Addition of Exogenous Fibrolytic Enzymes to the Feed of Confined Steers Modulates Fat Profile in Meat

Simple Summary

We used male dairy cattle (Holstein) as an experimental model for testing exogenous enzymes. The addition of fibrolytic enzymes to the diet of feedlot cattle did not significantly enhance digestibility, but there was a tendency for a higher NDF coefficient. The consumption of fibrolytic enzymes altered the ruminal fatty acid profile, especially a higher amount of volatile fatty acids (VFA). A higher quantity and proportion of acetic acid in the ruminal fluid may have been precursors of higher fat content in the meat, in addition to modulating the fatty acid profile, reducing SFA and increasing UFA in the meat.

Abstract

The objective of this study was to evaluate whether the addition of fibrolytic enzymes to the diet of cattle in confinement improves feed digestibility weight gain; as well as evaluating the profile of fatty acids in the ruminal environment and in meat fatty acids profile. In total, 24 male dairy cattle (Holstein) aged 8 months and weighing an average of 212 kg, were divided into 2 groups: control (without additive) and treatment (0.25 g of enzymes/kg of dry matter of total diet). The experiment lasted 120 days. The first 20 days are allocated for the adaptation period. During the study, samples of blood, ruminal fluid, and feces were collected, as well as weighing the cattle and measuring their daily feed consumption. There was no effect of treatment on body weight, feed intake, feed efficiency, and nutrient digestibility (p > 0.05). Cholesterol levels were higher in the serum of cattle that consumed the enzyme; serum amylase activity was higher in cattle that received the additive only on day 120 of the experiment (p < 0.05). There was a greater amount of volatile fatty acids in the ruminal fluid, combined with a greater amount of acetic acid. The amount of fat in the meat of cattle that consumed fibrolytic enzymes was higher compared to the control group (p < 0.05). Meat from cattle in the treatment group had lower amounts of saturated fatty acids and higher amounts of unsaturated fatty acids (p < 0.05). The fibrolytic enzymes addition altered rumen fermentation in such a way that lipid metabolism was changed, which had a serious impact on cholesterol and tissue levels, that is, in the meat that had a greater amount of total lipids, an unsaturated fat.

1. Introduction

Feed additives are adopted in 99.8% of confinement systems in Brazil [1]. With the primary objective of modulating rumen fermentation, the main group of additives are ionophores, which act preferentially on Gram-positive bacteria, favoring the growth of Gram-negative bacteria [2]. Research on the use of exogenous fibrolytic enzymes in ruminants dates back to the 1960s [3]. These additives aim to improve nutrient availability in the digestive system and break specific chemical bonds in raw material [4]. Previous studies have highlighted the positive effects of fibrolytic enzymes in ruminant diets, resulting in improvements in animal performance [5,6,7]. Exogenous fibrolytic enzymes, such as xylanases and cellulases, are widely used in ruminant feeding, with a focus on optimizing the digestibility of fiber present in low quality roughages [8]. Bureenok et al. [9] concluded that the addition of fibrolytic enzymes improves the digestibility of fiber and proteins.

Several studies have demonstrated that adding fibrolytic enzymes to ruminant diets can increase milk production in cows [7]. Liu et al. [10] observed that the combination of cellulase, xylanase, β-glucanase, and amylase in dairy cows at the beginning of lactation increases enzymatic activity in the rumen, resulting in improvements in productive performance, with a positive trend for feed efficiency. However, animal responses to enzyme addition vary considerably [5]. This variation can be attributed to a number of factors, such as variations in enzymatic activity, rate and method of application, substrate specificity, and physiological stage of the animals [11]. Therefore, the main objective of this study was to evaluate whether the addition of fibrolytic enzymes improves digestibility and growth performance, as well as the meat quality of steers finished in confinement.

2. Materials and Methods

2.1. Enzymatic Blend

The commercial product (Smizyme Multi-Nzymes—Salus) contains corn starch, 2.000 U/g of beta glucanase (Trichoderma longibrachiatum—CGMCC 7290), 300 U/g of cellulase (Trichoderma longibrachiatum—CGMCC 7289), and 20.000 U/g of xylanase (Aspergillus oryzae—CGMCC 7.70).

