and diluted in dehydrated glucose.

The enzymatic activity of arazyme was evaluated using the azocasein method. The azocasein method is specific for proteases, such as arazyme, which are enzymes that degrade proteins. In this method, azocasein, a casein-derived protein, is used as a substrate. The enzyme hydrolyzes azocasein, releasing protein fragments and azo compounds, which have an intense yellow color. The absorbance of the solution after incubation of the enzymatic solution with azocasein is measured at 436 nm. The intensity of the color is directly related to the enzymatic activity, allowing the quantification of the enzyme’s effectiveness in degrading azocasein. This analysis is crucial to evaluate the enzyme’s performance in its specific catalytic function and to ensure that it is active and ready for the desired applications.

To perform the assay, 500 μL of the diluted sample was mixed with 500 μL of 0.5% (w/v) azocasein in a 0.2 M C₂H₃NaO₂ buffered solution at pH 5.0. The mixture was incubated at 37.5 °C (SSD-5L, SolidSteel, Piracicaba, SP, Brazil) and, after 40 min, the reaction was stopped with 500 μL of 10% (w/v) TCA. Centrifugation was performed (L-1202, Loccus, Cotia, Brazil) at 10,000× g for 15 min. 1 mL of the supernatant was mixed with 1 mL of 5 M KOH to form an orange color. The absorbance of the solution was measured at 428 nm. The reaction blank used was 1000 μL of 0.5% (w/v) azocasein solution. One protease activity unit (U) was defined as the amount of enzyme that causes a change in absorbance of 0.001 per minute under the assay conditions [14,15]. As a result, we verified that the enzymatic activity obtained was 1,209,346 U/g.

2.2. Animals and Facilities

The study was carried out at the feedlot facilities of the ruminant sector of the experimental farm of the Santa Catarina State University (UDESC–FECEO), located in the municipality of Guatambu, Santa Catarina, Brazil. Animals were housed in individual pens (15 m²) with concrete floors (3.00 × 5.00 m), equipped with an automatic drinker and an individual feed bunk that allowed the measurement of feed intake. A total of 12 Holstein bulls were used, with an average initial body weight of 353.1 kg (±29 kg) and approximately 13 months of age.

2.3. Experimental Design and Diets

Animals were assigned to a completely randomized design (CRD) and divided into two groups with six animals each: Control (animals that did not receive the additive) and Treatment (animals fed concentrate containing the additive). The arazyme additive was incorporated into the concentrate during its manufacturing, 1 kg of arazyme per ton, according to the manufacturer’s recommendation; the dose was defined based on a previous pilot study. The experiment lasted 63 days. Prior to the experimental period, animals were adapted to the diet for 20 days, with a gradual increase in concentrate levels. The diet was formulated according to the nutritional requirements described by Valadares Filho et al. [16] using a forage/concentrate ratio of 40:60. Feed was offered twice daily at 09:00 and 16:00.

2.4. Data and Sample Collection

Feed intake was recorded daily by weighing feed refusals in the morning before the first feeding. Total mixed ration (TMR) samples were collected weekly to determine dry matter content and calculate dry matter intake (DMI). Animals were weighed on days 1, 28, and 63 using a calibrated digital scale (WT3000-I, RINNERT®, Chapecó, SC, Brazil)).

On the same days, blood samples (15 mL per animal) were collected from the coccygeal vein using needles and vacuum tubes. One tube contained EDTA anticoagulant for complete blood count analysis, and another tube without anticoagulant was used for serum separation and metabolic analyses.

Ruminal fluid samples were collected on days 28 and 63 using an ororuminal silicone tube (1.5 m length and 11 mm diameter; PROVAR^®, São Carlos, SP, Brazil) connected to a suction pump, as described by Signor et al. [17]. The collected material was used for pH measurement and volatile fatty acid analysis.

Fecal samples were collected for three consecutive days [18,19,20], i.e., days 60, 61 and 62 at three time points per day (07:00, 11:00, and 16:00), totaling nine samples per animal. Samples were frozen (−20 °C) immediately after collection. After the collection period, the nine samples from each animal were homogenized and a 400 g subsample was used for chemical analysis. During the same period, TMR samples were also collected for chemical composition analysis to calculate apparent digestibility coefficients.

