Use of a Blend of Exogenous Enzymes in the Diet of Lactating Jersey Cows: Ruminal Fermentation In Vivo and In Vitro, and Its Effects on Productive Performance, Milk Quality, and Animal Health

Abstract

The use of exogenous enzymes in the nutrition of dairy cows is an innovative and efficient strategy to maximize productivity and milk quality, with positive applications in the economic and environmental aspects of dairy farming. Therefore, the objective of this study was to evaluate whether the addition of a blend of exogenous enzymes to the diet of lactating Jersey cows has a positive effect on productive performance, milk quality, animal health, ruminal environment, and digestibility. Twenty-one primiparous Jersey cows, with 210 days in lactation (DL), were used. The exogenous enzymes used were blends containing mainly protease, in addition to cellulase, xylanase, and beta-glucanase. The animals were divided into three groups with seven replicates per group (each animal being the experimental unit), as follows: Control (T-0), basal diet without enzyme addition; Treatment (T-80), animals fed enzymes in the diet at a daily dose of 80 mg per kg of dry matter (DM); Treatment (T-160), animals fed enzymes in the diet at a daily dose of 160 mg per kg of DM. The study lasted 84 days, during which higher milk production was observed in the treated groups (T-80 and T-160) compared to the control group (p = 0.04). When calculating feed efficiency from days 1 to 84, greater efficiency was observed in both groups that received the blend compared to the control (p = 0.05). In the centesimal composition of the milk, it was observed that the percentage of protein in the milk of the T-160 group was higher compared to the control group (p = 0.03). The effect of the enzymes was verified for butyric (p = 0.05) and palmitic (p = 0.05) fatty acids. We also observed the effect of the enzyme blend on the amount of volatile fatty acids (VFAs), which were higher in the ruminal fluid of cows that received the enzymes (p = 0.01). Cows that consumed enzymes showed a higher apparent digestibility coefficient of crude protein (p = 0.01). In vitro, the main result is related to lower gas production in 24 and 48 h at T-160. We concluded that the use of a blend of exogenous enzymes in the diet of lactating Jersey cows was able to increase milk production in these animals, resulting in greater feed efficiency and also an increase in milk protein content, positively modulating the fatty acid profile in the rumen and improving the apparent digestibility of nutrients.

1. Introduction

In Brazil, approximately 160 million hectares are used as pastures for raising ruminants. Pastures are one of the main sources of feed in milk production systems [1]. However, one of the biggest challenges this system faces is how to efficiently transform pasture into milk, with greater milk production in a smaller amount of cultivated area [2]. Consequently, nitrogen excretion into the environment is reduced, with better use of nutrients by the animal.

The demand for milk production has increased considerably due to population growth, which is why there is a need to intensify production systems. However, there is a considerable challenge associated with this intensification, which is producing milk in a profitable and sustainable way to meet demand [3]. As a result, the aim is to increase productivity in order to have highly productive animals; nutritional strategies can enhance milk efficiency and meet the demands of these animals [4].

The use of exogenous enzymes has been an option in the search for increased animal performance through increased nutrient digestibility [5,6]. Among the main enzymes are proteases, which help degrade the protein fractions of feed in the rumen, leaving amino acids free for use by ruminal microorganisms. Thus, this protease enzyme, alone or in combination with other enzymes, promotes improvements in cattle performance, resulting in better nutrient utilization [7].

The results of studies with enzymes are still divergent. However, it is known that when researchers used exogenous proteolytic enzymes in the diet of cows receiving high-concentrate diets, they observed improved nutrient digestibility in the digestive tract and positive changes in milk composition [8]. Furthermore, when a mixture of enzymatic additives was also used in cattle, an increase in short-chain fatty acids in the rumen was observed [7,9]. More recently, Bugoni et al. [10] reported improved performance and higher nutrient digestibility in cows fed exogenous enzymes. However, in this study, we aim to understand the effect of using a combination of exogenous enzymes, composed mainly of protease, in addition to cellulase, xylanase, and beta-glucanase. Therefore, the objective of this study was to evaluate whether the addition of a blend of exogenous enzymes to the diet of lactating Jersey cows exerts a positive effect on productive performance, milk quality, animal health, ruminal environment, and nutrient digestibility, by analyzing biomarkers that help to understand the mechanisms involved. Furthermore, in vitro, we aim to understand what occurs in ruminal fermentation in the presence of exogenous enzymes.