2.2. Animals, Installation and Experimental Design

The experiment was carried out at the Experimental Farm of the State University of Santa Catarina (UDESC), in the municipality of Guatambu, Santa Catarina, southern Brazil. Twenty-four Holstein cattle (not-castrated) aged 8 months, and weighing approximately 212 (±4.62) kg were confined in a shed with individual stalls, with feeders and drinkers, which allowed daily measurement of feed intake. The feeding of these animals was based on corn silage and concentrate (ground corn, soybean meal, soybeans, wheat bran and mineral, and vitamin core) in a proportion of 30:70%, respectively. The diet was calculated in BRCORTE 2016 [12], in order to meet the animal’s nutritional demands so that it has an average daily gain of 1.5 kg during the experimental period of 120 days. It is important to make it clear that the cattle’s diet was calculated individually for each animal, based on its initial body weight, and provided in a restrictive manner throughout the experiment, that is, what was necessary for the animal to gain 1.5 kg per day. Adjustments to the quantity of feed available to the animals were made at intervals of 15 days, to meet physiological demands, and also allow the animal to gain weight and accumulate fat in the carcass. The adaptation period lasted 20 days, followed by 100 experimental days. The animals were divided into two homogeneous groups with twelve animals each, considering body weight to form the groups: Control group (n = 12—without additive) and treatment group (n = 12–0.25 g of enzyme blend/kg DM of total mixed ration (TMR)). The enzymes were added to the animals’ diet via mashed concentrate, being added during production.

2.3. Data and Sample Collection

The animals were weighed at 30-day intervals; feed intake was measured daily. Based on weight gain and consumption information, feed efficiency and feed conversion were calculated.

Blood collection was carried out on days 1, 20, 60, and 120 of the experiment, in order to evaluate the health of the steers. Blood samples were collected from the coccygeal vein, in tubes with anticoagulant for blood count analysis; another blood sample was stored in tubes without anticoagulant to obtain the serum that was used in metabolic biochemistry analyses. To obtain serum, the tubes were centrifuged (800× g) for 10 min; then the serum was collected and stored in Eppendorf tubes (−20 °C) until analysis.

Rumen fluid collection was performed using an esophageal probe coupled to the vacuum system at the end of the adaptation period (day 20), as well as on days 60 and 120 of the study. The first 100 mL of the collected liquid were discarded. This material was filtered using a double layer of gases and then frozen (−20 °C) to measure the volatile fatty acid profile.

During 5 consecutive days (between days 101 and 105 of the experiment), feces were collected directly from the rectum. During the collection period, samples were collected at different times of the day (08:00, 10:00; 13:00; 15:00, and 17:00) to have a homogeneous final sample. At the end of the collection, a pool of five collections per animal was made. The material was used for analyses to determine the apparent digestibility coefficient of nutrients.

At the end of the experiment, the animals were sent to the slaughterhouse, and the hot carcass weight was obtained on the slaughter line, as well as a fragment of muscle (Longissimus thoracic) was collected to evaluate the fat and fatty acid profile in the meat.

2.4. Feed Composition Analysis

The feed and feces samples were pre-dried in a forced ventilation oven at 55 °C for 72 h, after being removed from the oven, weighed to determine the partial dry matter content, and then ground in a Wiley mill (Marconi, model: MA340), using a 2 mm mesh sieve. The pre-dried and ground samples were heated at 105 °C to obtain the MS and the mineral material in a muffle furnace at 600 °C [13]. Samples were analyzed according to AOAC [14]: dry matter (DM), method 930.15; crude protein (CP), method 976.05; ether extract (EE), method 920.39 and ash, method 942.05. The concentrations of neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined according to the methodology of Van Soest et al. [15] without the addition of sodium sulfite or alpha-amylase.