2.5. Animal Performance

Body weight data were used to calculate total weight gain (final body weight − initial body weight) and average daily gain (ADG), calculated as weight gain divided by the number of experimental days. Feed efficiency was calculated using the ratio between ADG and dry matter intake (ADG/DMI).

2.6. Laboratory Analyses

2.6.1. Chemical Composition of Diet and Feces

Feed and fecal samples were pre-dried in a forced-air oven at 55 °C for 72 h. Samples were then weighed to determine partial dry matter and ground in a Wiley-type mill (model MA340, Marconi, Piracicaba, SP, Brazil) using a 1 mm screen. The samples were subsequently dried at 105 °C to determine final dry matter and then incinerated in a muffle furnace at 600 °C to determine ash content [21]. Chemical composition analyses followed AOAC [22] procedures: dry matter (method 930.15), nitrogen by micro-Kjeldahl (method 984.13), crude protein calculated as N × 6.25 (method 976.05), ether extract (method 920.39), and ash (method 942.05). Neutral detergent fiber (NDF) was determined using the filter bag technique according to Komarek and Robertson [23]. Samples were treated with neutral detergent solution in an autoclave at 110 °C for 40 min [24] with the addition of alpha-amylase [25]. Results in

Table 1. Chemical composition of the total mixed ration (TMR) provided to cattle.

Variables	TMR 1
Dry matter (DM), g/kg	411
Mineral matter, g/kg DM	64.4
Crude protein, g/kg DM	167
Neutral detergent fiber, g/kg DM	349
Acid detergent fiber, g/kg DM	186
Ether extract, g/kg DM	25.0

¹ TMR (DM) was 60:40 concentrate/corn silage, respectively. Concentrate (in dry matter) was formulated based on 350 g/kg ground corn, 280 g/kg soybean meal, 200 g/kg soybean hulls, 120 g/kg wheat bran and 50 g/kg mineral and vitamin supplement (Bovigold^®, Tortuga®, DSM-firmenich, São Paulo, SP, Brazil).

.

2.6.2. Hematological Analysis

Hemoglobin concentration, erythrocytes, total leukocytes, hematocrit, and leukocyte differential counts were determined immediately after sample arrival at the laboratory using an automatic hematology analyzer (EQUIP VET 3000®, Equip Vet Diagnósticos, Curitiba, PR, Brazil).

2.6.3. Serum Biochemical Analysis

Blood samples collected without an anticoagulant were centrifuged (7000 rpm for 10 min) to obtain serum. The supernatant was transferred to 1.5 mL microtubes and stored at −20 °C until analysis. Serum concentrations of albumin, cholesterol, gamma-glutamyl transferase (GGT), total protein, globulin, aspartate aminotransferase (AST), triglycerides, and urea were measured using a biochemical analyzer (Zybio EXC 200^®, Zybio Inc., Chongqing, China) with commercial kits (Analisa^®, Belo Horizonte, Brazil).

2.6.4. Ruminal pH and Volatile Fatty Acids

To determine the fatty acid profile of the rumen fluid, we followed the methodology described by Simon et al. [3], in which the samples were thawed at 5 °C and manually homogenized. From each sample, a 1 mL aliquot of the supernatant was collected and centrifuged for 5 min at 12,300× g in 2 mL polypropylene microtubes. After centrifugation, 250 μL of the supernatant was transferred to another microtube containing 250 μL of formic acid. The mixture was manually shaken and centrifuged again for 3 min. From this second supernatant, 250 μL were collected and mixed with 500 μL of a 3-octanol solution (665 μg mL⁻¹ in methanol), used as the internal standard. The samples were analyzed using a gas chromatograph equipped with a flame ionization detector (GC-FID; Varian Star 3600, Palo Alto, CA, USA) and an automatic sampler (Varian 8200CX, Palo Alto, CA, USA). One microliter of the extract was injected in split mode (1:10). Hydrogen was used as the carrier gas at a constant pressure of 20 psi. Acetic, propionic, and butyric acids were separated using a CP-WAX 52CB (Agilent Technologies, Santa Clara, CA, USA) capillary column (60 m × 0.25 mm; stationary phase thickness of 0.25 μm). The column temperature was initially set at 80 °C for 1 min, then increased to 120 °C at a rate of 8 °C min⁻¹, followed by a ramp to 230 °C at 15 °C min⁻¹, where it was held for 1 min. Injector and detector temperatures were set to 250 °C.