2. Materials and Methods

2.1. Blend of Exogenous Enzymes

The blend of exogenous enzymes (protease (min.) 7500 U/g, cellulase (min.) 2700 U/g, xylanase (min.) 1200 U/g, beta glucanase (min.) 300 U/g, and alpha-amylase (min.) 1000 U/g) was used in this study (Tecmax Pro-Ruminantes^®; Toledo, PR, Brazil), with composition described by Simon et al. [7]. The protease is a mixture of 50% acid (pH 2.5 to 3.5) and 50% alkaline (pH 6.7 to 7.5), derived from the fermentation of Bacillus and Aspergillus. The other enzymes present in the additive had a pH of approximately 5.5. One unit of enzyme activity is equivalent to the amount of enzyme that liberates 1 micromole of reducing sugar (excluding protease) per minute from the specific substrate at the ideal pH and 37 °C. For the proteases, the hydrolysis potential of 1 microgram of casein at 40 °C, pH 3.0, and the amount of enzyme that liberates 1 microgram of tyrosine per minute at pH 7.2 at 40 °C were evaluated. Xylanase and amylase are resistant to the protease present in the blend.

2.2. Animals and Installation

The experiment was developed in the ruminant sector—dairy cattle farming of the Experimental Farm of the Higher Education Center of the West (FECEO) of the State University of Santa Catarina (UDESC), located in the city of Guatambu, Santa Catarina State, Brazil. Twenty-one primiparous Jersey cows, pregnant, and with 210 days in lactation (DL) were used. The herd was housed in a confinement system with sawdust bedding, compost barn type, with access to pasture for a specific period per day. The study took place during the Brazilian winter, when temperatures ranged from 4 to 18 °C during the cows’ grazing hours. The animals were milked by a robotic milking system of the Delaval^® brand, model VMS™ V300 (DeLaval, Tumba, Sweden), whose process is fully automatic.

2.3. Experimental Design and Diets

The study lasted 84 days (14 days of adaptation + 60 days of data collection), in which the animals were randomly divided into three groups of 7 cows each: Treatment 0 (T-0): the control group, which was fed the basal concentrate without the addition of additives; Treatment 80 (T-80), in which the concentrate had the addition of 80 mg of the blend of enzymes per kg of dry matter; and Treatment 160 (T-160), in which the concentrate had the addition of 160 mg of the blend of enzymes per kg of dry matter.

The diets were formulated according to the nutritional requirements of the animals NASEM [11] considering the following feeds: concentrate and corn silage, which were mixed and provided in individual feeders (animals contained by head restraint), divided into 2 daily feds (07:00 and 16:00 h). For a period of 4 h (10:00 to 14:00), the cows had access to a pasture paddock, in a consortium of grasses (oats and ryegrass). Water was provided ad libitum in the barn, and also during feeding. The grazing paddocks had a water trough.

We used methodologies described by Silva and Queiroz [12] to analyze the chemical composition of the feed. A forced ventilation oven at 55 °C for 72 h was used to measure partial dry matter, followed by grinding in a Wiley mill (Marconi, model: MA340, Piracicaba, Brazil), using a 1 mm mesh sieve. Then, the samples were placed for 24 h in an oven at 105 °C. Ash results were obtained using a muffle furnace at 600 °C [12]. Crude protein percentage was measured using Method 984.13 [13]. The proportion of neutral detergent fiber (NDF) followed the methodology of Komarek [14] and Senger et al. [15]. The percentage of acid detergent fiber (ADF) was verified by Method 973.18 [13]. Ether extract was measured using an automatic fat extractor (VELP^® Scientifica, Usmate, Italy). The results are presented in

Table 1. Chemical composition of feed and total diet (PMR) provided to cows during the experimental period.

Variables, %	Silage in Individual Feeders	Basal Concentrate in Individual Feeders	Pelletized Concentrate—Robot	Oat and Ryegrass Pasture	Tifton Hay	PMR
Dry matter	25.9	91	88.2	14.3	86.9	41.5
Crude protein	9.8	16.6	20.4	17.3	11.7	14.6
Eter extract	2.9	2.2	4.4	2.6	1.56	3.54
Ash	3.8	7.1	6.8	9.5	6.88	7.09
NDF	40.4	-	20	56.4	75.1	30.7
ADF	19.3	-	9.1	22.3	35.3	12.1

The guaranteed levels of the mineral and vitamin core are as follows: Ca min. 231.40, max. 250 g; P min 40 g; S min. 20 g; Mg min. 25 g; K min. 10 g; Na min. 70 g; Co min. 15 mg; Cu. min. 15 mg; Cr min. 20 mg; I min. 40 mg; Mg min. 2000 mg; Se min. 22 mg; Zn min 2850 mg; Vit A 350,000 IU; Vit D3 min. 100,000 IU; Vit E 2000 IU.

.

2.4. Productive Performance

To determine production performance, the automatic milking system measures the daily milk production of each cow; the data is stored on a computer connected to the robot. The amount of feed provided is also measured, and the remaining amount is weighed after feeding to determine daily consumption. The robotic milking system provides information on the daily concentrate intake of each cow during milking. Based on this data, milk efficiency is calculated: milk production/feed intake.

2.5. Samples

Blood was collected on days 1, 21, 42, 63, and 84. Samples were collected in tubes with a clot activator (4 mL) to separate blood serum for biochemical analysis and in tubes with 10% EDTA anticoagulant (4 mL) for complete blood count analysis. The blood was kept refrigerated during collection and transportation from the farm to the laboratory. Blood collected in tubes without anticoagulants was centrifuged (QUIMIS^®, Diadema, SP, Brazil) for 10 min at 8000 rpm, and the serum was separated and stored in a freezer (−20 °C) until analysis.