2.5. Hemogram

The total erythrocyte and leukocyte count, hemoglobin concentration, hematocrit percentage, and leukocyte differentiation were performed with an automatic hematologic analyzer VET3000 (EQUIP^®), obtained using blood tubes with EDTA. In addition to the variables mentioned above, the determination of hemoglobin concentration (g/dL), hematocrit (%), mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and amplitude red blood cell distribution (RDW-SD).

2.6. Serum Biochemistries

The concentrations of total protein, albumin, cholesterol, fructosamide, urea, and uric acid were evaluated, as well as the activity of amylase in a semi-automatic analyzer (Bio-2000^®, Bioplus Produtos para Laboratórios Ltd.a, Barueri, SP, Brazil) and using specific commercial kits for each variable (Analisa^®, Belo Horizonte, Brazil). Globulin levels were obtained using the formula total protein—albumin.

2.7. Determination of Short Chain Fatty Acids (SCFA)

The preparation of samples for analysis and measurement of the SCFA profile used in this study was described in detail by Brunetto et al. [16]. A volume of 650 μL of the sample was inserted into a 2 mL injection vial; 1 μL of extract 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) up to 250 °C to measure acetic, propionic, butyric, valeric, and isovaleric acids. Accuracy was determined by recovering known amounts of the standard substances added to a diluted sample (Table S1). The results were showed in mol 100 mol/L of each short-chain SCFA in the ruminal fluid.

2.8. Apparent Digestibility Coefficient

Indigestible neutral detergent fiber (iNDF) was used as an internal marker to calculate apparent feed digestibility [17,18]. 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 [19], and dried in a forced-air ventilation oven at 55 °C. Digestibility was calculated as 1 − (iNDF in feed (% of DM)/iNDF in feces (% of DM)).

2.9. Fatty Acid (FA) Profile in Meat and Feed

To analyze the fatty acid profile in meat and feed, extraction was performed using the Bligh and Dyer [20] method with some modifications. FA methylation was also performed using a transesterification method proposed by Hartman and Lago [21]. Both methodologies have been previously described in detail by Brunetto et al. [16]. A TRACE 1310 gas chromatograph with a flame ionization detector was used to determine FAME, where one microliter of sample was injected into a split less injector operated in 1:10 split mode at 250 °C [16]. The chromatographic separation was performed using a CP-Sil 88 capillary column (100 m × 0.25 mm i.d., 0.20 μm film thickness) with helium as the carrier gas at a flow rate of 1 mL/min. The oven temperature program consisted of an initial temperature of 50 °C, held for 1 min, followed by an increase to 175 °C at 13 °C/min, held for 27 min, and then increased to 215 °C at 4 °C/min, held for 30 min. The results were presented as a percentage of each FA identified in the lipid fraction to according with Visentainer and Franco [22]. The results of FA at feed (TMR) are presented in

Table 1. Feed composition and fatty acid profile of diets fed to experimental animals.

Variables, %	TMR: Control	TMR: Treatment
Dry matter (%)	90.25	90.44
Organic matter, % MS	93.38	93.36
Crude protein, % MS	15.78	15.76
Ether extract, % MS	2.87	3.07
Ash, % MS	6.62	6.64
NDF, % MS	28.80	25.68
Fatty acid, %
C14:0 (Myristic)	0.313	0.331
C14:1 (Myristoleic)	0.003	0.022
C15:0 (Pentadecanoic)	0.174	0.192
C16:0 (Palmitic)	21.84	21.78
C16:1 (Palmitoleic)	0.300	0.318
C17:0 (Heptadecanoic)	0.295	0.313
C18:0 (Stearic)	4.679	4.682
C18:1n9t (Elaidic)	0.346	0.364
C18:1n9c (Oleic)	32.09	32.00
C18:2n6c (Linoleic)	34.47	34.37
C20:0 (Arachidic)	0.793	0.810
C20:1n9 (cis-11-Eicosenoic)	0.304	0.321
C18:3n3 (a-Linolenic)	2.407	2.417
C20:2 (cis-11,14-Eicosadienoic)	0.121	0.139
C22:0 (Behenic)	0.789	0.806
C22:1n9 (Erucic)	0.071	0.089
∑ Saturated fatty acids (SFA)	29.88	29.95
∑ Unsaturated fatty acids (UFA)	70.12	70.05

Note: The concentrate was formulated based on ground corn (63.04%), soybean hulls (25.5%), soybean meal (7.45%), mineral core (2.87%), and urea (1.15%).