Method validation included parameters such as selectivity, linearity, linear range, repeatability, precision, limit of detection (LOD), and limit of quantification (LOQ) for acetic, propionic, and butyric acids. Linearity was evaluated through regression equation analysis using the least squares method. LOD and LOQ values were obtained by serial dilutions until signal-to-noise ratios of 3:1 and 6:1, respectively, were reached (Table S1). Precision was assessed through repeatability analysis using eight replicate samples and was also determined by the recovery of known quantities of standard substances spiked into a diluted sample. The results were expressed in mmol L⁻¹ of each volatile fatty acid in the ruminal fluid.

2.6.5. Apparent Digestibility Coefficient

Feed and fecal samples were weighed in duplicate into polyester bags with 12 µm porosity and incubated in the rumen of a cannulated steer for 288 h [26]. Animals received the same basal diet (Table 1) for 14 days prior to incubation, for adaptation purposes. After incubation, samples were washed, treated with neutral detergent solution in an autoclave [24] and dried in an oven at 105 °C for 24 h to obtain the iNDF. Digestibility was calculated as: 1–(iNDF in feed (% DM)/iNDF in feces (% DM)).

2.7. Statistical Analysis

All data were analyzed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA; version 9.4), with the Satterthwaite approximation used to determine denominator degrees of freedom for fixed effects. Growth performance variables (except body weight) were tested for the fixed effect of treatment using animal (treatment) as a random effect. Body weight, hematology, serum biochemistry and ruminal biomarkers were analyzed as repeated measures and tested for fixed effects of treatment, day and treatment × day interaction using animal (treatment) as a random effect. Results from day 1 were included as a covariate. To generate treatment means, day 1 values were removed from the dataset but maintained as covariates. A first-order autoregressive covariance structure was selected based on the lowest Akaike information criterion. Means were compared using the PDIFF method and results were reported as LSMEANS followed by SEM. Statistical significance was declared at p ≤ 0.05.

3. Results

The results of zootechnical performance are presented in

Table 2. Body weight, weight gain, daily weight gain, feed intake, and feed efficiency of cattle fed exogenous enzymes (arazyme).

Variables	Treatment	Control	SEM	p-Value
Body Weight, kg				0.26
d1	352	353	3.37
d28	412	398	4.76
d63	460	448	4.52
Weight gain, kg
d1-63	107 a	94.5 b	2.98	0.05
Average daily gain, kg
d1-63	1.69 a	1.50 b	0.06	0.05
Feed intake DM, kg
d1-63	9.44	9.38	0.11	0.92
Feed efficiency, kg/kg
d1-63	0.181 a	0.158 b	0.006	0.03

NOTE 1: Statistical difference between groups (p < 0.05) was illustrated by different lowercase letters (^a,b) on the same line. The animals underwent a prior adaptation period of 20 days, during which they all received the same diet (silage and concentrate), without the additive. NOTE 2: Treatment group (n = 6), animals that received 1 kg of arazyme per ton of concentrate.

. There was a treatment effect on total weight gain, average daily gain, and feed efficiency, which were higher in the animals from the treatment group. Body weight and dry matter intake did not differ statistically between treatments. Based on the daily feed intake, the daily consumption of arazyme was estimated at 6.36 g per animal. Furthermore, we found that 0.67 g of arazyme per kg of dry matter was consumed.

Although the number of animals per treatment was limited (n = 6), the observed treatment effect on average daily gain was biologically relevant (12.5 kg) and statistically detectable (p = 0.05). Post hoc power estimation based on the observed variance indicated moderate statistical power (72%), which reflects the inherent logistical limitations of cattle feeding trials while still allowing detection of large treatment effects.

The hematological and biochemical results are presented in

Table 3. Hematological and serum biochemical biomarkers in cattle fed the exogenous enzyme arazyme.