Milk collection for analysis of centesimal composition and somatic cell count (SCC) was performed on days 1, 21, 42, 63, and 84 of the study, using a milk collector specific to the robotic milking machine. Milk collection for fatty acid profiling was performed on day 84 of the experiment. A collector attached to the robot, originating from the factory, collected 50 mL of milk, representing the entire milking.

Ruminal fluid collection was performed on three different days (days 42, 43, and 44), during the digestibility period. Collection was performed 3 h after the start of feeding. A silicone esophageal tube connected to a vacuum system allowed the collection of rumen fluid. Samples were refrigerated and stored frozen (−20 °C).

Feces were collected directly from the rectal ampulla on days 42, 43, and 44 of the experiment (three samples per day—a total of nine samples per animal over three days) and stretched and frozen (−20 °C). This material was used to determine nutrient digestibility coefficients.

During these three days of collection for analysis of digestibility and ruminal liquid, the cows received all the feed in individual feeders (hay, silage, concentrate, and chopped pasture) at three times of the day (07:00; 15:00, and 22:00 h). This methodology was used to allow us to know the exact feed intake of each cow.

2.6. Hemogram

Using collected EDTA blood tubes, the complete blood count was determined on the VET3000 automated hematology analyzer (EQUIP^® São Paulo, SP, Brazil). The equipment allowed counting of the number of total leukocytes, lymphocytes, granulocytes, monocytes, and erythrocytes, as well as determination of hemoglobin levels and hematocrit percentage.

2.7. Serum Biochemistry

Serum levels of total protein, albumin, urea, uric acid, cholesterol, and glucose were measured using Kits (ANALISA^® São Paulo, SP, Brazil) and automatic equipment (Zybio EXC-200^® Chongqing, China). Globulin levels were calculated (Total Protein − Albumin).

2.8. Biomarkers in Rumen Fluid

Ruminal fluid was slowly thawed (5 °C) and then shaken to homogenize, followed by centrifugation for 5 min (12,300× g). A volume of 250 μL of the sample supernatant was used for extraction of short-chain fatty acids according to the methodology described by Simon et al. [7]. A 1 μL aliquot of the sample was injected into a gas chromatograph equipped with a flame ionization detector (GC-FID; Varian Star 3400, Palo Alto, CA, USA) and an autosampler (Varian 8200CX, Palo Alto, CA, USA) in split mode (1:10) according to the literature [7]. The determination of the short-chain fatty acid profile was dependent on the standardization of methodologies and equipment, as detailed in Table S1. The final data were expressed in mmol per L of each VFA in the ruminal fluid.

2.9. Milk

2.9.1. Somatic Cell Composition and Count (SCC)

The analysis of the chemical composition of milk and the somatic cell count (SCC) were measured in a commercial laboratory. We used the Mid-Infrared Spectrometry Method to determine the chemical composition, and the Flow Cytometry Method for SCC, according to the methodology described in detail by de Vitt et al. [16].

2.9.2. Fatty Acid Profile

Lipid extraction was performed using the method of Bligh and Dyer [17] with adaptations described in detail by de Vitt et al. [16]. Fatty acid methylation was performed using the transesterification method proposed by Hartman and Lago [18]. For FAME determination, a TRACE 1310 gas chromatograph equipped with a flame ionization detector (Thermo Scientific, Waltham, MA, USA) was used, following the methodology described in detail by de Vitt et al. [16]. The results are presented as a percentage, considering the factor equivalent to the size of the FAME chain for FID and the conversion factor of the ester to the respective acid [19].

2.10. Apparent Digestibility Coefficients

Fecal samples (average of 150 g/day) from the three days of collection were homogenized (average of 450 g), and then the chemical composition of the fecal samples was analyzed according to the methodology previously described for feed. Based on the methodology described by Cochran et al. [20], indigestible neutral detergent fiber (iNDF) was used to determine apparent digestibility. For this purpose, feed and feces samples were incubated in the bovine rumen for 288 h, and washed and dried in a forced ventilation oven. In this material, NDF and ADF concentrations—information used to calculate nutrient digestibility [21]—were determined.

2.11. In Vitro Fermentation and Ammonia Nitrogen

For incubation, ruminal fluid was collected from two rumen-fistulated Red Angus steers (675 ± 12.4 kg) who were consuming the same diet as the cows at 14 days. The collection was performed on the day of incubation, filtered through four layers of cheesecloth, and subsequently transported to the laboratory. Temperature and pH were measured, and the ruminal fluid was mixed with a previously prepared buffer solution containing macro- and microminerals according to Menke and Steingass [22], at a ratio of 1:2, respectively. Each bottle received 1 g of total mixed diet with the enzyme blend doses and 150 mL of the solution and was inoculated with CO₂ for 20 s. Afterwards, the bottles were placed in an incubator with a shaker table, at a temperature of 39 °C and agitation at 90 RPM.