.

2.10. Statistical Analysis

The experimental data were first analyzed descriptively; measures of central tendency (median) and data dispersion (range that stands for the interval between the minimum and maximum values in the data) were computed. Further, all variables were subjected to the Shapiro–Wilk test, which revealed a normal data distribution. Skewness, kurtosis, and homogeneity were evaluated using the Levene test, and linearity was used using linear regression. All data were analyzed using the ‘MIXED procedure’ of SAS (SAS Inst. Inc., Cary, NC, USA; version 9.4, 2013), with Satterthwaite approximation to determine the denominator degrees of freedom for the test of fixed effects. The weight gain and feed efficiency were tested for treatment-fixed effects using a pen (treatment) and animal (pen) as random variables (i.e., pen within treatment and animal within pen). The data of body weight, all blood, and rumen fluid results were analyzed as repeated measurements and tested for fixed effects of treatment, day, and treatment × day, and pen (treatment) and animal (pen) as random variables. Post-mortem variable analysis (meat, carcass, and fatty acid profile) was tested using animal (treatment) as random variables for treatment-fixed effects. The d1 results were included as an independent covariate. The first-order autoregressive covariance structure was selected according to the lowest Akaike information criterion. Means were separated using PDIFF method (Student test) and all results were reported as LSMEANS followed by SEM. Significance was defined when p ≤ 0.05.

3. Results

3.1. Performance

The results of weight and intake measurements are presented in

Table 2. Fibrolytic enzymes impact on experimental animal’s performance.

Variables	Control	Treatment	SEM	p: Treat	p: Treat × Day
Body weight, kg				0.80	0.72
Initial	208	216	4.62
Final	389	399	4.29
Average daily gain, kg	1.56	1.57	0.25	0.94	-
Feed intake, kg DM	8.18	8.39	0.18	0.66	0.48
Feed convertion	5.23	5.34	0.15	0.79	-
Feed efficiency	0.19	0.18	0.02	0.91	-

Note: There was no treatment effect or treatment × day interaction (p > 0.05).

. No difference was observed between groups for body weight, average daily gain, feed intake, feed conversion, and feed efficiency (p > 0.05).

3.2. Hemogram and Biochemistry Seric

Hemogram results are presented in

Table 3. Impact of fibrolytic enzymes on blood hemogram and seric biochemistry of the experimental animals.

Variables	Control	Treatment	SEM	p: Treat	p: Treat × Day
Hemogram
Leukocytes (×103 µL)	10.1	9.19	0.74	0.63	0.31
Granulocyte (×103 µL)				0.59	0.05
D20	1.30	1.29	0.21
D60	1.67	1.38	0.21
D120	3.77 a	2.56 b	0.25
Lymphocytes (×103 µL)	6.33	6.23	0.52	0.87	0.91
Monocyte (×103 µL)	1.47	1.30	0.41	0.72	0.80
Erythrocyte (×106 µL)	7.13	6.98	0.08	0.89	0.73
Hemoglobin	11.4	11.5	0.36	0.95	0.97
Hematocrit, %	30.1	30.3	0.76	0.92	0.95
Platelet (×103 µL)	241	268	9.74	0.41	0.13
Seric biochemistry
Amylase (U/L)				0.25	0.05
D20	88.1	97.3	4.74
D60	106	105	4.68
D120	116 b	131 a	4.71
Globulin (g/dL)	4.13	4.06	0.35	0.74	0.81
Albumin (g/dL)	2.71	2.69	0.12	0.91	0.85
Total protein (g/dL)	6.85	6.75	0.39	0.88	0.79
Fructosamine (mg/dL)	2.11	2.12	0.09	0.96	0.98
Cholesterol (mg/dL)	87.6	98.9	1.25	0.05	0.11
Uric acid (mg/dL)	0.88	0.90	0.06	0.96	0.92
Urea (mg/dL)	15.9	16.1	0.63	0.97	0.99

Note: Different letters on the same line show statistical differences between groups (p ≤ 0.05) and also trend (p > 0.05 and p ≤ 0.10).