Variables	Treatment	Control	SEM	P-Treat 1	P-Day 2	P-Treat × Day 2
Hemogram
Total leukocyte (×103/µL)	7.40	8.71	0.23	0.74	0.63	0.61
Lymphocyte (×103/µL)	4.45	5.76	0.19	0.15	0.42	0.35
Granulocytes (×103/µL)	2.62	2.49	0.13	0.69	0.75	0.81
Monocyte (×103/µL)	0.32	0.45	0.16	0.92	0.87	0.86
Erythrocytes (×106/µL)	7.04	6.30	0.09	0.47	0.92	0.32
Hemoglobin (mg/dL)	10.7	10.2	0.12	0.94	0.95	0.90
Hematocrit (%)	30.7	28.2	0.34	0.52	0.78	0.63
Platelets (×103/µL)	351	364	8.21	0.89	0.94	0.91
Seric biochemistry
Albumin (g/dL)	2.64	2.68	0.12	0.96	0.95	0.93
Total protein (g/dL)	7.87	8.06	0.25	0.95	0.92	0.91
Globulin (g/dL)	5.02	5.37	0.23	0.89	0.84	0.83
GGT (U/L)	20.4	18.1	1.54	0.66	0.80	0.72
AST (U/L)	72.3	72.2	3.06	0.91	0.88	0.90
Cholesterol (mg/dL)	72.7 a	63.9 b	1.76	0.02	0.27	0.07
Triglycerides (mg/dL)	10.2	11.9	0.95	0.42	0.55	0.31
Urea (mg/dL)	28.6	29.4	0.39	0.90	0.86	0.76

¹ When p ≤ 0.05, there was a treatment effect, illustrated by different letters (^a,b) on the same line. ² There was no treatment × day interaction and day effect for serum biomarkers of biochemistry and hematology (p > 0.05).

. Among the hematological variables analyzed, no statistical differences were observed between groups for total leukocyte count, lymphocytes, granulocytes, monocytes, erythrocytes, and platelets, as well as hemoglobin concentration and hematocrit percentage (p > 0.05). A treatment effect was observed for serum cholesterol levels, with the treatment group showing a higher concentration of this lipid compared with the control group. There was no treatment effect or treatment × day interaction for albumin, total protein, globulins, triglycerides, and urea concentrations, nor for the activities of GGT and AST (p > 0.05).

Table 4. Fatty acid profile in ruminal fluid of cattle fed arazyme.

Variables	Treatment	Control	SEM	P-Treat 1	P-Day 3	P-Treat × Day 2
pH				0.37	0.25	0.05
d28	6.61	6.55	0.03
d63	6.85 A	6.42 B	0.03
Total SCFA (mmol L−1)				0.03	0.01	0.01
d28	95.7	87.0	3.60
d63	119 A,*	96.6 B	3.74
Acetic acid (mmol L−1)				0.04	0.03	0.05
d28	64.9 A	56.3 B	2.36
d63	73.0 A,*	66.5 B	2.57
Propionic acid (mmol L−1)
d28	19.1	18.2	1.18	0.28	0.01	0.01
d63	28.3 A,*	18.1 B	1.23
Butyric acid (mmol L−1)	11.5 a	9.22 b	0.53	0.05	0.21	0.10
Isovaleric acid (mmol L−1)	1.56	1.59	0.09	0.98	0.97	0.96
Valeric acid (mmol L−1)	1.24	1.42	0.08	0.55	0.49	0.32

¹ When p ≤ 0.05, there was a treatment effect, illustrated by different letters (^a,b) on the same line. ² There was a treatment × day interaction when p ≤ 0.05, with the difference between groups illustrated by uppercase letters (^A,B) on the same line. ³ The day effect was illustrated by an asterisk (*) and was only observed in the treatment group for SCFA, acetate, and propionate, increasing over time.

presents the results for ruminal pH and volatile fatty acids (VFAs) in ruminal fluid. There was a treatment × day interaction for ruminal pH, with higher values in the treatment group on day 63. A treatment effect was observed for total SCFA concentration, which was higher in the treatment group, as well as a treatment × day interaction, with greater SCFA concentrations on day 63 in animals receiving the additive. A higher concentration of acetic acid in the rumen of cattle from the treatment group was observed on days 28 and 63 (p < 0.05), reflecting an overall treatment effect compared with the control group. Only a treatment × day interaction was observed for propionic acid levels, which were higher in animals receiving the additive on day 63. The concentration of butyric acid showed only a treatment effect, with higher levels in the ruminal fluid of cattle supplemented with arazyme. No differences between groups were observed for valeric and isovaleric acid concentrations.