Gas Pressure Monitor software (Software GEN 3, Macedon, NY, USA) was used to determine gas production during incubation. Results were recorded every 30 min, and the valves released the gas when the pressure reached 5 kPa. The cumulative pressure over 48 h was converted to mL (GP (mL) = (Pc/Po) × Vo), with Pc representing the cumulative pressure change (kPa) in the flask headspace, Vo corresponding to the flask headspace volume (170 mL), and Po being the atmospheric pressure read by the equipment at the beginning of the measurement [23].

The bicompartmental Gompertz model was used to estimate the cumulative gas production curve [24], as well as the first and second pools, characterized as fast and slow, respectively:

PG₍t₎ = V₁ × e^(–e^[1 + k₁ × e × (L − t)]) + V₂ × e^(–e^[1 + k₂ × e × (L − t)])

where PG(t) = gas production at time t; V₁, k₁, and L = asymptotic cumulative gas volume, degradation rate for the first pool (fast), and lag time; V₂, k₂ = asymptotic cumulative gas volume and degradation rate for the second pool (slow); and t = incubation time (hours).

Metabolizable energy (ME) was calculated according to Menke and Steingass [22], using the equation: ME (MJ/kg DM) = 1.06 + 0.157GP + 0.084CP + 0.22EE − 0.081Ash, where GP represents net gas production (mL/200 mg DM) at 48 h, and CP, EE, and Ash correspond to crude protein, ether extract, and crude ash (%), respectively.

In vitro organic matter digestibility (IVOMD) was calculated according to Menke and Steingass [22], using the equation: IVOMD (g/kg DM) = 31.55 + 0.8343GP, where GP represents net gas production at 24 h (mL/200 mg DM). At the beginning (0 h) and end (48 h) of incubation, the pH of the solution was measured. After the incubation process was completed, subsamples of the liquid were collected, filtered through gauze, and stored in Falcon tubes containing 0.02 mL of 50% sulfuric acid (H₂SO₄). Two samples of 15 mL per bottle were used for the analysis of volatile fatty acids and ammonia nitrogen (NH₃-N). The concentrations of SCFA and NH₃-N were determined according to Chaney and Marbach [25].

2.12. Statistical Analysis

A descriptive analysis of the data was performed initially, followed by analysis of normality of residuals and homogeneity of variances. The results of some blood variables (leukogram) required transformation to achieve normality and homogeneity. All data were analyzed using the SAS ‘MIXED’ procedure (SAS Inst. Inc., Cary, NC, USA; version 9.4), with the Satterthwaite approximation to determine the denominator degrees of freedom for the fixed effects tested. In the experimental model, treatment, day, and the interaction between treatment and day were used as fixed effects, while the animal was considered to have a variable effect. Means were compared using orthogonal contrasts with 5% significance: contrast C1 = [T-0 vs. (T-80 + T-160)]; contrast C2 = [T-0 vs. T-80]; contrast C3 = [T-0 vs. T-160]. All data obtained on day 1 for each variable were included as covariates in each analysis, and initial weight was included as a variable in the model. The first-order autoregressive covariance structure was selected according to the lowest Akaike information criterion. Significance was defined as p ≤ 0.05.

3. Results

3.1. Productive Performance

The results of the cows’ productive performance are presented in

Table 2. Performance of cows fed a blend of exogenous enzymes.

Variables	T-0	T-80	T-160	SEM	C1 2	C2 2	C3 2
Milk production, kg/cow/day
d 0 *	17.3	17.0	17.1	0.11	0.95	0.93	0.96
d 1–14	17.5	18.3	18.1	0.10	0.12	0.32	0.45
d 15–84	17.2	17.6	18.0	0.11	0.05	0.72	0.04
d 1–84	17.3	17.5	18.1	0.11	0.05	0.74	0.11
Milk production, 4%FCM 1
d 15–84	17.8	17.9	19.1	0.10	0.45	0.82	0.02
Feed intake, kg DM/day
d 15–84	15.2	15.2	15.2	0.03	0.94	0.95	0.96
Feed efficiency, kg/kg
d 15–84	1.17	1.17	1.25	0.01	0.49	0.78	0.05

* Average milk production of the cows in the last 7 days, before the experiment. Note ¹: Milk production corrected for 4% fat (4% FCM) was estimated by the equation proposed by the NRC [26] FCM = 0.4 × (kg of milk produced) + 0.15 × (% fat) × (kg of milk produced). Note ²: Probabilities for orthogonal contrast effect: C1 = [T-0 vs. (T-80 + T-160)]; C2 = [T-0 vs. T-80]; C3 = [T-0 vs. T-160].