. There was no effect of treatment and treatment interaction for the variables: total leukocytes, lymphocytes, monocytes, erythrocytes, hematocrit and hemoglobin, and platelets (p > 0.05). However, there was an interaction effect for granulocyte count, which was lower in the blood of cattle in the treatment group on day 120.

The results regarding serum biochemistry are presented in Table 3. There was no effect of treatment and treatment interaction for the variables: globulins, albumin, total protein, fructosamine, uric acids, and urea (p > 0.05). However, there was an interaction effect for amylase activity, which was greater in the serum of cattle in the treatment group on day 120. The effect of treatment for cholesterol concentration was observed; being higher in the serum of cattle that consumed the enzymes.

3.3. VFAs in Ruminal Liquid

Volatile fatty acid (VFA) profile results in rumen fluid are presented in

Table 4. Ruminal fluid in bovines fed with fibrillytic enzyme blend: results presented in percentage and absolute values.

Variables	Control	Treatment	SEM	p: Treat	p: Treat × Day
Volatile fatty acids (mmol/L)				0.05	0.01
D20	75.6	70.2	2.29
D60	87.5 b	102.7 a	2.88
D120	79.0 b	90.9 a	1.76
Quantity results
Acetic acid (mmol/L)				0.05	0.01
D20	52.6	48.3	1.50
D60	53.6 b	66.7 a	1.52
D120	51.2 b	61.3 a	1.33
Butyric acid (mmol/L)				0.35	0.05
D20	7.79 a	6.11 b	0.28
D60	11.9	13.8	0.68
D120	9.46	10.7	0.36
Isovaleric acid (mmol/L)				0.68	0.02
D20	1.24	1.02	0.17
D60	2.59	2.64	0.18
D120	1.00 b	1.37 a	0.09
Propionic acid (mmol/L)	15.8	16.3	0.66	0.83	0.46
Valeric acid (mmol/L)	1.05	0.91	0.05	0.65	0.71
Proportion results
Acetic acid (%)				0.05	0.03
D20	69.5	69.1	0.42
D60	61.6 b	65.1 a	0.47
D120	64.0 b	67.7 a	0.39
Propionic acid (%)				0.70	0.05
D20	16.9 b	19.1 a	0.66
D60	20.8	17.9	0.81
D120	20.8	18.3	0.48
Butyric acid (%)				0.86	0.03
D20	10.3 a	8.73 b	0.22
D60	13.4	13.4	0.51
D120	11.9	11.7	0.33
Isovaleric acid (%)	1.91	1.84	0.11	0.55	0.69
Valeric acid (%)				0.12	0.01
D20	1.56	1.53	0.01
D60	1.29 a	0.87 b	0.08
D120	1.04 a	0.85 b	0.05
Acetic/propionic				0.79	0.01
D20	4.28	3.72	0.29
D60	3.13 b	3.66 a	0.14
D120	3.14 b	3.73 a	0.11

Note: Different letters on the same line show statistical differences between groups (p ≤ 0.05) and also trend (p > 0.05 and p ≤ 0.10).