The apparent digestibility coefficient (ADC) of nutrients is presented in

Table 5. Apparent digestibility coefficient (ADC) of dietary nutrients in feedlot cattle with the addition of arazyme to the diet.

ADC	Treatment	Control	SEM	p-Value 1
Dry matter	0.752	0.706	0.02	0.19
Organic matter	0.795	0.780	0.03	0.89
Crude protein	0.765 b	0.679 a	0.02	0.01
Neutral detergent fiber	0.494 b	0.407 a	0.02	0.05
Acid detergent fiber	0.412	0.428	0.02	0.92
Ether extract	0.743	0.763	0.03	0.85

¹ When p ≤ 0.05, there was a treatment effect was illustrated by different letters on the same line.

. There was a treatment effect on the digestibility of crude protein (CP) and neutral detergent fiber (NDF), with a higher digestibility coefficient in the treatment group. Regarding the digestibility coefficient of dry matter (DM), organic matter (OM), acid detergent fiber (ADF), and ether extract (EE), there was no statistically significant difference between the groups (p > 0.05).

4. Discussion

Arazyme is a metalloprotease produced by the bacterium Aranicola proteolyticus, isolated from the digestive tract of the spider Nephila clavata. It is characterized by broad substrate specificity, high catalytic efficiency, and remarkable stability over a wide pH and temperature range [8,9]. These properties distinguish arazyme from conventional proteases and support its potential application in complex biological systems, such as the gastrointestinal tract of ruminants, where physicochemical conditions are highly variable. In general, the addition of this exogenous enzyme to the diet of cattle improves digestibility and enhances weight gain, without apparent or laboratory harm to the cattle, despite the short experimental period of 63 days.

The addition of the exogenous enzyme arazyme significantly improved the zootechnical performance of feedlot cattle, as evidenced by the greater total weight gain, higher average daily gain, and improved feed efficiency observed in animals receiving the enzyme in the diet, without changes in dry matter intake. These combined results indicate a positive effect of the additive associated with a more efficient utilization of ingested nutrients such as protein and fiber, without increasing feed consumption, which is desirable in intensive production systems. Ferreira et al. [27] demonstrated in their review that exogenous enzymes can increase feed efficiency and performance in beef cattle, particularly when used in diets with high concentrate levels or fibrous ingredients of lower digestibility. Ingestion of arazyme significantly increased weight gain, but did not significantly increase body weight; this is justified by the animals having great variability in initial weight (d1), with a high standard error. The weight data on days 28 and 63 overlap, as weight gain occurs in both groups, but more promisingly in the treatment group.

The weight gain of cattle receiving arazyme during the experimental period was 13.2% higher than that of animals in the control group. Considering the improvement in feed efficiency, it can be suggested that the addition of the exogenous enzyme enhanced digestive and fermentative processes, resulting in greater conversion of feed and its nutrients into body tissue. This effect has been reported in recent studies highlighting the synergistic action between enzymes and ruminal microbiota, which promotes greater availability of fermentable substrates [27,28]. The hypothesis that the enzyme arazyme modulates the ruminal microbiota is a knowledge gap that needs to be investigated in future studies in order to understand the mechanisms involved.

Regarding the ruminal fermentation profile, the higher concentration of total volatile fatty acids observed in the treatment group, particularly acetic, propionic, and butyric acids, indicates enhanced ruminal fermentative activity. The increase in propionic acid is particularly relevant, and one possible explanation is that this response may have contributed to improved energy metabolism, since propionate is the main precursor for hepatic gluconeogenesis in ruminants and could potentially increase energy availability for growth and muscle tissue deposition [29]. Propionate concentrations were observed in ruminal fluid only on day 63, which may indicate a cumulative effect of the enzyme over time on ruminal microorganisms and their products. That increased propionate fermentation improves the utilization of available carbon, as it reduces the availability of substrate for methane production, improving energy efficiency and reducing the production of unwanted gases [30]. A recent study demonstrated that exogenous enzymes can modulate ruminal fermentation toward greater production of SCFA, including propionate, thereby improving performance in feedlot systems [3]. These results with the arazyme additive are particularly interesting and should be further explored in future studies to better understand how ruminal microbiota responds to the presence of this enzyme.