. The contrast analysis allowed us to verify the effect of blend on milk production when considering the two periods (d15–87 and d1–84) when compared to the control. When evaluating milk production corrected for 4% fat percentage, the effect of the dose was verified, i.e., the cows in T-160 produced 7.3% more milk daily compared to those in T-0. There was no effect of the treatment on feed consumption by the cows; however, there was an effect of the treatment on feed efficiency, with the cows in T-160 being 6.83% higher when compared to those in the other groups. There was an interaction treatment × day for daily milk production (Figure S1), with higher production in cows that consumed the highest dose of enzymes (T-160) on some days of the experiment. There was also an effect of the day in the three groups, and throughout the experiment the cows reduced their milk production, which is related to the animals being in the final third of lactation at the end of the research, as well as the lower bromatological quality of the pastures at the end of the study. Knowing the dry matter intake of the cows, the daily dose of the enzyme blend consumed per animal was calculated, i.e., 1.2 g/animal (T-80) and 2.4 g/animal (T-160).

3.2. Milk Quality

Results of the analysis of centesimal composition, somatic cell count (SCC), and fatty acid profile are presented in

Table 3. Composition and quality of milk from cows fed a blend of exogenous enzymes.

Variables	T-0	T-80	T-160	SEM	C1 1	C2 1	C3 1
Fat 2 (g/100 g)	4.27	4.12	4.39	0.05	0.82	0.57	0.94
Protein 2 (g/100 g)	3.68	3.71	3.76	0.03	0.25	0.46	0.05
Lactose 2 (g/100 g)	4.7	4.62	4.69	0.05	0.85	0.87	0.91
Total solids 2 (g/100 g)	12.6	12.4	12.8	0.15	0.89	0.78	0.91
Urea 2 (mg/dL)	14.5	15.0	15.7	0.51	0.22	0.83	0.56
Urea/protein ratio	3.94	4.04	4.17	0.04	0.21	0.65	0.15
SCC 2 (×1000/mL)	67.6	160	102	24.1	0.31	0.24	0.43
Fatty acid, %
C4:0 (Butyric)	0.51	0.58	0.60	0.02	0.05	0.28	0.16
C6:0 (Caproic)	0.90	0.87	0.96	0.02	0.95	0.91	0.88
C8:0 (Caprylic)	0.83	0.83	0.87	0.01	0.52	0.92	0.31
C10:0 (Capric)	2.64	2.68	2.71	0.05	0.24	0.86	0.63
C11:0 (Undecanoic)	0.23	0.24	0.25	0.01	0.77	0.85	0.69
C12:0 (Lauric)	3.57	3.67	3.70	0.08	0.55	0.64	0.57
C14:0 (Myristic)	12.3	12.5	12.9	0.12	0.81	0.89	0.83
C14:1 (Myristoleic)	0.62	0.72	0.67	0.02	0.12	0.23	0.35
C15:0 (Pentadecanoic)	1.19	1.20	1.20	0.02	0.96	0.97	0.96
C16:0 (Palmitic)	35.5	36.8	36.9	0.34	0.05	0.35	0.28
C16:1 (Palmitoleic)	1.05	1.20	1.03	0.04	0.72	0.31	0.97
C17:0 (Heptadecanoic)	0.51	0.51	0.50	0.01	0.98	0.99	0.98
C17:1 (cis-10-Heptadecenoic)	0.14	0.15	0.15	0.00	0.97	0.97	0.98
C18:0 (Stearic)	15.4	14.3	14.0	0.26	0.05	0.15	0.09
C18:1n9t (Elaidic)	2.04	1.77	1.91	0.08	0.82	0.25	0.83
C18:1n9c (Oleic)	18.9	18.6	17.9	0.31	0.88	0.93	0.62
C18:2n6c (Linoleic)	2.02	1.93	1.92	0.03	0.33	0.56	0.61
C20:0 (Arachidic)	0.22	0.21	0.20	0.00	0.97	0.98	0.94
C20:1n9 (cis-11-Eicosenoic)	0.09	0.10	0.07	0.01	0.95	0.97	0.91
C18:3n3 (a-Linolenic)	0.38	0.38	0.36	0.01	0.66	0.98	0.84
C21:0 (Henicosanoic)	0.41	0.39	0.38	0.02	0.49	0.56	0.51
C22:0 (Behenic)	0.10	0.10	0.09	0.00	0.98	0.99	0.98
C20:3n6 (cis-8,11,14-Eicosatrienoic)	0.07	0.06	0.06	0.00	0.81	0.87	0.86
C20:4n6 (Arachidonic)	0.06	0.05	0.05	0.00	0.98	0.96	0.96
C20:5n3 (cis-5,8,11,14,17-Eicosapentaenoic)	0.06	0.05	0.05	0.00	0.98	0.98	0.97
∑ Saturated fatty acids (SFA)	74.4	74.9	75.3	0.37	0.86	0.94	0.65
∑ Unsaturated fatty acids (UFA)	25.4	25.1	24.2	0.29	0.44	0.89	0.18
∑ Monounsaturated fatty acids (MUFA)	20.8	20.8	19.9	0.26	0.78	0.96	0.35
∑ Polyunsaturated fatty acids (PUFA)	2.58	2.46	2.44	0.04	0.86	0.89	0.87
UFA/SFA	0.34	0.33	0.32	0.01	0.94	0.95	0.91

Note ¹: Probabilities for orthogonal contrast effect: C1 = [T-0 vs. (T-80 + T-160)]; C2 = [T-0 vs. T-80]; C3 = [T-0 vs. T-160]. Note ²: No effect of day or treatment x day interaction was observed for milk composition and quality variables (p > 0.05).