. Treatment effect and treatment × day interaction for total VFA were observed, being higher in the rumen fluid of cattle that consumed the enzyme blend on days 60 and 120. The concentration (nmol/mL) and proportion (%) of acetic acid in the rumen was higher in cattle that consumed exogenous enzymes on days 60 and 120 compared to the control. There was no treatment or interaction effect for the concentration (nmol/mL) of propionic acid; but there was a treatment × day interaction in the proportion (%) of this acid, being higher in the rumen fluid of the animals in the treatment group on day 20. A treatment × day interaction (day 20) was observed for the concentration and proportion of butyric acid, being lower in cattle who consumed the fibrolytic enzyme blend. The concentration of isovalaric acid was higher in animals in the treatment group on day 120, which was not observed in the proportion analysis. The proportion (%) of valeric acid only had a treatment × day interaction, with the percentage of valeric acid being lower on days 60 and 120 in the rumen fluid of cattle in the treatment group. The acetic/propionic ratio was higher in the rumen fluid of cattle that consumed the fibrolytic enzyme blend on days 60 and 120.

3.4. Apparent Digestibility Coefficient (ADC)

ADC results are presented in

Table 5. Impact of fibrolytic enzymes addition on animal’s apparent digestibility coefficient.

Variables	Control	Treatment	SEM	p: Treat
Dry matter	0.41	0.49	0.05	0.10
Organic matter	0.46	0.50	0.04	0.40
Crude protein	0.45	0.48	0.03	0.42
Ether extract	0.52	0.50	0.02	0.85
NDF	0.29	0.37	0.05	0.07

Note: There was no treatment effect to variables (p > 0.05).

. There was no treatment effect for ADC of dry matter, organic matter, crude protein, ethereal extract, and NDF (p > 0.05). However, if we consider the trend (p > 0.05 to p ≤ 0.10), we have a difference between groups for the dry matter and NDF coefficient.

3.5. Meat Quality

The results of total lipids and fatty acid profile in meat are presented in

Table 6. Total lipids and fatty acid in meat of bovines fed with the fibrolytic enzyme blend.

Variables	Control	Treatment	SEM	p-Value
Total lipids (g/kg)	12.2 b	14.7 a	0.64	0.02
Fatty acid (%)
C14:0 (Myristic)	2.07 a	1.66 b	0.072	0.02
C14:1 (Myristoleic)	0.37	0.32	0.024	0.35
C15:0 (Pentadecanoic)	0.22	0.21	0.005	0.96
C16:0 (Palmitic)	30.00 a	25.75 b	0.166	0.01
C16:1 (Palmitoleic)	3.71	3.33	0.104	0.43
C17:0 (Heptadecanoic)	1.27	1.44	0.176	0.65
C18:0 (Stearic)	15.22	15.15	0.128	0.97
C18:1n9t (Elaidic)	1.83	1.71	0.096	0.82
C18:1n9c (Oleic)	34.08 b	37.66 a	0.276	0.01
C18:2n6c (Linoleic)	7.18	8.12	0.436	0.73
C20:0 (Arachidic)	0.07	0.07	0.002	0.99
C18:3n6 (Linolenic)	0.05	0.06	0.003	0.95
C20:1n9 (cis-11-Eicosenoic)	0.16	0.17	0.003	0.96
C18:3n3 (a-Linolenic)	0.32	0.35	0.018	0.26
C21:0 (Henicosanoic)	0.29	0.32	0.012	0.15
C20:2 (cis-11.14-Eicosadienoic)	0.10	0.11	0.005	0.95
C20:3n6 (cis-8,11,14-Eicosatrienoic)	0.47	0.51	0.029	0.36
C20:4n6 (Arachidonic)	2.27 b	2.69 a	0.055	0.01
C22:2 (cis-13,16-Docosadienoic)	0.03	0.03	0.002	0.99
C24:0 (Lignoceric)	0.04	0.04	0.002	0.99
C20:5n3 (cis-5,8,11,14,17-Eicosapentaenoic)	0.16	0.19	0.012	0.16
C24:1n9 (Nervonic)	0.04	0.04	0.003	0.98
C22:6n3 (cis-4.7.10.13.16.19-Docosahexaenoic)	0.05	0.06	0.006	0.95
Other variables
∑ Saturated fatty acids (SFA)	49.17 a	43.64 b	0.388	0.01
∑ Unsaturated fatty acids (UFA)	50.83 b	55.36 a	0.388	0.01
∑ Monounsaturated fatty acids (MUFA)	40.19 b	43.24 a	0.270	0.05
∑ Polyunsaturated fatty acids (PUFA)	10.64 b	12.12 a	0.249	0.09
UFA/SFA	1.04 b	1.27 a	0.011	0.01

Note: Different letters on the same line show statistical difference between groups (p ≤ 0.05) and also trend (p > 0.05 and p ≤ 0.10).