Regarding hematological and serum biochemical parameters, the absence of significant differences between groups for most variables suggests that arazyme intake did not adversely affect metabolic, hepatic, or immunological status in feedlot cattle; however, this needs to be investigated further, due to the short experimental period in a preliminary study. Nevertheless, animals receiving arazyme exhibited higher serum cholesterol concentrations compared with the control group. This response may be associated with the greater ruminal fermentative activity observed in the treatment group, characterized by increased total SCFA production, particularly acetate and propionate. In ruminants, acetate serves as an important precursor for de novo fatty acid and lipid synthesis, whereas propionate contributes to hepatic gluconeogenesis and overall energy metabolism, potentially influencing lipid transport and cholesterol metabolism [31,32]. Therefore, the increase in circulating cholesterol observed in the present study may reflect an adaptive metabolic response linked to enhanced nutrient utilization and hepatic lipid metabolism rather than a pathological alteration, especially considering that other serum biomarkers related to liver function and protein metabolism remained unchanged. Similar interpretations have been proposed in studies evaluating nutritional strategies that stimulate ruminal fermentation and improve metabolic efficiency in cattle [27,33].

The apparent digestibility results demonstrated a positive treatment effect on the digestibility of crude protein (CP) and neutral detergent fiber (NDF), indicating that the enzyme acted efficiently in increasing the degradation of these dietary components. Hristov et al. [34] and Monteiro et al. [35] report that more efficient hydrolysis of the protein fraction can stimulate positive interactions between the ruminal microbiota and the host, reflected in the greater SCFA production and productive performance. Improvements in NDF digestibility are particularly relevant in intensive feeding systems, where limitations in fiber degradation can restrict feed intake, ruminal function, and animal performance [31,36].

Likewise, increased CP digestibility may contribute to more efficient utilization of dietary nitrogen, reducing nitrogen excretion into the environment and improving overall protein efficiency in ruminants [34]. Conversely, the absence of effects on dry matter, organic matter, acid detergent fiber, and ether extract digestibility suggests that the enzyme exerted a specific mode of action depending on the nutrient, rather than promoting a generalized increase in overall diet digestibility. Nevertheless, even without changes in total dry matter digestibility, improvements in CP and NDF digestibility are nutritionally advantageous and may translate into improved productive responses, as observed in the present study through the higher average daily gain of cattle. However, we would like to make it clear to readers that the sample size is a limitation of the research, as is the number of days of stool collection for digestibility assessment; however, I emphasize that the standard error between experimental units was small, and the variability of analyses was small, which reinforces that our data reflect the effect of this protease.

The higher ruminal pH observed in cattle receiving arazyme on day 63 was an unexpected but biologically relevant finding, particularly because it occurred concomitantly with greater SCFA production. Although increased ruminal fermentation is often associated with lower pH due to acid accumulation, ruminal pH is determined not only by acid production but also by the balance among SCFA absorption, liquid turnover, and buffering capacity derived mainly from saliva. In the present study, the greater digestibility of neutral detergent fiber observed in the treatment group may have favored rumination activity and salivary secretion, thereby increasing bicarbonate buffering and contributing to maintenance of ruminal homeostasis. Furthermore, the increase in acetate, propionate, and total SCFA concentrations without reductions in dry matter intake suggests that arazyme enhanced fermentative efficiency rather than inducing excessive acidogenic fermentation. The delayed increase in pH, observed only on day 63, may also indicate progressive adaptation of the ruminal microbiota to enzyme intake, resulting in a more stable fermentative environment and improved regulation of acid production and utilization. Similar responses have been discussed in studies evaluating exogenous enzymes and fiber digestion in ruminants, where improved fermentative efficiency and buffering dynamics contributed to maintaining favorable ruminal conditions despite increased fermentation end-products [5,37].

These findings reinforce the potential use of exogenous enzymes as a strategic nutritional tool in intensive production systems. However, further studies are necessary to confirm whether these results are reproducible, to determine whether the applied dose is optimal for maximizing cattle performance, and to better understand the influence of this additive on ruminal microbiota.

5. Conclusions

The preliminary results of this study are promising and suggest that the inclusion of 0.67 g arazyme per kg of DM in the diet of feedlot cattle improves the efficiency of nutrient utilization, enhances ruminal fermentation in a beneficial manner, and positively reflects on productive performance.