. No effect of day or treatment × day interaction was observed for centesimal composition (fat, protein, lactose, and total solids) and SCC during the experiment. The contrast analysis showed an effect of the dose (T-160) for protein in the milk, being higher in the milk of cows that consumed the blend at the highest dose compared to those in T-0. No effect of treatment was observed for fat, lactose, total solids, urea, and SCC in the milk. The effect of enzymes was observed for butyric, palmitic, and stearic fatty acids; butyric and palmitic were higher in the milk of cows that consumed enzymes; while stearic was at a lower percentage in the milk of cows that consumed enzymes. There was no effect of treatment for the other fatty acids quantified in the milk (Table 3), as well as for the sum of saturated and unsaturated fatty acids.

3.3. Ruminal Environment

The profile of volatile fatty acids (VFAs) in the ruminal fluid was presented in

Table 4. Profile of volatile fatty acids (VFAs) in ruminal fluid and apparent digestibility coefficients of nutrients supplied to cows fed a blend of exogenous enzymes.

Variables	T-0	T-80	T-160	SEM	C1 1	C2 1	C3 1
VFAs in ruminal liquid
Total VFAs (mmol/L)	92.1	97.3	110.2	1.52	0.01	0.05	0.01
Acetic acid (mmol/L)	58.2	61.9	69.8	1.12	0.09	0.21	0.03
Propionic acid (mmol/L)	17.6	19.2	20.9	0.54	0.26	0.38	0.05
Butyric acid (mmol/L)	13.7	13.5	16.3	0.21	0.75	0.94	0.02
Isovaleic acid (mmol/L)	1.11	1.34	1.58	0.04	0.05	0.18	0.01
Valeric acid (mmol/L)	1.37	1.27	1.42	0.07	0.89	0.39	0.83
Acetic/propionic (mmol/L)	3.30	3.22	3.33	0.03	0.94	0.82	0.92
Apparent digestibility coefficient
Dry matter	0.74	0.78	0.81	0.06	0.15	0.52	0.05
Organic matter	0.76	0.79	0.82	0.06	0.22	0.61	0.08
Crude protein	0.70	0.77	0.83	0.05	0.05	0.22	0.01
NDF	0.63	0.67	0.78	0.09	0.43	0.82	0.12
ADF	0.61	0.66	0.73	0.08	0.63	0.79	0.15
EE	0.75	0.79	0.81	0.06	0.58	0.84	0.32

Note ¹: Probabilities for orthogonal contrast effect: C1 = [T-0 vs. (T-80 + T-160)]; C2 = [T-0 vs. T-80]; C3 = [T-0 vs. T-160].

. We verified the effect of blend on the quantity of SCFA (C1), as well as of the two doses evaluated individually (C2 and C3), being higher in the fluid of the cows that consumed the enzymes. The effect of the dose of 160 mg/kg DM (C3) impacted the concentration of acetic, propionic and butyric acid, and in the cows of T-160, higher levels of these fatty acids were observed when compared to T-0. Regarding the levels of isovaleric acid, an effect of enzymes (C1), and effect of the dose (T-160) was observed, being higher in these animals fed a blend when compared to T-0. No effect of treatment was observed for valeric acids in the ruminal fluid, as well as in the acetic/propionic ratio.

3.4. Digestibility Coefficient

The results regarding the apparent digestibility coefficients (ADCs) were presented in Table 4. The dose effect was observed for the ADC of the dry matter of the TMR, with greater digestibility of the cows of the T-160 group when compared to those of the T-0 group. The digestibility of the crude protein was influenced by the enzymes (C1), as well as a higher dose of the enzyme (C3), i.e., cows that consumed the blend had a higher ADC of the CP. There was no effect of the treatment for the ADCs of organic matter, ADF, NDF, and EE (p > 0.05).

3.5. Hematology and Serum Biochemistry

The results of hemogram and serum biochemistry are presented in

Table 5. Hemogram and serum biochemistry of cows fed a blend of exogenous enzymes.