. Cattle in the treatment group had a higher amount of lipids in meat compared to the control. Lower percentages of myristic and palmitic acids, as well as a higher proportion of oleic and arachidonic acids, were observed in beef from cattle that consumed fibrolytic enzymes. As a result, we found a lower amount of saturated fatty acids and a higher amount of unsaturated fatty acids in the meat of cattle in the treatment group compared to the control group. Both MUFA and PUFA sums were higher in the meat of cattle that consumed enzyme blends. A higher UFA/SFA ratio was observed in the meat of cattle in the treatment group.

4. Discussion

In the current study, the ingestion of fibrolytic enzymes by cattle altered the fatty acid profile of the meat. Although we did not identify a significant impact on the animals’ weight gain, the addition of enzymes resulted in modulation of rumen fermentation. Abid et al. [23] stated that the effectiveness of fibrolytic enzymes is influenced by the chemical composition of feed; as fibrolytic enzymes are most effective for sorghum straw, molasses grass, elephant grass, and sugar cane, respectively [24]. Furthermore, it was known that the greatest effects of fibrolytic enzymes are in diets composed of low quality forage; which would not be the case here in the present work, as the diet is based on concentrate (70%) and good quality silage (30%).

Short-chain fatty acids are the main source of energy for ruminant animals, increasing the production of these acids in the rumen has a positive correlation with performance. In our research, an increase in the total production of short-chain fatty acids and acetic and isovaleric acids was observed for animals fed with exogenous enzymes. Giraldo et al. [25] observed that in diets rich in forage, the addition of fibrolytic enzyme cellulase increases the production of butyrate and isovalerate. Another study demonstrated that the addition of exogenous fibrolytic enzymes improved the total digestibility [8] of NDF and ADF, but without affecting the total production of short-chain fatty acids [26,27,28]. With the addition of fibrolytic enzymes [8], they found an increase in ruminal ammonia due to better use of nitrogen for microbial growth. Added fibrolytic enzymes is a strategy to mitigate methane emissions without compromising meat quality [29].

Neutrophils, which are granulocyte leukocyte cells of fundamental importance, are considered a front line in the defense of animal organisms. They perform antimicrobial, pro- and anti-inflammatory functions. They perform antimicrobial, pro- and anti-inflammatory functions [30,31]. Neutrophils have the ability to attract and activate others of their species, modulating the secretions of mediators, such as platelet-activating factor, leukotriene B4 [32], and acid glycoprotein [33]. Immunosuppression, including neutrophil dysfunction, is considered a contributing cause and a target for therapeutic intervention [34,35,36]. Based on our observations, the greater number of granulocytes in the animals in the control group at the end of the experimental period suggests that the animals fed with the fibrolytic enzyme blend had a less activated immune system, consequently with lower energy expenditure. This could have resulted in better zootechnical performance.

Acetate is a precursor substance for fatty acid synthesis in bovine mammary epithelial cells (BMECs), and the mTOR signaling pathway plays an important role in milk fat synthesis. However, the mechanism of the regulatory effects of acetate on lipogenic genes via the mTOR signaling pathway in BMEC remains unknown. The ingestion of fibrolytic enzymes mixture (cellulase, xylanase, pectinase, and laccase) in the diet of Holstein bulls resulted in a lower rumen pH value and the ratio of acetate to propionate, and a higher concentration of VFA, but the content of ammonia-N was not influenced with increasing dose of enzyme mixture [37]. Based on our results, it is clear that the ingestion of fribrolytic enzymes changes the profile of volatile fatty acids in the rumen, with emphasis on the increase in acetic acid, which leads to the hypothesis that a similar mechanism occurs in lipid metabolism that impacts fat in meat. The modulation of the fatty acid profile in meat was beneficial to consumer health, since cattle that consumed fibrolytic enzymes had a lower proportion of saturated fatty acids, and a greater amount of unsaturated fatty acids; i.e., it increased the omegas. The recommendation for ingesting a smaller amount of saturated fat is to protect the cardiovascular system, preventing the deposition of fat in the blood vessels.

The animals in this study consumed restrictive feed, formulated to meet physiological and nutritional requirements, as well as an average daily weight gain of 1.5 kg. It is important to make it clear that if more feed was provided, the Holstein cattle would consume it. However, production costs are unsustainable according to research already carried out by our group when using cattle of the Holstein breed (unpublished data). We argue that most of the studies that tested fibrolytic enzymes and had positive results provided feed ad libitum. In dairy cows, researchers found that the ingestion of fibrolytic enzyme enhances the digestibility of DM, NDF, potentially digestible NDF (pdNDF), and organic matter [38]; but in our study we only found a trend towards better digestibility of NDF.

The fatty acid profile of meat not only reveals the nutritional value of the product, but also directly influences its flavor and contributes to the promotion of human health [39]. In ruminant animals, polyunsaturated fatty acids in the diet undergo the biohydrogenation process in the rumen, being converted into monounsaturated and saturated fatty acids. Only about 10% of polyunsaturated fatty acids remain available for incorporation into lipid tissues [39]. Based on the changes observed in our study, it is possible to state that fibrolytic enzymes modulate ruminal fermentation, improving the lipid profile of the meat. Simon et al. [40] observed a significant increase in the percentage of polyunsaturated fatty acids with exogenous enzyme fed. Furthermore, they concluded that the inclusion of digestive enzymes not only increased the amount of unsaturated fatty acids, and reduced saturated ones, but also had a positive effect on the oxidative stability of the meat and improved the animals’ performance.

The mechanisms that lead to changes in the fatty acid profile in meat are indirect, involving the modulation of rumen modification. biohydrogenation and, consequently, lipid metabolism [40]. In agreement with our data. Simon et al. [40] concluded that the addition of exogenous enzymes to the cattle diet increases polyunsaturated fatty acids and reduces saturated ones. This is extremely beneficial for human health as these acids have a potential protective effect against cancer [41]. Enriching meat with polyunsaturated fatty acids not only improves human nutrition but also has the potential to contribute to the health and well-being of impoverished populations [41]. Among saturated fatty acids, palmitic acid (C16:0) is the most common in diets, followed by stearic acid (C18:0) and myristic acid (C14:0) [42]. There is strong evidence that even-numbered saturated fatty acids increase the concentration of total and LDL cholesterol, in addition to promoting clotting, inflammation and insulin resistance, and are strongly associated with a greater risk of cardiovascular disease and type 2 diabetes [42].

Oleic acid (C18:1) is the most abundant monounsaturated fatty acid in the human diet [42]. Replacing saturated fatty acids with oleic acid has a lowering effect on total and LDL cholesterol [43,44], in addition to potentially reducing blood pressure [45]. Replacing saturated fatty acids with oleic acid can also improve glucose control and insulin sensitivity [46]. These findings prove the improvement in the quality of meat from animals fed with exogenous enzymes for human consumption, since there was a reduction in palmitic acid and the sum of saturated fatty acids. Therefore, the addition of exogenous fibrolytic enzymes, by improving the fatty acid profile of the meat, provides better quality food for human consumption.

5. Conclusions

In conclusion, under these experimental conditions, there was no effect of adding fibrolytic enzymes to the diet of cattle in the confinement phase, which may be related to not having improved nutrient digestibility. However, the consumption of fibrolytic enzymes changed the rumen fatty acid profile, especially a greater amount of VFA. A greater quantity and proportion of acetic acid in rumen fluid may have been the precursor to greater fat in meat; as well as modulating the fatty acid profile, reducing SFA and increasing UFA in meat.