Variables	T-0	T-80	T-160	SEM	C1 1	C2 1	C3 1
Hemogram
Erythrocytes (×106 µL)	5.42	5.03	5.15	0.12	0.35	0.29	0.58
Hemoglobin (g/dL)	9.59	9.44	9.45	0.17	0.87	0.82	0.82
Hematocrit (%)	26.2	25.7	25.9	0.52	0.46	0.59	0.63
Leukocytes (×103 µL)	6.25	7.04	6.50	0.24	0.51	0.45	0.79
Lymphocytes (×103 µL)	3.58	4.21	3.90	0.21	0.32	0.48	0.67
Granulocytes (×103 µL)	1.58	1.71	1.55	0.14	0.84	0.77	0.91
Monocyte (×103 µL)	1.08	1.10	1.03	0.25	0.92	0.87	0.85
Biochemistry
Albumin (g/dL)	3.16	2.99	3.17	0.08	0.85	0.68	0.93
Cholesterol (mg/dL)	160	161	151	3.21	0.79	0.93	0.22
Glucose (mg/dL)	71.3	69.7	70.0	1.05	0.96	0.95	0.97
Uric acid (mg/dL)	1.70	1.93	1.96	0.07	0.16	0.24	0.21
Total protein (g/dL)	7.32	7.52	7.19	0.26	0.54	0.62	0.73
Urea (mg/dL)	39.7	37.6	38.3	1.25	0.72	0.78	0.87
Globulin (g/dL)	4.15	4.53	4.02	0.21	0.76	0.66	0.81

Note ¹: Probabilities for orthogonal contrast effect: C1 = [T-0 vs. (T-80 + T-160)]; C2 = [T-0 vs. T-80]; C3 = [T-0 vs. T-160]. Note: There was no effect of treatment, day, or treatment x day interaction for hematological and biochemical variables (p > 0.05).

. No effect of treatment, day, or treatment × day interaction was observed for variables related to the blood analysis (total erythrocytes, hemoglobin, hematocrit, platelets, total leukocytes, lymphocytes, granulocytes, and monocytes) and serum biochemistry (albumin, total protein, globulin, urea, cholesterol, glucose, and uric acid).

3.6. In Vitro Fermentation and Ammonia

In vitro fermentation and ammonia data are described in

Table 6. In vitro fermentation and ammonia to cows fed a blend of exogenous enzymes.

Variables	T-0	T-80	T-160	SEM	C1 1	C2 1	C3 1
Total gas production, mL/g DM
24 h	163.9	143.9	132	4.59	0.01	0.04	0.01
48 h	181.2	166.0	147.5	5.56	0.01	0.02	0.01
pH	6.45	6.49	6.47	0.01	0.86	0.89	0.92
Metabolizable energy, MJ/kg DM	7.51	7.70	7.19	0.23	0.57	0.65	0.43
Digestibility of OM, g/kg DM	643.6	633.2	628.1	8.65	0.04	0.32	0.05
NH3-N, mg/dL	26.2	27.1	19.9	1.94	0.71	0.94	0.03

Note ¹: Probabilities for orthogonal contrast effect: C1 = [T-0 vs. (T-80 + T-160)]; C2 = [T-0 vs. T-80]; C3 = [T-0 vs. T-160]. OM: Organic Matter, DM: Dry Matter.

. We observed an effect of the enzymes on total gas production, with lower production in the groups that received the blend at 24 h, lower production in the T-160 group compared to the others at 48 h, and also lower gas production in T-80 compared to the T-0 group. The dose of 160 mg/kg DM was lower than the control, although dose T-80 did not differ from the other groups (Figure S2). Regarding ammonia production, an effect of the higher dose (T-160) was observed, with lower levels in the group of animals that received this dose. No treatment effects were observed for pH and metabolizable energy.

4. Discussion

Our hypothesis was confirmed, as the use of the enzyme blend containing protease led to improvements in milk production and composition, increasing the digestibility of dry matter and crude protein, as well as the levels of VFAs. The presence of exogenous protease combined with other enzymes in the diet of lactating Jersey cows enhanced productivity, due to modulation of the volatile fatty acid profile along with improved dietary protein digestibility, as expected. Similar results had already been reported when protease was combined with another enzyme and provided to lactating cows [10,27]. Eun and Beauchemin [8], on the other hand, observed that the use of a single proteolytic enzyme in different diets for Holstein cows reduced milk production—an outcome opposite to that found in the present study. These conflicting results are still not fully understood, but one possible explanation may lie in the different diets used in the studies. When correcting milk for fat, we found that milk production remained higher in the groups receiving protease, even without differences in dry matter intake (DMI). The study showed greater feed efficiency in the cows that consumed protease, as milk production was higher in that group. Researchers have found that lactating Holstein cows fed proteolytic enzymes showed better feed efficiency [28], a result similar to what was observed in this study.

Milk composition can be influenced by several factors, and animal feeding is directly associated with these changes [29]. The enzymatic blend had a positive effect on milk composition, increasing the protein content in milk. This result is due to the fact that the animal makes better use of nutrients, leading to greater protein absorption which, through metabolism, is converted into milk protein. Our result is consistent with studies in which a proteolytic enzyme was used in association with another exogenous enzyme in lactating cows, also resulting in increased milk protein [27]. Milk fatty acids can also vary according to the animal’s diet and microbial activity in the rumen [30]. Short- and medium-chain fatty acids (C4:0 to C16:0) are mainly synthesized by fatty acid synthase in the mammary gland [31]. Acetate is the precursor of acetyl-CoA, which is the main substrate for the synthesis of new fatty acids [32]. Based on this, the increase in acetate observed in our experiment may be one of the factors that enhanced the presence of C4:0 and C16:0 fatty acids in the cows fed exogenous enzymes in this study.

The production of VFAs was higher in cows that consumed the enzyme blend; we observed an increase in the production of acetic, propionic, butyric, and isovaleric acids. This increase in SCFA was also identified in another study that used the same mixture of exogenous enzymes in the diet of feedlot cattle [7]. In addition, when the protease enzyme was provided in isolation to lactating cows, an increase in the production of these acids was also observed [27]. This effect may have occurred in our study due to the action of the enzymes in the rumen, which increased the availability of substrates for microbial proliferation. As expected, we found greater nutrient digestibility in the group that received the enzyme blend, a result similar to that reported by [27], who observed better digestibility of dry matter (DM) and organic matter (OM) in cows fed only with protease. However, that study did not evaluate crude protein (CP), which theoretically would also show positive results. When exogenous enzymes were used, improvements in DM and OM digestibility [8], as well as in CP digestibility [7], were observed. This improvement in digestibility was the main expected outcome, and, as a consequence, other beneficial effects emerged, such as improved performance and milk quality. We believe there was no effect on animal health because the animals were not facing any health challenges. As previously mentioned, studies on exogenous enzymes are still scarce, and we suggest further research to confirm and explore new effects of this additive in the feeding of lactating cows.

The synergy between protease and fibrolytic enzymes improves feed degradation, resulting in greater nutrient availability [10,33]. Fibrolytic enzymes act on the plant cell wall, breaking the linkages and exposing the protein fraction of the plants, thereby facilitating protease access to the fiber-bound proteins. Consequently, there is greater release of amino acids to the rumen microorganisms, which produce more microbial protein and modulate fermentation [34]. This may help us understand that the increase in crude protein digestibility observed in this study led to higher concentrations of VFAs.

We hypothesized that a blend of exogenous enzymes could modulate ruminal fermentation, thereby enhancing the production of VFAs and improving nutrient availability. These effects are expected to positively influence animal performance and potentially reduce enteric methane emissions per unit of product in ruminants [34]. Notably, in the in vivo trial, the group receiving the T-160 enzyme dose exhibited a higher concentration of VFAs, particularly propionate. In the in vitro assay, this treatment also resulted in the lowest gas production at both 24 and 48 h. It is well established that methane (CH₄) and carbon dioxide (CO₂) are the primary gases produced during ruminal fermentation [35]. Moreover, metabolic pathways leading to propionate synthesis act as hydrogen sinks and do not release CO₂, thereby diverting substrates away from methanogenesis [36]. These findings suggest that exogenous enzyme fed may contribute to the mitigation of greenhouse gas emissions. However, it is important to note that CH₄ and CO₂ were not directly measured in this study, and future research should include direct quantification of these gases to confirm this hypothesis. Conversely, the lower NH₃-N concentrations observed at the higher enzyme dose were unexpected, as ammonia nitrogen is typically associated with protein degradation. However, this reduction could also indicate enhanced microbial growth, since ruminal ammonia is a key nitrogen source for microbial protein synthesis [37]. The incorporation of ammonia into microbial biomass is energy-dependent and is favored when sufficient carbon chain is available, which may have been influenced by the enzymatic treatment [37]. This hypothesis could be confirmed if we had measured the rate of microbial nitrogen flux; in addition, purine derivatives in urine or microbial markers could help to substantiate this inference of “increased microbial protein synthesis”.

The experiment lasted 84 days, without any effect on blood count or metabolic biochemistry, which were complementary data in this research and help to confirm that this combination of exogenous enzymes with a higher proportion of protease, tested for the first time in cows, had no negative effects on the animals’ health and metabolism at the tested doses.

5. Conclusions

We conclude that the use of the combination of exogenous enzymes in the diet of lactating Jersey cows was able to increase milk production of these animals, reflecting in greater feed efficiency and also increasing protein in the milk. These results are related to the presence of enzymes in the gastrointestinal tract of the cows having been able to positively modulate the profile of rumen fatty acids, as well as improving the apparent digestibility of crude protein. The addition of the highest dose of the enzyme increased the concentration of total VFAs in the rumen, due to the greater amounts of acetic, propionic, butyric, and isovaleric acids when compared to the ruminal liquid of animals that did not consume the exogenous enzyme blend. Therefore, in an applicable way, we verified that the dose of 160 mg/kg of DM consumed by cows in the final third of lactation is a viable alternative for milk producers in semi-confinement conditions, where the cows had access to oat and ryegrass pasture.